Alice Nomura

CD133+ Tumor Initiating Cells in a Syngenic Murine Model of Pancreatic Cancer Respond to Minnelide

Purpose: Pancreatic adenocarcinoma is the fourth leading cause for cancer-related mortality with a survival rate of less than 5%. Late diagnosis and lack of effective chemotherapeutic regimen contribute to these grim survival statistics. Relapse of any tumor is largely attributed to the presence of tumor-initiating cells (TIC) or cancer stem cells (CSC). These cells are considered as hurdles to cancer therapy as no known chemotherapeutic compound is reported to target them. Thus, there is an urgent need to develop a TIC-targeted therapy for pancreatic cancer. Experimental Design: We isolated CD133+ cells from a spontaneous pancreatic ductal adenocarcinoma mouse model and studied both surface expression, molecular markers of pancreatic TICs. We also studied tumor initiation properties by implanting low numbers of CD133+ cells in immune competent mice. Effect of Minnelide, a drug currently under phase I clinical trial, was studied on the tumors derived from the CD133+ cells. Results: Our study showed for the first time that CD133+ population demonstrated all the molecular markers for pancreatic TIC. These cells initiated tumors in immunocompetent mouse models and showed increased expression of prosurvival and proinvasive proteins compared to the CD133− non-TIC population. Our study further showed that Minnelide was very efficient in downregulating both CD133− and CD133+ population in the tumors, resulting in a 60% decrease in tumor volume compared with the untreated ones. Conclusion: As Minnelide is currently under phase I clinical trial, its evaluation in reducing tumor burden by decreasing TIC as well as non-TIC population suggests its potential as an effective therapy. Clin Cancer Res; 20(9); 2388–99. ©2014 AACR.

Microenvironment in determining chemo-resistance in pancreatic cancer: Neighborhood matters

Every year, nearly 300,000 people are diagnosed with pancreatic cancer worldwide, and an equivalent number succumb to this disease. One of the major challenges of pancreatic cancer that contributes to its poor survival rates is the development of resistance to the standard chemotherapy. Heterogeneity of the tumor, the dense fibroblastic stroma, and the aggressive biology of the tumor all contribute to the chemoresistant phenotype. In addition, the acellular components of the tumor microenvironment like hypoxia, stress pathways in the stromal cells, and the cytokines that are secreted by the immune cells, have a definitive role in orchestrating the chemoresistant property of the tumor. In this review, we systematically focus on the role played by the different microenvironmental components in determining chemoresistance of pancreatic tumors.

Microenvironment mediated alterations to metabolic pathways confer increased chemo-resistance in CD133 + tumor initiating cells

Chemoresistance in pancreatic cancer has been attributed to tumor-initiating cells (TICs), a minor sub-population of tumor cells. However, the mechanism of chemo-resistance in these cells is still unclear. In the current study, immunohistochemical analysis of LSL-KrasG12D; LSL-Trp53R172H; PdxCre (KPC) murine tumors indicated that hypoxic regions developed through tumor progression. This hypoxic “niche” correlated with increased CD133+ population that had an increased HIF1A activity. Consistent with this observation, CD133+ cells had increased glucose uptake and activity of glycolytic pathway enzymes compared to CD133− cells. Mass spectrometric analysis (UPLC-TQD) following metabolic labeling of CD133+ cells with [13C]-U6 glucose confirmed this observation. Furthermore, although both populations had functionally active mitochondria, CD133+ cells had low mitochondrial complex I and complex IV activity and lesser accumulation of ROS in response to standard chemotherapeutic compounds like paclitaxel, 5FU and gemcitabine. CD133+ cells also showed increased resistance to all three chemotherapeutic compounds and treatment with Glut1 inhibitor (STF31) reversed this resistance, promoting apoptotic death in these cells similar to CD133− cells. Our study indicates that the altered metabolic profile of CD133+ pancreatic TIC protects them against apoptosis, by reducing accumulation of ROS induced by standard chemotherapeutic agents, thereby confering chemoresistance. Since resistance to existing chemotherapy contributes to the poor prognosis in pancreatic cancer, our study paves the way for identifying novel therapeutic targets for managing chemoresistance and tumor recurrence in pancreatic cancer.

