Patched 1 Expression Correlates with Biochemical Relapse in High-Risk Prostate Cancer Patients.

There is an unmet clinical need for adequate biomarkers to aid risk stratification and management of prostate cancer (PCa) patients. Even within the high-risk PCa category, not all patients will invariably have a poor prognosis, and improved stratification of this heterogeneous group is needed. In this context, components of the hedgehog (Hh) pathway may have promise as biomarkers, because the available evidence suggests increased Hh pathway activity may confer a poorer outcome in advanced and castrate-resistant PCa. In this study, potential associations between Hh pathway protein expression and clinicopathological factors, including time to biochemical recurrence (BCR), were investigated using a tissue microarray constructed from benign and malignant prostate samples from 75 predominantly high-risk PCa patients who underwent radical prostatectomy. Hh signaling activity was found to differ between benign and malignant prostate tissue, with a greater amount of active Hh signaling present in malignant than benign prostate epithelium. High expression of Patched 1 in malignant prostate epithelium was found to be an independent predictor of BCR in high-risk PCa patients. Glioma-associated oncogene 1 may potentially represent a clinically useful biomarker of an aggressive tumor phenotype. Evaluation of Hh signaling activity in PCa patients may be useful for risk stratification, and epithelial Patched 1 expression, in particular, may be a prognostic marker for BCR in high-risk PCa patients.

Tissue microarray analysis indicates hedgehog signaling as a potential prognostic factor in intermediate-risk prostate cancer.

BACKGROUND: Prostate cancer (PCa) is a heterogeneous disease with a variable natural history, genetics, and treatment outcome. The Hedgehog (Hh) signaling pathway is increasingly recognized as being potentially important for the development and progression of PCa. In this retrospective study, we compared the activation status of the Hh signaling pathway between benign and tumor tissue, and evaluated the clinical significance of Hh signaling in PCa. METHODS: In this tissue microarray (TMA) study, the protein expression of several Hh signaling components and Hh target proteins, along with microvessel density, were compared between benign (n = 64) and malignant (n = 170) prostate tissue, and correlated with PCa clinicopathological characteristics and biochemical recurrence (BCR). RESULTS: The Hh signaling pathway appeared to be more active in PCa than in benign prostate tissue, as demonstrated by lower expression of the negative regulators PTCH1 and GLI3 in the tumor tissue compared to benign. In addition, high epithelial GLI2 expression correlated with higher pathological Gleason score. Overall, higher epithelial GLI3 expression in the tumor was shown to be an independent marker of a favorable prognosis. CONCLUSION: Hh signaling activation might reflect aggressive tumoral behavior, since high epithelial GLI2 expression positively correlates with a higher pathological Gleason score. Moreover, higher epithelial GLI3 expression is an independent marker of a more favorable prognosis.

The Effect of Metformin and GANT61 Combinations on the Radiosensitivity of Prostate Cancer Cells.

The anti-diabetes drug metformin has been shown to have anti-neoplastic effects in several tumor models through its effects on energy metabolism and protein synthesis. Recent studies show that metformin also targets Hedgehog (Hh) signaling, a developmental pathway re-activated in several tumor types, including prostate cancer (PCa). Furthermore, we and others have shown that Hh signaling is an important target for radiosensitization. Here, we evaluated the combination of metformin and the Hh inhibitor GANT61 (GLI-ANTagonist 61) with or without ionizing radiation in three PCa cell lines (PC3, DU145, 22Rv1). The effect on proliferation, radiosensitivity, apoptosis, cell cycle distribution, reactive oxygen species production, DNA repair, gene and protein expression was investigated. Furthermore, this treatment combination was also assessed in vivo. Metformin was shown to interact with Hh signaling by inhibiting the effector protein glioma-associated oncogene homolog 1 (GLI1) in PCa cells both in vitro and in vivo. The combination of metformin and GANT61 significantly inhibited PCa cell growth in vitro and enhanced the radiation response of 22Rv1 cells compared to either single agent. Nevertheless, neither the growth inhibitory effect nor the radiosensitization effect of the combination treatment observed in vitro was seen in vivo. Although the interaction between metformin and Hh signaling seems to be promising from a therapeutic point of view in vitro, more research is needed when implementing this combination strategy in vivo.

