Hedgehog is involved in prostate basal cell hyperplasia formation and its progressing towards tumorigenesis

The role of Hedgehog signaling in human basal cell hyperplasia formation and its progressing towards tumorigenesis was investigated. Hedgehog signaling members including PTCH1, GLI1, GLI2, and GLI3 were found co-localized with p63 expression in most hyperplastic basal cells, but rarely in normal basal cells, suggesting Hedgehog involvement in basal cell hyperplasia formation. Both CK-14 and CK-8 markers were found co-localized in the majority of hyperplastic basal cells, but relatively few in the normal basal cells, indicating a Hedgehog-promoted transitory differentiation. Furthermore, CK-14 and PTCH1 were found co-localized with CD44 in the hyerplastic basal cells, in a way similar to the CD44 co-localization with PTCH1 and GLI1 in the cancer cells. Together, the present study indicated Hedgehog involvement in forming basal cell hyperplasia and its progressing towards cancer, presumably by transforming the normal basal stem cells into the cancer stem cells where persistent Hedgehog activation might be mandatory for tumorigenesis.     

Expression of prostate specific membrane antigen (PSMA) in prostatic adenocarcinoma and prostatic intraepithelial neoplasia

The prostatic membrane antigen (PSMA) is a protein that is expressed in the prostatic epithelium. We studied the expression of PSMA in a series of 55 patients with different stages of prostate cancer and we compared the PSMA staining in prostate cancer cells, in high-grade prostatic intraepithelial neoplasia (PIN) and in histologically benign prostatic epithelium for the same specimen. For this purpose archival paraffin-embedded specimens were studied by immunohistochemistry with a monoclonal antibody 7E11-C5.3 against PSMA using the streptavidin-biotin method. The mean percentage of PSMA immunoreactivity was 56.67% in prostate cancer (CaP) cells, and 48.6% in PIN cells, which was significantly higher than benign-appearing prostatic epithelium (5.72%) (for each pair, p<0.001). PSMA expression was greater in CaP with a higher Gleason score (p=0.01), but no relationship was found with serum PSA value. We conclude that PSMA overexpression is detected in high-grade PIN and is associated with a higher Gleason score of prostate cancer. It is a potential marker for studying carcinogenesis and progression of prostate cancer.     

Acquisition of epithelial–mesenchymal transition and cancer stem cell phenotypes is associated with activation of the PI3K/Akt/mTOR pathway in prostate cancer radioresistance

Radioresistance is a major challenge in prostate cancer (CaP) radiotherapy (RT). In this study, we investigated the role and association of epithelial–mesenchymal transition (EMT), cancer stem cells (CSCs) and the PI3K/Akt/mTOR signaling pathway in CaP radioresistance. We developed three novel CaP radioresistant (RR) cell lines (PC-3RR, DU145RR and LNCaPRR) by radiation treatment and confirmed their radioresistance using a clonogenic survival assay. Compared with untreated CaP-control cells, the CaP-RR cells had increased colony formation, invasion ability and spheroid formation capability (P<0.05). In addition, enhanced EMT/CSC phenotypes and activation of the checkpoint proteins (Chk1 and Chk2) and the PI3K/Akt/mTOR signaling pathway proteins were also found in CaP-RR cells using immunofluorescence, western blotting and quantitative real-time PCR (qRT-PCR). Furthermore, combination of a dual PI3K/mTOR inhibitor (BEZ235) with RT effectively increased radiosensitivity and induced more apoptosis in CaP-RR cells, concomitantly correlated with the reduced expression of EMT/CSC markers and the PI3K/Akt/mTOR signaling pathway proteins compared with RT alone. Our findings indicate that CaP radioresistance is associated with EMT and enhanced CSC phenotypes via activation of the PI3K/Akt/mTOR signaling pathway, and that the combination of BEZ235 with RT is a promising modality to overcome radioresistance in the treatment of CaP. This combination approach warrants future in vivo animal study and clinical trials.     

