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Musashi-2 promotes c-MYC expression through IRES-dependent translation and self-renewal ability in hepatocellular carcinoma

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Musashi-2 promotes c-MYC expression through IRES-dependent translation and self-renewal ability in hepatocellular carcinoma

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Content Musashi-2 promotes c-Myc expression through IRES-
dependent translation and self-renewal ability in
hepatocellular carcinoma
By
Padmini Narayanan

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
In Partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

August 2015

2

ACKNOWLEDGEMENT

“The elevator to success is always out of order. You will have to use the
stairs…One step at a time and the people you meet on stairs decides how
far you will climb.” So I would like to thank all those people who
encouraged and helped me in this thesis. This dissertation would not have
been possible without the guidance and help of several individuals who in
one way or another contributed in the preparation and completion of this
study.
I am grateful to my professor Dr. Keigo Machida for giving me this
opportunity to work in the excellent and stimulating research environment
of his Lab. It was his guidance and encouragement that helped in the
successful completion of my thesis.
I am most obliged to Dr. Hifzur Siddiqui, Research Assistant at Dr.
Machida’s Lab without whom my research wouldn’t have been possible. He
taught, trained and guided me in all the possible techniques and at the
same time made learning fun. I would also like to thank all the former and
current members of the Lab for their support and co-operation: Dr. Douglas
Feldman, Ms. Chialin Chen, Mr. Chad Nakagawa, Mr. Dinesh Babu, Ms.
Ambika Ramrakhiani, Mr. Ahmed Rokan and Mr. Joe Chen.
I would also like to extend my gratitude to Ms. Michelle Mac Veigh from the
Cell and Tissue Imaging core of the USC Research Center for Liver
Diseases and Dr. Vasu Punj from the USC genomic center for their help.
I would like to thank my parents and my sister who have always been a
source of inspiration for me. I am also grateful to my friends for their love
and support.
My sincere gratitude and thanks goes to Ms. Silvina Campos and Ms.
Aileen Calimlim for their help.
Finally, I wish to extend a warm thanks to everybody involved directly or
indirectly with my work.

3

TABLE OF CONTENTS

List of figures…………………………………………………………………………………..5
Abstract………………………………………………………………………………………….6
Chapter 1: Introduction……………………………………………………………………….7
1.1 Hepatocellular Carcinoma………………………………………………...……………7
1.2 Tumor Initiating stem-like cells……………………………………………...…………8
1.3 m-RNA binding protein: Musahsi-2 (MSI-2)…….………………………...……….…8
1.4 m-RNA: c-MYC…………………………………………………...………………..……9
1.5 c-MYC Internal Ribosome Entry-Site………………………...……………….……..10

Chapter 2: Materials and Methods………………………………………………………...12
2.1 Cell Culture…………………………………………………...…………………………12
2.2 Mice…………………………………………………...…………………………………12
2.3 Lentiviral Transduction…………………………………………………...……………13
2.4 Western Blot…………………………………………………...……………………….13
2.5 IRES plasmid constructs…………………………………………………...………….13
2.6 Luciferase Assay…………………………………………………...…………………..14
2.7 Soft Agar Assay…………………………………………………...……………………14
2.8 Spheroid Assay…………………………………………………...……………………14
2.9 Subcutaneous xenograft transplantation…………………………………………….15
2.10 Tumor collection and analysis…………………………………………………...…..15
2.11 RNA-Immunoprecipitation…………………………………………………...……….15
2.12 qRT-PCR…………………………………………………...…………………………..15
2.13 Confocal Immunofluorescence staining……………………………………………..16
2.14 CLIP-sequencing and RNA-sequencing…………………………………………….16
2.15 Bioinformatic Analysis…………………………………………………...……………17
4

2.16 Statistical Analysis…………………………………………………...………………..17
Chapter 3: Results………………………………………………………………………...…18
3.1 MSI-2 and its target m-RNAs obtained from CLIP-Sequencing…..………………18
3.2 MSI-2 binds the c-Myc m-RNA…………………………………………………….....20
3.3 MSI-2 regulates c-MYC at post-transcriptional level………...……………………..21
3.4 MSI-2 expression increases colony formation and self-renewal of Huh7 cells….22
3.5 MSI-2 expression increases self-renewal and tumorigenesis, in vivo…...……….25
3.6 MSI-2 and c-MYC co-expression in HCC specimen………..……………………...26
3.7 MSI-2 can stimulate the c-MYC IRES, in vitro……………………………...……….27

Chapter 4: Discussion……………………………………………………………………….30

Chapter 5: Future Directions……………………………………………………….………32

References…………………………………………………………………………………….33

5

LIST OF FIGURES

Figure 1: MSI-2 binds to different regions of its target genes
Figure 2: MSI-2 specifically binds c-MYC m-RNA.
Figure 3: MSI-2 regulates c-MYC post transcriptionally in Huh7
Figure 4 : MSI-2 expression increases colony formation and self-renewal of Huh7 cells
Figure 5: MSI-2 expression increases self-renewal and tumorigenesis, in vivo
Figure 6: MSI-2 and c-MYC co-expression in HCC specimen
Figure 7: MSI-2 can stimulate the c-MYC IRES, in vitro
Figure 8: Summary Diagram.

