Growth factor regulation of estrogen receptor coregulator PELP1 functions via Protein Kinase A pathway.

PELP1 (proline-rich, glutamic acid-rich, and leucine-rich protein-1) is a potential proto-oncogene that functions as a coregulator of estrogen receptor (ER), and its expression is deregulated during breast cancer progression. Emerging evidence suggests growth factor signaling crosstalk with ER as one possible mechanism by which breast tumors acquire resistance to therapy. In this study, we examined mechanisms by which growth factors modulate PELP1 functions, leading to activation of ER. Using in vivo labeling assays, we have found that growth factors promote phosphorylation of PELP1. Utilizing a panel of substrate-specific phosphorylated antibodies, we discovered that growth factor stimulation promotes phosphorylation of PELP1 that is recognized by a protein kinase A (PKA) substrate-specific antibody. Accordingly, growth factor-mediated PELP1 phosphorylation was effectively blocked by PKA-specific inhibitor H89. Utilizing purified PKA enzyme and in vitro kinase assays, we obtained evidence of direct PELP1 phosphorylation by PKA. Using deletion and mutational analysis, we identified PELP1 domains that are phosphorylated by PKA. Interestingly, site-directed mutagenesis of the putative PKA site in PELP1 compromised growth factor-induced activation and subnuclear localization of PELP1 and also affected PELP1-mediated transactivation function. Utilizing MCF-7 cells expressing a PELP1 mutant that cannot be phosphorylated by PKA, we provide mechanistic insights by which growth factor signaling regulates ER transactivation in a PELP1-dependent manner. Collectively, these findings suggest that growth factor signals promote phosphorylation of ER coactivator PELP1 via PKA pathway, and such modification may have functional implications in breast tumors with deregulated growth factor signaling.

ssDNA-binding protein 2 is frequently hypermethylated and suppresses cell growth in human prostate cancer.

PURPOSE: Prostate cancer is a major cause of cancer death among men and the development of new biomarkers is important to augment current detection approaches. EXPERIMENTAL DESIGN: We identified hypermethylation of the ssDNA-binding protein 2 (SSBP2) promoter as a potential DNA marker for human prostate cancer based on previous bioinformatics results and pharmacologic unmasking microarray. We then did quantitative methylation-specific PCR in primary prostate cancer tissues to confirm hypermethylation of the SSBP2 promoter, and analyzed its correlation with clinicopathologic data. We further examined SSBP2 expression in primary prostate cancer and studied its role in cell growth. RESULTS: Quantitative methylation-specific PCR results showed that the SSBP2 promoter was hypermethylated in 54 of 88 (61.4%) primary prostate cancers versus 0 of 23 (0%) in benign prostatic hyperplasia using a cutoff value of 120. Furthermore, we found that expression of SSBP2 was down-regulated in primary prostate cancers and cancer cell lines. Hypermethylation of the SSBP2 promoter and its expression were closely associated with higher stages of prostate cancer. Reactivation of SSBP2 expression by the demethylating agent 5-aza-2'-deoxycytidine in prostate cancer cell lines confirmed epigenetic inactivation as one major mechanism of SSBP2 regulation. Moreover, forced expression of SSBP2 inhibited prostate cancer cell proliferation in the colony formation assay and caused cell cycle arrest. CONCLUSION: SSBP2 inhibits prostate cancer cell proliferation and seems to represent a novel prostate cancer-specific DNA marker, especially in high stages of human prostate cancer.

LOXL1 and LOXL4 are epigenetically silenced and can inhibit ras/extracellular signal-regulated kinase signaling pathway in human bladder cancer.

Promoter hypermethylation is one of the common mechanisms leading to gene silencing in various human cancers. Using a combination of pharmacologic unmasking and microarray techniques, we identified 59 candidate hypermethylated genes, including LOXL1, a lysyl oxidase-like gene, in human bladder cancer cells. We further showed that LOXL1 and LOXL4 are commonly silenced genes in human bladder cancer cells, and this silence is predominantly related to promoter methylation. We also found LOXL1 and LOXL4 gene methylation and loss of expression in primary bladder tumors. In addition, somatic mutations were identified in LOXL4, but not in LOXL1 in bladder cancer. Moreover, reintroduction of LOXL1 and LOXL4 genes into human bladder cancer cells leads to a decrease of colony formation ability. Further studies indicated that the overexpression of LOXL1 and LOXL4 could antagonize Ras in activating the extracellular signal-regulated kinase (ERK) signaling pathway. Thus, our current study suggests for the first time that lysyl oxidase-like genes can act as tumor suppressor genes and exert their functions through the inhibition of the Ras/ERK signaling pathway in human bladder cancer.

