SPINT2 deregulation in prostate carcinoma

SPINT2 is a tumor suppressor gene that inhibits proteases implicated in cancer progression, like HGFA, hepsin and matriptase. Loss of SPINT2 expression in tumors has been associated with gene promoter hypermethylation; however, little is known about the mechanisms of SPINT2 deregulation in prostate cancer (PCa). We aimed to analyze SPINT2 expression levels and understand the possible regulation by SPINT2 promoter hypermethylation in PCa. In a cohort of 57 cases including non-neoplastic and PCa tissues, SPINT2 expression and promoter methylation was analyzed by immunohistochemistry and methylation-specific PCR, respectively. Methylation status of the SPINT2 promoter was also evaluated by bisulfite sequencing and 5-aza-2’-deoxycytidine treatment. Oncomine and TCGA databases were used to perform in silico PCa analysis of SPINT2 mRNA and methylation levels. A reduction in SPINT2 expression levels from nonneoplastic to PCa tissues was observed; however, none of the cases exhibited SPINT2 promoter methylation. Both bisulfite sequencing and 5-aza demonstrated that SPINT2 promoter is not methylated in PCa cells. Bioinformatics approaches did not show downregulation of SPINT2 at the mRNA level and, in corroboration with our results, SPINT2 promoter region is reported to be unmethylated. Our study suggests an involvement of SPINT2 in PCa tumorigenesis, probably in association with a post-translational regulation of SPINT2.The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the ICVS internal research funds of participating authors and by FCT project, ref. PTDC/SAUONC/115513/2009. F.P. received fellowship from the FCT, ref. SFRH/BD/81369/2011 and M.VP from the ON.2 SR&TD Integrated Program (N-01-01-01-24-01-07), ref. UMINHO/ BPD/36/2013

Novel Molecular Targets of Azadirachta indica Associated with Inhibition of Tumor Growth in Prostate Cancer

Advanced prostate cancer has significant long-term morbidity, and there is a growing interest in alternative and complimentary forms of therapy that will improve the outcomes of patients. Azadirachta indica (common name: neem) contains multiple active compounds that have potent anti-inflammatory and anticancer properties. The present study investigates the novel targets of the anticancer activity of ethanol extract of neem leaves (EENL) in vitro and evaluates the in vivo efficacy in the prostate cancer models. Analysis of the components in the EENL by mass spectrometry suggests the presence of 2′,3′-dehydrosalannol, 6-desacetyl nimbinene, and nimolinone. Treatment of C4-2B and PC-3M-luc2 prostate cancer cells with EENL inhibited the cell proliferation. Genome-wide expression profiling, using oligonucleotide microarrays, revealed genes differentially expressed with EENL treatment in prostate cancer cells. Functional analysis unveiled that most of the up-regulated genes were associated with cell death, and drug metabolism, and the down-regulated genes were associated with cell cycle, DNA replication, recombination, and repair functions. Quantitative PCR confirmed significant up-regulation of 40 genes and immunoblotting revealed increase in the protein expression levels of HMOX1, AKR1C2, AKR1C3, and AKR1B10. EENL treatment inhibited the growth of C4-2B and PC-3M-luc2 prostate cancer xenografts in nude mice. The suppression of tumor growth is associated with the formation of hyalinized fibrous tumor tissue and the induction of cell death by apoptosis. These results suggest that EENL-containing natural bioactive compounds could have potent anticancer property and the regulation of multiple cellular pathways could exert pleiotrophic effects in prevention and treatment of prostate cancer

Identification of potential therapeutic targets in prostate cancer through a cross-species approach.

Genetically engineered mouse models of cancer can be used to filter genome-wide expression datasets generated from human tumours and to identify gene expression alterations that are functionally important to cancer development and progression. In this study, we have generated RNAseq data from tumours arising in two established mouse models of prostate cancer, PB-Cre/PtenloxP/loxP and p53loxP/loxPRbloxP/loxP, and integrated this with published human prostate cancer expression data to pinpoint cancer-associated gene expression changes that are conserved between the two species. To identify potential therapeutic targets, we then filtered this information for genes that are either known or predicted to be druggable. Using this approach, we revealed a functional role for the kinase MELK as a driver and potential therapeutic target in prostate cancer. We found that MELK expression was required for cell survival, affected the expression of genes associated with prostate cancer progression and was associated with biochemical recurrence

A novel tumor suppressor gene ECRG4 interacts directly with TMPRSS11A (ECRG1) to inhibit cancer cell growth in esophageal carcinoma

