Melanoma accounts for only 5% of all skin cancer variants, but is the most common cause of skin cancer related deaths, and is responsible for killing more young (<40yrs) people than any other cancer. Despite initial success of drugs that target specific enzyme mutations, melanomas rapidly acquire drug resistance. To understand baseline protein processes in melanomas with different genetic mutational status, mitogen activated protein kinase (MAPK) pathway mutant melanomas were compared to MAPK wild type (wt) using liquid chromatography (LC) coupled mass spectrometry (MS) for data independent MS (SWATH-MS) profiling.
Ten patient-derived melanomas with known MAPK mutational status (3xBRAFmut, 3xNRASmut, 3xMAPKwt, 1xMEKmut) were cultured. Tryptic digests (5 µg) were submitted to LC-MS/MS on a TripleTOF 5600 MS (SCIEX). Experiments were performed utilizing a novel microfluidic chip-based 2D chromatography (strong cation exchange coupled with reveres phase (RP)) for ion library generation and a rapid 1D RP for SWATH-MS quantitation (1h per cell line). Ten-day cell viability in presence of the MEK inhibitor AZD6244 was assessed by a Presto Blue Assay.
The approach enabled comprehensive protein detection identifying 3200 proteins (FDR<1%) across all 10 cell lines, with 2500 proteins quantifiable. Principal component analysis demonstrated segregation of melanomas based on sensitivity to MAPK inhibition (MEKi 2µM AZD6244), whereas genotype did not. In total, we show 57 proteins whose abundance correlates (r²>0.75) with MEKi cell viability, revealing changes in cell pigmentation, lipid metabolism, and adherence and inter-cell communication. Kaplan – Meier survival analysis using post-surgery patient survival data demonstrated untreated patients with MEKi sensitive melanoma cells showed significantly (p=0.01) lower mortality rates than patients with MEKi resistant tumours (mean survival >7.5 years MEKi sensitive versus 1.7 years MEKi resistant).