Methylation of the DFNA5 increases risk of lymph node metastasis in human breast cancer.

The pathogenesis of breast cancer involves multiple genetic and epigenetic events. In this study, we report an epigenetic alteration of DFNA5 in human breast cancer. DFNA5 gene was silenced in breast cancer cell lines that were methylated in the DFNA5 promoter, and restored by treatment with the demethylating agent, 5-aza-dC, and gene knock-down of DFNA5 increased cellular invasiveness in vitro. The mRNA expression of DFNA5 in breast cancer tissues was down-regulated as compared to normal tissues. Moreover, the DFNA5 promoter was found to be methylated in primary tumor tissues with high frequency (53%, 18/34). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary breast cancer tissues from normal breast tissues (15.3%, 2/13). Moreover, methylation status of DFNA5 was correlated with lymph node metastasis in breast cancer patients. Our data implicate DFNA5 promoter methylation as a novel molecular biomarker in human breast cancer.

Profiling the expression pattern of GPI transamidase complex subunits in human cancer.

The glycosylphosphatidylinositol transamidase complex (GPIT) consists of five subunits: PIG-U, PIG-T, GPAA1, PIG-S and GPI8, and is important in attaching GPI anchors to target proteins. On the basis of our previous reports incriminating PIG-U as an oncogene in bladder cancer and PIG-T and GPAA1 as oncogenes in breast cancer, we evaluated the expression pattern of the GPIT subunits in 19 different human cancers at both mRNA and protein levels. In general, our results demonstrate a more frequent expression of GPIT subunits in cancers than in normal. Among the 19 anatomic sites compared; breast, ovary and uterus showed consistent evidence of overexpression of specific GPIT subunits. There was also overexpression of PIG-U and GPI8 in lymphoma. In addition, non-small cell lung carcinoma showed significant overexpression of the GPIT subunits as compared to small cell lung carcinoma and normal lung tissue. Also, deregulation of specific GPIT subunits was seen in various other cancers. Forced overexpression of two GPIT subunits; PIG-S and GPI8 alone or in combination induced increased proliferation and invasion of breast cancer cells. Collectively, our study defines a trend involving the deregulated expression and the functional contribution of the GPIT subunits in various cancers with potential implications in diagnosis, prognosis and therapeutic intervention.

Oral cancer: reviewing the present understanding of its molecular mechanism and exploring the future directions for its effective management.

The present review aims to analyze the information available regarding the molecular mechanisms of Oral Carcinogenesis and explore the future directions where the field of Cancer Biology is venturing. Oncologists have excellently followed the proverb "Necessity is the mother of Invention". The desire to be more precise and comprehensive in their studies has led to the invention of some of the most innovative techniques like laser capture microdissection, comparative genomic hybridization, microarrays, and protein chips etc. Various Biotech companies and Cancer Institutes are on a hunt for anti-cancer drugs and molecular markers for cancers. These revolutionary approaches and the new breed of Oncologists have made the field very exciting and have generated the hope that finally the war against cancer would be won. In the end it is urged that the lead taken in other cancers like colon, breast, leukemia will be emulated in oral cancer. This is expected to provide a molecular blueprint for HNSCC, thus helping to identify suitable markers for the early detection of pre-neoplastic lesions, as well as novel targets for its pharmacological intervention.

Cysteine dioxygenase 1 is a tumor suppressor gene silenced by promoter methylation in multiple human cancers.

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.

Neurofilament heavy polypeptide regulates the Akt-beta-catenin pathway in human esophageal squamous cell carcinoma.

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.

U-box-type ubiquitin E4 ligase, UFD2a attenuates cisplatin mediated degradation of DeltaNp63alpha.

