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TLR8-transferred miR-192 acts as a tumor suppressor in neuroblastoma by inhibiting CTCF

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TLR8-transferred miR-192 acts as a tumor suppressor in neuroblastoma by inhibiting CTCF

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TLR8-TRANSFERRED MIR-192 ACTS AS A TUMOR
SUPPRESSOR IN NEUROBLASTOMA BY
INHIBITING CTCF

By
Mariam Murtadha

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the Requirements of the Degree
DOCTOR OF PHILOSOPHY
(Cancer Biology and Genomics)
August 2018

Copyright 2018 Mariam Murtadha

ii

Table of Contents
Acknowledgements ........................................................................................................................................... iii
List of Figures ....................................................................................................................................................iv
Abstract ............................................................................................................................................................. v
Chapter I. Identification of miR-192 in NB .......................................................................................................... 1
Introduction .................................................................................................................................................... 1
I. Neuroblastoma (NB) overview ............................................................................................................. 1
II. Undruggable MYCN in NB ................................................................................................................... 1
III. Role of tumor associated macrophages in NB ................................................................................. 2
IV. Exosomic microRNAs- regulators of intercellular communication in TME (19) ................................. 2
V. miR based therapeutics ................................................................................................................... 6
VI. Goal of the project ............................................................................................................................ 6
Materials and Methods ................................................................................................................................... 6
Results ........................................................................................................................................................... 8
Chapter II. Tumor suppressor role of miR-192 in NB ....................................................................................... 12
Introduction .................................................................................................................................................. 12
I. Dual role of miR-192 in cancer .......................................................................................................... 12
II. Goal of the project ............................................................................................................................. 12
Materials and Methods ................................................................................................................................. 12
Results ......................................................................................................................................................... 13
Chapter III. miR-192 suppresses NB growth by inhibiting CTCF ...................................................................... 15
Introduction .................................................................................................................................................. 15
I. Role of CTCF in cancer including NB ................................................................................................ 15
II. Goal of the study ............................................................................................................................... 16
Materials and Methods ................................................................................................................................. 16
Results ......................................................................................................................................................... 19
Chapter IV. Discussion, conclusion, and future work ....................................................................................... 26
References ...................................................................................................................................................... 28

iii

Acknowledgements

I would like to thank my mentor Dr. Muller Fabbri for all the academic support and guidance during those past
years. Despite the challenges posed by the project and technical issues, he motivated me to continue and
focus. He always supported me in all my career goals. I would like to especially thank him for being supportive
and very understanding for the motherhood role that I have besides a graduate student. Additionally, I have no
words of appreciation for the support he provided me during my mother’s illness.
I would like to thank my committee members Dr. Ite Offringa and Dr. Michael Stallcup. Ite, despite her very
busy schedule as a PI and PIBBS director, has provided me scientific mentorship and career counseling. I
turned to her whenever things were falling apart. I would like to thank Dr. Stallcup for all the scientific advising
he provided during my committee meetings and I truly appreciate him always being supportive of my career
goals.
Also, I would like thank my lab members Dr. Petra Wise and Dr. Paolo Neviani. Petra is another mentor I had
during those years, she motivated me as a scientist and as individual to continue. I would turn to her for any
lab and personal issues. Without Paolo, many experimental designs would have not been possible. Also I
would like to acknowledge other member of Dr. Fabbri’s lab: Jamie Frediani, Simona Barbato, Anna
Constanzini and Kishore Challagundla.
Without parents, I believe nothing in the world is possible. My father inspired me to become a scientist and my
mother supported me to continue my path of higher education. Though life has taken its turns and both my
parents are in the better place, their dreams are alive. I also would like to acknowledge my brother Murtaza
and my sisters Ameena and Sahar for always appreciating my goals and dreams.
The love filled appreciation goes to my husband and especially my cutest wonderful caring son Umer, they
stood beside me during those challenging and wonderful years. Umer inspires me to become a better scientist
and role model.

iv

List of Figures
Figure 1. Heatmap of miRs upregulated in SK.N.BE(2) cells in TLR8-dependent manner.
Figure 2. Transfer of miR-192 in TLR8 dependent manner from murine macrophages to NB cells in two NB cell
lines.
Figure 3. Immunophenotyping of M1 and M2 macrophages in a representative donor.

Figure 4. Healthy donors polarized M1 and M2 macrophages confer growth benefit to two NB cell lines.
Figure 5. Healthy donors polarized M1 and M2 macrophages confer NB cells with resistance to 13-cis-RA.
Figure 6. (A) miR-192 is upregulated in IMR32 following co-culture with M1 and M2 macrophages. (B) miR-192
is upregulated in IMR32 following co-culture with M1 and M2 macrophages in presence of 13-cis-RA.
Figure 7. miR-192 R2 database (dataset tumor neuroblastoma SEQC 498 RPM seqcnb1) analysis.
Figure 8. miR-192 induces NB growth suppression in vitro.
Figure 9. Expression of miR-192 in NB cell lines assayed with TaqMan miR-192 qRT-PCR.
Figure 10. miR-192 modulates CTCF and MYCN protein levels in NB cells.
Figure 11. miR-192 directly targeted 3’-UTR of CTCF.
Figure 12. miR-192 suppresses NB growth by modulation of CTCF.
Figure 13. miR-192 induces NB growth suppression regardless of MYCN status.
Figure 14. miR-192 suppresses NB growth in orthotopic NB xenograft model.
Figure 15. Correlation analysis between expression of CTCF and miR-192 in primary NB tissues.
Figure 16. A, protocol for developing antibody coated nanoparticle with carrier miR (97). B, Delivery of miR-186
to NB cells with GD2 coated nanoparticle in vitro. C, Delivery of miR-186 to NB cells with GD2 coated
nanoparticle in vivo

v

Abstract
Neuroblastoma (NB) is the most common solid extracranial malignancy in children. MYCN gene amplification
is detected in 25% of NB cases and is associated with an unfavorable outcome. Currently, MYCN is un-
druggable. Several therapies have been developed and being enhanced for targeting MYCN. Recently, higher
infiltration of tumor associated macrophages (TAMs) has been shown in metastatic NB cases compared to
locoregional NB tumors. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression and act
as ligands of the Toll like receptor 8 (TLR8). MiRs can be transferred in microvesicles called exosomes (exo-
miRs). Current evidence shows that exo-miRs orchestrate the intercellular communication within the tumor
microenvironment (TME). Our lab has recently shown TLR8-dependent transfer of miR-155 from TAMs to NB
cells in exosomes. TLR8-dependent transfer of miR-155 induces NB resistance to cisplatin by targeting
telomerase inhibitor TERF1. In this study, we investigated other miRs that are transferred from TAMs to NB
cells in a TLR8-dependent manner and how those TLR8-dependent exo-miRs can modulate MYCN in NB. We
found that miR-192 is transferred in a TLR8-dependent manner from TAMs to NB cells suggesting an
oncogenic role for miR-192 in NB. However, our gain of function studies of miR-192 in NB and R2-based
transcriptomic data analysis of miR-192 in NB primary tissues showed a tumor suppressor role for miR-192.
We investigated the tumor suppressor mechanism of miR-192 and how it can modulate MYCN.
CCCTC-binding factor (CTCF), a multifunctional protein with key a role in transcription regulation and genome
topology, has recently been identified as an independent prognostic factor for progressive NB. Additionally,
CTCF binding of the MYCN promoter stimulates MYCN expression in NB by recruitment of histone activating
marks and inducing chromatin remodeling. Taking into account that MYCN is un-druggable and CTCF induces
MYCN expression, we investigated if CTCF is a target of miR-192. Furthermore, CTCF plays an oncogenic
role in NB and there are no drugs to inhibit CTCF. Our in silico analysis predicts the CTCF mRNA 3’-UTR as a
putative target of miR-192. We investigated if miR-192 mediates NB growth suppression by inhibiting CTCF.
The overexpression of miR-192 in NB cell lines downregulated CTCF and MYCN at the protein level. Equally,
the inhibition of miR-192 upregulated CTCF and MYCN protein in NB cell lines. Dual luciferase assays showed
direct targeting of CTCF 3’-UTR by miR-192. The co-expression of miR-192 and CTCF cDNA without the 3’-
UTR abrogated miR-192-mediated NB growth suppression suggesting that miR-192-mediated killing of NB
cells is in part caused by the inhibition of CTCF. Therapeutic delivery of miR-192 via nanoparticles in an
orthotopic NB model significantly suppressed NB growth. In clinical NB tissues, we observed a negative
correlation of CTCF with miR-192. Together, these results indicate that miR-192 suppresses NB growth by
inhibiting CTCF.
The upregulation of miR-192 in NB cells by pro-tumoral TAMs is currently not understood in our study. TAMs
promote miR-192 expression by 2-9 folds in NB cells, whereas overexpression of miR-192 with mimic miR-192
overexpresses the miR beyond physiological concentrations. It is possible that miR exhibits an oncogenic role
at lower concentrations. Currently, there are no studies that correlate miR concentration with effect on its
target. Additionally, we need to investigate if miR-192 upregulation in NB cells is mediated through the transfer
of miR-192 in exosomes to NB cells, or if cytokines or growth factors induce transcription of miR-192 in NB
cells, when surrounded by TAMs.