<p>Abstract</p> <p>Background</p> <p>The esophageal carcinoma related gene 4 (ECRG4) was initially identified and cloned from human normal esophageal epithelium in our laboratory (GenBank accession no.<ext-link ext-link-id="AF325503" ext-link-type="gen">AF325503</ext-link>). ECRG4 has been described as a novel tumor suppressor gene associated with prognosis in esophageal squamous cell carcinoma (ESCC).</p> <p>Methods</p> <p>In this study, binding affinity assay in vitro and co-immunoprecipitation experiment in vivo were utilized to verify the physical interaction between ECRG4 and transmembrane protease, serine 11A (TMPRSS11A, also known as ECRG1, GenBank accession no. <ext-link ext-link-id="AF 071882" ext-link-type="gen">AF 071882</ext-link>). Then, p21 protein expression, cell cycle and cell proliferation regulations were examined after ECRG4 and ECRG1 co-transfection in ESCC cells.</p> <p>Results</p> <p>We revealed for the first time that ECRG4 interacted directly with ECRG1 to inhibit cancer cell proliferation and induce cell cycle G1 phase block in ESCC. Binding affinity and co-immunoprecipitation assays demonstrated that ECRG4 interacted directly with ECRG1 in ESCC cells. Furthermore, the ECRG4 and ECRG1 co-expression remarkably upregulatd p21 protein level by Western blot (P < 0.001), induced cell cycle G1 phase block by flow cytometric analysis (P < 0.001) and suppressed cell proliferation by MTT and BrdU assay (both P < 0.01) in ESCC cells.</p> <p>Conclusions</p> <p>ECRG4 interacts directly with ECRG1 to upregulate p21 protein expression, induce cell cycle G1 phase block and inhibit cancer cells proliferation in ESCC.</p

Comprehensive resequence analysis of a 97 kb region of chromosome 10q11.2 containing the MSMB gene associated with prostate cancer

Genome-wide association studies of prostate cancer have identified single nucleotide polymorphism (SNP) markers in a region of chromosome 10q11.2, harboring the microseminoprotein-β (MSMB) gene. Both the gene product of MSMB, the prostate secretory protein 94 (PSP94) and its binding protein (PSPBP), have been previously investigated as serum biomarkers for prostate cancer progression. Recent functional work has shown that different alleles of the significantly associated SNP in the promoter of MSMB found to be associated with prostate cancer risk, rs10993994, can influence its expression in tumors and in vitro studies. Since it is plausible that additional variants in this region contribute to the risk of prostate cancer, we have used next-generation sequencing technology to resequence a ~97-kb region that includes the area surrounding MSMB (chr10: 51,168,025–51,265,101) in 36 prostate cancer cases, 26 controls of European origin, and 8 unrelated CEPH individuals in order to identify additional variants to investigate in functional studies. We identified 241 novel polymorphisms within this region, including 142 in the 51-kb block of linkage disequilibrium (LD) that contains rs10993994 and the proximal promoter of MSMB. No sites were observed to be polymorphic within the exons of MSMB

ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma

<p>Abstract</p> <p>Background</p> <p>Cancer cells display widespread changes in DNA methylation that may lead to genetic instability by global hypomethylation and aberrant silencing of tumor suppressor genes by focal hypermethylation. In turn, altered DNA methylation patterns have been used to identify putative tumor suppressor genes.</p> <p>Methods</p> <p>In a methylation screening approach, we identified <it>ECRG4 </it>as a differentially methylated gene. We analyzed different cancer cells for <it>ECRG4 </it>promoter methylation by COBRA and bisulfite sequencing. Gene expression analysis was carried out by semi-quantitative RT-PCR. The <it>ECRG4 </it>coding region was cloned and transfected into colorectal carcinoma cells. Cell growth was assessed by MTT and BrdU assays. ECRG4 localization was analyzed by fluorescence microscopy and Western blotting after transfection of an <it>ECRG4-eGFP </it>fusion gene.</p> <p>Results</p> <p>We found a high frequency of <it>ECRG4 </it>promoter methylation in various cancer cell lines. Remarkably, aberrant methylation of <it>ECRG4 </it>was also found in primary human tumor tissues, including samples from colorectal carcinoma and from malignant gliomas. <it>ECRG4 </it>hypermethylation associated strongly with transcriptional silencing and its expression could be re-activated <it>in vitro </it>by demethylating treatment with 5-aza-2'-deoxycytidine. Overexpression of <it>ECRG4 </it>in colorectal carcinoma cells led to a significant decrease in cell growth. In transfected cells, ECRG4 protein was detectable within the Golgi secretion machinery as well as in the culture medium.</p> <p>Conclusions</p> <p><it>ECRG4 </it>is silenced via promoter hypermethylation in different types of human cancer cells. Its gene product may act as inhibitor of cell proliferation in colorectal carcinoma cells and may play a role as extracellular signaling molecule.</p

GOLPH2 protein expression as a novel tissue biomarker for prostate cancer: implications for tissue-based diagnostics

