Altered Transcriptional Control Networks with Trans-Differentiation of Isogenic Mutant-KRas NSCLC Models

The capacity of cancer cells to undergo epithelial mesenchymal trans-differentiation has been implicated as a factor driving metastasis, through the acquisition of enhanced migratory/invasive cell programs and the engagement of anti-apoptotic mechanisms promoting drug and radiation resistance. Our aim was to define molecular signaling changes associated with mesenchymal trans-differentiation in two KRas mutant NSCLC models. We focused on central transcription and epigenetic regulators predicted to be important for mesenchymal cell survival.

We have modeled trans-differentiation and cancer stemness in inducible isogenic mutant-KRas H358 and A549 non-small cell lung cell backgrounds. As expected, our models show mesenchymal-like tumor cells acquire novel mechanisms of cellular signaling not apparent in their epithelial counterparts. We employed large-scale quantitative phosphoproteomic, proteomic, protein-protein interaction, RNA-Seq, and network function prediction approaches to dissect the molecular events associated with the establishment and maintenance of the mesenchymal state.

Gene-set enrichment and pathway prediction indicated BMI1, KDM5B, RUNX2, MYC/MAX, NFκB, LEF1, and HIF1 target networks were significantly enriched in the trans-differentiation of H358 and A549 NSCLC models. Physical overlaps between multiple networks implicate NR4A1 as an overlapping control between TCF and NFκB pathways. Enrichment correlations also indicated marked decrease in cell cycling, which occurred early in the EMT process. RNA abundance time course studies also indicated early expression of epigenetic and chromatin regulators within 8-24 h, including CITED4, RUNX3, CMBX1, and SIRT4.

Multiple transcription and epigenetic pathways where altered between epithelial and mesenchymal tumor cell states, notably the polycomb repressive complex-1, HP1γ, and BAF/Swi-Snf. Network analysis suggests redundancy in the activation and inhibition of pathway regulators, notably factors controlling epithelial cell state. Through large-scale transcriptional and epigenetic cell reprograming, mesenchymal trans-differentiation can promote diversification of signaling networks potentially important in resistance to cancer therapies.