6

ABSTRACT

RNA-binding protein MSI-2 has been shown to be elevated in several cancers types such as
leukemia, hepatocellular carcinoma (HCC) and breast and has been linked with poor prognosis.
In Mixed Myeloid Leukemia (MLL), MSI-2 maintains efficient translation of the oncogene c-MYC
and maintains MLL self-renewal activity. In this study, we sought to elucidate the role of MSI-2 in
regulating the expression of proto-oncogenes in HCC and thereby determine the prognostic
significance of MSI-2 and proto-oncogenes in HCC. We performed RIP-seq using anti-MSI2
antibody in tumor-initiating stem-like cells (TICs). Among the MSI2-bound RNA, c-MYC mRNA
binding was confirmed by RIP-qPCR analysis. MSI-2 does not have any effect on level of c-MYC
m-RNA using RT-qPCR and immunoblots, but significantly increases the c-MYC protein levels,
indicating that MSI-2 promotes c-MYC protein in the post-transcriptional levels. Overexpression
of MSI-2 promotes self-renewal and tumor-initiation property (tumor formation) in xenograft tumor
mouse models while silencing MSI2 reduces them. Immunohistochemical analysis were also
performed. Overexpression of MSI2 promotes internal ribosome entry site (IRES)-dependent
mechanisms. In conclusion, we demonstrate that MSI-2 promotes liver tumorigenesis by
maintaining the c-MYC expression. Both MSI-2 and c-MYC may be useful prognostic factors of
HCC and MSI-2 targeted therapy may be beneficial in treatment of HCC patients.

7

Chapter 1: INTRODUCTION

1.1 Hepatocellular Carcinoma
Liver cancer is the fifth most common malignancy globally. Primary liver cancers include
cholangiocarcinomas, hepatoblastomas, and hepatocellular carcinomas (HCC), of which the
latter accounts for >90% of primary liver cancers with 500,000–1,000,000 new cases being
diagnosed globally annually
1
. HCC is the fifth most common cancer and second cause of
cancer-related mortality. HCC, is the second and sixth most lethal cancer in men and women,
respectively
2
. Several key risk factors contribute to the development of HCC .Hepatitis B virus
(HBV) and Hepatitis C virus infection (HCV) are the main factors causing HCC. Environmental
factors such as chronic alcoholic consumption and high fat diet, dietary aflatoxin and cigarette
smoking are some of the other causes of HCC
3
.
On a molecular level, HCC genomes commonly have alterations in the DNA genome, including
DNA mutations in multiple oncogenes and tumor suppressor genes and changes in
chromosomal copy number. One of the canonical molecular changes associated with HCC is
the differential expression of the Wnt pathway, which regulates the expression of many
proliferation, metabolism, and ECM remodeling-associated genes, such as c-MYC, c-jun,
glycogen synthase kinase-3β, cyclin D1 and matrix metalloproteinases (MMP) .The Wnt
pathway has been reported to be up-regulated in up to 90% of HCC tumors. Another pathway
altered in HCC is the Hedgehog signaling pathway, which is up-regulated in up to 60% of HCC
tumors. Similar changes are seen in the JAK/STAT pathway with the activating phosphorylation
of the key transcription factor STAT3 being reported in 50%–100% of HCC tumors
1
. While there
does not appear to be any single pathway that is sufficient or necessary for HCC development,
these pathways commonly result in up-regulation of genes involved in cell cycle progression,
proliferation and escape from apoptosis .Transcriptomic analysis has shown that changes in
these common pathways occur late in the tumor development
1
.
Potentially curative treatments are suitable for very-early- and early-stage HCC. However, the
vast majority of HCC patients are diagnosed in later stages, where the tumor characteristics or
progress of liver disease prevent curative interventions. There is currently no effective systemic
chemotherapy, immunologic, or hormonal therapy for HCC, and sorafenib (a popular drug
targeting multiple kinases) is the only approved molecular-targeted treatment for advanced
HCC. Other targeted agents are under investigation; trials comparing new agents in
8

combination with sorafenib are ongoing. Combinations of systemic targeted therapies with local
treatments are being evaluated for further improvements in HCC patient outcomes
2
.

1.2 Tumor initiating stem-like cells (TICs)
Recent research efforts in the fields of stem cell and cancer biology have resulted
in a ‘‘stem cell model of carcinogenesis’’ that postulates that the capability to maintain tumor
formation and growth is found in a small population of cells called tumor-initiating cells (TICs) or
cancer stem cells (CSCs)
4
. Occurrence of mutations during chronic liver injury likely to prompt
an expansion of altered stem cells, leading to the genesis of TICs for tumor development and
progression. The stem cell- and cancer cell-like characteristics of these cells are believed to
render these cells resistant to conventional therapies and allow them to drive tumorigenesis
Tumor-initiating stem-like cells (TICs) represent a major factor in chemotherapy resistance in
the treatment of hepatocellular carcinoma
5
. Roughly 40% of HCC are considered clonal, so
early tumor initiation may be stem-like in origin. Therefore, it is important to understand the key
functional pathways used by TICs for their self-renewal and identify new therapeutic targets for
HCC.

1.3 m-RNA binding protein: Musahsi-2 (MSI-2)
It Belongs to Musashi family of proteins, which are m-RNA binding proteins. Musashi has two
protein homologs Musashi-1(MSI-1) and Musahsi-2 (MSI-2). Both these proteins are highly
similar in expression pattern and structure, however differ in their functional roles in different
cancer types. MSI1 and MSI2 are located on chromosomes 12 and 17, respectively and encode
for a group of RNA-binding proteins that bind, via 2 ribonucleoprotein-type RNA recognition
motifs in their N-termini, to consensus motifs in mRNAs
6
. They specifically target
developmental transcriptional factors and cell cycle regulators. These proteins can take part in
post-transcriptional regulation via binding to specific mRNA and they play a role in regulating
proliferation and differentiation of stem cells
7
. They are expressed in stem cells, and their
expression has been associated with aggressive behavior in a range to tumors, including
gliomas, pediatric brain tumors, breast cancer and colorectal cancer
8
.

