The effect of human papillomavirus oncoproteins expression on RECK gene regulation

Abstract

Up regulation of metalloproteinases type 2 and 9 (MMP-2 and MMP-9) expression and activity are the most common extracellular matrix (ECM) related modifications in precursor cervical lesions and invasive carcinoma. We reported that this event correlated with the expression of MMP inhibitors, including REversion-inducing Cysteine-rich protein with Kazal motifs (RECK). Reduced RECK mRNA and protein levels have been reported in several types of cancer, where detectable expression of RECK gene has been associated with better overall prognosis. We already showed that RECK expression is down regulated in most cervical cancer derived cell lines and in cervical lesions. We also observed that HPV16 oncogenes expression was associated with reduced RECK protein levels in primary human keratinocytes (PHK) cultures. In a recent report, we showed that RECK overexpression reduced the tumorigenic potential of HPV-transformed cells in vivo and altered the profile of the tumor inflammatory infiltrate. Besides, we showed that reduced RECK levels were associated with cervical lesions progression, presence of metastasis and treatment response in clinical samples. RECK is a serine protease inhibitor that regulates various physiological events including the structural maintenance of ECM, correct formation of blood vessels during embryogenesis, organogenesis, tissue integrity, early neuronal differentiation, and limb formation. RECK mRNA may undergo alternative splicing, which generates variant mRNA sequences, such as RECK B isoform (NM_001316346.2). RECK B mRNA encodes a protein of theoretical mass of 37kDa, in which the serine protease inhibiting activity is lost and its functions are not yet fully defined. Preliminary data from our laboratory pointed to an early and progressive RECK gene hyper methylation in CIN lesions and invasive cervical carcinomas when compared with normal cervical tissues. Besides, we observed that higher methylation levels in RECK gene promoter (cg01258587 probe) inversely correlated with RECK mRNA expression in both cervical cancer and HNSCC samples deposited in TCGA database. Finally, we demonstrated that HPV16 E6 and E7 expression is associated with reduced activation of RECK gene promoter. Ras oncogenic function is associated with the negative regulation of RECK gene expression, in part through the repression complex formed by HDAC1 and either Sp1 alone or together with pRbAp46. Our initial in silico analysis on RECK gene promoter showed potential NFkB, ER, Sp1, HIF1a and E2F binding sequences. The identification of E2F binding sites in RECK promoter is particularly interesting once several members of this family of proteins are altered in HPV transformed cells. Previous findings showed that HPV16 E7 can induce the formation of a protein complex with NFkB-p50-p65, ERa, HDAC1 and JARID1B which was associated with the repression of TLR9 gene expression. We hypothesize that this mechanism may be involved in the negative regulation of RECK gene expression associated with HPV16 E7 expression in PHK. Here, we aim to define whether the negative effect of E6/E7 expression on the activation of RECK promoter is dependent on their physical bind to RECK promoter and describe its molecular partners with use of chromatin immunoprecipitation (ChIP) and PCR based promoter occupancy assays. Moreover, we will assess if E6 and/or E7 expression impact on the mRNA levels of different RECK isoforms and their stability in primary, immortalized and transformed epithelial cell models. The candidate would perform these experiments in Lawrence Banks´ laboratory in the International Centre for Genetic Engineering and Biotechnology/ICGEB, Italy. This study will provide answers on the mechanisms associated with RECK gene downregulation in HPV associated transformation, which may contribute to the better understanding of HPV driven cancers and to define a RECK gene target therapeutic protocol for these tumors in the future.