Role of chaperone-mediated autophagy in DNA repair and its implications in Breast Cancer

• Grant number: 22/04325-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Effective date (Start): August 01, 2023
• Effective date (End): December 31, 2026
• Field of knowledge: Biological Sciences - Genetics - Mutagenesis
• Principal Investigator: Nicolas Carlos Hoch
• Grantee: Isabeli Yumi Araújo Osawa
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 19/19435-3 - The role of DNA damage and mitochondrial function in vascular, immune and neurological ageing (DNA MoVINg), AP.TEM

Abstract

Chaperone-mediated autophagy (CMA) is a proteolytic system essential for maintaining cellular homeostasis, through which only intracellular proteins bearing a specific motif (KFERQ-like) are selectively degraded in lysosomes. This is accomplished through the coordinated action of cytosolic chaperones and a translocation complex of proteins located at the lysosome membrane. In addition to acting in quality control of proteins and in the energetic balance, this pathway is also responsible for the cellular response to multiple forms of stress, such as oxidative stress and DNA damage. In this sense, it is known that CMA is activated in response to genotoxic insults and that its activation can increase genomic instability, a well known hallmark of tumor development. Thus, dysfunctions in CMA and DNA repair mechanisms have been associated with cancer. However, the CMA role in DNA repair mechanisms was very little explored until this moment. Among the different types of cancer, breast cancer stands out because, besides being the most frequent one, it is the leading cause of cancer death between worldwide women. Furthermore, breast tumors are among the few types of tumors in which the role of CMA has already been explored, so that the high activity of this autophagic pathway is positively correlated with the progression of breast tumors. In this context, the present project intends to investigate the role of CMA in the DNA damage response, specifically in DNA repair, throughout the process of breast tumor progression. For this, non-tumorigenic and tumorigenic (with different degrees of malignancy) human breast cell lines will be used as the study model, knocked out for the lysosomal-associated membrane protein 2A (LAMP-2A), the limiting key protein for CMA performance. First, the sensitivity of these cells to different types of DNA damage and, consequently, the activation of distinct DNA repair pathways will be characterized. Hence, these cells will be exposed to different model DNA damage inducers such as ionizing radiation, UVC and H2O2, and to classical chemotherapeutic agents as doxorubicin, cisplatin and temozolomide (TMZ). The role of CMA in cell repair capacity will be evaluated on a large-scale in an unprecedented way, through Fluorescence Multiplex Host Cell Reactivation (FM-HCR) assays, allowing the analysis of the six main pathways of DNA repair in living cells. The HCR data will be further validated through specific biochemical assays for the repair pathways indicated as potentially modulated by CMA activity. The identification of CMA substrates that are involved in DNA repair mechanisms will be performed through comparative lysosome proteomics. Some of the most interesting proteins from the list obtained through the lysosome proteome will be chosen for inhibition. Thus, we will seek to identify the candidates able to reverse the phenotype of sensitivity to DNA damage-inducing agents. In this way, altogether, the results to be obtained by the present proposal will provide a solid overview of CMA role in the control of genomic instability, cancer development and response to chemotherapy, supporting the future use of CMA modulators as a potential anticancer therapeutic target. (AU)