Proteomic analysis reveals a proteolytic feedback loop in murine seminal fluid.

BACKGROUND: Matrix metalloproteinase 9 (MMP9) has been implicated in extracellular matrix (ECM) remodelling, angiogenesis and inflammation. However, the targets for proteolysis that lead to these physiological consequences are often undefined as is the regulation of MMP9 itself. Therefore, identification of both the potential direct and indirect targets of MMP9 is critical for further understanding the effects of its proteolytic cascades. METHODS: To study these cascades on a wider scale, transgenic mouse "knock-out" models and ultra-high performance liquid chromatography mass spectroscopy (UPLC-MS(E) ) were used to elucidate the MMP9 targets, inhibitors, and interactors found in mouse seminal vesicle fluid (SVF). RESULTS: Proteomics analysis of SVF from wild type, mmp9-/- or pn1-/- mice detected differences in serine protease inhibitors (serpins), reproductive proteins, developmental regulators, and cancer proto-oncogenes, including Renin 1/2. Protease nexin 1 (PN1), an ECM-based inhibitor of urokinase, was elevated in the SVF of mmp9-/- mice. We observed that MMP9-mediated N-terminal cleavage of PN1 reduces this serpin's functional activity. Our data also suggest a feedback loop in which inhibition of PN1 is a critical step in permitting greater activity of MMP9. CONCLUSION: This study extends the degradome of MMP9 and examines components relevant to seminal fluid physiology. PN1 is proposed to be a novel inhibitor of MMP9 activity and a block to collagen cleavage, a frequent antecedent to cancer cell invasion. The interaction of MMP9 with PN1 and other serpins may lead to a better understanding of seminal vesicle function and possible impacts on fertility, as well as provide novel therapeutic targets.

The hedgehog inhibitor GANT61 sensitizes prostate cancer cells to ionizing radiation both in vitro and in vivo.

Limited data exists regarding the combination of Hedgehog signaling (Hh) inhibition and radiotherapy, even though there are several indications that this might be a promising treatment strategy. In this study, we evaluated the combination of two Hh inhibitors, the SMO inhibitor GDC-0449 and the GLI inhibitor GANT61 with radiotherapy in different prostate cancer (PCa) models. In vitro, GANT61 was able to sensitize 22Rv1 PCa cells but not PC3 and DU145 PCa cells. The lack of radiosensitization in the latter cell lines was shown to be dependent on the presence of mutated p53. Introduction of WT p53 into PC3 cells resulted in radiosensization following GANT61 treatment, suggesting that the p53 transcription factor plays an important role in the GANT61-induced radiosensitization in vitro. Targeting at the level of SMO (GDC-0449) did not show cytotoxicity or synergy with radiation. Furthermore, we confirmed the radiosensitization effect of GANT61 in two in vivo xenograft PCa models. The decrease in tumor growth was associated with decreased proliferation and increased apoptosis. In conclusion, we provide evidence that GANT61 in combination with radiation treatment might represent a promising therapeutic strategy for enhancing the radiation response of PCa patients.

Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein.

Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-κB pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis.

Protease nexin 1 inhibits hedgehog signaling in prostate adenocarcinoma.

Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

Matrix metalloproteinase-9 regulates tumor cell invasion through cleavage of protease nexin-1.

