Mechanisms of Apoptosis Regulation in Carcinogenesis and Chemoresistance: The Role of Inhibitor of Apoptosis Proteins as Therapeutic Targets in Melanoma

• Grant number: 24/14964-6
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: June 01, 2025
• End date: May 31, 2027
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Leticia Veras Costa Lotufo
• Grantee: Catarina Sofia Mateus Reis e Silva
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 14/50926-0 - INCT 2014: biodiversity and natural products, AP.BTA.TEM

Abstract

Apoptosis is a physiological process of programmed cell death regulated by pro- and anti-apoptotic proteins, essential for normal embryonic development, genome integrity, immune system function, and tissue homeostasis. Dysregulation of the apoptotic process allows tumor cells to survive and proliferate, maintaining the tumor microenvironment. Consequently, research targeting members of the Inhibitor of Apoptosis Proteins (IAP) family is abundant, exploring various substances and techniques. Despite progress, questions regarding the biology of IAPs in cancer remain unanswered, presenting opportunities for future treatment discoveries. In this project, we extracted data from 33 cancer types using the TIMER 2.0 platform to identify IAPs that are highly expressed in tumors. Focusing on cutaneous melanoma cohort data from TCGA deposited in cBioPortal, we analyzed IAP expression in melanoma stages and survival outcomes using GraphPad Prism 8.0. RNA-seq data were pre-processed for differential expression using limma-voom in Galaxy. GSEA v.4.0, using Hallmark gene sets from MSigDB, was performed with a false discovery rate (FDR) of 25% and a p-value <0.05. Cell viability was measured using an MTT assay, with a total of 5 × 10³ cells per well seeded in a 96-well plate and exposed to increasing concentrations of doxorubicin, cefalochromine, YM155, PLX4032, prodigiosin, or tolinapant. For treatment analysis, melanoma cell lines SK-MEL-28, SK-MEL-147, SK-MEL-173, 501-mel, and HT-144 were treated with graded doses of the mentioned drugs for 72 hours, and IC50 values were calculated using nonlinear regression analysis in GraphPad Prism 8. Based on our TIMER 2.0 analysis, we selected melanoma as our focus due to the overexpression of 5 out of 8 IAPs in melanoma patients. High expression of BIRC5 was identified as a poor prognostic predictor, aligning with existing research and showing enrichment in MYC, pyrimidine, and purine metabolic pathways. On the other hand, NAIP, BIRC3, and XIAP were considered protective factors, raising questions about their role in cancer maintenance. BIRC2, BIRC6, BIRC7, and BIRC8 had no significant impact on survival outcomes (p > 0.05). Notably, BIRC3 was enriched in pathways related to inflammatory responses, hematopoietic cell lineage, and KRAS and JAK-STAT signaling. It was also positively regulated in processes involving humoral immune responses, including B cell proliferation, activation, differentiation, and lymphocyte proliferation. To validate these bioinformatic findings, we performed in vitro assays in melanoma cell lines. YM155 and cefalochromine, both associated with IAP suppression (particularly BIRC5), produced the best results in reducing viability.