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Neuronal master regulator SRRM4 in breast cancer cells facilitates CNS-acclimation and colonization leading to brain metastasis
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Neuronal master regulator SRRM4 in breast cancer cells facilitates CNS-acclimation and colonization leading to brain metastasis
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NEURONAL MASTER REGULATOR SRRM4 IN BREAST CANCER CELLS FACILITATES
CNS-ACCLIMATION AND COLONIZATION LEADING TO BRAIN METASTASIS
by
Krutika Tushar Deshpande, M.S
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MEDICAL BIOLOGY)
MAY 2021
Copyright 2021 Krutika Tushar Deshpande
ii
Dedication
To my husband Tejas, and my parents
for their unwavering belief in my abilities and their unending love for me.
iii
Acknowledgements
Thesis Committee Members
Dr. Alan Epstein (Chair)
Dr. Josh Neman (Mentor)
Dr. Yves DeClerck
Medical Biology Program
Dr. Martin Kast (Program Director)
I want to first express my sincerest thanks to Dr. Josh Neman for being a great mentor over the
last five years. His academic and emotional support has been instrumental in my progress. I
started out as a curious student ready to embark on my journey towards getting my Phd, and Dr.
Neman has been with me every step of the way, encouraging my scientific curiosity. He taught
me how to organize my thoughts into tangible hypotheses and then to test them thoroughly. He
always encouraged independent thought and discussion, while providing me with the best
knowledge and resources required to succeed. Dr. Neman firmly believes in merging the
objectivity of scientific research with true empathy for the patients that are affected by the diseases
we study. I will be eternally grateful to him for helping me align my scientific pursuit with humanity
and clinical relevance. His dedication towards conducting high quality research continues to
inspire me, and I will take his teachings with me as I embark on the next stage of my professional
and personal life.
I want to express deep gratitude to the members of my committee, Dr. Alan Epstein and
Dr. Yves DeClerck. Since my qualifying exam, they have steadily guided me through my graduate
research, making sure I remained on track. Their invaluable insights and constructive feedback
shaped how I conducted my studies. I especially want to thank Dr. Alan Epstein, who welcomed
iv
all my questions and provided me with vital advice about my projects, and also about scientific
writing, publishing and professional growth. His mentorship was instrumental in motivating me
during the final phase of my thesis research.
Working with my colleagues in the Neman lab has been a joy. I especially want to thank
Vahan Martirosian, who started with his PhD studies in the lab the same time that I did. He helped
me with planning and troubleshooting my experiments, and it was always a treat to have long
discussions with him on Friday evenings in the lab when we would bounce ideas off each other.
Some of these ideas eventually became full-fledged experiments which led to valuable data.
One of the highlights of my graduate studies was being able to collaborate with scientists
from different departments and fields and to learn from their expertise. I thank Dr. Ling Shao and
Ms. Brooke Nakamura for contributing their knowledge and also their resources to my project and
helping me with establishing primary neuronal cultures from mice. I also want to thank Dr. Frank
Attenello at USC Neurosurgery for his help with establishing patient-derived brain-metastatic
tumor cells that were instrumental in providing me a mechanistic perspective in my studies.
I also want to thank the many cores and resources at USC that facilitated my research. I
am immensely grateful to Ms. Ivetta Vorobyova who helped me with intracardiac injections for our
brain metastasis mouse model, and then imaged the animals over many months so that we could
successfully complete our in vivo experiments, even during the COVID-19 pandemic. I thank Ms.
Bernadette Masinsin and Mr. Jeffery Boyd at the FACS core who helped me establish and select
stably transduced cell lines. I also want to thank Ms. Heather Johnson and Ms. Angela Garrison
who helped me schedule the confocal microscope innumerable times during the week and
weekends, so that I could get best quality images and quantification data.
Our lab works very closely with breast cancer survivors and patient advocates. It was an
honor for me to be able to interact with these champions and their families, and to be privy to their
powerful stories. Their resilience, and desire to learn and grow even in the face of adversity
continues to inspire me. I especially want to thank Ms. Michelle Rakoff, Ms. Michelle Atlan, Ms.
v
Andrea Hutton, and Ms. Sharon Schlesinger for encouraging me to merge the clinical and the
biological sides of science to get a human perspective on cancer.
My experience of graduate school was made richer by the presence of many friends that
I made over the years. I especially thank my friends Urvi Shroff, Kaivalya Shevade, and Rucha
Bapat with whom I shared the common goal of earning a PhD, and who were always with me in
happy as well as tough times through the last 5 years. I also thank my best friend Nupur Kathikar,
who was source of much needed comfort and laughter even though she was in a different country.
I have lived away from my home in India for almost a decade, and I want to convey my
indebtedness to my parents Dr. (Mrs) Sushama Deshpande, and Mr. Tushar Deshpande for
giving me the wings to fly and to pursue my dreams. They have been a constant source of love
and encouragement from afar, making sure I was safe, healthy and happy while I worked towards
achieving my goals. They always set great examples for me since childhood, and I love them for
putting their belief in me, always.
Last but not least, I want to express my deepest gratitude and love for my husband Tejas
Narsule. He stood by me, supported my dreams, and was the person I drew strength from all
through my graduate career. Even though my admission to USC meant that we lived in two
different cities for the duration of my graduate studies, he made sure the distance was never
bothersome. On his visits to LA, he would keep me company as I worked in the lab late nights on
weekdays, and even on weekends. He cared for me like a partner, friend and parent put together,
and I could not have asked for a better person with whom to share my journey and
accomplishments.
vi
Table of Contents
Dedication ..................................................................................................................................... ii
Acknowledgements ...................................................................................................................... iii
List of Figures ............................................................................................................................. viii
Abstract ......................................................................................................................................... x
Chapter I: Introduction .................................................................................................................. 1
1.1 Brain metastasis- a significant clinical complication: .......................................................... 1
1.2 Cancer type and propensity of brain metastasis ................................................................ 2
1.2.1 Lung Cancer ................................................................................................................ 2
1.2.2 Breast Cancer .............................................................................................................. 3
1.2.3 Melanoma .................................................................................................................... 3
1.2.4 Other cancers .............................................................................................................. 3
1.3 Diagnosis and current treatment paradigms ....................................................................... 4
1.4 Current knowledge of affected processes and pathobiology in brain metastasis: .............. 5
1.5 Challenges and areas of advancement in brain metastasis research: ............................... 6
1.6 Scope of the current dissertation research ......................................................................... 7
1.7 Summary: ........................................................................................................................... 9
References ............................................................................................................................... 9
Chapter 2: Neuronal exposure induces neurotransmitter signaling and synaptic plasticity
mediators in tumors early in brain metastasis ............................................................................ 12
2.1 Introduction ....................................................................................................................... 13
2.2 Materials and Methods ..................................................................................................... 14
2.2.1 Cell culture ................................................................................................................. 14
2.2.2 Animals ...................................................................................................................... 15
2.2.3 RNA Isolation and qPCR ........................................................................................... 16
2.2.4 Immunofluorescence ................................................................................................. 16
2.2.5 Microscopy and Imaging ............................................................................................ 17
2.2.6 Bioinformatics ............................................................................................................ 17
2.2.7 Statistics .................................................................................................................... 18
2.3 Results .............................................................................................................................. 18
2.3.1 Neuronal exposure induces NT responsiveness and synaptic plasticity in tumor cells
............................................................................................................................................ 18
2.3.2 Breast cancer cells display enhanced reliance on paracrine microenvironmental
GABA in presence of neurons ............................................................................................ 22
2.3.3 Neuron and neurotransmitter exposure induces resurgence of Reelin expression in
tumor cells .......................................................................................................................... 28
vii
2.4 Discussion ........................................................................................................................ 36
References ............................................................................................................................. 38
Chapter 3: SRRM4-mediated REST to REST4 dysregulation facilitates CNS-acclimation and
colonization in breast-to-brain metastases ................................................................................. 41
3.1 Introduction ....................................................................................................................... 41
3.2 Materials and Methods ..................................................................................................... 44
3.2.1 Cell culture ................................................................................................................. 44
3.2.2 Animals ...................................................................................................................... 45
3.2.3 Intracardiac injection of tumor cells and animal imaging ........................................... 45
3.2.4 RNA isolation and qPCR analysis ............................................................................. 46
3.2.5 Immunofluorescence ................................................................................................. 46
3.2.6 Microscopy and Imaging ............................................................................................ 47
3.2.7 Bioinformatics ............................................................................................................ 47
3.2.8 Statistical analysis ..................................................................................................... 48
3.3 Results .............................................................................................................................. 48
3.3.1 Breast to brain metastases show reduced expression and nuclear localization of
REST .................................................................................................................................. 48
3.3.2 REST dysregulation in BBMs is mediated by SRRM4 and alternative splice product
REST4 ................................................................................................................................ 51
3.3.3 Enhanced SRRM4 expression confers less dormant and more proliferative phenotype
in breast cancer cells .......................................................................................................... 57
3.3.4 SRRM4 facilitates CNS-acclimation and provides proliferative advantage to BC cells
in the neuronal microenvironment ...................................................................................... 60
3.4 Discussion ........................................................................................................................ 63
References ............................................................................................................................. 65
Chapter 4: Conclusions and future directions ............................................................................ 68
4.1 Recap ............................................................................................................................... 68
4.2 Limitations and future directions ....................................................................................... 68
4.3 Therapeutic avenues ........................................................................................................ 70
References ............................................................................................................................. 71
Appendix .................................................................................................................................... 73
Bibliography ................................................................................................................................ 75
viii
List of Figures
Figure 1.1 Scope of the current dissertation research ................................................................. 8
Figure 2. 1 Characterization of Neurotransmitter signaling and synaptic plasticity mediators in
tumor cells exposed to neurons. ................................................................................................ 19
Figure 2. 2 Target validation in tumor cells. ............................................................................... 21
Figure 2. 3 Neuronal exposure activates transcription factor CREB in tumor cells .................... 22
Figure 2. 4 Autocrine Dopaminergic cells persist within the neuronal microenvironment .......... 23
Figure 2. 5 Breast cancer cells display enhanced reliance on paracrine microenvironmental
GABA in presence of neurons. ................................................................................................... 25
Figure 2. 6 Reliance on microenvironmental GABA is an early CNS acclimation strategy in
breast cancer. ............................................................................................................................. 26
Figure 2. 7 Tumor cells show functional localization of NT vesicle protein SNAP25 in presence
of neurons. ................................................................................................................................. 27
Figure 2. 8 Establishment of dormant SKBR3 breast cancer cells. ............................................ 29
Figure 2. 9 NT stimulation induces Reelin expression in dormant breast cancer cells. ............. 30
Figure 2. 10 Reelin expression is lost in primary metastatic breast and lung cancer. ................ 31
Figure 2. 11 Reelin is expressed in brain-metastatic breast and lung tumor tissue. .................. 32
Figure 2. 12 Enhanced Reelin expression is associated with worse prognosis and brain relapse
in breast cancer. ......................................................................................................................... 33
Figure 2. 13 Neuron exposure lowers Histone De-acetylase (HDAC) activity in tumor cells,
facilitating epigenetic upregulation of RELN. .............................................................................. 33
Figure 2. 14 Neuronal contact enhances Reelin protein expression in tumor cells. ................... 34
Figure 2. 15 Paracrine Reelin-mediated induction of autocrine Reelin signaling in breast cancer
cells is crucial in breast-to-brain metastasis. .............................................................................. 35
Figure 3. 1 REST expression is not prognostic in breast and lung cancer and melanoma. ....... 49
Figure 3. 2 BM breast cancer and melanoma tissues show reduced REST expression. ........... 49
Figure 3. 3 Breast-to-brain metastatic cells show reduced nuclear REST. ................................ 50
Figure 3. 4 Nuclear REST/RILP remains unchanged between non-BM and BM tumor cells. .... 51
Figure 3. 5 High SRRM4 is significantly associated with worse overall survival in breast cancer.
................................................................................................................................................... 52
Figure 3. 6 BBM tissues show enhanced SRRM4 and REST4 activity. ..................................... 53
Figure 3. 7 SRRM4 expression and nuclear localization is enhanced in BBMs, resulting in splice
isoform REST4. .......................................................................................................................... 54
Figure 3. 8 Establishment of SRRM4 overexpressed (SKBR3
OE
) and SRRM4 knockdown
(SKBR3
KD
) cell lines. .................................................................................................................. 55
ix
Figure 3. 9 SRRM4 overexpression facilitates accelerated brain metastasis and contributes to
worse overall survival in vivo. ..................................................................................................... 56
Figure 3. 10 Analysis of BM lesions from xenografted animals. ................................................. 57
Figure 3. 11 SRRM4 regulates proliferative capacity in breast cancer cells. ............................. 58
Figure 3. 12 Low SRRM4 expression confers dormant phenotype and reduced REST4
expression in breast cancer cells. .............................................................................................. 59
Figure 3. 13 SRRM4
KD
cells show enhanced chemoresistance. ................................................ 60
Figure 3. 14 Neuronal exposure enhances SRRM4 activity in breast cancer cells. ................... 60
Figure 3. 15 SRRM4 promotes expression of CNS-specific synaptic plasticity mediators in
breast cancer cells, within the neuronal microenvironment. ....................................................... 62
Figure 3. 16 Enhanced SRRM4 expression provides proliferative advantage in the neuronal
microenvironment in vitro. .......................................................................................................... 63
Figure 3. 17 Role of SRRM4 in facilitating breast-to-brain metastasis ....................................... 65
Table 1 Genes of interest and primer sequences in Chapter 2 .................................................. 73
Table 2 Genes of interest and primer sequences in Chapter 3. ................................................. 74
x
Abstract
Brain metastases (BMs), diagnosed in 20-40% of cancer patients, remain a significant
complication of advanced carcinomas. In order to successfully combat the rising incidence of BM
induced mortality, it is imperative to understand the affected processes and molecular
mechanisms that facilitate the establishment of brain metastases.
To successfully form BMs, tumor cells that extravasate into the brain parenchyma must
adapt to and survive within the new microenvironment. This includes interaction with the various
cells that comprise the neural metastatic niche including neurons, glia, and cells of the blood
brain/ blood cerebrospinal fluid barriers (BBB and BCSFB). Furthermore, tumor cells must also
alter their own genetic and metabolic landscape to augment metastatic competency within the
central nervous system (CNS).
Previous studies have reported that activated astrocytes promote tumor cell proliferation
in the brain by releasing pro-metastatic cytokines and chemokines (IL-6, TNFa). Microglia have
also been known to facilitate CNS colonization by tumor cells through activation of Wnt signaling.
However, despite the fact that neurons contribute to a significant portion of cellular signaling within
the CNS, there are currently no studies investigating the role of neuronal input in facilitating tumor
colonization in the brain. Additionally, even though acquisition of neuronal attributes has been
reported in brain-metastatic tumor cells, and presence of CNS-primed cells within the primary
tumor has been described in breast cancer, the mechanisms underlying the gain of CNS-
metastatic competency in tumor cells are largely unexplored.
This dissertation aims to investigate the early stages of brain-metastatic colonization in
two parts: (Part I) Characterizing early tumor-neuron interactions and the induced CNS-adaptive
changes in tumor cells; and (Part II) Elucidating a novel mechanism that promotes CNS
acclimation and colonization competency in brain-seeking breast cancer cells.
xi
The results from part I show that early interaction with neurons induces neurotransmitter
(NT) responsiveness and synaptic plasticity mediators in tumor cells. On NT-dependent
characterization of tumors, we see that breast cancer cells become reliant on paracrine
microenvironmental GABA to facilitate CNS colonization; while autocrine Dopaminergic tumor
cells persist in the brain-metastatic niche. We also show that neuron-mediated upregulation of
ECM glycoprotein Reelin in tumor cells is crucial early in brain metastatic progression, particularly
in breast cancer.
In part II of this dissertation, we investigate the atypical activation of neuron-specific splice
protein Serine/Arginine Repetitive Matrix protein 4 (SRRM4) in breast-to-brain metastases. Our
studies show that SRRM4-mediated dysregulation of neuronal gene suppressor REST into its
splice isoform REST4 promotes CNS colonization in breast cancer. Specifically, enhanced
SRRM4 expression promotes enhanced proliferation, supports expression of CNS-specific target
genes, and facilitates tumor cell growth in the neuronal microenvironment in vitro. In vivo, high
SRRM4 expression and activity augments brain metastasis, and contributes to worse overall
survival in breast cancer; while low SRRM4 expression is associated with tumor dormancy, and
provides no CNS-metastatic advantage.
Taken together, the results from this dissertation research comprehensively advance our
knowledge of the early mediators of brain-metastatic colonization, and also identify potential
therapeutic targets for future use in at-risk patients.
1
Chapter I: Introduction
1
1.1 Brain metastasis- a significant clinical complication:
Brain metastases are the most common cause of intracranial neoplasms in adults with invasive
cancers. 20-45% of cancer patients are diagnosed with brain metastases in their lifetime
1
.
Metastatic brain tumors occur at a much higher rate than both adult and pediatric primary brain
tumors, and are the major cause of mortality from malignant brain disease Intracranial metastasis
is associated with worse prognosis (< 1 year), moderate to severe neurodegeneration, and overall
reduction in quality of life
2,3
. The incidence and severity of brain metastatic disease varies
according to the origin of the primary tumor, and the treatment strategy followed for the patient.
Lung, breast, melanoma, colorectal, and renal cancers show most proclivity for the brain, followed
more uncommonly by thyroid, gastro-intestinal (GI), and prostate cancers
4
.
Due to advances in control of primary tumors and extra-cranial metastases, as well as
superior methods of early detection, patient survival has increased. This has however led to rising
numbers of patients being diagnosed with brain metastases, with or without concomitant
extracranial disease. Tumors originating from different tissues show varying latency to
metastasize to the brain. This can be explained by the aggressiveness of the tumor type, modes
of dissemination, development of resistance to therapy, or molecular affinity for the neuronal
niche. Despite considerable advances in elucidating the cellular and molecular events underway
in metastasis, few treatments have been realized and as such prognoses remains poor. The
current standard of care for brain metastases includes surgical excision, whole brain radiation
therapy (WBRT), and stereotactic radiation surgery (SRS) combined with steroids
5
. Targeted
1
This chapter is modified from my first author publication in Cold Spring Harbor Perspectives in
Medicine. (Deshpande K, Buchanan I, Martirosian V, Neman J. Clinical perspectives in brain
metastasis. Cold Spring Harbor Perspectives in Medicine 2020 Jun 1;10(6):a037051. doi:
10.1101/cshperspect.a037051)
2
molecular therapies show little or no effect mainly because of their inability to penetrate the elusive
blood brain barrier (BBB)
6
. Thus, with limited options available, malignant tumors take refuge in
the brain and escape most forms of intervention, contributing to patient mortality.
The management of brain metastases is an urgent unmet clinical need and warrants
immediate attention and investigation.
1.2 Cancer type and propensity of brain metastasis:
Metastasis is a carefully orchestrated process involving breakage of cells from the primary
tumor, gain of invasive properties, and interaction with the microenvironmental niche to establish
tumors at distant sites. Clinically, different kinds of tumors show varying proclivities in their ability
to successfully metastasize within the central nervous system (CNS). The most common primary
sites are lung, breast, and melanoma in that order. These are followed more uncommonly by
thyroid, GI, and prostate cancers. Propensity of occurrence of brain metastasis can also be further
ranked by clinical subtypes of solid cancers.
1.2.1 Lung Cancer:
Lung cancer is the primary tumor in about 40-50% of patients diagnosed with brain metastases
(BM). 50% of lung cancer BMs occur at disease presentation/diagnosis, and sometimes the CNS
is the only location of dissemination. This highlights the aggressive nature of the primary tumor,
and the short latency period seen for lung-to-brain metastasis. Due to higher incidence, non-small
cell lung cancer (NSCLC) comprises a higher percentage of brain metastases than small cell LC
(SCLC), but recent studies show that SCLC has a higher biological propensity for CNS spread
7
.
About 7% of patients with NSCLC present with brain BMs at diagnosis and about 25-30% develop
BMs over the course of their disease
8
. For Small Cell Lung Carcinoma (SCLC) patients with brain
metastases, the median overall survival (OS) is 4.9 months, and 1.9-2.4 months for
leptomeningeal metastases. SCLC usually manifests as multiple brain lesions, and thus surgery
is not a common therapeutic avenue for patients.
3
1.2.2 Breast Cancer:
Up to 30% of all breast cancer patients are diagnosed with brain metastases within their lifetime.
Out of these, patients diagnosed with the triple-negative subtype (TNBC) show increased risk of
brain metastasis, followed by the Her2
+
and hormone positive (ER
+
/PR
+
) subtypes
9,10
. Successful
control of extra-cranial breast carcinoma, and emergence of advanced diagnostics has increased
the incidence and diagnosis of breast-to-brain metastases (BBMs). Breast cancer cells that
escape chemotherapy or surgical extraction can persist in the patient’s body and eventually lead
to BBMs. Breast cancer is unique in its longer latency to forming brain metastases
11
and this has
been associated with the acquisition of neuronal characteristics
12,13
. A recent clinical study also
substantiated that breast-to-brain metastases are able to acquire mutations in clinically targetable
genes (HER2) and that about 20% of Her2-negative diagnosed breast tumors showed a switch
to Her2-positivity in the brain
14
. This highlights the need for molecular characterization of highly
invasive breast tumors for patients over the course of their disease, to avoid non-relevant
therapeutic interventions.
1.2.3 Melanoma:
Brain metastases are a significant complication in patients diagnosed with advanced melanoma.