9

Msi2 is well known for its function as a translation inhibitor, but previous studies have shown
that Msi2 can also act as a translation activator depending on the cell cycle status
10
. Increased
expression of MSI2 may predict aggressiveness in a variety of cancers. This has been shown
particularly in Acute Myeloid Leukemia, where it functions as an independent adverse
prognostic marker. MSI-2 has been shown to maintain the self-renewal program in myeloid
leukemia by directly increasing the protein translation of some critical transcriptional factors and
epigenetic modulators such as IKZF2, MYC, and HOXA9. MSI-2 alters only the translation of
these proteins without significantly increasing their m-RNA levels
11
.

MSI2 as a potential predictive biomarker of HCC invasion and prognosis. However, MSI-2 has
been shown to be co-related with HCC more than MSI-1. Musahi-2 is known to inhibit NUMB,
an inhibitor of Notch, Hedgehog and p53 signaling pathways, and thereby regulate stem cell
proliferation
12
. However, MSI-2 acts in a NUMB independent manner to promote HCC
7
.Therefore, alternative signaling pathways needs to be explored by which MSI-2 mediates its
biological effects in HCC. These findings can help in understanding the prognostic significance
of MSi-2 in HCC and also develop efficient therapy targeting MSI-2.

1.4 m-RNA: c-MYC
c-MYC belongs to MYC family of proteins which includes- N-myc and L-myc. MYC is a potent
oncogene that can promote tumorigenesis in a wide range of tissues. MYC is the most
frequently amplified oncogene and the elevated expression of its gene product, the transcription
factor c-MYC, correlates with tumor aggression and poor clinical outcome. Elevated expression
of c-MYC occurs through multiple mechanisms in tumor cells, including gene amplification,
chromosomal translocation, single nucleotide polymorphism in regulatory regions, mutation of
upstream signaling pathways, and mutations that enhance the stability of the protein
13
. In
normal cells, c-MYC links growth factor stimulation and cellular proliferation .Mitogenic growth
factor signaling induces MYC expression, and c-MYC is thought to enhance transcription of
proliferation-associated genes. In tumor cells that express high levels of c-MYC, cellular
proliferation is no longer dependent on growth-factor stimulation, and this uncoupling from
growth factor regulation leads to the uncontrolled proliferation characteristic of cancer cells.
Elevated expression of c-MYC also causes changes in chromatin structure, ribosome
10

biogenesis, metabolic pathways, cell adhesion, cell size, apoptosis and angiogenesis, among
others.

c-MYC has been observed to be a potent initiating oncogene of liver tumors and inactivation of
c-MYC is sufficient to induce sustained regression of MYC-initiated liver tumors in mice. c-MYC
regulates several cellular processes and is crucial for stem cell maintenance. It is also essential
for normal growth and proliferation since its inactivation produces lethal effects, indicating its
level has to be tightly regulated. Down-regulation of c-MYC both in vitro and in vivo has been
shown to induce growth inhibition and differentiation of HCC
15
. The transcription initiation of
MYC starts from multiple promoters – P0, P1, P2 and P3. The two major promoters, P1 and P2
contribute 75±90% and 10±25% of the cytoplasmic c-MYC mRNAs respectively. In normal and
transformed cells the majority of mRNAs initiate at the P2 promoter
16
.P1 and P2 give rise to m-
RNA’s with 5’UTR of approximately 400-600 nucleotide long. These promoters are activated
based on the function the translated protein needs to perform. P2 promoter gives rise to 2 major
c-MYC proteins-c-MYC-1 (67kDa) and c-MYC-2 (64kDa). The most abundant c-MYC protein, c-
MYC-2, initiates translation in exon 2 from the first AUG codon, whereas the c-MYC-1 protein
initiates upstream in exon 1 at a non-AUG site (a CUG in human c-MYC). There are no
apparent differences between these two proteins in subcellular localization, stability, or post-
translational modifications
17
. A functional significance for the non-AUG-initiated c-MYC- 1
protein in regulating cell growth, however, is suggested by its absence in tumor- derived cell
lines that have an alteration at the c-MYC locus, such as in human Burkitt's lymphomas
18
.

1.5 c-MYC Internal Ribosome Entry-Site
5’UTR (untranslated region) of the c-MYC proto-oncogene is highly structured as it plays an
important in maintaining role of c-MYC protein. For the overwhelming majority of eukaryotic
mRNAs initiation of protein synthesis occurs via a cap-dependent mechanism (involving binding
of the eukaryotic initiation factor (eIF) 4E, to the 7methyl G cap of the mRNA)
19
.In case of some
m-RNA’s like, c-MYC, structured 5’UTR contains an internal ribosome entry segment (IRES)
which allows cap-independent translation. The c-MYC protein can be translated under situations
where initiation from the 5' cap structure and ribosome scanning is reduced. There are a
number of situations where modulation in the levels of c-MYC protein via internal ribosome entry
11

may be required including the onset of proliferation, during mitosis where cap-dependent
translation is reduced and following DNA damage
20
.

These IRESs are capable of directing ribosomes to an internal start codon which may be some
considerable distance (600 ± 1000 nts) from the 5' end of the message. The c-MYC IRES is 340
nucleotide long (2500-2840 ntds of exon 1) and is located downstream of the P2 promoter. A
consistent point mutation(C T) at 2756
th
nucleotide of the c-MYC IRES was identified in
Multiple Myeloma Cell lines.

Several IRES-trans acting factors (ITAFs) have been identified that bind to c-MYC IRES. Four
ITAFs have been identified such as- PSF (PTB-associated splicing factor); its binding partner,
p54nrb; GRSF-1 (G-rich RNA sequence binding factor 1) and YB-1 (Y-box binding protein 1).
They bind and stimulate the IRES activity
21
.

