Potential Prognostic Role of SPARC Methylation in Non-Small-Cell Lung Cancer.

The silencing of SPARC (secreted protein acid and rich in cysteine) gene through methylation of its promoter region represents a common event in many solid tumors and it is frequently associated with tumor progression and an aggressive clinical outcome. Anyhow, the data concerning the epigenetic mechanism of SPARC deregulation and its prognostic value in lung cancer are still incomplete. We explored the aberrant methylation of SPARC and its effects in 4 non-small cell lung cancer (NSCLC) cell lines and 59 NSCLC tissues and correlated the methylation levels with clinical-pathological features and disease outcome of patients. In 3 out of 4 tumor cell lines high SPARC methylation levels were observed. An inverse correlation between the epigenetic silencing and SPARC expression was confirmed by 5-Aza-2'-deoxycytidine ((5-Aza-CdR) treatment that also significantly induced a reduction in cell viability, proliferation and tumor cell migration. In tissues, the DNA methylation levels of the SPARC gene were significantly lower in paired non-neoplastic lungs (NLs) and normal lungs distant from tumor (NLDTs) than in NSCLCs (p = 0.002 and p = 0.0034 respectively). A promoter hypermethylation was detected in 68% of squamous cell carcinoma (SqCCs, 17/25) and 56% of adenocarcinoma (ADCs, 19/34), with SqCC showing the highest levels of methylation. Higher SPARC methylation levels were significantly associated with higher mortality risk both in all NSCLCs early stage patients (Hazard Ratio, HR = 1.97; 95% Confidence Interval, CI: 1.32-2.93; p = 0.001) and in those with SqCC (HR = 2.96; 95% CI: 1.43-6.12; p = 0.003). Promoter methylation of SPARC gene should represent an interesting prognostic biomarker in NSCLC, with potential application in the squamous early-stage context. Further research in this setting on larger independent cohorts of lung patients with different histologies and stages of disease are warranted.

Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors.

BACKGROUND: The KEAP1/NRF2 pathway has been widely investigated in tumors since it was implicated in cancer cells survival and therapies resistance. In lung tumors the deregulation of this pathway is mainly related to point mutations of KEAP1 and NFE2L2 genes and KEAP1 promoter hypermethylation, but these two genes have been rarely investigated in low/intermediate grade neuroendocrine tumors of the lung. METHODS: The effects of KEAP1 silencing on NRF2 activity was investigated in H720 and H727 carcinoid cell lines and results were compared with those obtained by molecular profiling of KEAP1 and NFE2L2 in a collection of 47 lung carcinoids. The correlation between methylation and transcript levels was assessed by 5-aza-dC treatment. RESULTS: We demonstrated that in carcinoid cell lines, the KEAP1 silencing induces an upregulation of NRF2 and some of its targets and that there is a direct correlation between KEAP1 methylation and its mRNA levels. A KEAP1 hypermethylation and Loss of Heterozygosity at KEAP1 gene locus was also observed in nearly half of lung carcinoids. CONCLUSIONS: This is the first study that has described the effects of KEAP1 silencing on the regulation of NRF2 activity in lung carcinoids cells. The epigenetic deregulation of the KEAP1/NRF2 by a KEAP1 promoter hypermethylation system appears to be a frequent event in lung carcinoids.

Frequent NRG1 fusions in Caucasian pulmonary mucinous adenocarcinoma predicted by Phospho-ErbB3 expression.

NRG1 fusions were recently reported as a new molecular feature of Invasive Mucinous Adenocarcinoma (IMA) of the lung. The NRG1 chimeric ligand acts as a strong inductor of phosphorylation and tyrosine kinase activity of the ErbB2/ErbB3 heterodimer, thus enhancing the PI3K-AKT/MAPK pathways. The NRG1 fusions were widely investigated in Asian IMA cohorts, whereas just anecdotal information are available about the occurrence of NRG1 fusions in IMA Caucasian population. Here we firstly explored a large Caucasian cohort of 51 IMAs and 34 non-IMA cases for the occurrence of NRG1 rearrangements by fluorescent in situ hybridization (FISH) and RNA target sequencing. FISH results were correlated to the immunohistochemical expression of phosphorylated-ErbB3 (pErbB3) receptor and the mutational status of KRAS, EGFR and ALK genes. The NRG1 rearrangements were detected in 31% IMAs and 3% non-IMAs and the CD74-NRG1 fusion transcript variant was characterized in 4 NRG1-positive IMAs. Moreover, pErbB3 expression was found to be strictly associated to the mucinous pattern (p = 0.012, Chi-square test) and all IMA cases showing aberrant expression of pErbB3 demonstrated NRG1 rearrangements. No significant correlation between NRG1 rearrangements and EGFR, KRAS or ALK mutations respectively, was observed. We report for the first time that NRG1 fusions are driver alterations clearly associated with mucinous lung adenocarcinoma subtype of Caucasian patients and not exclusive of Asiatic population. pErbB3 immunostaining may represent a strong predictor of NRG1 fusions, pointing out the detection of pErbB3 by IHC as a rapid and effective pre-screening method to select the NRG1-positive patients.

Keap1/Nrf2 pathway in kidney cancer: frequent methylation of KEAP1 gene promoter in clear renal cell carcinoma.

The Keap1/Nrf2 pathway is a master regulator of the cellular redox state through the induction of several antioxidant defence genes implicated in chemotherapeutic drugs resistance of tumor cells. An increasing body of evidence supports a key role for Keap1/Nrf2 pathway in kidney diseases and renal cell carcinoma (RCC), but data concerning the molecular basis and the clinical effect of its deregulation remain incomplete.Here we present a molecular profiling of the KEAP1 and NFE2L2 genes in five different Renal Cell Carcinoma histotypes by analysing 89 tumor/normal paired tissues (clear cell Renal Carcinoma, ccRCCs; Oncocytomas; Papillary Renal Cell Carcinoma Type 1, PRCC1; Papillary Renal Cell Carcinoma Type 2, PRCC2; and Chromophobe Cell Carcinoma).A tumor-specific DNA methylation of the KEAP1 gene promoter region was found as a specific feature of the ccRCC subtype (18/37, 48.6%) and a direct correlation with mRNA levels was confirmed by in vitro 5-azacytidine treatment. Analysis of an independent data set of 481 ccRCC and 265 PRCC tumors corroborates our results and multivariate analysis reveals a significant correlation among ccRCCs epigenetic KEAP1 silencing and staging, grading and overall survival.Our molecular results show for the the first time the epigenetic silencing of KEAP1 promoter as the leading mechanism for modulation of KEAP1 expression in ccRCCs and corroborate the driver role of Keap1/Nrf2 axis deregulation with potential new function as independent epigenetic prognostic marker in renal cell carcinoma.