Immune evasion tactics of liver cancer cells revealed

A faulty cancer gene helps tumours evade immunity by stopping tumour cells releasing message-containing cargoes called exosomes.

These findings provide what the editors say is fundamental insight into the underlying biology of hepatocellular carcinoma (HCC), and compelling data supporting the role of beta-catenin in immune evasion. They suggest this work will be of special interest to researchers investigating the basic biology of liver function, as well as those investigating novel pathways for therapeutic targeting of liver cancer.

HCC is the most frequent form of primary liver cancer, accounting for around 80% of cases. It is one of a subset of tumours caused by faulty beta-catenin genes. These beta-catenin-activated tumours – which also include bowel, womb and melanoma skin cancer – all tend to have tumour microenvironments containing few immune cells. This means that immunotherapy treatments which rely on unleashing an influx of immune cells into the tumour will not work. This is a type of treatment resistance known as ‘immune escape’.

“As immunotherapies are now the first-line treatment for HCC and other cancers, it’s important to better understand the mechanisms underlying immune escape,” says co-author Violaine Moreau, research scientist at the University of Bordeaux, INSERM, France. “Exosomes – small vesicles that transport messages between cells – are known to control infiltration of immune cells into the tumour microenvironment, but their regulation by beta-catenin in tumour cells has not been explored.”

To investigate this, the team studied the effects of silencing the mutated beta-catenin gene on liver cancer cells. They first identified two exosome-associated genes that looked to be influenced by the faulty beta-catenin gene – called syndecan-4 and Rab27A. Next, they used a nanoparticle tracking method and a fluorescent label to monitor the release of exosomes within liver cancer cells. When mutant-beta-catenin was silenced, they saw an increase in the number of exosomes released. This led them to speculate that in tumour cells where mutant beta-catenin is active, it might reduce the release of exosomes carrying important signals within the tumour microenvironment.

Next, they tested whether silencing the activity of mutant-beta-catenin would affect immune cell infiltration, by using 3D tumour spheroids from two different liver cancer cells lines. This showed that when faulty beta-catenin was silenced, not only did it enhance the infiltration of immune cells into the tumour spheroids, it was also associated with increased tumour cell death. This suggests that silencing mutant beta-catenin could be a viable therapeutic strategy for triggering the influx of immune cells into these hard-to-treat tumours.

Activated ß-catenin represses immune infiltration in liver cancer spheroids through exosomes

(a) Peripheral Blood Mononuclear Cells (PBMC) infiltration analysis protocol. Created with BioRender.com. (b) Images of HepG2 spheroids expressing a control shRNA incubated or not with PBMC for 24 hours. (c) Analysis of PBMC infiltration and tumor cell survival in Huh7 spheroids treated with DMSO or CHIR99021. (d) Analysis of PBMC infiltration and tumor cell survival in HepG2 spheroids expressing control shRNA (shCtrl) or shRNA targeting mutated ß-catenin (shßcat MUT). (e) Analysis of PBMC infiltration and tumor cell survival in HepG2 spheroids co-expressing shRNA targeting mutated ß-catenin (shßcat MUT) and control siRNA (siCtrl) or siRNA targeting Rab27A (siRab27A). (c-e) Graphs show the quantification of six independent experiments. Results are expressed as Mean ± SEM, one or two-tailed Student’s t-test analysis. *P<0.05; **P<0.01; ***P<0.001.

To explore whether this mechanism might hold true in patients, the team verified whether human liver cancer samples had dysregulated levels of the exosome-associated genes, syndecan-4 and Rab27A. As hoped – in two independent sets of human liver cancer samples – the levels of these genes were markedly reduced when beta-catenin was faulty. Moreover, other markers of beta-catenin activity were also linked to low levels of Rab27A protein in the liver tumour samples.

“Our study is the first description of the cancer gene beta-catenin as a key regulator of exosome release and provides new insights into control of tumour-derived exosome production and its involvement in immune escape,” concludes co-author Clotilde Billottet, a Professor of Cell Biology at the University of Bordeaux, INSERM. “Given that beta-catenin signalling is also activated in melanoma, womb and bowel cancers, these new findings may have broad significance for cancer immunotherapy. Our results suggest that exosomes could be an important marker of treatment response, and that restoring tumour exosome release in beta-catenin-mutated tumours could increase tumour-to-immune cell communication, and make immunologically ‘cold’ tumours, ‘hot’.”

SourceEurekalert

Camille D, Justine V, Aude B et al. (2024) Emerging role of oncogenic ß-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma. eLife 13:RP95191. [article]

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