Creted PTHrP is known to mediate its cellular effects via interaction with the G-protein-coupled PTH/PTHrP receptor. Coexpression of PTHrP and its receptor has previously been identified in prostate cancer primary tumors and their corresponding bone metastases. Additionally, Freemont et al have previously reported an increase in expression of PTHrP receptor in prostate cancer bone metastases compared to primary tumors, suggesting a potential role 1676428 of the receptor-mediated pathway in the formation of skeletal metastases. Epithelial-to-mesenchymal transition is a process in which epithelial cells undergo cytoskeletal and morphological changes to acquire a mesenchymal phenotype and is important in normal processes such as fibrosis. Due to its effects on cell adhesion and mobility EMT is also critically involved in cancer metastasis and invasion. EMT may be characterized by loss of epithelial Felypressin markers such as E-cadherin and increased expression of mesenchymal proteins including vimentin and Ncadherin. The transcription factors Snail, Slug and Twist are known to repress E-cadherin expression and induce EMT. Other oncogenic pathways including Src, Ras, Wnt/bcatenin, PI3K/Akt, MAPK, and TGF-b have all been linked to EMT. Multiple studies have shown that cancer cells become more invasive and metastatic after undergoing EMT. In addition, 1 PTHrP Promotes Prostate Cancer EMT EMT has been shown to confer stem cell properties to breast cancer cells. Given that PTHrP has a role in promoting invasion and metastasis in prostate cancer and that EMT is one of the main regulators of these properties in cancer, the crucial question presented is whether PTHrP is capable of promoting EMT in cancer cells. PTHrP has been shown to induce EMT in a few contexts, including during parietal endoderm formation and renal fibrogenesis, although the ability of PTHrP to regulate EMT in cancer has remained uninvestigated. In breast cancer, the pro-metastatic effects of TGF-b, a potent inducer of EMT, has been shown to be mediated by PTHrP. Taken together, the existing literature suggests that SC1 site regulation of EMT by PTHrP in cancer is highly likely. In this study we sought to determine the role of PTHrP in regulating EMT in prostate cancer cells along with invasion and metastasis. Establishing a role of PTHrP in regulating bone metastasis cancer and EMT provides both basic and clinical rationales for elucidating the molecular mechanism of PTHrP’s actions in many common cancers like prostate. DU145 cells were expanded. It has previously been shown that wild type and vector-transfected DU 145 exhibit a similar phenotype. Wild type parental DU 145 cells were thus used as a control for RT-qPCR and matrigel invasion experiments, however parallel experiments were performed with an empty vector-transfected control derivative. The PC-3 prostate cells underwent a stable knockdown of PTHrP via lentiviral siRNA. Control cells were subjected to lentiviral siRNA transduction with a non-targeting sequence construct. Quantitative Reverse-Transcription PCR Cells were harvested two days after being passaged at about 70 80% confluence. Total cell lysate was collected and RNA was extracted using an RNeasy kit. cDNA was synthesized using Superscript III Reverse Transcriptase according to the manufacturer’s instructions. Real-time PCR reaction mixes were prepared using Power SYBR Green, and run on the 7300 Real-time PCR System using the following program: 95uC for 10 min, 95uC for 30 s, and 60uC.Creted PTHrP is known to mediate its cellular effects via interaction with the G-protein-coupled PTH/PTHrP receptor. Coexpression of PTHrP and its receptor has previously been identified in prostate cancer primary tumors and their corresponding bone metastases. Additionally, Freemont et al have previously reported an increase in expression of PTHrP receptor in prostate cancer bone metastases compared to primary tumors, suggesting a potential role 1676428 of the receptor-mediated pathway in the formation of skeletal metastases. Epithelial-to-mesenchymal transition is a process in which epithelial cells undergo cytoskeletal and morphological changes to acquire a mesenchymal phenotype and is important in normal processes such as fibrosis. Due to its effects on cell adhesion and mobility EMT is also critically involved in cancer metastasis and invasion. EMT may be characterized by loss of epithelial markers such as E-cadherin and increased expression of mesenchymal proteins including vimentin and Ncadherin. The transcription factors Snail, Slug and Twist are known to repress E-cadherin expression and induce EMT. Other oncogenic pathways including Src, Ras, Wnt/bcatenin, PI3K/Akt, MAPK, and TGF-b have all been linked to EMT. Multiple studies have shown that cancer cells become more invasive and metastatic after undergoing EMT. In addition, 1 PTHrP Promotes Prostate Cancer EMT EMT has been shown to confer stem cell properties to breast cancer cells. Given that PTHrP has a role in promoting invasion and metastasis in prostate cancer and that EMT is one of the main regulators of these properties in cancer, the crucial question presented is whether PTHrP is capable of promoting EMT in cancer cells. PTHrP has been shown to induce EMT in a few contexts, including during parietal endoderm formation and renal fibrogenesis, although the ability of PTHrP to regulate EMT in cancer has remained uninvestigated. In breast cancer, the pro-metastatic effects of TGF-b, a potent inducer of EMT, has been shown to be mediated by PTHrP. Taken together, the existing literature suggests that regulation of EMT by PTHrP in cancer is highly likely. In this study we sought to determine the role of PTHrP in regulating EMT in prostate cancer cells along with invasion and metastasis. Establishing a role of PTHrP in regulating bone metastasis cancer and EMT provides both basic and clinical rationales for elucidating the molecular mechanism of PTHrP’s actions in many common cancers like prostate. DU145 cells were expanded. It has previously been shown that wild type and vector-transfected DU 145 exhibit a similar phenotype. Wild type parental DU 145 cells were thus used as a control for RT-qPCR and matrigel invasion experiments, however parallel experiments were performed with an empty vector-transfected control derivative. The PC-3 prostate cells underwent a stable knockdown of PTHrP via lentiviral siRNA. Control cells were subjected to lentiviral siRNA transduction with a non-targeting sequence construct. Quantitative Reverse-Transcription PCR Cells were harvested two days after being passaged at about 70 80% confluence. Total cell lysate was collected and RNA was extracted using an RNeasy kit. cDNA was synthesized using Superscript III Reverse Transcriptase according to the manufacturer’s instructions. Real-time PCR reaction mixes were prepared using Power SYBR Green, and run on the 7300 Real-time PCR System using the following program: 95uC for 10 min, 95uC for 30 s, and 60uC.