DeltaNp63alpha, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, including cisplatin. But little is understood about the post-translational protein stability of DeltaNp63alpha. In this present study we demonstrate for the first time that DeltaNp63alpha physically interacts with U-box-type E4 ubiquitin ligase UFD2a. UFD2a stabilizes DeltaNp63alpha, and ubiquitylation of DeltaNp63alpha is attenuated by UFD2a both in the presence and absence of cisplatin. Ectopic expression of UFD2a increased the half-life of DeltaNp63alpha in association with a significant enhancement of the repressive transcriptional activity of DeltaNp63alpha. Downregulation of endogenous UFD2a by RNAi resulted in degradation of DeltaNp63alpha. Taken together, our current study provides an insight onto the regulation of DeltaNp63alpha protein levels in response to cisplatin and also suggests that UFD2a might play an important role in the regulation of cisplatin mediated cell death mediated by p63.

Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer.

BACKGROUND: DNA methylation has emerged as a promising biomarker for prostate cancer detection. In this report, we screened 36 candidate genes generated by a bioinformatic analysis of the human genome, and found that the melanoma cell adhesion molecule (MCAM) was an excellent candidate for cancer-specific methylation in prostate cancer. METHODS: Direct sequencing of bisulfite-treated genomic DNA, conventional methylation-specific PCR (MSP), real-time quantitative methylation-specific PCR, immunohistochemistry, colony formation assay, and statistical analysis. RESULTS: We found that the melanoma cell adhesion molecule (MCAM) gene promoter was specifically methylated in prostate cancer cell lines and primary prostate cancer (PCa) but not in non-neoplastic prostate (BPH) tissues by direct sequencing of bisulfite-treated genomic DNA and conventional methylation-specific PCR (MSP). Further analysis with quantitative MSP showed greater hypermethylation of the MCAM promoter (80%, 70/88) in primary prostate cancer compared to 12.5% (3/24) in BPH. Prostatic intraepithelial neoplasias (PIN), potential precursors of prostate carcinoma, showed an intermediate methylation rate of 23% (7/30). We further observed that MCAM promoter methylation was directly correlated with tumor stage (pT3+pT4) (P = 0.001) and Gleason score (P = 0.018) in primary prostate carcinoma. CONCLUSIONS: Our results suggest that MCAM promoter hypermethylation deserves further attention as a potential diagnostic prostatic DNA marker in human prostate cancer.

Polymorphism in IGF-2 as a surrogate marker for predisposition towards tobacco chewing-mediated oral cancer.

Insulin and insulin-like growth factors (IGFs) are major determinants of proliferation and apoptosis, thereby playing a significant role in carcinogenesis. Epidemiological evidence associates high levels of INS and IGFs with an increased risk of cancer. Polymorphism of the genes involved in insulin-signaling pathways has been associated with a variable risk for neoplasms in different ethnic and environmental backgrounds. In this study, using PCR-RFLP-based assays, we investigated the distribution of genetic polymorphism in INS and IGF-2 genes in tobacco chewing-mediated oral cancer patients (n = 60) and healthy controls (n = 45) of Indian ethnic origin. The genotyping was performed for +1127 INS-Pst1 in INS and +3580 IGF-2-Msp1 in IGF-2. The frequencies of the IGF-2 genotypes AG, GG and AA found in oral cancer patients were 0.68, 0.2 and 0.12, respectively, whereas in noncancer controls these frequencies were 0.27, 0.71 and 0.02. Frequencies of each allele, i.e. CT, TT and CC of INS gene, were found to be nearly equal in the tumor (0.22, 0.75 and 0.03) as well as the normal (0.27, 0.67 and 0.06) population. A significant difference was observed in genotypic frequencies of IGF-2 and INS in the Indian ethnic population as compared to the Caucasian, African and Hispanic populations. Polymorphism at +1127 INS-Pst1 locus of INS gene does not show an implication in oral cancer, whereas the genotype AG or AA at +3580 IGF-2-Msp1 locus of IGF-2 is associated with progression and increased risk of oral cancer. From our study we can conclude that single nucleotide polymorphisms in the IGF-2 gene can be used as a marker for prediction of the risk of oral carcinogenesis.

Understanding the biology of oral cancer.

