Alejandro H. Corvalán

Mismatch repair expression in testicular cancer predicts recurrence and survival.

We investigated mismatch repair (MMR) gene expression in testicular cancer as a molecular marker for clinical outcome (recurrence, response to chemotherapy and death) using protein expression and specific genetic alterations associated with the presence or absence of MMR activity. One hundred sixty‐two cases of paraffin‐embedded testis cancer specimens were subjected to immunohistochemical analysis using monoclonal antibody for MLH1 and MSH2 MMR proteins and genetic analysis using specific polymorphic markers. The degree of MMR immunoreactivity and genetic instability in the form of loss of heterozygosity (LOH) and/or microsatellite instability (MSI) were determined by comparing matched normal and tumor tissue. The degree of immunohistochemical staining for MMR expression was associated with a shorter time to tumor recurrence, resistance to chemotherapy and death. Furthermore, clinical relapse and cancer specific death was also associated with tumors exhibiting a high degree of MSI, p = 0.01 and 0.04, respectively. In contrast, LOH was not associated with recurrence, resistance to chemotherapy or death. Therefore, MMR expression defines testis cancers with distinct molecular properties and clinical behavior, such that tumors with decreased MMR immunostaining and/or increased frequency of MSI have a shorter time to recurrence and death despite chemotherapy.

Baseline assessment of prevalence and geographical distribution of HPV types in Chile using self-collected vaginal samples

BackgroundChile has broad variations in weather, economics and population from the far desert north (Region 1) to the cold, icy south (Region 12). A home-based self-collected vaginal sampling was nested in the 2003 Chilean population-based health survey in order to explore the possibility of a type-specific geographical variation for human papillomavirusMethodsThe population was a national probability sample of people 17 years of age and over. Consenting women provided self-collected cervicovaginal swabs in universal collection media (UCM). DNA was extracted and typed to 37 HPV genotypes using PGMY consensus PCR and line blot assay. Weighted prevalence rates and adjusted OR were calculated.ResultsOf the 1,883 women participating in the health survey, 1,219 (64.7%) provided a cervicovaginal sample and in 1,110 (56.2% of participants and 66.5% of those eligible) the samples were adequate for analysis. Refusal rate was 16.9%. HPV prevalence was 29.2% (15.1% high-risk HPV and 14.1% low-risk HPV). Predominant high-risk types were HPV 16, 52, 51, 56 and 58. Predominant low-risk HPVs were HPV 84, CP6108, 62, 53 and 61. High-risk and low-risk HPV rates were inversely correlated between the regions. High-risk HPV prevalence was highest among the youngest women, whereas low-risk HPV increased slightly with age.ConclusionSelf-obtained vaginal sampling is adequate for monitoring HPV in the community, for identifying high-risk areas, and for surveying the long term impact of interventions.

DNA methylation profile in diffuse type gastric cancer: evidence for hypermethylation of the BRCA1 promoter region in early-onset gastric carcinogenesis

Diffuse type gastric carcinoma is the most aggressive type of gastric cancer. This type of tumor is not preceded by precancerous changes and is associated with early-onset and hereditary syndromes. To test the hypothesis that DNA methylation profile would be useful for molecular classification of the diffuse type gastric carcinoma, DNA methylation patterns of the CpG Island of 17 genes were studied in 104 cases and 47 normal adjacent gastric mucosa by Methylation-specific PCR, Immunohistochemistry and Hierarchical clustering analysis. The most frequent methylated genes were FHIT, E-cadherin, BRCA1 and APC (>50%), followed by p14, p16, p15, p73, MGMT and SEMA3B (20-49%). Hierarchical clustering analysis reveals four groups with different clinical features. The first was characterized by hypermethylation of BRCA1 and younger age (<45 years old), and the second by hypermethylation of p14 and p16 genes, male predominance and Epstein-Barr virus infection. The third group was characterized by hypermethylation of FHIT and antrum located tumors and the fourth was not associated with any clinical variables. In normal adjacent mucosa only the p73 gene was significantly less methylated in comparison to tumor mucosa. DNA methylation identified subgroups of diffuse type gastric cancer. Hypermethylation of BRCA1 associated with young age suggests a role in early-onset gastric carcinoma.

