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.