MiR-23b/-27b Cluster Suppresses the Metastatic Phenotype of Aggressive Prostate Cancer Cells

105 Pages·2015·3.05 MB·English

University of Miami
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2013-08-23
MiR-23b/-27b Cluster Suppresses the Metastatic
Phenotype of Aggressive Prostate Cancer Cells
Reema A. Ishteiwy
University of Miami, [email protected]
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UNIVERSITY OF MIAMI

MIR-23B/-27B CLUSTER SUPPRESSES THE METASTATIC PHENOTYPE OF
AGGRESSIVE PROSTATE CANCER CELLS

By

Reema A. Ishteiwy

A DISSERTATION

Submitted to the Faculty
of the University of Miami
in partial fulfillment of the requirements for
the degree of Doctor of Philosophy

Coral Gables, Florida

December 2013
©2013
Reema A. Ishteiwy
All Rights Reserved
UNIVERSITY OF MIAMI

A dissertation submitted in partial fulfillment of
the requirements for the degree of
Doctor of Philosophy

MIR-23B/-27B CLUSTER SUPPRESSES THE METASTATIC PHENOTYPE OF
AGGRESSIVE PROSTATE CANCER CELLS

Reema A. Ishteiwy

Approved:

________________ _________________
Mary Lou King, Ph.D. Kerry Burnstein, Ph.D.
Professor of Cell Biology Professor of Molecular
and Anatomy and Cellular Pharmacology

________________ _________________
Derek Dykxhoorn, Ph.D. Pedro Salas, Ph.D.
Assistant Professor of Professor of Cell Biology
Human Genetics and Anatomy

________________ ________________
Yehia Daaka, Ph.D. M. Brian Blake, Ph.D.
Professor of Urology Dean of the
University of Florida Graduate School
ISHTEIWY, REEMA A. (Ph.D., Molecular and Cellular Pharmacology)
MiR-23b/-27b Cluster Suppresses the (December 2013)
Metastatic Phenotype of Aggressive
Prostate Cancer Cells

Abstract of a dissertation at the University of Miami.

Dissertation supervised by Professor Kerry L. Burnstein.
No. of pages in text. (93)

MicroRNAs (miRs) are small, endogenous, non-coding RNAs that regulate
gene expressing by inhibiting the stability and/or translation of complementary
mRNA targets. Expression of miR-23b/-27b, which are encoded in tandem as a
cluster on chromosome 9, are specifically downregulated in metastatic prostate
cancer tissue samples compared to benign prostate. MiR-23b/-27b levels are
also lower in aggressive prostate cancer cell lines compared to more indolent
cells. We show miR-23b/-27b suppress migration and invasion of two aggressive
human prostate cancer cell lines. Furthermore, inhibition of miR-23b/-27b
increases the migration and invasion potential of a less aggressive prostate
cancer cell line. Interestingly, we found that the miR-23b/-27b cluster does not
affect prostate cancer cell proliferation suggesting miR-23b/-27b are metastatic
suppressors. We provide evidence that miR-23b/-27b suppress additional key
tumorigenic processes. To understand the mechanism of miR-23b/-27b
mediated suppression of metastatic cell processes, we examined the effects of
miR-23b/-27b on Rac1, a Rho GTPase essential for the metastatic phenotype of
prostate cancer cells. We show ectopic expression of miR-23b/-27b decrease
Rac1 activity of aggressive prostate cancer cell lines while inhibition of these
miRs increase Rac1 activity in a less aggressive prostate cancer cell line. In
addition, we show that expression of miR-23b/-27b increase the cell adhesion
protein E-cadherin, in two independent aggressive prostate cancer cell lines.
Conversely inhibition of miR-23b/-27b in a less aggressive prostate cancer cell
line decreases E-cadherin levels.
These data establish an important role for miR-23b/-27b mediated-
suppression of invasion and migration of aggressive prostate cancer cells. Since
miRs have great potential use as biomarkers and drug targets or mimics,
elucidating the role and mechanism of miR-23b/-27b in prostate cancer is critical
in the development of novel and effective therapies and prognostic indicators.
TABLE OF CONTENTS

Page
LIST OF FIGURES........................................................................................... v
ABBREVIATIONS…………………………………………………………………….viii

Chapter

1 INTRODUCTION ..................................................................................... 1
MicroRNA biogenesis and mechanism of action ...………….……………. 1
MicroRNA and Cancer ….………………………………..………………….. 4
Prostate Cancer and Metastasis ...………………………………………… 8
MiR-23b/-27b in Prostate Cancer………………………………………….. 12
Statement of purpose ...…………………………………………………….. 13

2 MATERIALS AND METHODS .............................................................. 14
Cell Culture ……..………………………………………………………..…. 14
RNA Isolation and Quantitative RT-PCR ………………………………….15
Immunoblotting ..………………...…………………………………………. 16
Plasmids and Lentiviral Production ………………………………………. 16
Flow Cytometry …………………………………………………………….. 18
Cell Transfections ………………………………………………………….. 18
Microarray …………………………………………………………………... 19
Cell Proliferation Assays ……………………………………………………19
Scratch Assays ………………………………………………………………20
Boyden Chamber and Matrigel Invasion Assays ………………………. .20
Rac1 Activity Assays…………………………………………………….…. 21
Soft Agar Assays……………………………………………………………. 22
Zymography Assays………………………………………………………… 22