Merkel cell polyomavirus in non-small cell lung carcinomas from Chile

Lung cancer is a leading pathology strongly associated with the smoking habit. However, a viral etiology for a subset of patients developing lung cancer has been suggested. Polyomaviruses (PyVs) are small double stranded DNA viruses associated with the development of some human diseases. However, a causal role of these viruses in human cancer has been difficult to demonstrate. In this study, eighty-six non-small cell lung carcinomas (NSCLCs), including adenocarcinomas (AdCs) and squamous cell lung carcinomas (SQCs) from Chile were analyzed for the presence of PyVs using polymerase chain reaction (PCR). All of the specimens were positive for a fragment of the betaglobin gene. We found that 4/86 (4.7%) of lung carcinomas were positive for PyVs. After sequencing and BlastN alignment, all four cases were identified as Merkel cell polyomaviruses (MCV) that corresponded to two AdCs and two SQCs. A non-significant statistical association was found between the presence of MCV and clinic-pathological features of the patients and tumors. In addition, 1/4 (25%) of the carcinomas were actively expressing large T antigen (LT) transcripts, as demonstrated by reverse-transcriptase PCR (RT-PCR). Thus a possible role of MCV in a very small subset of patients with lung cancer cannot be ruled out and warrants more investigation.

Helicobacter pylori—Induced Loss of the Inhibitor-of-Apoptosis Protein Survivin Is Linked to Gastritis and Death of Human Gastric Cells

Helicobacter pylori infects the human stomach and modifies signaling pathways that affect gastric epithelial cell proliferation and viability. Chronic exposure to this pathogen contributes to the onset of gastric atrophy, an early event in the genesis of gastric cancer associated with H. pylori infection. Susceptibility to H. pylori-induced cell death ultimately depends on the presence of protective host cell factors. Although expression of the inhibitor-of-apoptosis protein survivin in adults is frequently linked to the development of cancer, evidence indicating that the protein is present in normal gastric mucosa is also available. Thus, we investigated in human gastric tissue samples and cell lines whether H. pylori infection is linked to loss of survivin and increased cell death. Our results show that infection with H. pylori decreased survivin protein levels in the mucosa of patients with gastritis. Furthermore, survivin down-regulation correlated with apoptosis and loss of cell viability in gastrointestinal cells cocultured with different H. pylori strains. Finally, overexpression of survivin in human gastric cells was sufficient to reduce cell death after infection. Taken together, these findings implicate survivin as an important survival factor in the gastric mucosa of humans.

Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

Loss of Expression of Reprimo, a p53-induced Cell Cycle Arrest Gene, Correlates with Invasive Stage of Tumor Progression and p73 Expression in Gastric Cancer.

Reprimo (RPRM), a downstream effector of p53-induced cell cycle arrest at G2/M, has been proposed as a putative tumor suppressor gene (TSG) and as a potential biomarker for non-invasive detection of gastric cancer (GC). The aim of this study was to evaluate the epigenetic silencing of RPRM gene by promoter methylation and its tumor suppressor function in GC cell lines. Furthermore, clinical significance of RPRM protein product and its association with p53/p73 tumor suppressor protein family was explored. Epigenetic silencing of RPRM gene by promoter methylation was evaluated in four GC cell lines. Protein expression of RPRM was evaluated in 20 tumor and non-tumor matched cases. The clinical significance of RPRM association with p53/p73 tumor suppressor protein family was assessed in 114 GC cases. Tumor suppressor function was examined through functional assays. RPRM gene expression was negatively correlated with promoter methylation (Spearman rank r = -1; p = 0.042). RPRM overexpression inhibited colony formation and anchorage-independent growth. In clinical samples, RPRM gene protein expression was detected in 75% (15/20) of non-tumor adjacent mucosa, but only in 25% (5/20) of gastric tumor tissues (p = 0.001). Clinicopathological correlations of loss of RPRM expression were significantly associated with invasive stage of GC (stage I to II-IV, p = 0.02) and a positive association between RPRM and p73 gene protein product expression was found (p<0.0001 and kappa value = 0.363). In conclusion, epigenetic silencing of RPRM gene by promoter methylation is associated with loss of RPRM expression. Functional assays suggest that RPRM behaves as a TSG. Loss of expression of RPRM gene protein product is associated with the invasive stage of GC. Positive association between RPRM and p73 expression suggest that other members of the p53 gene family may participate in the regulation of RPRM expression.

