Maria Luana Poeta

The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies

Colorectal cancer (CRC) is a major health problem in industrialized countries. Although inflammation-linked carcinogenesis is a well accepted concept and is often observed within the gastrointestinal tract, the underlying mechanisms remain to be elucidated. Inflammation can indeed provide initiating and promoting stimuli and mediators, generating a tumour-prone microenvironment. Many murine models of sporadic and inflammation-related colon carcinogenesis have been developed in the last decade, including chemically induced CRC models, genetically engineered mouse models, and xenoplants. Among the chemically induced CRC models, the combination of a single hit of azoxymethane (AOM) with 1 week exposure to the inflammatory agent dextran sodium sulphate (DSS) in rodents has proven to dramatically shorten the latency time for induction of CRC and to rapidly recapitulate the aberrant crypt foci–adenoma–carcinoma sequence that occurs in human CRC. Because of its high reproducibility and potency, as well as the simple and affordable mode of application, the AOM/DSS has become an outstanding model for studying colon carcinogenesis and a powerful platform for chemopreventive intervention studies. In this article we highlight the histopathological and molecular features and describe the principal genetic and epigenetic alterations and inflammatory pathways involved in carcinogenesis in AOM/DSS–treated mice; we also present a general overview of recent experimental applications and preclinical testing of novel therapeutics in the AOM/DSS model.

Nonrandom Distribution of Aberrant Promoter Methylation of Cancer-Related Genes in Sporadic Breast Tumors

Purpose: In an effort to additionally determine the global patterns of CpG island hypermethylation in sporadic breast cancer, we searched for aberrant promoter methylation at 10 gene loci in 54 primary breast cancer and 10 breast benign lesions. Experimental Design: Genomic DNA sodium bisulfate converted from benign and malignant tissues was used as template in methyl-specific PCR for BRCA1, p16, ESR1, GSTP1, TRβ1, RARβ2, HIC1, APC, CCND2, and CDH1 genes. Results: The majority of the breast cancer (85%) showed aberrant methylation in at least 1 of the loci tested with half of them displaying 3 or more methylated genes. The highest frequency of aberrant promoter methylation was found for HIC1 (48%) followed by ESR1 (46%), and CDH1 (39%). Similar methylation frequencies were detected for breast benign lesions with the exception of the CDH1 gene (P = 0.02). The analysis of methylation distribution indicates a statistically significant association between methylation of the ESR1 promoter, and methylation at CDH1, TRβ1, GSTP1, and CCND2 loci (P < 0.03). Methylated status of the BRCA1 promoter was inversely correlated with methylation at the RARβ2 locus (P < 0.03). Conclusions: Our results suggest a nonrandom distribution for promoter hypermethylation in sporadic breast cancer, with tumor subsets characterized by aberrant methylation of specific cancer-related genes. These breast cancer subgroups may represent separate biological entities with potential differences in sensitivity to therapy, occurrence of metastasis, and overall prognosis.

Changes in CpG Islands Promoter Methylation Patterns during Ductal Breast Carcinoma Progression

Aberrant promoter methylation of several known or putative tumor suppressor genes occurs frequently during carcinogenesis, and this epigenetic change has been considered as a potential molecular marker for cancer. We examined the methylation status of nine genes (APC, CDH1, CTNNB1, TIMP3, ESR1, GSTP1, MGMT, THBS1, and TMS1), by quantitative methylation specific PCR. Synchronous preinvasive lesions (atypical ductal hyperplasia and/or ductal carcinoma in situ) and invasive ductal breast carcinoma from 52 patients, together with pure lesions from 24 patients and 12 normal tissues paired to tumor and 20 normal breast distant from tumor were analyzed. Aberrant promoter methylation was detected in both preinvasive and invasive lesions for genes APC, CDH1, CTNNB1, TIMP3, ESR1, and GSTP1. However, hierarchical mixed model and Generalized Estimating Equations model analyses showed that only APC, CDH1, and CTNNB1 promoter regions showed a higher frequency and methylation levels in pathologic samples when compared with normal breast. Whereas APC and CTNNB1 did not show differences in methylation levels or frequencies, CDH1 showed higher methylation levels in invasive tumors as compared with preinvasive lesions (P < 0.04, Mann-Whitney test with permutation correction). The analysis of APC, CDH1, and CTNNB1 methylation status was able to distinguish between normal and pathologic samples with a sensitivity of 67% (95% confidence interval, 60-71%) and a specificity of 75% (95% confidence interval, 69-81%). Our data point to the direct involvement of APC, CDH1, and CTNNB1 promoter methylation in the early stages of breast cancer progression and suggest that they may represent a useful tool for the detection of tumor cells in clinical specimens. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2694–700)

