The issue at hand Mike is you have not posted anything to back up your assertions. How can you prove that you are correct without evidence? Your word means nothing without proof. Plain and simple.
Try that in a court of law or even any college classes, and see how far you get.
Does this study gives us to clues as to what Mike is referring to?
Metformin represses androgen-dependent and androgen-independent prostate cancers by targeting androgen receptor. - PubMed - NCBI
Prostate. 2015 Aug 1;75(11):1187-96. doi: 10.1002/pros.23000. Epub 2015 Apr 20.
Metformin represses androgen-dependent and androgen-independent prostate cancers by targeting androgen receptor.
Wang Y1, Liu G1, Tong D1, Parmar H2, Hasenmayer D2, Yuan W1, Zhang D2, Jiang J1.
Author information
Abstract
BACKGROUND:
Metformin has been reported to inhibit the growth of different types of cancers, including prostate cancer. We were interested to understand if the effect of metformin on prostate cancer is AR-dependent and, if so, whether metformin could act synergistically with the other anti-AR agents to serve as a therapeutic regimen with high efficacy and low toxicity.
METHODS:
Cell viabilities and apoptosis were determined by MTT assay and annexin V-FITC staining, respectively, when the two human prostate cancer cell lines, the androgen-dependent LNCaP and the androgen-independent 22RV1 were treated with metformin alone or in combination with bicalutamide. Quantitative RT-PCR and western blotting assays were conducted to examine metformin effects on AR mRNA and protein levels, respectively. Chromatin immunoprecipitation (ChIP) assays were conducted to confirm the recruitment of AR to the ARE(s) located on the promoter region of the AR target gene PSA.
RESULTS:
Metformin treatment reduced cell viability and enhanced apoptosis for both cell lines and additive effects were observed when LNCaP cells were treated with combined metformin and bicalutamide. Metformin down-regulated full-length AR protein in LNCaP cells. Both full-length and the truncated AR (AR-v7) were down-regulated by metformin in CWR22Rv1 cells. In both LNCaP and CWR22Rv1 cells, metformin repressed AR signaling pathway not by affecting AR protein degradation/stability, but rather through down-regulating the levels of AR mRNAs.
CONCLUSIONS:
Metformin represses prostate cancer cell viability and enhances apoptosis by targeting the AR signaling pathway. Combinations of metformin and other anti-AR agents pose a potentially promising therapeutic approach for treatment of prostate cancers, especially the castrate-resistant prostate cancer, with high efficacy and low toxicity.
© 2015 Wiley Periodicals, Inc.
http://www.cancerletters.info/article/S0304-3835(14)00499-6/abstract
SMILE upregulated by metformin inhibits the function of androgen receptor in prostate cancer cells
Highlights
•Metformin reduces the androgen-dependent growth of prostate cancer cells and the expression of endogenous AR target genes.
•Metformin induces SMILE expression in prostate cancer cells, of which knockdown with shRNA abolished the inhibitory effect of metformin on AR function.
•SMILE suppresses AR transactivation through physical interaction with AR.
•SMILE inhibits the expression of AR target genes and the androgen-dependent growth of prostate cancer cells.
Abstract
Metformin, a diabetes drug, has been reported to inhibit the growth of prostate cancer cells. In this study, we investigated the effect and action mechanism of metformin on the function of androgen receptor (AR), a key molecule in the proliferation of prostate cancer cells. Metformin was found to reduce androgen-dependent cell growth and the expression of AR target genes by inhibiting AR function in prostate cancer cells such as LNCaP and C4-2 cells. Interestingly, metformin upregulated the protein level of small heterodimer partner-interacting leucine zipper (SMILE), a coregulator of nuclear receptors, and knockdown of SMILE expression with shRNA abolished the inhibitory effect of metformin on AR function. Further studies revealed that SMILE protein itself suppressed the transactivation of AR, and its ectopic expression resulted in the repressed expression of endogenous AR target genes, PSA and NKX3.1, in LNCaP cells. In addition, SMILE protein physically interacted with AR and competed with the AR coactivator SRC-1 to modulate AR transactivation. As expected, SMILE repressed androgen-dependent growth of LNCaP and C4-2 cells. Taken together, these results suggest that SMILE, which is induced by metformin, functions as a novel AR corepressor and may mediate the inhibitory effect of metformin on androgen-dependent growth of prostate cancer cells.