About 20% of these patients present with BMs at diagnosis, and about 40-50% develop BMs
during the course of the disease
15
. Melanoma BMs (MBMs) can present as single or multiple intra-
cranial lesions, with better prognostic index for patients presenting with single BM at melanoma
diagnosis. The median survival for melanoma patients with BMs is 3 months (without treatment)
to about 9 months (with treatment)
16
.
1.2.4 Other cancers:
Brain metastases from renal cell cancers (RCC) largely occur asynchronous with primary disease,
and about 6% of patients eventually develop CNS lesions after initial nephrectomy. Out of all
4
gastrointestinal (GI) cancers, patients with colorectal cancer most commonly develop BMs. About
40-70% of the time, these BMs are diagnosed simultaneously with significant extracranial tumor
load, particularly in the lung
17
. BMs can present as solitary or multiple lesions. Most BMs are
solitary with 20% of diagnoses having two or less lesions, although 30% of cases have three or
more lesions. Lung and melanoma primarily lead to multiple BMs, whereas breast, renal, colon
usually present as single lesions.
1.3 Diagnosis and current treatment paradigms:
Sixty to 75% of brain metastases (BMs) are symptomatic, whereas a smaller number of patients
harbor central nervous system (CNS) metastases without any neurological signs. Imaging is
crucial in the detection and diagnosis of BMs. It is used to confirm previously undiagnosed CNS
metastases in patients with neurological symptoms, to confirm brain involvement in a person with
systemic metastatic disease, and for staging and monitoring of BMs over the course of therapy.
Imaging is also essential before surgery to plan safe excision of the tumor from the brain
18
. Using
conventional therapy to treat brain metastases (BMs) has limited to no efficacy. Personalization
of metastatic cancer treatment using molecular profiling to select therapies that benefit the
patients while avoiding irrelevant therapies with potential toxicity is limited by poor penetration of
most chemotherapeutic agents across the BBB. In addition, the high sensitivity of brain and its
significance limit radiotherapy and radiation dosage and constrain resection margins. Therefore,
neurosurgical resection of individual BMs, followed by standard chemotherapy or whole brain
radiotherapy (WBRT) is the current standard of care
19
. However, these interventions are
associated with risk of cognitive decline, and recurrence of the tumor leads to the failure of
radiotherapy.
Each stage of brain metastasis process can be a target for personalized intervention or,
at least, better prognosis regarding the molecular properties of the metastases. Metastatic tumor
5
cells evolve after establishing themselves within the brain parenchyma. Genomic studies have
shown that the molecular profile of BMs is distinctive from the primary tumor. These alterations
are clinically critical in patients with inoperative BMs in which primary tumors remain as the only
tissue available for genomic analysis for the selection of the proper therapy
20,21
. In coming years,
further DNA- and RNA-based high- throughput sequencing comparing primary and BMs genomic
profiling will reveal addition- al information on the metastatic process and, subsequently, the
potential individual therapies.
Research is currently also focused on understanding mechanisms involved in
metastatic/tumor dormancy, which confers chemoresistance to tumor cells causing them to
persist in the patient for extended periods of time. These tumor cells which can then be reactivated
in suitable microenvironmental conditions, have been implicated as ‘seeds’ for brain metastasis,
particularly in breast cancer
22,23
. Thus, future therapeutic success also relies on complete
characterization and subsequent targeting of this dormant cell population.
1.4 Current knowledge of affected processes and pathobiology in brain metastasis:
According to the “seed and soil” hypothesis, dynamic interplay between tumor cells and a
permissive microenvironment is crucial for the successful establishment of metastases. Once
tumor cells extravasate into the brain through the cells lining the fluid barriers of the BBB or the
BCSFB, they can go through immune clearance, apoptosis or dormancy
24
. Only cells that can
interact with the neural niche and alter their own genetic/proteomic landscape can subsequently
colonize the brain to form clinical macrometastases.
The CNS metastatic niche is a well-regulated mileu of cells, each with essential functions.
Cellular homeostasis is maintained in the brain with the help of glial cells like astrocytes and
oligodendrocytes, as well as microglia
25,26
. Recent studies have described the pro-metastatic role
of reactive astrocytes through direct interaction with tumor cells via gap junctions
27
, or through
secreted molecules that induce tumor cell proliferation
28,29
. Microglia, the resident immune cells
6
in the brain, can secrete growth factors that augment tumor cell growth and aid in acquiring
chemoresistance
30
. A previous study conducted by Dr. Josh Neman also showed that brain-
trophic tumor cells create a tumor conducive metastatic niche by facilitating the differentiation of
neural progenitor cells into tumor-associated astrocytes via a BMP-2 secretion
12
. Thus, the brain
microenvironment facilitates tumor invasion and colonization.
Furthermore, as resident brain cells react to metastatic invasion, tumor cells also need to
adapt to the neural niche in order to successfully colonize it. Zeng et al recently described the
presence of a sub-population of aggressive tumor cells within primary breast cancers that express
increased levels of Glutamate receptor GluN2B. These cells, now primed for brain metastasis,
can respond favorably to paracrine Glutamate widely available in the brain, facilitating
colonization
31
. Dr. Josh Neman described the gain of neuronal characteristics in breast-to-brain
metastases (BBMs) that were not observed in primary tumors. These BBMs acquire expression
of variables (GABA receptors, ECM glycoprotein Reelin) otherwise only expressed by GABAergic
neurons within the brain
13
. Howe et al also reported the upregulation of cell surface proteome
regulator Rab11b in BBMs early in brain metastasis, which allows them to bind to the brain ECM
via integrin B1
32
. Taken together, these studies show that neural acclimation is requisite for
successful brain metastasis, particularly in breast cancer.
1.5 Challenges and areas of advancement in brain metastasis research:
Neurons constitute a large portion of the cellular bulk in the brain, apart from glial and
endothelial cells. Neurons transmit information between cells through electrochemical impulses
mediated by neurotransmitters (NT) like Glutamate, GABA, Serotonin, Acetylcholine, Dopamine,
etc
33
. The short- and long-term neuronal responses resulting from these communications
constitute neuronal synaptic plasticity, which is essential for CNS homeostasis and the formation
of memory
34
. Moreover, as described in the previous section, the expression of NT signaling
genes in brain metastatic tumors suggests that response to neuronal biochemical cues is
7
important for BM progression. Despite neuron-mediated cell signaling being critical in normal and
pathological brain function, there are no current studies that investigate the interactions between
tumor cells and neurons during brain metastasis. Since tumors from different primary sites show
varying proclivities for CNS metastasis, it is essential to characterize the commonalities and
differences in their interactions with cells of the neural niche, particularly with neurons. Thus, to
advance our current understanding of the CNS tumor microenvironment, it is essential to elucidate
the influence of early tumor-neuron interaction on the establishment and progression of brain
metastasis.
The brain is structurally and functionally isolated from the rest of the body, and thus
constitutes a metastatic niche that is unique in many ways. To successfully colonize the CNS,
brain-seeking tumor cells must overcome the blood brain/CSF barriers, tumor dormancy,
apoptosis, and potential metastatic suppression by brain resident cells. Furthermore, they must
adapt to the neural microenvironment by altering the expression of genes that enable CNS-
acclimation. Although the induction of neural characteristics in tumor cells has been described,
there are no studies that investigate the drivers of CNS metastatic competency in tumor cells.
Identification of transcriptional or functional regulators of brain-metastatic colonization will
facilitate therapeutic intervention or prevention early in the course of brain metastasis.
1.6 Scope of the current dissertation research
Through the research presented in this dissertation, I aim to elucidate the affected
processes and mechanisms that affect CNS colonization by tumor cells. To this effect, the
research was conducted in two parts, examining different aspects facilitating brain-metastatic
colonization:
In the first part of dissertation, I characterized early tumor-neuron interactions, and the
induced CNS-adaptive changes in tumor cells. Utilizing pure neuronal cultures, and brain-naïve
and patient -derived brain-metastatic cells in vitro, I assessed the induction of neurotransmitter
8
(NT) and synaptic plasticity mediators in breast and lung cancer cells upon neuronal exposure,
particularly on direct contact. Reliance on microenvironmental GABA in brain-trophic breast
cancer cells was examined in vivo. Neuron-mediated upregulation of ECM glycoprotein Reelin in
breast cancer cells was found to be critical for breast-to-brain colonization. This characterization
of early tumor-neuron interactions will be discussed in chapter two.
In the second part of the dissertation, I investigated a novel mechanism that promotes
CNS-metastatic competency and neuronal acclimation in breast cancer cells. We demonstrated
the enhanced activity of neural-specific splicing factor Ser/Arg Repetitive Matrix Protein 4
(SRRM4) in breast-to-brain metastases. Our results show that SRRM4 regulates CNS
competency, proliferative capacity, and CNS acclimation in breast cancer cells and ultimately
promotes accelerated brain metastasis. A detailed study of the role of SRRM4 in breast-to-brain
metastasis will be elaborated in chapter three.
Figure 1.1 Scope of the current dissertation research
9
1.7 Summary:
In this chapter, we establish that although the control of extracranial disease has improved
tremendously in recent years, the management of brain metastases (BMs) still remains an urgent
unmet clinical need. Limited knowledge of the pathobiology of BMs, lack of actionable clinical
targets, combined with the sensitive nature of the CNS as a metastatic site makes the treatment
or prevention of brain metastasis complex.
In recent years, studies have focused on elucidating the microenvironmental mediators of
BM progression, however the role of neuronal input, and the identification of drivers of CNS-
metastatic competency in tumor cells remains understudied. This is of particular importance in
cancers like breast cancer, that manifest with brain metastases even years after primary diagnosis
and treatment. This indicates that acclimation to the neural niche is requisite for successful
colonization of the brain.
Through the research conducted as a part of this dissertation, I aim to advance our
knowledge of the microenvironmental as well as tumor-inherent regulators of CNS metastasis, in
order to derive better targeted therapies for BM patients.
References
1. Achrol AS, Rennert RC, Anders C, et al. Brain metastases. Nature Reviews Disease
Primers. 2019; 5(1):5.
2. Tabouret E, Chinot O, Metellus P, Tallet A, Viens P, Gonçalves A. Recent trends in
epidemiology of brain metastases: an overview. Anticancer Res. 2012; 32(11):4655-4662.
3. Owonikoko TK, Arbiser J, Zelnak A, et al. Current approaches to the treatment of
metastatic brain tumours. Nat Rev Clin Oncol. 2014; 11(4):203-222.
4. Valiente M, Ahluwalia MS, Boire A, et al. The Evolving Landscape of Brain Metastasis.
Trends Cancer. 2018; 4(3):176-196.
5. Lin X, DeAngelis LM. Treatment of Brain Metastases. J Clin Oncol. 2015; 33(30):3475-
3484.
10
6. Fortin D. The blood-brain barrier: its influence in the treatment of brain tumors metastases.
Curr Cancer Drug Targets. 2012; 12(3):247-259.
7. Lukas RV, Gondi V, Kamson DO, Kumthekar P, Salgia R. State-of-the-art considerations
in small cell lung cancer brain metastases. Oncotarget. 2017; 8(41).
8. Owen S, Souhami L. The management of brain metastases in non-small cell lung cancer.
Front Oncol. 2014; 4:248.
9. Niwińska A, Murawska M, Pogoda K. Breast cancer brain metastases: differences in
survival depending on biological subtype, RPA RTOG prognostic class and systemic
treatment after whole-brain radiotherapy (WBRT). Ann Oncol. 2010; 21(5):942-948.
10. Witzel I, Oliveira-Ferrer L, Pantel K, Müller V, Wikman H. Breast cancer brain metastases:
biology and new clinical perspectives. Breast Cancer Res. 2016; 18(1):8.
11. Saunus JM, Momeny M, Simpson PT, Lakhani SR, Da Silva L. Molecular aspects of breast
cancer metastasis to the brain. Genet Res Int. 2011; 2011:219189.
12. Neman J, Choy C, Kowolik CM, et al. Co-evolution of breast-to-brain metastasis and
neural progenitor cells. Clin Exp Metastasis. 2013; 30(6):753-768.
13. Neman J, Termini J, Wilczynski S, et al. Human breast cancer metastases to the brain
display GABAergic properties in the neural niche. Proceedings of the National Academy
of Sciences. 2014; 111(3):984-989.
14. Priedigkeit N, Hartmaier RJ, Chen Y, et al. Intrinsic Subtype Switching and Acquired
ERBB2/HER2 Amplifications and Mutations in Breast Cancer Brain Metastases. JAMA
Oncol. 2017; 3(5):666-671.
15. Vosoughi E, Lee JM, Miller JR, et al. Survival and clinical outcomes of patients with
melanoma brain metastasis in the era of checkpoint inhibitors and targeted therapies.
BMC Cancer. 2018; 18(1):490.
16. Chukwueke U, Batchelor T, Brastianos P. Management of Brain Metastases in Patients
With Melanoma. J Oncol Pract. 2016; 12(6):536-542.
17. Nayak L, Lee EQ, Wen PY. Epidemiology of brain metastases. Curr Oncol Rep. 2012;
14(1):48-54.
18. Fink KR, Fink JR. Imaging of brain metastases. Surg Neurol Int. 2013; 4(Suppl 4):S209-
219.
19. Ewend MG, Elbabaa S, Carey LA. Current Treatment Paradigms for the Management of
Patients with Brain Metastases. Neurosurgery. 2005; 57(suppl_5):S4-66-S64-77.
20. Han CH, Brastianos PK. Genetic Characterization of Brain Metastases in the Era of
Targeted Therapy. Front Oncol. 2017; 7:230.
21. Liao L, Ji X, Ge M, et al. Characterization of genetic alterations in brain metastases from
non-small cell lung cancer. FEBS Open Bio. 2018; 8(9):1544-1552.
11
22. Neophytou CM, Kyriakou TC, Papageorgis P. Mechanisms of Metastatic Tumor Dormancy
and Implications for Cancer Therapy. Int J Mol Sci. 2019; 20(24).
23. Boire A, Coffelt SB, Quezada SA, Vander Heiden MG, Weeraratna AT. Tumour Dormancy
and Reawakening: Opportunities and Challenges. Trends in Cancer. 2019; 5(12):762-765.
24. Singh M, Manoranjan B, Mahendram S, McFarlane N, Venugopal C, Singh SK. Brain
metastasis-initiating cells: survival of the fittest. Int J Mol Sci. 2014; 15(5):9117-9133.
25. Sofroniew MV, Vinters HV. Astrocytes: biology and pathology. Acta Neuropathol. 2010;
119(1):7-35.
26. Butovsky O, Weiner HL. Microglial signatures and their role in health and disease. Nature
Reviews Neuroscience. 2018; 19(10):622-635.
27. Chen Q, Boire A, Jin X, et al. Carcinoma–astrocyte gap junctions promote brain
metastasis by cGAMP transfer. Nature. 2016; 533(7604):493-498.
28. Wasilewski D, Priego N, Fustero-Torre C, Valiente M. Reactive Astrocytes in Brain
Metastasis. Front Oncol. 2017; 7:298-298.
29. Gong X, Hou Z, Endsley MP, et al. Interaction of tumor cells and astrocytes promotes
breast cancer brain metastases through TGF-β2/ANGPTL4 axes. npj Precision Oncology.
2019; 3(1):24.
30. You H, Baluszek S, Kaminska B. Supportive roles of brain macrophages in CNS
metastases and assessment of new approaches targeting their functions. Theranostics.
2020; 10(7):2949-2964.
31. Zeng Q, Michael IP, Zhang P, et al. Synaptic proximity enables NMDAR signalling to
promote brain metastasis. Nature. 2019; 573(7775):526-531.
32. Howe EN, Burnette MD, Justice ME, et al. Rab11b-mediated integrin recycling promotes
brain metastatic adaptation and outgrowth. Nature Communications. 2020; 11(1):3017.
33. Lovinger DM. Neurotransmitter roles in synaptic modulation, plasticity and learning in the
dorsal striatum. Neuropharmacology. 2010; 58(7):951-961.
34. Citri A, Malenka RC. Synaptic Plasticity: Multiple Forms, Functions, and Mechanisms.
Neuropsychopharmacology. 2008; 33(1):18-41.
12
Chapter 2: Neuronal exposure induces neurotransmitter signaling and
synaptic plasticity mediators in tumors early in brain metastasis
2
Abstract:
Brain metastases (BM) are responsible for neurological decline and poor overall survival.
Although the pro-metastatic roles of glial cells, and the acquisition of neuronal attributes in
established BM tumors have been described, there are no studies that investigate the initial
interplay between neurons and brain-seeking tumor cells. The aim of this study was to
characterize early tumor-neuron interactions and the induced CNS-adaptive changes in tumor
cells prior to macro-colonization. Utilizing pure neuronal cultures and brain-naïve and patient-
derived BM tumor cells, we surveyed the early induction of mediators of neurotransmitter (NT)
signaling and synaptic plasticity in breast and lung tumor cells. Reliance on microenvironmental
GABA in breast-to-brain metastatic cells (BBMs) was assessed in vivo. Our results show that
direct neuronal contact induces early expression of classical NT receptor genes (GRIA2, GRMs
3,7,8, GABBR1, GABRB1, HTR4) and mediators of neuronal synaptic plasticity (GRIN1, FOS,
NTF3, CNR1, EGR2, and RELN) in breast and lung cancer cells. NT-dependent classification of
tumor cells within the neuronal niche shows breast cancer cells become GABAergic responsive
brain metastases (GRBMs) and transition from relying on autocrine GABA, to paracrine GABA
from adjacent neurons; while autocrine Dopaminergic breast and lung tumor cells persist. In vivo
studies confirm reliance on paracrine microenvironmental GABA is an early CNS-acclimation
strategy in breast cancer. Moreover, neuronal contact induces early resurgence in Reelin
expression in tumor cells through epigenetic activation, facilitating CNS acclimation and synaptic
2
This chapter is modified from my first author original research article currently under review at
Neuro-Oncology. Deshpande K, Martirosian V, Nakamura B, Julian A, Eisenbarth R, Shao L,
Attenello F, Neman J. Neuronal exposure induces neurotransmitter signaling and synaptic
plasticity mediators in tumors early in brain metastasis. (In revision, Neuro-Oncology March 2021)
13
plasticity. Taken together, the results in this chapter establish that early tumor-neuron interactions
allow for CNS-adaptation early in the course of brain metastasis.
2.1 Introduction:
Brain metastases (BMs) are the most common cause of intra-cranial neoplasms and related
morbidity in cancer patients. Based on the “seed and soil” hypothesis, it has now been established
that once tumor cells extravasate from systemic circulation into the brain parenchyma, they can
respond to microenvironmental cues and also alter their own cellular landscape in order to
favorably adapt and develop a CNS-metastatic niche
1
. For example, the pro-metastatic role of
astrocytes and microglia in the successful establishment of brain metastases has been
investigated before
12,35,36
. Moreover, previous studies have reported the acquisition of neuronal
characteristics in brain-metastatic tumors, that are not observed in primary tumor masses
13
.
Others have demonstrated the upregulation of NMDA receptors in aggressive primary breast
tumors, which primes them for brain metastasis
31
. These studies examine inherent transcriptomic
and proteomic attributes in tumor cells that are already selected for effective CNS-colonization.
However, there are currently no reports characterizing neuron-mediated CNS-acclimation in
tumor cells, early in the brain-metastatic cascade.
Neurons contribute to a significant portion of cellular signaling within the brain through
electro-chemical communication mediated by classical neurotransmitters including GABA,
Glutamate, Acetylcholine, Dopamine and Serotonin
37
. NT-induced responses in neurons and their
recipient cells are integral to synaptic plasticity in the central nervous system (CNS)
38
. We
postulate that initial interactions between brain-seeking tumor cells and neurons induces CNS-
adaptation in tumor cells and facilitates the establishment of BMs.
In this study, we recapitulated early tumor-neuron interaction in the brain through co-
cultures using primary neurons and brain-naïve/brain-metastatic breast and lung cancer cells. We
characterized neuron-mediated neurotransmitter responsiveness and early synaptic plasticity in
14
tumor cells upon exposure to neurons. We examined NT-dependent classification of tumor cells
within the neuronal microenvironment. Furthermore, we assessed the effect of neuronal cues on
reactivation of dormant breast cancer cells, which persist long-term in the circulation or at distant
metastatic sites in patients, and have been implicated in the eventual establishment of clinical
brain metastases
39
. The current study thus provides insight into the effects of neuronal input in
facilitating microenvironmental adaptation in tumor cells early in the course of brain metastasis,
before macro-colonization.
2.2 Materials and Methods:
2.2.1 Cell culture:
Low passage Patient-derived Brain-metastatic (BM) breast (BBM 3.1) and lung (LuBM5) cells
were propagated in our lab from surgically resected BM tissue from consenting patients. On
receipt of resected tumors from USC Neurosurgery, the tissue was mechanically dissociated,
trypisinized at 37C for 10-15 minutes, and triturated. The tissue was centrifuged (500g for 2
minutes) and resuspended in cell culture media (50% DMEM-F12, 50% Neurobasal-A, 20%FBS,
2% glutamine, 1%Antibacterial-Antimycotic). MD-MB-231Br, a commercially available brain-
trophic breast cancer cell line, was lentivirally transduced to overexpress Her2 and was also used
in our study. For studies with non-brain metastatic cells, commercially available breast (BT474,
SKBR3) and lung (A549) cell lines were used. RELN gene was knocked down lentivirally in BT474
cells using shRNA (Genecopoiea CS-HSH015094-LVRU6MP). All cell lines were maintained in
DMEM-F12 (Thermo, 12634028) supplemented with 10%FBS, 1%Glutamine (Thermo,
35050061) and 1%Antibacterial-Antimycotic agent (Sigma, 15240062), and were tested for
mycoplasma using the DAPI test before use.