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Chapter I. Identification of miR-192 in NB
Introduction
I. Neuroblastoma (NB) overview
NB is the most common extracranial solid malignancy diagnosed in children. It accounts for 6% of all
childhood cancers and the age-adjusted incidence in the United States is 10.5 cases/million/year in children
younger than 15 years (1,2). NB originates during the embryonal neural crest specification and differentiation.
The programmed epithelial-to-mesenchymal transition (EMT) of multipotent early neural crest cells gives rise to
plethora of tissues including enteric ganglia, parasympathetic and sympathetic nervous system, and adrenal
gland. Disruption of this normal developmental stage with oncogenic drivers at different times can lead to
multiple subtypes of NB (3). NB tumors generally arise in tissues of the sympathetic nervous system typically in
the adrenal medulla or paraspinal ganglia and thus can present as lesions in the neck, chest, abdomen, or
pelvis (1).
NB is characterized by its heterogeneity and variable clinical presentation. A patient’s age, stage of disease
at diagnosis, and MYCN gene amplification are key three determinants of clinical outcome. These
determinants along with loss of chromosome 11q, histology, and ploidy are the basis of risk group stratification
(4). Patients with favorable tumors are more likely to be less than 1 year of age, have localized tumors with
good prognosis, and have near-triploid karyotypes with whole chromosome gains (5). On the other hand,
unfavorable tumors are characterized by structural changes including deletions of 1p or 11q, unbalanced gain
of 17q and/or amplification of the MYCN proto-oncogene. The high-risk group with unfavorable outcome is
usually older than 1 year of age and has worse prognosis (5). Interestingly, a distinct subset of highly
undifferentiated NB (Stage IVS or M4S) presents with metastatic disease in very young infants. Remarkably,
the vast majority of M4S patients spontaneously regress within months (3).
Currently, there is a multimodal therapy for high risk NB patients, which includes high-dose chemotherapy
with autologous hematopoietic stem cell transplant (ASCT), radiotherapy, the differentiating agent 13-cis-
retinoic acid (13-cis-RA), and immunotherapy with anti-GD2 monoclonal antibodies (4). Gangliosides, such as
GD2, are complex glycolipids found on the outer cell membrane. Antenatally, GD2 is expressed on neural and
mesenchymal stem cells, whereas postnatally, the expression of GD2 is restricted to peripheral neurons,
central nervous system, and skin melanocytes. NB cells express high levels of GD2 with an estimated 5-10
million molecules per cell (6).
II. Undruggable MYCN in NB
Amplification of MYCN is found in 25% of NB cases and correlates with high-risk disease (7). MYCN is a
member of the MYC family of proto-oncogenes that also comprises c-MYC and MYCL. MYCN was first
discovered in NB cell lines as amplified DNA with homology to viral myc. MYCN amplification or
overexpression has now been described in several other cancers, specifically cancers of embryonic and/or
neuroendocrine origin such as retinoblastoma, Wilms’ tumor, rhabdomyosarcoma, medulloblastoma,
glioblastoma, and small cell lung cancer (8).
Like the other MYC proteins, MYCN is a transcription factor that controls expression of several genes.
MYCN promotes tumorigenesis by activating transcription of genes involved in metastasis, survival,
proliferation, pluripotency, self-renewal, and angiogenesis. Furthermore, MYCN can also suppress the
expression of genes that promote differentiation, cell cycle arrest, immune surveillance, and genes that
antagonize metastasis and angiogenesis (7).
MYCN protein structure resembles structures of most transcription factors. At the amino terminal lies the
transcriptional activation domain (TAD), a region that is sufficient for transcriptional activation once fused to
heterologous DNA binding domain. At the carboxy-terminal of MYCN is a 100 amino acid basic helix-loop-helix
leucine zipper (BR-HLH-LZ) domain that serves as DNA binding domain. BR-HLH-LZ proteins bind DNA as
2

dimers (in complex with Max) and recognize a consensus sequence CACGTG which is termed Enhancer box
(E-box) (9).
Alignment of MYC family members across species reveals regions of conserved motifs called ‘MYC boxes’
that serve as docking sites for protein-protein interactions (9). The stability of MYC proteins is regulated by
phosphorylation within Myc box I (MBI) that spans the N-terminal TAD domain. MYCN is first phosphorylated
on Serine 62 (Ser62) by Cdk1/cyclin B and is then phosphorylated on Threonine 58 (Thr58) by Gsk3β.
Dephosphorylation of Ser62 by PP2A directs that activity of the E3 ubiquitin ligase SCF
Fbxw7
to modify MYCN
with K48 linked ubiquitin chains and subject to ubiquitin proteasome degradation. In NB cells, the Ser/Thr
protein kinase Aurora-A blocks this process resulting in excess MYCN protein. Aurora-A binds to the MYCN-
SCF
Fbxw7
complex and reduces K48 linkages in the polyubiquitin chains and stabilizes MYCN (10).
MYCN is currently considered as undruggable because its DNA binding domains are composed of two
extended alpha helices with no apparent surface for small molecule binding. Additionally, proteolytic
degradation of MYCN protein is regulated in part by a kinase independent function of Aurora-A kinase (11).
Some groups have explored downregulating MYCN by RNA interference. Most groups have observed that
targeting MYCN expression in NB cells leads to growth arrest in the G1 phase of the cell cycle, apoptosis
and/or morphological differentiation. These studies suggest that targeting of MYCN could be promising for NB
therapy. However, RNA interference as therapeutic strategy in clinic is hindered by insufficient delivery to the
target tissue due to poor RNA stability (8). Furthermore, the Aurora-A kinase inhibitors have shown minimum
efficacy (10,11).
III. Role of tumor associated macrophages in NB
The association of MYCN gene amplification with poor prognosis in NB has led to anticipation of other
genetic events. And recently, the role of the tumor microenvironment (TME) in high-risk NB has been
investigated and it is now recognized that tumor growth goes beyond cancer cell genetic abnormalities (12).
Physiologically, the bi-directional communication between cells and their stroma is critical for development and
homeostasis. Tumor cells, on the other hand, are utilizing the microenvironment to promote tumor growth. TME
consists of leukocytes, fibroblasts, and vascular endothelial cells. Tumor associated macrophages (TAMs),
specifically, are the prominent immune cells that orchestrate various factors in TME (13).
Generally, macrophages can be polarized to M1 or M2 macrophages. Classically activated macrophages,
also known as M1-polarized macrophages, are activated by cytokines such as interferon-γ, produce pro-
inflammatory cytokines (e.g. interleukin [IL]-12 and IL-23) and are involved in helper T cell (Th) 1 responses to
infection. TAMs are thought to resemble M2 polarized macrophages, also known as alternatively activated
macrophages which are activated by Th2 cytokines (e.g. IL-4, IL-10, and IL-13) (13,14).
A recent study showed that metastatic MYCN non-amplified NB cases had higher infiltration of TAMs than
locoregional tumors. Further, the study also showed that metastatic tumors diagnosed in patients at age ≥ 18
months had higher expression of inflammatory related genes than those in patients diagnosed at age < 18
months (15). Infiltration with Th2 driven macrophages expressing CD163 and CD206 was also recently
observed in a subset of high risk NB cases with deletion of chromosome 11q (16). Additionally, blocking
macrophage stimulatory factor (CSF-1) in CSF-1 negative xenotransplanted NB tumors extends survival in
tumor bearing mice (17). Another study identified that soluble IL-6 receptor produced by TAMs promotes IL-6
activation of STAT3 pathway in NB cells (18).
IV. Exosomic microRNAs- regulators of intercellular communication in TME (19)
MicroRNAs (miRs) are endogenously expressed small non-coding RNAs (19-24 nucleotides(nt)) that play
key roles in gene regulation at the post-transcriptional level (20). Over the last decade, the active role of miRs
in many normal and pathological biological processes has been extensively studied. It is now known that miRs
are frequently dysregulated in all cancer types where they have been examined, including both hematological
and solid cancers (21).
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Mature miRs bind to their target messenger RNA (mRNA) by imperfect complementary base pairing (22),
typically binding to the 3’ untranslated region (3’ UTR) of the target mRNA by a seed sequence (2-7 nt long)
that is located near the 5’ end of the miR. In most cases, this interaction leads to a decrease in protein
expression by mRNA destabilization, mRNA degradation, and/or translation repression (23). The base
pairing between the miR and the mRNA is imperfect; this allows each miR to target several mRNAs, and
individual mRNAs to be regulated by several miRs (24). More recent studies have expanded our
understanding of miR-mediated regulation by showing that, in some cases, miRs can bind the 5’ UTR region
or the coding sequence of target mRNAs. Furthermore, some miR-mRNA interactions result in the up-
regulation of protein expression, challenging the current paradigm of miRs as exclusively negative regulators
of gene expression. Three mechanisms through which miRs up-regulate protein expression are: (1) miRs
increase the recruitment of protein complexes to AU-rich elements of mRNA, increasing translation of target
mRNA (25); (2) miRs interact with proteins that suppress the translation of the associated mRNA (26); and
(3) miRs lead to overall increase in protein synthesis by enhancing ribosome biogenesis (27) (22).
Nearly 50% of known miRs are located at fragile sites on chromosomes known for carrying genetic
alterations in cancer. Thus, it is not surprising that miRs are dysregulated in cancer and associated with
cancer development, metastasis, and drug resistance (28). Additionally, 60% of protein coding genes are
predicted to be subject to regulation by miRs, further emphasizing their role in cellular processes, normal and
otherwise. (29). miRs act as tumor suppressors or oncogenes depending on the function of their target
genes, therefore cancer cells modulate miR expression by different mechanisms (mutations, epigenetic
marks, etc.) to promote tumorigenicity (22,24). The most recognized oncogenic miRs include the miR-17~92
cluster, as well miR-21, and miR-155 (24); overexpression of these miRs has been documented in several
cancers. The miR17~92 cluster is located in intron 3 of the C13orf25 gene at 13q31.3, a genomic region
commonly amplified in most hematological and solid malignancies. This cluster of miRs has been shown to
act with the c-myc oncogene to accelerate B lymphomagenesis in an in vivo B cell lymphoma model (30).
Commonly recognized tumor suppressor miRs include miR-34a and let-7a (24). It was shown that inhibition
of the RNA binding protein Lin28 increased expression of let-7a and radio-sensitized A549 lung cancer cell
line (31). Strikingly, some miRs can act as tumor suppressors in one cancer and as oncogenes in another
cancer (22). For example, a recent study demonstrated that overexpression of miR-192 inhibits metastasis to
liver in an orthotopic colon cancer murine model by repressing oncogenic Bcl-2 and VEGFA (32). In another
study, miR-192 was shown to be upregulated in high-risk neuroblastomas, where it silenced the tumor
suppressor Dicer1 (33).
In recent years, the role of extracellular vesicles (EVs) in cancer biology has attracted increasing
attention. Despite a fundamental lack on consensus on the detailed classification of EVs, a general
classification system has been devised, where EVs are classified into 3 groups based on their size:
exosomes (40-120 nm), microvesicles (50-1000 nm) and apoptotic bodies (500-2000 nm) (34). The lipidic
bilayer structure of the vesicles (as shown by electron microscopy) and the specific expression of surface
markers also contribute to the characterization of EVs, according to the recently published guidelines by the
International Society of Extracellular Vesicles (35). Exosomes consist of a lipidic bilayer surrounding a small
cytosol that is devoid of cellular organelles and is enriched in proteins and nucleic acids (36). Exosomes
were first discovered as transferrin associated 50 nm vesicles extruding from reticulocytes (37). Exosomes
are important mediators of intercellular communication both in disease and healthy states due to their ability
to deliver protein and nucleic acid cargo between cells (38). In 2007, Valadi et al. showed that exosomes
contain miRs, which can be transferred inter-cellularly and are biologically active in the recipient cell (39).
Further studies supported the transfer of functional exosomal miRs (exo-miRs) between cells as mediators of
conserved intercellular communication (40,41). As an example, T-cells uni-directionally transfer miRs to
antigen presenting cells (APCs) by delivery of CD63+ exosomes on the immune synapse, and transferred
miRs modulate gene expression in APCs (42). The first evidence implicating exo-miR involvement in the
4

