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Gene Expression Regulation and Cancer

Final Report Summary - MISWI (Gene Expression Regulation and Cancer)

Gene expression regulation is one of the most fascinating and intricate aspects of biology. Chromatin-structure and microRNAs (miRNAs) both play important roles on gene expression regulation and have been found to be critical in the development of human pathologies.
DNA is packaged inside cells through a scaffold of proteins, constituting the chromatin. The chromatin not only has a structural role, but it also plays an active role in other processes including gene expression regulation. The SWI/SNF complex is an ATP-dependent chromatin-remodelling complex that uses the energy from ATP hydrolysis to modify the interactions between DNA and histones. In this way, the SWI/SNF complex can render the DNA accessible or inaccessible to the transcription machinery. Ever increasing evidence demonstrates that some components of the SWI/SNF complex act as tumour suppressors and their deregulation is involved in human cancer development. Importantly, functional alterations of SWI/SNF complex factors are ubiquitous in tumor types, underlining the importance of this complex in carcinogenesis .The human SWI/SNF is a complex composed of a catalytic subunit that has chromatin remodeling activity itself and a group of 9 to15 variable subunits that modulate its activity. SMARCA4 (also known as BRG1) is a catalytic ATPase/helicase subunit of the SWI/SNF complex. SMARCA4 has been widely studied for its association with cancer. Previously I determined the importance of the inactivation of SMARCA4 in lung cancer cell lines. Remarkably, we found that 35% of non-small cell lung cancer (NSCLC) cell lines bear homozygous SMARCA4 inactivating mutations. These results reveal SMARCA4 to be one of the genes most frequently mutated in NSCLC cancer cell lines, highlighting the tumor suppressor activity of SWI/SNF. We also described the first somatic mutation of SMARCA4 in primary tumors. However, although SMARCA4 mutations were found frequently in lung cancer cell lines, we were unable to find this same frequency of mutations in primary tumors. However, a tissue microarray of 245 NSCLC tumors detected the loss of expression of SMARCA4 in the 48% of the tumors, in contrast with normal tissue that express SMARCA4 in all cases. Intriguingly, the loss of the expression of SMARCA4 in primary tumors is similar to that observed in cell lines. Thus, as there exists an unknown mechanism that contributes to the functional inactivation of SMARCA4.
During carcinogenesis, cells develop different mechanisms to inactivate gene expression of tumor suppressor genes. Promoter hypermethylation is one of the most studied mechanisms of epigenetic silencing in tumor development. We previously studied if SMARCA4 expression was inactivated by this mechanism by studying the promoter hypermethylation status using methylation-specific PCR (MSP) and bisulfite sequencing. However, we concluded that promoter hypermethylation was not a mechanism of SMARCA4 expression inactivation.
MicroRNAs (miRNAs) are a class of small RNA molecules that negatively regulate gene expression at the posttranscriptional level by binding to complementary sequences in 3’ untranslated regions (UTRs), ushering in a renewed appreciation of the regulative capabilities of non-coding RNA (ncRNA). Today, miRNAs are increasingly seen as important regulators of gene expression, and they have seen to play an important role in human pathology, including canc.. Importantly, aberrant levels of miRNAs often result in loss of differentiation, a hallmark of cancer. Not surprisingly, therefore, dysfunctions of the miRNA pathway affect many cellular processes that are routinely altered in cancer, such as differentiation, proliferation, apoptosis, metastasis, and senescence. Interestingly, recent studies have observed that miRNAs are able to very tightly control expression of SWI/SNF complex protein members. Therefore is plausible to believe that miRNAs could contribute to the loss of expression of SMARCA4 during lung development. One of the main aims of this work consisted of determining if SMARCA4 expression is lost in lung cancer primary tumors because of repression by miRNAs. Interestingly, we have found that the loss of expression of SMARCA4 observed in some primary lung tumors, whose mechanism was largely unknown, can be explained, at least partially by the activity of microRNAs (miRNAs). We reveal that SMARCA4 expression is regulated by miR-101, miR-199 and especially miR-155 through their binding to two alternative 3’UTRs. Importantly, our experiments suggest that the oncogenic properties of miR-155 in lung cancer can be largely explained by its role inhibiting SMARCA4. This new discovered functionally relationship could explain the poor prognosis displayed by patients that independently have high miR-155 and low SMARCA4 expression levels. In addition, these results could lead to application of incipient miRNA technology to the synthetic lethal therapeutic strategies.