Investigating the roles of p63 and p73 isoforms to therapeutically treat p53-altered cancers

224 Pages·2016·10.88 MB·English

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The University of Texas MD Anderson Cancer
The University of Texas MD Anderson Cancer
Center UTHealth Graduate School of
Center UTHealth Graduate School of
Biomedical Sciences Dissertations and Theses
Biomedical Sciences
(Open Access)
5-2015
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Avinashnarayan Venkatanarayan
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Venkatanarayan, Avinashnarayan, "Investigating the roles of p63 and p73 isoforms to therapeutically treat
p53-altered cancers" (2015). The University of Texas MD Anderson Cancer Center UTHealth Graduate
School of Biomedical Sciences Dissertations and Theses (Open Access). 576.
https://digitalcommons.library.tmc.edu/utgsbs_dissertations/576
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INVESTIGATING THE ROLES OF P63 AND P73 ISOFORMS TO
THERAPEUTICALLY TREAT P53-ALTERED CANCERS
By
Avinashnarayan Venkatanarayan, M.S.

APPROVED:

______________________________
Elsa R. Flores, Ph.D.
Advisory Professor

______________________________
Mong Hong Lee, Ph.D.

______________________________
Michael J. Galko, Ph.D.

______________________________
Kenneth Y. Tsai, M.D., Ph.D.

______________________________
Sendurai Mani, Ph.D.

______________________________
Preethi H. Gunaratne, Ph.D.

APPROVED:

____________________________
Dean, The University of Texas
Graduate School of Biomedical Sciences at Houston
INVESTIGATING THE ROLES OF P63 AND P73 ISOFORMS TO
THERAPEUTICALLY TREAT P53-ALTERED CANCERS

A
DISSERTATION

Presented to the Faculty of
The University of Texas
Health Science Center at Houston
and
The University of Texas
MD Anderson Cancer Center
Graduate School of Biomedical Sciences
in Partial Fulfillment
of the Requirements
for the Degree of

DOCTOR OF PHILOSOPHY

By
Avinashnarayan Venkatanarayan, M.S.
Houston, Texas
May, 2015
Dedication
To my grandmothers Janakavalli A & Shanthakumari P who have
been a tremendous source of will power, dedication and humility

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Acknowledgements
My training and success in the graduate school would have not been possible
without the support, mentorship and blessings of many people.
First and foremost, I would like to thank God, the Almighty for giving me the
energy and health to complete this endeavor successfully.
I would like to thank my mentor, Dr. Elsa Flores for accepting me as a
graduate student in her laboratory; training me and importantly helping me achieve
my goals. Dr. Flores has not only been a great mentor, but also a role model from
whom I often draw inspiration, sometimes as my friend with whom I tend to chat and
sometimes as my scientific mom for the care and affection she has provided me. I
learnt to not only appreciate and perform science but more importantly the purpose
we serve as scientists and the hope we give to the people by advancing cancer
research.
I am also fortunate to have the support and guidance of an excellent
committee. Dr. Mong Hong Lee, Dr. Michael Galko, Dr. Sendurai Mani, Dr. Kenneth
Tsai and Dr. Preethi Gunaratne have been excellent mentors and always provided
me with constructive suggestions and helped me progress in my scientific career. I
also wanted to to acknowledge three people, Dr. Elsa Flores, Dr. Michael Galko and
Dr. Andreas Bergmann who offered me rotation positions in there labs and made my
dream of pursuing a PhD program a reality.
Also, I had the support and guidance of a small but highly talented group of
people in the lab. I would like to thank all the present and former members of the
Flores Lab. I would like to thank Dr. Min Soon Cho for helping me learn the basics
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when I was a rotation student. I had lot of help with the mouse work from Young Jin
Gi, Lingzhi Liu and Payal Raulji. Dr. Xiaohua Su is an expert in our lab and she has
always been my go to person. Dr. Marco Napoli has been a good friend and
excellent person to bounce ideas from him. Marlese Pisegna has been our always-
positive lab manager and my bench buddy. She has provided me with moral support
and she was always there to share with me my failures and success. And finally, I
would like to acknowledge my fellow graduate students Dr. Ramon Flores Gonzalez,
Andrew Davis, Ngoc Bui and Sarah Wu. I think we started as lab mates and now we
have become friends for life. Our repeated discussions on performing and improving
experiments have made our lab a happening place.
I would also like to thank and acknowledge the rotation and summer students
who worked closely with me. Particularly, I would like to thank Pruthali Kulkarni and
Eliot F. Sananikone for their motivation and enthusiasm while performing
experiments in the lab. I would also like to thank my collaborators, Dr. William
Norton (Veterinary Medicine) for his help with the mouse surgeries. Dr. James
Bankson and members of the Small Animal Imaging Facility (SAIF) for their help with
mouse imaging. Dr. Cristian Coarfa for his help with Bioinformatic analysis and Dr.
Anil Sood, Dr. Mangala Selenere and Aguayo Cristian for their help with the
liposomal siRNA particles.
My success in graduate school is also greatly attributed to help and guidance
from Dr. Abinav Jain and Dr. Srikanth Appikonda (Barton Lab), Dr. James Jackson
and Dr. Vinod Pant (Lozano Lab) and Dr. Parameshwar Djardiane. We have
become good friends over the last few years and I really cherish the time I spent with
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all of them. I would also like to thank Elisabeth Lindheim, the Genes and
Development program coordinator for her help and support for the last 6 years.
Also, I would like to thank and acknowledge my family for their unconditional
love and support during my training as a graduate student. My parents Mr. A.
Venkatanarayan and Mrs. V. Ashalatha have always given me the freedom to
pursue my interests and never doubted my actions. My brother Dr. Ajay
Venkatanarayan, in many ways made sacrifices on my behalf so that I could
complete my graduate school, by supporting my parents and being self-less. I also
would like to thank my uncle Dr. A. Srinivasan who has always reminded me that
hard work leads to success. Finally, I would like to thank my fiancée Ms. Amritha
Sathish for her love, support and positive attitude. She has been highly
understanding of my busy schedule and helped me stay focused.
Finally, I would like to thank my friends who have been my pillars of strength
throughout my graduate school. My friends have become like my family and I am
excited to see what the future holds for each of us. I would like to thank Dr. Rajesha
Rupaimoole my friend and roommate for the past 8 years. Rajesha and myself have
a special understanding and we both have taken similar steps in our life at the same
time. Also, I would like to thank Dr. Anantha and Anil Chukkapalli, Dr. Zeynep and
Dr. Kadir Akdemir, Uphanya and Dr. Sridhar Eshwaran for their love, friendship and
support. Also, a special thanks to Dr. Shankar Venugopalan and my buddies Bharat
Chaganty and Dr. Kalyan Nallaparaju for always giving the best and positive advice.