communication among cancer cells came from Skog et al., who demonstrated that glioblastoma-derived
exosomes contain miR-21 that is taken up by normal host cells and modulates target genes leading to
increased proliferation of cancer cells (43).
It is now well understood that cancer is not a disease limited to the cancer cells, but that cancer cells
orchestrate changes to the surrounding tumor microenvironment (TME) to promote cancer growth (44).
Recent evidence has shown two important roles of miRs in the development and maintenance of the TME:
dysregulated miRs in cancer cells mold the composition of the TME, and miRs in the TME can act in a pro-
tumoral manner (45). A plethora of studies is currently investigating the role of exo-miRs as direct
bidirectional mediators of intercellular TME crosstalk. Cancer cells or the cells of TME directly and
reciprocally transfer exosomic miRs to mediate phenotype changes in the TME and in the cancer cell. There
is evidence that exosome secretion is increased in cancer, and exosomes represent one of the main
mechanisms of crosstalk in the TME and in priming the metastatic niche (46,47).
The TME is composed of cellular and non-cellular components, and plays a fundamental role in the
progression of cancer and in the acquisition of resistance to therapy (48,49). The cellular components are
derived from both the tumor and the host. Host cells in the TME include Tumor-Associated Macrophages
(TAMs), blood endothelial cells (BECs), lymphatic endothelial cells (LECs), carcinoma-associated fibroblasts
(CAFs), and bone marrow derived mesenchymal stem cells (MSCs). The immune cells mediate anti-tumoral
or pro-tumoral functions depending on their specific phenotype, such as anti-tumoral Th1 vs pro-tumoral
Th17 subsets of CD4(+) T cells, anti-tumoral type I vs cancer promoting type II NKT cells, and M1 vs tumoral
M2 macrophages. The non-cellular components of the TME include the extracellular matrix (ECM), as well
as the physical and chemical parameters (pH, oxygen tension, interstitial pressure, and fluid flux) (50).
Tumor progression requires cooperative and continuous intercellular communication between cancer cells
and host cells (50-52).
Intercellular communication in multicellular organisms is generally accomplished through direct cell-cell
contact (e.g. nibbling) or transfer of secreted molecules (e.g. hormones, cytokines, chemokines) (40).
Secreted molecules can act over a short distance (autocrine and paracrine mechanisms) or travel long
distances circulating in blood and body fluids (endocrine mechanism). Over the last few years, EVs have
emerged as important mediators of intercellular communication in cancer, and are being investigated as
essential mediators of intercellular communication in cancer and as cancer biomarkers. Specifically,
exosomes are secreted in the extracellular space carrying miR cargo; they can then be transferred between
cells or enter the general circulation.
Exosome production is generally increased in cancer cells, and the cargo content of cancer
exosomes is distinct from the exosome cargo of normal cells. For instance, a study showed that in exosome-
free medium, breast cancer cells produced 53.2 x 10
8
exosomes per 10
6
cells, whereas normal mammary
epithelial cells produced 4.5 x 10
8
exosomes per 10
6
cells in a 24-hour period (53). Interestingly, breast
cancer exosomes isolated from metastatic breast cancer cells MDA-MB-231 and 4T1 showed significantly
higher miR enrichment when compared to exosomes derived from the non-metastatic breast cancer cell line
MCF7 (54). Furthermore, breast cancer cell-derived exosomes contained pre-miRs that were associated with
the RISC loading complex and displayed cell-independent capacity to process those pre-miRs into mature
miRs; this is in contrast to exosomes isolated from normal cells, which lacked the miR processing machinery.
Pre-miRs along with Dicer and Ago2 are present in breast cancer exosomes. Exosomes extracted from sera
and cells of patients with breast cancer are able to transform non-tumorigenic epithelial cells into malignant
cancer cells that are capable of forming tumors in a Dicer-dependent manner (54).
Most current evidence supports a pro-tumorigenic role for exo-miRs. However, one group showed
that exosomes from the metastatic gastric cancer cell line AZ-P7a were enriched with the let-7 family of miRs
that mostly act as tumor suppressors in a variety of malignancies. This study proposed that AZ-P7a released
let-7 miRs via exosomes into the extracellular environment to maintain their oncogenic potential (55).
5

Cancer cell-derived exosomes and their miR cargo can be functionally transferred to the cells of the
TME, where they modulate gene expression and lead to amplified malignant potential of the tumor. Some of
the pro-tumoral roles of cancer cell exo-miRs in the recipient cells include: inactivation or shift of immune cell
responses, transfer of drug resistance, increased angiogenesis, increased invasiveness, priming of the pre-
metastatic niche, and enhanced epithelial to mesenchymal (EMT) transition. In the TME, dendritic cells
(DCs) shift from effective antigen presenting cells to negative regulators of immune responses, and this shift
seems mediated by exo-miRs. Pancreatic cancer-derived exosomes downregulate the expression of TLR4 in
DCs via transfer of miR-203 (56). TLR4 has an important role in the recognition of the damage-associated
molecular patterns (DAMPs). Pancreatic cancer exosomes have increased expression of miR-203, and DCs
treated with those exosomes show significant downregulation of TLR4. In another study, the exosomes from
docetaxel (doc)-resistant breast cancer cell lines were isolated and co-cultured with doc-sensitive breast
cancer cells, leading to the upregulation of 20 miRs in the sensitive breast cancer cells. Target gene
prediction and pathway analysis showed that the up-regulated miRs might be responsible for the lack of
response to therapy. Those results suggest that drug-resistant cancer cells may spread chemoresistance to
sensitive cells by releasing exosomes, and these effects could be partly attributed to the miR cargo of
exosomes (57). Yamada et al. found that the incubation of colorectal cancer cell-derived microvesicles with
human endothelial cells (HUVECs) increased proliferation, migration, and tube formation of HUVECs. The
colorectal cancer cells shuttled miR-1246 into HUVECs, which silenced promyelocytic leukemia protein
(PML) and activated Smad 1/5/8 signaling (not Smad 2/3 which inactivates HUVEC proliferation) (58). miR-
10b was highly expressed in MDA-MB-231 cells compared to non-metastatic breast cancer cells or non-
malignant breast cells. Additionally, miR-10b was highly secreted into medium via exosomes. nSMase2
promotes the loading of miR-10b into exosomes of breast cancer cells (59). Upon co-culturing of those
tumoral miR-10b loaded exosomes with non-metastatic mammary epithelial breast cells (HMLE cells), the
HMLE cells acquired invasion ability (59).
The pre-metastatic niche supports the soil and seed theory proposed by Paget, where tumors
prepare selected organs for metastasis. Tumor cells utilize exo-miRs to educate the selected host tissues
toward a pro-metastatic phenotype. Metastatic rat adenocarcinoma BSp73ASML (ASML)-derived exosomes
carry levels of miR-494 and miR-542-3p significantly higher than the secreting cells. Those two miRs
enhanced the transcription of matrix metalloproteinases and downregulated cadherin-17 (cdh-17) in a
preferential site of metastasis: the lymph node (60). The miR-200 family is specifically involved in the
regulation of EMT. It was found that EVs of breast cancer cells were enriched with miR-200. Murine and
human breast cancer cell EVs transferred miR-200 to non-metastatic cells and promoted EMT (61).
Most studies focused on exo-miRs secreted by cancer cells to modulate cells of the TME. However,
there is some evidence that exo-miRs secreted by TME can alter gene expression of cancer cells and
promote their malignancy (62). TAMs are important cellular players in the TME, involved in tumor
progression and metastasis. However, the mechanisms underlying the pro-tumoral interactions of TAMs and
cancer cells are not fully understood. One possible explanation is that TAM-released exosomes deliver
oncogenic miRs to breast cancer cells. MiR-223 is upregulated in TAMs and is secreted in TAM-derived
exosomes. Upon co-culture of breast cancer cells (MDA-MB-231 and SKBR3) with TAMs without direct cell-
cell contact, miR-223 was significantly up-regulated in co-cultured breast cancer cells. The results of
functional assay showed that miR-223 increased the invasive potential of breast cancer cells via activation of
the Mef2c-β-catenin pathway (63). Two studies showed an important role for exo-miRs from MSCs in
promoting tumor growth. MSC exosomes isolated from bone marrow of patients with multiple myeloma
promoted tumor growth and metastasis. Those MSC-derived exosomes showed significant down-regulation
of an important tumor suppressor, miR-15a (64). EVs derived from serum-deprived human MSCs (SD-
MSCs) were enriched with miR-21 and -34a compared to MSCs. Furthermore, co-injection of EVs from SD-
MSCs with breast cancer cells (MCF-7) into immunodeficient mice promoted breast cancer growth (65).
6

The crosstalk between cancer cells and host cells within the TME appears to be affected by
additional miR-mediated mechanisms, beyond the alteration of gene expression in recipient cells by binding
to their target mRNA. Our lab has shown that some of the exo-miRs secreted by cancer cells act as ligands
of Toll-like Receptor 8 (TLR8) in TAMs, promoting tumor growth, dissemination, and resistance to therapy
(66,67). Thus, TLR8 can be considered the founding member of a class of molecules we call the miRceptors
(defined as a receptor for miRs) (68). In 2012, we showed that Non-Small Cell Lung Cancer (NSCLC)-
derived exosomes were enriched for miR-21 and -29a. The NSCLC exosomes were taken up by surrounding
TAMs at the tumor interface and transferred to TAM endosomes, where miR-21 and -29a were directly
bound to TLR8 and triggered a TLR8-mediated activation of the NF- B signaling. The NF- B activation led to
increased expression of pro-inflammatory cytokines IL-6 and TNF- , further promoting the metastatic
potential of NSCLC (66). Recently, we have shown that this mechanism of “education” of TAMs also occurs
in NB and several other types of cancers (67,69). NB-secreted exosomes were enriched in miR-21 (but not
in miR-29a), which was taken up by surrounding TAMs. miR-21 then bound to TLR8, leading to increased
expression of the oncogenic miR-155 in a TLR8- and NF- B- dependent manner. miR-155 was then shuttled
back to NB cells, where miR-155 targeted the inhibitor of telomerase TERF1, thus increasing telomerase
activity and resistance of NB cells to cisplatin (CDDP). These studies confirm a central role of exosomic miR
exchange between cancer cells and the TME in orchestrating the biology of cancer growth and the
development of mechanisms of resistance to treatments.
V. miR based therapeutics
miRs act as tumor suppressors or oncogenes and this has inspired the field to consider them as
therapieutics. miR based therapies are divided into miR mimics and miR inhibitors. miR mimics are synthetic
double-stranded small molecules that match the corresponding miR sequence that aim to replenish the lost
miR expression in disease. On the other hand, antimiRs are single stranded antisense oligonucleotides
(ASOs) that aim to inhibit the oncogenic miR (22,70).
Several miR based therapeutics have reached clinical development, including mimic of the tumor
suppressor miR-34, which was tested in phase I clinical trials for treating cancer and anti-miR targeting miR-
122, which reached phase II trials for treating hepatitis (70).
One of the challenges of RNA based therapies is that single or double stranded oligonucleotides have
the potential for degradation by RNases in the serum or the endocytic compartment of cells. Two different
strategies have been developed to circumvent oligonucleotide degradation. The first includes altering
oligonucleotide chemistry by modifying the nucleotides or RNA backbone through methylation. The second
strategy is to deliver the oligonucleotides in delivery vehicles such as nanoparticles (70).
VI. Goal of the project
MYCN gene amplification and infiltration of TAMs correlate with poor prognosis in NB. TLR8 dependent
exo-miRs are key regulators of intercellular communication in TME. Our lab has shown that TLR8-dependent
upregulation of miR-155 induces NB cell resistance to CDDP. Currently, the role of TLR8 transferred miRs
from TAMs to NB cells in modulating MYCN in NB remains unknown. In this study, we identified miRs that
are transferred in a TLR8-dependent manner from TAMs to NB cells. Additionally, we investigated the role of
TLR8 restricted miRs in modulating MYCN expression in NB. This study will pave the way for the use of
exosome modulators in cancer and also identify the role of the TME in regulating MYCN expression in NB.
Materials and Methods
Cell lines. SK.N.BE(2), a MYCN amplified (MYCN-A) NB cell line, was obtained from American Type Culture
Collection (ATCC) and grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin-streptomycin (P/S) (5000 U/ml). IMR32, a second MYCN-A NB cell line
was obtained from Dr. Yves DeClerck’s lab and grown similarly. CHLA255, a MYC overexpressing and MYCN-
7