Role of epithelial cell fibroblast growth factor receptor substrate 2alpha in prostate development, regeneration and tumorigenesis

The fibroblast growth factor (FGF) regulates a broad spectrum of biological activities by activation of transmembrane FGF receptor (FGFR) tyrosine kinases and their coupled intracellular signaling pathways. FGF receptor substrate 2alpha (FRS2alpha) is an FGFR interactive adaptor protein that links multiple signaling pathways to the activated FGFR kinase. We previously showed that FGFR2 in the prostate epithelium is important for branching morphogenesis and for the acquisition of the androgen responsiveness. Here we show in mice that FRS2alpha is uniformly expressed in the epithelial cells of developing prostates, whereas it is expressed only in basal cells of the mature prostate epithelium. However, expression of FRS2alpha was apparent in luminal epithelial cells of regenerating prostates and prostate tumors. To investigate FRS2alpha function in the prostate, the Frs2alpha alleles were ablated specifically in the prostatic epithelial precursor cells during prostate development. Similar to the ablation of Fgfr2, ablation of Frs2alpha disrupted MAP kinase activation, impaired prostatic ductal branching morphogenesis and compromised cell proliferation. Unlike the Fgfr2 ablation, disrupting Frs2alpha had no effect on the response of the prostate to androgens. More importantly, ablation of Frs2alpha inhibited prostatic tumorigenesis induced by oncogenic viral proteins. The results suggest that FRS2alpha-mediated signals in prostate epithelial cells promote branching morphogenesis and proliferation, and that aberrant activation of FRS2-linked pathways might promote tumorigenesis. Thus, the prostate-specific Frs2alpha(cn) mice provide a useful animal model for scrutinizing the molecular mechanisms underlying prostatic development and tumorigenesis.     

Caveolin-1 upregulation contributes to c-Myc-induced high-grade prostatic intraepithelial neoplasia and prostate cancer

Previously we reported caveolin-1 (Cav-1) overexpression in prostate cancer cells and showed that it promotes prostate cancer progression. Here, we report that Cav-1 was overexpressed in 41.7% (15 of 36) of human high-grade prostatic intraepithelial neoplasia (HGPIN) specimens obtained during radical prostatectomies. Positive correlations exist between Cav-1-positive (Cav-1(+)) HGPIN and Cav-1(+) primary prostate cancer (rho = 0.655, P < 0.0001) and between Cav-1 and c-Myc expression in HGPIN (rho = 0.41, P = 0.032). To determine whether Cav-1 cooperates with c-Myc in development of premalignant lesions and prostate cancer in vivo, we generated transgenic mice with c-Myc overexpression driven by the ARR(2)PB promoter. In this ARR(2)PB-c-myc model, Cav-1 overexpression was found in mouse PIN (mPIN) lesions and prostate cancer cells and was associated with a significantly higher ratio of proliferative to apoptotic labeling in mPIN lesions than in the Cav-1-negative epithelia adjacent to those lesions (10.02 vs. 4.34; P = 0.007). Cav-1 overexpression was also associated with increased levels of P-Akt and VEGF-A, which were previously associated with Cav-1-induced prostate cancer cell survival and positive feedback regulation of cellular Cav-1 levels, respectively. In multiple prostate cancer cell lines, Cav-1 protein (but not mRNA) was induced by c-Myc transfection, whereas VEGF siRNA transfection abrogated c-Myc-induced Cav-1 overexpression, suggesting a c-Myc-VEGF-Cav-1 signaling axis. Overall, our results suggest that Cav-1 is associated with c-Myc in the development of HGPIN and prostate cancer. Furthermore, Cav-1 overexpression in HGPIN is potentially a biomarker for early identification of patients who tend to develop Cav-1(+) primary prostate cancer.     

Conditional expression of the androgen receptor induces oncogenic transformation of the mouse prostate

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.     

Forkhead‐box R2 promotes metastasis and growth by stimulating angiogenesis and activating hedgehog signaling pathway in ovarian cancer

Overexpression of forkhead‐box R2 (FoxR2) is related to metastasis and progression of tumor. However, its biological functions in ovarian cancer (OC) progression remain unclear. Herein, we aimed to explore the changes in biological functions and molecular events related to FoxR2 overexpression. We found that FoxR2 is upregulated frequently in OC where these events are associated with worse histologic grade and poor survival. Enhanced expression of FoxR2 was related to cell growth, migration, and epithelial‐mesenchymal transition, whereas silencing of FoxR2 suppressed these malignant phenotypes. In addition, angiogenesis was stimulated by FoxR2 overexpression by enhancing vascular endothelial growth factor expression. Further mechanistic investigations revealed that the increase in cell surface FoxR2 promoted sonic hedgehog binding and signaling. Inhibiting hedgehog pathway with sonidegib decreased FoxR2‐induced migration and lung metastasis of OC cells, establishing the critical role of hedgehog signaling in mediating the effects of FoxR2 expression. Taken together, our results indicate that FoxR2 overexpression in OC contributes to malignant behavior in cancer cells, at least in part through stimulating angiogenesis and activation of the hedgehog signaling pathway. Hedgehog signaling pathway activation may be the key in tumor progression mediated by FoxR2.     

