BACKGROUND The majority of the prostatic cancers are adenocarcinomas characterized by glandular formation and the expression of luminal differentiation markers androgen receptor (AR) and prostate-specific antigen (PSA). Most adenocarcinomas are indolent and androgen-dependent. Hormonal therapy that inhibits AR signaling produces symptomatic relief in patients with advanced and metastatic adenocarcinomas. Prostatic small cell neuroendocrine carcinoma (SCNC) is a variant form of prostate cancer (PC). In contrast to adenocarcinoma, the tumor cells of SCNC do not form glands and are negative for AR and PSA. SCNC is extremely aggressive and does not respond to hormonal therapy. The purpose of this study was to compare the important and relevant features of two most commonly used PC cell lines, LNCaP and PC3, with prostatic adenocarcinoma and SCNC. METHODS Xenograft tumors of LNCaP and PC3 were prepared and compared with human prostatic adenocarcinoma and SCNC for the expression of key signaling molecules by immunohistochemistry and Western blot analysis. RESULTS LNCaP cells express AR and PSA and their growth is inhibited by androgen withdrawal, similar to human prostatic adenocarcinoma. PC3 cells do not express AR and PSA and their proliferation is independent of androgen, similar to SCNC. Adenocarcinoma cells and LNCaP cells are negative for neuroendocrine markers and stem cell-associated marker CD44 while SCNC and PC3 cells are positive. LNCaP cells have identical cytokeratin profiles to adenocarcinoma while PC3 cells have cytokeratin profiles similar to SCNC. CONCLUSION LNCaP cells share common features with adenocarcinoma while PC3 cells are characteristic of SCNC.

Drosophila Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the Drosophila transforming growth factor ␤ (TGF-␤)͞bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-␤ signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-␤ receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-␤ treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-␤-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-␤ in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-␤ signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-␤ receptor complex is an essential step in signal transduction by TGF-␤ for both inhibition of cell proliferation and activation of the PAI-1 promoter.

Transforming growth factor ␤ (TGF-␤) regulates a variety of physiologic processes, including growth inhibition, differentiation, and induction of apoptosis. Some TGF-␤-initiated signals are conveyed through Smad3; TGF-␤ binding to its receptors induces phosphorylation of Smad3, which then migrates to the nucleus where it functions as a transcription factor. We describe here the association of Smad3 with the nuclear protooncogene protein SnoN. Overexpression of SnoN represses transcriptional activation by Smad3. Activation of TGF-␤ signaling leads to rapid degradation of SnoN and, to a lesser extent, of the related Ski protein, and this degradation is likely mediated by cellular proteasomes. These results demonstrate the existence of a cascade of the TGF-␤ signaling pathway, which, upon TGF-␤ stimulation, leads to the destruction of protooncoproteins that antagonize the activation of the TGF-␤ signaling.

TGF-beta treatment of cells induces a variety of physiologic responses, including growth inhibition, differentiation, and induction of apoptosis. TGF-beta induces phosphorylation and nuclear translocation of Smad3. We describe here the association of Smad3 with the nuclear protooncogene protein Ski in response to the activation of TGF-beta signaling. Association with Ski represses transcriptional activation by Smad3, and overexpression of Ski renders cells resistant to the growth-inhibitory effects of TGF-beta. The transcriptional repression as well as the growth resistance to TGF-beta by overexpression of Ski can be overcome by overexpression of Smad3. These results demonstrate that Ski is a novel component of the TGF-beta signaling pathway and shed light on the mechanism of action of the Ski oncoprotein.

Summary The ERG gene is fused to TMPRSS2 in approximately 50% of prostate cancers (PrCa) resulting in its overexpression. However, whether this is the sole mechanism underlying ERG elevation in PrCa is currently unclear. Here we report that ERG ubiquitination and degradation is governed by the Cullin 3-based ubiquitin ligase SPOP and that deficiency in this pathway leads to aberrant elevation of the ERG oncoprotein. Specifically, we find that truncated ERG (ΔERG), encoded by the ERG fusion gene, is stabilized by evading SPOP-mediated destruction, while prostate cancer-associated SPOP mutants are also deficient in promoting ERG ubiquitination. Furthermore, we show that SPOP/ERG interaction is modulated by CKI-mediated phosphorylation. Importantly, we demonstrate that DNA damage drug, topoisomerase inhibitors, can trigger CKI activation to restore the SPOP/ΔERG interaction and its consequent degradation. Thus SPOP functions as a tumor suppressor to negatively regulate the stability of the ERG oncoprotein in prostate cancer.

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