Does androgen-ablation therapy (AAT) associated autophagy have a pro-survival effect in LNCaP human prostate cancer cells?

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Androgen-ablation therapy (AAT) and chemotherapy are commonly used to treat incurable prostate cancer. To improve outcome, there is major on-going research to develop more effective treatments with less toxicity. Autophagy has been suggested from previous studies to play a potential role in cell survival and may be associated with resistance to chemotherapy. Autophagy is known to be upregulated by nutrient starvation or AAT in prostate cancer. However, its functional impact is not fully known. The present study describes the potential synergism between the blockade of autophagy and AAT alone or AAT combined with taxane chemotherapy. Hence, future combined treatment options are warranted to further investigate the clinical impact of autophagy suppression as a treatment strategy. OBJECTIVE: To study the cellular effects of the anti-androgen bicalutamide on autophagy and its potential impact on response to androgen-ablation therapy (AAT) alone or combined with docetaxel chemotherapy in human prostate cancer LNCaP cells. MATERIALS AND METHODS: LNCaP cells were treated with bicalutamide ± docetaxel, and cellular effects were assayed: lipidated LC3 (a microtubule-associated protein) for autophagy and its trafficking to fuse with lysosome; flow cytometry using propidium iodide or caspase 3 for cell death; and sulforhodamine B assay for cell growth. RESULTS: Bicalutamide treatment enhanced autophagy in LNCaP cells with increased level of autophagosome coupled with an altered cellular morphology reminiscent of neuroendocrine differentiation. Consistent with the literature on the interaction between androgen receptor activation and taxane chemotherapy, bicalutamide diminished docetaxel mediated cytotoxicity. Significantly, pharmacological inhibition of autophagy with 3-methyladenine significantly enhanced the efficacy cell kill mediated by AAT ± docetaxel. CONCLUSION: Autophagy associated with bicalutamide treatment in LNCaP cells may have a pro-survival effect and strategy to modulate autophagy may have a potential therapeutic value.

GRP78 up-regulation is associated with androgen receptor status, Hsp70-Hsp90 client proteins and castrate-resistant prostate cancer.

GRP78/BiP is a key member of the molecular chaperone heat shock protein (Hsp) 70 family. It has a critical role in prostate cancer (PC) including Pten loss-driven carcinogenesis, but the molecular basis of this remains unclear. We investigated the effect of GRP78 and its putative client proteins, including androgen receptor (AR) in clinical PC. Expression of GRP78 and key Hsp70-hsp90 client proteins (HER2, HER3, AR and AKT) were studied in an incidence tissue microarray (TMA) of prostate cancer. The relationship of GRP78 and AR was further tested in in vitro cell models (LNCaP and its derived LNCaP-CR subclone) and a matched TMA of hormone-naïve (HNPC) and castrate-resistant prostate cancer (CRPC). In vitro and in vivo expression of GRP78 and client proteins were assessed by western blotting and immunohistochemistry, respectively, using the weighted histoscore method. Significant co-expression of GRP78, pAKT, HER2, HER3 and AR was observed in PC. Abnormal AR, GRP78 and pAKT expression have significant impact on patient survival. GRP78 expression in AR(+) tumours was significantly higher than in AR(-) tumours. In keeping with our clinical data, activation of AR by dihydrotestosterone (DHT) potently activated GRP78 expression in both LNCaP and LNCaP-CR cells. For the first time, using a matched HNPC and CRPC TMA, enhanced cytoplasmic and membranous GRP78 expression was observed in CRPC. Future prospective studies are therefore warranted to validate GRP78 as prognostic marker and therapeutic target, in the context of the AR and pAKT status. In summary, GRP78 is co-expressed with Hsp70-hsp90 client proteins. Up-regulated expression of AR and GRP78 expression in untreated prostate cancer predicts a less favourable outcome. This points to the importance of understanding in the molecular interaction among AR, GRP78 and AKT.

Estrogen receptor expression in lumbosacral dorsal root ganglion cells innervating the female rat urinary bladder.