12

Chapter 2: MATERIALS AND METHODS

2.1 Cell culture
Huh7, HEK293T and Mouse TIC cells were used for this study. Huh7 is a well differentiated
hepatocyte derived cellular carcinoma cell line that was originally taken from a liver tumor in a
57-year-old Japanese male. HEK293T is a variant type of the Human embryonic kidney 293
cells containing the large T-antigen of the SV40 virus. This variant helps achieve episomal
replication of transfected plasmids and generally used in retroviral or lentiviral vectors. These
cells form the basis for a lot of lentiviral packaging cell lines. Huh7 and HEK293T cells were
cultured in Dulbecco’s Modified Essential Medium (Caisson Labs) supplemented with 10% heat-
inactivated fetal bovine serum, 1% antibiotics and non-essential medium. Cell Line was cultured
at 37°C in a 5% CO2 humidified atmosphere. Mouse TICs were cultured in DMEM Ham’sF12
(Caisson Labs) supplemented with 10% heat-inactivated fetal bovine serum, antibiotics, non-
essential amino acids, Nucleosides, mEGF.

2.2 Mice
In the animal studies, Immunodeficient NSG mice (Jackson Lab) were used.

13

2.3 Lentiviral transduction
To prepare si-RNA and over expressing lentiviral particle for MSI-2 and c-MYC, HEK293T cells
were seeded in T75 flasks, 24 hours before transfection. The following day cells reach 70%
confluency. 293T cells are then transfected with 6.5 ug of psPAX2, 3.5 ug of pMD2G, 31.5 ul of
BioT transfection reagent (Bioland Scientific LLC) and finally with serum free DMEM medium.
Then 2ml of the respective transfection agent was added to the T75 flask. Medium was changed
after 16 hours. Every 24 hours and 48 hours supernatants were pooled, filtered through 0.45 um
filter and ultra-centrifuged at 20,000 rpm for 2 hours at 4
o
c. Pellets, which has the viral particle,
were then suspended in media and stored at -80
o
c. Next day the Huh7 cells were infected with
viral particle. The transduced cells were then selected on 5ug/ml of puromycin to create stable
shMSI-2 and MSI-2 over-expressing cell line. The stable MSI-2 over-expressing cells were
infected with si-c-MYC viral particle and used for respective experiment 3 days later.

2.4 Western Blot
To prepare the samples for western blot analysis, cells were harvested and lysed using RIPA
lysis buffer (sodium chloride, NP-40 or Triton X-100, sodium deoxycholate, SDS, protease and
phosphatase inhibitors). Protein Extracts were separated by 10% SDS-PAGE and transferred in
to nitrocellulose membranes probed with antibodies against c-Myc (9E10, Santa Cruz
Biotechnology, Santa Cruz, CA), MSI-2 (sc-83160, 1:250, Santa Cruz Biotechnology, Santa
Cruz, CA) and β-actin (sigma). Horseradish peroxidase–conjugated IgG (Santa Cruz
Biotechnology; 1:2,000) was used to treat the membranes for 1 hour at room temperature, and
enhanced with a SuperSignal® West PicoChemiluminescent substrate (Thermo). The bands
were detected in Premium Clear Blue X-Ray films
(Bioland Scientific LLC).

2.5 IRES Plasmid constructs
The plasmids pRF, pRMF, pRLsF and pRNF which harbor the myc family of IRES, pRHCV
harbors the Hepatitis C virus IRES, pRAF and pRBF harbor the Apaf-1 and BAG-1 IRES were
obtained from Dr. Anne E Willis (University of Leicester,UK).

14

2.6 Luciferase assay
Luciferase-based genetic reporter assays provide sensitive methods for assaying gene
expression, enabling the accurate quantification of small changes in transcription resulting from
subtle changes in biology. Cells are inherently complex, and the data available from a single
reporter may be insufficient for achieving reliable results. Dual-reporter assays enable
researchers to obtain additional information from complex systems with minimal effort. The
Dual-Luciferase® Reporter (DLR™) Assay and Dual-Glo™ Assay enable the sequential
measurement of both firefly and Renilla luciferases from one sample. The DLR™ and Dual-
Glo™ Assays provide rapid and convenient means for achieving greater control over the
biological significance of reporter data by differentiating genetic responses of interest from non-
relevant influences in the experimental system. The pRF vector containing the MYC family IRES
regions and HCV IRES were transfected into MSI-2 over-expressing, si-MSI-2 and scrambled
Huh7 cells. After 24 hours incubation, the luciferase activity was assessed with the Dual-
Luciferase Reporter Assay Kit (Promega). All experiments were performed in triplicate on three
independent occasions

2.7 Soft Agar Assay
Cells (2,500) were seeded in 0.35% agarose in DMEM medium on a layer of 0.5% agar in the
DMEM medium. Cells were incubated for 28 days at 37 ℃ in a humidified atmosphere at 5%
CO2 in air and 500 μl DMEM medium were added twice a week. At the end of the incubation
period, colonies were stained with crystal violet (CV) followed by scanning for colony counts.

2.8 Spheroid Assay
50 of TICs were seeded onto Ultra low attachment 96-well plates (Corning Inc.), followed by
incubating at 37 ℃ in a humidified atmosphere at 5% CO2 in the air for 14 days and 100 μl TIC
growth medium were added twice a week. The number of colonies were counted under optical
microscope and the proliferation was measured using Luminescent Cell Viability Assay
(Promega) followed by manufacturer’s instructions. All experiments were repeated thrice at least
in 24 wells.

15

2.9 Subcutaneous xenograft transplantation of the TICs into immunodeficient mice
Cells (10,000) in 100 μl solution were mixed with 100 μl Matrigel (BD Biosciences) and injected
into the dorsal flanks of NSG mice. Mice were anesthetized with isofluorene during the
procedure. The tumor volume was measured with a caliper and calculated according to the
formula V=a × b
2
, where “V” represents tumor volume, “a” presents the largest, and “b” the
smallest superficial diameter. All the animal experiments were approved by the IACUC
Committee of the University of Southern California.