non amplified (MYCN-NA) cell line, was provided by Dr. Seeger’s lab and grown in Iscove's Modified
Dulbecco's Medium (IMDM) supplemented with 10% FBS and 1% P/S (5000 U/ml). The cell lines used were
confirmed to have been STR genotyped and Mycoplasma free before usage for the experiments.
Mouse strains and isolation of peritoneal macrophages. C57BL/6J wildtype and TLR7 knockout
(B6.129S1-Tlr7
tm1Flv
/J) mouse strains were obtained from The Jackson Laboratory. Mouse TLR7
is a homolog of human TLR8. For each experiment, five wildtype and five TLR7 knockout mice were used
to isolate the peritoneal macrophages (F4/80 cells). Briefly, the peritoneal cavity was washed with cold Miltenyi
MACs buffer and cells were collected. The collected cells were stained with APC anti-mouse F4/80 Antibody
(Biolegend) for 30 minutes on ice. Following cell washes, the APC labelled cells were isolated with magnetic
APC microbeads (Miltenyi Biotec).
NB-peritoneal macrophage co-culture setup. SK.N.BE(2) cells were plated overnight in 6 well plates at
density of 0.5 million cells per well. The isolated peritoneal macrophages were added to 0.4 µm cell culture
inserts (Greiner bio-one, 657641) at ratio of 1:1 of cancer cell:macrophage. The inserts were added to
SK.N.BE(2) plated cells for 48 hours. At 48 hours, SK.N.BE(2) cells were harvested and suspended in TRIzol®
reagent (Invitrogen) for RNA extraction.
miR profiling. RNA was extracted from SK.N.BE(2) cells alone, SK.N.BE(2) cells in co-culture with wildtype
peritoneal macrophages, and SK.N.BE(2) cells in co-cultures with TLR7 knockout peritoneal macrophages.
The extracted RNA was subjected to miR profiling using the TaqMan Array MicroRNA 384-well Cards
following manufacturer’s protocol (Thermo Fisher Scientific).
qRT-PCR for validation of TLR8 (mouse TLR7) transferred miR-192-5p. miR192 identified in high
throughput profiling was validated with quantitative reverse transcriptase polymerase chain reaction (qRT-
PCR). cDNA was synthesized using TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems
TM
).
qPCR reaction for miR-192 was done using TaqMan® MicroRNA assay using U6 as endogenous control
(Thermo Fisher Scientific).
Human monocyte isolation and M1/M2 polarization. Monocytes were isolated from peripheral blood
mononuclear cells (PBMCs) using EasySep human monocytes enrichment kit (Stemcell Technologies). Briefly,
human PBMCs were isolated by gradient centrifugation on histopaque (Sigma) from discarded leukocyte filters
obtained during platelet collection from healthy adult donors at Children’s Hospital Los Angeles Blood
collection center (in accordance to a protocol approved by the CHLA’s Institutional Review Board). PBMCs
were incubated with antibody cocktail against non-monocytes followed by incubation with dextran coated
magnetic particles. Labeled non-monocytes were removed using an EasySep magnet and monocytes were
enriched. The isolated monocytes were suspended in monocyte attachment medium (PromoCell) and
incubated at 37
◦
C for 2 hours. The attached monocytes were washed with monocyte attachment medium and
M1/M2 Macrophage Generation Medium DXF (PromoCell) supplemented with provided cytokines and human
serum was added according manufacturer’s instructions.
Immunophenotyping of macrophages. To confirm purity of monocytes and macrophage differentiation,
CD68 and CD14 expression markers were analyzed by flow cytometry. To check the polarization of
macrophage subtypes, CD274 (PD-L1) and HLA-DR were used for classically activated M1 macrophages and
CD163 and CD16 were used for M2 macrophages. The antibodies used were: anti-human CD68 PE-Cy7, anti-
human CD14 APC Cy7, anti-human CD16 PE, anti-human HLA-DR-FITC, anti-human CD163-APC, and anti-
human CD274 BV421 (BioLegend). Samples were acquired and analyzed with an LSRII flow cytometer (BD
Biosciences, San Jose, CA).
8

M1/M2-NB co-culture growth curve analysis. NB cells were plated overnight at density of 0.1-0.2 million
cells per well in complete medium. Triplicate wells were considered for each time point of the following
samples: NB cells alone, NB cells co-culture with M1, and NB cells in co-culture with M2. The polarized M1
and M2 macrophages were harvested, counted, and added to 0.4 µm cell culture inserts (Greiner bio-one,
657641) at ratio of 1:1 of cancer cell : macrophage. The inserts were added to NB cells and incubated up to 96
hours. At each time point, the NB cells alone and in co-culture with M1 and M2 were harvested and counted
with Vi-Cell cell counter.

M1/M2-NB co-culture growth curve analysis in presence of 13-cis-retinoic acid. NB cells were plated
overnight at density of 0.1-0.2 million cells per well in complete medium. Triplicate wells were considered for
each time point of the following samples: NB cells alone, NB cells alone with 13-cis-RA, NB cells co-culture
with M1 in presence of 13-cis-RA, and NB cells in co-culture with M2 in presence of 13-cis-RA. The polarized
M1 and M2 macrophages were harvested, counted, and added to 0.4 µm cell culture inserts (Greiner bio-one,
657641) at ratio of 1:1 of cancer cell : macrophage. The macrophage inserts were added to NB cells and 13-
cis-RA was added daily at concentration of 25 µM (determined through dose-response curve) and cells were
incubated up to 96 hours. At each time point, the NB cells alone and in co-culture with M1 and M2 in presence
of 13-cis-RA were harvested and counted with Vi-Cell cell counter.

Results

TLR8 transferred miRs from TAMs to NB cells. To identify miRs that are transferred in a TLR8-dependent
manner from TAMs to NB cells in exosomes, we set up a non-contact transwell-based co-culture of
SK.N.BE(2) (MYCN-A) with murine wildtype peritoneal macrophages and TLR7 knockout macrophages. RNA
was extracted for high throughput TaqMan® array microRNAs cards from SK.N.BE(2) cells alone and
SK.N.BE(2) cells in co-culture with wildtype and TLR7 knockout peritoneal macrophages. We identified miR-
155, -192, and -33b as most upregulated in SK.N.BE(2) cells co-cultured with wildtype macrophages compared
to NB cells alone (Figure 1). Additionally, no change in expression of these miRs was observed in NB cells co-
cultured with TLR7 knockout macrophages indicating a TLR7-dependent transfer of these miRs from
macrophages to NB cells.

Figure 1. Heatmap of miRs upregulated in SK.N.BE(2) cells in a TLR8-dependent manner (TLR7 of mouse is
ortholog of human TLR8).
We validated the transfer of miR-192 in a TLR8-dependent manner from macrophages to NB cells with other
NB cell lines. We setup a co-culture of IMR32 (MYCN-A) and CHLA255 (MYCN-NA) cell lines with murine
9

wildtype peritoneal macrophages and TLR7 knockout macrophages. qRT-PCR analysis showed upregulation
of miR-192 by fold-change of 1.81 and 3.68 in CHLA255 and IMR32 co-cultured with wildtype macrophages
compared to NB cells alone respectively (Figure 2). Additionally, miR-192 remained at basal levels in
CHLA255 cells co-cultured with TLR7 knockout macrophages compared to CHLA255 cells alone. Interestingly,
miR-192 was downregulated in IMR32 cells co-cultured with TLR7 knockout macrophages (Figure 2).

Figure 2. Transfer of miR-192 in a TLR8-dependent manner from murine macrophages to NB cells in two NB
cell lines. The co-culture of IMR32 with wildtype peritoneal macrophages and TLR7
-/-
knockout macrophages
was done twice, and similar co-culture experiment was done once for CHLA255. (CC= co-culture, PM=
peritoneal macrophages, n.s.=not significant, *p<0.05, **p<0.01, and ***p<0.001).
M1 and M2 macrophages confer a growth benefit on NB cells. Next, we wanted to determine and validate
if human macrophages upregulate miR-192 in NB cells as observed with wildtype murine peritoneal
macrophages. Before validating miR-192 transfer from human macrophages to NB cells, we validated the
purity of our polarized macrophages and whether they confer a growth advantage to cancer cells as
phenotypic check of TAMs. Currently, there is no established protocol for the polarization and study of TAMs.
Some groups polarize the macrophages to M1 and M2 using commercially available M1 and M2 polarization
media and then follow with co-culture with tumor cells as in our study. TAMs have been shown to exhibit an
M2-like phenotype. Other groups co-culture monocytes with cancer cells and allow the cancer cell to polarize
monocytes to TAMs as has been done in our previously published study (67). Additionally, some investigators
pre-differentiate monocytes to macrophages using GM-CSF (for M1) and M-CSF (for M2) for 6 days and then
supplement with subtype cytokines for 24 hours followed by co-culture with cancer cells (71). Some groups
utilize cancer cell-conditioned medium to polarize macrophages into TAMs (72). In our first step, we analyzed
the polarized macrophages for macrophage and monocyte lineage markers (CD68 and CD14) and M1 and M2
specific surface markers. The percentage of CD163 positive M2 macrophages varied between donors,
however, we observed polarization of M1 and M2 macrophages to their respective subtypes (representative
donor, Figure 3). The representative donor M1 macrophages express a higher percentage of HLA-DR and
CD274, whereas M2 macrophages express higher CD163 and CD16.
10

Figure 3. Immunophenotyping of M1 and M2 macrophages from a representative donor.