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Investigating the roles of p63 & p73 isoforms to therapeutically treat
p53-altered cancers
Avinashnarayan Venkatanarayan, M.S.
Supervisory Professor: Elsa R. Flores, Ph.D.
The TP53 tumor suppressor is mutated in approximately 50% of human
cancers rendering cancer therapies ineffective. p53 reactivation suppresses tumor
formation in mice. However, this strategy has proven difficult to implement
therapeutically. An alternate approach to overcome p53 loss is to manipulate the
p53-family members, p63 and p73, which interact and share structural similarities to
p53. p63 and p73, unlike p53 are less frequently mutated and have two major
isoforms with distinct functions which makes them unique targets for therapeutic
intervention. The full-length acidic transactivation (TA) isoforms of p63 and p73
function similar to p53. While the deltaN (ΔN) isoforms of p63 and p73, which lack
the acidic transactivation domain, are overexpressed in cancers and function in a
dominant negative manner against p53, TAp63 and TAp73. As result of the
opposing isoform-specific function, the roles of p63 and p73 in tumorigenesis
requires further characterization. In an attempt to identify novel therapeutic
approaches to treat p53-altered cancers by utilizing the p53-family members, I aim
to delineate the roles of ΔN isoforms of p63 and p73 in tumorigenesis.
I have demonstrated that deletion of ΔNp63 or ΔNp73 in p53-deficient tumors
mediates tumor regression through the upregulation of tumor suppressive isoforms,
TAp63 and TAp73. Upon loss of ΔNp63 or ΔNp73, TAp63 and TAp73 activate IAPP
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a metabolic regulator, which induces metabolic reprogramming resulting in tumor
regression in p53-deficient mice. I have shown that IAPP, which encodes amylin, a
37-amino acid peptide functions as a tumor suppressor in p53-deficient cancers.
IAPP functions through the calcitonin and RAMP3 receptors to limit glucose uptake
and reduce glycolysis in the cancer cells resulting in ROS accumulation and
apoptosis. Additionally, I have also shown that use of Pramlintide, a synthetic analog
of IAPP, mediates tumor regression in p53-deficient mice and apoptosis in multiple
p53-mutant human cancer cell lines.
Further, to therapeutically treat p53-deficient cancers in vivo, liposomal
nanoparticle siRNA’s targeting ΔNp63 and ΔNp73 were administered into p53-
deficient mouse thymic lymphomas, which resulted in tumor regression. Taken
together, my work has defined the isoform specific functions of p63 and p73 in
tumorigenesis. Importantly, I have also demonstrated the use of pramlintide, a
diabetic drug to treat p53-altered cancers. Thus, by understanding the interplay
among the p53-family members, novel therapeutic methods could be designed to
treat p53-altered human cancers.

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Table of Contents
Approvals ................................................................................................................ i
Title ........................................................................................................................ ii
Dedication ............................................................................................................. iii
Acknowledgements ............................................................................................... iv
Abstract ................................................................................................................ vii
Table of Contents .................................................................................................. ix
List of Figures........................................................................................................ xi
List of Tables ........................................................................................................ xv
List of Appendices ............................................................................................... xvi
Chapter 1: Introduction ......................................................................................... 1
Chapter 2: Material and Methods ........................................................................ 26
Chapter 3: ΔNp63 and ΔNp73 function as oncogenes in a p53-deficient
model of thymic lymphoma ................................................................ 43
Chapter 4: IAPP driven metabolic reprogramming induces regression of
p53-deficient tumors in vivo ............................................................. 71
Chapter 5: IAPP functions through the calcitonin and RAMP3 receptors to
suppress tumorigenesis ..................................................................... 98
Chapter 6: Therapeutic approaches to treat p53-mutated human cancers ...... 105
Chapter 7: Therapeutically targeting the oncogenic isoforms of p63 and p73
to treat p53-deficient tumors ........................................................... 123
Chapter 8: Investigating the roles of p63 and p73 regulated lncRNAs upon
DNA damage and in p53-deficient tumors ...................................... 130
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