2.10 Tumor collection and analysis
Tumor-bearing animals were sacrificed at day 60, and tumors were collected and measured as
the volume and weight. The tumor tissues were divided for (1) fixation with neutrally buffered
10% formalin for H&E staining and histological evaluation of the tumor; (2) fixation with 4%
paraformaldehyde followed by sucrose treatment for subsequent immune-staining; and (3)
snap-freezing for mRNA and protein analysis of the targeted genes.

2.11 RNA-Immunoprecipitation
2 million cells overexpressing Flag-MSI2 and si-MSI-2 were used for RIP using the MBL RIP
RNA binding protein immunoprecipitation kit. In brief, cells were washed with cold PBS and
lysed with RIP lysis buffer provided from the kit. 5 μg anti–Flag M2 Ab (Sigma-Aldrich), anti–
rabbit Ab, or anti-Msi2 Ab (EMD Millipore), which were incubated with magnetic beads, were
used to immunoprecipitate Flag–MSI2–RNA complexes. Immunoprecipitated complexes were
washed and treated with proteinase K. Finally RNA was extracted using the reagents provided
in the kit.

2.12 Quantitative Real-Time PCR (qRT-PCR)
Total RNA was isolated from the cells using Qiagen RNeasy mini kit (Qiagen, Inc., Venlo,
Netherlands) according the manufacturer's protocol and the RNA concentration was measured
using Thermo Scientific NanoDropTM Spectrophotometer. cDNA was synthesized from the
RNA templates using Random primers and 10 mM dNTPs under the following conditions- 16
o
C
16

for 30 min, 42
o
C for 30 min and 85
o
C for 5 min. Real time PCR analysis was performed on ABI
7900 HT QPCR system (Life Technologies, Carlsbad, CA) using SYBR Green QPCR Master
Mix (Stratagene) according to the manufacturers' instructions. β-actin and GAPDH was used as
endogenous reference control.

2.13 Confocal Immunofluorescence Staining
Immunofluorescence staining of cryosections or paraffin sections was performed using primary
antibodies against MSI-2 (Goat monoclonal antibody, Santa Cruz) and c-MYC (Mouse, Santa
Cruz) based on the standard protocol with their respective secondary antibodies. Slides were
mounted using the mounting media, including DAPI for nuclei counterstaining (Vector
Laboratories) according to the manufacturer’s recommendations. The staining was subjected to
morphometric analysis. To determine the specificity of immunofluorescent staining, serial
sections were similarly processed, except primary antibodies were omitted in controls.
Fluorescence images were captured on a Zeiss confocal microscope LSM510, using sequential
acquisition to give separate image files. The degree of staining was categorized by the extent
and intensity of the staining. Image analysis of nuclear translocation was performed using
Metamorph or Image J v3.91 software (http://rsb.info.nih.gov/ij). Ten high power fields were
selected for analysis of each stain. The sections were then evaluated and photographed under
a fluorescence microscope and expression of MSI-2 and c-MYC were co related.

2.14 CLIP-Sequencing and RNA Sequencing
Mouse Tumor Initiating Cells (mTICs) samples were used for both HITS-CLIP and RNA
Sequencing Analysis. HITS-CLIP involves UV-crosslinking protein, MSI-2 with the sample
followed by immunoprecipitation. This method has an advantage over RNA Sequencing based
analysis as the amount of input RNA needed for maximum yield is more.

17

2.15 Bioinformatics Analysis
Mouse mm9 genome was used to map the CLIP-Sequencing reads, using the Burrows-Wheeler
Aligner (BWA). The genome was divided into non-overlapping bins of 20bp and a sliding
window approach was used to find regions of enrichment. A read is considered to be with in a
window if the midpoint of its estimated fragment is within the window. The center of the peak will
have maximal enrichment. Zero- truncated negative binomial model (ZNTB) was used to
determine the statistically significantly enriched peaks / regions and a peak cut off of FDR
adjusted p value (p<0.05) was used. Further a Mann and Whitney U test was used determine
the differential enrichment Msi2 IP from input sample. Further ranking of peaks/ genes were
performed using Fold enrichment (>2 fold).

2.16 Statistical Analysis
Experimental data are presented as the mean ±standard deviation (SD). All statistical analysis
was performed using a two-tailed Student’s t test and Chi squared test. Differences were
considered statistically significant when P values were less than 0.05. Error bars reflect
standard errors. All graphs and statistical analysis were performed using Prism 6 software.

18

Chapter 3: RESULTS

3.1 MSI-2 and its target m-RNAs obtained from CLIP-Sequencing Analysis.

CLIP-Sequencing reads obtained for MSI-2 were aligned to mouse mm9 genome using the
Burrows-Wheeler Aligner (BWA). The data was arranged in an excel sheet (not shown), which
shows the start and stop codons, the peak summit, p-value, target site on m-RNA and the gene
symbol for the target m-RNAs. We observed that MSI-2 mainly binds to the intronic regions of
its target m-RNAs followed by their coding regions (Fig1.a). Venn diagram representing genes
sets obtained by RNA-Sequencing and CLIP-Sequencing (done previously in lab) and a large
reference genome, shortlists 12 genes as the top targets for MSI-2,which includes c-MYC
(Fig1.b). A sliding window approach which was used to find regions of enrichment within the c-
MYC m-RNA, shows maximum enrichment in the coding areas of c-MYC (Fig1.c). Enrichment
for MSI-2 was also observed in the non-coding RNA, miR-22 (Fig1.d). These findings suggest
that MSI-2 may play an important role in regulating the protein expression of c-MYC. Also, the
mouse genome, mm9 and human genome show 95% homology.

a. b.

19

c.

d.