Garcinone C suppresses colon tumorigenesis through the Gli1-dependent hedgehog signaling pathway

BackgroundAlthough garcinone C, a natural xanthone derivative identified in the pericarp of Garcinia mangostana, has been demonstrated to exert different health beneficial activities in oxidative stress and β-amyloid aggregation, the role of garcinone C in colon tumorigenesis has not been investigated. In addition, aberrant Hedgehog (Hh) signaling activation is associated with tumorigenesis including colon cancer. Here, we hypothesized that garcinone C can prevent colon tumorigenesis through regulating the Hh signaling pathway.MethodColony formation assay and flow cytometry were used to evaluate the effect of garcinone C on the proliferation and cell cycle progression of colon cancer cells. Protein expression of cell cycle related markers and Hh/Gli1 signaling mediators were determined. The regulatory effect of orally administered garcinone C on the Hh/Gli1 signaling pathway and colon tumorigenesis was evaluated in an azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colon cancer animal model.ResultsGarcinone C suppressed the proliferation of colon cancer cells, induced G0/G1 cell cycle arrest, as well as regulated the expression of cell cycle-related markers such as cyclin D1, cyclin E, CDK6, and p21. Garcinone C inhibited the expression of Gli1, a key mediator of Hedgehog signaling, and protein kinase B (AKT) phosphorylation in Smo-independent colon cancer cells. In the AOM/DSS-induced colon tumorigenesis model, garcinone C significantly inhibited tumor development, regulated the expression of cell cycle markers and Gli1, and reduced AKT phosphorylation in colon tumor tissues, which is consistent with our in vitro results.ConclusionGarcinone C can suppress colon tumorigenesis in vitro and in vivo through Gli1-dependent non-canonical Hedgehog signaling, suggesting that it may serve as a potent chemopreventive agent against colon tumorigenesis.     

Combination Therapy with the Histone Deacetylase Inhibitor LBH589 and Radiation Is an Effective Regimen for Prostate Cancer Cells

Radiation therapy (RT) continues to be one of the most popular treatment options for localized prostate cancer (CaP). The purpose of the study was to investigate the in vitro effect of LBH589 alone and in combination with RT on the growth and survival of CaP cell lines and the possible mechanisms of radiosensitization of this combination therapy. The effect of LBH589 alone or in combination with RT on two CaP cell lines (PC-3 and LNCaP) and a normal prostatic epithelial cell line (RWPE-1) was studied by MTT and clonogenic assays, cell cycle analysis, western blotting of apoptosis-related and cell check point proteins, and DNA double strand break (DSB) repair markers. The immunofluorescence staining was used to further confirm DSB expression in treated CaP cells. Our results indicate that LBH589 inhibited proliferation in both CaP and normal prostatic epithelial cells in a time-and-dose-dependent manner; low-dose of LBH589 (IC₂₀) combined with RT greatly improved efficiency of cell killing in CaP cells; compared to RT alone, the combination treatment with LBH589 and RT induced more apoptosis and led to a steady increase of sub-G1 population and abolishment of RT-induced G2/M arrest, increased and persistent DSB, less activation of non-homologous end joining (NHEJ)/homologous recombination (HR) repair pathways and a panel of cell cycle related proteins. These results suggest that LBH589 is a potential agent to increase radiosensitivity of human CaP cells. LBH589 used either alone, or in combination with RT is an attractive strategy for treating human CaP.     

