Olivia McGinn

Triptolide-induced Cell Death in Pancreatic Cancer Is Mediated by O-GlcNAc Modification of Transcription Factor Sp1

Background: Preclinical evaluation of triptolide shows pancreatic tumor regression in animal models. Results: Triptolide deregulates glycosylation of Sp1, leading to its decreased activity and causing pancreatic cancer cell death associated with down-regulating HSP70. Conclusion: Triptolide down-regulation of HSP70 is associated with inhibition of Sp1 activity in pancreatic cancer. Significance: This mechanism is of relevance, as its water-soluble prodrug, Minnelide, is currently under Phase 1 clinical trial. Pancreatic cancer, the fourth most prevalent cancer-related cause of death in the United States, is a disease with a dismal survival rate of 5% 5 years after diagnosis. One of the survival proteins responsible for its extraordinary ability to evade cell death is HSP70. A naturally derived compound, triptolide, and its water-soluble prodrug, Minnelide, down-regulate the expression of this protein in pancreatic cancer cells, thereby causing cell death. However, the mechanism of action of triptolide has not been elucidated. Our study shows that triptolide-induced down-regulation of HSP70 expression is associated with a decrease in glycosylation of the transcription factor Sp1. We further show that triptolide inhibits glycosylation of Sp1, inhibiting the hexosamine biosynthesis pathway, particularly the enzyme O-GlcNAc transferase. Inhibition of O-GlcNAc transferase prevents nuclear localization of Sp1 and affects its DNA binding activity. This in turn down-regulates prosurvival pathways like NF-κB, leading to inhibition of HSF1 and HSP70 and eventually to cell death. In this study, we evaluated the mechanism by which triptolide affects glycosylation of Sp1, which in turn affects downstream pathways controlling survival of pancreatic cancer cells.

Impaired synthesis of stromal components in response to Minnelide improves vascular function, drug delivery and survival in pancreatic cancer

Purpose: Pancreatic cancer stromal microenvironment is considered to be the major reason for failure of conventional and targeted therapy for this disease. The desmoplastic stroma, comprising mainly collagen and glycosaminoglycans like hyaluronan (HA), is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis. Minnelide, a water-soluble prodrug of triptolide currently in phase I clinical trial, has been very effective in multiple animal models of pancreatic cancer. However, whether Minnelide will have efficacious delivery into the tumor despite the desmoplastic stroma has not been evaluated before. Experiment Design: Patient tumor-derived xenografts (PDX) and spontaneous pancreatic cancer mice were treated with 0.42 and 0.21 mg/kg body weight for 30 days. Stromal components were determined by IHC and ELISA-based assays. Vascular functionality and drug delivery to the tumor were assessed following treatment with Minnelide. Result: Our current study shows that treatment with Minnelide resulted in reduction of ECM components like HA and collagen in the pancreatic cancer stroma of both the spontaneous KPC mice as well as in patient tumor xenografts. Furthermore, treatment with Minnelide improved functional vasculature in the tumors resulting in four times more functional vessels in the treated animals compared with untreated animals. Consistent with this observation, Minnelide also resulted in increased drug delivery into the tumor compared with untreated animals. Along with this, Minnelide also decreased viability of the stromal cells along with the tumor cells in pancreatic adenocarcinoma. Conclusions: In conclusion, these results are extremely promising as they indicate that Minnelide, along with having anticancer effects is also able to deplete stroma in pancreatic tumors, which makes it an effective therapy for pancreatic cancer. Clin Cancer Res; 22(2); 415–25. ©2015 AACR.

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 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.

Abstract B46: Low mitochondrial activity of CD133+ population enables evasion of apoptosis leading to increased chemo-resistance in tumor initiating cells

Background: Pancreatic ductal adenocarcinoma is currently the fourth most frequent cause of cancer-related death in the United States with poor survival. Tumor recurrence in this devastating disease adds to its grim survival. Relapse of any tumor is largely attributed to the presence of tumor-initiating cells (TIC). Chemo-resistance is considered a hallmark of TICs. Standard chemotherapy typically targets rapidly growing cells as a result of which the quiescent TICs often evade cell death by these agents. However, the mechanism involved in conferring chemo-resistance to these cells is still unclear. Our study shows that CD133+ pancreatic cancer TICs have increased glycolysis and low mitochondrial activity compared to CD133- non TIC. Our study further shows that CD133+ cells have increased resistance to chemotherapeutic agents like gemcitabine, 5FU and paclitaxel resulting in decreased apoptotic cells and decreased accumulation of reactive oxygen species (ROS). This altered metabolic profile offers a survival advantage to the quiescent TICs. Methods: CD133+ pancreatic TICs were isolated from KPC tumor tissues , KPC cell lines isolated from primary tumors and patient tumor derived tumor xenografts using MACS beads (Miltnyi Biotech). Expression of genes controlling glucose metabolism and mitochondrial activity were evaluated using PCR array (SA biosciences). Activity assays for glycolytic enzymes (Sigma), Pentose phosphate pathway enzyme (Sigma) and mitochondrial complex I and Complex IV (AbCam) was done using kits from indicated vendors according to manufacturers instruction. Apoptosis assays were done using Caspase 3/7 assay kit (Promega). Cell viability was assessed using Trypan Blue exclusion method. Results: CD133+ pancreatic TIC (from KPC tumor, KPC cell lines and human patient derived xenografts) had increased glucose uptake (4 fold higher) compared to CD133- cells. These cells also showed increased expression of glycolytic enzymes compared to CD133- cells (4-20 fold higher). Consistent with this, the CD133+ cells had increased HK2 (3 fold) and LDH activity (8 fold). CD133+ pancreatic TIC showed 10-fold lower activity of mitochondrial enzyme complex 1 and mitochondrial enzyme complex IV compared to CD133- genes. Consistent with this, the expression of genes in these enzyme complexes was also significantly decreased. This indicated that CD133+ TICs had increased glycolysis but low mitochondrial activity Accumulation of ROS is one of the major inducers of apoptosis in cells. Treatment with standard chemotherapeutic agents results in generation of free radicals of oxygen, which trigger mitochondrial apoptotic pathways. Low mitochondrial complex activity prevents accumulation of ROS. Treatment of CD133+ population with standard chemotherapeutic agents like 5FU, Gemcitatbine and paclitaxel showed increased cell viability compared to CD133- cells. These treatments also resulted in decreased apoptosis in CD133+ population compared to CD133- population. Consistent with this, CD133+ cells also showed increased expression of anti-apoptotic proteins like Bcl2 and Survivin compared to CD133- cells. Further, CD133+ population showed decreased accumulation of ROS compared to CD133- cells. These results indicated that low mitochondrial activity in CD133+ TICs offered survival advantages to these cells, allowing them to be resistant to standard chemotherapeutic compounds. Conclusion: Understanding the mechanism behind persistence of TIC population in a tumor following chemotherapy is one of the prime thrust area of cancer research. Our study indicates that altered metabolic profile in CD133+ pancreatic TIC protect the cells from apoptosis induced by standard chemotherapeutic agents by reducing accumulation of ROS and decreasing apoptosis, thereby confering chemoresistance to these cells. Citation Format: Sulagna Banerjee, Alice Nomura, Olivia McGinn, Kelsey Jensen, Veena Sangwan, Vikas Dudeja, Ashok Saluja. Low mitochondrial activity of CD133+ population enables evasion of apoptosis leading to increased chemo-resistance in tumor initiating cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B46.