Figure 1: MSI-2 binds to different regions of its target genes. (a) The pie diagram shows the
genomic annotations of the MSI-2 binding sites, obtained by the Burrows-Wheeler Aligner
(BWA) as compared to the whole genome analysis. According to the diagram, MSI-2 binds
mainly binds to the intronic region of its target m-RNAs followed by the coding sequence (b)
Venn diagram represents the overlap of data sets obtained by RNA-Sequencing, CLIP-
sequencing and a large reference genome. 12 gene sets overlapped between them, c-MYC
being one of them. (c) Enrichment Analysis from BWA data for MSI-2 shows enriched binding in
the exonic region of c-MYC. Red peaks shows the c-MYC coding regions while the green peaks
show the input sequences. (p<0.05 FC >1.5) (d) Enrichment analysis for MSI-2 is also observed
within non-coding RNA, miR-22.
20

3.2 MSI-2 binds the c-Myc m-RNA.

In order to determine if MSI-2 affects the c-MYC expression by specifically binding to its m-RNA,
RNA-Immunoprecipitation experiment was performed. MSI-2 overexpressing and MSI-2
silenced Huh7 cell extracts were used for the analysis. The m-RNA binding protein (MSI-2) and
the m-RNA(c-MYC) complex were isolated from the cell extracts by immunoprecipitation using
RIP certified Anti-RBP Antibodies (Fig2.a).The c-MYC m-RNA was then isolated from the
complex by guanidine hydrochloride and analyzed by RT- PCR. From the analysis in MSI-2
overexpressing Huh7 cells, we found an enrichment in the binding of MSI-2 to its target m-RNA
transcript, c-MYC. To validate if the binding was specific, RIP was also performed in MSI-2
silenced Huh7 cells and we found a reduced enrichment of c-MYC (Fig2.b). Thus, these data
suggested that MSI-2 binds to its target m-RNA, c-MYC.

a. b.

RIP-qPCR protocol

N e g a tiv e C o n tro l
M S I-2 O E
s i-M S I-2
0
1
2
3
4
m -R N A E N R IC H M E N T
21

Figure 2: MSI-2 specifically binds c-MYC m-RNA. (a) RIP was performed with anti-FLAG Ab
and Ab specific for MSI-2 in MSI-2 overexpressed and si-MSI-2 Huh-7 cells and c-MYC m-RNA
was validated using RT-qPCR (b) RT-qPCR on the RNA isolated showed enrichment for target
c-MYC in MSI-2 overexpressing cells and reduced enrichment in the si-MSI-2 Huh7 cells.
Gapdh m-RNA was used for normalization. Values represent the mean of three independent
experiments and error bars the standard deviations. Normal Rabbit Ig was used as negative
control. N=3 *p<0.05.

3.3 MSI-2 regulates c-MYC at post-transcriptional level.

To examine if MSI-2 regulates c-MYC expression at post-transcriptional level, we
overexpressed and silenced MSI-2 in human Huh7 hepatocytes by Lentiviral transduction. The
cells were harvested after 48 hours, lysed for Western Blot analysis and RNA was isolated for
RT-qPCR analysis. The overexpression of MSI-2 did not significantly increase m-RNA for c-
MYC as compared to control vector (Fig3.a). However, we observe an increase in the protein
expression for c-MYC in MSI-2 overexpressing cells and reduced expression in MSI-2 silenced
cells (Fig3.b).These data suggest that MSI-2 expression alters the translation of c-MYC.

a.

H u h 7
C o n tro l V e c to r
M S I-2 O E
s i-s c ra m b le d
s i-M S I-2
0 .0
0 .2
0 .4
0 .6
0 .8
R E L A T IV E E X P R E S S IO N
H uh7
C o n tro l V e c to r
M S I-2 O E
s i-s c ra m b le d
s i-M S I-2
*
*
22

b.

Figure 3: MSI-2 regulates c-MYC post transcriptionally in Huh7 Quantitative real time PCR
(qPCR) and Western Blot results. After lentiviral transfection of Huh7 cells with MSI-2 silencing
and overexpressing plasmids, the m-RNA and protein level of c-MYC was measured. (a) RT-
qPCR results shows that MSI-2 overexpression does not significantly alter c-MYC m-RNA level
as compared to control vector and si-MSI-2 m-RNA level. Gapdh m-RNA was used for
normalization. Values represent the mean of three independent experiments and error bars the
standard deviations, n=3 *P < 0.05 (b) Western Blot results shows an increase in c-MYC protein
expression in MSI-2 overexpressing cells whereas a decrease in protein level is seen in MSI-2
silenced cells. β-Actin was used as loading control.

3.4 MSI-2 expression increases colony formation and self-renewal of Huh7 cells.

Next, the functionality of MSI-2 in Huh7 cells were assessed by overexpressing and silencing
MSI-2 and also by silencing c-MYC in MSI-2 overexpressing cells. By Soft Agar Assay, we
observed that MSI-2 overexpressing cells showed increase in number of colonies and when c-
MYC was abrogated with si-RNA a decrease in colony number was observed.MSI-2 silenced
cells also showed reduced colony formation (Fig4.a). Next in order to check in vitro self-
renewing capacity of the cells, we performed Spheroid Assay in mTICs. The spheroid formation
in Methylcellulose was observed for a period of 90 days. After serial passages, we observed an
increase in the formation of spheroids in MSI-2 overexpressing cells, while the number of

α-MSI-2
p38

α-c-MYC
p67

β-ACTIN
p35
23

spheroids formed in si-MSI-2 and si-c-MYC cells were less (Fig4.b). Thus, MSI-2 increases the
tumor forming capacity of Huh7 cells in vitro, by regulating c-MYC expression.

a.

24

b.