Metformin anti-tumor effect via disruption of the MID1 translational regulator complex and AR downregulation in prostate cancer cells - Springer
Metformin anti-tumor effect via disruption of the MID1 translational regulator complex and AR downregulation in prostate cancer cells
Abstract
Background
Metformin is an approved drug prescribed for diabetes. Its role as an anti-cancer agent has drawn significant attention because of its minimal side effects and low cost. However, its mechanism of anti-tumour action has not yet been fully clarified.
Methods
The effect on cell growth was assessed by cell counting. Western blot was used for analysis of protein levels, Boyden chamber assays for analyses of cell migration and co-immunoprecipitation (CoIP) followed by western blot, PCR or qPCR for analysis of protein-protein and protein-mRNA interactions.
Results
Metformin showed an anti-proliferative effect on a wide range of prostate cancer cells. It disrupted the AR translational MID1 regulator complex leading to release of the associated AR mRNA and subsequently to downregulation of AR protein in AR positive cell lines. Inhibition of AR positive and negative prostate cancer cells by metformin suggests involvement of additional targets. The inhibitory effect of metformin was mimicked by disruption of the MID1-α4/PP2A protein complex by siRNA knockdown of MID1 or α4 whereas AMPK activation was not required.
Conclusions
Findings reported herein uncover a mechanism for the anti-tumor activity of metformin in prostate cancer, which is independent of its anti-diabetic effects. These data provide a rationale for the use of metformin in the treatment of hormone naïve and castration-resistant prostate cancer and suggest AR is an important indirect target of metformin.
**broken link removed**
Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling
Roberta Malaguarnera*, Antonella Sacco*, Alaide Morcavallo, Sebastiano Squatrito, Antimo Migliaccio, Andrea Morrione, Marcello Maggiolini, and Antonino Belfiore
We have previously demonstrated that, in prostate cancer cells, androgens up-regulate IGF-I receptor (IGF-IR) by inducing cAMP-response element-binding protein (CREB) activation and CREB-dependent IGF-IR gene transcription through androgen receptor (AR)-dependent membrane-initiated effects. This IGF-IR up-regulation is not blocked by classical antiandrogens and sensitizes cells to IGF-I-induced biological effects. Metformin exerts complex antitumoral functions in various models and may inhibit CREB activation in hepatocytes. We, therefore, evaluated whether metformin may affect androgen-dependent IGF-IR up-regulation. In the AR+ LNCaP prostate cancer cells, we found that metformin inhibits androgen-induced CRE activity and IGF-IR gene transcription. CRE activity requires the formation of a CREB-CREB binding protein-CREB regulated transcription coactivator 2 (CRTC2) complex, which follows Ser133-CREB phosphorylation. Metformin inhibited Ser133-CREB phosphorylation and induced nuclear exclusion of CREB cofactor CRTC2, thus dissociating the CREB-CREB binding protein-CRTC2 complex and blocking its transcriptional activity. Similarly to metformin action, CRTC2 silencing inhibited IGF-IR promoter activity. Moreover, metformin blocked membrane-initiated signals of AR to the mammalian target of rapamycin/p70S6Kinase pathway by inhibiting AR phosphorylation and its association with c-Src. AMPK signals were also involved to some extent. By inhibiting androgen-dependent IGF-IR up-regulation, metformin reduced IGF-I-mediated proliferation of LNCaP cells. These results indicate that, in prostate cancer cells, metformin inhibits IGF-I-mediated biological effects by disrupting membrane-initiated AR action responsible for IGF-IR up-regulation and suggest that metformin could represent a useful adjunct to the classical antiandrogen therapy.
Androgen stimulation is critical for growth and resistance to apoptosis in most early-stage prostate carcinomas, which, therefore, are responsive to androgen deprivation. However, the clinical benefits of androgen deprivation are temporary, and these carcinomas may eventually progress to castration-resistant tumors, for which no effective treatment is currently available. The molecular basis of androgen independency is incompletely understood.
In response to androgens, androgen receptors (ARs) regulate transcription by interacting with the androgen response elements located within the promoter regions of target genes and forming a multiprotein complex, which contains coactivators, corepressors, histone acetyltransferases, and histone deacetylases (1). However, increasing evidence suggests that the biological responses to androgens can be additionally mediated by membrane-initiated signals, which trigger rapid intracellular transduction pathways like ERK, phosphoinositide 3-kinase, protein kinase A, and protein kinase C, that may eventually activate gene transcription (2). Membrane-initiated androgen signals appear to be enhanced in malignant prostate cells by various mechanisms, including increased proportion of membrane-associated ARs and increased expression of kinases (eg, c-Src) and/or adaptors that contribute to the formation of multiprotein complexes with AR at the membrane level and trigger the activation of intracellular pathways (3).