Following immunophenotyping of macrophages, we setup a co-culture of NB cells with M1 and M2
macrophages and monitored the NB cell growth as it has been reported that TAM infiltration is associated with
poor NB prognosis (15). Both the M1 and M2 macrophages conferred a growth benefit to NB cells (CHLA255
and IMR32) (Figure 4A and 4B). This is in contrast to some studies that have reported M1 mediate killing of
cancer cells (73,74).

Figure 4. Polarized M1 and M2 macrophages from healthy donors confer growth benefit to two NB cell lines
(based on three different experiments for each cell line and best representation shown). (CC = co-culture,
*p<0.05, **p<0.01, and ***p<0.001).
M1
M2
11

M1 and M2 macrophages confer resistance to 13-cis-RA on NB cells. 13-cis-RA is used as differentiating
therapy in NB (75). We analyzed if our polarized TAMs confer resistance to 13-cis-RA on NB cells. We setup a
co-culture of M1 and M2 macrophages with NB cells in the presence of 13-cis-RA. Both SK.N.BE(2) and
IMR32 cells showed enhanced growth in the presence of M1 and M2 macrophages despite 13-cis-RA
treatment implying M1 and M2 macrophages confer resistance to NB cells (Figure 5).

Figure 5. Polarized M1 and M2 macrophages from healthy donors confer resistance to 13-cis-RA on NB cells
(based on three different experiments for each cell line with best representation shown) (CC = co-culture,
*p<0.05, **p<0.01, and ***p<0.001).

miR-192 is upregulated in NB cells co-cultured with M1 and M2 macrophages. At each time point of the
growth curve both with and without 13-cis-RA, NB cells alone and NB cells co-cultured with M1 and M2
macrophages were harvested and RNA was extracted to check for transfer of miR-192 from macrophages to
NB cells. M1 and M2 macrophages upregulated miR-192 in IMR32 cells compared to IMR32 cells alone
(Figure 6).

Figure 6. (A) miR-192 is upregulated in IMR32 following co-culture with M1 and M2 macrophages (based on
three experiments). (B) miR-192 is upregulated in IMR32 following co-culture with M1 and M2 macrophages in
presence of 13-cis-RA (based on two experiments). (CC = co-culture, *p<0.05, **p<0.01, and ***p<0.001).

12

Chapter II. Tumor suppressor role of miR-192 in NB
Introduction
I. Dual role of miR-192 in cancer
The tumor suppressor role of miR-192 has been documented in several cancers. miR-192
overexpression induced apoptotic death in bladder cancer cells by increasing the levels of p21, p27, and Bax
(76). Wu et al. showed miR-192 overexpression exhibiting potent anti-angiogenic effect in cancer cells by
downregulating angiogenic pathways through regulation of EGR1 and HOXB9 (77). miR-192 suppresses
tumorigenicity of prostate cancer cells by targeting and inhibiting nin one binding protein (NOB1) (78). Current
studies have also shown an oncogenic role of miR-192. In gastric cancer, miR-192 positively correlated with
lymph node metastasis and inhibition of miR-192 reduced gastric cancer cell invasion (79). Interestingly, the
role of miR-192 has been controversial in hepatocellular carcinoma. Lian et al. have shown through gain and
loss of function analysis that miR-192 suppresses metastasis of hepatocellular carcinoma cell lines in vitro and
in vivo through targeting of the oncogenic SLC39A6/SNAIL pathway (80). The Wen group showed that ectopic
expression of miR-192 promotes proliferation and metastasis of hepatocellular carcinoma cells by targeting of
angiogenesis suppressor SEMA3A (81).
II. Goal of the project
TAMs upregulate miR-192 in NB cells in a TLR8-dependent manner. In this part of the study, we
investigated the effect of miR-192 on NB cell growth. We utilized the NB transcriptome data available on R2
database for Kaplan Meier survival curve analysis relating to expression of miR-192. Additionally, we carried
out gain of function analysis of miR-192 in NB cell lines. Both in silico and in vitro analyses suggest a tumor
suppressor role of miR-192 in NB.
Materials and Methods

Cell lines SK.N.BE(2), a MYCN-A NB cell line, was obtained from American Type Culture Collection (ATCC)
and grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin-streptomycin (P/S) (5000 U). CHLA136, MYCN-A Fluc NB cell line expressing firefly
luciferase was obtained from Dr. Robert Seeger’s lab and grown in Iscove's Modified Dulbecco's Medium
(IMDM) supplemented with 10% FBS and 1% P/S (5000 U/ml). CHLA255, a MYC overexpressing and MYCN-
non amplified (MYCN-NA) cell line, was provided by Dr. Seeger’s lab and grown similarly to CHLA136-Fluc.
The cell lines used were confirmed to have been STR genotyped and Mycoplasma free before usage for the
experiments.
Growth curve analysis Cells were seeded at a density of 1.5 x 10
5
cells/well in a 6-well plate in complete
medium (2.0 ml per well) and transfected the following day. On the day of transfection, complete medium was
replaced and transfected with 25 nM or 50 nM miR-192 and scrambled (scr) negative mimics (Ambion, Thermo
Fisher Scientific) up to 72 or 96 hours using RNAiMax lipofectamine transfection reagent (Thermo Fisher
Scientific). The mimics were transfected only once at the start of the experiment. Cells were harvested and
counted each time point using Vi-CELL cell counter (Beckman Coulter). Data were plotted as mean of
triplicates ± SD for each experiment and more than three experiments were performed for each cell line with
best representation included.
miR-192 qRT-PCR To analyze miR-192 overexpression in NB cells, RNA was extracted using TRIzol®
reagent (Invitrogen) from harvested cells and cDNA was synthesized using TaqMan® MicroRNA Reverse
Transcription Kit (Applied Biosystems
TM
). qPCR reaction for miR-192 was done using TaqMan® MicroRNA
assay using U6 as endogenous control (Thermo Fisher Scientific).
13

Bioinformatics We used R2 database dataset tumor neuroblastoma SEQC 498 RPM seqcnb1
(https://hgserver1.amc.nl/cgi-bin/r2/main.cgi) for Kaplan Meier survival curve analysis of miR-192 in NB.
Results

Expression levels and Kaplan Meier survival curve analysis of miR-192 in NB Utilizing the R2 database,
we compared expression levels of miR-192 in high risk and low risk NB cases. There is no significant
difference in expression levels of miR-192 between low and high risk NB cases (Figure 7A). However, the
Kaplan Meier survival curve analysis show that miR-192 increases event free survival (EFS) in high risk NB (p
= 0.020) (Figure 7B) and overall survival (OS) in MYCN-A NB (p = 0.029) (Figure 7C).

Figure 7. R2 database (dataset tumor neuroblastoma SEQC 498 RPM seqcnb1) analysis. A, Expression
levels of miR-192 in low and high risk NB patients. B, Kaplan Meier survival curve of miR-192 in high risk NB
on basis of EFS C, Kaplan Meier survival curve of miR-192 in MYCN-A NB on basis of OS.
miR-192 suppresses NB growth in vitro The effect of miR-192 on NB growth was studied in vitro in both
MYCN-A and MYCN-NA cell lines. NB cell lines were transfected with miR-192 mimic or scrambled (scr) mimic
RNA and cell growth was monitored. We observed significant suppression of NB cell growth in three MYCN-A
cell lines with miR-192 overexpression (Figure 8A). The overexpression of miR-192 in IMR32 decreased the
cell viability by 45% (p = 0.016) and 40% (p = 0.009) at 48 and 72 respectively compared to scr control group.
In SK.N.BE(2) and CHLA136-Fluc cell lines, overexpression of miR-192 suppressed NB cell viability by 37% (p
= 0.038) and 34% (p = 0.032) respectively at 96H compared to scr control group. The overexpression of miR-
192 in CHLA255, MYCN-NA cell line, decreased the cell viability by 56% (p = 0.009) at 48H and 50% (p =
0.006) at 72H compared to scr control group (Figure 8B). The overexpression of miR-192 was confirmed in all
the transfected cell lines by qRT-PCR (Figure 9).

14

Figure 8. miR-192 induces NB growth suppression in vitro. A, Growth curves of three MYCN-A NB cell lines
transfected with miR-192 mimic or scr mimic control on day 0. B, Growth curve of CHLA255, MYCN-NA cell
line, transfected with miR-192 mimic or scr mimic control on day 0. (*P < 0.05, **P < 0.01)

Figure 9. Expression of miR-192 in NB cell lines assayed with TaqMan miR-192 qRT-PCR. A, Expression of
miR-192 in three MYCN-A NB cell lines up to 72 or 96 hours following transfection with miR-192 mimic on day
0 compared to scr control. B, Expression of miR-192 in CHLA255 transfected with miR-192 mimic compared to
scr control up to 72 hours. (*P < 0.05, **P < 0.01, and ***P<0.001)

