Abstract 5039: Novel insight into mechanisms of parathyroid hormone-related protein (PTHrP) action in prostate cancer growth and skeletal metastasis: Altered anoikis and angiogenesis

Abstract

Abstract PTHrP was first discovered as an etiologic factor in malignancy-induced hypercalcemia, and was later shown to regulate many important functions in tumor progression, particularly in prostate and breast cancer skeletal metastasis. However, the underlying mechanisms are understudied and controversial. In this study, the roles of PTHrP in prostate cancer growth and skeletal metastasis were examined in vitro and in vivo, focusing on anoikis and angiogenesis. PC-3 (expressing high endogenous PTHrP) and ACE-1 (expressing low endogenous PTHrP) prostate carcinoma cells were used in these studies. Luciferase-labeled human prostate cancer cells (PC-3L) were transduced with PTHrP-directed shRNA lentiviral vectors or empty vector (EV), and stable clones (PC-3L-shPTHrP and PC-3L-EV, respectively) were subsequently isolated and expanded. ACE-1 cells were stably transfected with a PTHrP overexpression vector. For the in vitro anoikis assay, cells were seeded in agarose-covered plates to prevent attachment and harvested 12 hours later for flow cytometric analysis of Annexin V+ apoptotic cells. For in vivo experiments, PC-3L-shPTHrP or PC-3L-EV cells were transplanted into male athymic mice via subcutaneous (to examine tumor growth and angiogenesis) or intra-cardiac (to examine metastasis) routes. Tumor growth and/or metastasis were monitored by weekly bioluminescence imaging. Subcutaneous tumor tissues were harvested for determination of angiogenesis (via mean vessel density quantification) and recruitment of hematopoietic stem/progenitor cells to the tumor (via enumeration of Lin−CD150+ cells). Reduction of PTHrP expression in PC-3L cells resulted in a significant increase in detachment-induced apoptosis (i.e. anoikis) in vitro that was rescued when PTHrP was overexpressed. In addition, intra-cardiac injection of PC-3L-shPTHrP clones resulted in significantly fewer tumor cells (as quantified by bioluminescence) localized in the mandibular and hind limb skeletal regions at an early time point (day-5) compared to the empty vector control, suggesting an anoikis-mediated event in the bloodstream. PC-3L-shPTHrP cells were also found to produce smaller subcutaneous tumors in vivo compared to PC-3L-EV control tumors, while the in vitro proliferation rate was not affected. Interestingly, PTHrP-knockdown tumor tissues resulted in a significant reduction in CD31+ mean vessel density, and significantly fewer Lin−CD150+ stem cells in the tumor tissues compared with control tumors. In conclusion, these findings suggest that tumor-derived PTHrP contributes to prostate cancer skeletal metastasis by conferring resistance to anoikis. Additionally, PTHrP promotes tumor growth in vivo at least partially by increasing angiogenesis and recruitment of hematopoietic stem/progenitor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5039.