Dataset Information

Differential patterns of protein expression in monolayer or spheroid melanoma cell models of acquired resistance to BRAF and MEK1/2 inhibitors

ABSTRACT: Mitogen activated protein kinase (MAPK) inhibitors are important therapies for treating many cancers. However, the development of acquired resistance to most protein kinase inhibitors limit their ability to provide durable responses. Approximately 50% of malignant melanomas contain activating mutations in the BRAF kinase, which promote cancer cell survival by activating the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway through direct phosphorylation of the MAPK/ERK kinase-1/2 (MEK1/2). Although combination treatment with BRAF and MEK1/2 inhibitors is a recommended approach to treat melanoma, the development of drug resistance remains a barrier to achieving long term patient benefits. Using high-resolution label-free mass spectrometry, the current studies compared relative protein changes in BRAF and MEK1/2 inhibitor resistant A375 melanoma cells grown as monolayers or 3D spheroids. While approximately 66% of proteins identified were common in both monolayer and spheroid cultures, only 6.2% or 3.6% of proteins that significantly increased or decreased, respectively, compared to drug sensitive cells were common between the drug-resistant monolayer and spheroid cells. Major changes in drug-resistant monolayers suggested upregulation of alternative kinase signaling pathways that promote growth and metastasis. In contrast, the major changes in drug-resistant spheroids indicated increased catabolic metabolism to support oxidative phosphorylation and energy requirements.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Weiliang Huang

LAB HEAD: Maureen Kane

PROVIDER: PXD026952 | Pride | 2022-02-17

REPOSITORIES: Pride

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Publications

Proteomic Changes in the Monolayer and Spheroid Melanoma Cell Models of Acquired Resistance to BRAF and MEK1/2 Inhibitors.

Martinez Ramon R Huang Weiliang W Buck Heather H Rea Samantha S Defnet Amy E AE Kane Maureen A MA Shapiro Paul P

ACS omega 20220118 4

Extracellular signal-regulated kinase-1/2 (ERK1/2) pathway inhibitors are important therapies for treating many cancers. However, acquired resistance to most protein kinase inhibitors limits their ability to provide durable responses. Approximately 50% of malignant melanomas contain activating mutations in BRAF, which promotes cancer cell survival through the direct phosphorylation of the mitogen-activated protein kinase MAPK/ERK 1/2 (MEK1/2) and the activation of ERK1/2. Although the combinatio ...[more]

PMID: 35128241

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Project description:Analysis of the transcriptional profiles of mRNA and microRNA in Rasless fibroblasts. 4-Hydroxy-tamoxifen (4-OHT) treatment triggers removal of K-Ras expression in [H-Ras-/-;N-Ras-/-;K-Raslox/lox;RERTert/ert] mouse fibroblasts (named K-Raslox) generating M-bM-^@M-^\RaslessM-bM-^@M-^] MEFs which are unable to proliferate, but recover proliferative ability after ectopic expression of constitutively active downstream kinases such as BRAF and MEK1. Using Affymetrix microarrays, we compared the transcriptional profiles of Rasless fibroblasts to those of K-Raslox (MEFs lacking only H-Ras and N-Ras). We also compared the transcriptional profile of Rasless cells with those of BRAF and MEK1-transfected Rasless cells which recovered proliferative ability. Re-entry of the Rasless cells into cell cycle progression through ectopic expression of activated BRAF or MEK1 resulted in full reversion of most transcriptional alterations identified in cells lacking the three canonical Ras proteins. Pre-confluent cell cultures corresponding to the different Ras genotypes were harvested for extraction of total RNA and miRNA samples, and subsequent hybridization on Affymetrix microarrays. The different sets of experimental mRNA samples analyzed here included (i) 8 samples K-Raslox (proliferating cells expressing only K-Ras, considered as Control): 4 biological replicates, technical duplicates each; (ii) 7 samples Rasless: the same cells after treatment with 4-OHT for 12 days (to become non-proliferating fibroblasts), including 4 biological replicates and technical duplicates from 3 of them; (iii) 3 samples BRAF-rescued MEFs (Rasless cells harboring activated BRAF constructs, with regained proliferative ability): 3 biological replicates; (iv) 3 samples MEK1-rescued MEFs (Rasless cells harboring activated MEK1 constructs, with regained proliferative ability): 3 biological replicates; and their respective controls harbouring the vempty vectors (v) 2 samples Hygromycin-resistant (control for BRAF-transfected Rasless cells) including 2 biological replicates, and (vi) 4 samples Puromycin-resistant (control for MEK1-transfected Rasless cells) including 2 biological replicates, technical duplicates each. The gene expression study contains a total of 27 samples. Regarding the miRNA expression study, the experimental samples analyzed included (i) 4 samples samples K-Raslox (used as Control): 2 biological replicates, technical duplicates each; (ii) 8 samples Rasless: the same cells after treatment with 4-OHT for 6 days (2 biological replicates, technical duplicates each) and 12 days (2 biological replicates, technical duplicates each); (iii) 4 samples BRAF-rescued, including 2 biological replicates, technical duplicates each; (iv) 4 samples MEK1-rescued,including 2 biological replicates, technical duplicates each, and (v) 4 samples Puromycin-resistant empty vector, containing 2 biological replicates, technical duplicates each. The miRNA expression study contains a total of 24 samples.

Dataset Information

Differential patterns of protein expression in monolayer or spheroid melanoma cell models of acquired resistance to BRAF and MEK1/2 inhibitors

Publications

Proteomic Changes in the Monolayer and Spheroid Melanoma Cell Models of Acquired Resistance to BRAF and MEK1/2 Inhibitors.

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