Activation of the mammalian target of rapamycin signalling pathway in prostate cancer and its association with patient clinicopathological characteristics

OBJECTIVE

To evaluate the activation level of the mammalian target of rapamycin (mTOR) signalling pathway in Chinese patients with prostate cancer, as this pathway is over‐activated in many human cancers and is an attractive target for cancer therapy.

PATIENTS AND METHODS

We used immunohistochemistry to investigate the activation level of five important markers of the mTOR pathway, including PTEN, p‐Akt, p‐mTOR, p‐p70S6K and p‐4E‐BP1, in tissues from 182 patients with prostate cancer, 20 with benign prostatic hyperplasia (BPH) and 10 with high‐grade prostatic intraepithelial neoplasia (HGPIN). The expression levels of these five markers were associated with patient clinical and pathological characteristics.

RESULTS

Expression levels of p‐Akt, p‐mTOR, p‐4E‐BP1 and p‐p70S6K were significantly higher in prostate cancer tissues than in BPH and HGPIN tissues. In 182 patients with prostate cancer the p‐mTOR expression level significantly and positively correlated with its upstream p‐Akt and downstream p‐4E‐BP1 and p‐p70S6K expression levels. The cancer Gleason score was significantly correlated with p‐Akt and p‐mTOR expression level but not with p‐4E‐BP1 and p‐p70S6K expression level. However, the p‐4E‐BP1and p‐p70S6K expression levels in primary cancer lesions were statistically significantly correlated with patient T stage and distant metastases.

CONCLUSIONS

Most patients with prostate cancer have at least one component of the mTOR signalling pathway activated. The activation of the mTOR pathway might be involved in prostate cancer development and progression. The association between activation of mTOR pathway and patient clinicopathological variables suggested that not all patients are equally amenable to treatment strategies targeting the mTOR pathway.     

Expression of mTOR signaling pathway markers in prostate cancer progression

BACKGROUND: The PI3K/AKT/mTOR pathway is central to prostate cancer progression. A preliminary investigation of immuno-histochemical expression of mammalian target of rapamycin (mTOR) pathway markers was undertaken to identify patterns of expression in prostate tissue. METHODS: Immunohistochemistry was performed on a custom-made prostate tissue array. Mean long scores and variability of long scores for each marker were recorded for normal lumenal cells, prostate intraepithelial neoplasia (PIN), and cancer. RESULTS: Expression of PTEN decreased and mTOR signaling pathway markers increased in PIN and in cancer as compared to normal cells in the majority of samples. Overexpression of 4E-BP1 and p-4E-BP1 was observed in PIN and cancer. However, in cancer, the overexpression of 4E-BP1 was significantly higher than with any other marker. DISCUSSION: Results suggest that 4E-BP1 overexpression is strongly associated with prostate cancer, especially when combined with PTEN and mTOR expression data. Hierarchical clustering analysis utilizing PTEN, mTOR, and 4E-BP1 separated normal from cancer cell populations in most cases.     

Interleukin-4 stimulates androgen-independent growth in LNCaP human prostate cancer cells

BACKGROUND: Clinical data showed that the levels of interleukin-4 (IL-4) are significantly elevated in serum of patients with ablation resistant prostate cancer. Previous studies demonstrated that IL-4 enhances androgen receptor (AR) activation mediated by NF-kappaB in the absence or in the very low levels of androgen in prostate cancer cells. In this study, the role of IL-4 in promoting the growth of androgen-independent prostate cancer cells was examined. METHODS: LNCaP cells were transfected with a full-length IL-4 cDNA and stable clones expressing IL-4 were selected. The growth of LNCaP cells expressing IL-4 was analyzed in vitro and in vivo both in the presence and absence of androgen. RESULTS: Overexpression of IL-4 enhances the growth of androgen-sensitive LNCaP cells in culture media containing charcoal-stripped FBS condition (CS-FBC), and increases the sensitivity of LNCaP cells in response to androgen stimulation. The DHT-mediated cell growth could not be blocked by bicalutamide in IL-4 overexpressing LNCaP cells, but can be neutralized by bicalutamide in parental LNCaP and neo control cells. Furthermore, overexpression of IL-4 stimulates tumor growth of androgen-sensitive LNCaP cells both in intact and castrated male mice. CONCLUSIONS: Overexpression of IL-4 increases the sensitivity of androgen-sensitive LNCaP prostate cancer cells in response to androgen stimulation and enhances the growth of LNCaP cells both in the presence and absence of androgen in vitro and in vivo. These studies suggest that IL-4 plays an important role in promoting androgen-independent prostate cancer growth.     