Primary neuron cultures:
Neural cells were isolated from whole brain tissue of postnatal day 1-4 mice using the Worthington
Papain dissociation system kit (LK003150), plated on Poly-D Lysine (Sigma, P7280-5MG), and
15
maintained in complete neuron culture medium (Neurobasal-A medium Gibco, 10888022;
supplemented with B27 (Thermo 17504044), Glutamine and Anti-Anti). On the third day post
plating, the mixed neural cell cultures were treated with Cytosine Arabinoside (Sigma, C6645-
25MG), to eliminate miscellaneous neural cell populations leaving behind pure neuronal cultures.
50% media change with complete neuron culture medium was performed every 3 days to maintain
healthy pure neurons.
Establishment of dormant breast cancer cells:
SKBR3 cells were gradually acclimated to dormancy medium over one week. Dormancy media
composition was as follows: (DMEM with no glucose, no glutamine, Thermo A1413004)
supplemented with 5% Fetal Bovine Serum (FBS), and 1%Antibacterial-Antimycotic agent
(Sigma, 15240062).
Tumor-neuron co-cultures:
Tumor cells were resuspended in complete neuron culture medium and seeded onto established
neuronal cultures (1:60 tumor cell to neuron ratio) to model tumor-neuron interaction. At 48h post
seeding, tumor cells were collected from co-cultures for qPCR, or co-cultures were fixed with 4%
Formaldehyde for immunofluorescence studies.
2.2.2 Animals:
Female athymic mice on BalbC background were purchased from Jackson laboratories The
animals were maintained in sterile cages under 12 hour light/dark cycles and were provided with
food and water ad libitum. Animal procedures were performed under approved IACUC protocols
and guidelines. Before surgical implantation of tumor cells, each animal was anesthetized using
Isofluorane for the duration of the procedure. 1x10
5
MDA-MB231Br cells (2ul volume) were
injected intracranially into the cerebellum of the mice. Animal health was monitored and visible
distress/10% loss of BW was specified as humane experimental endpoint. Injected animals in the
study were divided into three groups: untreated (n=5), and animals treated with early
16
administration of Valproic acid (VPA; n=5), vs. late administration of VPA (n=5). Mice were treated
with 200mg/kg VPA intraperitoneally for 14 days post-transplantation.
2.2.3 RNA Isolation and qPCR:
Cells from various conditions were harvested by trypsinization for 3-5 minutes at 37C. Trypsin
(Thermo, 25300-120) was neutralized with culture medium supplemented with FBS, and the cell
suspension was centrifuged at 1000rpm for 1 minute. The supernatant was discarded, and the
pellet was processed immediately, or frozen for subsequent use. RNA isolation was performed
using the Qiagen RNeasy mini kit (Qiagen, 74136). RNA concentration was determined using a
Nanodrop (Varioskan) and cDNA was synthesized using the Maxima First Strand cDNA synthesis
kit (Thermo, K1642). PowerUp SYBR Green Master Mix (Thermo, A25918) was used for all qPCR
reactions performed using the QuantStudio VI thermocycler. All primers used for qPCR analysis
were purchased from IDT (Table 1). Human Neurotransmitter receptor (330231 PAHS-060ZA)
and Synaptic Plasticity (330231 PAHS-126Z) arrays were purchased from Qiagen to conduct the
mRNA expression screens for all cell lines and conditions.
2.2.4 Immunofluorescence:
Human primary tumor tissue microarrays were obtained from Biomax US (Breast PM2a-ER, Lung
LC241l). Patient-consented surgically resected brain-metastatic tumor tissues acquired via USC
Neurosurgery were formalin fixed, paraffin embedded, and processed into 10um thick sections
by the histology core at USC. Standard immunohistochemistry protocol was followed for paraffin-
embedded tissue sections and acidic antigen retrieval was performed as necessary (Citrate Buffer
(10mM, pH 6.0) at >80C for 20 minutes, and then RT for 10 minutes), followed by blocking (50%
Seablock in 1XPBS with 0.3M Glycine) at RT for 1 hour. For IF studies with cells, tumor cells/co-
cultures were fixed with 4% paraformaldehyde for 10 minutes at RT, and then washed twice with
1X PBS before staining. Membrane permeabilization was performed as necessary with 0.3%
17
Triton X-100 for 20 minutes, followed by blocking at RT for 1 hour. Antibody incubations on cells
and tissues were performed overnight at 4C. The antibodies used were as follows: phospho-
CREB Ser 133 (Abcam ab32096, 1:500), HER2 (Thermo MA5-14057, 1:500), GABA (Genetex
GTX125988, 1:100), SNAP25 (Abcam ab31281, 2ug/ml), Dopamine (Abcam ab6427, 1:500),
Reelin (Genetex GTX37552, 1:100) SOX9 (Novus Biologicals AF3075, 10ug/ml) , SOX2 (Novus
Biologicals AF2018, 15ug/ml), p16-ARC (Abcam ab51243 1:100), phospho-p38 (Biorbyt orb9426,
1:35), phospho-ERK(Cell Signaling Technology 9101S, 1:500). All secondary antibodies
(fluorophore conjugated) were purchased from Jackson ImmunoResearch and used at 1:300
(Goat Anti-rabbit Cy3, Goat Anti-rabbit 647, Donkey Anti-Goat 488). Phalloidin (488 or 647
conjugated) was used to stain cellular actin where necessary. Stained cells and tissue sections
were mounted in Prolong Gold Antifade reagent with DAPI (Invitrogen) for imaging and long-term
storage.
2.2.5 Microscopy and Imaging:
Confocal imaging was performed using the Leica SP8 microscope. For quantification of GABA
and dormancy markers, mean intensity per cell was measured in ImageJ, by drawing a region of
interest (ROI) around each cell. Mean intensity for the different groups/conditions measured was
then plotted in GraphPad Prism for statistical analysis.
2.2.6 Bioinformatics:
Gene expression data from TCGA datasets (normal tissue, primary tumor, metastatic tissue)
(Breast: TARGET GTEx, n=1212; and Lung: TARGET GTEx, n=1122) were visualized and
analyzed using UCSC Xena, a platform for multi-omic clinical/phenotypic data. Kaplan-Meier
survival analysis on breast and lung cancer datasets - TCGA (overall survival) and Bos-Massague
(morbidity from brain relapse)- for RELN was performed using SurvExpress, an online biomarker
validation and survival analysis tool. High vs. Low gene expression thresholds were determined
18
internally by the SurvExpress data analysis software. Gene expression values from the 84-gene
arrays were visualized as clustergrams using Heatmapper, a web-based interactive data
visualization tool. For the clustergrams, Ct values obtained from qPCR analysis were normalized
to the same housekeeping gene and plotted in Heatmapper as Log10 of absolute expression
values. Row Z scores for each map were based on internal controls from Heatmapper. Venn
diagrams were constructed using a freely available web tool designed by the Bioinformatics and
Evolutionary Genomics (BEG) department at Ghent University, Belgium.
2.2.7 Statistics:
Statistics were performed using GraphPad Prism. t-tests were used to compare expression
between two different cell types/conditions. One/Two-way Anova was used to compare more
than 2 groups/conditions. Statistical significance and Hazard Ratio for survival data from in vivo
experiments was calculated using Log-Rank Test.
2.3 Results:
2.3.1 Neuronal exposure induces NT responsiveness and synaptic plasticity in tumor
cells
Within the CNS, neurons communicate primarily through classical neurotransmitters (NTs)
Glutamate, GABA, Dopamine, Serotonin, Acetylcholine. Neuronal activity resulting from these
chemical inputs constitutes synaptic plasticity
34,40
. We wanted to characterize tumor cells as
responders to neurotransmitter (NT) and synaptic cues and determine changes which lead to
colonization through acclimation to the neural microenvironment. We first conducted a wide
survey of NT receptor and synaptic plasticity gene expression in brain-metastatic (BM) and
primary (non-BM) breast and lung cancer cells that were either co-cultured with neurons, or
acclimated to neuron-conditioned media (NCM; Figure 2.1 A,B). Non-BM breast (SKBR3, BT474)
and lung (A549) cell lines, as well as patient-derived BM breast (BBM 3.1) and lung (LuBM5) cells
were used for our study. We then identified NT receptor mRNA (GRIA2, GRM3, GRM7, GABBR1,
19
GABRB1) and synaptic plasticity mRNA (CNR1, EGR2, FOS, GRIN1, GRM4, GRM8, NTF3,
RELN) commonly upregulated in both BM and non-BM breast and lung cancer cells in co-culture
with neurons (Figure 2.1 C,D). We then validated selected target mRNA to confirm whether their
regulation was reliant on direct/indirect tumor-neuron contact.
Figure 2. 1 Characterization of Neurotransmitter signaling and synaptic plasticity mediators in tumor cells
exposed to neurons. Clustergram representing mRNA expression of human neurotransmitter receptor (A) and (B)
synaptic plasticity genes in non-BM and BM breast and lung cancer cells in three conditions -tumor only control, tumors
treated with NCM, tumors co-cultured with neurons. (C, D) Venn diagram and graphical representation of genes
commonly upregulated in all samples co-cultured with neurons, relative to their respective controls (tumor only). Black
bars represent breast cancer cells (SKBR3, BT474, BBM 3.1) and red bars represent lung cancer cells (A549, LuBM5).
Data in graph for each cell line represented as fold change in expression (Log10 scale) (mean and SEM) relative to
tumor cells only.
GRIA2 and GRIN2B encoding ionotropic AMPA- and NMDA-type Glutamate receptor subunits
respectively
41,42
, displayed significantly enhanced expression in all tested tumor cells only upon
direct co-culture with neurons (Figure 2.2 A,B). GRMs 3, 7, 8, encoding three metabotropic
Glutamate receptor subunits
43
were also upregulated significantly in all tumor cells in co-culture
with neurons (Figure 2.2 C,D,E). GABBR1, a component of a heterodimeric GPCR (type B) for
GABA
44
increased significantly in SKBR3, BBM 3.1 and LuBM5 cells grown in NCM, but was
20
found to be downregulated in BT474 and A549 cells exposed to neurons (Figure 2.2 F). GABRB1,
encoding GABA receptor type-A subunit B1
45
showed less distinct proclivity for direct versus
indirect neuronal contact and showed increases in both conditions in all tumor cells (Figure 2.2
G). In contrast, Serotonin receptor subunit HTR4
46
was upregulated only in tumor cells in co-
culture (Figure 2.2 H).
EGR2 (Figure 2.2 I) which encodes for a transcription factor involved in early synaptic
plasticity
47
was upregulated in cells on both direct and indirect neuron exposure. ARC (Figure 2.2
K), a master regulator of synaptic plasticity
48
showed significant upregulation in BBM 3.1 and
A549 cancer cells only on direct co-culture with neurons, and was downregulated in cells treated
with NCM. MMP9 (Figure 2.2 J) a metalloprotease with novel functions in regulating pathological
glutamate-dependent neuronal plasticity
49
was significantly upregulated in all breast cancer cells
grown in NCM, but remained unchanged in co-culture. In contrast, MMP9 was significantly
downregulated in A549 cells and remained unchanged in LuBM5 cells upon neuronal exposure,
highlighting differential responses of breast and lung cancer cells within the neuronal
microenvironment. NRXN1, encoding a cell adhesion molecule critical to Glutamate and GABA-
mediated synaptic plasticity
50,51
was upregulated in all BM and non-BM tumor cells in co-culture
indicating cell-to-cell connection between tumor cells and neurons (Figure 2.2 L).
21
Figure 2. 2 Target validation in tumor cells. Commonly upregulated targets were validated by qPCR to investigate
differences in mRNA expression in tumor cells upon indirect (NCM; blue bars) vs. direct neuronal exposure (co-culture;
black bars). Data for each cell line represented as fold change in expression relative to tumor cells alone (Mean and
SEM).
Activity-induced gene expression is dependent on a variety of transcription factors
including cAMP Response Element Binding Protein (CREB), a well-studied regulator of neuronal
gene expression and synaptic plasticity in the brain e.g. EGR2, ARC, FOS, GABRB1
52
. Ser133-
phosphorylated CREB can bind to CRE elements on target genes to promote their transcription.
We wanted to determine if neuronal exposure activates CREB in tumor cells.
Immunofluorescence (IF) results showed enhanced Ser133-phosphorylated CREB staining in
both non-BM and BM breast (Figure 2.3 A,B) and lung (Figure 2.3 C,D) cancer cells upon
exposure to neurons indicating increased CREB activity and target gene transcription in the
neuronal microenvironment.
22
Figure 2. 3 Neuronal exposure activates transcription factor CREB in tumor cells. Immunofluorescence for
Ser133-phosphorylated CREB (Cy3-Red) in non-BM and BM breast cancer (BT47, 231Br-Her2
+
) (A,B) and lung cancer
cells (A549, LuBM5) (C,D) in 3 conditions- tumor only control, tumors treated with NCM, tumors co-cultured with
neurons (T= tumor cell, N=neuron; Magnification 63x; Scale 20µm).
Our results thus suggest induction of contact-dependent responsiveness to NTs
Glutamate, GABA and Serotonin in tumor cells, upon neuron exposure. Taken together, NT and
synaptic responsiveness are initiated early in tumor cells, particularly on direct contact with
neurons, during the establishment of BMs.
2.3.2 Breast cancer cells display enhanced reliance on paracrine microenvironmental
GABA in presence of neurons:
In the brain, neurons are classified by their utilization of the 5 classical NTs Glutamate,
GABA, Dopamine, Serotonin, and Acetylcholine. We sought to determine whether breast and lung
cancer cells showed a similar NT-dependent classification in the CNS-microenvironment.
Specifically, we investigated whether acquisition of neuronal attributes in tumors is supported by
23
autocrine NT signaling, or by responses to paracrine neurotransmitters from neurons. Overall,
Serotonin, Acetylcholine and Glutamate NT were not detected in tumor cells in all tested
conditions. Furthermore, endogenous Dopamine expression in breast and lung cancer cells
remained unchanged upon co-culture with neurons; suggesting autocrine expression for this
neurotransmitter (Figure 2.4 A-D). This is corroborated by mRNA expression analysis which
showed elevated endogenous levels of Dopamine receptors DRD1,2, and 5 in BBM 3.1 cells; and
that Dopamine receptor expression did not significantly change in tumor cells upon neuronal
exposure (Figure 2.1A).
Figure 2. 4 Autocrine Dopaminergic cells persist within the neuronal microenvironment. Immunofluorescence
for Dopamine (Cy3-red) in non-BM and BM breast cancer (SKBR3 and BBM 3.1) (A,B) and lung cancer cells (A549
and LuBM5) (C,D) in 2 conditions- tumor only and tumors in co-culture with neurons (T= tumor cell, N=neuron;
Magnification 63x; Scale 20µm).
We observed a converse effect for inhibitory NT GABA in tumor cells when interacting with
neurons. Specifically, endogenous GABA levels in BBM3.1 cells were significantly reduced when
co-cultured with neurons (Figure 2.5 Ai). Correspondingly, mRNA expression of GABA synthetic
enzyme GAD1 was significantly reduced in BBM 3.1 cells co-cultured with neurons, suggesting a
diminished autocrine GABA response in these tumor cells in the neuronal niche (Figure 2.5 D).
24
Considering this, we postulated whether neurons might be a paracrine source of GABA for these
brain-trophic tumor cells. Since paracrine and synaptic signaling occurs between cells located in
close proximity, we quantified GABA signal in neurons adjacent to tumor cells, as well as in those
located distally within the co-culture. We compared these signals to GABA intensity in pure
neuronal cultures without any tumor cells. Indeed, our results showed an increase in GABA
intensity in neurons in immediate proximity of BBM 3.1 cells, compared to neurons that were
situated farther away (Figure 2.5 Aii). Increase in neuronal GABA upon proximity to BBMs, along
with enhanced GABA receptor expression in BBMs on neuronal exposure as previously described
(Figure 2.1), indicates that breast cancer cells become GABAergic responsive brain metastases
(GRBMs) within the neuronal microenvironment, showing reliance on GABA from adjacent
neurons. In contrast, both non-BM (A549) and BM (LuBM5) lung cancer cells showed an increase
in autocrine GABA intensity on co-culture with neurons), suggesting increased reliance on
autocrine GABA in these cells (Figure 2.5 Bi, Ci). GAD1 expression remained unchanged in
A549 cells and showed slight reduction in LuBM5 cells, upon co-culture with neurons (Figure 2.5
E). Moreover, neurons both proximal and distant to lung cancer cells in co-culture showed no
change in GABA intensity (Figure 2.5 Bii; Cii).
25
Figure 2. 5 Breast cancer cells display enhanced reliance on paracrine microenvironmental GABA in presence
of neurons. Immunofluorescence of GABA in breast cancer (A) and lung cancer cells (B,C) in 2 conditions- tumor only
and tumor co-cultured with neurons (T= tumor cell, N=neuron; Magnification 63x; Scale 20µm). Mean GABA intensity
was measured for tumor cells (Ai, Bi, Ci), and for neurons proximal and distant to tumor cells (Aii, Bii, Cii) in all co-
cultures. Individual intensity values were plotted for all groups for comparison (mean and SEM). qPCR comparing
relative GAD1 expression in breast (D) and lung (F) tumor cells in 2 conditions- tumor only, and tumor in co-culture with
neurons (48h).
Previous studies show breast cancer utilize GABA through the GABA metabolic shunt for
proliferative advantage
13
. TCGA datasets results show primary breast cancer patients with high
GABA shunt mRNA expression (ABAT, ALDH5A1, SLC6A1, SLC6A11)) are associated with
significantly worse overall survival and 2.16 hazard ratio (Figure 2.6 A). Furthermore, enhanced
expression of GABA shunt mediators also correlates with significantly worse prognosis from brain
relapse, with patients at 6.6-fold greater risk of death from CNS metastasis (Figure 2.6 B). From
this, we postulated that inhibiting GABA availability in brain-trophic tumor cells will lead to better
prognostic index. Valproic Acid (VPA) used in the treatment of neurological disorders and as an
adjuvant in cancer therapy, has been shown to block GABA metabolism by inhibiting ABAT and
ALDH5A1
53
. Xenograft studies showed BBM-bearing animals treated with early VPA regimen (3
days post-transplantation) have significantly enhanced overall survival compared to both
untreated and late -VPA treated (21 days post-transplantation) groups (Figure 2.6 C). However,
26
late-VPA treatment group show no significant difference in survival compared to untreated group.
Thus, our results indicate that reliance on paracrine GABA in the brain is an early acclimation
strategy in breast cancer, and that targeting this property earlier in disease progression provides
significant survival benefit.
Figure 2. 6 Reliance on microenvironmental GABA is an early CNS acclimation strategy in breast cancer.
Kaplan-Meier survival analysis in breast cancer by mRNA expression of genes involved in GABA metabolism (ABAT,
ALDH5A1, SLC6A1, SLC6A11). (A) represents overall survival in patients, whereas (B) represents probability of
mortality from brain relapse in breast cancer patients, both categorized by differential expression of GABA shunt
mediator genes. Analysis was performed using publicly available TCGA and Bos-Massague datasets for breast cancer
respectively. Low (blue) and high (red) gene expression were plotted on the graph with Y-axis representing % patient
survival (100-0), and X-axis displaying time of data collection. (C)Kaplan-Meier survival analysis for in vivo mouse
model studying CNS-colonization in breast cancer. Blue, red and green lines represent survival data from control
(untreated; n=5), early-stage VPA treated (n=5), and late-stage VPA treated groups (n=5), respectively. Hazard Ratios
were calculated for each KS plot, significance was calculated using Log-Rank test.
Given that tumor cells show distinct NT expression and responsiveness within the
neuronal microenvironment, we next examined the expression of regulators of neurotransmission
in tumor cells in co-culture. SNAP25, a neuronal vesicle protein which facilitates neurotransmitter
release at chemical synapses and NT receptor trafficking at the plasma membrane
54,55
. Therefore,
27
we investigated whether neuron exposure would alter SNAP25 expression and location in tumor
cells. In breast cancer, endogenous SNAP25 was elevated in BBM 3.1 cells relative to SKBR3
cells, consistent with its mRNA expression. SNAP25 staining was mainly peri-nuclear in both cell
populations. However, in co-culture with neurons, SNAP25 localized to the plasma membrane in
BBM 3.1 cells, whereas a moderate increase in cytoplasmic SNAP25 staining was observed in
SKBR3 cells (Figure 2.7 A,B). Both A549 and LuBM5 lung cancer cells exhibited strong peri-
nuclear and cytoplasmic SNAP25 expression in control conditions. In neuronal co-cultures, both
cell types showed SNAP25 localized towards the plasma membrane (Figure 2.7 C,D). Taken
together, these results indicate direct interaction with neurons induces membrane localization,
and potential functionality of NT-regulators like SNAP25 in breast cancer cells. In contrast,
SNAP25 expression/localization does not appear to be a CNS-adaptive mechanism in lung
cancer cells but might in fact be an inherent property facilitating their rapid metastatic competency.
Figure 2. 7 Tumor cells show functional localization of NT vesicle protein SNAP25 in presence of neurons.