Matrix metalloproteinase-9 (MMP-9) expression is known to enhance the invasion and metastasis of tumor cells. In previous work based on a proteomic screen, we identified the serpin protease nexin-1 (PN-1) as a potential target of MMP-9. Here, we show that PN-1 is a substrate for MMP-9 and establish a link between PN-1 degradation by MMP-9 and regulation of invasion. PN-1 levels increased in prostate carcinoma cells after downregulation of MMP-9 and in tissues of MMP-9-deficient mice, consistent with PN-1 degradation by MMP-9. We identified three MMP-9 cleavage sites in PN-1 and showed that mutations in those sites made PN-1 more resistant to MMP-9. Urokinase plasminogen activator (uPA) is inhibited by PN-1. MMP-9 augmented uPA activity in the medium of PC3-ML cells by degrading PN-1. Prostate cancer cells, overexpressing PN-1 or treated with MMP-9 shRNA, had reduced cell invasion in Matrigel. PN-1 siRNA restored uPA activity and the invasive capacity. PN-1 mutated in the serpin inhibitory domain, the reactive center loop, failed to inhibit uPA and to reduce Matrigel invasion. This study shows a novel molecular pathway in which MMP-9 regulates uPA activity and tumor cell invasion through cleavage of PN-1.

Testicular germ cell sensitivity to TRAIL-induced apoptosis is dependent upon p53 expression and is synergistically enhanced by DR5 agonistic antibody treatment.

The ability of the TRAIL/DR5 signaling pathway to induce apoptosis has generally been limited to tumor cells. Here we report that in primary testis explants, addition of TRAIL (0.5 mug/ml) caused a three-fold increase in germ cell apoptosis. Furthermore, exposure of C57BL/6 mice to the testicular toxicant, mono-(2-ethylhexyl) phthalate (MEHP), caused an increased p53 stability and elevated DR5 mRNA levels coincident with increases in the levels of apoptosis in spermatocytes. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo TRAIL/DR5-mediated apoptosis, we used the germ cell lines GC-1spg and GC-2spd(ts) (a temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32 degrees C but not 37 degrees C). Addition of TRAIL and the anti-DR5 monoclonal antibody, MD5-1, triggered a robust synergistic increase of apoptosis in p53 permissive GC-2 cells (32 degrees C) but not in GC-1 cells. In addition, DR5 levels on the plasma membrane of permissive cells were considerably enhanced concomitant with p53 expression and after MD5-1 treatment. These data represent the first indication that testicular germ cells, specifically spermatocytes, can undergo TRAIL-mediated apoptosis and the clinically relevant observation that pretreatment with a DR5 monoclonal antibody can greatly sensitize their apoptotic response to TRAIL.

Influence of TRP53 status on FAS membrane localization, CFLAR (c-FLIP) ubiquitinylation, and sensitivity of GC-2spd (ts) cells to undergo FAS-mediated apoptosis.

Previously we reported that testicular germ cells undergo FAS-mediated apoptosis after exposure of mice to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP) and that this process is partially dependent on the TRP53 protein (p53). Recent reports have suggested that TRP53 may influence the ubiquitinylation and consequent proteosomal degradation of a negative regulator of FAS, CFLAR (L) (c-FLIP [L]), in human colon cancer cells. To further characterize the relationship between CFLAR and TRP53, we used the transformed germ cell line GC-2spd (ts), which harbors a temperature-sensitive Trp53 mutation that allows for TRP53 activation at 32 degrees C. We report here that GC-2 cells expressed a 10-fold increase in basal cell membrane FAS levels and an increased sensitivity to FAS agonistic antibody (JO2)-triggered apoptosis only when they were maintained at the permissive TRP53 temperature. After JO2 exposure, CFLAR (L) protein levels were enhanced only at the nonpermissive TRP53 temperature (37 degrees C) while real-time PCR results indicated an absence of Cflar (L) mRNA changes in GC-2 cells regardless of the temperature. Furthermore, transfection of GC-2 cells at 37 degrees C with siRNA against Cflar resulted in reduction of CFLAR (L) protein levels and increased sensitivity to JO2-mediated apoptosis. The CFLAR (L) protein was also more strongly ubiquitinylated in response to JO2 treatment at the permissive TRP53 temperature. Taken together, these data suggest that the TRP53 protein influences the sensitivity of GC-2 cells to undergo FAS-mediated apoptosis by modulating the expression of FAS on their cell membranes and subsequently influencing the degradation of the antiapoptotic protein CFLAR (L).