GEMCITABINE RESISTANCE IN PANCREATIC CANCER CELLS IS ASSOCIATED WITH INCREASED EXPRESSION OF STEM CELL-LIKE MARKERS AND A CONCOMITANT DOWN-REGULATION OF PTEN AND ACTIVATION OF AKT

Abstract

Gemcitabine is currently the standard chemotherapeutic for patients with advanced pancreatic cancer. Despite the rapid development of tumor cell resistance, the specific mechanisms of gemcitabine resistance remain largely unknown and may involve highly resilient cancer stem cells. Whether such cells have relevance in chemoresistance in pancreatic tumors remains unknown. Therefore, the objective of our study was to isolate gemcitabine resistant pancreatic tumor cells from more sensitive parental cells and to determine if they possessed stem cell-like properties. Two resistant cell lines, ASPC1 and L3.6pl, were isolated by growing them in increasing concentrations of gemcitabine. Remaining cells demonstrated effective chemoresistance and were subsequently grown in media containing therapeutic concentrations of gemcitabine. When compared to respective parental cells, resistant cells demonstrated morphologic and biochemical properties of Epidermal to Mesenchymal Transition (EMT). In addition, relative to parental cells, these cells expressed an increased level of surface proteins associated with cancer stem cells, including CD44, CD24, and ESA. Recently, decreased expression of PTEN has been associated with self-renewal of cancer stem cells in hematopoietic tumors, but not normal hematopoietic stem cells. We therefore compared expression of PTEN in our gemcitabine sensitive and resistant cells. We observed that PTEN was decreased >2 fold in both ASPC1 resistant cells and L3.6p1 resistant cell lines relative to their respective parental cells. Consistent with a decreased expression of functional PTEN, we found an increase in pAKT expression but not total Akt in the gemcitabine-resistant cells relative to their respective parental cell lines. Collectively, these results suggest that gemcitabine resistance is associated with an increased cancer stem cell-like population and that decreased PTEN might be involved in pancreatic tumor stem cell renewal, in part through de-repression of the Akt survival pathway. Our results further suggest that overcoming gemcitabine resistance may require novel strategies that inhibit pancreatic stem cell renewal.