Immunofluorescence for cellular SNAP25 protein expression (Alexa Fluor 488-green) in non-BM and BM breast cancer
(SKBR3, BBM 3.1) (A,B) and lung cancer cells (A549, LuBM5) (C,D) in 2 conditions- tumor only, and tumor co-cultured
with neurons (T= tumor cell, N=neuron; Magnification 63x).
28
2.3.3 Neuron and neurotransmitter exposure induces resurgence of Reelin expression in
tumor cells:
Breast cancer shows slower progression to the CNS, with metastases sometimes
becoming evident years after primary diagnosis
56
. This is in part due to tumor dormancy, where
mesenchymal-type tumor cells show quiescence characterized by slower growth, stem cell-like
characteristics, and resistance to chemotherapy after extravasation into secondary sites. Dormant
tumor cells though non-proliferative, can interact with their microenvironment, and can be
reactivated to eventually form metastases
57
.
Given this, we asked whether dormant tumor cells are able to respond to NT and establish initial
synaptic plasticity. First, we established dormant breast cancer cells
58
in vitro (Figure 2.8 A,B,C),
and then treated them with exogeneous GABA, Dopamine and Serotonin to study their response
to classical NTs secreted by neurons. Results show that tumor cells remained dormant post-NT
exposure (Figure 2.9 D,E,F), but showed a dose dependent increase in the expression RELN,
an extracellular matrix glycoprotein (Figure 2.9 A,B,C), indicating an immediate early response
for CNS synaptic plasticity.
29
Figure 2. 8 Establishment of dormant SKBR3 breast cancer cells. (A) mRNA expression of cell cycle mediators in
dormant breast cancer cells. Data plotted in bar graph as fold change in expression relative to control (SKBR3 cells
grown in non-stress conditions). (B) mRNA expression of epithelial-to-mesenchymal transition (EMT) genes in dormant
SKBR3 cells, plotted as fold change relative to control SKBR3 cells. (C) IF and quantification for dormancy markers
(SOX9, P16-ARC, p-P38, p-ERK. SOX2) in SKBR3 cells in normal and dormant conditions (Magnification 63x; Scale
10µm). Mean intensity for all markers was measured and values were plotted as bar graphs (mean and SEM). Along
with significantly enhanced expression of SOX9, P16, SOX2, dormant cells also showed higher p38/ERK ratio relative
to control SKBR3 cells, confirming dormant phenotype.
30
Figure 2. 9 NT stimulation induces Reelin expression in dormant breast cancer cells. qPCR for Reelin expression
in dormant SKBR3 cells on treatment with GABA (A), Dopamine (B) and, Serotonin (C) respectively for 48 hours. mRNA
expression of cell cycle mediators in dormant SKBR3 cells treated with (D) GABA, (E ) Dopamine, and (F) Serotonin
respectively. Data plotted as fold change in expression relative to untreated dormant cells.
Additionally, synaptic plasticity mRNA expression analysis revealed significantly
enhanced RELN expression in both breast and lung cancer cells upon neuronal contact (Figure
2.1 B). We thus postulated that neuron exposure stimulates Reelin expression in tumor cells early
in brain metastasis, to facilitate CNS acclimation and metastatic colonization. Although astrocyte-
mediated Reelin expression has been shown to regulate Her2
+
breast cancer proliferation in the
brain
59
, there are currently no studies that evaluate the role of neuronal input in inducing Reelin
expression in tumor cells for CNS acclimation.
To investigate the importance of tumor Reelin expression in the neuronal niche, we first
determined Reelin expression in patient breast and lung tumors. RELN expression was
significantly reduced in primary and metastatic breast and lung tumors compared to that in normal
tissue (Figure 2.10 A,B), consistent with previous reports that loss of RELN expression promotes
invasiveness in epithelial tumors
31-33
and correspondingly, negligible endogenous RELN
expression seen in non-BM BT474, SKBR3 and A549 cells (Figure 2.10 C,D).
31
Figure 2. 10 Reelin expression is lost in primary metastatic breast and lung cancer. RELN expression in patient
normal (non-pathological), primary and metastatic breast (A) and lung (B) tissue. Expression values were obtained
from UCSC Xena, and were normalized to the same internal control of that database (TCGA TARGET GTEx breast,
and TCGA TARGET GTEx lung) for comparison. (C,D) RELN mRNA expression in brain-metastatic (BBM 3.1 and
LuBM5) cells, compared to their non-BM counterparts (SKBR3, BT474 and A549) respectively. Data displayed as
relative RELN expression in all cells. Statistical significance for breast cancer cells was calculated using One-way
ANOVA, and for lung cancer cells using t-test.
Immunofluorescence studies also confirmed the presence of Reelin in brain-metastatic
breast and lung cancer, with the protein being distinctly expressed, and localized towards the
plasma-membrane in these tumors (Figure 2.11 A,B).
32
Figure 2. 11 Reelin is expressed in brain-metastatic breast and lung tumor tissue. Immunofluorescence for Reelin
(Cy3) in primary and brain-metastatic breast (A) and lung (B) cancer patient tissues respectively (Magnification 63x;
Scale 20µm).
We next determined whether Reelin expression was prognostic in breast and lung cancer.
While not prognostic in lung cancer, increased RELN expression in breast cancer was significantly
associated with worse overall survival and patients with high expression were at 1.8 times greater
risk of death (Figure 2.12 A,B). Moreover, elevated expression of mediators of the Reelin
signaling cascade (RELN, LRP8, DAB1) was significantly associated with worse prognosis from
brain relapse in breast cancer with a hazard ratio of 3.68 (Figure 2.12 C). Consistent with patient
data, brain-metastatic breast (BBM 3.1) and lung (LuBM5) cells used in this study showed
significantly enhanced endogenous RELN expression relative to their non-BM counterparts as
shown previously (Figure 2.10 C,D).
33
Figure 2. 12 Enhanced Reelin expression is associated with worse prognosis and brain relapse in breast
cancer. Kaplan-Meier analysis for association between RELN expression and overall survival in lung (A) and breast
(B) cancers performed using SurvExpress. TCGA datasets for both cancers were used for this study. Low (blue) and
high (red) RELN expression were plotted on the graph with Y-axis representing percent survival (100-0), and X-axis
displaying time of data collection. (C) Kaplan-Meier survival analysis in breast cancer for expression of mediators of
Reelin signaling (RELN, LRP8, DAB1) and risk of mortality from brain relapse. Analysis was performed using Bos-
Massague dataset for breast cancer. Low gene expression (blue) and high gene expression (red) were plotted on the
graph with Y-axis representing % patient survival (100-0), and X-axis displaying time of data collection.
RELN is known to be epigenetically suppressed in many cancers
63
and we postulated that
exposure to neurons will reverse this blockade. We first confirmed the epigenetic modulation of
the RELN gene in non-BM tumor cells by treating BT474 and A549 cells with HDAC inhibitor
Trichostatin A (TSA). BT474 and A549 cells, showed large increases in RELN post-treatment
(Figure 2.13 A). Next, HDAC activity analysis revealed that exposure to neurons for 48 hours
was able to significantly reduce Histone De-Acetylase activity in breast and lung cancer cells
(Figure 2.13 B) indicating that neuron exposure can reverse epigenetic suppression in incoming
tumor cells, allowing the expression of RELN.
Figure 2. 13 Neuron exposure lowers Histone De-acetylase (HDAC) activity in tumor cells, facilitating epigenetic
upregulation of RELN. (A) Reelin mRNA expression in BT474 and A549 cell treated with TSA (100nM). Data displayed
as fold increase in RELN over untreated cells. (B) HDAC activity as measured by luminescence, in breast (BT474,
SKBR3, 231Br-Her2
+
, BBM 3.1) and lung (A549 and LuBM5) cells in 2 conditions- tumor cells only (control), and tumor
cells grown in NCM for 48 hours.
34
Correspondingly, non-BM BT474 cells showed enhanced Reelin upon exposure to
neurons for 48 hours (Figure 2.14 A). Brain-trophic MDA-MB-231Br-Her2
+
cells showed large
increases in Reelin expression only on direct co-culture with neurons, and slight increase when
grown in NCM (Figure 2.14 B). Similarly, both A549 and LuBM5 lung cancer cells showed an
increase in Reelin expression on exposure to neurons (Figure 2.14 C,D). Overall, our results
indicate neuronal exposure, particularly direct contact, enhances Reelin expression in tumor cells.
Figure 2. 14 Neuronal contact enhances Reelin protein expression in tumor cells. Immunofluorescence for Reelin
(Cy3) in in non-BM and BM breast cancer (BT474, 231Br-Her2
+
) (A,B) and lung cancer cells (A549, LuBM5) (C,D) in 3
conditions- tumor only control, tumors treated with NCM, tumors co-cultured with neurons (T= tumor cell, N=neuron;
Magnification 63x; Scale 20µm).
Reelin released by GABAergic and glutamatergic neurons in the adult brain is necessary
for normal neurotransmission and synaptic plasticity and is thus ubiquitous in the neuronal
microenvironment
64
. We wanted to specifically examine whether paracrine Reelin from the
neuronal microenvironment influences metastatic tumor cells. We first knocked down RELN in
35
BT474 cells (BT474
KD
) to mitigate the effect of tumor-derived Reelin. BT474
control
and BT474
KD
cells were then treated with 2.5nM human recombinant Reelin for 24 hours. BT474
control
cells (red
bars) showed upregulation of endogenous RELN and its target gene RARA (Figure 2.15 A,B).
Moreover, reduced expression of genes involved in epithelial-to-mesenchymal transition (VIM,
SNAI1, MMP9) (Figure 2.15 C,D,E), and upregulation of mediators of synaptic plasticity (NTRK2,
NGFR, NRXN1, NLGN4X) (Figure 2.15 F,G,H,I) in BT474
control
cells indicated their ability to
upregulate autocrine Reelin signaling upon stimulation with paracrine Reelin. In contrast, BT474
KD
cells (blue bars) were unable to respond to exogenous Reelin.
Figure 2. 15 Paracrine Reelin-mediated induction of autocrine Reelin signaling in breast cancer cells is crucial
in breast-to-brain metastasis. BT474-control (red) and RELN knock-down (blue) cells were treated with exogenous
human recombinant Reelin (2.5nM) for 24 hours. Change in mRNA expression of Reelin response genes RELN (K),
RARA (L), EMT genes MMP9 (M), SNAI1 (N) VIM (O), and synaptic plasticity genes NTRK2 (P), NGFR (Q), NRXN1
(R), NLGN4X (S) after exogenous Reelin treatment. Data displayed as fold change in mRNA over untreated cells.
These results suggest that although loss of Reelin in the primary tumor mediates
metastasis, the ability of tumor cells to upregulate autocrine Reelin signaling, even in the presence
of microenvironmental neuronal Reelin, is crucial in breast-to-brain metastasis.
36
2.4 Discussion:
The investigation of initial interactions of tumor cells with the CNS microenvironmental
niche is imperative in understanding the mediators of tumor colonization and BM progression.
Most studies have focused on tumor masses that have already successfully established
themselves in the brain. But initial interactions of tumor cells with neurons and the ensuing CNS-
adaptive responses in these tumor cells are largely unexplored. Given that primary tumors show
varying proclivity to the CNS, it is essential to determine commonalities and differences in their
interaction with the neuronal milieu.
Overall, we provide evidence that neurons induce the expression of CNS-specific genes
in tumor cells, that are crucial for successful metastatic colonization in the brain. We show
neuronal contact is able to induce enhanced NT responsiveness in both breast and lung cancer
cells, compared to neuron-conditioned media exposure alone. These results highlight the
acclimatory effects of early direct interactions between tumor cells and neurons within the CNS.
Furthermore, our results corroborate previous observation of GRIN2B expression in established
BBMs
31
. However, we now show that it is the early neuronal contact which significantly increases
GRIN2B expression in breast and lung cancer cells.
In synaptic plasticity, the change in synapse strength according to stimuli can induce
memory through short-term (STP) or long-term potentiation (LTP)
34
. STP changes occur very
rapidly, while LTP can last anywhere from mere seconds to even years and is the main route the
brain takes to store information and create memory. Mediators of synaptic plasticity have been
identified, and their role in STP/LTP have been determined (e.g BDNF, Reelin, CREB, Glutamate
and GABA receptors, metalloproteases like MMP9, ADAMs, etc). The current study demonstrates
that exposure to neurons enhances expression of LTP and STP-like mediators in both breast and
lung cancer cells. Moreover, in neuronal co-cultures, tumor cells show upregulation of ARC, an
immediate-early LTP mediator present on dendritic spines and postsynaptic densities
65
,
suggesting neurons can regulate tumor cells by transferring signals at direct points of contact.
37
Whether these tumor-neuron synaptic plasticity interactions form true-like memory state as seen
in neuron-neuron interactions remains to be further elucidated.
Our results show reduced GABA in breast cancer cells upon neuronal contact,
accompanied by enhanced GABA levels in tumor-adjacent neurons, which corroborates the
reliance of brain-trophic breast cancer cells on microenvironmental GABA within the CNS
13
.
Reduced GABA levels in breast cancer cells has been associated with worse prognosis
66
. Thus,
GABA levels in breast cancer cells in the neuronal microenvironment could be correlated to their
aggressiveness and ability to form clinical metastases. Furthermore, we show in vivo that early
administration of VPA (3dpi) blocks CNS colonization of breast cancer and provides significant
survival benefit. This could be attributed in part to inhibition of GABA metabolism mediators ABAT
and ALDH5A1 by VPA
53
. Once brain-metastases have been established (by 21 dpi), the inability
of pharmacological intervention in prolonging survival may be due to enhanced proliferative
properties in the tumor cells, and reduced reliance on the microenvironment once CNS-
acclimation is accomplished. Thus, a chemotherapeutic regimen with VPA as an adjuvant may
be clinically beneficial to potentially prevent CNS metastasis in patients with advanced breast
cancer.
Our data indicates that induction of Reelin expression in tumor cells on neuronal exposure
is important for CNS acclimation. In the brain, Reelin is secreted by GABAergic neurons
64
,
indicating its importance in cells that utilize GABA as a neurotransmitter. In breast cancer cells,
enhanced expression of Reelin is concomitant with reduced Epithelial-Mesenchymal Transition
and increased expression of synaptic plasticity genes. This indicates that upregulation of Reelin
helps breast cancer cells transition back to a more epithelial phenotype and establish themselves
in the brain. This data could be correlated with the higher mortality from brain relapse in breast
cancer patients who show enhanced activity of the Reelin signaling pathway. Furthermore, we
also show that dormant breast cancer cells can respond to neurotransmitter signals to activate
RELN transcription. Reactivation of quiescent dormant cells in a fertile microenvironment
38
contributes to their progress to macro-metastases. It remains to be seen whether this Reelin
expression aids in the reactivation of dormant cells in the CNS metastatic niche.
Tumor metastasis is a complex, inefficient process where only a few competent tumor cell
progress to clinical metastases. Our results demonstrate that early tumor-neuron interaction is
important in facilitating CNS acclimation in tumor cells. Furthermore, acquisition of neuronal gene
and protein expression is a requisite acclimation strategy in breast cancer. Exploiting the reliance
of incoming breast cancer cells on the neuronal microenvironment can lead to potentially effective
control of brain metastasis. Moreover, understanding the inherent and acquired properties in
metastatic cells from different primary tumors can provide insight into niche mechanistic targets.
References:
1. Achrol AS, Rennert RC, Anders C, et al. Brain metastases. Nature Reviews Disease
Primers. 2019; 5(1):5.
12. Neman J, Choy C, Kowolik CM, et al. Co-evolution of breast-to-brain metastasis and
neural progenitor cells. Clin Exp Metastasis. 2013; 30(6):753-768.
13. Neman J, Termini J, Wilczynski S, et al. Human breast cancer metastases to the brain
display GABAergic properties in the neural niche. Proceedings of the National Academy
of Sciences. 2014; 111(3):984-989.
31. Zeng Q, Michael IP, Zhang P, et al. Synaptic proximity enables NMDAR signalling to
promote brain metastasis. Nature. 2019; 573(7775):526-531.
34. Citri A, Malenka RC. Synaptic Plasticity: Multiple Forms, Functions, and Mechanisms.
Neuropsychopharmacology. 2008; 33(1):18-41.
35. Kaverina N, Borovjagin AV, Kadagidze Z, et al. Astrocytes promote progression of breast
cancer metastases to the brain via a KISS1-mediated autophagy. Autophagy. 2017;
13(11):1905-1923.
36. Pukrop T, Dehghani F, Chuang H-N, et al. Microglia promote colonization of brain tissue
by breast cancer cells in a Wnt-dependent way. Glia. 2010; 58(12):1477-1489.
37. Hyman SE. Neurotransmitters. Current Biology. 2005; 15(5):R154-R158.
38. Huang EP, Stevens CF. Neurotransmitter Release and Synaptic Plasticity. In: Bittar EE,
ed. Advances in Organ Biology. Vol 2: Elsevier; 1997:171-191.
39
39. Flüh C, Mafael V, Adamski V, Synowitz M, Held-Feindt J. Dormancy and NKG2D system
in brain metastases: Analysis of immunogenicity. Int J Mol Med. 2020; 45(2):298-314.
40. Lovinger DM. Neurotransmitter roles in synaptic modulation, plasticity and learning in the
dorsal striatum. Neuropharmacology. 2010; 58(7):951-961.
41. Isaac JT, Ashby MC, McBain CJ. The role of the GluR2 subunit in AMPA receptor function
and synaptic plasticity. Neuron. 2007; 54(6):859-871.
42. Akashi K, Kakizaki T, Kamiya H, et al. NMDA Receptor GluN2B (GluRε2/NR2B) Subunit
Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin
Cytoskeleton at Hippocampal CA3 Synapses. The Journal of Neuroscience. 2009;
29(35):10869-10882.
43. Crupi R, Impellizzeri D, Cuzzocrea S. Role of Metabotropic Glutamate Receptors in
Neurological Disorders. Front Mol Neurosci. 2019; 12:20.
44. Jones KA, Tamm JA, Craig DA, Yao W-J, Panico R. Signal Transduction by GABAB
Receptor Heterodimers. Neuropsychopharmacology. 2000; 23(1):S41-S49.
45. Duka T, Nikolaou K, King SL, et al. GABRB1 Single Nucleotide Polymorphism Associated
with Altered Brain Responses (but not Performance) during Measures of Impulsivity and
Reward Sensitivity in Human Adolescents. Front Behav Neurosci. 2017; 11:24-24.
46. Rebholz H, Friedman E, Castello J. Alterations of Expression of the Serotonin 5-HT4
Receptor in Brain Disorders. Int J Mol Sci. 2018; 19(11):3581.
47. Poirier R, Cheval H, Mailhes C, et al. Distinct functions of egr gene family members in
cognitive processes. Front Neurosci. 2008; 2(1):47-55.
48. Korb E, Finkbeiner S. Arc in synaptic plasticity: from gene to behavior. Trends Neurosci.
2011; 34(11):591-598.
49. Lepeta K, Kaczmarek L. Matrix Metalloproteinase-9 as a Novel Player in Synaptic
Plasticity and Schizophrenia. Schizophr Bull. 2015; 41(5):1003-1009.
50. Graf ER, Zhang X, Jin SX, Linhoff MW, Craig AM. Neurexins induce differentiation of
GABA and glutamate postsynaptic specializations via neuroligins. Cell. 2004;
119(7):1013-1026.
51. Dean C, Dresbach T. Neuroligins and neurexins: linking cell adhesion, synapse formation
and cognitive function. Trends Neurosci. 2006; 29(1):21-29.
52. Sakamoto K, Karelina K, Obrietan K. CREB: a multifaceted regulator of neuronal plasticity
and protection. J Neurochem. 2011; 116(1):1-9.
53. Johannessen CU, Johannessen SI. Valproate: past, present, and future. CNS Drug Rev.
2003; 9(2):199-216.
54. Antonucci F, Corradini I, Fossati G, Tomasoni R, Menna E, Matteoli M. SNAP-25, a Known
Presynaptic Protein with Emerging Postsynaptic Functions. Frontiers in Synaptic
Neuroscience. 2016; 8(7).
40
55. Fan HP, Fan FJ, Bao L, Pei G. SNAP-25/syntaxin 1A complex functionally modulates
neurotransmitter gamma-aminobutyric acid reuptake. J Biol Chem. 2006; 281(38):28174-
28184.
56. Pedrosa RMSM, Mustafa DA, Soffietti R, Kros JM. Breast cancer brain metastasis:
molecular mechanisms and directions for treatment. Neuro Oncol. 2018; 20(11):1439-
1449.
57. Montagner M, Sahai E. In vitro Models of Breast Cancer Metastatic Dormancy. Frontiers
in Cell and Developmental Biology. 2020; 8(37).
58. Yadav AS, Pandey PR, Butti R, et al. The Biology and Therapeutic Implications of Tumor
Dormancy and Reactivation. Front Oncol. 2018; 8:72-72.
59. Jandial R, Choy C, Levy DM, Chen MY, Ansari KI. Astrocyte-induced Reelin expression
drives proliferation of Her2(+) breast cancer metastases. Clin Exp Metastasis. 2017;
34(2):185-196.
60. Stein T, Cosimo E, Yu X, et al. Loss of reelin expression in breast cancer is epigenetically
controlled and associated with poor prognosis. Am J Pathol. 2010; 177(5):2323-2333.
61. Serrano-Morales JM, Vázquez-Carretero MD, Peral MJ, Ilundáin AA, García-Miranda P.
Reelin-Dab1 signaling system in human colorectal cancer. Mol Carcinog. 2017; 56(2):712-
721.