We have investigated whether bladder afferent neurons are likely to be targets for circulating estrogens by mapping estrogen receptor (ER) distribution in lumbosacral dorsal root ganglia (DRG) of adult female rats. Sensory neurons innervating either the detrusor or trigone regions were identified by application of fluorescent retrograde tracer dyes to the bladder wall. Labelled neurons were classified by their immunoreactivity for either type of ER (ERalpha or ERbeta) and further compared with subpopulations of neurons containing substance P, calcitonin gene-related peptide and vanilloid receptor (a marker of polymodal nociceptors). Both ER types were expressed in numerous sensory neurons of either upper lumbar (L1/L2) or lower lumbar/sacral (L6/S1) ganglia and there was almost complete coexpression of ERalpha and ERbeta. ER-positive neurons were mainly small-medium size (18-25-microm diameter), indicating that they may be nociceptors and/or supply visceral targets. Most bladder-projecting neurons expressed ERs and the majority of these also expressed neuropeptides or vanilloid receptor. Afferent neurons supplying detrusor and trigone regions had similar immunohistochemical features. About a third of the bladder-projecting neurons expressed both ER and vanilloid receptor, suggesting a mechanism by which estrogens could influence bladder pain. The prevalence of different chemical classes of ER-positive bladder-projecting neurons was reflected throughout the entire population of neurons in dorsal root ganglia of these spinal levels, suggesting that neurons supplying other pelvic visceral targets may have similar chemical profiles. These results suggest that many functional classes of sensory neurons innervating the lower urinary tract are likely to be targets for circulating estrogens, including many nociceptor neurons. The coexistence of ERalpha and ERbeta suggests a broad range of potential mechanisms by which estrogens may exert their genomic effects in this system.

Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation.

The docking protein Gab2 is overexpressed in several human malignancies, including breast cancer, and is associated with increased metastatic potential. Here we report that Gab2 overexpression in MCF-10A mammary epithelial cells led to delayed cell spreading, a decrease in stress fibers and mature focal adhesions, and enhanced cell migration. Expression of a Gab2 mutant uncoupled from 14-3-3-mediated negative feedback (Gab2(2xA)) led to a more mesenchymal morphology and acquisition of invasive potential. Expression of either Gab2 or Gab2(2xA) led to decreased activation of RhoA, but only the latter increased levels of Rac-GTP. Expression of constitutively active RhoA in MCF-10A/Gab2 cells restored stress fibers and focal adhesions, indicating that Gab2 signals upstream of RhoA to suppress these structures. Mutation of the two Shp2-binding sites to phenylalanine (Gab2(ΔShp2)) markedly reduced the effects of Gab2 on cellular phenotype and RhoA activation. Expression of Gab2 or Gab2(2xA), but not Gab2(ΔShp2), promoted Vav2 phosphorylation and plasma membrane recruitment of p190A RhoGAP. Knockdown of p190A RhoGAP reversed Gab2-mediated effects on stress fibers and focal adhesions. The identification of a novel pathway downstream of Gab2 involving negative regulation of RhoA by p190A RhoGAP sheds new light on the role of Gab2 in cancer progression.

Increased proliferation and altered growth factor dependence of human mammary epithelial cells overexpressing the Gab2 docking protein.

The docking protein Gab2 is a proto-oncogene product that is overexpressed in primary breast cancers. To determine the functional consequences of Gab2 overexpression, we utilized the immortalized human mammary epithelial cell line MCF-10A. In monolayer culture, expression of Gab2 at levels comparable with those detected in human breast cancer cells accelerated epidermal growth factor (EGF)-induced cell cycle progression and was associated with increased basal Stat5 tyrosine phosphorylation and enhanced and/or more sustained EGF-induced Erk and Akt activation. Three-dimensional Matrigel culture of MCF-10A cells resulted in the formation of polarized, growth-arrested acini with hollow lumina. Under these conditions, Gab2 increased cell proliferation during morphogenesis, leading to significantly larger acini, an effect dependent on Gab2 binding to Grb2 and Shp2 and enhanced by recruitment of the p85 subunit of phosphatidylinositol 3-kinase. Pharmacological inhibition of MEK revealed that, in addition to direct activation of phosphatidylinositol 3-kinase, increased Erk signaling also contributed to Gab2-mediated enhancement of acinar size. In addition, Gab2 overcame the proliferative suppression that normally occurs in late stage cultures and conferred independence of the morphogenetic program from exogenous EGF. Finally, higher levels of Gab2 expression led to the formation of large disorganized structures with defective luminal clearance. These findings support a role for Gab2 in mammary tumorigenesis.