Abstract 2456: External uPA overexpresses and interacts with Lhx-2 and induces a pancreatic cancer stem cell phenotype

Abstract

Abstract Although the body of knowledge surrounding the molecular profile of pancreatic cancer has substantially increased, survival rates have not changed for over four decades. Previous studies have demonstrated that urokinase plasminogen activator (uPA) is frequently present in a higher concentration in the serum of pancreatic cancer patients. uPA is involved in extracellular matrix (ECM) degradation and has been correlated with the malignant transformation of cancer cells. Additionally, recent reports have also shown the translocation of uPA to the nucleus without binding to uPAR. However, its role in the nucleus is still unclear. Existing evidence suggest that a hypoxic environment forces cancer cells to acquire stem cell characteristics, causing recurrence after chemotherapy and radiation. In the present study, we have attempted to correlate the roles of uPA and hypoxia in cancer stem cell transformation. Initially, to determine the interaction of uPA with Lhx-2, we conducted immunoprecipitation and immunolocalization studies in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines. Further, to determine the role of exogenous uPA on Lhx-2 expression in an uPA-null background, cells were downregulated for uPA and followed by supplementation with purified uPA under normoxic as well as hypoxic conditions. To further resolve whether Lhx-2 is involved in the activation of CD133 and STRO-1 expression in pancreatic cells, uPA and/or Lhx-2 were knocked down under normoxic or hypoxic conditions and the expression levels of CD133 and STRO-1 were determined. In addition, cell lysates were screened for stem cell markers using stem cell-specific antibody arrays. To determine the role of uPA in tumorigenicity after hypoxia, MIA PaCa-2 or PANC-1 hypoxic cells were subcutaneously implanted in nude mice followed by injection of siRNA for uPA or Lhx-2 and 100 nM of uPA at the tumor site, and tumor growth was determined. From the immunoprecipitation and immunolocalization studies, we observed that uPA interacts with Lhx-2 both in the cytoplasm and the nucleus of MIA PaCa-2 and PANC-1 pancreatic cancer cells. Further, we observed that the addition of external uPA caused an increase in the expression of Lhx-2. The addition of uPA under hypoxic conditions resulted in increased expression of CD133 and STRO-1 whereas suppression of Lhx-2 retarded the overexpression of CD133 and STRO-1. Antibody array analysis indicated that hypoxic cells had increased expression of stem cell markers when supplemented with uPA, which was abolished by suppression of Lhx-2. From the animal studies, we observed that hypoxic MIA PaCa-2 or PANC-1 cells supplemented with external uPA were able to form subcutaneous tumors within 30 days of implantation. Taken together, our results demonstrate that in a hypoxic environment, uPA activates the expression of stem cell markers mediated by the expression of Lhx-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2011-2456