62. Okamura Y, Nomoto S, Kanda M, et al. Reduced expression of reelin (RELN) gene is
associated with high recurrence rate of hepatocellular carcinoma. Ann Surg Oncol. 2011;
18(2):572-579.
63. Chen Y, Sharma RP, Costa RH, Costa E, Grayson DR. On the epigenetic regulation of
the human reelin promoter. Nucleic Acids Res. 2002; 30(13):2930-2939.
64. Campo CG, Sinagra M, Verrier D, Manzoni OJ, Chavis P. Reelin secreted by GABAergic
neurons regulates glutamate receptor homeostasis. PLoS One. 2009; 4(5):e5505.
65. Messaoudi E, Kanhema T, Soulé J, et al. Sustained Arc/Arg3.1 synthesis controls long-
term potentiation consolidation through regulation of local actin polymerization in the
dentate gyrus in vivo. The Journal of neuroscience : the official journal of the Society for
Neuroscience. 2007; 27(39):10445-10455.
66. Brzozowska A, Burdan F, Duma D, Solski J, Mazurkiewicz M. γ-amino butyric acid (GABA)
level as an overall survival risk factor in breast cancer. Ann Agric Environ Med. 2017;
24(3):435-439.
41
Chapter 3: SRRM4-mediated REST to REST4 dysregulation facilitates
CNS-acclimation and colonization in breast-to-brain metastases
3
Abstract:
Although the importance of neural acclimation in breast-to-brain metastases (BBMs) has been
described, the mechanisms that govern the acquisition of neuronal characteristics and the
resulting brain-metastatic competency are poorly understood. Herein, we show BBMs display
enhanced expression of neural-specific splicing factor Ser/Arg Repetitive Matrix Protein 4
(SRRM4) relative to primary breast cancers. This leads to loss of neuronal gene repressor REST,
and enhanced expression and nuclear localization of SRRM4-regulated alternative splice isoform
REST4. Enhanced SRRM4 expression promotes increased proliferation and acquisition of
neurotransmitter and synaptic plasticity mediators, while reduced SRRM4 confers dormant
characteristics in breast cancer cells. SRRM4 overexpression facilitates accelerated breast-to-
brain metastasis and leads to worse overall survival in vivo, while SRRM4 knockdown provides
no CNS-metastatic advantage. Thus, SRRM4 mediated REST dysregulation promotes CNS-
adaptation and brain-metastatic competency in breast cancer.
3.1 Introduction:
Brain metastases (BMs) are the main cause of intracranial neoplasms in adults with invasive
cancers. Lung, breast, melanoma, colorectal, and renal cancers show most proclivity for the brain,
followed more uncommonly by thyroid, gastrointestinal (GI) and prostate cancers. Breast cancer
is unique in its longer latency in forming BMs
11
indicating that tumor cells which successfully form
detectable metastases, are able to overcome dormancy, immune clearance, and cell death and
3
This chapter is modified from my first author original research article currently in preparation.
Deshpande K, Martirosian V, Nakamura B, Shao L, Buckley N, Reed M, Neman J. SRRM4-
mediated REST dysregulation acilitates CNS-acclimation and colonization in breast-to-brain
metastases (in preparation)
42
can acclimate to the neural microenvironment
24
. Previous studies have reported the acquisition
of neuronal characteristics in breast-to-brain metastases (BBMs)
13
, and have also described the
presence of sub-populations within the primary tumor in basal-like breast cancers, that show
enhanced expression of neurotransmitter receptors that prime them to successfully metastasize
to the brain
31
. Therefore, adaptation to the CNS microenvironment appears to be requisite for
successful breast-to-brain metastasis.
The expression of CNS-specific genes in BBMs indicates that neural regulatory pathways
may be activated in these tumor cells. Currently, there are no studies investigating the contribution
of neuro-developmental programs in promoting brain-metastatic capabilities in breast cancer. RE-
1 Silencing Transcription Factor (REST) is a well-studied regulator of the neuronal phenotype. It
represses target genes by binding to the Neuron Restrictive Silencer Element (RE-1 element)
sequence on those genes
67
. REST represses the expression of neural genes in non-neuronal
cells
68
and is also involved in maintaining pluripotency and self-renewal in CNS-progenitor cells
69
.
Owing to its dual functions, REST is an oncogene in many primary brain tumors e.g.
medulloblastoma and glioma
70,71
, but is known to be a tumor suppressor in epithelial cancers like
breast cancer
72
. Loss of REST expression and a corresponding increase in invasive capabilities
has been observed in breast, lung and colon cancers
73,74
. Thus, regulation of REST function has
implications in advanced carcinomas.
REST expression is regulated by Serine/Arginine Repetitive Matrix Protein 4 (SRRM4),
an RNA splicing factor that generates neural specific splice variants of its target genes
75
. SRRM4
regulates REST transcription and function by inserting a neural specific exon N into the REST
transcript, resulting in alternative splice variants including the truncated isoform REST4. REST4,
although able to enter the nucleus, only shows weak binding to the RE-1 element, and thus is
able to reverse the repression on genes targeted by full-length REST
76
. SRRM4-mediated
regulation of REST function is crucial during neuronal differentiation in the developing brain,
where a balance shift in favor of REST4 allows progenitor cells to mature into functional neurons
43
by allowing the expression of neuronal genes
77
. Recent studies have shown that SRRM4
expression promotes the development of an aggressive subtype of castration-resistant prostate
cancer called Neuro-endocrine prostate cancer (NePC) by activating the neural-specific splicing
of specific genes
78
. Thus, SRRM4 is implicated in the acquisition of neuronal attributes in
aggressive epithelial cancers.
Although loss of REST expression is implicated in increased invasiveness in breast cancer
cells, a role for REST dysregulation has not been reported in the context of CNS metastasis in
breast cancer. Herein, we show that brain-metastatic breast cancers (BBMs) show enhanced
expression of REST-regulator SRRM4 relative to primary breast cancers. This is accompanied
by enhanced expression and nuclear localization of the alternative splice form REST4, concurrent
with reduced expression of full-length REST. This SRRM4-mediated REST dysregulation is not
seen in brain-metastatic tumor cells from lung cancer or melanoma, suggesting that this
mechanism is breast cancer-specific. SRRM4 overexpression in brain-naïve BC cells confers
increased proliferative capacity, and enhanced expression of CNS-specific genes, many of which
are regulated by the SRRM4/REST axis. In contrast, SRRM4
knockdown
BC cells show a more
quiescent phenotype consistent with slower proliferation, enhanced expression of dormancy
genes, and increased chemoresistance. Furthermore, SRRM4 overexpression in BC cells
promotes accelerated brain metastasis and leads to worse overall survival in vivo, while SRRM4
knockdown provides no CNS-metastatic advantage. We also show that this is attributed to
SRRM4-mediated CNS acclimation in breast cancer cells within the neuronal microenvironment.
Our results thus reveal a novel role for SRRM4-mediated REST dysregulation in tumor
cells, that promotes CNS-adaptation and brain-metastatic competency in breast cancer.
44
3.2 Materials and Methods:
3.2.1 Cell culture: Low passage Patient-derived Brain-metastatic (BM) breast (BBM 3.1), lung
(LuBM5), and melanoma (MBM2) cells were propagated in our lab from surgically resected BM
tissue from consenting patients. On receipt of resected tumors from USC Neurosurgery, the tissue
was mechanically dissociated, trypisinized at 37C for 10-15 minutes, and triturated. The tissue
was centrifuged (500g for 2 minutes) and resuspended in cell culture media (50% DMEM-F12,
50% Neurobasal-A, 20%FBS, 2% glutamine, 1%Antibacterial-Antimycotic). For non-brain
metastatic cells the following commercially available cell lines were used: Breast cancer (SKBR3,
BT474, MDA-MB231), Lung cancer (A549), and Melanoma (A2058). All cell lines were maintained
in DMEM-F12 (Thermo, 12634028) supplemented with 10%FBS, 1%Glutamine (Thermo,
35050061) and 1%Antibacterial-Antimycotic agent (Sigma, 15240062), and were tested for
mycoplasma using the DAPI test before use.
Primary neuron cultures: Neural cells were isolated from whole brain tissue of postnatal day 1-
4 mice using the Worthington Papain dissociation system kit (LK003150), plated on Poly-D Lysine
(Sigma, P7280-5MG), and maintained in complete neuron culture medium (Neurobasal-A
medium Gibco, 10888022; supplemented with B27 (Thermo 17504044), Glutamine and Anti-
Anti). On the third day post plating, the mixed neural cell cultures were treated with Cytosine
Arabinoside (Sigma, C6645-25MG), to eliminate miscellaneous neural cell populations leaving
behind pure neuronal cultures. 50% media change with complete neuron culture medium was
performed every 3 days to maintain healthy pure neurons.
Lentiviral transduction: Complete lentiviral vectors were constructed in 293T cells using
SRRM4 overexpression ORF cDNA clone (Genecopoeia EX-Y3278-Lv224), and SRRM4
knockdown shRNA clone set (Genecopoeia HSH114130-LVRU6GP). SKBR3 breast cancer cells
were transduced with these vectors to stably overexpress (SKBR3
OE
) or knockdown (SKBR3
KD
)
45
SRRM4. These cells were further stably transduced with lentivirus to express GFP-FF Luciferase
to facilitate bio-luminescent imaging of tumors in vivo
Establishment of dormant breast cancer cells: SKBR3 (control, SKBR3
OE
and SKBR3
KD
) cells
were gradually acclimated to dormancy medium over one week. Dormancy media composition
was as follows: (DMEM with no glucose, no glutamine, Thermo A1413004) supplemented with
5% Fetal Bovine Serum (FBS), and 1%Antibacterial-Antimycotic agent (Sigma, 15240062).
Tumor-neuron co-cultures: Tumor cells were resuspended in complete neuron culture medium
and seeded onto established neuronal cultures (1:60 tumor cell to neuron ratio) to model tumor-
neuron interaction. At 48h post seeding, tumor cells were collected from co-cultures for qPCR, or
co-cultures were fixed with 4% Formaldehyde for immunofluorescence studies.
3.2.2 Animals: NSG (NOD SCID) mice on BalbC background were purchased from Jackson
laboratories and used for in vivo experiments in this study. 11-week old adult NSG animals were
used for intra-cardiac injection experiments to model brain metastasis. The animals were
maintained in sterile cages under 12 hour light/dark cycles and were provided with food and water
ad libitum. Animal procedures were performed under approved IACUC protocols and guidelines.
Humane endpoints were specified and followed for all experimental animals.
3.2.3 Intracardiac injection of tumor cells and animal imaging: Tumor cells were injected
intra-cardiac into mice to evaluate the role of SRRM4 in the development of breast-to-brain
metastases. Adherent cells were trypsinized, re-suspended in FBS-free cell culture media and
counted. Each mouse was injected with 1x10
5
tumor cells in 100ul of sterile 1X PBS, with a 25-
gauge needle. Development of brain lesions and other distant metastases was monitored by
optical imaging of injected animals. Luciferin (1ul/gram bodyweight of animal) was injected into
46
the tail vein of each mouse and bioluminescence was imaged (dorsal and ventral) 1.5 minutes
after injection. Optical signal from brain-metastases was quantified by measuring the same brain
ROI for all experimental animals on all imaging days. Bodyweights were monitored for all animals,
and 20% loss of bodyweight and/or visible distress were designated as experimental endpoints.
3.2.4 RNA isolation and qPCR analysis: Cells from various conditions were harvested by
trypsinization for 3-5 minutes at 37C. Trypsin (Thermo, 25300-120) was neutralized with culture
medium supplemented with FBS, and the cell suspension was centrifuged at 1000rpm for 1
minute. The supernatant was discarded, and the pellet was processed immediately, or frozen for
subsequent use. RNA isolation was performed using the Qiagen RNeasy mini kit (Qiagen, 74136).
RNA concentration was determined using a Nanodrop (Varioskan) and cDNA was synthesized
using the Maxima First Strand cDNA synthesis kit (Thermo, K1642). PowerUp SYBR Green
Master Mix (Thermo, A25918) was used for all qPCR reactions performed using the QuantStudio
VI thermocycler. All primers used for qPCR analysis were purchased from IDT (Table 2). Human
Neurotransmitter receptor (330231 PAHS-060ZA) and Synaptic Plasticity (330231 PAHS-126Z)
arrays were purchased from Qiagen to conduct the mRNA expression screens for all cell lines
and conditions.
3.2.5 Immunofluorescence: Human primary tumor tissue microarrays were obtained from
Biomax US (Breast PM2a-ER, Lung LC241l, Melanoma ME242c). Brain-metastatic tumor tissues
from patients were acquired via USC Neurosurgery. These tissues were formalin fixed, paraffin
embedded, and processed into 10um thick sections by the histology core at USC. Standard
immunohistochemistry protocol was followed for paraffin-embedded tissue sections and acidic
antigen retrieval was performed as necessary (Citrate Buffer (10mM, pH 6.0) at >80C for 20
minutes, and then RT for 10 minutes), followed by blocking (50% Seablock in 1XPBS with 0.3M
Glycine to reduce background) at RT for 1 hour.
47
For IF studies with cells, tumor cells/neuron co-cultures were fixed with 4% paraformaldehyde for
10 minutes at RT, and then washed twice with 1X PBS. Membrane permeabilization was
performed as necessary with 0.3% Triton X-100 for 20 minutes, followed by blocking at RT for 1
hour. Antibody incubations on cells and tissues were performed overnight at 4C. The antibodies
used were as follows: Rabbit Anti-REST (1:100, LSBio), Rabbit Anti-REST4 (1:300 IHC, 1:500
ICC, generous gift from Dr. Noel J. Buckley at Oxford University), Rabbit Anti-SRRM4 (1:500,
Biorbyt), Rabbit Anti-RILP (1:500, Abcam), Goat Anti-SNAP25 (3ug/ml, Abcam), Goat Anti-SOX2
(1:500, Novus Biologicals), Goat Anti-SOX9 (1:500, Novus Biologicals), Rabbit Anti-Ki67 (1:500,
Biocare Medical). All fluorophore conjugated secondary antibodies were purchased from Jackson
ImmunoResearch and used at 1:300 (Goat Anti-rabbit Cy3, Goat Anti-rabbit 647, Donkey Anti-
Goat 647). Phalloidin (488 or 647 conjugated) was used to stain cell membrane where necessary.
Stained cells and tissue sections were mounted in Prolong Gold Antifade reagent with DAPI
(Invitrogen) for imaging and long-term storage.
3.2.6 Microscopy and Imaging: Confocal imaging was performed using the Leica SP8
microscope. Nuclear colocalization studies were performed by analyzing co-localization between
protein of interest (REST, RILP, SRRM4, REST4, SOX9) and DAPI, which stains cellular nuclei.
Channel background and thresholds were set so as to avoid false positive results. Settings were
normalized so that different groups could be compared to one another for statistical analysis.
3.2.7 Bioinformatics: Kaplan Meier survival curve analyses for target genes were performed
using UCSC Xena, an online exploration tool for public/private multi-omic and clinical/phenotypic
data. Publicly available TCGA data for breast cancer, lung cancer and melanoma were analyzed
using this open source platform.
48
3.2.8 Statistical analysis: Statistics were performed using GraphPad Prism. t-tests were used to
compare expression between two different cell types/conditions. One-way Anova was used to
compare more than 2 groups/conditions. Statistical significance and Hazard Ratio for survival
data from in vivo experiments was calculated using Log-Rank Test.
3.3 Results:
3.3.1 Breast to brain metastases show reduced expression and nuclear localization of
REST:
Previous studies have demonstrated the gain of neuronal cell characteristics in BBMs
13,79
. Of
these, several genes (BDNF, RELN, GRM8) are normally repressed by RE-1 Silencing
Transcription Factor (REST)
80,81
. Loss of REST function has been implicated in invasiveness in
breast, lung and colon cancer, but its role in brain metastasis is currently unknown.
We hypothesized that REST would be downregulated in BBMs, allowing for the expression
of CNS-specific genes. First, we examined whether REST expression in tumors was associated
with overall patient survival. Kaplan-Meier survival analysis from TCGA datasets show REST was
not prognostic in breast and lung cancer, or in melanoma (Figure 3.1 A,B,C). Correspondingly,
in vitro REST expression was not drastically different between BM vs. non-BM breast, lung or
melanoma tumor cells (Figure 3.1 D,E,F).
49
Figure 3. 1 REST expression is not prognostic in breast and lung cancer and melanoma. Kaplan-Meier analysis
for association between REST expression and overall survival in breast (A), lung (B), and melanoma (C) cancers.
TCGA datasets for all cancers were used for this study. Low (blue) and high (red) REST expression were plotted on
the graph with Y-axis representing probability of survival (1-0), and X-axis displaying time of data collection (days).
REST mRNA expression in brain-metastatic breast BBM 3.1 (A), lung LuBM5 (B), and melanoma MBM2 (C) cancer
cells, relative to their primary (non-BM) counterparts (mean and SEM).
However, patient tissues revealed reduced levels of REST protein in brain-metastatic breast
cancer and melanoma, compared to their non-BM counterparts (Figure 3.2 A, C). This difference
was not observed in lung cancer (Figure 3.2 B).
Figure 3. 2 BM breast cancer and melanoma tissues show reduced REST expression. Representative IF images
for REST (Cy3) in primary and brain-metastatic patient tissues from breast (A), lung (B) and melanoma (C) cancer
respectively (Magnification 63x)
50
REST acts as a transcriptional repressor in cellular nucleus, with loss of nuclear REST promoting
the expression of its target genes
82
. We thus evaluated nuclear REST in non-BM and BM tumor
cells. Results showed significantly reduced nuclear REST in BBMs, compared to non-BM breast
cancer cells (Figure 3.3 A). In contrast, nuclear REST remained unchanged between BM vs non-
BM cells in lung cancer and melanoma (Figure 3.3 B,C). These results suggest that REST is
dysregulated at a translational and functional level in BBMs.
Figure 3. 3 Breast-to-brain metastatic cells show reduced nuclear REST. Representative IF images, and
quantification of nuclear REST (Cy3) localization in primary and patient-derived BM cells in breast (SKBR3, BBM 3.1)
(A), lung (A549, LuBM5) (B), and melanoma (A2058, MBM2) cancer cells respectively (Magnification 63x). Graphs
show comparison of percent nuclear localization of REST, with data plotted as individual values with mean and SEM.
Nuclear localization of REST in BBM 3.1 cells was compared to that in 3 different kinds of non-BM breast cancer cell
lines (BT474, MDA-MB-231, SKBR3).
51
3.3.2 REST dysregulation in BBMs is mediated by SRRM4 and alternative splice product
REST4:
REST expression is regulated by two mediators, REST/NRSF-Interacting LIM Domain Protein
(RILP), or Serine/Arginine Repetitive Matrix Protein 4 (SRRM4). RILP has been shown to bind to
REST and facilitate its function as a transcription factor by importing it into the nucleus
83
. Our
results show that ratio of REST/RILP nuclear colocalization remained unchanged between BM
versus non-BM cells in breast and lung cancer and melanoma (Figure 3.4 A,B,C). This suggests
that reduced nuclear import of REST in BBMs is not mediated by RILP.
Figure 3. 4 Nuclear REST/RILP remains unchanged between non-BM and BM tumor cells. Representative IF
images, and quantification of nuclear REST/RILP in primary and patient-derived BM breast (SKBR3, BBM 3.1) (A), lung
(A549, LuBM5) (B) and melanoma (A2058, MBM2) (C) cells respectively (Magnification 63X). Graphs show comparison
of percent nuclear co-localization of REST/RILP in individual cells, with data plotted as individual values with mean and
SEM.
52
Thus, we next investigated the involvement of SRRM4, a neural-specific exon splicing
factor that regulates the alternative splicing of REST into its truncated isoform REST4, which in
turn reverses REST-mediated repression of target genes. We first evaluated whether SRRM4
expression in tumors was associated with overall survival. Kaplan-Meier survival analysis from
TCGA datasets showed that although not prognostic in lung cancer or melanoma (Figure 3.5
A,B), high SRRM4 expression was significantly associated with worse overall survival (OS) in
patients with breast cancer (Figure 3.5 C). Furthermore, the effect of SRRM4 expression on OS
in breast cancer patients becomes significant later in the course of the disease (>6 years follow
up), suggesting a role for SRRM4 in advanced breast cancer, and potentially brain metastasis.
Figure 3. 5 High SRRM4 is significantly associated with worse overall survival in breast cancer. Kaplan-Meier
analysis for association between SRRM4 expression and overall survival in lung (A), melanoma (B), and breast (C)
cancers performed using UCSC Xena. TCGA datasets for all cancers were used for this study. Low (blue) and high
(red) SRRM4 expression were plotted on the graph with Y-axis representing probability of survival (1-0), and X-axis
displaying time of data collection (days).
Given the association of SRRM4 with poor survival and the reduced expression and nuclear
localization of REST in BBMs, we postulated that SRRM4 expression would be enhanced in
BBMs, accompanied by increased expression REST4. Expression and nuclear localization of both
proteins remained unchanged between BM and non-BM lung cancer tissues (Figure 3.6 A,B),
while both proteins showed significantly reduced nuclear localization in BM melanoma patient
tissues compared to primary melanoma (Figure 3.6 C,D). In contrast, total SRRM4 and REST4
expression were greatly enhanced in BBM tissues compared to non-BM breast cancer (Figure
3.6 E,F). Quantification of nuclear SRRM4 did not show statistically significant increase in BBMs
53
compared to primary breast cancer tissues (Figure 3.6 E). However, nuclear localization of
REST4 was significantly increased in BBMs, indicating enhanced SRRM4 expression mediates
REST4 functionality in these tumors (Figure 3.6 F).