Aberrant Keap1 methylation in breast cancer and association with clinicopathological features

Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein that mediates the ubiquitination/degradation of genes regulating cell survival and apoptosis under oxidative stress conditions. We determined methylation status of the KEAP1 promoter in 102 primary breast cancers, 14 pre-invasive lesions, 38 paired normal breast tissues and 6 normal breast from reductive mammoplasty by quantitative methylation specific PCR (QMSP). Aberrant promoter methylation was detected in 52 out of the 102 primary breast cancer cases (51%) and 10 out of 14 pre-invasive lesions (71%). No mutations of the KEAP1 gene were identified in the 20 breast cancer cases analyzed by fluorescence based direct sequencing. Methylation was more frequent in the subgroup of patients identified as ER positive-HER2 negative tumors (66.7%) as compared with triple-negative breast cancers (35%) (p = 0.05, Chi-square test). The impact of the interactions between Er, PgR, Her2 expression and KEAP1 methylation on mortality was investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple-negative breast cancer patients with KEAP1 methylation had higher mortality risk than patients without triple-negative breast cancer (HR = 14.73, 95%CI: 3.65–59.37). Both univariable and multivariable COX regressions analyses showed that KEAP1 methylation was associated with a better progression free survival in patients treated with epirubicin/cyclophosfamide and docetaxel as sequential chemotherapy (HR = 0.082; 95%CI: 0.007–0.934). These results indicate that aberrant promoter methylation of the KEAP1 gene is involved in breast cancerogenesis. In addition, identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients.

High Specificity of Quantitative Methylation-Specific PCR Analysis for MGMT Promoter Hypermethylation Detection in Gliomas

Normal brain tissue from 28 individuals and 50 glioma samples were analyzed by real-time Quantitative Methylation-Specific PCR (QMSP). Data from this analysis were compared with results obtained on the same samples by MSP. QMSP analysis demonstrated a statistically significant difference in both methylation level (P = .000009 Mann Whitney Test) and frequencies (P = .0000007, Z-test) in tumour samples as compared with normal brain tissues. Although QMSP and MSP showed similar sensitivity, the specificity of QMSP analysis was significantly higher (93%; CI95%: 84%–100%) as compared with MSP (64%; 95%CI: 46%–82%). Our results suggest that QMSP analysis may represent a powerful tool to identify glioma patients that will benefit from alkylating agents chemotherapy.

Association of Promoter Methylation of VGF and PGP9.5 with Ovarian Cancer Progression

Purpose To elucidate the role of biological and clinical impact of aberrant promoter hypermethylation (PH) in ovarian cancer (OC). Experimental Design PH of PGP9.5, HIC1, AIM1, APC, PAK3, MGMT, KIF1A, CCNA1, ESR1, SSBP2, GSTP1, FKBP4 and VGF were assessed by quantitative methylation specific PCR (QMSP) in a training set. We selected two genes (VGF and PGP9.5) for further QMSP analysis in a larger independent validation (IV) set with available clinical data. Biologic relevance of VGF gene was also evaluated. Results PH frequency for PGP9.5 and VGF were 85% (316/372) and 43% (158/366) respectively in the IV set of samples while no PH was observed in controls. In 372 OC cases with available follow up, PGP9.5 and VGF PH were correlated with better patient survival [Hazard Ratios (HR) for overall survival (OS) were 0.59 (95% Confidence Intervals (CI)  = 0.42–0.84, p = 0.004), and 0.73 (95%CI = 0.55–0.97, p = 0.028) respectively, and for disease specific survival (DSS) were 0.57 (95%CI 0.39–0.82, p = 0.003) and 0.72 (95%CI 0.54–0.96, p = 0.027). In multivariate analysis, VGF PH remained an independent prognostic factor for OS (HR 0.61, 95%CI 0.43–0.86, p<0.005) and DSS (HR 0.58, 95%CI 0.41–0.83, p<0.003). Furthermore, PGP9.5 PH was significantly correlated with lower grade, early stage tumors, and with absence of residual disease. Forced expression of VGF in OC cell lines inhibited cell growth. Conclusions Our results indicate that VGF and PGP9.5 PH are potential biomarkers for ovarian carcinoma. Confirmatory cohorts with longitudinal follow-up are required in future studies to define the clinical impact of VGF and PGP9.5 PH before clinical application.