Androgen activity itself may contribute to the progression to castration-resistant prostate cancer by up-regulating autocrine loops involving peptide growth factors and their cognate receptors (4).
In this context, we have previously found that androgens induce a selective up-regulation of the IGF-I receptor (IGF-IR) in prostate cancer cells and increase, in this way, cell proliferation and invasiveness in response to IGF-I (5). This effect occurs through the activation of membrane-initiated signals, which require the recruitment of membrane-bound AR to c-Src and subsequent activation of a downstream signaling pathway involving c-Src/ERK/cAMP-response element-binding protein (CREB) that eventually stimulates the activity of the IGF-IR promoter (5, 6). This mechanism may open a new approach to prostate cancer therapy, because it is poorly affected by classic antiandrogens but can be blocked by CREB silencing or by inhibitors of the c-Src/ERK pathway (6). The transcriptional activity of CREB-dependent target genes requires the formation of the CREB-CREB binding protein (CBP)-CREB regulated transcription coactivator 2 (CRTC2) complex (7). In particular, AMPK phosphorylates CRTC2 at Ser171 causing its interaction with 14–3-3 proteins and sequestration in the cytoplasm. Glucose and hormones lead to the dephosphorylation of CRTC2, its dissociation from 14–3-3 proteins, and as a consequence, its translocation to the nucleus, where it binds CREB and promotes CREB-dependent transcription. Metformin may additionally disrupt the CREB-CBP-CRTC2 complex by inducing CBP phosphorylation at Ser436 (7).
In recent years, the biguanide metformin, widely used as antidiabetic drug, has raised much interest for its anticancer potential (8, 9). Indeed, metformin has shown antiproliferative effects in several cancer cells, including prostate cancer cells (10, 11). Interestingly, prostate cancer cells appear to be more sensitive to metformin than normal epithelial prostate cells. In vivo, metformin increases the response of prostate cells xenografts to the antiandrogen bicalutamide (12). Anticancer effects of metformin are mostly attributed to its ability to activate AMPK, which, in turn, down-regulates mammalian target of rapamycin complex 1 (mTORC1) signaling, an essential regulator of cell growth and proliferation (8). Metformin has, however, pleiotropic effects and may additionally inhibit mTORC1 through AMPK-independent pathways (13, 14). Metformin may also target ERK signaling (15), reduce Ca(2+)-dependent protein kinase Cα/ERK and c-Jun N-terminal kinase/activator protein-1 signaling pathways (16, 17), and also inhibit Akt (protein kinase B [PKB]) activity through serine phosphorylation of insulin receptor substrate-1 (18).
Because metformin is able to inhibit multiple signaling pathways, we aimed at evaluating whether, in prostate cancer cells, metformin may also affect the membrane-initiated effects of androgens, which lead to IGF-IR up-regulation and increased sensitivity to IGF-I. Here, we show that metformin inhibits androgen-induced IGF-IR up-regulation and the resulting mitogenic and invasive effects of IGF-I through multiple mechanisms. These include the inhibition of transcriptional activity of CREB-CBP-CRTC2 complex, the inhibition of mTORC1/p70S6 kinase (p70S6K) pathway, and the disruption of AR/c-Src interaction.
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In conclusion, we have identified a novel potential antitumor effect of metformin operating in AR+ prostate cancer cells and involving the inhibition of membrane-initiated androgen effects, adding to the list of the numerous antitumor effects of metformin observed in several in vitro and in vivo model systems. We have especially characterized the action of metformin in blocking androgen-induced IGF-IR up-regulation and IGF-I-mediated biological effects. However, given that metformin inhibits a very early step of membrane-initiated androgen effects, ie, the association between Src and AR and AR phosphorylation, there is the possibility that metformin may also block other membrane-initiated effects of androgens. Several evidences indicate that membrane-initiated androgen effects are enhanced in cancer cells and may play a role in tumor progression (1–3). Our present findings, therefore, open the way to a deeper understanding of the potential effects of metformin in prostate cancer.
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