Escaping Antiangiogenic Therapy: Strategies Employed by Cancer Cells

Tumor angiogenesis is widely recognized as one of the “hallmarks of cancer”. Consequently, during the last decades the development and testing of commercial angiogenic inhibitors has been a central focus for both basic and clinical cancer research. While antiangiogenic drugs are now incorporated into standard clinical practice, as with all cancer therapies, tumors can eventually become resistant by employing a variety of strategies to receive nutrients and oxygen in the event of therapeutic assault. Herein, we concentrate and review in detail three of the principal mechanisms of antiangiogenic therapy escape: (1) upregulation of compensatory/alternative pathways for angiogenesis; (2) vasculogenic mimicry; and (3) vessel co-option. We suggest that an understanding of how a cancer cell adapts to antiangiogenic therapy may also parallel the mechanisms employed in the bourgeoning tumor and isolated metastatic cells delivering responsible for residual disease. Finally, we speculate on strategies to adapt antiangiogenic therapy for future clinical uses.

Functional Interaction between Human Papillomavirus Type 16 E6 and E7 Oncoproteins and Cigarette Smoke Components in Lung Epithelial Cells

The smoking habit is the most important, but not a sufficient cause for lung cancer development. Several studies have reported the human papillomavirus type 16 (HPV16) presence and E6 and E7 transcripts expression in lung carcinoma cases from different geographical regions. The possible interaction between HPV infection and smoke carcinogens, however, remains unclear. In this study we address a potential cooperation between tobacco smoke and HPV16 E6 and E7 oncoproteins for alterations in proliferative and tumorigenic properties of lung epithelial cells. A549 (alveolar, tumoral) and BEAS-2B (bronchial, non-tumoral) cell lines were stably transfected with recombinant pLXSN vectors expressing HPV16 E6 and E7 oncoproteins and exposed to cigarette smoke condensate (CSC) at different concentrations. HPV16 E6 and E7 expression was associated with loss of p53 stability, telomerase (hTERT) and p16INK4A overexpression in BEAS-2B cells as demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). In A549 cells we observed downregulation of p53 but not a significant increase of hTERT transcripts. In addition, the HPV16 E6/E7 transfected cell lines showed an increased proliferation rate and anchorage-independent growth in a HPV16 E6 and E7 expression-dependent manner. Moreover, both HPV16 E6/E7 and mock transfected cells showed an increased proliferation rate and anchorage-independent growth in the presence of 0.1 and 10 µg/mL CSC. However, this increase was significantly greater in HPV16 E6/E7 transfected cells (p<0.001). Data were confirmed by FCSE proliferation assay. The results obtained in this study are suggestive of a functional interaction between tobacco smoke and HPV16 E6/E7 oncoproteins for malignant transformation and tumorigenesis of lung epithelial cells. More studies are warranted in order to dissect the molecular mechanisms involved in this cooperation.

High frequency of p 16 promoter methylation in non-small cell lung carcinomas from Chile

The inactivation of tumour suppressor genes by aberrant methylation of promoter regions has been described as a frequent event in neoplasia development, including lung cancer. The p16 gene is a tumour suppressor gene involved in the regulation of cell cycle progression that has been reported to be inactivated by promoter methylation in lung carcinomas at variable frequencies around the world in a smoking habit dependent manner. The purpose of this study was to investigate the methylation status of the promoter region of the p16 gene in 74 non-small cell lung carcinomas from Chile. The frequency of p16 gene inactivation by promoter methylation was determined as 79.7% (59/74). When we considered histological type, we observed that p16 promoter methylation was significantly higher in squamous cell carcinomas (30/33, 91%) compared with adenocarcinomas (21/30, 70%) (p=0.029). In addition, no association between p16 promoter methylation and gender, age or smoking habit was found (p=0.202, 0.202 and 0.147 respectively). Our results suggest that p16 promoter hypermethylation is a very frequent event in non-small cell lung carcinomas from Chile and could be smoking habit-independent.