Figure 3. 6 BBM tissues show enhanced SRRM4 and REST4 activity. Representative IF images, and quantification
of nuclear SRRM4 and REST4 localization in primary and brain-metastatic tumor tissue from lung (A,B), melanoma
(C,D), and breast (E,F) cancer respectively (Magnification 63x). Graphs show comparison of percent nuclear
localization of SRRM4 or REST4, with data plotted as individual values with mean and SEM.
In vitro, while SRRM4 and REST4 expression remained unchanged between primary and BM in
lung cancer and melanoma cells (Figure 3.7 A,B), there was a significant increase in the
expression of both mRNAs in BBM 3.1 cells compared to all non-BM breast cancer cells (Figure
3.7 C). Furthermore, there was no significant difference in nuclear colocalization of SRRM4 in BM
versus non-BM cells in lung cancer (Figure 3.7 D), while BM melanoma showed a significant
reduction of nuclear SRRM4 compared to its primary (Figure 3.7 E). However, nuclear SRRM4
54
was significantly enhanced in BBM 3.1 cells compared to non-BM SKBR3 breast cancer cells
(Figure 3.7 F).
Figure 3. 7 SRRM4 expression and nuclear localization is enhanced in BBMs, resulting in splice isoform REST4.
SRRM4 and REST4 mRNA expression in brain-metastatic lung LuBM5 (A), melanoma MBM2 (B), and breast BBM 3.1
(C) cancer cells, relative to their primary (non-BM) counterparts (mean and SEM). SRRM4 and REST4 expression in
BBM 3.1 cells was compared to 3 different non-BM breast cancer cell lines (BT474, SKBR3, MDA-MB-231).
Representative IF images, and quantification of nuclear SRRM4 (Cy3) localization in primary and patient-derived BM
cells in lung (A549, LuBM5) (D), melanoma (A2058, MBM2) (E), and breast (SKBR3, BBM 3.1) (F) cancer cells
respectively (Magnification 63x). Graphs show comparison of percent nuclear localization of SRRM4, with data plotted
as individual values/cell with mean and SEM.
We concluded that REST to REST4 dysregulation is mediated by SRRM4 and is exclusive to
BBMs. To validate this, we established stably transduced SKBR3 primary breast cancer cells with
SRRM4 overexpressed (SKBR3
OE
) or knocked down (SKBR3
KD
). Expression and nuclear
localization of SRRM4 was significantly enhanced in SKBR3
OE
, and reduced in SKBR3
KD
cells,
55
compared to parental SKBR3 cells (Figure 3.8 A,B). Appropriately, REST4 analysis showed
increase in SKBR3
OE
cells and diminished expression in SKBR3
KD
cells. (Figure 3.8 C,D)
Figure 3. 8 Establishment of SRRM4 overexpressed (SKBR3
OE
) and SRRM4 knockdown (SKBR3
KD
) cell lines.
Confirmation of successful transduction of SKBR3 cells by mRNA and protein analysis of SRRM4 (K,L) and REST4
(M,N). mRNA overexpression or knockdown was confirmed by qPCR, with data represented in bar graphs as fold
change in expression relative to parental SKBR3 cells. Overexpression or knockdown of protein was assessed by
immunofluorescence. Graphs show comparison of percent nuclear localization of SRRM4 and REST4 in SKBR3 cells
(parental, SKBR3
OE
and KSBR3
KD
), with data plotted as individual values with mean and SEM.
We next elucidated the contribution of SRRM4 in BBM colonization in vivo. All SKBR3
OE
xenografted mice developed brain metastases by day 98 post intracardiac injection, compared to
two mice from the SKBR3
CTRL
(control) group and only one from the SKBR3
KD
group (Figure 3.9
A,B,C). Extracranial tumor load remained comparable between SKBR3
CTRL
and SKBR3
OE
xenografts, whereas SKBR3
KD
showed lower tumor load overall. Although 4 out of 5 mice in the
control group developed brain metastases eventually, this was a significantly delayed event
compared to SKBR3
OE
group (p=0.022), while mice from SKBR3
KD
group remained BM-free
(Figure 3.9 D). Moreover, SKBR3
OE
xenografts showed significantly worse overall survival
compared to control (Figure 3.9 E), suggesting that even in the presence of comparable
56
extracranial tumor load, increased SRRM4 expression in tumor cells contributes to worse
prognosis by triggering enhanced BM competency in breast cancer.
Figure 3. 9 SRRM4 overexpression facilitates accelerated brain metastasis and contributes to worse overall
survival in vivo. Representative images of bio-luminescent imaging (BLI) for brain-metastasis and overall tumor load,
post intracardiac injection of SKBR3
ctrl
(control) (A), SKBR3
OE
(B), and SKBR3
KD
(C) cells in immune-compromised
mice. (D) Graph representing percentage of animals that developed brain metastases in each group over time (control,
SKBR3
OE
, SKBR3
KD
). (E) Kaplan-Meier survival analysis SKBR3
ctrl
and SKBR3
OE
xenografted mice (n=5 per group).
Survival curve shows significantly worse overall survival (OS) in mice bearing tumor load from SKBR3
OE
cells (blue
line) compared to SKBR3
ctrl
cells (red line) (p=0.0013, HR=5.321).
Analysis of SKBR3
CTRL
xenografts showed areas of cells with high expression and nuclear
localization of SRRM4 and REST4, and low REST in an otherwise heterogenous brain-metastatic
tumor population. In contrast, SKBR3
OE
BMs showed uniform and significantly higher nuclear
localization of SRRM4 and REST4, concurrent with diminished REST expression (Figure 3.10
57
A,B,C). Overall these results show enhanced SRRM4 expression augments brain-metastatic
potential in breast cancer cells and contributes to worse overall survival in vivo.
Figure 3. 10 Analysis of BM lesions from xenografted animals. Representative IF images for expression and
comparison of nuclear localization of SRRM4 (A), REST4 (B) and REST (C) in brain-metastatic lesions from SKBR3
ctrl
and SKBR3
OE
xenografted mice respectively. Graphs show comparison of percent nuclear localization of SRRM4 and
REST4 for both groups, with data plotted as individual values with mean and SEM.
3.3.3 Enhanced SRRM4 expression confers less dormant and more proliferative
phenotype in breast cancer cells
Clinically, breast cancer patients have longer latency to brain metastasis, compared to more
aggressive cancers like lung cancer
84
. This can be attributed partly to the presence of dormant
tumor cells (DTCs) which can eventually be reactivated for successful colonization
85,86
. Therefore,
we wanted to determine whether the lack of SRRM4 expression which resulted in reduced
metastatic competency we observed in vivo was because of a DTC phenotype in SRRM4-low
breast cancer cells. First, BrDU incorporation analysis showed enhanced proliferation in SKBR3
OE
58
cells, but slower growth in SKBR3
KD
cells, relative to control SKBR3 cells (Figure 3.11 A). This
was further confirmed by the presence of significantly enhanced Ki67 positivity in SKBR3
OE
cells,
compared to SKBR3
KD
and control SKBR3 cells (Figure 3.11 B). These results indicate that
SRRM4 expression regulates proliferative potential in breast cancer cells.
Figure 3. 11 SRRM4 regulates proliferative capacity in breast cancer cells. (A) relative BrdU incorporation in
control, SKBR3
OE
, SKBR3
KD
cells. (B) Representative IF images and quantification of proliferative marker Ki67 (red) in
control, SKBR3
OE
, SKBR3
KD
cells. Data represented as percent Ki67 positive cells per field (mean and SEM)
(Magnification 63x).
Next, tumor cells were acclimated to dormant conditions to model a nutrition-deficient metastatic
microenvironment. Indeed, control and SKBR3
KD
cells showed enhanced mRNA expression of
dormancy markers p53, SOX9, RARB and TGFB3 compared to SKBR3
OE
cells (Figure 3.12 A).
Consistent with mRNA data, control
and SKBR3
KD
cells showed enhanced SOX9 expression and
nuclear localization compared to SKBR3
OE
cells in dormant conditions (Figure 3.12 B), indicating
propensity for metastatic dormancy in cells with low SRRM4/REST4 expression. Interestingly,
SKBR3
OE
cells showed sustained nuclear REST4, while control
and SKBR3
KD
cells displayed
significant loss of nuclear REST4 in dormant conditions (Figure 3.12 C).
59
Figure 3. 12 Low SRRM4 expression confers dormant phenotype and reduced REST4 expression in breast
cancer cells. (A) Relative mRNA expression (mean and SEM) of dormancy markers p53, SOX9, RARb, TGFB3 in
control, SKBR3
OE
, SKBR3
KD
cells acclimated to dormant conditions. Representative IF images and comparison of
nuclear colocalization of SOX9 (B) and REST4 (C) in control, SKBR3
OE
, SKBR3
KD
cells in normal (NC) and dormant
(DC) conditions respectively (Magnification 63x). Data for both markers presented as percent nuclear colocalization
per cell, with individual values represented (mean and SEM) in both conditions.
Next, we treated SKBR3 cells with 5-Fluorouracil and Paclitaxel which are standard
chemotherapeutic drugs used for breast cancer patients
87
. Slower growing SKBR3
KD
cells were
most resistant to both drugs, followed by control SKBR3 cells, whereas SKBR3
OE
cells showed
enhanced sensitivity to Paclitaxel (Figure 3.13 A,B). Taken together, these results suggest that
low SRRM4 expression confers a dormant phenotype, whereas enhanced SRRM4 expression
leading to REST4 activity increases proliferative capacity and chemo-sensitization.
60
Figure 3. 13 SRRM4
KD
cells show enhanced chemoresistance. Response to chemotherapeutic drugs 5-Fluorouracil
(A) and Paclitaxel (B) in control, SKBR3
OE
, SKBR3
KD
cells. Data represented on graph as percent cell viability (mean
and SEM) upon treatment with drug for 48 hours.
3.3.4 SRRM4 facilitates CNS-acclimation and provides proliferative advantage to BC cells
in the neuronal microenvironment.
Because in vivo results revealed upregulation of SRRM4 provides a proclivity for breast-to-brain
metastasis, we asked whether this neuronal master regulator facilitates tumor-CNS acclimation
and colonization through enhanced tumor-neuron interaction. We observed an increase of
SRRM4 and REST4 in control SKBR3 cells cultured with neuronal conditioned media (Figure
3.14 A). Furthermore, SKBR3
OE
cells co-cultured with neurons had significantly enhanced nuclear
SRRM4 and REST4 in (Figure 3.14 B), while control SKBR3 cells in co-culture showed gain of
nuclear SRRM4 but not REST4 (Figure 3.14 C). These results indicate that exposure to the
neuronal microenvironment promotes increased SRRM4 and REST4 in breast cancer cells.
Figure 3. 14 Neuronal exposure enhances SRRM4 activity in breast cancer cells. (A) mRNA expression of SRRM4
and REST4 in SKBR3 cells treated with neuron conditioned media. Data represented as fold change in expression
relative to untreated SKBR3 cells. Quantification and comparison of nuclear localization of SRRM4 and REST4 in
control SKBR3 cells (B), and SKBR3
OE
(C) cells in 2 conditions (tumor only, tumor-neuron co-culture). Individual values
for both markers represented in graph as percent nuclear colocalization per cell (mean and SEM) for both conditions
tested.
61
Furthermore, on exposure to neuron-conditioned media, SKBR3
OE
cells maintained their
inherently high expression of SRRM4 and REST4, while SKBR3
KD
cells were unable to upregulate
the expression of either gene (Figure 3.15 A). Since SRRM4 regulates the expression of CNS-
specific genes, we next determined which targets in tumors are regulated by SRRM4
augmentation in presence of neurons to promote brain metastasis competency. To investigate
this, we first interrogated the expression of 153 genes involved in neurotransmitter and synaptic
signaling in SKBR3 cells (control, SKBR3
OE
, SKBR3
KD
) cultured in the neuronal
microenvironment. Of these 153 targets, 45 were determined to be direct SRRM4-mediated
based on association overlap of tumor-inherent SRRM4 regulated genes, and neural
microenvironment-induced SRRM4 regulated genes (Figure 3.15 B). We then validated some of
the targets identified from our screen by qPCR.
SKBR3
ctrl
and SKBR3
OE
cells showed upregulation of BDNF, a neurotrophin involved in
regulating long-term synaptic plasticity
88
(Figure 3.15 C). SKBR3
ctrl
cells also upregulated
vesicular trafficking protein RAB3A involved in regulating BDNF-mediated plasticity
89
, GRIN2B,
an NMDAR-type glutamate receptor
90
, presynaptic regulator cannabinoid receptor CNR1
91
, and
TACR3 a receptor for neurokins
92
(Figure 3.15 D,E,F,G). SKBR3
OE
cells showed inherently
enhanced expression of these genes, and thus retained the expression of most genes within the
neuronal microenvironment. Furthermore, only SKBR3
OE
cells showed upregulation of synaptic
master regulator RELN
93
, and metabotropic glutamate receptor GRM8 on exposure to the
neuronal microenvironment (Figure 3.15 H,I). In contrast, SKBR3
KD
were unable to respond to
the neuronal exposure and did not show upregulation of any CNS acclimatory genes. These data
suggest that increased SRRM4 expression promotes the transcription of downstream CNS-
specific target genes; and that exposure to the neuronal microenvironment is also able to induce
SRRM4 and REST4 expression, which subsequently drives CNS-acclimation in brain-trophic
breast cancer cells.
62
Figure 3. 15 SRRM4 promotes expression of CNS-specific synaptic plasticity mediators in breast cancer cells,
within the neuronal microenvironment. (A) mRNA expression of SRRM4 and REST4 in SKBR3
OE
and SKBR3
KD
cells in two conditions – tumor only, and tumor cells treated with neuron conditioned media (48h). Data represented in
graph as relative expression of SRRM4 and REST4 in both cell types in all represented conditions. Red bars represent
SKBR3
OE
cells and blue bars represent SKBR3
KD
cells. (B) Venn diagram classification of tumor-inherent vs.
microenvironment-mediated SRRM4 dependent and independent synaptic plasticity genes expressed in SKBR3
OE
and
SKBR3
KD
cells in two conditions – tumor only, and tumor cells treated with neuron conditioned media (48h). SRRM4
dependent targets were identified, and then validated by qPCR. Target validation by qPCR for expression of SRRM4-
dependent target genes BDNF (C), RAB3A (D), GRIN2B (E), CNR1 (F), TACR3 (G), RELN (H), GRM8 (I) in SKBR3
cells (SKBR3
ctrl
, SKBR3
OE
and SKBR3
KD
) in two conditions - tumor only, and tumor cells treated with neuron conditioned
media (48h). Data represented in graph as relative expression of target genes in each cell type in all represented
conditions. Red bars represent SKBR3
ctrl
cells, blue bars represent SKBR3
OE
cells, and black bars represent SKBR3
KD
cells.
We next asked whether interaction with neurons, along with enhanced SRRM4 in tumor
cells provides a colonization and growth advantage to breast cancer cells in the early CNS
metastatic microenvironment. To determine this, we established competitive in vitro colonization
model where combination of either: 1) SKBR3 and SKBR3
OE
(Figure 3.16 A), 2) SKBR3
OE
and
SKBR3
KD
(Figure 3.16 B), or 3) SKBR3 and SKBR3
KD
(Figure 3.16 C) were co-cultured with
primary neurons and evaluated for growth. Results show SKBR3
OE
cells have significant
63
proliferative advantage over both control and SKBR3
KD
cells (Figure 3.16 A,B). Additionally,
control SKBR3 cells which can upregulate SRRM4 in the neuronal microenvironment as
previously shown, have significant growth advantage compared to SKBR3
KD
cells (Figure 3.16
C). Furthermore, SKBR3
KD
remained quiescent throughout the various competitive models. Taken
together, increased SRRM4 promotes CNS acclimation in breast cancer cells, resulting in
proliferative advantage in the neuronal microenvironment.
Figure 3. 16 Enhanced SRRM4 expression provides proliferative advantage in the neuronal microenvironment
in vitro. Competitive in vitro colonization model was established with SKBR3 cells co-cultured with neurons in the
following groups: (1)SKBR3 and SKBR3
OE
(2) SKBR3
OE
and SKBR3
KD
or (3) SKBR3 and SKBR3
KD
. Tumor cells in
these co-cultures were then evaluated for growth. Representative IF images and quantification of total cell numbers for
each tumor cells type in 3 different combination co-cultures with primary neurons: SKBR3 (GFP)/ SKBR3
OE
(mCherry)
(A), SKBR3
OE
(mCherry)/ SKBR3
KD
(GFP) (B), SKBR3 (mCherry)/ SKBR3
KD
(GFP) (C). Data was collected over 120
hours of co-culture. Individual values in graph represent number of cells of each subtype per image quantified (red vs
blue) (mean and SEM).
3.4 Discussion:
Successful brain metastases can be established only by tumor cells that are competent to
colonize the foreign metastatic landscape of the brain. Our study sought to determine the
regulators of CNS competency and neural acclimation in brain-seeking tumors cells.
64
We uncovered enhanced expression of neural-specific splicing protein SRRM4 in breast-to-brain
metastases (BBMs), resulting in reduced functionality of neuronal gene suppressor REST,
concomitant with increased expression and nuclear localization of its alternative splice isoform
REST4. SRRM4-mediated REST to REST4 splicing is critical in the developing brain for normal
neuronal development and maturation. Our results thus suggest that breast cancer cells co-opt
neurodevelopmental pathways to successfully adapt to the CNS-metastatic niche.
CNS-metastatic competency relies on inherent properties of the tumor cells, as well as
their ability to interact with the neural microenvironment
94
. In vivo results revealed that SRRM4
overexpression in brain-naïve breast cancer cells promotes accelerated brain metastasis and
contributes to worse overall survival. In contrast, lack of SRRM4 expression provides no CNS-
metastatic advantage, concomitant with low tumor load. Correspondingly, we also determined
that SRRM4 regulates proliferative potential and reactivation of dormancy in breast cancer cells,
via nuclear REST4. These data indicate, cells with low SRRM4 expression which show quiescent
phenotype might persist as dormant tumor cells for extended periods of time in breast cancer
patients. However, only cells that can upregulate SRRM4 inherently or on exposure to the neural
microenvironment will eventually colonize the brain.
Appropriately, we next show that neuronal contact induces enhanced expression and
nuclear localization of SRRM4 and REST4 in parental and SKBR3
OE
cells, allowing for expression
of CNS-specific mediators of synaptic plasticity in these tumor cells. SKBR3
KD
cells however are
unable to modulate SRRM4/REST4 within the neuronal microenvironment. We thus provide
evidence that elevated SRRM4 and REST4 levels regulate the gain of neural attributes in breast-
to-brain metastases (BBMs) as described in previous studies
13,31
. Differential mRNA expression
analysis also revealed specific SRRM4/REST4 regulated genes that warrant further investigation
as clinically actionable targets to manage or prevent BBM colonization, early in brain metastasis.
Ultimately, results from our competitive in vitro colonization model confirm that breast
cancer cells that can upregulate SRRM4 activity are able to successfully colonize a permissive
65
early neural niche, while SKBR3
KD
cells remain quiescent. Altogether, we conclude that enhanced
SRRM4 promotes CNS-metastatic colonization in breast cancer.
This study thus identifies a novel mechanism that governs BM competency, dormancy
and CNS acclimation in breast cancer cells, providing clues into why breast cancer cells are able
to establish successful brain metastases after a long latency period.
Figure 3. 17 Role of SRRM4 in facilitating breast-to-brain metastasis
References
11. Saunus JM, Momeny M, Simpson PT, Lakhani SR, Da Silva L. Molecular aspects of breast
cancer metastasis to the brain. Genet Res Int. 2011; 2011:219189.
13. Neman J, Termini J, Wilczynski S, et al. Human breast cancer metastases to the brain
display GABAergic properties in the neural niche. Proceedings of the National Academy
of Sciences. 2014; 111(3):984-989.
31. Zeng Q, Michael IP, Zhang P, et al. Synaptic proximity enables NMDAR signalling to
promote brain metastasis. Nature. 2019; 573(7775):526-531.
67. Chong JA, Tapia-Ramírez J, Kim S, et al. REST: a mammalian silencer protein that
restricts sodium channel gene expression to neurons. Cell. 1995; 80(6):949-957.
66
68. Ballas N, Battaglioli E, Atouf F, et al. Regulation of neuronal traits by a novel transcriptional
complex. Neuron. 2001; 31(3):353-365.
69. Ballas N, Grunseich C, Lu DD, Speh JC, Mandel G. REST and Its Corepressors Mediate
Plasticity of Neuronal Gene Chromatin throughout Neurogenesis. Cell. 2005; 121(4):645-
657.
70. Lawinger P, Venugopal R, Guo ZS, et al. The neuronal repressor REST/NRSF is an
essential regulator in medulloblastoma cells. Nat Med. 2000; 6(7):826-831.
71. Kamal MM, Sathyan P, Singh SK, et al. REST regulates oncogenic properties of
glioblastoma stem cells. Stem Cells. 2012; 30(3):405-414.
72. Wagoner MP, Gunsalus KT, Schoenike B, Richardson AL, Friedl A, Roopra A. The
transcription factor REST is lost in aggressive breast cancer. PLoS Genet. 2010;
6(6):e1000979.