Figure 4 : MSI-2 expression increases colony formation and self-renewal of Huh7 cells (a)
In vitro oncogenecity of MSI-2 was tested via colony formation, overexpressing MSI-2 in Huh7
significantly increases the formation of colonies as compared to control vector whereas
silencing MSI-2 reduces the colony formation. When c-MYC was silenced in MSI-2
overexpressing cells a significant reduction in colony formation is observed. (b) In vitro self-
renewal was tested by Spheroid Formation Assay, overexpressing MSI-2 resulted in larger
number of spheroids as compared to MSI-2 silenced Huh7 cells. Also, smaller spheroids were
observed in c-MYC silenced MSI-2 overexpressing cells. Colony formation and Spheroid
Formation data are summarized as graphical annotation, n=3 *P < 0.05.

25

3.5 MSI-2 expression increases self-renewal and tumorigenesis, in vivo.

We next studied the tumor growth and self-renewing capacity of MSI-2, in vivo. NSG mice were
subcutaneously transplanted with MSI-2 overexpressed and MSI-2 and c-MYC silenced cells
and tumor growth was monitored over a period of 60 days. An increase in tumor growth was
observed in MSI-2 overexpressing NSG mice, while tumor growth was slow in MSI-2 silenced
(Fig5.a and b) .The tumor volume was bigger in MSI-2 overexpressing mice compared to the
silenced tumor volumes. Thus, from these experiments we could conclude that MSI-2 has an
effect on tumor forming capacity of Huh7 cells and increases its self-renewing capacity, by
regulating the c-MYC expression.
a.

26

b.

Figure 5: MSI-2 expression increases self-renewal and tumorigenesis, in vivo. Image of
subcutaneous tumors on mice at day 60 post-xenograft injection (a) Tumor volume and weight
of subcutaneously injected Huh7 with MSI-2 over-expression, si-MSI-2 and si-scrambled was
measured weekly. On day 60, the mice were sacrificed and the left and right tumor was
dissected and its final weight was measured. (b) Tumor volume was monitored for a period of
60 days. *p<0.05

3.6 MSI-2 and c-MYC co-expression in HCC specimen.

We then assessed the clinical relevance of our findings. The expression of c-MYC and MSI-2
proteins were analyzed in HCC samples obtained from 10 different patients and made the
comparison to their corresponding non-cancerous liver tissues by immunofluorescence.
Immunofluorescent staining showed increased expression and co-expression of MSI-2 and c-
MYC only in cancerous tissue. The non-cancerous tissue showed minimal staining for c-MYC
and did not stain for MSI-2. (Fig.6 a and b).

s i-M S I-2
s i-s c ra m b le d
M S I-2 O E
0
1
2
3
4
5
T u m o r w e ig h (in g m s )
*
27

a. b.

Figure 6: MSI-2 and c-MYC co-expression in HCC specimen. (a) Immunofluorescent
microscope demonstrates increased expression of MSI-2 and c-MYC in HCC specimens. (b)
Number of cells stained positive for tumor specimens as compared with corresponding non-
tumorous specimen. 20X and 40X oil N=10. *p<0.05

3.7 MSI-2 can stimulate the c-MYC IRES, in vitro

In order to examine, if MSI-2 regulates c-MYC expression through its IRES, luciferase assay
was performed using plasmid construct for c-MYC (pRMF) IRES as well as the construct that
initiates cap-dependent translation of c-MYC (without c-MYC IRES) (pRF) (Fig7.a). The IRES
constructs of Apaf-1 (pRAF), BAG-1 (pRBF) proteins (other cellular IRESs) and HCV (pRHCV)
were used as specificity control. Huh7 cells were transfected with overexpressing and silencing
plasmids for MSI-2 and then transfected with IRES-containing plasmids and cells were
harvested after 48hours for performing the luciferase assay. Without MSI-2 in pRMF plasmid, no
L iv e r T u m o r
L iv e r N o n -T u m o r
0
1 0
2 0
3 0
4 0
5 0
N o .o f c e lls s ta in e d p o s itiv e / 2 0 X fie ld
28

appreciable luciferase activity was detected beyond that produced by pRF construct. However,
In the presence of MSI-2 overexpression, we observed an increase in c-MYC IRES mediated
activity whereas in si-MSI-2 the luciferase activity was reduced. The IRES related activity of N-
myc L-myc (data not shown), Apaf-1 and BAG-1 were not significant, meaning that MSI-2 binds
with different binding affinity to other Myc family IRESs and it does not show appreciable binding
to other cellular IRESs (Fig7.b). Thus, we suggested that MSI-2 maybe required for regulating
cap-independent translation of c-MYC.

a.

b.

pRMF
29

Figure 7: MSI-2 can stimulate the c-MYC IRES, in vitro. Huh7 cells were transfected with
plasmids plasmids that reduce and over-express MSI-2 and then transfected with luciferase
plasmids-pRF and pRMF. Cells were harvested after 48 hours and lysed and the luciferase
activity was assayed. (a) Schematic representation of the reporter constructs pRF and pRMF,
where pRF is the construct that does not contain c-MYC IRES and pRMF is the construct with
the c-MYC IRES. (b) The luciferase activity for pRMF when MSI-2 was silenced showed
significant reduction while a significant increase in IRES activity was observed when MSI-2 was
overexpressed. No appreciable increase in activity was observed for c-MYC without its IRES,
Apaf-1 and BAG-1 proteins.

