UNIVERSITY OF CALGARY by

UNIVERSITY OF CALGARY
Roles of the ING1 Epigenetic Regulator in Breast Cancer
by
Satbir Singh Thakur
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
GRADUATE PROGRAM IN BIOCHEMISTRY AND MOLECULAR BIOLOGY
CALGARY, ALBERTA
OCTOBER, 2014
© Satbir Singh Thakur 2014

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Abstract
ING proteins are epigenetic “readers” that can target various chromatin modifying
complexes to chromatin. They are involved in various cellular processes such as DNA
repair, apoptosis and cellular senescence. This study focuses on examining the potential
role of ING1 as a therapeutic agent and prognostic marker for breast cancer.
We began by asking whether dysregulating epigenetic pathways with different
chemical inhibitors could show synergistic effects with ING1 on killing cancer cells. We
tested whether ING1 could synergize better with chemotherapeutics that target the same
epigenetic mechanism or a different epigenetic mechanism. Combination treatment of
ING1b with LBH589 (HDAC inhibitor) showed synergy, but the combination of ING1b
with 5azaC (DNMT inhibitor), thus targeting two distinct epigenetic mechanisms, was
more effective. Adenoviral delivery of ING1b combined with 5azaC also inhibited cancer
cell growth in a xenograft model and led to tumor regression. These data showed that
targeting distinct epigenetic pathways in our model was more effective in blocking cancer
cell line growth than targeting the same pathway with multiple agents.
Since ING1 expression is frequently repressed in breast carcinomas, but its
mechanistic role in breast cancer development and metastasis was unknown, we analyzed
ING1 levels in patient samples and correlated it to patient outcome. We also studied the
effects of altering ING1 levels in metastasis assays in vitro and mouse metastasis model
in vivo. ING1 levels were lower in tumors compared to adjacent normal breast tissue and
correlated with tumor size and distant recurrence. ING1 could also predict disease-
specific and distant metastasis-free survival in these patients. Decreasing levels of ING1

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increased, and increasing levels decreased migration and invasion of MDA-MB231 cells
in vitro. ING1 overexpression also blocked cancer cell metastasis in vivo and eliminated
tumor-induced mortality in mouse models.
Lastly, we determined if ING1 expression could predict breast cancer patient
outcome. We found that stromal cell expression of ING1 showed an inverse correlation
with patient survival. ING1 also correlated with tumor grade in these patients and
multivariate analysis showed that ING1 was an independent prognostic marker in the
breast cancer cohort we tested. This study provides important pre-clinical data that could
help establish ING1 as a prognostic and therapeutic agent for breast cancer.

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Acknowledgements
I would like to express my sincere gratitude to my supervisor and mentor, Dr. Karl
Riabowol for providing me with the opportunity to pursue my doctoral studies under his
guidance. His constant support, encouragement and belief in me have been the major
reasons that have brought this project to its fruition. The freedom Dr. Riabowol provided,
to try my own ideas in his laboratory has taught me to think independently and bravely
face difficulties.
I would also like to thank my committee members Dr. Aru Narendran and Dr.
Olga Kovalchuk for their support and guidance throughout the program. Their valuable
advice and suggestions were really helpful to the work I did. I would also like to thank
Dr. Oliver Bathe and Dr. Samuel Benchimol for agreeing to serve as examiners on my
thesis and Dr. William Brook for serving as neutral chair.
This work would not have been possible without the support of various
collaborations that I had during my stay in the department. I would like to thank Dr. Don
Fujita, Dr. Frank Jirik, Dr. Don Morris, Dr. Joe Dort, Dr. Tony Magliocco and Dr. Alex
Klimowicz for collaborating with us and providing reagents and suggestions for
experiments. Special thanks to Dr. Manoj Mishra and Dr. Arvind Singla for their
guidance regarding experiments and life in general.
Past and present members of the Riabowol lab have been instrumental in making
this work possible. I would like to thank Dr. Pinaki Bose for his guidance and support
during the early days in the lab. I would also like to thank Annie and Donna for the help

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regarding reagents and managing the lab. Thanks to Arash, Mahsa, Yang and Alex for
their friendship and support in the lab.
Staying in Calgary would not have been possible without my friends. Thanks to
Abhishek, Saurav, Soumya, Kunal, Ranjan, Manoj, Sabarish and Sarvan for the good
time and I look forward to a lifetime of friendship and working together.
I would like to acknowledge financial support from the Alberta Cancer
Foundation for the studentship they provided during my doctoral study.
Special thanks to my Mom and my Sister for their constant care, encouragement
and support throughout my life. Thanks for listening to me and supporting me during my
frustrations and difficulties. Lastly, and most importantly, thanks to my Master without
whom I hold no existence.