In vivo and in vitro studies of anticancer actions of alpha-TEA for human prostate cancer cells

BACKGROUND: Vitamin E analog, 2,5,7,8-tetramethyl-2R-(4R,8R, 12-trimethyltridecyl) chroman-6-yloxyacetic acid, referred to as alpha-TEA induces apoptosis in a variety of human cancer cells in cell culture and reduces tumor burden and metastases in preclinical animal models of breast and ovarian cancer. The goal of this study was to determine in vivo anticancer efficacy of alpha-TEA against human prostate cancer cells and identify mechanisms of action. METHODS: A PC-3-GFP xenograft model was used to assess the effects of alpha-TEA formulated in liposomes and administered orally on tumor burden and metastases. Tumor tissue was examined by immunohistochemical staining for percentage of cells undergoing apoptosis by TUNEL or cell proliferation by Ki-67. In vitro analyses of mechanisms employed western immunoblotting to examine effects of alpha-TEA-treatments in LNCaP and PC-3-GFP cells on levels of pro-survival and pro-death factors. Functional significance was determined using ectopically expressed constitutively active forms, inhibitors, or siRNA. RESULTS: alpha-TEA significantly reduced tumor burden and metastases, increased apoptosis and decreased proliferation of tumor cells (P < 0.05). alpha-TEA treatment of both LNCaP and PC-3-GFP cells in vitro reduced levels of pAkt1, pAkt2; FOXO1, c-FLIP(L) and survivin. Constitutively active Akt1, Akt2, c-FLIP or survivin reduced alpha-TEA-induced apoptosis. PI3K inhibitor enhanced apoptosis. Constitutively active FOXO1 enhanced alpha-TEA induced Fas ligand expression; whereas, FOXO1 siRNA reduced alpha-TEA induced Fas ligand expression. CONCLUSIONS: alpha-TEA is an effective anticancer agent for human prostate cancer cells. Downregulation of pro-survival and upregulation of pro-death factors play roles in alpha-TEA-induced apoptosis.     

Akt down-modulation induces apoptosis of human prostate cancer cells and synergizes with EGFR tyrosine kinase inhibitors

BACKGROUND: PTEN is a well-characterized tumor suppressor that negatively regulates cell growth and survival through the modulation of PI3K/Akt pathway. METHODS: In this paper, we investigated the effects of an PI3K/Akt inhibitor, perifosine, in human prostate cancer (PCa) cells analyzing cell proliferation, apoptosis, and the synergy with EGFR inhibitors. RESULTS: Clinically achievable concentrations of perifosine, as well as Akt gene knockdown, induced a G0/G1 arrest and apoptosis in PTEN defective PCa cells. Although PTEN introduction was able to restore the control of Akt activity and to reduce cell proliferation, the manipulation of PTEN gene was not able alone to influence apoptosis. Perifosine induced apoptotic program also in PTEN positive cells when Akt activity was augmented by EGF suggesting the possibility that this drug could be used in combination with EGFR inhibitors. The combination treatment between erlotinib and pharmacological or molecular Akt knockdown, indeed, showed synergistic effects. This is the first demonstration that a pharmacological compound against Akt activity can restore the efficacy against EGFR inhibitors in PCa and has important therapeutic fallout since EGFR inhibitors have demonstrated very low effectiveness in PCa patients. CONCLUSIONS: Taken together our data have an important clinical relevance in the treatment of advanced prostate tumors. However, further studies in the setting of combination therapies in advanced PCas are necessary.     

Down-regulation of human X-box binding protein 1 (hXBP-1) expression correlates with tumor progression in human prostate cancers

BACKGROUND: In our previous study, the gene encoding the human X-box binding protein 1 (hXBP-1) was isolated as a down-regulated gene in advanced prostate cancers using cDNA-representational difference analysis (RDA). In the present investigation, we characterized alterations of hXBP-1 in prostate cancer specimens. METHODS: Expression patterns of hXBP-1 in a series of human prostate cancers were examined by Northern blotting, mRNA in situ hybridization or immunohistochemistry. Loss of heterozygosity (LOH) analysis using microsatellite markers and gene mutation analysis in the hXBP-1 region were also performed. RESULTS: Expression of hXBP-1 was localized in epithelial and adenocarcinoma cells of the prostate. An inverse correlation between hXBP-1 expression and histological differentiation was found in a series of prostate cancers without hormonal therapy. Majority of refractory cancer cases exhibited weak hXBP-1 expression. No allelic loss or gene mutations were found in the hXBP-1 region and its open reading frame, respectively, in the prostate cancer examined. CONCLUSIONS: These results suggest that reduction of hXBP-1 expression may be a useful marker for prostate adenocarcinoma differentiation and progression.     

Preferential chemosensitization of PTEN-mutated prostate cells by silencing the Akt kinase

BACKGROUND: In prostate cancer, mutations of the phosphatase PTEN can activate the kinase cascade PI3K/Akt/mTOR which induces drug resistance. METHODS: Chemosensitization by siRNA targeting Akt was studied in HEK293 cells forced to express CA-Akt or kinase-dead DN-Akt. To decrease drug resistance, Akt was silenced with siRNA in human prostate DU-145 cell line expressing the normal PTEN or in LNCaP and PC3 cell lines expressing mutated-PTEN. Taxol was used for the chemosensitization studies. RESULTS: Silencing Akt in the drug-resistant CA-Akt cells efficiently sensitized cells to antitubule agents, whereas silencing drug-responsive DN-Akt cells did not. Only minor effects were obtained in wild-type HEK293 cells. Potentiation by siRNA of taxol cytotoxicity was significantly greater in mutated-PTEN cells than in prostate cells expressing wild-type PTEN. The apoptotic program induced by taxol was preferentially potentiated by Akt siRNA in PTEN-mutated cell lines as regards the DU-145 cell line. CONCLUSIONS: Silencing Akt in PTEN-mutated prostate cancer cells enhances the antitumor effects of taxol. No siRNA chemosensitization was obtained in prostate cells with wild type PTEN.