ldn-193189 and Prostatic-Neoplasms

Angiomotin (AMOT) is a family of proteins found to be a component of the apical junctional complex of vertebrate epithelial cells and is recently found to play important roles in neurofibromatosis type 2 (NF-2). Whether AMOT plays a role in prostate cancer (PCa) is unknown. AMOT is expressed as two isoforms, AMOTp80 and AMOTp130, which has a 409 aa N-terminal domain that is absent in AMOTp80. Both AMOTp80 and AMOTp130 are expressed in LNCaP and C4-2B4, but at a low to undetectable level in PC3, DU145, and BPH1 cells. Further study showed that AMOTp130 and AMOTp80 have distinct functions in PCa cells. We found that AMOTp80, but not AMOT p130, functioned as a tumor promoter by enhancing PCa cell proliferation. Mechanistic studies showed that AMOTp80 signaled through the Hippo pathway by promoting nuclear translocation of YAP, resulting in an increased expression of YAP target protein BMP4. Moreover, inhibition of BMP receptor activity by LDN-193189 abrogates AMOTp80-mediated cell proliferation. Together, this study reveals a novel mechanism whereby the AMOTp80-Merlin-MST1-LATS-YAP-BMP4 pathway leads to AMOTp80-induced tumor cell proliferation.

Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Angiomotins; Animals; Antineoplastic Agents; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Hippo Signaling Pathway; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice, SCID; Microfilament Proteins; Phosphoproteins; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Pyrazoles; Pyrimidines; RNA Interference; Signal Transduction; Time Factors; Transcription Factors; Transfection; YAP-Signaling Proteins

Although hyperactivated mTOR is well recognized as being pivotal to prostate cancer growth and progression, the underlying mechanisms by which it promotes such responses remain incompletely understood. Here, we show that rapamycin activates Smads 1 and 5 in human prostate cancer cells and tissues through blocking mTORC1 kinase. Small hairpin RNA-based gene silencing and gene overexpression approaches reveal that Smads 1 and 5 mediate, whereas Smad8 represses, rapamycin-induced cell death and expression of the bone morphogenetic protein (BMP) transcriptional target Id1 in human prostate cancer cell lines. Moreover, such phospho-Smad1/5-mediated rapamycin responses were blocked by LDN-193189 (a BMPRI kinase inhibitor) or Noggin (a BMP antagonist) in LNCaP prostate cancer cells. Likewise, the mTOR kinase inhibitors Ku-0063794 and WYE-354 each enhanced phosphorylation of Smad1/5. Intriguingly, silencing raptor alone enhanced, whereas silencing rictor repressed, the phosphorylation of Smad1/5, indicating that mTORC1 represses, whereas mTORC2 activates, BMP signaling. Immunohistochemical analysis showed increased levels of phospho-Smad1/5 concomitant with suppression of phospho-S6 and survivin levels in PC3 human prostate cancer xenografts in athymic mice administered rapamycin (intraperitoneally, 5 mg/kg/d, 2-6 days). Moreover, we show that compared with prostate tumor tissue from untreated patients, levels of phospho-Smad1/5 were significantly elevated in the prostate tumor tissue of patients with high-risk prostate cancer who received 8 weeks of the rapalog everolimus as part of a neoadjuvant clinical trial before undergoing local definitive therapy by radical prostatectomy. Taken together, our data implicate Smads 1, 5 and 8 as potential prognostic markers and therapeutic targets for mTOR inhibition therapy of prostate cancer.

Topics: Animals; Blotting, Western; Bone Morphogenetic Proteins; Cell Line, Tumor; Cell Survival; Cytostatic Agents; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Mice; Mice, Nude; Morpholines; Phosphorylation; Prostatic Neoplasms; Pyrazoles; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Sirolimus; Smad1 Protein; Smad5 Protein; Smad8 Protein; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Induction of new bone formation is frequently seen in the bone lesions from prostate cancer. However, whether osteogenesis is necessary for prostate tumor growth in bone is unknown. Recently, 2 xenografts, MDA-PCa-118b and MDA-PCa-133, were generated from prostate cancer bone metastases. When implanted subcutaneously in severe combined immunodeficient (SCID) mice, MDA-PCa-118b induced strong ectopic bone formation while MDA-PCa-133 did not. To identify the factors that are involved in bone formation, we compared the expression of secreted factors (secretome) from MDA-PCa-118b and MDA-PCa-133 by cytokine array. We found that the osteogenic MDA-PCa-118b xenograft expressed higher levels of bone morphogenetic protein BMP4 and several cytokines including interleukin-8, growth-related protein (GRO), and CCL2. We showed that BMP4 secreted from MDA-PCa-118b contributed to about a third of the osteogenic differentiation seen in MDA-PCa-118b tumors. The conditioned media from MDA-PCa-118b induced a higher level of osteoblast differentiation, which was significantly reduced by treatment with BMP4 neutralizing antibody or the small molecule BMP receptor 1 inhibitor LDN-193189. BMP4 did not elicit an autocrine effect on MDA-PCa-118b, which expressed low to undetectable levels of BMP receptors. Treatment of SCID mice bearing MDA-PCa-118b tumors with LDN-193189 significantly reduced tumor growth. Thus, these studies support a role of BMP4-mediated osteogenesis in the progression of prostate cancer in bone.

Topics: Adenocarcinoma; Animals; Autocrine Communication; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type I; Bone Neoplasms; Cell Line, Tumor; Culture Media, Conditioned; Cytokines; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, SCID; Neoplasm Proteins; Ossification, Heterotopic; Osteoblasts; Osteogenesis; Prostatic Neoplasms; Pyrazoles; Pyrimidines; Recombinant Proteins; Subcutaneous Tissue; Xenograft Model Antitumor Assays