Mutational analysis of hedgehog signaling pathway genes in human malignant mesothelioma

Our plain-English summary of Mutational analysis of hedgehog signaling pathway genes in human malignant mesothelioma, by C.B. Lim, C.M. Prêle, H.M. Cheah, et al (PloS One, 2013).

The study

• Authors: C.B. Lim, C.M. Prêle, H.M. Cheah, Y.Y. Cheng, S. Klebe, G. Reid, D.N. Watkins, S. Baltic, P.J. Thompson, S.E. Mutsaers
• Journal: PloS One
• Published: 2013;8(6):e66685
• Cited by: 29 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: The Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown.

Methodology/principal Findings: Real-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69_70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour.

Conclusions/significance: We identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy.

In plain English

This laboratory study looked for mutations in 13 genes of the Hedgehog (HH) signaling pathway in human malignant mesothelioma (MMe). The authors sequenced all the exons of these genes in 7 MMe cell lines. They then checked 4 more cell lines, and tumor samples from 14 Sydney patients with malignant pleural mesothelioma, for the mutations they had found. They also measured expression of three pathway genes in 7 cell lines and in normal mesothelial cells. Asbestos exposure was not studied. GLI1, GLI2 and PTCH1 were highly expressed in the cancer cell lines but very low or undetectable in normal cells. Three mutations were found, in only two cell lines: a SUFU point mutation (p.T411M) and a SMO insertion in LO68, and a deletion of PTCH1 exons 18 to 23 in JU77. One tumor, from a man with biphasic MMe, carried the SMO insertion. The article contradicts itself on whether the SUFU mutation changed SUFU function in a laboratory test. The authors concluded that HH pathway activation in most MMe is unlikely to be driven by mutations, and that the pathway may be a treatment target.

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 genetic study: exon sequencing and gene-expression measurement in mesothelioma cell lines, checking of the mutations found in archived tumor samples, computer prediction of mutation effects, and a cell-based reporter test of one mutation
• Who was studied: Human MMe cell lines: 7 had all exons of the 13 genes sequenced (MSTO-211H, NCI-H28, JU77, LO68, NO36, ONE58, STY51), and 4 more (NCI-H226, NCI-H2052, NCI-H2452, REN) were genotyped only for the mutations identified. Normal mesothelial cells came from patients undergoing thoracic surgery. Tumor samples were archived formalin-fixed tissue from 14 patients with malignant pleural mesothelioma (MPM) who had extrapleural pneumonectomy at Royal Prince Alfred or Strathfield Private Hospitals, Sydney, between 1994 and 2009 (12 male, 2 female; 11 epithelioid, 3 biphasic).
• Exposure: No asbestos or other exposure was studied. The study examined genetic changes in 13 Hedgehog pathway genes (GLI1, GLI2, GLI3, IHH, PTCH1, SHH, SMO, PTCH2, STK36, DHH, SUFU, HHIP, KIF7). The introduction's statement about asbestos as a risk factor cites other work.
• Main results:
  • GLI1, GLI2 and PTCH1 mRNA were expressed in the 7 MMe cell lines. In normal mesothelial cells, PTCH1 and GLI2 were at extremely low levels and GLI1 was undetectable. The authors took this to suggest active HH signaling in MMe cells.
  • Across the cell lines, three mutations were found, in 3 of the 13 genes, and in only two cell lines.
  • In cell line LO68, a point mutation in SUFU (p.T411M, exon 10) was found. The prediction programs SIFT and PolyPhen2 judged it damaging; Table 3 lists the PolyPhen2 result as 'Possibly damaging'.
  • In cell line JU77, exons 18 to 23 of PTCH1 were deleted on both copies (homozygous deletion).
  • In cell line LO68, a 3-base insertion (69_70insCTG) in exon 1 of SMO was found, adding one leucine to the SMO protein. Its functional role is unknown.
  • Of the 14 patient tumors, one carried the SMO CTG insertion. That patient was a male Caucasian diagnosed at age 51 with biphasic MMe who survived 3.4 months. None of the 14 tumors had the PTCH1 exon deletions or the SUFU mutation.
  • No mutations were found in the exonic regions of SHH, DHH, IHH, PTCH1, PTCH2, HHIP, KIF7, GLI1, GLI2 and GLI3. No point mutations were found in PTCH1; its change was the exon deletion.
  • In the 7 sequenced cell lines, 35 SNPs (common gene variants) were found. All had been reported before, and 16 would change an amino acid.
  • Reporter test of the SUFU p.T411M mutation: the Discussion and the Figure 7 title state the mutation did not alter SUFU's inhibitory function. The Results text states the mutant failed to suppress Gli1 reporter activity. The article is internally inconsistent on this point.
• The authors' conclusion: The authors concluded that, unlike some other cancers, aberrant HH signaling activation in MMe is unlikely to be driven by mutations in most tumors. They suggested it is instead activated through autocrine signaling, and that the pathway may be a therapeutic target for HH pathway inhibitors. They also stated that HH pathway mutations in a subset of MMe tumors cannot be discounted.
• Limitations the authors state:
  • The patient tumor cohort was small, which may explain why the PTCH1 deletion was not found in tumors.
  • No blood was collected from the patient whose tumor had the SMO insertion, so it could not be determined whether the mutation was inherited (germline) or arose in the tumor (somatic).
  • The two prediction programs disagreed, and the real functional significance of the non-synonymous SNPs needs to be confirmed by biochemical studies.
  • The role of HH pathway mutations in a subset of MMe needs investigation in a larger sample set.

Topics

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

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

Related studies

• CK2α, over-expressed in human malignant pleural mesothelioma, regulates the Hedgehog signaling pathway in mesothelioma cells — S. Zhang, Y.L. Yang, et al. (2014)
• Targeting of the Hedgehog signal transduction pathway suppresses survival of malignant pleural mesothelioma cells in vitro — M. You, J. Varona-Santos, et al. (2014)
• Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma — M. Meerang, K. Bérard, et al. (2016)
• Inhibition of hedgehog signaling reduces the side population in human malignant mesothelioma cell lines — H.A. Kim, M.C. Kim, et al. (2015)
• Novel EPHB4 Receptor Tyrosine Kinase Mutations and Kinomic Pathway Analysis in Lung Cancer — B.D. Ferguson, Y.H. Carol Tan, et al. (2015)
• Asbestos-induced activation of cell signaling pathways in human bronchial epithelial cells — X. Wang, J.M. Samet, et al. (2006)

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