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.
Highlights
Resistance to chemotherapy involves multiple genes and multiple mechanisms, including a rearrangement of cell populations endowed with an adaptive ability to therapyinduced stress [1,2,3,4]
Pathway- and promoter-enrichment analysis strengthen the relevance of the NFkB pathway in mediating the survival of the ALDHbright cells and have shown how butein treatment modulates DNA damage and proliferation-associated pathways, thereby supporting the observed chemo-sensitizing effects of the drug when co-administered with chemotherapy, at least partially through NFkB modulation [24,27,28,29,30,31,32,33]
To date and to the best of our knowledge, a full gene expression profiling of FACS-sorted ALDHbright cells is unprecedented in malignant pleural mesothelioma (MPM)
Summary
Resistance to chemotherapy involves multiple genes and multiple mechanisms, including a rearrangement of cell populations endowed with an adaptive ability to therapyinduced stress [1,2,3,4]. Despite important progress at identifying mediators of resistance at the genetic and epigenetic level [5,6,7], what mediates the dynamic remodeling of cell subpopulations within drug-challenged tumors remains relatively unexplored. ALDHbright cells represent, quantitatively, the main chemoresistant cell subpopulation in several tumors in a conspicuous number of developmentally unrelated tumors [22,23] Both in vitro and ex vivo, we and others have shown that the ALDH activity is largely responsible for the chemoresistance of MPM cells, and MPM ALDHbright cells are enriched for the expression of the ALDH1A3 isoform. By taking into consideration both mRNAs enriched in the ALDHbright cells and those more deeply downregulated in the latter cells by butein treatment, we identified an eighteen-gene signature that held prognostic potential in 84 MPM patients from the TGCA database
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