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

Our plain-English summary of 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, by M. Cioce, A. Sacconi, H.I. Pass, et al (International Journal of Molecular Sciences, 2021).

The study

• Authors: M. Cioce, A. Sacconi, H.I. Pass, C. Canino, S. Strano, G. Blandino, V.M. Fazio
• Journal: International Journal of Molecular Sciences
• Published: 2021;22(21)
• Cited by: 12 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This laboratory study used one malignant pleural mesothelioma (MPM) cell line, NCI-H2595, and public patient data; it did not study asbestos. The authors sorted the cells by activity of the enzyme ALDH into high (ALDHbright) and low groups, treated both for 7 hours with butein or a control, and measured gene activity. In 84 MPM patients from The Cancer Genome Atlas, ALDH1A3 gene levels were linked to differences in overall survival (p = 0.014). In the cells, 924 genes differed by ALDH status, treatment, or both. Genes raised in ALDHbright cells were linked to NFkB-related pathways, and genes butein lowered more in ALDHbright cells were linked to DNA repair. From these the authors built an eighteen-gene signature; among the 84 patients, high-signature and low-signature groups differed in median survival by almost 60 months. In a separate dataset of 40 MPM and 9 normal samples, 15 signature genes were detectable. Only two changed significantly and consistently with the sorted cells. The authors suggested studying specific cell subgroups may give clinically useful results. As a limitation, they did not profile cell lines of the epithelioid, biphasic and sarcomatoid types; theirs came from a mainly epithelioid specimen.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) study of a mesothelioma cell line. Cells were sorted by ALDH activity, treated with butein or control, and profiled for gene expression. This was combined with analysis of public patient gene-expression datasets.
• Who was studied: The human MPM cell line NCI-H2595 (sorted ALDHbright and ALDHlow cells, duplicate samples). Also 84 MPM patients from the TCGA database (written 'TGCA' in the article), and a dataset (GSE2549) of 40 MPM and 9 normal samples.
• Exposure: No asbestos exposure was studied. The laboratory treatment was butein (10 micromol/L for 7 hours), compared with a control of 0.05% DMSO for 7 hours.
• Main results:
  • In 84 MPM patients from TCGA, ALDH1A3 mRNA levels stratified patients by overall survival (p = 0.014).
  • After 7 hours, butein-treated and control cell samples did not differ significantly in the percentage of dead cells.
  • In the sorted cells, 924 genes changed according to ALDH status, treatment, or both (false discovery rate-corrected p of 0.25 or less).
  • The 76 genes enriched more than two-fold in control-treated ALDHbright cells, compared with ALDHlow cells, showed enrichment for NFkB-related pathways.
  • Genes that butein lowered more strongly in ALDHbright cells than in ALDHlow cells showed enrichment for DNA damage repair pathways.
  • In the 84 TCGA patients, those with high and low levels of the eighteen-gene signature differed in median survival by almost 60 months.
  • The GSE2549 dataset compared 40 MPM samples with 9 normal samples. In it, 15 of the eighteen signature genes were detectable and six reached statistical significance. Only two (TSPAN13, DDIT4) were significantly changed (p < 0.05) in a direction consistent with the ALDHbright cell findings.
  • QRT-PCR of 34 genes agreed with the microarray values (r square 0.7943).
• The authors' conclusion: The authors concluded that isolating and characterizing specific cell subpopulations to study intra-tumor heterogeneity may provide clinically useful results. They also concluded that most of the genes they identified would have been missed by analysis of unfractionated samples.
• Limitations the authors state:
  • The authors did not obtain gene expression data from cell lines representing the epithelioid, biphasic and sarcomatoid MPM types. The single cell line used came from a mainly epithelioid specimen. The authors reported that the signature appeared to perform equally well across histotypes in the TCGA patients, but did not show those data.
  • Too few treated patients were available to draw robust conclusions about ALDH1A3 levels and outcome after radio- or chemotherapy (an unpublished observation).

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Pleural Mesothelioma, Mesothelioma, Apoptosis.

Related studies

• A STAT3-NFkB/DDIT3/CEBPβ axis modulates ALDH1A3 expression in chemoresistant cell subpopulations — C. Canino, Y. Luo, et al. (2015)
• Isolation of Chemoresistant Cell Subpopulations — C. Canino, M. Cioce (2016)
• Multiplexed single-cell and spatial profiling reveal B cells and tertiary lymphoid structures as prognostic indicators in pleural mesothelioma — A. Rigutto, N.G. Núñez, et al. (2026)
• Insights Into Genomic Drivers, Transcriptomic Heterogeneity, and Therapeutic Vulnerabilities From Novel Mesothelioma Cell Lines — Y. Wu, F. Cao, et al. (2026)
• A Novel BRCA1-Associated Protein-1 Isoform Affects Response of Mesothelioma Cells to Drugs Impairing BRCA1-Mediated DNA Repair — R. Parrotta, A. Okonska, et al. (2017)
• DNA methylation profiling of asbestos-treated MeT5A cell line reveals novel pathways implicated in asbestos response — E. Casalone, A. Allione, et al. (2018)

Browse more research in International Journal of Molecular Sciences, or return to medical research.