73. Kreisler A, Strissel PL, Strick R, Neumann SB, Schumacher U, Becker CM. Regulation of
the NRSF/REST gene by methylation and CREB affects the cellular phenotype of small-
cell lung cancer. Oncogene. 2010; 29(43):5828-5838.
74. Coulson JM. Transcriptional Regulation: Cancer, Neurons and the REST. Current Biology.
2005; 15(17):R665-R668.
75. Raj B, O'Hanlon D, Vessey JP, et al. Cross-regulation between an alternative splicing
activator and a transcription repressor controls neurogenesis. Mol Cell. 2011; 43(5):843-
850.
76. Tabuchi A, Yamada T, Sasagawa S, Naruse Y, Mori N, Tsuda M. REST4-mediated
modulation of REST/NRSF-silencing function during BDNF gene promoter activation.
Biochem Biophys Res Commun. 2002; 290(1):415-420.
77. Lunyak VV, Rosenfeld MG. No Rest for REST: REST/NRSF Regulation of Neurogenesis.
Cell. 2005; 121(4):499-501.
78. Li Y, Donmez N, Sahinalp C, et al. SRRM4 Drives Neuroendocrine Transdifferentiation of
Prostate Adenocarcinoma Under Androgen Receptor Pathway Inhibition. Eur Urol. 2017;
71(1):68-78.
79. Klotz R, Thomas A, Teng T, et al. Circulating Tumor Cells Exhibit Metastatic Tropism and
Reveal Brain Metastasis Drivers. Cancer Discovery. 2020; 10(1):86-103.
80. Hara D, Fukuchi M, Miyashita T, et al. Remote control of activity-dependent BDNF gene
promoter-I transcription mediated by REST/NRSF. Biochem Biophys Res Commun. 2009;
384(4):506-511.
81. Warburton A, Savage AL, Myers P, Peeney D, Bubb VJ, Quinn JP. Molecular signatures
of mood stabilisers highlight the role of the transcription factor REST/NRSF. Journal of
Affective Disorders. 2015; 172:63-73.
82. Shimojo M. Characterization of the nuclear targeting signal of REST/NRSF. Neurosci Lett.
2006; 398(3):161-166.
67
83. Shimojo M, Hersh LB. Characterization of the REST/NRSF-interacting LIM domain protein
(RILP): localization and interaction with REST/NRSF. J Neurochem. 2006; 96(4):1130-
1138.
84. Kodack DP, Askoxylakis V, Ferraro GB, Fukumura D, Jain RK. Emerging strategies for
treating brain metastases from breast cancer. Cancer Cell. 2015; 27(2):163-175.
85. Banys M, Hartkopf AD, Krawczyk N, et al. Dormancy in breast cancer. Breast Cancer
(Dove Med Press). 2012; 4:183-191.
86. Lorger M, Felding-Habermann B. Capturing changes in the brain microenvironment during
initial steps of breast cancer brain metastasis. Am J Pathol. 2010; 176(6):2958-2971.
87. Nicholson BP, Paul DM, Hande KR, et al. Paclitaxel, 5-fluorouracil, and leucovorin (TFL)
in the treatment of metastatic breast cancer. Clin Breast Cancer. 2000; 1(2):136-143;
discussion 144.
88. Lu B, Nagappan G, Lu Y. BDNF and synaptic plasticity, cognitive function, and
dysfunction. Handb Exp Pharmacol. 2014; 220:223-250.
89. Thakker-Varia S, Alder J, Crozier RA, Plummer MR, Black IB. Rab3A is required for brain-
derived neurotrophic factor-induced synaptic plasticity: transcriptional analysis at the
population and single-cell levels. J Neurosci. 2001; 21(17):6782-6790.
90. Shin W, Kim K, Serraz B, et al. Early correction of synaptic long-term depression improves
abnormal anxiety-like behavior in adult GluN2B-C456Y-mutant mice. PLoS Biol. 2020;
18(4):e3000717.
91. Carey MR, Myoga MH, McDaniels KR, et al. Presynaptic CB1 receptors regulate synaptic
plasticity at cerebellar parallel fiber synapses. J Neurophysiol. 2011; 105(2):958-963.
92. Cui W-Q, Zhang W-W, Chen T, et al. Tacr3 in the lateral habenula differentially regulates
orofacial allodynia and anxiety-like behaviors in a mouse model of trigeminal neuralgia.
Acta Neuropathologica Communications. 2020; 8(1):44.
93. Beffert U, Weeber EJ, Durudas A, et al. Modulation of Synaptic Plasticity and Memory by
Reelin Involves Differential Splicing of the Lipoprotein Receptor Apoer2. Neuron. 2005;
47(4):567-579.
94. Lorusso G, Rüegg C. The tumor microenvironment and its contribution to tumor evolution
toward metastasis. Histochem Cell Biol. 2008; 130(6):1091-1103.
68
Chapter 4: Conclusions and future directions
4.1 Recap
In the previous chapters, we discussed two different aspects that ultimately promote CNS
metastatic colonization in tumor cells. We established that early tumor-neuron interactions are
crucial within the neural niche and induce neurotransmitter responsiveness and synaptic plasticity
mediators in tumor cells. As we built on the foundation of previous studies that demonstrate the
gain of CNS-adaptive attributes in tumor cells, we asked whether this was due to the activation of
transcriptional regulatory pathways in brain-seeking tumor cells. We subsequently uncovered that
enhanced expression of neural-specific splicing protein SRRM4 in breast cancer cells promoted
CNS-metastatic competency.
This chapter reviews my thesis work and focuses on the limitations and future directions arising
from the study.
4.2 Limitations and future directions
Since our study establishes SRRM4 as a driver of BM-competency, it is now important to identify
factors that regulate SRRM4 expression in breast cancer cells. Brain-naïve SKBR3 cells (along
with other non-BM breast cancer cell lines) show negligible endogenous expression of SRRM4.
However, as described in chapter 3, neuronal exposure can significantly enhance its expression
and nuclear localization in these cells. A recent study by Serrano et al showed that SRRM4
expression is epigenetically suppressed by promoter hypermethylation in tumors of non-neuronal
origin including breast cancer
95
. Our results from chapter 2 indicate that breast cancer cells show
a global reduction in Histone De-acetylase (HDAC) activity on exposure to neuron conditioned
media facilitating the expression of CNS-specific genes (Figure 2.13). Thus, it is possible that
early interaction with the brain microenvironment can result in epigenetic upregulation of SRRM4
in brain-seeking breast cancer cells.
69
Our studies also show however, that cells that endogenously over-express SRRM4 i.e SKBR3
OE
cells, show enhanced BM competency and proclivity for CNS-acclimation in vivo and in vitro.
Thus, owing to tumor heterogeneity, a sub-population of tumor cells might be able to increase
SRRM4 expression before extravasating into the brain, that then allows them to successfully
traverse the metastatic cascade. Analysis of matched breast cancer cells/tissue in various stages
of the disease can aid in identifying the molecular processes underlying SRRM4 activation.
An important early event in brain metastasis, is the extravasation of metastatic cells
through the cells lining the blood-brain barrier (BBB)
96
. For our in vivo studies, we xenografted
mice via intracardiac injection, to model the natural occurrence of BMs through the circulation.
The incidence of CNS metastases was accelerated in SKBR3
OE
group (figure 3.9 D), perpetuating
two hypotheses – a) enhanced SRRM4 expression allowed them to persist in the CNS after
migration through the BBB compared to SKBR3
ctrl
or SKBR3
KD
cells, or b) SRRM4 can enhance
migration of breast cancer cells through the BBB.
We elucidated that SRRM4 regulates proliferation (figure 3.9E) and CNS-acclimation
(figure 3.15 C-I) in breast cancer cells, indicating that they can persist in the CNS better than cells
that show low/no SRRM4 expression. However, the effect of SRRM4 on tumor migration remains
to be studied. REST has been implicated as a transcriptional repressor of Neuropilin-1 (NRP-1),
a receptor for vascular endothelial growth factor (VEGF)
97
. NRP-1 expression in breast cancer is
directly correlated with increased survival, migration and invasiveness
98
. Since brain-metastatic
breast cancer cells show SRRM4-mediated loss of REST activity, this could be associated with
the gain of expression of genes that mediate migration though endothelial cells, including NRP-
1. Differential expression analysis of genes involved in tumor invasion and migration, combined
with in vitro assays comparing migration/invasion capacity of breast cancer cells with high vs. low
SRRM4 expression will help in answering this question.
A recent retrospective clinical research article of which Dr. Neman and I were a part,
shows that brain-metastatic tumor cells from different primary cancers show differential positional
70
propensities of brain metastases (Predictive spatial modeling reveal primary cancers have
distinct central nervous system topography patterns of brain metastasis, Accepted to Journal of
Neurosurgery, January 2021). Specifically, breast cancer metastases are statistically more likely
to grow in the left cerebellar hemisphere. This is particularly interesting because of two reasons.
First, our results in chapter 2 show that reliance on microenvironmental GABA is an early
CNS-acclimatory strategy in breast cancer (figure 2.6). GABA, the predominant inhibitory
neurotransmitter in the adult brain, is crucial for the development of cerebellar function and
GABAergic synapses
99
. Although GABA is distributed throughout the parenchyma, the GABA
concentration in the cerebellum is higher than that in the cortex. This indicate that the cerebellum
provides a conducive microenvironment for incoming breast cancer cells that can adapt to a
GABA rich soil.
Second, microarray analysis of SRRM4 expression in normal human tissues shows
highest expression in the brain, particularly in cerebellar neurons. This indicates that the
cerebellar microenvironment supports enhanced SRRM4 functionality. It remains to be seen
whether the particular proclivity of breast-to-brain metastases to the cerebellum is influenced by
the permissiveness of this site for tumor cells that can increase their SRRM4 expression/activity
within this niche. To assess this, sparse numbers of breast cancer cells (low vs. high SRRM4
expression) can be injected intracranially into different lobes of the brain, and the development of
macro-metastases can be monitored, to analyze with nuance whether SRRM4 regulates
colonization within the CNS as a whole, or if it also affects growth of lesions within specific sites.
4.3 Therapeutic avenues
The research presented in this dissertation identifies various targets that are upregulated
in tumor cells within the neuronal microenvironment. We also identify SRRM4 as a metastatic
driver in breast cancer cells. We hope that future studies conducted within the realm of these
discoveries, can facilitate the recognition of clinically actionable targets. Therapeutic intervention
71
in brain metastasis presents with complications including ineffective penetration of small
molecules through the BBB, and lack of targeted therapies
100
. A recent study proposed the use
of antisense oligos targeting SRRM4 for use in aggressive small cell lung cancer
101
. This oligo
does not cross the BBB, and thus cannot yet be studied for use in overt brain metastases, but
could potentially be used to target circulating tumor cells in breast cancer that are confirmed to
express SRRM4. This can be effective after CTC biopsies in at-risk patients with advanced breast
cancer.
Many of the targets identified in this study are also expressed endogenously by healthy
neural cells, particularly by neurons (neurotransmitter receptors, and neurodevelopmental
regulators like SRRM4 and REST4). This poses an issue because attempting to target these
mediators in brain-metastatic tumor cells, poses a threat of potential off target effects on healthy
brain cells. Thus, it might be prudent to run differential expression analysis on breast cancer cells
with high vs low SRRM4 expression and determine targets (cell surface/intracellular) that are
more tumor specific, and can be targeted so as to limit harmful off target effects. We also propose
conducting small molecule screens on these tumor cells which can identify existing/new anti-
cancer drugs that are able to specifically target tumor cells that show a gene signature amenable
to brain metastasis.
In summary, this chapter highlights the discoveries discussed at length in the dissertation,
and identifies areas of future research that can further our knowledge of the mechanisms involved
in CNS colonization, particularly in breast cancer. I hope that addressing the questions that arise
from this work can aid the development of innovative therapeutic interventions for patients with
brain metastasis.
References
95. Head SA, Hernandez-Alias X, Yang J-S, et al. Silencing of SRRM4 suppresses microexon
inclusion and promotes tumor growth across cancers. PLOS Biology. 2021;
19(2):e3001138.
72
96. Arshad F, Wang L, Sy C, Avraham S, Avraham HK. Blood-Brain Barrier Integrity and
Breast Cancer Metastasis to the Brain. Pathology Research International. 2011;
2011:920509.
97. Kurschat P, Bielenberg D, Rossignol-Tallandier M, Stahl A, Klagsbrun M. Neuron
restrictive silencer factor NRSF/REST is a transcriptional repressor of neuropilin-1 and
diminishes the ability of semaphorin 3A to inhibit keratinocyte migration. J Biol Chem.
2006; 281(5):2721-2729.
98. Ferrario C, Hostetter G, Bouchard A, Huneau M-C, Mamo A, Basik M. Expression of
neuropilin-1 and related proteins in breast cancer. Cancer Research. 2006; 66(8
Supplement):1182-1182.
99. Leto K, Rolando C, Rossi F. The Genesis of Cerebellar GABAergic Neurons: Fate
Potential and Specification Mechanisms. Frontiers in Neuroanatomy. 2012; 6(6).
100. Ren D, Cheng H, Wang X, et al. Emerging treatment strategies for breast cancer brain
metastasis: from translational therapeutics to real-world experience. Ther Adv Med Oncol.
2020; 12:1758835920936151.
101. Shimojo M, Kasahara Y, Inoue M, et al. A gapmer antisense oligonucleotide targeting
SRRM4 is a novel therapeutic medicine for lung cancer. Scientific Reports. 2019;
9(1):7618.
73
Appendix
Table 1 Genes of interest and primer sequences in Chapter 2
Gene (Human) Primer Sequence
GRM3 Primer 1: 5’-GATGCGTAGCTGATCTGAGG-3’
Primer 2: 5’-CCTATGCCATTCAAGAAAACATCC-3’
GRM4 Primer 1: 5’-CATAAGCTGAATCCTGCCCAA -3’
Primer 2: 5’-GCTCCAAGATTGCACCTGT -3’
GRM7 Primer 1: 5’-CCCAGTATTCGGCAAACCATA -3’
Primer 2: 5’-CCAGATGAAGATATCGCAGA -3’
GRM8 Primer 1: 5’-ATTGGCAAGAGTTCGGCTT -3’
Primer 2: 5’-ATAGCACCTGTCTATCAGCAAG -3’
GRIA2 Primer 1: 5’-CGTAGTCCTCACAAACACAGA -3’
Primer 2: 5’-GAACATTAGACTCTGGCTCCA -3’
GRIN2B Primer 1: 5’-CTTCATAGAGACAGGCATCAGT -3’
Primer 2: 5’-CATCACAAACATCATCACCCATAC -3’
HTR4 Primer 1: 5’-CAGATGTCTTGAACCAGCTCA -3’
Primer 2: 5’-GGTGATGGTGGCTGTGTG -3’
GABRB1 Primer 1: 5’-AATGTCATCAGTGGTATAGCCA -3’
Primer 2: 5’-ATGATTCGAACTGCATCCTGAT -3’
GABBR1 Primer 1: 5’-ATCTCAATTCCAGCCTCCTTC -3’
Primer 2: 5’-CCACACTCCACAACCCTAC -3’
GAD1 Primer 1: 5’-AACCAACACTCCCTCATGTC -3’
Primer 2: 5’-TTTCAACCAGCTCTCCACTG -3’
ARC Primer 1: 5’-CTGAGCTCTGATTCTTCTCCAG -3’
Primer 2: 5’-GAGAGTAGAAGTCGCACACG -3’
EGR2 Primer 1: 5’-AGATCCAACGACCTCTTCTC -3’
Primer 2: 5’-TCTTTCCCAATGCCGAACTG -3’
MMP9 Primer 1: 5’-ACATCGTCATCCAGTTTGGTG -3’
Primer 2: 5’-CGTCGAAATGGGCGTCT -3’
RELN Primer 1: 5’-GTGATGCCTGAACACTTGTAGA -3’
Primer 2: 5’-CAACCCCACCTACTACGTTC -3’
VIMENTIN Primer 1: 5’-GTGAATCCAGATTAGTTTCCCTCA -3’
Reverse: 5’-CAAGACCTGCTCAATGTTAAGATG -3’
SNAI1 Primer 1: 5’-AGTCCCAGATGAGCATTGG -3’
Primer 2: 5’-CCAATCGGAAGCCTAACTACAG-3’
RARA Primer 1: 5’-CCGAGAAGGTCATGGTGTC -3’
Primer 2: 5’-ACCAGATCACCCTCCTCAA -3’
NTRK2 Primer 1: 5’-GACTTTCCTTCCTCCACAGTG -3’
Primer 2: 5’-CACTCAGGATTTGTACTGCCT -3’
NGFR Primer 1: 5’-GTTGGCTCCTTGCTTGTTC -3’
Primer 2: 5’-CCTGTCTATTGCTCCATCCTG -3’
NRXN1 Primer 1: 5’-CTGTCCCAACATTAAACTTAACTCC -3’
Primer 2: 5’-GCCATAGGTTTTAGCACTGTTC -3’
NLGN4X Primer 1: 5’-CTCCCACATTCTCCTCAATCC -3’
Primer 2: 5’-TGGCAAGCTACGGAAACG -3’
CDK4 Primer 1: 5’-TTCAGAGTTTCCACAGAAGAGAG -3’
Primer 2: 5’-TACCGAGCTCCCGAA -3’
CDK6 Primer 1: 5’-CCAACACTCCAGAGATCCAC -3’
Primer 2: 5’-GCCCGCATCTATAGTTTCCAG -3’
P16-ARC (CDKN2A) Primer 1: 5’-AGCTGTCGACTTCATGACAAG -3’
Primer 2: 5’-TGAGCTTTGGTTCTGCCATT -3’
CCND1 Primer 1: 5’-AGCGGTCCAGGTAGTTCA -3’
Primer 2: 5’-GTGTCCTACTTCAAATGTGTGC -3’
GAPDH Primer 1: 5’-TGAGTGTGGCAGGGACT -3’
Primer 2: 5’-AGGGTGGTGGACCTCAT -3’
74
Table 2 Genes of interest and primer sequences in Chapter 3.
Gene (Human) Primer Sequence
REST Primer 1: 5’-ACTCAGCGTCGTAGAACCTCA-3’
Primer 2: 5’-CGAAAGGGTTTGGTCTTCGAG -3’
REST4 Primer 1: 5’-ACTCATACAGGAGAACGCCCA -3’
Primer 2: 5’-GGCTTCTCACCCATCTAGATCAC -3’
SRRM4 Primer 1: 5’-CCTCTCATGACAAAGACTTGAC -3’
Primer 2: 5’-TTTCTTGACAGGCGATGGG -3’
P53 Primer 1: 5’-CTTACATCTCCCAAACATCCCT -3’
Primer 2: 5’-CCAGGACTTCCATTTGCTTTG -3’
SOX9 Primer 1: 5’-CGTTCTTCACCGACTTCCTC -3’
Primer 2: 5’-CTGGGCAAGCTCTGGAG -3’
RARb Primer 1: 5’- GTCTCCTTCTTTTTCTTGTTCCTG -3’
Primer 2: 5’- GATGCCAATACTGTCGACTCC -3’
TGFb3 Primer 1: 5’-GAGTGGCTGTCCTTTGATGT -3’
Primer 2: 5’-AGTGAATGCTGATTTCTAGACCT -3’
BDNF Primer 1: 5’- CAAACATAGGTCCTTCCGTCA -3’
Primer 2: 5’- AATGCTCACACTCCACATCC -3’
RAB3A Primer 1: 5’-TGCCTCAAAGAACTCGAACC -3’
Primer 2: 5’-ACCCAGATCAAGACCTACTCA -3’
GRIN2B Primer 1: 5’-CTTCATAGAGACAGGCATCAGT -3’
Primer 2: 5’-CATCACAAACATCATCACCCATAC -3’
CNR1 Primer 1: 5’-CCTGGTCTGCTGGGACTA -3’
Primer 2: 5’-CACCATCACCACTGACCTC -3’
TACR3 Primer 1: 5’-CCGTACTCTCTGCTTTGTGC -3’
Primer 2: 5’-TGTATGTAATACCCATGATGAGCA -3’
GRM8 Primer 1: 5’-ATTGGCAAGAGTTCGGCTT -3’
Primer 2: 5’-ATAGCACCTGTCTATCAGCAAG -3’
RELN Primer 1: 5’-GTGATGCCTGAACACTTGTAGA -3’
Primer 2: 5’-CAACCCCACCTACTACGTTC -3’
75
Bibliography
1. Achrol AS, Rennert RC, Anders C, et al. Brain metastases. Nature Reviews Disease
Primers. 2019; 5(1):5.
2. Tabouret E, Chinot O, Metellus P, Tallet A, Viens P, Gonçalves A. Recent trends in
epidemiology of brain metastases: an overview. Anticancer Res. 2012; 32(11):4655-4662.
3. Owonikoko TK, Arbiser J, Zelnak A, et al. Current approaches to the treatment of
metastatic brain tumours. Nat Rev Clin Oncol. 2014; 11(4):203-222.
4. Valiente M, Ahluwalia MS, Boire A, et al. The Evolving Landscape of Brain Metastasis.
Trends Cancer. 2018; 4(3):176-196.
5. Lin X, DeAngelis LM. Treatment of Brain Metastases. J Clin Oncol. 2015; 33(30):3475-
3484.
6. Fortin D. The blood-brain barrier: its influence in the treatment of brain tumors metastases.
Curr Cancer Drug Targets. 2012; 12(3):247-259.
7. Lukas RV, Gondi V, Kamson DO, Kumthekar P, Salgia R. State-of-the-art considerations
in small cell lung cancer brain metastases. Oncotarget. 2017; 8(41).