Collecting duct carcinoma of the kidney is associated with CDKN2A deletion and SLC family gene up-regulation

The genetic landscape and molecular features of collecting duct carcinoma (CDC) of the kidney remain largely unknown. Herein, we performed whole exome sequencing (WES) and transcriptome sequencing (RNASeq) on 7 CDC samples (CDC1 −7). Among the 7 samples, 4 samples with matched non-tumor tissue were used for copy number analysis by SNP array data. No recurrent somatic SNVs were observed except for MLL, which was found to be mutated (p.V297I and p.F407C) in 2 samples. We identified somatic SNVs in 14 other cancer census genes including: ATM, CREBBP, PRDM1, CBFB, FBXW7, IKZF1, KDR, KRAS, NACA, NF2, NUP98, SS18, TP53, and ZNF521. SNP array data identified a CDKN2A homozygous deletion in 3 samples and SNV analysis showed a non-sense mutation of the CDKN2A gene with unknown somatic status. To estimate the recurrent rate of CDKN2A abnormalities, we performed FISH screening of additional samples and confirmed the frequent loss (62.5%) of CDKN2A expression. Since cisplatin based therapy is the common treatment option for CDC, we investigated the expression of solute carrier (SLC) family transporters and found 45% alteration. In addition, SLC7A11 (cystine transporter, xCT), a cisplatin resistance associated gene, was found to be overexpressed in 4 out of 5 (80%) cases of CDC tumors tested, as compared to matched non-tumor tissue. In summary, our study provides a comprehensive genomic analysis of CDC and identifies potential pathways suitable for targeted therapies.

Aberrant promoter methylation of beta-1,4 galactosyltransferase 1 as potential cancer-specific biomarker of colorectal tumors

Epigenetic alterations, such as CpG islands methylation and histone modifications, are recognized key characteristics of cancer. Glycogenes are a group of genes which epigenetic status was found to be changed in several tumors. In this study, we determined promoter methylation status of the glycogene beta‐1,4‐galactosyltransferase 1 (B4GALT1) in colorectal cancer patients. Methylation status of B4GALT1 was assessed in 130 colorectal adenocarcinomas, 13 adenomas, and in paired normal tissue using quantitative methylation specific PCR (QMSP). B4GALT1 mRNA expression was evaluated in methylated/unmethylated tumor and normal specimens. We also investigated microsatellite stability and microsatellite instability status and KRAS/BRAF mutations. Discriminatory power of QMSP was assessed by receiving operating curve (ROC) analysis on a training set of 24 colorectal cancers and paired mucosa. The area under the ROC curve (AUC) was 0.737 (95% confidence interval [CI]:0.591–0.881, P = 0.005) with an optimal cutoff value of 2.07 yielding a 54% sensitivity (95% CI: 35.1%–72.1%) and a specificity of 91.7% (95% CI: 74.1%–97.7%). These results were confirmed in an independent validation set where B4GALT1 methylation was detected in 52/106 patients. An inverse correlation was observed between methylation and B4GALT1 mRNA expression levels (r = −0.482, P = 0.037). Significant differences in methylation levels and frequencies was demonstrated in invasive lesions as compared with normal mucosa (P = 0.0001) and in carcinoma samples as compared with adenoma (P = 0.009). B4GALT1 methylation is a frequent and specific event in colorectal cancer and correlates with downregulation of mRNA expression. These results suggest that the glycogene B4GALT1 represent a valuable candidate biomarker of invasive phenotype of colorectal cancer.