15

Chapter III. miR-192 suppresses NB growth by inhibiting CTCF
Introduction
I. Role of CTCF in cancer including NB
CCCTC-binding factor (CTCF) is a highly conserved protein in eukaryotes that was initially described
as a transcription factor (82). CTCF consists of three major domains: N-terminal domain, C-terminal domain,
and a central domain of 11 zinc fingers that bind a 20-bp nonpalindromic DNA sequence (83). CTCF is present
at 55,000-65,000 sites in mammalian genome with 50% of CTCF binding sites are in intergenic regions (82).
Recent advances have shown that CTCF is a multifunctional protein that acts as a transcription activator, a
transcription repressor, and an insulator protein by mediating and regulating interactions between promoters
and enhancers (84). Furthermore, it has been shown that CTCF interacts with cohesins at the borders of
topological associated domains (TADs) in chromosomes, facilitating interactions within domains and affecting
genome topology (85).
Notably, CTCF has recently been shown as an independent prognostic factor for progressive
unfavorable NB (86). There is decreased overall survival in NB cases with higher expression of CTCF.
Additionally, CTCF promotes MYCN expression in NB (86). MYCNOS, an antisense noncoding RNA derived
from transcription unit located on the DNA strand opposite to MYCN, has been shown to facilitate the
recruitment of CTCF to the MYCN promoter by binding to CTCF zinc fingers (86). The ectopic expression of
CTCF in NB cell lines leads to increased binding of RNA polymerase II (RNA pol II), histone H3 lysine 4
dimethylation (H3K4me2) and histone H3 lysine 4 trimethylation (H3K4me3) to the MYCN promoter and
decreased recruitment of repressive histone markers (H3K9me3 and H3K27me3) (86).
Several studies have shown a tumor suppressor role for CTCF in cancer. De La Rosa-Velazquez et al.
showed that CTCF is involved in epigenetic regulation of the retinoblastoma (Rb) gene. The study identified a
58-base pair (bp) GC-rich sequence in the Rb gene promoter that had significant homology to previously
known CTCF binding sites (CTS). The mutation of this 58-bp CTS, named cts rb, diminished RB promoter
activity (87). The authors demonstrated that CTCF binding of cts rb is dependent on the DNA methylation status
of RB promoter. The methylation of RB promoter abrogates CTCF binding of cts rb and induces recruitment of
Kaiso, a methyl-CpG binding protein involved in epigenetic silencing at various gene promoters. INK4, another
tumor suppressor gene that is frequently inactivated in several human cancers either by deletion or aberrant
DNA methylation, is regulated by CTCF. INK4 encompasses a 42-kb region on chromosome 9 and encodes
three distinct tumor suppressor proteins p15INK4b, p14ARF, and p16INK4a (referred to as p15, p14, and p16).
Witcher and Emerson showed the presence of a chromosomal boundary at ~2kb upstream of p16 transcription
start site (TSS) that separates the p16 gene locus into discrete domains characterized by presence or absence
of repressive epigenetic marks. The authors observed CTCF association with this chromosomal boundary in
several p16-expressing cell lines and the absence of CTCF at this boundary in several p16 non-expressing
breast cancer cell lines and myeloma cancer cell lines (88). CTCF also binds the p53 gene promoter both in
vitro and in vivo at cts p53 and promotes p53 transcription. CTCF prevents repressive histone post-translation
modifications (histone PTMs) at the p53 promoter. The transcriptional activation of p53 is abolished in HeLa
cells with PARP inhibitor 3-ABA implying that CTCF PARylation is necessary for p53 activation (89).
Recent evidence shows an oncogenic role of CTCF in other cancers in addition to NB. In the majority of
cancers, the ability of cancer cells to divide infinitely is mediated by the activation of the gene encoding the
telomerase reverse transcriptase catalytic subunit (TERT). In lung cancer, the knockdown of CTCF by siRNA
downregulated TERT mRNA. A novel enhancer element was found 4.5 kb upstream of the TERT TSS with the
CTCF binding site (90). In another study, the binding of CTCF to the Bax promoter in breast cancer cell lines is
associated with resistance of breast cancer cells to apoptosis (91).
MYC is an oncogene in several cancers (92). CTCF was discovered as a repressor of chicken MYC
(82). There are several CTCF binding sites in the MYC promoter, however, the deletion of the CTCF binding
16

sites on the MYC promoter has repressed or activated MYC transcription in different normal and cancer cell
lines suggesting a tumor suppressor or an oncogenic role for CTCF (93). The ectopic overexpression of CTCF
in K562, a leukemia cell line, downregulates MYC and induces differentiation of myeloid leukemia cells into
erythroid lineage suggesting a negative MYC regulator role for CTCF in leukemia (94). Gombert and Krumm
transferred a series of mutant human chromosomal alleles lacking two CTCF binding sites of the MYC
promoter into murine melanoma cells. Contrary to the leukemia cell model, the authors observed
downregulation of MYC and increased DNA methylation of the MYC promoter implying requirement of CTCF
for enhanced MYC expression (95). The current findings indicate that CTCF regulation of its targets depends
on several cell-type features such as DNA methylation at the promoter, post translational modifications of
CTCF such as PARYlation, and the differential binding of the CTCF DNA sequences by the different 11 zinc
fingers (82,93).
II. Goal of the study
The Kaplan Meier survival curve analysis of miR-192 in NB cases and gain of function studies of miR-192
in NB cell lines suggest a tumor suppressor role of miR-192 in NB. A recent study showed enhanced
expression of CTCF in NB is associated with progressive NB. Additionally, CTCF promotes MYCN expression
in NB (86). Our in silico analysis predicted the CTCF mRNA 3’-UTR as putative target of miR-192. In this part
of the study, we investigated if miR-192 suppresses NB growth by silencing of CTCF.
Materials and Methods

Cell lines SK.N.BE(2), a MYCN-A NB cell line, was obtained from American Type Culture Collection (ATCC)
and grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin-streptomycin (P/S) (5000 U/ml). IMR32, a second MYCN-A NB cell line was obtained
from Dr. Yves DeClerck’s lab and grown similarly. SHEPN21 cell line is a subclone of SHEP NB cells that is
transfected with a doxycycline (dox) regulated (tet-off) MYCN expression vector. SHEP and SHEPN21 cell
lines were also obtained from Dr. DeClerck’s lab and grown similarly. To inhibit MYCN expression in SHEPN21
cells, doxycycline was added at 10 ng/ml as previously described (96). CHLA136 MYCN-A Fluc NB cell line
expressing firefly luciferase for in vivo experiment was obtained from Dr. Robert Seeger’s lab and grown in
Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS and 1% P/S (5000 U/ml).
CHLA255, a MYC overexpressing and MYCN-non amplified (MYCN-NA) cell line, was provided by Dr.
Seeger’s lab and grown similarly to CHLA136-Fluc. The cell lines used were confirmed to have been STR
genotyped and Mycoplasma free before usage for the experiments.
Transfection of miR-192 mimic and inhibitor Cells were seeded at a density of 1.5 x 10
5
cells/well in a 6-
well plate in complete medium (2.0 ml per well) and transfected the following day. On the day of transfection,
complete medium was replaced and transfected with 25 nM or 50 nM miR-192 and scrambled (scr) negative
mimics (Ambion, Thermo Fisher Scientific) or the 50 nM miR-192 inhibitor (α-miR-192) and inhibitor control (α-
Scr) (Exigon) up to 72 or 96 hours using RNAiMax lipofectamine transfection reagent (Thermo Fisher
Scientific).
Gene expression analysis The overexpression of miR-192 and modulation of targets (CTCF and MYCN) was
analyzed using qRT-PCR. To analyze miR-192 overexpression in NB cells, RNA was extracted using TRIzol®
reagent (Invitrogen) from harvested cells and cDNA was synthesized using TaqMan® MicroRNA Reverse
Transcription Kit (Applied Biosystems
TM
). qPCR reaction for miR-192 was done using TaqMan® MicroRNA
assay using U6 as endogenous control (Thermo Fisher Scientific). To determine expression changes of CTCF,
cDNA was synthesized using iScript
TM
Reverse Transcription Supermix (BioRad). qPCR reaction for CTCF
was setup with CTCF TaqMan® Gene Expression Assay (Hs00902016_m1) and TaqMan® GAPDH as
internal control. For MYCN gene expression analysis, cDNA was synthesized using iScript
TM
Reverse
17

Transcription Supermix (BioRad) and qPCR reaction was setup with iTaq
TM
Universal SYBR® Green Supermix
(BioRad) using GAPDH as internal control. The primer sequences for the targets are the following: GAPDH
forward (Fwd) 5′-GATTCCACCCATGGCAAATTC-3’ and reverse (Rev) 5′-AGCATCGCCCCACTTGATT-3′;
MYCN Fwd 5’-ACCCGGACGAAGATGACTTCT-3’and Rev 5’-CAGCTCGTTCTCAAGCAGCAT-3’. All qPCR
reactions were performed in triplicates. Relative gene or miR-192 expression levels were calculated using the
ΔCτ method.

Western blot analysis Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris (pH
8.0)) supplemented with 0.2 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, and 0.001 M DTT. After
incubation on ice for 30 minutes, lysates were cleared by centrifugation at maximum speed (15 minutes, 4
◦
C),
denatured, and subjected to SDS-PAGE and western blot. The antibodies used were the following: a mouse
MAb against human CTCF (G-8; Santa cruz #271474; 1:1000 dilution in 2.5% milk at 4
◦
C overnight), a mouse
MAb against human MYCN (Abcam #16898, 1:1000 dilution in TBST at 4
◦
C overnight), and a mouse β-Actin
HRP conjugated MAb (Cell Signaling #8H10D10; 1:10,000 dilution in 2.5% milk for one hour at room
temperature). The secondary antibody used was goat anti mouse (BioRad) at dilution of 1:10,000 in 2.5% milk
for one hour at room temperature.
Constructs Predicted binding site (BS) of miR-192 within the 3’-UTR of CTCF was cloned into psiCHECK2
vector downstream of the control reporter gene (Renilla luciferase-hRluc) and upstream of firefly luciferase
reporter gene (hluc+). The forward and reverse primers spanning the binding site of miR-192 in 3’-UTR of
CTCF were designed with forward restriction site being the XhoI and reverse restriction site NotI. The primers
were annealed in equimolar amounts at 95
◦
C for 15 minutes in heat block and removed when the heat block
temperature reached room temperature. The annealed primers were digested with XhoI and NotI, cleaned with
PCR cleanup kit (Denville), and ligated with XhoI/NotI digested psiCHECK2 vector overnight and transformed
into 5-alpha E. coli competent cells (NEB). The primer sequences are the following: Fwd 5’-
CAATCTCGAGCCAGAAAATTTCACTAGGTCAGGCGGCCGC-3’ and Rev 5’-
ATGCGGCCGCCTGACCTAGTGAAATTTTCTGGCTCGAG-3’. The construct was designated as psiCHECK2-
CTCF 3’UTR miR-192 BS wt (wildtype). To determine that CTCF 3’-UTR is a direct target of miR-192, the
miR-192 seed region of 7 base pairs within the miR-192 binding site of the CTCF 3’-UTR was deleted using
QuikChange II site directed mutagenesis kit (Agilent Genomics). The construct was labeled as psiCHECK2-
CTCF 3’UTR miR-192 BS mut (mutated). The primers utilized for mutagenesis are the following: Fwd 5'-
GCCAGAAAATTTCACGGCGGCCGCTGGC-3’ and Rev 5'-GCCAGCGGCCGCCGTGAAATTTTCTGGC-3'.
Dual luciferase assay A total of 16 million cells were suspended in 800 µl of complete RPMI (20% FBS and
1% P/S) and divided equally into four micropulser electroporation cuvettes (BioRad). The first cuvette was co-
transfected with 200 nM scr and 5 µg of psiCHECK2-CTCF 3’UTR miR-192 BS wt; second cuvette was co-
transfected with 200 nM miR-192 and 5 µg of psiCHECK2-CTCF 3’UTR miR-192 BS wt; third cuvette was co-
transfected with 200 nM scr and 5 µg of psiCHECK2-CTCF 3’UTR miR-192 BS mut; and fourth cuvette was
co-transfected with 200 nM scr and 5 µg of psiCHECK2-CTCF 3’UTR miR-192 BS mut. The electroporation
protocol used was the following: exponential, 250 volts, 925 µF capacitance, 4 mm cuvette width, and ∞
resistance. Following electroporation, contents of each cuvette were suspended in 4.0 ml of complete RPMI
(20% FBS and 1% P/S) and divided equally on 4 wells of 12-well plate. After 24 hours, cells were assayed for
luciferase activities using Dual-Luciferase® reporter assay system (Promega) and plate was read on
GloMax®-Multi Detection System (Promega). The Relative Luciferase Unit (RLU) was calculated as the ratio of
expression of firefly luciferase to renilla luciferase. The assay was repeated more than three times per cell line
with best representation shown.
18

