Browsing by Subject "Pancreatic Cancer"

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    Endoplasmic Reticulum Stress-Mediated Signaling in Pancreatic Cancer
    (2018-04) Dauer, Patricia
    Pancreatic ductal adenocarcinoma (PDAC) ranks among the poorest prognoses for cancer patients, with an estimated 5-year survival of just 8%. The stagnant survival rates are a result of late detection, chemoresistance, and an aggressive tumor phenotype. Too few patients are eligible for surgery, which results in an urgent need for more effective chemotherapeutic treatment options. One promising pharmacological advancement is currently undergoing a Phase II clinical trial and has been studied by our laboratory. Triptolide is a Chinese herb, which has shown to be very effective in eliminating pancreatic cancer cells in vitro and in vivo. In conjunction with the Medicinal Chemistry Department at the University of Minnesota, a prodrug of triptolide, named Minnelide™, has been synthesized. Our laboratory has since studied triptolide and Minnelide™ extensively, in order to determine the mechanisms of action. The initial study in this dissertation precipitated based on an earlier finding in the Saluja laboratory that triptolide not only downregulates heat shock protein 70 (HSP70) and specificity protein 1 (SP1), but also causes chronic endoplasmic reticulum (ER) stress and cell death. Our study shows that downregulating SP1, a transcription factor that is overexpressed in pancreatic cancer, activates the unfolded protein response (UPR) and results in chronic ER stress. We further show that inhibition of SP1, as well as inducing ER stress, leads to lysosomal membrane permeabilization (LMP), a sustained accumulation of cytosolic calcium, and eventually cell death in pancreatic cancer. Even though ER stress can result in cell death, it is initially a homeostatic mechanism, which aims to protect cells. This led us to ask what role acute ER stress and UPR plays in pancreatic cancer. We show that modulating glucose regulatory protein 78 (GRP78), the master regulator of the UPR, can have a profound effect on multiple pathways that mediate chemoresistance. Our study showed for the first time that knockdown of GRP78 can diminish efflux activity of ATP-binding cassette (ABC) transporters, and it can decrease the antioxidant response resulting in an accumulation of reactive oxygen species (ROS). We also show that these effects can be mediated by the activity of SP1. Our investigation into acute ER stress led to further studies to characterize the UPR signaling in pancreatic cancer. We show that shGRP78 dysregulates multiple transcriptomic and proteomic pathways important in cancer (proliferation, survival, fatty acid metabolism). GRP78 downregulation decreases stemness and self-renewal properties in vitro. In vivo studies demonstrate that GRP78 knockdown results in delayed tumor initiation, and decreased tumor growth. Further, downregulation of GRP78 results in fatty acid metabolism dysregulation. The last study in this dissertation focuses on the tumor microenvironment and SP1 oncogenic signaling. We evaluated the transcriptomic profiling conducted after treatment with triptolide revealed deregulation of the transforming growth factor beta (TGF-β) signaling pathway in cancer-associate fibroblasts (CAFs), resulting in an apparent reversal of their activated state to a quiescent, non-proliferative state. The neighboring epithelial cells exhibited a decrease in oncogenic signaling as manifested by downregulation of SP1. Our findings suggest that approaches to inactivate CAFs and prevent tumor-stromal crosstalk may offer a viable strategy to treat pancreatic cancer. These studies underscore the importance of ER stress and understanding the complex balance of adaptation versus cell death in pancreatic cancer. We have identified SP1 and GRP78 as potential targets for future PDAC therapies. These findings have clinical relevance as both SP1 and GRP78 are overexpressed in pancreatic cancer patients and increased expression of these proteins are indicative of poor prognosis.