30

Chapter 4: DISCUSSION

In this study, we studied the prognostic significance of MSI-2 in Hepatocellular Carcinoma.
Studies included understanding the post-transcriptional regulation of c-Myc by MSI-2. We also
observed how MSI-2 increases cell proliferation and self-renewal of TICs by regulating the c-
MYC expression. Using NSG mouse model, we showed an increase in tumor formation under
conditions of MSI-2 overexpression and a decrease in tumor formation under conditions of MSI-
2 and c-MYC knockdown.
Next, we try to find the mechanism MSI-2 uses to control c-MYC expression. The initiation of
translation of c-MYC can occur by cap-dependent mechanisms as well as by the internal
ribosome entry structure of the c-MYC IRES. C-MYC IRES is regulated by several trans-acting
factors. We try to elucidate if MSI-2 is one of the trans-acting factors that c-MYC uses to
increase its translation, in HCC. We show that when MSI-2 is over-expressed, an increase in c-
MYC translation by its IRES is observed, while no appreciable increase in c-MYC translation is
observed under conditions of MSI-2 knockdown. But we also need to examine if MSI-2 affects
the c-Myc transcript nuclear export. We also, show that MSI-2 binds with different affinities to
other Myc family protein-N-myc and L-myc. Thus, from these results we suggested that MSI-2
may bind to the c-MYC IRES and thereby increase its translation. However, nothing is known
yet of a mutation within the c-MYC IRES that causes binding of MSI-2.
We also show clinical significance of the regulation of c-MYC by MSI-2 in Grade 3 HCC
patients. We observe an increased expression of MSI-2 and c-MYC and also their co-
expression.
Several studies have shown that miR-22 (micro RNA) is activated by Myc transcript
22, 23
.Micro-
RNAs are known to modify and reshape the organization of the transcripts they bind to and
recruit neighborhood proteins to that complex. It would be interesting to study the interaction
between c-Myc, MSI-2 and miR-22.
Current failure with cancer treatment is not usually due to a lack of primary response or initial
induction of remission, but due to relapse or tumor recurrence after therapy, in which TICs are
thought to have crucial roles. However, targeting TIC’s have several implications such as
elimination of normal stem cells, that can lead to chronic loss of normal regeneration. The best
therapeutic strategy would be to target survival pathways used by TIC’s for self-renewal.
31

Combining agents that target the TIC’s with conventional strategies can reduce the bulk of the
tumor and target the niche. Currently there are no drugs against the Musashi family of RNA-
binding proteins. A novel high-throughput drug screen to identify inhibitors for the MSI family of
RNA binding proteins is essential.

MSI-2 binds the c-Myc m-RNA

Increased translation

Increase cell proliferation Increased in vitro and in vivo self-renewal of TICs

Tumor progression in HCC

Figure 8: Summary Diagram

MSI-2
c-Myc m-RNA
32

Chapter 5: FUTURE DIRECTIONS

Now that we know that MSI-2 positively regulates c-MYC expression in Hepatocellular
carcinoma, both in vitro and in vivo, our next step will be to find the binding sequences
for MSI-2 in the c-MYC m-RNA and mutate those sequences to reverse the binding
effect of MSI-2. Since, we show that MSI-2 affects the c-MYC IRES mediated
translation, one of the future approaches would be to detect any mutation within the c-
Myc IRES region.
We can further examine the translational regulation of c-MYC by MSI-2 by performing
Western Blot on c-MYC nuclear extracts .Also, one other group that could be included in
the tumorigenic assay, in vivo and in vitro would be knockdown of MSI-2 under
conditions of c-MYC expression. These results would give a clearer picture of the c-MYC
regulation by MSI-2.
As immunofluorescence was performed only for 10 different tissue samples, it would be
essential to perform staining on tissue array sample slides that contains tissues of
different clinical and pathological grades of liver cancer. This can provide a better
understanding of the co-expression of MSI-2 and c-MYC in different tissue grade
samples and it will also help establish the clinical significance of this regulation in HCC
specimens.
Since the gene enrichment analysis for MSI-2, shows enrichment for a gene, Tmtc2,
which has sequence that overlaps with micro-RNA, miR-22, future experiment would be
to perform RT-qPCR for miR-22, when MSI-2 is overexpressed and knockdown in Huh7
cells. If a significance difference is observed in its expression, it could mean that MSI-2
also regulates the non-coding RNA, miR-22. And based on these results, we would
prepare a hypothetical model to show the interaction between c-MYC, MSI-2 and miR-
22.

33

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Abstract (if available)

Abstract RNA-binding protein MSI-2 has been shown to be elevated in several cancers types such as leukemia, hepatocellular carcinoma (HCC) and breast and has been linked with poor prognosis. In Mixed Myeloid Leukemia (MLL), MSI-2 maintains efficient translation of the oncogene c-MYC and maintains MLL self-renewal activity. In this study, we sought to elucidate the role of MSI-2 in regulating the expression of proto-oncogenes in HCC and thereby determine the prognostic significance of MSI-2 and proto-oncogenes in HCC. We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). Among the MSI2-bound RNA, c-MYC mRNA binding was confirmed by RIP-qPCR analysis. MSI-2 does not have any effect on level of c-MYC m-RNA using qRT-PCR and immunoblots, but significantly increases the c-MYC protein levels, indicating that MSI-2 promotes c-MYC protein in the post-transcriptional levels. Overexpression of MSI-2 promotes self-renewal and tumor-initiation property (tumor formation) in xenograft tumor mouse models while silencing MSI2 reduces them. Immunohistochemical analysis were also performed. Overexpression of MSI2 promotes internal ribosome entry site (IRES)-dependent mechanisms. In conclusion, we demonstrate that MSI-2 promotes liver tumorigenesis by maintaining the c-MYC expression. Both MSI-2 and c-MYC may be useful prognostic factors of HCC and MSI-2 targeted therapy may be beneficial in treatment of HCC patients.

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Asset Metadata

Creator Narayanan, Padmini (author)

Core Title Musashi-2 promotes c-MYC expression through IRES-dependent translation and self-renewal ability in hepatocellular carcinoma

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 07/24/2015

Defense Date 05/26/2015

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

Tag c-MYC,hepatocellular carcinoma,IRES,MSI-2,OAI-PMH Harvest,self-renewal

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Advisor Machida, Keigo (committee chair), Gao, Shou-Jiang (committee member), Stiles, Bangyan L. (committee member)

Creator Email padminin@usc.edu,padumini1207@gmail.com

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