Comparative proteomics of a model MCF10A-KRasG12V cell line reveals a distinct molecular signature of the KRasG12V cell surface

Abstract

Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry–based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.

Comparative Microsomal Proteomics of A Model Lung Cancer Cell Line NCI-H23 Reveals Distinct Differences Between Molecular Profiles of 3D and 2D Cultured Cells

“…[44] Our laboratory has pioneered and applied proteomics for profiling of the cell-surface proteome in cell lines and tissue specimens. [11,14,17,18,25,45,46] order to secure cell survival and growth in hypoxia. [49] The principal regulator of intensified glycolysis is the hypoxia inducible factor 1 (HIF-1), a transcription factor responsible for increased transcription/translation of glycolytic enzymes induced by hypoxia.…”

“…This remarkable phenotypical transformation captured by SGM proteomics is equivalent to hypoxia-induced vascular mimicry in melanoma [58,59] that we previously investigated using SGM proteomics. [45] Next, we focused specifically on differentially regulated CD molecules/antigens, primarily due to their importance in cancer diagnostics (i.e., cancer biomarkers) [60,61], cancer treatment (i.e., cancer immunotherapy) [62,63] and our interest in the cell surface proteomics of cancer cell lines [17] and tumor xenografts [11] harboring KRas mutants in the context of biomarker and drug target discovery. [47] We were the first show colocalization of CD147 (BSG) and CD318 (CDCP1) at the surface of cancer cell line expressing KRas4B mutant.…”

“…isolated as previously described. [17] Briefly, each cell pellet was thawed in 50 mM NH4HCO3 containing 1 mM PMSF. Cells were homogenized using tip sonication.…”

“…Towards this goal, we used SGM proteomic methodology which has been established as an efficient approach for profiling of complex membrane protein mixtures. [17,18,25] The SGM workflow is depicted in Figure 1.…”

“…Unlike canonical pathway and/or disease/function analysis that groups molecules based on known commonalities or what is accepted in the field, the network analysis groups clusters molecules into networks based on any direct or indirect biological relationship described in the literature. [11,17,32] Top networks are ranked by statistical significance and number of interacting proteins using Fisher's exact test. [33] The IPA network analysis showed enrichment of 25 functional protein networks in NCI-H23 cells grown in 3D and 2D cell culture ( Supplementary Table S15).…”