3 RESULTS ............................................................................................... 23
MiR-23b/-27b suppresses motility, invasion and anchorage-independent
growth of aggressive prostate cancer cells ………………………………. 23
MiR-23b/-27b regulate E-cadherin and Rac1 activity……………………. 30
Identifying the gene targets of miR-23b/-27b in prostate
cancer………………………………………………………………………….38
HIP1R is a target of miR-23b/-27b in prostate cancer cells…………….. 41
Ectopic expression of HIP1R increases migration and invasion potential of
LNCaP cells without affecting proliferation……………………………….. 43
Knockdown of HIP1R decreases the migration and invasion potential of
aggressive prostate cancer cells…………………………………………... 45
iii
HIP1R rescues miR-23b/-27b mediated suppression of migration of
prostate cancer cells……………………………………………………..…. 51
Knockdown of HIP1R results in increased E-cadherin ………………... 53
Depletion of HIP1R decreases Rac1 activity, but not total Rac1
levels in ALVA31 cells…………………………….……………………..…..54
Ectopic expression of miR-23b, but not miR-27b or miR-23b/-27b
decrease cell proliferation ………………………………………………... 55

4 DISCUSSION ........................................................................................ 59
The role of miR-23b/-27b-mediated suppression of
metastatic phenotype of prostate cancer cells ………………………...... 59
Mechanism of miR-23b/-27b mediated suppression effects
may involve Rac1 activity and E-cadherin …………………………….... 61
Discovery of a novel target of miR-23b/-27b, HIP1R and its role
in prostate cancer cell migration and invasion …………………...…...… 66
Clinical applications for miR-23b/-27b in cancer prevention
and therapy ……………………………………….....…………………..... 69

Appendices………………………………………………………………………….. 72
References………… ........................................................................................ 81

iv
List of Figures

Chapter I
Figure 1. The biogenesis and mechanism of action of microRNA …. 4

Figure 2. The metastatic cascade…………………………………….….9

Chapter III
Figure 3. Assessment of transduction efficiency of ALVA31 and
PC3-ML cells………………………………..…………………24

Figure 4. MiR-27b expression levels in LNCaP cells transfected
with antagomiR-23b plus 27b…………………...……….. 25

Figure 5. MiR-23b/-27b expression decreases prostate cancer cell
migration while inhibition of miR-23b/-27b increases
migration of less aggressive prostate cancer cells……… 26

Figure 6. MiR-23b/-27b expression decreases the invasiveness of
prostate cancer cells while inhibition of miR-23b/-27b in
LNCaP cells increases invasiveness…………………...… 27

Figure 7. MiR-23b/-27b decreases anchorage-independent growth
of aggressive prostate cancer cell lines…………………. 29

Figure 8. MiR-23b/-27b does not regulate prostate cancer cell
proliferation………………………………………………….... 30

Figure 9. MiR-23b/-27b significantly increases E-cadherin
expression in aggressive prostate cancer cell
lines……………………………………………………………..31

Figure 10. MiR-23b/-27b increases E-cadherin mRNA levels in
aggressive prostate cancer cell lines……………………… 32

Figure 11. MiR-23b/-27b do not affect mRNA levels of Snail, an E-
cadhe3rin transcriptional repressor in prostate
cancer cells ……………………………………………….. 33

Figure 12. MiR-23b/-27b do not alter transcriptional activity of the
3’UTR of Zeb1 or Zeb2 in prostate cancer………………. 33

Figure 13. MiR-23b/-27b significantly decrease Rac1 activity but
do not alter total Rac1 levels in aggressive prostate
cancer cell lines…………………………………………….. 35
v
Figure 14. Inhibition of miR-23b/27b in LNCaP cells increases Rac1
activity, but not total Rac1 levels……………………………. 36

Figure 15. MiR-23b/-27b do not alter MMP13, MMP2 and MMP9 in
prostate cancer cell lines………………………………….. 36

Figure 16. MiR-23b/-27b decreased the level of actin based
protrusions of ALVA31 cells……………………………….. 37

Figure 17. MiR-23b levels in PC3-ML cells overexpressing
miR-23b/-27b and LNCaP cells used for microarray……. 40

Figure 18. MiR-23b/-27b regulated genes linked to metastatic
processes………………………………………………..……. 41

Figure 19. MiR-23b/-27b regulate HIP1R in prostate cancer cells…. 42

Figure 20. MiR-23b/-27b do not regulate the 3’UTR of HIP1R in
prostate cancer cells………………………………………….43

Figure 21. Ectopic expression of HIP1R increased the invasion and
migration potential of the poorly aggressive prostate
cancer cell line LNCaP……………………………………… 44

Figure 22. Knockdown of HIP1R, a miR-23b/-27b target,
decreased the invasion and migration potential of
ALVA31 cells…………………………………..…………….. .46

Figure 23. Knockdown of HIP1R, a miR-23b/-27b target, decreased
the invasion and migration potential of PC3-ML
cells…………………………………………………………… 48

Figure 24. Knockdown of HIP1R in prostate cancer cell lines is
specific …………………………………………………...… 49

Figure 25. HIP1R rescues miR-23b/-27b mediated suppression of
migration of prostate cancer cells……………………...… 52

Figure 26. Knockdown of HIP1R increased E-cadherin levels in
prostate cancer cells……………………………………… 54

Figure 27. Knockdown of HIP1R decrease Rac1 activity but not
total Rac1 levels in prostate cancer cells……………… 55

vi