Graduation Date

Spring 5-8-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Dr. Surinder K. Batra

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease that has one of the lowest 5-year survival rates among cancers, at just 9%. This grim prognosis is primarily due to the extensive metastatic spread of tumor cells beyond the pancreas at diagnosis and the inability of current therapeutic modalities to treat this aggressive disease effectively. Given that the cancer cells in pancreatic tumors are heterogeneous, the major culprit for cancer initiation, progression, and metastasis remains elusive. Recent studies provide evidence for the existence of highly tumorigenic and drug-resistant cells that are capable of tumor initiation, known as the cancer stem cells (CSCs). The resistance to standard chemotherapy, metastatic potential, and resultant aggressiveness of PDAC have been attributed to an inadequate understanding of how these pancreatic CSCs are maintained within the tumor and a lack of effective strategies to target them. In my doctoral research, I have attempted to address these knowledge gaps by studying a protein, RNA polymerase II-associated factor 1 (PAF1) that is more than 30-fold overexpressed in the aggressive, poorly differentiated tumors compared to well-differentiated tumors. Our previous work demonstrated that PAF1 or pancreatic differentiation 2 (PD2), is a novel CSC marker that mediates drug resistance and metastasis in PDAC. PAF1 is a core component of human PAF1 complex (PAF1C), which along with 4 other components (LEO1, CTR9, CDC73, and SKI8) recruits RNA polymerase II for transcriptional elongation. Paf1 also maintains the self-renewal of mouse embryonic stem cells and ovarian CSCs via its interaction with OCT3/4, a major regulator of pluripotency. However, the mechanistic role of PAF1 in CSC maintenance and CSC-mediated PDAC pathogenesis is poorly understood.

In this dissertation, my first goal was to delineate the underlying mechanism of PAF1-mediated CSC maintenance. I established that PAF1 downregulation decreased tumorigenesis and metastasis in xenograft mouse models using multiple PDAC cell lines. Moreover, PAF1 was overexpressed in isolated CSCs and was essential for regulating the phenotypic features of CSCs such as in vitro tumor sphere formation and colony formation. Interestingly, the other PAF1C subunits, LEO1, CTR9, and CDC73, appeared dispensable for the maintenance of stem cell state, as their downregulation affected neither the expression of CSC markers nor the formation of tumor spheres. Through IP-mass spectrometry, I identified PHD finger protein 5a (PHF5A) and DEAD-box RNA helicase 3 (DDX3) as unique interacting partners for PAF1 in CSCs. Using a global approach via chromatin immunoprecipitation sequencing (ChIP-Seq) with PAF1 and PHF5A-pulldown, I found that promoters of several stemness regulators were occupied by PAF1 and PHF5A in CSCs. Nanog was amongst the top genes whose promoters were jointly occupied by PAF1 and PHF5A in CSCs. Next, I investigated the effect of RK-33, a specific small-molecule inhibitor of DDX3 helicase activity on pancreatic CSCs. Treatment of CSCs with RK-33 led to a significant downregulation of CSC markers (b-CATENIN, CD44v6, SOX9, and NANOG), inhibition of tumor sphere formation, and impairment of PAF1 binding to Nanog promoter. Additionally, treatment with RK-33 resulted in apoptosis of CSCs, while minimal cell death was seen in normal human fibroblasts. Overall, the data indicated that PAF1 functions as the master regulator for stem cell maintenance by regulating the transcription of several stem cell-related genes via a PAF1C-independent mechanism.

My second goal was to explore the functional impact of Paf1 depletion on the pancreatic homeostasis using a CRISPR/Cas9-based conditional knockout mouse model. The deletion of Paf1 from the mouse pancreas caused a significant decrease in pancreas weight in young mice (up to 5-months old), without affecting the body weight. Histologically, Paf1 loss caused an extensive loss of acinar parenchyma with associated inflammation and appearance of ‘naked ducts’ embedded in fat. However, the ‘naked-duct like’ phenotype was restored in older mice (7-, 9-, and 12-months old). RNA-seq analyses of the pancreas from homozygous Paf1-deleted and floxed mice revealed pathways prevalent in system development and acinar cell survival. Based on candidate players identified via RNA-seq and other histochemical analyses, I showed that Paf1 plays a role in acinar lineage differentiation and acinar maintenance. Next, to understand the role of Paf1 in PDAC progression, I generated the KPCP (KrasG12D; Trp53R172H/+; Pdx1-Cre; Paf1fl/fl) PDAC mouse model. The loss of Paf1 in the context of endogenous expression of KrasG12D and Trp53R172H/+ significantly accelerated disease progression and decreased overall survival as compared to KPC mice that have Kras and p53 mutations. Cell lines derived from KPCP mice showed a significantly higher expression of mesenchymal markers (Vimentin and Snail) compared to KPC cell lines. Altogether, I demonstrated a previously unknown role for Paf1 in acinar lineage differentiation and provided molecular insights into Paf1-mediated cancer progression. In conclusion, studies in this dissertation delineated the mechanistic role of PAF1 in CSC maintenance and provided a means for selective targeting of pancreatic CSCs, as well as, elucidated the in vivo role of PAF1 in pancreatic homeostasis and PDAC pathogenesis.

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