Cellular and molecular responses to proteotoxic stress in melanoma cells

Protein folding and protein degradation constitute the two essential arms of the cellular protein quality control (PQC) system that preserves the integrity of functional proteins. Because a protein must adopt its native conformation to be functional, nascent proteins are subjected to co‑translational and post‑translational folding, throughout their lifetime unfolded or misfolded species are subjected to refolding, all these processes rely on a network of molecular chaperones. When a protein becomes terminally misfolded, it is targeted for selective degradation because its accumulation is proteotoxic. In addition, protein degradation also serves to regulate the cellular levels of many proteins. The heat‑shock protein 90 (HSP90) is a major chaperone that stabilizes a broad repertoire of client proteins, including kinases, transcription factors and E3 ligases. The ubiquitin‑proteasome system, centered on the 26S proteasome, is a principal machinery for selective protein degradation. Due to their indispensable roles in PQC, HSP90 and the proteasome have been pursued as therapeutic targets in diseases marked by elevated proteotoxic stress, such as neurodegenerative disorders and cancer. Cancer cells have a high number of protein‑altering mutations and sustain elevated rates of protein synthesis for their uncontrolled growth. As a result, they experience chronic folding stress and rely heavily on HSP90 and the proteasome for survival. Melanoma provides a suitable model for studying intrinsic proteotoxic stress because it is a highly mutated cancer with a rapid proliferation rate. In this project, I used two melanoma cell lines, Ma‑Mel‑94 and Ma‑Mel‑102, which contain 678 and 1123 missense mutations, respectively, and represent opposite ends of the melanoma‑lineage spectrum. I hypothesized that, in the presence of chronic intrinsic folding stress in these cancer cell lines, even a modest compromise of PQC capacity or a low level of extrinsic stress would expose the most responsive proteostasis factors. To test this hypothesis, I applied low‑dose inhibition of HSP90 with 17‑DMAG (50 nM) and of the proteasome with bortezomib (5 nM) to both cell lines. The treatment induced folding stress, as shown by upregulation of HSPA1A (the stress‑inducible form of heat shock protein 70, HSP70) in western blots, and the cells tolerated the stress for extended periods in MTS cell viability assays. Proteomic analysis revealed a shared response in both cell lines: upregulation of proteostasis factors such as heat‑shock proteins and of ubiquitin‑related components, including several E3 ligases. Among the identified ligases, TRIP12 stood out because it is highly abundant even at basal levels in both cell lines, suggesting a physiological role. TRIP12 is a HECT-type E3 ligase that has been implicated in various cellular processes, including DNA-damage repair, chromatin remodeling, cell differentiation, and cell-cycle progression. It has also been linked to immune signaling, epithelial-mesenchymal transition, and neurological disorders such as Parkinson's disease and intellectual disability. Recently, TRIP12 has been implicated in oxidative stress, highlighting its potential role in regulating cellular stress responses. To elucidate the functions of TRIP12 in melanoma cells and in the context of proteotoxic stress, I performed siRNA‑mediated knockdown of TRIP12, either alone or together with HSP90 inhibition (17‑DMAG), and analyzed the consequences by proteomics and transcriptomics. At basal conditions, loss of TRIP12 induced upregulation of epigenetic and transcription‑regulatory proteins in both cell lines, although the specific proteins differed between the two cell lines. A subset of proteins was detectable only after TRIP12 depletion, suggesting that they are normally turned over rapidly by TRIP12 under basal conditions. Strikingly, TRIP12 depletion generated more downregulated than upregulated proteins, and the most of these downregulations occurred without accompanying changes in mRNA levels, indicating post‑transcriptional regulation or indirect effects of TRIP12 on these targets. Among the downregulated proteins were proteostasis factors, most prominently E3 ligases. This overall pattern was observed in both cell lines, although the specific proteins affected differed between the lines. The combined perturbation of HSP90 and TRIP12 produced more extensive proteomic remodeling than each single perturbation. Many proteins changed exclusively in the combined condition, including downregulation of additional E3 ligases, indicating a synergistic relationship between HSP90 and TRIP12 in modulating the ubiquitin‑ligase network. Overall, this work demonstrates that a low‑dose approach can uncover the most stress‑responsive components of the proteostasis network while preserving the physiological state of cancer cells. It uncovers a role for TRIP12 in regulating epigenetic and transcriptional regulators, and demonstrates that TRIP12 also contributes to the broader proteostasis system, particularly the ubiquitin‑ligase landscape, and acts synergistically with HSP90 to modulate E3 ligases.