When I first arrived at MD Anderson Cancer Center, I knew relatively little about the complexities and dynamics of epigenetics. However, by the end of my research internship, I can safely say that I feel confident talking about this topic and my work. Though my project is far from complete, I know that I made strides towards answering some interesting questions about cancer biology.
I was able to successfully utilize gene editing methodology to analyze the levels of various genes, including vital tumor suppressor genes that are lost in pancreatic ductal adenocarcinoma (PDAC), that are targeted by enhancer of zeste homolog 2 (EZH2). By successfully knocking out EZH2, a histone methyltransferase involved in gene repression, using CRISPR-Cas 9 constructs, I was able to visualize an upregulation in expression of its targets, which was one of the goals of my project this summer. However, this was not the only goal of this summer. I also learned about how answering one small question in biology can lead to a myriad of more advanced, complicated questions. This other goal was to keep on questioning and knowing that a ten-week project is only just the beginning of this cascade of “what-ifs.”
The findings of my work suggest that the polycomb protein EZH2 could be a therapeutic target in PDAC, especially in samples with MLL3 (mixed lineage leukemia 3, involved in a complex of proteins that facilitate gene activation) inactivating mutations. A better understanding of the molecular mechanisms by which EZH2 functions will be necessary for the development of specific EZH2 methyltransferase inhibitors to combat pancreatic cancer.
These past ten weeks have been extremely vital in reaffirming my belief that there are many individuals who are dedicated to STEM. I feel very fortunate to have been able to spend my summer learning and working with such a talented and dedicated team. The opportunity to work with these scientists, side by side, deeply enhanced my thinking about my current and future academic, professional, and personal endeavors.
Academically, learning about the epigenetics underlying pancreatic cancer has augmented by commitment to cancer biology research. The time spent reading journal articles and attending seminars opened my eyes to what good science is truly about. Professionally, this experience has grounded me. I am critical and cognizant of the rigors of graduate school. Everyone in the lab was extremely hard working, and I aspire to embody that same attitude towards my work moving forward. And finally, personally, I think this summer experience was a good reminder that sometimes the problems we are trying to solve are literally right outside our doors. I want to embrace the philosophy behind bench-to-bedside research because I believe that our ultimate goal in all biomedical research is to help the patients. I hope to continue researching interesting problems in the future in order to find cures.
As I transition from this summer of intensive wet-lab work to integrating wet and dry-lab work in a systems biology lab (computationally modeling complex and dynamic biological systems), I hope to take the analytical skills I gained within the Maitra and Gupta lab and combine them with my budding computational skills to think about the emerging systems problems that are central to the areas of biology and disease.
On my morning commute to the lab a few weeks back, I saw a patient strolling slowly by the front of the main hospital. She took a moment to smell one of the roses that was in full bloom right outside the main door. She had on a hot pink bandanna that matched this color of the rose so perfectly. It was unfortunate for me to see the flowers completely demolished by bulldozers the following week. This infinitesimal moment is something I think about constantly, especially now.
My knockdown experiment was unsuccessful – it only showed about 40% knockdown efficiency. This was not nearly enough for me to continue culturing my cells and running subsequent experiments on EZH2 target genes. It was devastating to find out after multiple weeks of agonizing over what could have possible gone wrong. We personally think that it has something to do with the vector we used, which subsequently led to insufficient transfection and downstream problems. Because of this setback, I am not nearly as far along in my project as I would have hoped. I restarted transfecting the KMC mouse cell line with CRISPR-CAS9 constructs of EZH2 in the hope that this slightly different approach will be more successful, in this case, in knocking out EZH2. I will also be pursuing the knockdown again but with some modifications to the protocol.
Being at this institution and meeting many graduate students this summer has made me think about the prospects of graduate school as well. Alas, this too has made me feel unequivocally terrified. As a first generation college student, I, along with many others I have spoken with in the same situation, constantly feel as though we do not have a structured support system from our family to figure out how the whole “college and graduate school thing” works. Thankfully, I was met with kind support and advice from current students within UT-Health and MD Andersons’ Graduate School of Biomedical Sciences (GSBS) and Dr. Melinda Yates. They run a First Generation Students Organization to talk about these concerns, flesh out solutions, and share stories. One of the first things I was told was that you should never feel like you are alone in this process. There are others before you that have gone through the application process and the five to seven years of doctoral work. They will guide you when no one else can. Current students shared their diverse paths to getting to graduate school, including applying multiple times, taking gap years to pursue related and unrelated passions, post-baccalaureate programs, entrepreneurial endeavors, supportive mentorship from employers and principal investigators, and persistence through every single moment when someone told them they would not be able to make it through. Every fear I have had is a fear that every person in that room shared and they were still able to achieve exactly what they wanted, meaning that any first generation student is capable of doing the same.