GOLPH2 is coding the 73-kDa type II Golgi membrane antigen GOLPH2/GP73. Upregulation of GOLPH2 mRNA has been recently reported in expression array analyses of prostate cancer. As GOLPH2 protein expression in prostate tissues is currently unknown, this study aimed at a comprehensive analysis of GOLPH2 protein in benign and malignant prostate lesions. Immunohistochemically detected GOLPH2 protein expression was compared with the basal cell marker p63 and the prostate cancer marker α-methylacyl-CoA racemase (AMACR) in 614 radical prostatectomy specimens. GOLPH2 exhibited a perinuclear Golgi-type staining pattern and was preferentially seen in prostatic gland epithelia. Using a semiquantitative staining intensity score, GOLPH2 expression was significantly higher in prostate cancer glands compared with normal glands (P<0.001). GOLPH2 protein was upregulated in 567 of 614 tumours (92.3%) and AMACR in 583 of 614 tumours (95%) (correlation coefficient 0.113, P=0.005). Importantly, GOLPH2 immunohistochemistry exhibited a lower level of intratumoral heterogeneity (25 vs 45%). Further, GOLPH2 upregulation was detected in 26 of 31 (84%) AMACR-negative prostate cancer cases. These data clearly suggest GOLPH2 as an additional ancillary positive marker for tissue-based diagnosis of prostate cancer

Esophageal Cancer Related Gene-4 Is a Choroid Plexus-Derived Injury Response Gene: Evidence for a Biphasic Response in Early and Late Brain Injury

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24–72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6–8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation

Discovery of Molecular Mechanisms of Traditional Chinese Medicinal Formula Si-Wu-Tang Using Gene Expression Microarray and Connectivity Map

To pursue a systematic approach to discovery of mechanisms of action of traditional Chinese medicine (TCM), we used microarrays, bioinformatics and the “Connectivity Map” (CMAP) to examine TCM-induced changes in gene expression. We demonstrated that this approach can be used to elucidate new molecular targets using a model TCM herbal formula Si-Wu-Tang (SWT) which is widely used for women's health. The human breast cancer MCF-7 cells treated with 0.1 µM estradiol or 2.56 mg/ml of SWT showed dramatic gene expression changes, while no significant change was detected for ferulic acid, a known bioactive compound of SWT. Pathway analysis using differentially expressed genes related to the treatment effect identified that expression of genes in the nuclear factor erythroid 2-related factor 2 (Nrf2) cytoprotective pathway was most significantly affected by SWT, but not by estradiol or ferulic acid. The Nrf2-regulated genes HMOX1, GCLC, GCLM, SLC7A11 and NQO1 were upreguated by SWT in a dose-dependent manner, which was validated by real-time RT-PCR. Consistently, treatment with SWT and its four herbal ingredients resulted in an increased antioxidant response element (ARE)-luciferase reporter activity in MCF-7 and HEK293 cells. Furthermore, the gene expression profile of differentially expressed genes related to SWT treatment was used to compare with those of 1,309 compounds in the CMAP database. The CMAP profiles of estradiol-treated MCF-7 cells showed an excellent match with SWT treatment, consistent with SWT's widely claimed use for women's diseases and indicating a phytoestrogenic effect. The CMAP profiles of chemopreventive agents withaferin A and resveratrol also showed high similarity to the profiles of SWT. This study identified SWT as an Nrf2 activator and phytoestrogen, suggesting its use as a nontoxic chemopreventive agent, and demonstrated the feasibility of combining microarray gene expression profiling with CMAP mining to discover mechanisms of actions and to identify new health benefits of TCMs

DNA repair: the culprit for tumor-initiating cell survival?

The existence of “tumor-initiating cells” (TICs) has been a topic of heated debate for the last few years within the field of cancer biology. Their continuous characterization in a variety of solid tumors has led to an abundance of evidence supporting their existence. TICs are believed to be responsible for resistance against conventional treatment regimes of chemotherapy and radiation, ultimately leading to metastasis and patient demise. This review summarizes DNA repair mechanism(s) and their role in the maintenance and regulation of stem cells. There is evidence supporting the hypothesis that TICs, similar to embryonic stem (ES) cells and hematopoietic stem cells (HSCs), display an increase in their ability to survive genotoxic stress and injury. Mechanistically, the ability of ES cells, HSCs and TICs to survive under stressful conditions can be attributed to an increase in the efficiency at which these cells undergo DNA repair. Furthermore, the data presented in this review summarize the results found by our lab and others demonstrating that TICs have an increase in their genomic stability, which can allow for TIC survival under conditions such as anticancer treatments, while the bulk population of tumor cells dies. We believe that these data will greatly impact the development and design of future therapies being engineered to target and eradicate this highly aggressive cancer cell population