Androgen deprivation-induced elevated nuclear SIRT1 promotes prostate tumor cell survival by reactivation of AR signaling.

The NAD+-dependent deacetylase, Sirtuin 1 (SIRT1) is involved in prostate cancer pathogenesis. However, the actual contribution is unclear as some reports propose a protective role while others suggest it is harmful. We provide evidence for a contextual role for SIRT1 in prostate cancer. Our data show that (i) mice orthotopically implanted with SIRT1-silenced LNCaP cells produced smaller tumors; (ii) SIRT1 suppression mimicked AR inhibitory effects in hormone responsive LNCaP cells; and (iii) caused significant reduction in gene signatures associated with E2F and MYC targets in AR-null PC-3 and E2F and mTORC1 signaling in castrate-resistant ARv7 positive 22Rv1 cells. Our findings further show increased nuclear SIRT1 (nSIRT1) protein under androgen-depleted relative to androgen-replete conditions in prostate cancer cell lines. Silencing SIRT1 resulted in decreased recruitment of AR to PSA enhancer selectively under androgen-deprivation conditions. Prostate cancer outcome data show that patients with higher levels of nSIRT1 progress to advanced disease relative to patients with low nSIRT1 levels. Collectively, we demonstrate that lowering SIRT1 levels potentially provides new avenues to effectively prevent prostate cancer recurrence.

Characterization of PacMetUT1, a recently isolated human prostate cancer cell line.

BACKGROUND: Existing prostate cancer cell lines have limitations. METHODS: Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors. RESULTS: We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p. CONCLUSION: The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.

A 90 kDa fragment of filamin A promotes Casodex-induced growth inhibition in Casodex-resistant androgen receptor positive C4-2 prostate cancer cells.

Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with anti-androgen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD+FlnA1-15) and 110 kDa fragments (FlnA16-24); the latter is further cleaved to a 90 kDa fragment (repeats 16-23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4-2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16-24 cDNA in C4-2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4-2 cells proliferate in androgen-reduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4-2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4-2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.

Arginine vasopressin stimulates mesangial cell proliferation by activating the epidermal growth factor receptor.

The potent vasoconstrictor arginine vasopressin (AVP) is also a mitogen for mesangial cells. Treatment with AVP decreased transit time through the cell cycle. AVP-stimulated mesangial cell growth by activating both the Ras mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) cell signaling pathways. Both the selective PI3K inhibitor LY-294002 and the MAPK kinase (MEK) inhibitor PD-98059 inhibited AVP-stimulated mesangial cell proliferation. However, LY-294002 was more potent, indicating an important role for PI3K activation in AVP-stimulated mesangial cell proliferation. AVP appeared to exert its effect on MAPK and PI3K activation, as well as on cell proliferation, by activating the epidermal growth factor receptor (EGF-R). Pretreatment with the tyrphostin-derived EGF-R antagonist AG-1478 inhibited mesangial cell proliferation as well as the activation of extracellular signal-regulated kinase 1/2 (ERK1/2 or p42/p44(MAPK)), and p70S6 kinase, a downstream effector of PI3K, providing evidence that MAPK and PI3K activation, respectively, occurred downstream of EGF-R activation. Treatment with rapamycin, an inhibitor of the p70S6 kinase activator mTOR, also resulted in growth inhibition, further suggesting the importance of the PI3K signaling pathway in AVP-induced proliferation. AVP treatment appeared to transactivate EGF-R by inducing tyrosine phosphorylation of the Ca(2+)/protein kinase C (PKC)-dependent nonreceptor tyrosine kinase, Pyk2, leading to Pyk2/c-Src association and c-Src activation. This was followed by association of c-Src with EGF-R and EGF-R activation. These data suggested that AVP-stimulated Pyk2 tyrosine phosphorylation to activate c-Src, thereby leading to EGF-R transactivation.

Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells.

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.