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    Molecular Mechanisms of Stemness and Invasion in Pancreatic Cancer
    (2016-05) Nomura, Alice
    Pancreatic cancer remains a cancer with the lowest survival rate and shortest median survival. It is predicted to accelerate to the 2nd cause of cancer related death in the next 15 years. Poor survival rates stem from late disease detection, aggressive tumor biology, and unsuccessful treatments. These dismal statistics highlight the urgency for increased knowledge of the biology of pancreatic cancer and the need for effective therapies. Within the past decade, cancer stem cells, a population within the tumor, have been exposed for their role in tumor initiation, progression, chemoresistance, and metastasis. These studies focus to examine the role of CD133 positive cancer stem cells in stemness and metastasis within pancreatic cancer. We demonstrated that CD133 positive pancreatic cancer cells, from human pancreatic cancer cell lines and a spontaneous murine model of pancreatic cancer, are capable of tumor initiation at very low cell numbers in both immunocompromised and immunocompetent mice, respectively. These cells exhibited chemoresistance properties by the upregulation of drug transporters, active drug efflux capabilities, and resistance to cell death upon treatment with conventional chemotherapies, such as gemcitabine and 5-fluorouracil. CD133 positive cancer stem cell population was, however, sensitive to a novel therapy- Minnelide™. In both in vitro and in vivo studies, CD133 positive cells responded to Minnelide™ treatment and underwent apoptosis in addition to the CD133 negative population. These investigations led to questions regarding the function of this surface marker. CD133 played no functional role in conferring the cancer stem cell phenotype to this population. Through overexpression of CD133 in a pancreatic cancer cell line with very low endogenous CD133 expression, we determined that CD133 expression influences both stemness and invasiveness. Cells overexpressing CD133 were capable of initiating tumors at low cell numbers, as compared to control cells, and exhibited an upregulation in pluripotency and developmental signaling gene expression. Additionally, tumors derived from CD133 overexpressing cells demonstrated a marked increase in metastasis to several distant sites. This was shown to occur through the activation of NF-kB signaling and induction of the epithelial-mesenchymal transition; resulting in increased cellular invasiveness. Further, CD133 expressing cells displayed increased expression and secretion of the cytokine, interleukin-1 beta. Inhibition of IL-1 signaling through various methods established a significant role for IL-1 in the induction of the epithelial-mesenchymal transition and cellular invasiveness. Significantly, interleukin-1 beta positively correlates with CD133 gene expression in pancreatic cancer cell lines of varying aggressiveness. In cell lines with high levels of CD133 positive populations, inhibition of IL-1 signaling demonstrated its critical role in epithelial-mesenchymal transition induction and invasiveness. This exhibited that IL-1 signaling functions within CD133 positive populations during the metastasis process. Finally, these studies demonstrated the pivotal role of NF-kB activation in the induction of the epithelial-mesenchymal transition, cellular invasion, and metastasis. Using triptolide and NF-kB signaling inhibitor treatment, as well as, NF-kB signaling pathway modulation via constitutively active or inactive plasmid expression, this signaling pathway was decisively confirmed to mediate invasion and metastasis in pancreatic cancer. Taken together, this work establishes the functional role of the cancer stem cell marker, CD133; IL-1 signaling; and NF-kB activation in pancreatic cancer stemness and metastasis.
