POST-TRANSLATIONAL MODIFICATIONS IN CANCER AND DIABETES

Abstract

Background: In Qatar, non-communicable diseases like cancer and diabetes are leading causes of mortality. Cancer is caused by various environmental and hereditary factors, with metastasis from the primary site being the major cause of cancer related death. This calls the need for better diagnostics and treatments. Diabetes, on the other hand, can lead to severe complications such as diabetic peripheral neuropathy (DPN), which drastically reduces patients' quality of life by impairing pain sensation. Understanding the molecular mechanisms driving cancer metastasis and pain in DPN is essential for developing novel, targeted therapies. Post-translational modifications (PTMs) have emerged as key regulators of protein function, making them promising biomarkers and therapeutic targets in both cancer and diabetes. Methodology: The study was conducted in two phases. In the first, common PTMs of human serum albumin (HSA) were identified under normal conditions using water, DMEM medium, and serum albumin from a healthy individual. Common PTMs across these three conditions were designated as 'normal' and used as a baseline for comparison with those PTMs observed after exposing HSA to breast cancer cell lines (MCF-7, MDA-MB-231, and MDA-MB-468). PTMs were analyzed using 2- dimensional electrophoresis, mass spectrometry and three- dimensional structural modeling. In the second phase, a streptozotocin (STZ)-induced diabetic mouse model was developed to investigate the role of PTMs in DPN and pain signaling. Diabetic mice were treated with PTM-targeting drugs (AG1478, Afatinib, LY294002 and SU6656), and pain behavior was evaluated using the Hargreaves test, a measure of thermal nociception. In addition, intraepidermal nerve fiber density was assessed using immunohistochemistry (IHC) of paw skin biopsies to evaluate nerve damage. RNA sequencing of dorsal root ganglion (DRG) neurons from control, diabetic, and drugtreated groups was performed to identify differentially expressed genes related to PTMs and pain signaling pathways. Results: Mass spectroscopy analysis identified 61 new PTMs in HSA under normal conditions including, phosphorylation, glycosylation, and nitrosylation. Exposure to breast cancer cell lines led to 14 additional novel PTMs compared to those identified under normal condition (water, DMEM medium, and human serum albumin from healthy individual). Distinct patterns were observed between aggressive (MDAMB- 231, MDA-MB-468) and non-aggressive (MCF-7) cell lines. Three-dimensional modelling revealed that many PTMs were located in functional regions of HSA, such as Sudlow's site and lipid-binding regions, potentially altering albumin's drug-binding and transport capacities. In diabetic mice, the Hargreaves test revealed a significant delay in paw withdrawal latency, indicating impaired thermal nociception and reduced pain sensitivity-a hallmark of diabetic peripheral neuropathy. This pain deficit was effectively reversed by treatment with PTM-targeting drugs. However, no significant differences in intraepidermal nerve fiber density were observed across groups, suggesting that the observed pain relief may be independent of structural nerve restoration. RNA sequencing of DRG neurons provided critical insights into the molecular mechanisms underlying pain and PTMs in DPN. Gene ontology analysis highlighted key pathways involved in sensory perception of pain, regulation of membrane potential, and blood circulation as significantly enriched in diabetic mice. Differential gene expression analysis identified several notable genes involved in pain signaling, including Scn9a (Nav1.7), a key sodium channel in nociceptive neurons, Trpv1, associated with thermal pain sensitivity, and Faah, involved in endocannabinoid signaling. Additionally, Mgat4a, a glycosyltransferase implicated in PTMs, showed significant differential expression, underscoring the role of PTMs in modulating pain pathways. Conclusions: This study highlights the critical role of PTMs in albumin and receptor tyrosine kinases signaling in cancer and pain sensitivity in diabetic peripheral neuropathy. 3D structural analysis showed that many of the PTMs identified would have an impact on albumin functions. PTM-targeting drugs demonstrated strong efficacy in reversing pain behavior deficits in diabetic mice, despite no observable changes in nerve fiber density, pointing to the potential for modulating pain pathways at the molecular level. RNA sequencing further revealed the involvement of key pain-related genes and PTM-associated pathways, offering promising targets for therapeutic intervention. Developing PTMfocused therapies holds significant potential for addressing cancer and diabetes-induced complications.