Inhibition of NF-kappa B pathway leads to deregulation of epithelial-mesenchymal transition and neural invasion in pancreatic cancer

NF-κB has an essential role in the initiation and progression of pancreatic cancer and specifically mediates the induction of epithelial–mesenchymal transition and invasiveness. In this study, we demonstrate the importance of activated NF-κB signaling in EMT induction, lymphovascular metastasis, and neural invasion. Modulation of NF-κB activity was accomplished through the specific NF-κB inhibitor (BAY 11-7085), triptolide, and Minnelide treatment, as well as overexpression of IKBα repressor and IKK activator plasmids. In the classical lymphovascular metastatic cascade, inhibition of NF-κB decreased the expression of several EMT transcription factors (SNAI1, SNAI2, and ZEB1) and mesenchymal markers (VIM and CDH2) and decreased in vitro invasion, which was rescued by IKK activation. This was further demonstrated in vivo via BAY 11-7085 treatment in a orthotopic model of pancreatic cancer. In vivo NF-κB inhibition decreased tumor volume; decreased tumor EMT gene expression, while restoring cell–cell junctions; and decreasing overall metastasis. Furthermore, we demonstrate the importance of active NF-κB signaling in neural invasion. Triptolide treatment inhibits Nerve Growth Factor (NGF) mediated, neural-tumor co-culture in vitro invasion, and dorsal root ganglia (DRG) neural outgrowth through a disruption in tumor-neural cross talk. In vivo, Minnelide treatment decreased neurotrophin expression, nerve density, and sciatic nerve invasion. Taken together, this study demonstrates the importance of NF-κB signaling in the progression of pancreatic cancer through the modulation of EMT induction, lymphovascular invasion, and neural invasion.

Dynamic Tumor Growth Patterns in a Novel Murine Model of Colorectal Cancer

Colorectal cancer often arises from adenomatous colonic polyps. Polyps can grow and progress to cancer, but may also remain static in size, regress, or resolve. Predicting which polyps progress and which remain benign is difficult. We developed a novel long-lived murine model of colorectal cancer with tumors that can be followed by colonoscopy. Our aim was to assess whether these tumors have similar growth patterns and histologic fates to human colorectal polyps to identify features to aid in risk stratification of colonic tumors. Long-lived ApcMin/+ mice were treated with dextran sodium sulfate to promote colonic tumorigenesis. Tumor growth patterns were characterized by serial colonoscopy with biopsies obtained for immunohistochemistry and gene expression profiling. Tumors grew, remained static, regressed, or resolved over time with different relative frequencies. Newly developed tumors demonstrated higher rates of growth and resolution than more established tumors that tended to remain static in size. Colonic tumors were hyperplastic lesions (3%), adenomas (73%), intramucosal carcinomas (20%), or adenocarcinomas (3%). Interestingly, the level of β-catenin was higher in adenomas that became intratumoral carcinomas than those that failed to progress. In addition, differentially expressed genes between adenomas and intramucosal carcinomas were identified. This novel murine model of intestinal tumorigenesis develops colonic tumors that can be monitored by serial colonoscopy, mirror growth patterns seen in human colorectal polyps, and progress to colorectal cancer. Further characterization of cellular and molecular features is needed to determine which features can be used to risk-stratify polyps for progression to colorectal cancer and potentially guide prevention strategies. Cancer Prev Res; 7(1); 105–13. ©2013 AACR.

A Novel Immunocompetent Mouse Model of Pancreatic Cancer with Robust Stroma: a Valuable Tool for Preclinical Evaluation of New Therapies

A valid preclinical tumor model should recapitulate the tumor microenvironment. Immune and stromal components are absent in immunodeficient models of pancreatic cancer. While these components are present in genetically engineered models such as KrasG12D; Trp53R172H; Pdx-1Cre (KPC), immense variability in development of invasive disease makes them unsuitable for evaluation of novel therapies. We have generated a novel mouse model of pancreatic cancer by implanting tumor fragments from KPC mice into the pancreas of wild type mice. Three-millimeter tumor pieces from KPC mice were implanted into the pancreas of C57BL/6J mice. Four to eight weeks later, tumors were harvested, and stromal and immune components were evaluated. The efficacy of Minnelide, a novel compound which has been shown to be effective against pancreatic cancer in a number of preclinical murine models, was evaluated. In our model, consistent tumor growth and metastases were observed. Tumors demonstrated intense desmoplasia and leukocytic infiltration which was comparable to that in the genetically engineered KPC model and significantly more than that observed in KPC tumor-derived cell line implantation model. Minnelide treatment resulted in a significant decrease in the tumor weight and volume. This novel model demonstrates a consistent growth rate and tumor-associated mortality and recapitulates the tumor microenvironment. This convenient model is a valuable tool to evaluate novel therapies.