Rescue assay with CTCF cDNA overexpression SK.N.BE(2) cells were plated at a density of 2.5 x 10
5

cells/well overnight in 6-well plate (triplicate wells for each condition). The plated cells were co-transfected with
1 µg/well CTCF human ORF clone lentivector (CTCF-pLenti-C-mGFP) (origene #RC202416L2) or the empty
vector and scr or miR-192 mimic (50 nM) up to 72 hours using the RNAiMax lipofectamine transfection
reagent. The cells were harvested at each time point, counted with Vi-CELL and lysed for protein extraction
and western blot analysis.
Xenografts Animal experiments were performed under the protocol # 357-16 approved by the Institutional
Animal Care and Use Committee at CHLA. A total of 12 female and male NSG mice between ages of 6-8
weeks were used.
Orthotopic injection of the kidney Briefly, the mice were placed under general anesthesia and 5mm transverse
skin incision was made over the left shoulder, and the muscle overlying the retroperitoneum was sharply
divided with iris scissors to expose the left kidney. The kidney was gently exteriorized out of the wound and
gently placed on a sterile 2x2 gauze pad. The kidney was injected with 1 x 10
6
CHLA-136-Fluc cells in 100 µl
of 20% FBS RPMI. The kidney was gently replaced back into the body, the muscle and the skin were closed.
On day 4, the mice were randomized on basis of age and sex into two groups (6 mice per group).
Treatment groups Two groups received the following treatments: group I received miR-192 enclosed
nanoparticles, whereas group II received scr enclosed nanoparticles. The mice received treatment for three
weeks three times per week. Tumor growth was monitored weekly with bioluminescence imaging using a
Xenogen IVIS 100 instrument (IVIS Lumina XR System; Caliper Life Sciences).
Preparation of nanoparticles The preparation of anionic lipopolyplex nanoparticles enclosed with miR-192 and
scr control was done following the published protocol (97). A total of 3 mg of miR-192 and scr oligos were
synthesized (Bio-Synthesis), dissolved in 600 µl of nuclease free water to final concentration of 5 µg/µl. Briefly,
25 µl of synthetic oligos (scr and miR-192) in 112.5 µl Hepes (20 mM) were added to 43.75 µl of PEI (1mg/ml)
transfection reagent in 93.75 µl Hepes (20 mM) and incubated at room temperature (RT) for 2 minutes.
Following incubation, 350 µl of empty nanoparticles (2 mg/ml) were added, sonicated for 5 minutes, and further
incubated at RT for 20 minutes. Each mouse received 100 µl (20 µg) of respective (scr or miR-192)
nanoparticle treatment.
Patients Fresh frozen neuroblastoma primary tissues were collected from patients treated at CHLA (n = 21).
Informed consent was obtained and all the required procedures of institutional review board were followed.
RNA was extracted using TRIzol® reagent (Invitrogen), cDNA synthesized, and qRT-PCR was done for miR-
192 and CTCF as outlined above
Bioinformatics We used miRWalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/index.html),
targetscan (http://www.targetscan.org/vert_71/), and miRanda (http://www.microrna.org/microrna/home.do)
databases to determine miRs that target CTCF.
Statistics All the experiments were performed in triplicate and repeated more than three times with best
representation included. The data were plotted as mean of triplicates ± standard deviation. The statistical
significance was calculated by two-tailed Student’s t test. A value of p < 0.05 was considered statistically
significant. GraphPad Prism 7 software was used for all the statistical analyses. The Spearman correlation test
was used to investigate the correlation of miR-192 and CTCF in NB tissue samples.

19

Results

miR-192 downregulates CTCF and MYCN protein expression in NB cell lines The overexpression of miR-
192 in NB cell lines downregulated CTCF protein expression levels relative to scr control. In SK.N.BE(2) cell
line, there was down regulation of 47% (avg. downregulation of 36%, p = 0.041) and 41% (avg. downregulation
of 30.3%, p = 0.018) at 24 and 48 hours respectively of CTCF protein levels in miR-192 overexpressing cells
compared to scr control (Figure 10A). Likewise, the overexpression of miR-192 in IMR32 decreased CTCF
protein expression by 25% (avg. downregulation of 27.5%, p = 0.0082) and 30% (avg. downregulation of
25.5%, p = 0.029) at 24 and 48 hours respectively compared to scr control respectively (Figure 10A).
Additionally, CHLA255, a MYCN-NA cell line, also had decreased levels of CTCF upon miR-192
overexpression by 91% and 50% (avg. downregulation of 41%, p = 0.02) at 24 and 48 hours respectively
compared to scr control (Figure 10B).
Equally, the inhibition of miR-192 in SK.N.BE(2) cell line upregulated CTCF protein levels by 1.89, 1.18, and 4
fold at 24, 48, and 72 hours compared to anti-scr control group respectively (Figure 10C). The levels of CTCF
were also upregulated in CHLA255 by 3.5-fold upon inhibition of miR-192 (Figure 10D).
CTCF has been shown to promote MYCN expression (86); therefore, we also investigated if downregulation of
CTCF by miR-192 decreases MYCN protein expression in MYCN-A NB cell lines. The overexpression of miR-
192 decreased MYCN protein levels by 83% (avg. downregulation of 72%, p = 0.012) and 38% (avg.
downregulation of 43%, p = 0.038) at 24 and 48 hours in SK.N.BE(2) relative to scr control group respectively
(Figure 10A). Similarly, there was downregulation of MYCN protein upon miR-192 overexpression in IMR32 by
10% and 33% (avg. downregulation of 31%, p = 0.0041) at 24 and 48 hours compared to scr control group
respectively (Figure 10A). The inhibition of miR-192 increased MYCN levels in SK.N.BE(2) by 1.53, 1.34, and
1.58 fold at 24, 48, and 72 hours, respectively.
20

Figure 10. miR-192 modulates CTCF and MYCN protein levels in NB cells. A, Representative western blot for
CTCF and MYCN in two MYCN-A NB cell lines following miR-192 gain of function experiment (left) with
quantitation of CTCF and MYCN levels on right, n represents number of independent experiments. B,
Representative western blot for CTCF in CHLA255 with miR-192 gain of function experiment (left) and
quantitation of CTCF protein (right). C, Western blot for CTCF and MYCN in SK.N.BE(2) (left) and confirmation
of inhibition of miR-192 by qRT-PCR (right) each time point. D, Western blot for CTCF in CHLA255 (left) with
confirmation of loss of function of miR-192 by qRT-PCR (right). (*P < 0.05, **P < 0.01, ***P < 0.001)
CTCF 3’-UTR is a direct target of miR-192 The in silico analysis predict miR-192 to target the CTCF mRNA
3’-UTR between position 938-959 base pairs. We cloned the miR-192 binding site of the CTCF mRNA 3’-UTR
into the psiCHECK2 vector and determined the activity of CTCF with the dual luciferase assay (Figure 11A).
There was a decrease in RLU of CTCF in both MYCN-A and MYC-NA NB cell lines co-transfected with CTCF
3’-UTR and miR-192 compared to NB control cells co-transfected with CTCF 3’-UTR and scr. In CHLA255, the
co-transfection of CTCF 3’-UTR and miR-192 decreased CTCF RLU by 82% (p = 0.0005) compared to scr
control group (Figure 11B). There was a decrease of 45% (p =0.0034) and 23% (p = 0.022) of CTCF RLU in
IMR32 and SK.N.BE(2), respectively, co-transfected with CTCF 3’-UTR and miR-192 compared to scr control
group (Figure 11B).
21

To validate that CTCF 3’-UTR is a direct target of miR-192, we mutagenized the miR-192 seed sequence of
CTCF 3’-UTR in the psiCHECK2 vector (Figure 11A). The CTCF RLU returned to basal value with co-
transfection of mutagenized CTCF 3’-UTR and miR-192 in IMR32 and SK.N.BE(2) compared to scr control
group co-transfected with mutagenized CTCF 3’-UTR (Figure 11C).

Figure 11. miR-192 directly targeted 3’-UTR of CTCF. A, Two constructs were designed for luciferase reporter
assay. In the first construct, the predicted miR-192 BS of CTCF 3’-UTR (938-959 bp) was cloned in
psiCHECK2 dual luciferase reporter vector (top box), and in the second construct (bottom box), the miR-192
seed region within CTCF 3’-UTR was deleted (red line) from construct-1. B, Construct-1 was co-transfected
with scr or miR-192 mimic in three NB cell lines and RLU activity was assessed with dual luciferase reporter
assay. C, Construct-2 was co-transfected with scr or miR-192 mimic in two NB cell lines and RLU activity of
CTCF was measured with dual luciferase reporter assay. (*P < 0.05, **P < 0.01, n.s., not significant)
miR-192 mediates NB cell growth suppression through targeting of 3’-UTR CTCF miR-192
downregulates CTCF and MYCN protein levels and suppresses NB cell growth. Next, we investigated if miR-
192 suppression of NB growth is directly mediated by inhibition of CTCF. We overexpressed CTCF cDNA
without 3’-UTR and co-transfected SK.N.BE(2) with miR-192 and scr control. As expected, the ectopic
expression of CTCF cDNA with miR-192 abolished the miR-192 NB growth suppression compared to co-
22

transfection of miR-192 and empty vector (Figure 12A). The ectopic expression of CTCF increased NB cell
growth compared to empty vector control (p = 0.025) (Figure 12A). Consistent with previous results, the
overexpression of miR-192 with empty vector decreased SK.N.BE(2) cell viability by 25% (p = 0.037)
compared to co-transfection of empty vector and scr control (Figure 12A). Protein expression of CTCF is
decreased by 65% in empty vector and miR-192 co-transfected SK.N.BE(2) cells, whereas it is decreased only
27% in CTCF cDNA and miR-192 co-transfected cells (Figure 12B). Similarly, MYCN protein expression is
decreased by 51% in empty vector and miR-192 co-transfected SK.N.BE(2) cells, whereas there is no change
in MYCN expression in CTCF cDNA and miR-192 co-transfected cells (Figure 12B). Therefore, we conclude
that the repression of cell growth by miR-192 is at least in part through targeting of CTCF.

