purmorphamine and Cell-Transformation--Neoplastic

The SASH1 gene is discovered as a tumor suppressor recently. However, the molecular mechanisms of SASH1 in hepatocarcinoma (HCC) remain unclear. In present studies, we investigated the molecular mechanisms of SASH1 on cell invasion and metastasis of hepatocarcinoma in vivo and in vitro. In this study, SASH1 overexpression HCC cell lines were treated with purmorphamine (0, 0.5, 1, 2μmol/l). Western blot and qRT-PCR were used to examine the related gene expression of EMT markers and the Shh-Gli1 and PI3K/Akt-dependent pathway. Cell migration and invasion were assessed by Transwell assay. In addition, a mice SASH1 overexpression HCC orthotopic xenograft model was established and treated with purmorphamine or 740Y-P or PDGF. Tumor volume was assessed, and H&E staining was applied to histopathologic analysis. The results showed that purmorphamine exposure significantly increased the mRNA and protein expression levels of Shh and Gli1 in a dose-dependent manner in the SASH1 overexpression HepG2 and HCCLM3 cells. Besides, purmorphamine promoted the migration and invasion of SASH1 overexpression HCC cells, as well as the EMT progress. Moreover, purmorphamine significantly increased the synthesis of PI3K and pAkt in a dose-dependent manner. Furthermore, the invasion and migration abilities were also improved by treatment with 740Y-P or PDGF in the SASH1 overexpression HCC cells. Additionally, the agonists promoted tumor growth and intrahepatic and pulmonary metastasis of the orthotopic transplantation tumors grown from SASH1 overexpression HCC cells in vivo. In conclusion, SASH1 may inhibit hepatocarcinoma cell invasion and metastasis through down-regulating the Shh-Gli1 and PI3K-AKT pathways in vivo and in vitro.

Topics: Animals; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Down-Regulation; Epithelial-Mesenchymal Transition; Hedgehog Proteins; Hep G2 Cells; Humans; Liver Neoplasms; Lung Neoplasms; Male; Mice; Morpholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Purines; Signal Transduction; Zinc Finger Protein GLI1

Contribution of stromal Hedgehog (Hh) signaling is evident in the prostate gland in mice, but needs translation to human tissues if Hh therapeutics are to be used effectively. Our goal was to determine if primary human prostate fibroblasts contain cilia, and respond to prostate Hh signaling.. Primary human prostate cancer-associated (CAFs), and adjacent non-malignant (NPFs) fibroblasts isolated from human tissue specimens were analyzed using immunofluorescence, real-time PCR, and available array data. Cell culture and tissue recombination were used to determine responsiveness of human fibroblasts to Hh pathway manipulation and the paracrine effects of stromal Hh signaling, respectively.. Prostatic fibroblasts were capable of forming primary cilia, with the capacity for active Hh signaling as seen by Smo co-localization to the tip of the primary cilium. Expression of genes known to represent a signature of active Hh signaling in the prostate (especially Fgf5 and Igfbp6) were increased in CAFs compared to NPFs. The level of canonical Hh genes and prostate Hh signature genes were rarely synchronous; with lower doses of Purmorphamine/BMS-833923 regulating canonical transcription factors, and higher doses effecting prostate Hh signature genes. Grafts consisting of NPFs with constitutively active Hh signaling induced increased proliferation and dedifferentiation of adjacent non-malignant BPH-1 epithelial cells.. These data show that human prostatic fibroblasts have the capacity for Hh signaling and manipulation. Increased expression of a signature of prostatic Hh genes in the prostate tumor microenvironment suggests a role in the epithelial transformations driving prostate cancer (PCa).

Topics: Animals; Benzamides; Cell Communication; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Epithelium; Fibroblasts; Hedgehog Proteins; Heterografts; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; Morpholines; Prostatic Neoplasms; Purines; Quinazolines; Signal Transduction; Stromal Cells

Activation of the Hedgehog (Hh) pathway is known to drive development of basal cell carcinoma and medulloblastomas and to associate with many other types of cancer, but the exact molecular mechanisms underlying the carcinogenesis process remain elusive. We discovered that skin tumors derived from epidermal expression of oncogenic Smo, SmoM2, have elevated levels of IL-11, IL-11Rα, and STAT3 phosphorylation at Tyr(705). The relevance of our data to human conditions was reflected by the fact that all human basal cell carcinomas examined have detectable STAT3 phosphorylation, mostly in keratinocytes. The functional relevance of STAT3 in Smo-mediated carcinogenesis was revealed by epidermal specific knockout of STAT3. We showed that removal of STAT3 from mouse epidermis dramatically reduced SmoM2-mediated cell proliferation, leading to a significant decrease in epidermal thickness and tumor development. We also observed a significant reduction of epidermal stem/progenitor cell population and cyclin D1 expression in mice with epidermis-specific knockout of STAT3. Our evidence indicates that STAT3 signaling activation may be mediated by the IL-11/IL-11Rα signaling axis. We showed that tumor development was reduced after induced expression of SmoM2 in IL-11Rα null mice. Similarly, neutralizing antibodies for IL-11 reduced the tumor size. In two Hh-responsive cell lines, ES14 and C3H10T1/2, we found that addition of Smo agonist purmorphamine is sufficient to induce STAT3 phosphorylation at Tyr(705), but this effect was abolished after IL-11Rα down-regulation by shRNAs. Taken together, our results support an important role of the IL-11Rα/STAT3 signaling axis for Hh signaling-mediated signaling and carcinogenesis.

Topics: Animals; Blotting, Western; Carcinoma, Basal Cell; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Epidermal Cells; Epidermis; Female; Humans; Immunohistochemistry; Interleukin-11 Receptor alpha Subunit; Male; Mice; Mice, Knockout; Mice, Transgenic; Morpholines; Phosphorylation; Purines; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Skin Neoplasms; Smoothened Receptor; STAT3 Transcription Factor