The present review is an attempt to summarize the important advances made during the last decade in the molecular approach to oral cancer and its application for early, sensitive diagnosis, effective treatment, and improved prognosis. Cancer of the oral cavity is more prevalent in developing countries, where many people are addicted to tobacco chewing and maintain poor oral hygiene. Despite extensive research on the biological and molecular aspects of oral SCC, the problems of local-regional recurrence and distant metastasis still persist. Among the more pressing problems in clinical management is the lack of early detection, due to the absence of a potential diagnostic marker. Oncologists are now more aware of the challenges associated with the treatment of cancer of the oral cavity, and survival percentages are improving significantly. More trials are need in the area of improved surgical procedures, variations in dosages of radiotherapy, and the use of various combinations of chemotherapeutic agents with minimal side effects. Moreover, progress in the elucidation of the molecular genetic changes that lead to the development of these tumors should soon bring novel diagnostic and therapeutic procedures into clinical practice. The case of ONYX-015 is one example of success, which has shown the great potential in Phase-I and II clinical trials. Finally, the legislator should also impose some restrictions and bans on the easy availability of various forms of tobacco.

Activation of Stat-3 as one of the early events in tobacco chewing-mediated oral carcinogenesis.

BACKGROUND: The Jak/Stat signaling pathway transmits signals from many cytokines and growth factor receptors to target genes in the nucleus. Constitutive activation of Stat-3 recently has been observed in many tumor cells, and dysregulation of the Stat signaling pathway has been proposed to be implicated in malignant transformation. In the current study for the first time to the authors's knowledge, the expression of STAT-3 was analyzed in various stages and sites of squamous cell carcinoma of the head and neck (HNSCC). METHODS: Tissue samples from 90 patients of tobacco chewing-mediated HNSCC representing various stages, sites, and differentiation states were selected for studying STAT-3 protein and RNA expression. In vivo localization of STAT-3 was studied by immunohistochemistry of paraffin embedded sections. The presence of STAT-3 and its phophorylated and activated form pSTAT-3 was checked by Western blotting. mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Apoptosis analysis was conducted by in situ ENA nick end labeling assay and hematoxylin and eosin staining. RESULTS: Overall, 58.9% of HNSCC tumors showed very high Stat-3 protein accumulation, and 23.3% showed intermediate accumulation whereas 17.8% of HNSCC tumors were negative for Stat-3. No Stat-3 was detected in normal samples, and only one of eight premalignant lesions showed intermediate Stat-3 accumulation. On immunoblotting, very high protein accumulation was detected in T1 and T2 classification, moderate in T3 and T4 (P = 0.033, chi-square test), whereas no Stat-3 was detected in normal samples. Similar trend also was found in Stat-3 mRNA expression by RT-PCR analysis which was high in T1 and T2 (early stages), moderate in T3 and T4 (late stages), and no expression in normal samples. The mean apoptotic indices were 1.75, 1.88, and 1.66 for normal, premalignant lesions, and HNSCC cases, respectively. CONCLUSIONS: Stat-3 activation is an early event in head and neck carcinogenesis though its role in blocking the apoptosis in vivo in solid tumors was not observed.

Prevalence of high-risk human papilloma virus types and its association with P53 codon 72 polymorphism in tobacco addicted oral squamous cell carcinoma (OSCC) patients of Eastern India.

Human papillomavirus (HPV) infects the squamous epithelial cells of oral cavity and cervix leading to formation of warts that develops into the cancer. Human papillomavirus (HPV)-16 and 18 encode E6 oncoprotein, which binds to and induces degradation of the tumour suppressor protein p53. A common polymorphism of p53, encoding either proline (Pro) or arginine (Arg) at position 72, affects the susceptibility of p53 to E6 mediated degradation in vivo. Oral cancer is a pressing problem in India due to the widespread habit of chewing betel quid, which plays an important role in etiology of this disease. In the present study an attempt has been made to analyze the genetic predisposition of the Indian population to HPV infection and oral carcinogenesis. In our study a total of 110 cases of Oral Cancer highly addicted to betel quid and tobacco chewing are analyzed for HPV 16/18 infection and its association with polymorphism at p53 codon 72. Of these a total number of 37 patients (33.6%) have shown the presence of HPV, among which the presence of HPV-16, 18 and 16/18 coinfection is 22.7%, 14.5% and 10%, respectively. Our results also indicate that the p53 codon 72 genotype frequencies in Indian Oral Cancer patients are 0.55 (Arg) and 0.45 (Pro) as per Hardy-Weinberg equilibrium. In our study, striking reduction in Pro/Pro allele frequency has been found in HPV positive cases, indicating Arg/Arg genotype to be more susceptible to HPV infection and oral carcinogenesis.