Inhibition of hypoxic response decreases stemness and reduces tumorigenic signaling due to impaired assembly of HIF1 transcription complex in pancreatic cancer

Pancreatic tumors are renowned for their extremely hypoxic centers, resulting in upregulation of a number of hypoxia mediated signaling pathways including cell proliferation, metabolism and cell survival. Previous studies from our laboratory have shown that Minnelide, a water-soluble pro-drug of triptolide (anti-cancer compound), decreases viability of cancer cells in vitro as well as in vivo. However, its mechanism of action remain elusive. In the current study we evaluated the effect of Minnelide, on hypoxia mediated oncogenic signaling as well as stemness in pancreatic cancer. Minnelide has just completed Phase 1 trial against GI cancers and is currently awaiting Phase 2 trials. Our results showed that upon treatment with triptolide, HIF-1α protein accumulated in pancreatic cancer cells even though hypoxic response was decreased in them. Our studies showed even though HIF-1α is accumulated in the treated cells, there was no decrease in HIF-1 binding to hypoxia response elements. However, the HIF-1 transcriptional activity was significantly reduced owing to depletion of co-activator p300 upon treatment with triptolide. Further, treatment with triptolide resulted in a decreased activity of Sp1 and NF-kB the two major oncogenic signaling pathway in pancreatic cancer along with a decreased tumor initiating cell (TIC) population in pancreatic tumor.

Inhibition of Sp1 prevents ER homeostasis and causes cell death by lysosomal membrane permeabilization in pancreatic cancer

Endoplasmic reticulum (ER) stress initiates an important mechanism for cell adaptation and survival, named the unfolded protein response (UPR). Severe or chronic/prolonged UPR can breach the threshold for survival and lead to cell death. There is a fundamental gap in knowledge on the molecular mechanism of how chronic ER stress is stimulated and leads to cell death in pancreatic ductal adenocarcinoma (PDAC). Our study shows that downregulating specificity protein 1 (Sp1), a transcription factor that is overexpressed in pancreatic cancer, activates UPR and results in chronic ER stress. In addition, downregulation of Sp1 results in its decreased binding to the ER stress response element present in the promoter region of Grp78, the master regulator of ER stress, thereby preventing homeostasis. We further show that inhibition of Sp1, as well as induction of ER stress, leads to lysosomal membrane permeabilization (LMP), a sustained accumulation of cytosolic calcium, and eventually cell death in pancreatic cancer.

GRP78‐mediated antioxidant response and ABC transporter activity confers chemoresistance to pancreatic cancer cells

Chemoresistance is a major therapeutic challenge that plays a role in the poor statistical outcomes in pancreatic cancer. Unfolded protein response (UPR) is one of the homeostasis mechanisms in cancer cells that have been correlated with chemoresistance in a number of cancers including pancreatic cancer. In this study, 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 silencing 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 specificity protein 1 (SP1), a transcription factor overexpressed in pancreatic cancer. Thus, inhibition of SP1 negatively affects the UPR, deregulates the antioxidant response of NRF2, as well as ABC transporter activity by inhibiting GRP78‐mediated ER homeostasis. Sp1 and NRF2 have been classified as nononcogene addiction genes and thus are imperative to understanding the molecular mechanism of resistance. These finding have huge clinical relevance as both Sp1 and GRP78 are overexpressed in pancreatic cancer patients and increased expression of these proteins is indicative of poor prognosis. Understanding how these proteins may regulate chemoresistance phenotype of this aggressive cancer may pave the way for development of efficacious therapy for this devastating disease.

NFκB-Mediated Invasiveness in CD133+ Pancreatic TICs Is Regulated by Autocrine and Paracrine Activation of IL1 Signaling

Tumor-initiating cells (TIC) have been implicated in pancreatic tumor initiation, progression, and metastasis. Among different markers that define this cell population within the tumor, the CD133+ cancer stem cell (CSC) population has reliably been described in these processes. CD133 expression has also been shown to functionally promote metastasis through NF-κB activation in this population, but the mechanism is unclear. In the current study, overexpression of CD133 increased expression and secretion of IL1β (IL1B), which activates an autocrine signaling loop that upregulates NF-κB signaling, epithelial–mesenchymal transition (EMT), and cellular invasion. This signaling pathway also induces CXCR4 expression, which in turn is instrumental in imparting an invasive phenotype to these cells. In addition to the autocrine signaling of the CD133 secreted IL1β, the tumor-associated macrophages (TAM) also produced IL1β, which further activated this pathway in TICs. The functional significance of the TIC marker CD133 has remained elusive for a very long time; the current study takes us one step closer to understanding how the downstream signaling pathways in these cells regulate the functional properties of TICs. Implications: This study demonstrates the important role of tumor- and macrophage-derived IL1β stimulation in pancreatic cancer. IL1 signaling is increased in cells with CD133 expression, leading to increased NF-kB activity, EMT induction, and invasion. Increased invasiveness via IL1β stimulation is mediated by the upregulation of CXCR4 expression. The study highlights the importance of IL1-mediated signaling in TICs. Mol Cancer Res; 16(1); 162–72. ©2017 AACR.