Differential regulation of the sphere formation and maintenance of cancer-initiating cells of malignant mesothelioma via CD44 and ALK4 signaling pathways

Our plain-English summary of Differential regulation of the sphere formation and maintenance of cancer-initiating cells of malignant mesothelioma via CD44 and ALK4 signaling pathways, by Y. Ohno, S. Shingyoku, S. Miyake, et al (Oncogene, 2018).

The study

• Authors: Y. Ohno, S. Shingyoku, S. Miyake, A. Tanaka, S. Fudesaka, Y. Shimizu, A. Yoshifuji, Y. Yamawaki, S. Yoshida, S. Tanaka, K. Sakura, T. Tanaka
• Journal: Oncogene
• Published: 2018;37(49):6357-6367
• Cited by: 18 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Malignant mesothelioma (MM) has a poor prognosis and is largely resistant to standard treatments, so it is important to seek novel therapeutic strategies for this disease. Cancer-initiating cells (CICs) were previously identified in MM using stem cell-associated markers in combination with spheroid cultures. However, the mechanisms underlying the induction and maintenance of CICs in MM remain to be fully explored. Here we showed that the CICs, which had high aldehyde dehydrogenase levels (ALDHbright) and stem cell-associated genes, were expanded in MM cells cultured under sphere-forming conditions. The MM spheroids also initiated tumors in immunodeficient mice more efficiently than did conventional adherent MM cells. In the MM spheroids, the expression of hyaluronan (HA) synthases was upregulated. Inhibiting the HA synthesis or CD44 functions by gene knockdown or neutralizing antibody abolished the formation of large-sized spheroids and the expansion of ALDHbright CICs. The expression of activin-A was also increased in the spheroids, and type I activin-A receptor subunit (ALK4) was upregulated in the ALDHbright CICs. The neutralization of activin-A or functional inactivation of ALK4 diminished the ALDHbright CICs without affecting spheroid formation. The knockdown of CD44 or ALK4 strongly suppressed the tumor growth in immunodeficient mice. These results together suggest that the HA-CD44 and activin-A-ALK4 pathways differentially regulate the spheroid formation and maintenance of ALDHbright CICs in MM cells, and that both pathways play critical roles in tumor growth in immunodeficient hosts. Our findings provide a novel therapeutic option for MM that targets signaling pathways that promote the CIC compartment through CD44 and ALK4.

In plain English

This was a laboratory study of human malignant mesothelioma cell lines grown in culture and in immunodeficient mice. It did not study asbestos exposure. The authors grew five cell lines either flat on dishes or as floating cell clusters called spheroids. Four lines formed spheroids and NCI-H28 did not. In spheroids of the MSTO-211H line, cells with high activity of the enzyme ALDH expanded; the authors treated these as cancer-initiating cells. Such cells were barely detected in flat cultures. Spheroid cells also grew tumors in mice more readily. Blocking hyaluronan, or its receptor CD44, prevented large spheroids and reduced the high-ALDH cells. Blocking activin-A, or its receptor ALK4, reduced the high-ALDH cells but did not affect spheroid formation. Silencing CD44 or ALK4 substantially inhibited tumor growth in mice (6 per group). The text reports these results mainly through figures and P-value thresholds. The authors concluded that targeting these two pathways could be effective strategies for suppressing cancer-initiating cells in mesothelioma, while noting that some mechanisms remain unknown.

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: experiments on human malignant mesothelioma cell lines in culture (flat adherent culture compared with spheroid culture), plus tumor-growth experiments in immunodeficient mice
• Who was studied: Five human malignant mesothelioma cell lines: MSTO-211H (biphasic), NCI-H28 (epithelioid), NCI-H2052 (sarcomatoid), ACC-MESO-1 (sarcomatoid) and ACC-MESO-4 (epithelioid). Most later experiments used MSTO-211H only. Tumor-growth experiments used NK-cell-depleted NOD/SCID mice (3 per group, comparing spheroid and adherent cells) and SCID mice (6 per group, for the gene-knockdown experiments).
• Exposure: No asbestos exposure was studied; asbestos appears only as background in the introduction. The experimental treatments were: spheroid culture conditions; the hyaluronan synthesis inhibitor 4MU (100 μM); a neutralizing anti-CD44 antibody (10 μg/ml); a neutralizing anti-activin A antibody (10 μg/ml); the ALK4/ALK5/ALK7 inhibitor SB431542 (10 μM); and shRNA knockdown of CD44 or ALK4, with an LMNA shRNA as control.
• Main results:
  • MSTO-211H, MESO-4, MESO-1 and H2052 formed spheroids; H28 did not.
  • In MSTO-211H cells, the population of cells with high ALDH activity (ALDHbright) expanded in spheroids but was barely detected in conventional adherent culture.
  • In immunodeficient mice (3 per group), MSTO-211H cells grown as spheroids showed higher tumorigenicity than cells grown in adherent culture.
  • MSTO-211H spheroids showed higher mRNA expression of the hyaluronan synthases HAS2 and HAS3, but not HAS1, than adherent cells.
  • Blocking hyaluronan synthesis with 4MU, or blocking CD44 with an antibody or shRNA, inhibited the formation of large spheroids (over 100 µm). 4MU decreased the ALDHbright cells, and CD44 shRNA inhibited the rise in ALDH mRNA in spheroids.
  • ACTIVIN-A mRNA rose in MSTO-211H spheroids while TGF-β mRNA was unchanged. The activin receptor subunits, including ALK4, were preferentially expressed in the ALDHbright cells.
  • A neutralizing anti-activin A antibody did not affect spheroid formation but significantly decreased the ALDHbright population. ALK4 shRNA and SB431542 had similar effects.
  • In SCID mice (6 per group, measured to day 31), shRNAs for CD44 and ALK4 substantially inhibited growth of MSTO-211H tumors. The control LMNA shRNA appeared to have some inhibitory effect, but it was not statistically significant.
• The authors' conclusion: The authors concluded that the HA–CD44 pathway and the activin-A–ALK4 pathway differentially contribute to spheroid formation and to the maintenance of cancer-initiating cells in mesothelioma cells, that both pathways are critical for tumor growth in immunodeficient mice, and that targeting them could be effective strategies for suppressing the cancer-initiating cell compartment in mesothelioma.
• Limitations the authors state:
  • The detailed organization of the hyaluronan-rich matrix around the cells, and the direct interaction of CD44 and hyaluronan in place, had not been determined.
  • The signals that increase HAS2 and HAS3 expression in mesothelioma spheroids are unknown.
  • The roles of other TGF-β family members were not tested experimentally.
  • The animal experiments were not blinded, and sample sizes were not chosen by statistical methods.

Topics

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

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

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