Avaliação da relação entre a expressão de genes da via de reparo por excisão de bases e resistência tumoral em câncer de mama

Abstract

Breast cancer is the most common type of cancer worldwide and the second leading cause of death among women. Tumors identified in advanced stages may present regions of hypoxia, which makes chemotherapy less effective, in most cases, by activating molecular mechanisms related to drug resistance. Chemotherapy with Doxorubicin is one of the most used in the treatment of breast cancer, however, the effectiveness of the treatment is significantly reduced due to the activity of repair pathways for DNA damage, especially oxidative damage that is repaired by pathways such as the BER pathway. The APE1/Ref-1 enzyme is a multifunctional enzyme that acts in the repair of DNA lesions and plays an important role in redox regulation through the regulation of transcription factors, so it has been considered an important protein for the study of response to chemotherapy treatment in breast cancer. Therefore, this work proposes to investigate the BER pathway in breast cancer and its relationship to chemotherapy resistance in hypoxia. In silico analyzes of BER pathway gene transcript levels in tumor samples from breast cancer patients from the TCGA (BRCA) were performed using the XENA USCS platform. In silico analyzes were performed comparing tumors with and without hypoxia signature, for this an algorithm containing a panel of previously described genes was used. The investigation of transcript levels of BER pathway genes in MCF-7 and MDA-MB-231 cell cultures under normoxia and hypoxia conditions were performed by RT-qPCR amplification. Cell viability assays of breast cancer cells was performed by colorimetric assay, through Elisa's reader, with WST-1 reagent. The results of the analysis in silico in the TCGA BRCA patients indicate that the genes of the BER pathway are more expressed in tumors than normal samples (n=28 genes out of 28 analyzed), and in those that showed higher relationship with molecular hypoxia signature. The APEX2 and PARP3 genes differ in their expression between early and late stages, so they can be used as staging markers for auxiliary in diagnostic phase. The PARP3 gene expression showed a significant difference in patient survival. The basal subtype was the most common with the genetic signature of hypoxia, while the Luminal subtype was less common. The tumor size T2 was the most predominant in both normoxia and hypoxia. The results of in vitro analysis indicated that there was a difference in the transcript levels only of the NEIL3 and FEN1 genes in the MCF-7 cells, while in the MDA-MB-231 cells there was no difference in the expression of the genes. Furthermore, The MCF-7 and MDA-MB-231 cells were the greater survival to treatment with the inhibitor APX2009 and Doxorubicin when submitted to hypoxia condition. Thus, it was possible to conclude that the increased expression of genes in the BER pathway is associated with tumors and in tumors with hypoxia characteristics. And the condition of hypoxia directly influence cell survival to treatments with APX2009 and Doxorubicin in the MCF-7 and MDA-MB-231 cells