8. Owen S, Souhami L. The management of brain metastases in non-small cell lung cancer.
Front Oncol. 2014; 4:248.
9. Niwińska A, Murawska M, Pogoda K. Breast cancer brain metastases: differences in
survival depending on biological subtype, RPA RTOG prognostic class and systemic
treatment after whole-brain radiotherapy (WBRT). Ann Oncol. 2010; 21(5):942-948.
10. Witzel I, Oliveira-Ferrer L, Pantel K, Müller V, Wikman H. Breast cancer brain metastases:
biology and new clinical perspectives. Breast Cancer Res. 2016; 18(1):8.
11. Saunus JM, Momeny M, Simpson PT, Lakhani SR, Da Silva L. Molecular aspects of breast
cancer metastasis to the brain. Genet Res Int. 2011; 2011:219189.
12. Neman J, Choy C, Kowolik CM, et al. Co-evolution of breast-to-brain metastasis and
neural progenitor cells. Clin Exp Metastasis. 2013; 30(6):753-768.
13. Neman J, Termini J, Wilczynski S, et al. Human breast cancer metastases to the brain
display GABAergic properties in the neural niche. Proceedings of the National Academy
of Sciences. 2014; 111(3):984-989.
14. Priedigkeit N, Hartmaier RJ, Chen Y, et al. Intrinsic Subtype Switching and Acquired
ERBB2/HER2 Amplifications and Mutations in Breast Cancer Brain Metastases. JAMA
Oncol. 2017; 3(5):666-671.
15. Vosoughi E, Lee JM, Miller JR, et al. Survival and clinical outcomes of patients with
melanoma brain metastasis in the era of checkpoint inhibitors and targeted therapies.
BMC Cancer. 2018; 18(1):490.
76
16. Chukwueke U, Batchelor T, Brastianos P. Management of Brain Metastases in Patients
With Melanoma. J Oncol Pract. 2016; 12(6):536-542.
17. Nayak L, Lee EQ, Wen PY. Epidemiology of brain metastases. Curr Oncol Rep. 2012;
14(1):48-54.
18. Fink KR, Fink JR. Imaging of brain metastases. Surg Neurol Int. 2013; 4(Suppl 4):S209-
219.
19. Ewend MG, Elbabaa S, Carey LA. Current Treatment Paradigms for the Management of
Patients with Brain Metastases. Neurosurgery. 2005; 57(suppl_5):S4-66-S64-77.
20. Han CH, Brastianos PK. Genetic Characterization of Brain Metastases in the Era of
Targeted Therapy. Front Oncol. 2017; 7:230.
21. Liao L, Ji X, Ge M, et al. Characterization of genetic alterations in brain metastases from
non-small cell lung cancer. FEBS Open Bio. 2018; 8(9):1544-1552.
22. Neophytou CM, Kyriakou TC, Papageorgis P. Mechanisms of Metastatic Tumor Dormancy
and Implications for Cancer Therapy. Int J Mol Sci. 2019; 20(24).
23. Boire A, Coffelt SB, Quezada SA, Vander Heiden MG, Weeraratna AT. Tumour Dormancy
and Reawakening: Opportunities and Challenges. Trends in Cancer. 2019; 5(12):762-765.
24. Singh M, Manoranjan B, Mahendram S, McFarlane N, Venugopal C, Singh SK. Brain
metastasis-initiating cells: survival of the fittest. Int J Mol Sci. 2014; 15(5):9117-9133.
25. Sofroniew MV, Vinters HV. Astrocytes: biology and pathology. Acta Neuropathol. 2010;
119(1):7-35.
26. Butovsky O, Weiner HL. Microglial signatures and their role in health and disease. Nature
Reviews Neuroscience. 2018; 19(10):622-635.
27. Chen Q, Boire A, Jin X, et al. Carcinoma–astrocyte gap junctions promote brain
metastasis by cGAMP transfer. Nature. 2016; 533(7604):493-498.
28. Wasilewski D, Priego N, Fustero-Torre C, Valiente M. Reactive Astrocytes in Brain
Metastasis. Front Oncol. 2017; 7:298-298.
29. Gong X, Hou Z, Endsley MP, et al. Interaction of tumor cells and astrocytes promotes
breast cancer brain metastases through TGF-β2/ANGPTL4 axes. npj Precision Oncology.
2019; 3(1):24.
30. You H, Baluszek S, Kaminska B. Supportive roles of brain macrophages in CNS
metastases and assessment of new approaches targeting their functions. Theranostics.
2020; 10(7):2949-2964.
31. Zeng Q, Michael IP, Zhang P, et al. Synaptic proximity enables NMDAR signalling to
promote brain metastasis. Nature. 2019; 573(7775):526-531.
32. Howe EN, Burnette MD, Justice ME, et al. Rab11b-mediated integrin recycling promotes
brain metastatic adaptation and outgrowth. Nature Communications. 2020; 11(1):3017.
77
33. Lovinger DM. Neurotransmitter roles in synaptic modulation, plasticity and learning in the
dorsal striatum. Neuropharmacology. 2010; 58(7):951-961.
34. Citri A, Malenka RC. Synaptic Plasticity: Multiple Forms, Functions, and Mechanisms.
Neuropsychopharmacology. 2008; 33(1):18-41.
35. Kaverina N, Borovjagin AV, Kadagidze Z, et al. Astrocytes promote progression of breast
cancer metastases to the brain via a KISS1-mediated autophagy. Autophagy. 2017;
13(11):1905-1923.
36. Pukrop T, Dehghani F, Chuang H-N, et al. Microglia promote colonization of brain tissue
by breast cancer cells in a Wnt-dependent way. Glia. 2010; 58(12):1477-1489.
37. Hyman SE. Neurotransmitters. Current Biology. 2005; 15(5):R154-R158.
38. Huang EP, Stevens CF. Neurotransmitter Release and Synaptic Plasticity. In: Bittar EE,
ed. Advances in Organ Biology. Vol 2: Elsevier; 1997:171-191.
39. Flüh C, Mafael V, Adamski V, Synowitz M, Held-Feindt J. Dormancy and NKG2D system
in brain metastases: Analysis of immunogenicity. Int J Mol Med. 2020; 45(2):298-314.
40. Lovinger DM. Neurotransmitter roles in synaptic modulation, plasticity and learning in the
dorsal striatum. Neuropharmacology. 2010; 58(7):951-961.
41. Isaac JT, Ashby MC, McBain CJ. The role of the GluR2 subunit in AMPA receptor function
and synaptic plasticity. Neuron. 2007; 54(6):859-871.
42. Akashi K, Kakizaki T, Kamiya H, et al. NMDA Receptor GluN2B (GluRε2/NR2B) Subunit
Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin
Cytoskeleton at Hippocampal CA3 Synapses. The Journal of Neuroscience. 2009;
29(35):10869-10882.
43. Crupi R, Impellizzeri D, Cuzzocrea S. Role of Metabotropic Glutamate Receptors in
Neurological Disorders. Front Mol Neurosci. 2019; 12:20.
44. Jones KA, Tamm JA, Craig DA, Yao W-J, Panico R. Signal Transduction by GABAB
Receptor Heterodimers. Neuropsychopharmacology. 2000; 23(1):S41-S49.
45. Duka T, Nikolaou K, King SL, et al. GABRB1 Single Nucleotide Polymorphism Associated
with Altered Brain Responses (but not Performance) during Measures of Impulsivity and
Reward Sensitivity in Human Adolescents. Front Behav Neurosci. 2017; 11:24-24.
46. Rebholz H, Friedman E, Castello J. Alterations of Expression of the Serotonin 5-HT4
Receptor in Brain Disorders. Int J Mol Sci. 2018; 19(11):3581.
47. Poirier R, Cheval H, Mailhes C, et al. Distinct functions of egr gene family members in
cognitive processes. Front Neurosci. 2008; 2(1):47-55.
48. Korb E, Finkbeiner S. Arc in synaptic plasticity: from gene to behavior. Trends Neurosci.
2011; 34(11):591-598.
49. Lepeta K, Kaczmarek L. Matrix Metalloproteinase-9 as a Novel Player in Synaptic
Plasticity and Schizophrenia. Schizophr Bull. 2015; 41(5):1003-1009.
78
50. Graf ER, Zhang X, Jin SX, Linhoff MW, Craig AM. Neurexins induce differentiation of
GABA and glutamate postsynaptic specializations via neuroligins. Cell. 2004;
119(7):1013-1026.
51. Dean C, Dresbach T. Neuroligins and neurexins: linking cell adhesion, synapse formation
and cognitive function. Trends Neurosci. 2006; 29(1):21-29.
52. Sakamoto K, Karelina K, Obrietan K. CREB: a multifaceted regulator of neuronal plasticity
and protection. J Neurochem. 2011; 116(1):1-9.
53. Johannessen CU, Johannessen SI. Valproate: past, present, and future. CNS Drug Rev.
2003; 9(2):199-216.
54. Antonucci F, Corradini I, Fossati G, Tomasoni R, Menna E, Matteoli M. SNAP-25, a Known
Presynaptic Protein with Emerging Postsynaptic Functions. Frontiers in Synaptic
Neuroscience. 2016; 8(7).
55. Fan HP, Fan FJ, Bao L, Pei G. SNAP-25/syntaxin 1A complex functionally modulates
neurotransmitter gamma-aminobutyric acid reuptake. J Biol Chem. 2006; 281(38):28174-
28184.
56. Pedrosa RMSM, Mustafa DA, Soffietti R, Kros JM. Breast cancer brain metastasis:
molecular mechanisms and directions for treatment. Neuro Oncol. 2018; 20(11):1439-
1449.
57. Montagner M, Sahai E. In vitro Models of Breast Cancer Metastatic Dormancy. Frontiers
in Cell and Developmental Biology. 2020; 8(37).
58. Yadav AS, Pandey PR, Butti R, et al. The Biology and Therapeutic Implications of Tumor
Dormancy and Reactivation. Front Oncol. 2018; 8:72-72.
59. Jandial R, Choy C, Levy DM, Chen MY, Ansari KI. Astrocyte-induced Reelin expression
drives proliferation of Her2(+) breast cancer metastases. Clin Exp Metastasis. 2017;
34(2):185-196.
60. Stein T, Cosimo E, Yu X, et al. Loss of reelin expression in breast cancer is epigenetically
controlled and associated with poor prognosis. Am J Pathol. 2010; 177(5):2323-2333.
61. Serrano-Morales JM, Vázquez-Carretero MD, Peral MJ, Ilundáin AA, García-Miranda P.
Reelin-Dab1 signaling system in human colorectal cancer. Mol Carcinog. 2017; 56(2):712-
721.
62. Okamura Y, Nomoto S, Kanda M, et al. Reduced expression of reelin (RELN) gene is
associated with high recurrence rate of hepatocellular carcinoma. Ann Surg Oncol. 2011;
18(2):572-579.
63. Chen Y, Sharma RP, Costa RH, Costa E, Grayson DR. On the epigenetic regulation of
the human reelin promoter. Nucleic Acids Res. 2002; 30(13):2930-2939.
64. Campo CG, Sinagra M, Verrier D, Manzoni OJ, Chavis P. Reelin secreted by GABAergic
neurons regulates glutamate receptor homeostasis. PLoS One. 2009; 4(5):e5505.
79
65. Messaoudi E, Kanhema T, Soulé J, et al. Sustained Arc/Arg3.1 synthesis controls long-
term potentiation consolidation through regulation of local actin polymerization in the
dentate gyrus in vivo. The Journal of neuroscience : the official journal of the Society for
Neuroscience. 2007; 27(39):10445-10455.
66. Brzozowska A, Burdan F, Duma D, Solski J, Mazurkiewicz M. γ-amino butyric acid (GABA)
level as an overall survival risk factor in breast cancer. Ann Agric Environ Med. 2017;
24(3):435-439.
67. Chong JA, Tapia-Ramírez J, Kim S, et al. REST: a mammalian silencer protein that
restricts sodium channel gene expression to neurons. Cell. 1995; 80(6):949-957.
68. Ballas N, Battaglioli E, Atouf F, et al. Regulation of neuronal traits by a novel transcriptional
complex. Neuron. 2001; 31(3):353-365.
69. Ballas N, Grunseich C, Lu DD, Speh JC, Mandel G. REST and Its Corepressors Mediate
Plasticity of Neuronal Gene Chromatin throughout Neurogenesis. Cell. 2005; 121(4):645-
657.
70. Lawinger P, Venugopal R, Guo ZS, et al. The neuronal repressor REST/NRSF is an
essential regulator in medulloblastoma cells. Nat Med. 2000; 6(7):826-831.
71. Kamal MM, Sathyan P, Singh SK, et al. REST regulates oncogenic properties of
glioblastoma stem cells. Stem Cells. 2012; 30(3):405-414.
72. Wagoner MP, Gunsalus KT, Schoenike B, Richardson AL, Friedl A, Roopra A. The
transcription factor REST is lost in aggressive breast cancer. PLoS Genet. 2010;
6(6):e1000979.
73. Kreisler A, Strissel PL, Strick R, Neumann SB, Schumacher U, Becker CM. Regulation of
the NRSF/REST gene by methylation and CREB affects the cellular phenotype of small-
cell lung cancer. Oncogene. 2010; 29(43):5828-5838.
74. Coulson JM. Transcriptional Regulation: Cancer, Neurons and the REST. Current Biology.
2005; 15(17):R665-R668.
75. Raj B, O'Hanlon D, Vessey JP, et al. Cross-regulation between an alternative splicing
activator and a transcription repressor controls neurogenesis. Mol Cell. 2011; 43(5):843-
850.
76. Tabuchi A, Yamada T, Sasagawa S, Naruse Y, Mori N, Tsuda M. REST4-mediated
modulation of REST/NRSF-silencing function during BDNF gene promoter activation.
Biochem Biophys Res Commun. 2002; 290(1):415-420.
77. Lunyak VV, Rosenfeld MG. No Rest for REST: REST/NRSF Regulation of Neurogenesis.
Cell. 2005; 121(4):499-501.
78. Li Y, Donmez N, Sahinalp C, et al. SRRM4 Drives Neuroendocrine Transdifferentiation of
Prostate Adenocarcinoma Under Androgen Receptor Pathway Inhibition. Eur Urol. 2017;
71(1):68-78.
79. Klotz R, Thomas A, Teng T, et al. Circulating Tumor Cells Exhibit Metastatic Tropism and
Reveal Brain Metastasis Drivers. Cancer Discovery. 2020; 10(1):86-103.
80
80. Hara D, Fukuchi M, Miyashita T, et al. Remote control of activity-dependent BDNF gene
promoter-I transcription mediated by REST/NRSF. Biochem Biophys Res Commun. 2009;
384(4):506-511.
81. Warburton A, Savage AL, Myers P, Peeney D, Bubb VJ, Quinn JP. Molecular signatures
of mood stabilisers highlight the role of the transcription factor REST/NRSF. Journal of
Affective Disorders. 2015; 172:63-73.
82. Shimojo M. Characterization of the nuclear targeting signal of REST/NRSF. Neurosci Lett.
2006; 398(3):161-166.
83. Shimojo M, Hersh LB. Characterization of the REST/NRSF-interacting LIM domain protein
(RILP): localization and interaction with REST/NRSF. J Neurochem. 2006; 96(4):1130-
1138.
84. Kodack DP, Askoxylakis V, Ferraro GB, Fukumura D, Jain RK. Emerging strategies for
treating brain metastases from breast cancer. Cancer Cell. 2015; 27(2):163-175.
85. Banys M, Hartkopf AD, Krawczyk N, et al. Dormancy in breast cancer. Breast Cancer
(Dove Med Press). 2012; 4:183-191.
86. Lorger M, Felding-Habermann B. Capturing changes in the brain microenvironment during
initial steps of breast cancer brain metastasis. Am J Pathol. 2010; 176(6):2958-2971.
87. Nicholson BP, Paul DM, Hande KR, et al. Paclitaxel, 5-fluorouracil, and leucovorin (TFL)
in the treatment of metastatic breast cancer. Clin Breast Cancer. 2000; 1(2):136-143;
discussion 144.
88. Lu B, Nagappan G, Lu Y. BDNF and synaptic plasticity, cognitive function, and
dysfunction. Handb Exp Pharmacol. 2014; 220:223-250.
89. Thakker-Varia S, Alder J, Crozier RA, Plummer MR, Black IB. Rab3A is required for brain-
derived neurotrophic factor-induced synaptic plasticity: transcriptional analysis at the
population and single-cell levels. J Neurosci. 2001; 21(17):6782-6790.
90. Shin W, Kim K, Serraz B, et al. Early correction of synaptic long-term depression improves
abnormal anxiety-like behavior in adult GluN2B-C456Y-mutant mice. PLoS Biol. 2020;
18(4):e3000717.
91. Carey MR, Myoga MH, McDaniels KR, et al. Presynaptic CB1 receptors regulate synaptic
plasticity at cerebellar parallel fiber synapses. J Neurophysiol. 2011; 105(2):958-963.
92. Cui W-Q, Zhang W-W, Chen T, et al. Tacr3 in the lateral habenula differentially regulates
orofacial allodynia and anxiety-like behaviors in a mouse model of trigeminal neuralgia.
Acta Neuropathologica Communications. 2020; 8(1):44.
93. Beffert U, Weeber EJ, Durudas A, et al. Modulation of Synaptic Plasticity and Memory by
Reelin Involves Differential Splicing of the Lipoprotein Receptor Apoer2. Neuron. 2005;
47(4):567-579.
94. Lorusso G, Rüegg C. The tumor microenvironment and its contribution to tumor evolution
toward metastasis. Histochem Cell Biol. 2008; 130(6):1091-1103.
81
95. Head SA, Hernandez-Alias X, Yang J-S, et al. Silencing of SRRM4 suppresses microexon
inclusion and promotes tumor growth across cancers. PLOS Biology. 2021;
19(2):e3001138.
96. Arshad F, Wang L, Sy C, Avraham S, Avraham HK. Blood-Brain Barrier Integrity and
Breast Cancer Metastasis to the Brain. Pathology Research International. 2011;
2011:920509.
97. Kurschat P, Bielenberg D, Rossignol-Tallandier M, Stahl A, Klagsbrun M. Neuron
restrictive silencer factor NRSF/REST is a transcriptional repressor of neuropilin-1 and
diminishes the ability of semaphorin 3A to inhibit keratinocyte migration. J Biol Chem.
2006; 281(5):2721-2729.
98. Ferrario C, Hostetter G, Bouchard A, Huneau M-C, Mamo A, Basik M. Expression of
neuropilin-1 and related proteins in breast cancer. Cancer Research. 2006; 66(8
Supplement):1182-1182.
99. Leto K, Rolando C, Rossi F. The Genesis of Cerebellar GABAergic Neurons: Fate
Potential and Specification Mechanisms. Frontiers in Neuroanatomy. 2012; 6(6).
100. Ren D, Cheng H, Wang X, et al. Emerging treatment strategies for breast cancer brain
metastasis: from translational therapeutics to real-world experience. Ther Adv Med Oncol.
2020; 12:1758835920936151.
101. Shimojo M, Kasahara Y, Inoue M, et al. A gapmer antisense oligonucleotide targeting
SRRM4 is a novel therapeutic medicine for lung cancer. Scientific Reports. 2019;
9(1):7618.
Abstract (if available)
Abstract
Brain metastases (BMs), diagnosed in 20−40% of cancer patients, remain a significant complication of advanced carcinomas. In order to successfully combat the rising incidence of BM induced mortality, it is imperative to understand the affected processes and molecular mechanisms that facilitate the establishment of brain metastases. ❧ To successfully form BMs, tumor cells that extravasate into the brain parenchyma must adapt to and survive within the new microenvironment. This includes interaction with the various cells that comprise the neural metastatic niche including neurons, glia, and cells of the blood brain/ blood cerebrospinal fluid barriers (BBB and BCSFB). Furthermore, tumor cells must also alter their own genetic and metabolic landscape to augment metastatic competency within the central nervous system (CNS). ❧ Previous studies have reported that activated astrocytes promote tumor cell proliferation in the brain by releasing pro-metastatic cytokines and chemokines (IL-6, TNFa). Microglia have also been known to facilitate CNS colonization by tumor cells through activation of Wnt signaling. However, despite the fact that neurons contribute to a significant portion of cellular signaling within the CNS, there are currently no studies investigating the role of neuronal input in facilitating tumor colonization in the brain. Additionally, even though acquisition of neuronal attributes has been reported in brain-metastatic tumor cells, and presence of CNS-primed cells within the primary tumor has been described in breast cancer, the mechanisms underlying the gain of CNS-metastatic competency in tumor cells are largely unexplored. ❧ This dissertation aims to investigate the early stages of brain-metastatic colonization in two parts: (Part I) Characterizing early tumor-neuron interactions and the induced CNS-adaptive changes in tumor cells
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Creator
Deshpande, Krutika Tushar
(author)
Core Title
Neuronal master regulator SRRM4 in breast cancer cells facilitates CNS-acclimation and colonization leading to brain metastasis
School
Keck School of Medicine
Degree
Doctor of Philosophy
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Medical Biology
Publication Date
05/03/2021
Defense Date
03/16/2021
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brain metastasis,breast cancer,cancer,Colonization,mechanism,microenvironment,neuron,neurotransmitter,OAI-PMH Harvest,plasticity
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), Neman, Josh (
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Tags
brain metastasis
breast cancer
mechanism
microenvironment
neuron
neurotransmitter
plasticity