Calretinin promotes invasiveness and EMT in malignant mesothelioma cells involving the activation of the FAK signaling pathway

Our plain-English summary of Calretinin promotes invasiveness and EMT in malignant mesothelioma cells involving the activation of the FAK signaling pathway, by J. Wörthmüller, W. Blum, L. Pecze, et al (Oncotarget, 2018).

The study

• Authors: J. Wörthmüller, W. Blum, L. Pecze, V. Salicio, B. Schwaller
• Journal: Oncotarget
• Published: 2018;9(91):36256-36272
• Cited by: 20 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Calretinin (CR) is used as a positive marker for human malignant mesothelioma (MM) and is essential for mesothelioma cell growth/survival. Yet, the putative role(s) of CR during MM formation in vivo, binding partners or CR's influence on specific signaling pathways remain unknown. We assessed the effect of CR overexpression in the human MM cell lines MSTO-211H and SPC111. CR overexpression augmented the migration and invasion of MM cells in vitro. These effects involved the activation of the focal adhesion kinase (FAK) signaling pathway, since levels of total FAK and phospho-FAK (Tyr397) were found up-regulated in these cells. CR was also implicated in controlling epithelial-to-mesenchymal transition (EMT), evidenced by changes of the cell morphology and up-regulation of typical EMT markers. Co-IP experiments revealed FAK as a new binding partner of CR. CR co-localized with FAK at focal adhesion sites; moreover, CR-overexpressing cells displayed enhanced nuclear FAK accumulation and an increased resistance towards the FAK inhibitor VS-6063. Finally, CR downregulation via a lentiviral shRNA against CR (CALB2) resulted in a significantly reduced tumor formation in vivo in an orthotopic xenograft mouse model based on peritoneal MM cell injection. Our results indicate that CR might be considered as a possible target for MM treatment.

In plain English

This laboratory study tested what happens when the calcium-binding protein calretinin (CR) is raised or lowered in human malignant mesothelioma (MM) cells. Using lentiviral vectors, the authors overexpressed CR in the biphasic MM lines SPC111 and MSTO-211H (and in other MM lines). In scratch wound assays, CR-overexpressing SPC111 cells closed the gap in 18 h versus 38 h for wild-type cells (p less than or equal to 0.0001), and MSTO cells in 10 h versus 16 h (p less than or equal to 0.05); with a Matrigel barrier, only CR-overexpressing cells closed the gap. Total FAK and phospho-FAK (Tyr397) rose, nuclear FAK increased by about 30% (p less than or equal to 0.01), co-immunoprecipitation pulled FAK down with CR antibodies, and CR-overexpressing cells were more resistant to the FAK inhibitor VS-6063 at 5 to 10 micromolar. E-cadherin fell and N-cadherin rose, with EMT-gene changes. In mice injected intraperitoneally with MSTO-211H-Rluc cells, shRNA against CALB2 significantly reduced tumour burden at day 30 (p less than or equal to 0.01). The authors concluded CR might be considered a possible target for MM treatment.

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 study: in vitro experiments in human mesothelioma cell lines (lentiviral overexpression and shRNA knockdown) plus an orthotopic xenograft mouse experiment
• Who was studied: Human malignant mesothelioma cell lines MSTO-211H and SPC111 (biphasic), with additional lines ZL5, ZL55, SPC212 and ZL34; and NOD SCID gamma (NSG) mice, 4 per group, in Fribourg, Switzerland
• Exposure: Lentiviral overexpression of calretinin (pLV-CALB2), lentiviral shRNA knockdown of CR (CALB2) or GFP control, and treatment with the FAK inhibitor VS-6063 (Defactinib) at 2.5 to 10 micromolar; no asbestos exposure was studied
• Main results:
  • Wound closure was faster in CR-overexpressing cells: 18 h for SPC111-CR versus 38 h for SPC111-wt (p less than or equal to 0.0001), and 10 h versus 16 h for MSTO-211H (p less than or equal to 0.05)
  • In the Matrigel invasion assay, SPC111-CR closed the wound almost completely by 90 h and MSTO-CR by 60 h
  • Neither wild-type line invaded the gap space at any point up to 140 h
  • p-FAK (Tyr397) rose by approximately 50% in SPC111-CR cells (p less than or equal to 0.01); in MSTO-CR cells the increase was 27±12% and not significant, while the p-FAK/FAK ratio in MSTO-CR was 1.314±0.001 (p less than or equal to 0.0001)
  • Nuclear FAK in CR-overexpressing cells increased by about 30%, significant in both SPC111 and MSTO-211H
  • Co-immunoprecipitation with CR antibodies pulled down FAK, including in MSTO wild-type cells; the reverse pull-down with two FAK antibodies was negative
  • CR-overexpressing cells of both lines were more resistant to the FAK inhibitor VS-6063 at concentrations of 5 micromolar or above, most evident at 10 micromolar
  • EMT markers changed with CR overexpression: E-cadherin protein decreased and N-cadherin increased in both CR-overexpressing lines
  • In the mouse model, bioluminescence signals were equivalent at day 16, but at day 30 tumours had grown in the shGFP control group while the shCALB2 group showed a significant reduction in tumour burden (p less than or equal to 0.01)
  • shCALB2 reduced CR protein by about 90% in MSTO-211H cells in vitro with a similar decrease in total FAK; in shCALB2-injected mice no infiltration of the skeletal muscle layer was seen, unlike in shGFP controls
• The authors' conclusion: The authors concluded that CR up-regulation activates FAK signalling, inducing migration, invasion and EMT, and that CR downregulation is a possible strategy to treat MM, so CR might be considered a possible target for MM treatment
• Limitations the authors state:
  • The authors state the mechanism by which raised CR increases FAK expression or phosphorylation is unknown and that the possible explanations need further study
  • The reverse co-immunoprecipitation (CR pulled down by FAK antibodies) was negative; the authors attribute this to antibody interference rather than absence of interaction
  • The proposed binding of CR to FAK via an LD domain, competing with paxillin, is stated by the authors as a hypothesis

Topics

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

Related studies

• Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin — J. Wörthmüller, V. Salicio, et al. (2019)
• Overexpression of activin-A and -B in malignant mesothelioma - attenuated Smad3 signaling responses and ERK activation promote cell migration and invasive growth — J.A. Tamminen, M. Yin, et al. (2015)
• TAZ activation by Hippo pathway dysregulation induces cytokine gene expression and promotes mesothelial cell transformation — A. Matsushita, T. Sato, et al. (2019)
• Asbestos-induced activation of cell signaling pathways in human bronchial epithelial cells — X. Wang, J.M. Samet, et al. (2006)
• The extracellular matrix protein fibulin-3/EFEMP1 promotes pleural mesothelioma growth by activation of PI3K/Akt signaling — A. Roshini, C. Goparaju, et al. (2022)
• Low-dose photon irradiation induces invasiveness through the SDF-1α/CXCR4 pathway in malignant mesothelioma cells — Y. Yamauchi, S. Safi, et al. (2017)

Browse more research in Oncotarget, or return to medical research.