Abstract B57: Cell survival in pancreatic cancer is regulated by glycosylation of the transcription factor Sp1.

Background: Sp1 protein is a sequence-specific, DNA-binding protein that is a part of the Specificity protein/Kruppel-like factor family of transcription factors which is critical in the regulation of cell growth, differentiation and apoptosis. In pancreatic adenocarcinoma, Sp1 overexpression has been correlated with tumor differentiation, clinical tumor staging, tumor metastasis, and patient survival. Combination treatments with Sp1 inhibitor and antiangiogenic compounds have also shown promising results in pancreatic tumors. However, the underlying mechanism by which Sp1 regulates cell survival in pancreatic cancer cells has not been addressed. Sp1 activity is tightly regulated by several posttranslational modifications, one of which is glycosylation. The enzyme responsible for glycosylation of Sp1 is O-GlcNAc transferase (OGT) which adds a single GlcNAc. In the current study we have evaluated the regulation of Sp1 activity by glycosylation, which also affects downstream pathways controlling survival in pancreatic cancer cells. Methods: Expression of Sp1 was studied in the different pancreatic cancer cell lines and mouse models at the RNA level by qRT-PCR and in the protein level by Western blot. Sp1 expression and activity was inhibited using the specific inhibitor mithramycin or by using siRNA against Sp1. Effect of Sp1 inhibition on viability was studied using an MTT based assay. Apoptosis was evaluated by caspase activity assay. Glycosylation of Sp1 was inhibited either by decreasing expression of O-GlcNAC transferase (OGT) by siRNA or by alloxan, a specific inhibitor. The effect of Sp1 activity, NFkB activity and HSP activity following inhibition of glycosylation was measured by dual luciferase reporter assay. Effect of inhibition of OGT and Sp1 on pancreatic cancer cell proliferation and migration was studied using ECIS (electric cell-substrate impedance sensing).Inhibition of Sp1 translocation to nucleus in response to inhibitor treatment was studied by confocal microscopy. Results: Sp1 was found to be 5-15 fold overexpressed in pancreatic cancer cell lines over non-tumorigenic ductal cells and between 5-10 fold over normal pancreas in xenotransplanted patient tumors in mice. Inhibition of Sp1 by mithramycin, or by siRNA, resulted in a loss in viability (30% of control). Similar loss is viability was also seen following inhibition of the glycosyltransferase (OGT) by siRNA (32% of control).Inhibition of glycosylation of Sp1 resulted in its accumulation in the cytosol of the pancreatic cancer cells. Survival of pancreatic cancer cells is attributed to high expression of heat shock proteins and a constitutively active NF-kB pathway. In order to see if altering glycosylation of Sp1 had an effect on expression of these proteins, reporter activity assay was performed following inhibition of Sp1 activity (by mithramycin), or expression (siRNA) or by inhibiting glycosylating enzyme. Both altering glycosylation as well as inhibition of Sp1 resulted in lowered activity of several survival pathways like NF-kB (30% of control) and Heat shock proteins (40% of control) and resulted in downregulation of apoptosis evading genes like survivin and Bcl2. Inhibition of glycosylation of Sp1 also resulted in decreased proliferation and migration of pancreatic cancer cells. Conclusion: This study showed down-regulation of either Sp1 expression or activity leads to pancreatic cancer cell death. Furthermore, glycosylation of Sp1 by OGT plays a vital role in regulating proliferation, migration, and survival of pancreatic cancer cells. Citation Format: Sulagna Banerjee, Veena Sangwan, Olivia McGinn, Tara Kendall Krosch, Nameeta Mujumder, Vikas Dudeja, Mackenzie Tiffany, Vickers M. Selwyn, Ashok K. Saluja. Cell survival in pancreatic cancer is regulated by glycosylation of the transcription factor Sp1. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B57.