ATM activation is accompanied with earlier stages of prostate tumorigenesis

The ATM (ataxia telangiectasia mutated) kinase plays an essential role in maintaining genome integrity by coordinating cell cycle arrest, apoptosis, and DNA damage repair. Phosphorylation of ATM at serine 1981 (ATMpSer1981) by DNA damage activates ATM, which subsequently phosphorylates H2AX Ser139 (gammaH2AX), Chk2 Thr68 (Chk2pThr68), and p53 Ser15 (p53pSer15). To determine the role of the ATM pathway in prostate cancer tumorigenesis, we have analyzed 35 primary prostate cancer specimens for ATMpSer1981 (ATM activation), Chk2pThr68, gammaH2AX, and p53pSer15 by immunohistochemistry (IHC) in normal glands, prostatic intraepithelial neoplasias (PINs), and carcinomas. Increases in the intensities of ATMpSer1981, Chk2pThr68, and gammaH2AX and in the percentage of cells that are positive for ATMpSer1981, Chk2pThr68, or gammaH2AX were observed in PINs (p<0.001) compared to normal prostatic glands and carcinoma. However, this pattern of immunostaining was not seen for p53pSer15. Thus, ATM and Chk2 are specifically activated in PINs. As PINs are generally regarded as precursors of prostatic carcinoma, our results suggest that ATM and Chk2 activation at earlier stages of prostate tumorigenesis suppresses tumor progression, with attenuation of ATM activation leading to cancer progression.     

Interactions between Cells with Distinct Mutations in c-MYC and Pten in Prostate Cancer

In human somatic tumorigenesis, mutations are thought to arise sporadically in individual cells surrounded by unaffected cells. This contrasts with most current transgenic models where mutations are induced synchronously in entire cell populations. Here we have modeled sporadic oncogene activation using a transgenic mouse in which c-MYC is focally activated in prostate luminal epithelial cells. Focal c-MYC expression resulted in mild pathology, but prostate-specific deletion of a single allele of the Pten tumor suppressor gene cooperated with c-MYC to induce high grade prostatic intraepithelial neoplasia (HGPIN)/cancer lesions. These lesions were in all cases associated with loss of Pten protein expression from the wild type allele. In the prostates of mice with concurrent homozygous deletion of Pten and focal c-MYC activation, double mutant (i.e. c-MYC+;Pten-null) cells were of higher grade and proliferated faster than single mutant (Pten-null) cells within the same glands. Consequently, double mutant cells outcompeted single mutant cells despite the presence of increased rates of apoptosis in the former. The p53 pathway was activated in Pten-deficient prostate cells and tissues, but c-MYC expression shifted the p53 response from senescence to apoptosis by repressing the p53 target gene p21^Cip1. We conclude that c-MYC overexpression and Pten deficiency cooperate to promote prostate tumorigenesis, but a p53-dependent apoptotic response may present a barrier to further progression. Our results highlight the utility of inducing mutations focally to model the competitive interactions between cell populations with distinct genetic alterations during tumorigenesis.     

BMI1, Stem Cell Factor Acting as Novel Serum-biomarker for Caucasian and African-American Prostate Cancer

Background

Lack of reliable predictive biomarkers is a stumbling block in the management of prostate cancer (CaP). Prostate-specific antigen (PSA) widely used in clinics has several caveats as a CaP biomarker. African-American CaP patients have poor prognosis than Caucasians, and notably the serum-PSA does not perform well in this group. Further, some men with low serum-PSA remain unnoticed for CaP until they develop disease. Thus, there is a need to identify a reliable diagnostic and predictive biomarker of CaP. Here, we show that BMI1 stem-cell protein is secretory and could be explored for biomarker use in CaP patients.

Methodology/Principal Findings

Semi-quantitative analysis of BMI1 was performed in prostatic tissues of TRAMP (autochthonous transgenic mouse model), human CaP patients, and in cell-based models representing normal and different CaP phenotypes in African-American and Caucasian men, by employing immunohistochemistry, immunoblotting and Slot-blotting. Quantitative analysis of BMI1 and PSA were performed in blood and culture-media of siRNA-transfected and non-transfected cells by employing ELISA. BMI1 protein is (i) secreted by CaP cells, (ii) increased in the apical region of epithelial cells and stromal region in prostatic tumors, and (iii) detected in human blood. BMI1 is detectable in blood of CaP patients in an order of increasing tumor stage, exhibit a positive correlation with serum-PSA and importantly is detectable in patients which exhibit low serum-PSA. The clinical significance of BMI1 as a biomarker could be ascertained from observation that CaP cells secrete this protein in higher levels than cells representative of benign prostatic hyperplasia (BPH).

Conclusions/Significance

BMI1 could be developed as a dual bio-marker (serum and biopsy) for the diagnosis and prognosis of CaP in Caucasian and African-American men. Though compelling these data warrant further investigation in a cohort of African-American patients.