In the face of adversity, I have to remember that the flowers in front of the main hospital will eventually bloom again, just as promising results will eventually come from my project, and just as I will be able to pursue science like everyone else here has. There are always going to be setbacks in one’s health, studies, or educational endeavors. However, to overcome these obstacles, you must optimize, think critically, and practice again and again. It is about resilience. I have to remember that the little moments are what should matter most, like this patient smelling the flowers. I appreciate the techniques I have learned, the science that I am learning, the papers I have read, the seminars I have attended, and the budding scientists I have had the pleasure of interacting with so far.
One of the first things Dr. Ronald A. DePinho, the institution’s president, told me when I arrived at The University of Texas MD Anderson Cancer Center was that everyone here eats, sleeps, and breathes cancer research. Every person is reading journal articles, treating patients, attending seminars, leading discussions, and ultimately trying to find a cure. Every moment we spend at the cancer center allows the center to dictate what we think or talk about and what we want to define us. The macro-environment at MD Anderson is extremely impactful in cancer biology and oncology because the institution is devoted to the integration of basic science research, technology, and education. In my first few weeks here, I have been able to analyze many different dimensions of biological space because, in essence, I have become like everyone else at the institution: devoted to cancer abolition.
Since arriving on June 5th, even the lab I am working in has stressed the institution’s goals, among their many other goals. The joint lab of Dr. Anirban Maitra and Dr. Sonal Gupta puts an emphasis on educating students because the lab believes that young scientists will be the ones to eventually find a cure for pancreatic cancer. Thus, one of the things I was most awestruck by was the number of individuals working in the lab. The individuals ranged from visiting professors from other institutions, postdoctoral fellows, graduate students, and research technicians, to other summer trainees like myself. They were students of all ages in this macro-environment ready to learn from each other.
The Maitra lab has over ten ongoing projects and a myriad of instruments that serve to expedite data generation, collection, and analysis. The access to this type of equipment, I learned, gives the lab the ability to make meaningful conclusions at a rapid pace. However, most of their projects boil down to answering one overarching question: what drives epigenetic dysregulation in pancreatic cancer, and how can we combat it? Finding an answer to this question is extremely important because pancreatic cancer is the fourth-leading cause of death in the United States.
My project for the summer is to develop an answer to an aspect of this question. I am attempting to understand the significance of enhancer of zeste homolog 2 (EZH2) in pancreatic cancer. EZH2 is the catalytic subunit of polycomb repressive complex 2 (PRC2), and it functions to maintain transcriptional repression of certain genes. In early development, EZH2 is involved in repressing certain genes to allow differentiation to occur. This is the only time when large amounts of EZH2 are present in cells. Later on, in healthy adults that are fully developed, EZH2 levels are kept low. However, in many cancer types, EZH2 becomes overexpressed, meaning that it is repressing the transcription of many genes (such as tumor suppressor genes) abnormally, leading to increased proliferation and tumorigenesis. EZH2 does this without changing nucleotide sequences. In fact, this is what cancer epigenetics is all about – the modification of histones. More specifically, EZH2 can methylate, a type of histone modification, which can be integral in repressing the function of a gene. We have yet to understand by what and how EZH2 is regulated in cancer.
I am in the process of knocking down EZH2 using shRNA in a specific pancreatic cancer cell line (KMC cells which have mutant Kras and MLL3 deletion). I transformed E. Coli to incorporate foreign DNA into their genome, this foreign DNA being a vector containing the shRNA of interest. I allowed the E. Coli to produce plasmid. I then transfected competent 293T-cells, meaning that I allowed these mammalian cells uptake the plasmid. These cells produced lentiviral clones. I finally transduced the KMC cell line with the lentiviral clones in order to incorporate the shRNA into this line so that EZH2 expression is knocked down. I am currently in the process of performing qRT-PCR and immunoblotting to determine whether I was successfully able to knockdown EZH2 in these cells. If so, I will proceed forward in determining how this knockdown of EZH2 affects the expression of its target genes in KMC cells. I expect to eventually follow up with knockdowns in two other pancreatic cancer cell lines and knockouts of EZH2 using CRISPR-Cas9. The data I collect will hopefully corroborate functional assays done previously showing that if you inhibit EZH2, you will be able to obstruct pancreatic cancer cell growth, which has huge implications in translational medicine.
The techniques I have mastered and the concepts I have learned so far would not have been possible without the help and guidance of scientists in the lab. Therefore, one thing I understand now is that scientific research is more than just about generating data. Scientific research is about educating those around you devoted to the same cause. Moreover, I think that health disparities across the globe can only be overcome by educating individuals about disease. Science education should not be a luxury that only those with access to schooling should have. Rather, to level health disparities we must use technology to educate many people about treatments and preventative measures and demystify the myths that surround our health. It is particularly important now more than ever because in the last century life expectancy has doubled, but there has also been an equal or greater increase in disease, including cancer.