Analysis of Protein Expression Regulated by the Brn-2 Transcription Factor in Cells treated with Peptides-Derived from POU Domain of Brn-2 and Determination of the Internalization and Cell Localization of this Peptide

Abstract

The transcription factor Brn-2 is expressed in melanocyte cell lines and superexpressed in melanoma. The increase in Brn-2 expression is related to tumor formation, tumor progression and development of metastases. The three major intracellular signaling pathways that are involved in melanoma formation, the MAPK / BRAF, Wnt / ²-catenin and PI3K / Akt, Brn-2 regulates the expression of Brn-2. The constitutive activation of BRAF induces the increase in Brn-2 expression that induces MITF, which is a protein responsible for melanogenesis. The increase in MITF expression is related to cell survival by regulating the expression of the anti-apoptotic Bcl2 protein.However, the Brn-2 transcription factor when overexpressed independently of BRAF may also sub-rule MITF levels, suppressing the phenotype of melanocytic differentiation and consequently promoting tumor metastasis. Melanoma cells with low levels of MITF are more invasive, clearly implicating Brn-2 in melanoma tumorigenicity and in the formation of metastases. Brn-2 also down regulates expression of PDE5A leading to an increase of intracellular Ca+2, stimulating cell invasion. In addition, Brn-2 has been implicated in the regulation of the NOTCH signaling pathway another pathway that is directly related to melanoma progression. In melanoma, Brn-2 has been identified as the transcriptional repressor of the T-cadherin cell surface glycoprotein (cadherin 13). This protein has been identified as a tumor suppressor because its expression is significantly decreased in several types of cancer.Recent studies in our laboratory have shown that peptides derived from the DNA binding domain of Brn-2 transcription factor have important biological effects. Three peptides showed antitumor activity, and peptide R18H induces apoptosis in treated cells. However, other four peptides tested showed no significant antitumor effect, but caused inhibition of cell migration and increase in intracellular calcium levels, and possibly also interfered with the mechanisms of invasion and metastasis. Peptides derived from Brn-2 could display several inhibitory effects in melanoma development, including competition for the DNA binding site of the Brn-2 transcription factor. In addition, the mechanisms of cellular internalization and cellular location of these peptides should be investigated. We propose that these peptides could be altering the gene expression of Brn-2, MITF, T-Cadherin, PTEN, NOTCH1, JAG2, DLL1, Bax and Bcl2 proteins. Changes in the levels of these proteins will be analyzed by Western blotting in cells treated with the peptides. In addition, interference in the gene expression of peptides derived from the Brn-2 transcription factor will be assessed by the RNAseq technique using the Ilumina platform. This study will be important for these peptides to be used as precursors of promising cancer drugs. (AU)