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    Use of Oncolytic Adenovirus expressing Interferon (IFN) Alpha as a tool to improve IFN-based chemoradiation regimen to treat pancreatic cancer
    (2018-02) Salzwedel, Amanda
    Pancreatic Ductal Adenocarcinoma (PDAC) may soon become the third-leading cause of cancer-related death in the United States. Aside from curative resection of tumors, there is no highly effective therapy to treat PDAC. Because patients are usually diagnosed in the late stage of the disease, more than 80% of them are not eligible to undergo surgery, which results in a post-diagnosis survival rate of three to four months, and an extremely low five-year survival rate of 6%. Despite discouraging current clinical outcomes, clinical trials treating PDAC patients with adjuvant therapy combined with IFN- (IFN), Fluorouracil (5-FU), Cisplatin (CDDP), and radiation have shown to improve patients' two-year survival rates by 20-41%, and improve their five-year survival rates by 35%. These clinical trials show that IFN-therapy is now one of the few therapeutic regimens that can significantly improve the short- and long-term survival of PDAC patients. Despite its high efficacy, the drawbacks of IFN therapy included high IFN systemic toxicity, which resulted in increased patient drop-out rates in clinical trials, and low levels of the cytokine in tumors, which hampered the chemo-radio-sensitization capability of IFN during therapy. To attempt to improve the efficacy and overcome the drawbacks of IFN therapy, we studied the use of IFN-expressing replication competent oncolytic adenovirus (OAd) vectors in combination with chemoradiation mimicking the aforementioned chemoradiation and IFN-based clinical trials. Because IFN therapy can stimulate a tumor-specific immune response, we first evaluated the anti-tumor effect of oncolytic adenovirus vector expressing hamster IFN (OAd-hamIFN) in a syngeneic immunocompetent PDAC hamster model. To further understand the interaction between IFN-expressing OAd and chemotherapy, radiation, and chemoradiation, and to evaluate the therapeutic effect of the virus used in treatments mimicking the IFN therapy, we also tested the effect of a human IFN-expressing oncolytic adenovirus (OAd-IFN) in immunodeficient mice bearing human PDAC xenografts. To increase infectivity of OAd-hamIFN in hamster pancreatic cancer cell lines, we included the RGD-4C (Arginive-Glycine-Aspartic) motif in the HI loop of OAd-hamIFN fiber. This modification is known to shift viral tropism from the Coxsackie and Adenovirus Receptor (CAR) to integrins αvβ3 and αvβ5, which are widely expressed in hamster pancreatic cancer cell lines. To increase infectivity of OAd-IFN in human pancreatic cancer cells lines, we replaced the adenovirus type 5 fiber with the chimeric adenovirus 5/3 fiber, which consisted of the fiber of adenovirus type 5 with the knob of adenovirus type 3. This modification has been proven to shift viral tropism from CAR receptor, which shows low expression in human pancreatic cancer cells, to Desmoglein type-2 and CD46 proteins, which are widely expressed in human pancreatic cancer cell lines. To improve spreading in tumors and oncolytic effect of both OAd-hamIFN and OAd-IFN in PDAC cells, and to achieve replication-dependent expression of IFN, we included the Adenovirus Death Protein (ADP) and respective IFN genes in the adenovirus (Ad) E3 region. To restrict the replication of human IFN expressing OAd vector (OAd-IFN) in human PDAC cells, we added the cyclooxygenase-2 (Cox-2) promoter upstream of the Ad E1 region, which is the region responsible for initiating viral replication. Combinations of 5-FU, radiation, and 5-FU + Radiation with OAd-hamIFN in hamster PDAC cells, or with OAd-IFN in human PDAC cells, resulted in highly synergistic and cytotoxic combinations in vitro. Studies in the syngeneic hamster PDAC model showed that including OAd-hamIFN in combination with therapeutics used in IFN therapy improved treatment efficacy, and that using the virus in treatment mimicking the IFN therapy (OAd-hamIFN + 5-FU + Radiation) was the most effective treatment strategy in the study. When we analyzed the effect of OAd-IFN in combination with 5-FU + CDDP in human PDAC cells, the combinations were antagonistic and weakly cytotoxic. But adding radiation to the treatment (OAd-IFN + (5-FU + CDDP) + Radiation) overcame the chemotherapy antagonism, which resulted in highly synergistic and extremely potent treatments in vitro and in mice bearing PDAC xenografts. Because radiation eliminated the antagonism of chemotherapy to the virus in vivo, we tested different radiation protocols in combination with OAd-IFN in mice bearing PDAC tumors. We concluded that administering radiation before infecting tumors with OAd-IFN improved treatment efficacy and that using radiation before OAd-IFN infection should potentiate the effect of Cox-2 controlled IFN-expressing OAds in combination with chemoradiation. In summary, our data strongly support including an IFN expressing OAd in treatments mimicking IFN therapy. As this therapy is one of the few therapeutic regimens shown to improve patients' short- and long-term survival rates, developing a virus-based IFN treatment protocol may result in a highly effective means to treat PDAC.