Pancreatic ductal adenocarcinoma (PDAC) is the 3rd deadliest cancer in the United States with a projected 12% 5-year survival rate. Acinar cells have been proposed as a potential cell-of-origin for PDAC after undergoing acinar to ductal metaplasia (ADM). In the absence of oncogenic mutations (e.g. Kras), ADM lesions form as an adaptive response and eventually resolve to regenerate the acinar compartment, which we term as adaptive ADM. However, in the presence of oncogenic Kras mutations, the ADM lesions can transform to a pre-invasive state called pancreatic intraepithelial neoplasia (PanIN). Thus, a normally adaptive metaplastic response becomes maladaptive, which we term as oncogenic ADM. The mechanisms that drive PanIN formation in the context of injury and oncogenic mutations are poorly understood, resulting in an absence of targets to combat persistent ADM. This thesis investigates the role of FRA1 (gene name Fosl1) in acinar cell de-differentiation, PanIN transformation, and eventual PDAC tumorigenesis. Through CUT&RUN sequencing of mice undergoing recovery from caerulein-induced acute pancreatitis, we identify FRA1 as the most active transcription factor during KrasG12D mediated acute pancreatitis- mediated injury. We have elucidated a functional role of FRA1 by generating an acinar-specific Fosl1 knockout mouse expressing KrasG12D. Using a gene regulatory network and pseudotime trajectory inferred from single nuclei ATAC-seq and bulk-RNA seq, we hypothesize a regulatory model of the acinar-ADM-PanIN continuum and experimentally validate that Fosl1 knockout mice are delayed in the onset of ADM and PanIN. Furthermore, deletion of Fosl1 in an autochthonous PDAC mouse model revealed that this ADM-initiated delay eventually culminates in a significant survival advantage and a less aggressive tumor phenotype. Through investigation of upstream regulators of FRA1, we identified G-CSF as an ADM-promoting cytokine. Fosl1 depletion prevented the pro-inflammatory effects of G-CSF, indicating that the G-CSF/FRA1 signaling axis can modulate ADM. Using ex vivo acinar cultures, we also showed that G-CSF can induce FRA1 through MEK/ERK signaling. Our findings reveal that FRA1 is a mediator of acinar cell plasticity and contributes to acinar cell de-differentiation and malignant transformation. Although the majority of this thesis focuses on oncogenic ADM, we also include a chapter on the role of Prrx1 in adaptive ADM. Our comprehensive and unbiased approach identified previously the Paired-Related homebox1 (Prrx1) as the most upregulated transcription factor in the intersection of pancreatic ductal development, regeneration, and evolution of PanIN. We have demonstrated previously that Prrx1 can promote a ductal phenotype by binding the Sox9 promotor and inducing its expression during pancreatitis. In this body of work, we present a novel mechanism by which Prrx1 regulates maintenance of adaptive ADM. Using novel mouse models and ex vivo acinar culture systems, we demonstrate that Prrx1 can induce TGFβ signaling and reduce E-Cadherin expression to promote ADM. We do not know if there is any potential epistatic interaction between FRA1 and PRXX1. Overall, we reveal the rippling effects of FRA1 can have during the early stages of pre-neoplasia, and we unveil an alternative function of PRRX1 for stimulating an adaptive response to stress. This thesis presents a new understanding of how acinar cell de-differentiation occurs in the pancreas by revealing novel roles of two transcription factors, FRA1 and PRRX1, and furthers our understanding of tissue regeneration in an injured pancreas.

Indiana University-Purdue University Indianapolis (IUPUI) / Sequestration at the aggresome and degradation through autophagy are two approaches by which a cell can counteract the toxic effect of misfolded proteins. Tuberous sclerosis (TS) and cancer cells can become dependent on autophagy for survival due to the high demand for protein synthesis, thus making protein clearance a potential therapeutic target. Because of its histone deacetylase (HDAC) inhibitory activity, we hypothesized that 4-phenylbutyrate (4-PBA) inhibits HDAC6 and aggresome formation to induce TS cell death. We found that 4-PBA treatment increases cell death and reduces bortezomib-induced aggresome formation. To link these results with HDAC inhibition we used two other HDAC inhibitors, trichostatin A (TSA) and tubastatin, and found that they also reduce bortezomib-induced protein aggregation. Because tubulin is a target of HDAC6, we next measured the effect of the HDAC inhibitors and 4-PBA treatment on tubulin acetylation. As expected, tubastatin increased tubulin acetylation but surprisingly TSA and 4-PBA did not. Because 4-PBA did not significantly inhibit HDAC6, we next hypothesized that 4-PBA was alternatively inducing autophagy and increasing aggresome clearance. Surprisingly, autophagy inhibition did not prevent the 4-PBA-induced reduction in protein aggregation. In conclusion, we found 4-PBA to induce cell death and reduce aggresome levels in TS cells, but we found no link between these phenomena. We next hypothesized that loss of the Ral guanine nucleotide exchange factor Rgl2 induces cell death via autophagy inhibition in pancreatic adenocarcinoma (PDAC) cells. KRas is mutationally activated in over 90% of PDACs and directly activates Rgl2. Rgl2 activates RalB, a known regulator of autophagy, and Rgl2 has been shown to promote PDAC cell survival. We first confirmed that loss of Rgl2 does increase cell death in PDAC cells. Initial experiments using doubly tagged fluorescent p62 and LC3 (autophagy markers) suggested that loss of Rgl2 inhibited autophagosome accumulation, but after developing a more sophisticated quantitation method we found loss of Rgl2 to have no effect. We also measured endogenous LC3 levels, and these experiments confirmed loss of Rgl2 to have no effect on autophagy levels. Therefore, loss of Rgl2 increases cell death in PDAC cells, but does not have a significant effect on autophagy.