Figure 12. miR-192 suppresses NB growth by modulation of CTCF. A, Growth curve of SK.N.BE(2) cells co-
transfected with CTCF cDNA without 3’-UTR or the empty vector and miR-192 or scr control. B, Western blot
of CTCF and MYCN in SK.N.BE(2) co-transfected with CTCF cDNA without 3’-UTR or the empty vector and
miR-192 or scr control. (*P < 0.05, **P < 0.01, ***P < 0.001)
miR-192 suppression of NB growth is not modulated by MYCN status CTCF promotes MYCN expression
in NB (86). We therefore investigated if inhibition of MYCN prevents miR-192-mediated NB growth suppression
by using SHEP and SHEPN21 cells. The overexpression of miR-192 in SHEP cells reduced the growth rate by
70% at 48 hours (p = 2.32 x 10
-5
) and 87% at 72 hours (p = 1.52 x 10
-7
) compared to scr transfected SHEP
cells control group (Figure 13A). Similarly, the overexpression of miR-192 in SHEPN21 cells decreased cell
viability by 22% (p = 0.016), 60% (p = 0.0017), and 82% (p = 0.006) at 24, 48, and 72 hours respectively
compared to scr transfected SHEPN21 cells control group (Figure 13A). The treatment of SHEPN21 cells with
dox inhibits MYCN expression. We observed that overexpression of miR-192 decreased cell viability of
SHEPN21 treated with dox compared to scr control SHEPN21 cells treated with dox (Figure 13B). The
overexpression of miR-192 in SHEPN21 cells treated with dox decreased cell viability by 37% (p = 0.00037)
and 59% (p = 0.0024) at 48 and 72 hours respectively relative to SHEPN21 control scr group treated with dox
despite MYCN inhibition (Figure 13B).

23

Figure 13. miR-192 induces NB growth suppression regardless of MYCN status. A, Growth curves of SHEP
and SHEPN21 cells transfected with miR-192 mimic or scr mimic control on day 0. B, Growth curves of SHEP
and SHEPN21 cells transfected with miR-192 mimic or scr mimic control on day 0 with dox treatment daily to
inhibit MYCN in SHEPN21 cells. (*P < 0.05, **P < 0.01, and ***P<0.001)

miR-192 suppresses NB growth in vivo Taking the anti-tumoral role of miR-192 in vitro, we investigated if
miR-192 treatment can reduce tumor burden in an orthotopic NB model. We injected NSG mice intra-renally
with CHLA136-Fluc cell line and treated systemically with nanoparticles containing miR-192 or scr as a control
three times per week for three weeks. The delivery of miR-192 was confirmed ex-vivo, and there was 10.9 fold
upregulation of miR-192 in miR-192 treated group (p = 0.017) compared to scr treated mice group (Figure
14B).The treatment of mice with miR-192 reduced the kidney volume by 57% (p = 3.25 x 10
-7
) (Figure 14C),
kidney weight by 42% (p = 7.9 x 10
-5
) (Figure 14D), and total flux by 93% (p = 0.017) (Figure 14A).

24

Figure 14. miR-192 suppresses NB growth in orthotopic NB xenograft model. A, Bioluminsence imaging of
tumor growth in mice treated with miR-192 NP versus scr NP (left) and bioluminsence images of the two
groups of mice on the day before harvesting the tumors (right). B, qRT-PCR showing delivery of miR-192 ex-
vivo in group of mice treated with miR-192 nanoparticles (NP) compared to scr NP treated control group. C, D,
Kidney volume and weight of two groups following harvesting of the tumors. E, Images of kidneys of two
groups on day of harvesting the tumors. ((*P < 0.05, ***P < 0.001)
Negative Correlation of CTCF with miR-192 in NB tissues We analyzed by real time quantitative RT-PCR
the expression levels of miR-192 and CTCF in 21 NB primary tumors being blinded to the risk groups and
MYCN status. We found a statistically significant negative correlation between miR-192 and CTCF (R = -0.5, p
< 0.05) (Figure 15). These results further support that miR-192 acts as an inhibitor of CTCF.
25

Figure 15. Correlation analysis between expression of CTCF and miR-192 in primary NB tissues.

26

Chapter IV. Discussion, conclusion, and future work
MYCN gene amplification and subsequent MYCN protein expression is a prognostic factor for
unfavorable NB (98). MYCN constitutes one of the common oncogenic drivers of pediatric tumors that is
currently undruggable (92). There is an urgent need to develop enhanced therapies for inhibiting MYCN, and
our study investigated miR-based therapy. MYCNOS recruits CTCF to the MYCN promoter in NB and
stimulates MYCN expression (86). Currently, there are no available drugs for CTCF. Our study identified miR-
192 as key inhibitor of CTCF in NB. The inhibition of CTCF by miR-192 also downregulated MYCN protein
expression in NB. However, we observed that miR-192 targeting of CTCF suppressed NB growth regardless of
MYCN status. The growth of SHEPN21 cells, which stably express MYCN and MYCN is inhibited upon dox
treatment, was significantly suppressed by miR-192 despite dox treatment. Additionally, miR-192 suppressed
growth of CHLA255 NB cells, which is a MYCN-NA NB cell line that overexpresses homologous MYC protein.
TAMs infiltration is associated with unfavorable NB (15). Our study showed that TAMs upregulate miR-
192 in NB cell in TLR8 dependent manner suggesting an oncogenic role for miR-192 in NB. However, our gain
of function studies of miR-192 in NB and R2-based transcriptomic data of miR-192 in NB primary tissues
suggest tumor suppressor role for miR-192. TAMs upregulate miR-192 in NB cell lines by ~2-9 fold change,
whereas, the overexpression of miR-192 with mimic upregulates miR-192 levels by thousand folds. It is
possible that expression of the miR beyond the physiological concentrations conceals miR effect on cancer cell
growth. To better understand the role of miR-192 in NB, we need to express the miR at more physiological
levels.
Our study showed upregulation of miR-192 in NB cells by TAMs, however, we need to explore the
means of upregulation of miR-192 by TAMs in NB. Our future work will identify if miR-192 upregulation by
TAMs in NB is mediated through transfer of miR-192 in exosomes or whether there is a secretion of cytokine or
growth factor that upregulates miR-192 transcriptionally in NB cells. Additionally, we need to further understand
the role of TLR8 and TLR8 secreted exo-miRs in NB.
RNA based therapeutics are challenging due to ease of degradation of RNA molecules. Recently, many
groups have developed vehicles such as nanoparticles to deliver anti-miRs or miR mimics. Our group has
recently coated nanoparticles with anti GD2 antibodies to deliver the miR of interest specifically to NB cells that
overexpress GD2. There is an enhanced delivery of miR-186 with GD2 coated nanoparticles compared to IgG
coated miR-186 nanoparticles to NB cells in vivo (Figure 16). For future work, we will explore the delivery of
miR-192 to NB cells in GD2 coated nanoparticles.
27

Figure 16. A, protocol for developing antibody coated nanoparticle with carrier miR (97). B, Delivery of miR-
186 to NB cells with GD2 coated nanoparticle in vitro. C, Delivery of miR-186 to NB cells with GD2 coated
nanoparticle in vivo.

28

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Abstract (if available)

Abstract Neuroblastoma (NB) is the most common solid extracranial malignancy in children. MYCN gene amplification is detected in 25% of NB cases and is associated with an unfavorable outcome. Currently, MYCN is undruggable. Several therapies have been developed and being enhanced for targeting MYCN. Recently, higher infiltration of tumor associated macrophages (TAMs) has been shown in metastatic NB cases compared to locoregional NB tumors. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression and act as ligands of the Toll like receptor 8 (TLR8). MiRs can be transferred in microvesicles called exosomes (exo-miRs). Current evidence shows that exo-miRs orchestrate the intercellular communication within the tumor microenvironment (TME). Our lab has recently shown TLR8-dependent transfer of miR-155 from TAMs to NB cells in exosomes. TLR8-dependent transfer of miR-155 induces NB resistance to cisplatin by targeting telomerase inhibitor TERF1. In this study, we investigated other miRs that are transferred from TAMs to NB cells in a TLR8-dependent manner and how those TLR8-dependent exo-miRs can modulate MYCN in NB. We found that miR-192 is transferred in a TLR8-dependent manner from TAMs to NB cells suggesting an oncogenic role for miR-192 in NB. However, our gain of function studies of miR-192 in NB and R2-based transcriptomic data analysis of miR-192 in NB primary tissues showed a tumor suppressor role for miR-192. We investigated the tumor suppressor mechanism of miR-192 and how it can modulate MYCN. CCTC-binding factor (CTCF), a multifunctional protein with key a role in transcription regulation and genome topology, has recently been identified as an independent prognostic factor for progressive NB. Additionally, CTCF binding of the MYCN promoter stimulates MYCN expression in NB by recruitment of histone activating marks and inducing chromatin remodeling. Taking into account that MYCN is un-druggable and CTCF induces MYCN expression, we investigated if CTCF is a target of miR-192. Furthermore, CTCF plays an oncogenic role in NB and there are no drugs to inhibit CTCF. Our in silico analysis predicts the CTCF mRNA 3’-UTR as a putative target of miR-192. We investigated if miR-192 mediates NB growth suppression by inhibiting CTCF. The overexpression of miR-192 in NB cell lines downregulated CTCF and MYCN at the protein level. Equally, the inhibition of miR-192 upregulated CTCF and MYCN protein in NB cell lines. Dual luciferase assays showed direct targeting of CTCF 3’UTR by miR-192. The co-expression of miR-192 and CTCF cDNA without the 3’UTR abrogated miR-192-mediated NB growth suppression suggesting that miR-192-mediated killing of NB cells is in part caused by the inhibition of CTCF. Therapeutic delivery of miR-192 via nanoparticles in an orthotopic NB model significantly suppressed NB growth. In clinical NB tissues, we observed a negative correlation of CTCF with miR-192. Together, these results indicate that miR-192 suppresses NB growth by inhibiting CTCF. The upregulation of miR-192 in NB cells by pro-tumoral TAMs is currently not understood in our study. TAMs promote miR-192 expression by 2-9 folds in NB cells, whereas overexpression of miR-192 with mimic miR-192 overexpresses the miR beyond physiological concentrations. It is possible that miR exhibits an oncogenic role at lower concentrations. Currently, there are no studies that correlate miR concentration with effect on its target. Additionally, we need to investigate if miR-192 upregulation in NB cells is mediated through the transfer of miR-192 in exosomes to NB cells, or if cytokines or growth factors induce transcription of miR-192 in NB cells, when surrounded by TAMs.

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Asset Metadata

Creator Murtadha, Mariam Mumtaz (author)

Core Title TLR8-transferred miR-192 acts as a tumor suppressor in neuroblastoma by inhibiting CTCF

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Cancer Biology and Genomics

Publication Date 06/26/2020

Defense Date 03/07/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag microRNAs,neuroblastoma,OAI-PMH Harvest,TLR8

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Offringa, Ite (committee chair), Fabbri, Muller (committee member), Stallcup, Michael (committee member)

Creator Email mariammurtadha@yahoo.com,mmurtadh@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-507756

Unique identifier UC11267191

Identifier etd-MurtadhaMa-6344.pdf (filename),usctheses-c40-507756 (legacy record id)

Legacy Identifier etd-MurtadhaMa-6344.pdf

Dmrecord 507756

Document Type Dissertation

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Rights Murtadha, Mariam Mumtaz

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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