(233b) JNK-Inhibition Improves Viability in Cytidine-Analog-Treated Melanoma Cells

Melanoma stands as an increasingly pressing health concern within the United States. Over the past few decades, melanoma incidence has been on a steady incline. The American Cancer Society's estimates for 2024 indicate that melanoma will account for more than 8,000 deaths in the United States [1]. Notably, chemotherapy is less effective in treating melanoma compared to other cancers, and melanoma exhibits a recurrence rate of approximately 8.8% [1]. Cancer “persisters” have been recognized as a potential source of recurrent tumors [2-3]. The mechanisms behind the drug tolerance in these persister cells are complex and not well understood. Previously, we reported increased mitochondrial activity in melanoma persister cells following treatment with traditional chemotherapeutics [4]. In this study, we conducted proteomic analyses to elucidate the physiological alterations induced by chemotherapeutic agents, aiming to enhance our understanding of the underlying mechanisms of drug tolerance in melanoma.

Our initial analyses involved assessing protein expression in A375 melanoma cells (used as a model cell line) following treatment with gemcitabine (GEM) in comparison to untreated cells. Employing protein immunodetection arrays and untargeted proteomics analysis, we consistently observed an increased expression of c-Jun following GEM treatment. Moreover, the proteomics dataset identified networks of additionally upregulated and downregulated proteins (see Figure 1). The upregulated protein networks were related to DNA damage response (DDR), cell cycle regulation, apoptosis, the electron transport chain (ETC), mitochondrial ribosomal activity, protein folding, mRNA splicing, and multivesicular body (MVB) transport; downregulated networks included DDR, cell cycle regulation, mRNA and rRNA metabolism, tRNA aminoacylation, ribosome biogenesis, and Golgi body vesicular activity. Upon detecting the upregulation of c-Jun and its intriguing connections to the apoptotic proteins, we conducted co-treatment experiments with GEM and a JNK-inhibitor (hereafter JNK_i, which prevents c-Jun activation). Inhibiting JNK demonstrated a notable enhancement in the survival of GEM-treated cells, suggesting the involvement of c-Jun-related signaling in GEM-induced cell death. These findings were corroborated in similar experiments conducted with four additional melanoma cell lines and two other drugs falling into the same class as GEM (cytidine analogs) (see Figure 2). Proteomics analysis was also carried out on co-treated A375 samples (GEM + JNK_i), revealing JNK_i induced upregulations in sterol biosynthesis and cholesterol transport, as well as downregulations in cell cycle regulation and apoptosis. These findings identified multiple targets to improve chemotherapeutic treatment of melanoma, which could have broader implications for other malignancies.

References

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[2] S. V. Sharma et al., “A Chromatin-Mediated Reversible Drug-Tolerant State in Cancer Cell Subpopulations,” Cell, vol. 141, no. 1, pp. 69–80, Apr. 2010.

[3] M. Ramirez et al., “Diverse drug-resistance mechanisms can emerge from drug-tolerant cancer persister cells,” Nature Communications 2016 7:1, vol. 7, no. 1, pp. 1–8, Feb. 2016.

[4] P. Karki, V. Angardi, J. C. Mier, and M. A. Orman, “A Transient Metabolic State in Melanoma Persister Cells Mediated by Chemotherapeutic Treatments,” Front Mol Biosci, vol. 8, p. 780192, Jan. 2022.