Insights into Intra-Tumoral Heterogeneity: Transcriptional Profiling of Chemoresistant MPM Cell Subpopulations Reveals Involvement of NFkB and DNA Repair Pathways and Contributes a Prognostic Signature.

ABSTRACT

Chemoresistance is a hallmark of malignant pleural mesothelioma (MPM) management and the expression of ALDH1A3 is responsible for the survival and activity of MPM chemoresistant cell subpopulations (ALDHbright cells). We enriched mesothelioma ALDHbright cells to near homogeneity by FACS sorting and an Aldefluor assay and performed unbiased Affymetrix gene expression profiling. Viability and ELISA assays were used to rule out significant apoptosis in the sorted cell subpopulations and to assess target engagement by butein. Statistical analysis of the results, pathway enrichment and promoter enrichment were employed for the generation of the data. Q-RTPCR was used to validate a subset of the identified, modulated mRNAs In this work, we started from the observation that the mRNA levels of the ALDH1A3 isoform could prognostically stratify MPM patients. Thus, we purified MPM ALDHbright cells from NCI-H2595 cells and interrogated their gene expression (GES) profile. We analyzed the GES of the purified cells at both a steady state and upon treatment with butein (a multifunctional tetrahydroxy-chalcone), which abates the ALDHbright cell number, thereby exerting chemo-sensitizing effects in vitro and in vivo. We identified 924 genes modulated in a statistically significant manner as a function of ALDH status and of the response to the inhibitor. Pathway and promoter enrichment identified the molecular determinant of high ALDH status and how butein treatment altered the molecular portrait of those chemoresistant cell subpopulations. Further, we unraveled an eighteen-gene signature with high prognostic significance for MPM patients, and showed that most of the identified prognostic contributors escaped the analysis of unfractionated samples. This work proves that digging into the unexplored field of intra-tumor heterogeneity (ITH) by working at the cell subpopulation level may provide findings of prognostic relevance, in addition to mechanistic insights into tumor resistance to therapy.