Dysregulation of EGFR Pathway in EphA2 Cell Subpopulation Significantly Associates with Poor Prognosis in Colorectal Cancer

Purpose: EphA2 receptor is involved in multiple cross-talks with other cellular networks, including EGFR, FAK, and VEGF pathways, with which it collaborates to stimulate cell migration, invasion, and metastasis. Colorectal cancer (CRC) EphA2 overexpression has also been correlated to stem-like properties of cells and tumor malignancy. We investigated the molecular cross-talk and miRNAs modulation of the EphA2 and EGFR pathways. We also explored the role of EphA2/EGFR pathway mediators as prognostic factors or predictors of cetuximab benefit in patients with CRC. Experimental Design: Gene expression analysis was performed in EphA2high cells isolated from CRC of the AOM/DSS murine model by FACS-assisted procedures. Six independent cohorts of patients were stratified by EphA2 expression to determine the potential prognostic role of a EphA2/EGFR signature and its effect on cetuximab treatment response. Results: We identified a gene expression pattern (EphA2, Efna1, Egfr, Ptpn12, and Atf2) reflecting the activation of EphA2 and EGFR pathways and a coherent dysregulation of mir-26b and mir-200a. Such a pattern showed prognostic significance in patients with stage I–III CRC, in both univariate and multivariate analysis. In patients with stage IV and WT KRAS, EphA2/Efna1/Egfr gene expression status was significantly associated with poor response to cetuximab treatment. Furthermore, EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutation status. Conclusions: These results suggest that EphA2/Efna1/Egfr genes, linked to a possible control by miR-200a and miR-26b, could be proposed as novel CRC prognostic biomarkers. Moreover, EphA2 could be linked to a mechanism of resistance to cetuximab alternative to KRAS mutations. Clin Cancer Res; 23(1); 159–70. ©2016 AACR.

Bilateral consecutive rupture of the quadriceps tendon in a man with BstUI polymorphism of the COL5A1 gene : Reply to the letter by R. Dalgleish (Letter to the Editors)

Dear Editors, We thank Dr Dalgleish for his comments. Indeed, we agree with his concerns, and we believe that we clarified our position in the response to the letter by Collins et al. [4]. We can only reiterate that in our article [3] we do not state that the COL5A1 gene polymorphism is associated with the development of bilateral quadriceps tendon rupture: we agree with Dr Dalgleish that this statement would not have been valid and would have been misleading. Tendon pathology is consistent across sites, and the features of the failed healing response are common in all the studied tendons with tendinopathy or rupture [1, 5–7]. The intimate nature of tendon pathology is still largely unknown. New therapeutic options have been proposed, including biomaterials, platelet rich plasma and tissue engineering, but the therapeutic options are still limited. In our article [3], we did state that ‘‘it is possible that an interaction between the various intrinsic and extrinsic factors with the genetic make up of a given individual increases the likelihood of that individual developing tendinopathy’’ and that ‘‘the exact role of COL5A1 and TNC genes in the pathogenesis of tendinopathy is still debated, and the current evidence does not allow to clarify whether or not COL5A1 and TNC genes are the ideal markers of tendinopathy’’. ‘‘It is also possible that other genes, yet to be determined, may contribute towards the pathogenesis of tendinopathy, which could be a polygenic condition, given the multitude of the genes involved in maintaining normal tendon homeostasis. Based on current evidence, it is difficult to conceive that only a single gene and not multiple genes are involved in the pathogenesis of tendinopathy. Thus, additional investigation needs to be performed to identify these genes’’. Our investigation is not a genetic associations study, but a simple case report. Bilateral ruptures of the quadriceps tendon are difficult to study, especially because they are uncommon, with less then 110 patients identified worldwide in a recent meta-analysis [8]. Therefore, it is almost impossible to conduct a well-powered study in a single centre to detect the incidence of polymorphisms involved in the aetiopathogenesis of this complex pathology [2]. Only wide, multicentre studies will allow to collect a sufficient number of patients to clarify the gene involved in the aetiopathogenesis of this complex pathology. For this reason, we already stated that ‘‘Further studies on large populations are required to confirm this result’’. The letter by Dalgleish can be found at DOI 10.1007/s00167-011-1408-1.