The protein behind immunotherapy resistance

Immunotherapy is a cutting-edge approach to treating cancer by turning the patient’s immune system against their tumor. Our growing knowledge of the mechanisms by which the body regulates immune responses has transformed our fight against cancer.

But despite the success rates, immunotherapy has repeatedly come up against a stubborn obstacle: tumor cells often escape the “radar” of immune cells seeking to destroy them. This in turn leads to treatment resistance, which in many cases would benefit from a deeper understanding of the mechanisms that can help circumvent it.

A new study by EPFL scientists has discovered a protein that plays a key role in helping tumors evade immune destruction. The protein, called ‘fragile X mental retardation protein’ (FMRP), regulates a network of genes and cells in the tumor microenvironment that contribute to its ability to ‘hide’ from immune cells. Normally, FMRP is involved in the regulation of protein translation and mRNA stability in neurons. But researchers have found that it is aberrantly regulated in multiple forms of cancer.

The study, published in Science, was led by researchers from Douglas Hanahan’s group at the Swiss Institute for Experimental Cancer Research (ISREC) and the Lausanne branch of the Ludwig Institute for Cancer Research, as well as colleagues from the University Hospital Center of Lausanne (CHUV) and other Swiss institutions. The discovery also led to an EPFL spin-off, Opna Bio, whose staff were also involved in the research.

But why FMRP? The idea came from previous studies showing that cancer cells that naturally overexpress FMRP are invasive and metastatic. Other studies show that if, on the contrary, FMRP is not expressed in developing neurons, it can lead to cognitive defects (hence the “mental retardation” part of the protein’s name).

With this evidence in mind, researchers set out to study FMRP expression in human tumors. They then evaluated its tumor-promoting functions in mouse models of cancer, and finally studied its association with the prognosis of human cancer patients.

The study involved several stages of data collection. First, the scientists performed immunostaining for FMRP on tissue from human tumors. The majority of tumors tested positive, unlike the corresponding normal tissues. This means that FMRP is specifically and strongly expressed in cancer cells.

The team then moved on to the main part of their research, which was to determine the functional significance of FMRP in these tumors – they express the protein, but what does it do?

FMRP is involved in the immune system

To explore this, scientists have developed so-called “knockout” cancer cell lines. Knockout cells or organisms are genetically engineered to lose — “knock out” — a specific gene in order to find clues to its function. Essentially, any change that occurs in knockout cells compared to cells that still have the gene – called “wild type” – can usually be attributed to the missing gene.

In this case, scientists used the famous CRISPR-Cas9 gene-editing technique to knock out the gene (called FMR1) that produces FMRP in mouse cancer cells from melanocytes in the pancreas, colon, breast, and skin. They then compared FMRP-knockout cancer cells to cancer cells that still had the FMR1 gene and thus expressed the FMRP protein.

The researchers assessed survival rates between mice with tumors containing FMRP-knockout cancer cells and those with wild-type FMRP cells, first in mice whose immune systems had been compromised. The comparison revealed similar survival rates. In contrast, when they compared knockout tumors to wild-type tumors growing in mice with properly functioning immune systems, they found that tumors without FMRP grew more slowly and the animals survived longer. long time.

What this part of the study showed is that FMRP is not involved in stimulating tumor growth per se, and instead involved the adaptive immune system (the part of our immune system that we “train ” with vaccines).

This was confirmed by the observation that wild-type tumors had very few infiltrating T cells, whereas knockout tumors were highly inflamed. Depletion of T cells from FMRP knockout tumors caused them to start growing faster and reduced the survival rates of the mice, which means that FMRP is somehow involved in tumors evading the immune system.

How Tumors With FMRP Defend Against Immune Cells

The team continued molecular genetic profiling of knockout and wild-type tumors. This revealed significant differences in gene transcription across the genome, suggesting that FMRP interacts with multiple genes. Additionally, the tumors showed marked differences in the abundance of cancer cells, macrophages, and T cells, further implicating the role of FMRP in modulating components of the immune system.

The next phase of the study looked at the production of specific factors associated with distinctive immune responses – evasion versus attack. FMRP-expressing tumors have been shown to produce interleukin-33, a protein that induces the production of regulatory T cells, a specialized subpopulation of T cells that inhibit immune responses. They also produce protein S, a glycoprotein known to promote tumor growth. Finally, tumors produce exosomes – cellular organelles that have been shown to trigger the production of a type of macrophage cell that normally aids in wound healing and tissue repair. Together, these three factors are immunosuppressive and contribute to the tumor barrier against T cell attacks.

In contrast, FMRP knockout tumor cells actually downregulated all three factors (interleukin-33, protein S, and exosomes) whereas they upregulated a different chemokine called “CC motif chemokine ligand 7” (CCL7), which helps recruit and activate T cells. This process is further facilitated by the induction of immunostimulatory (not immunosuppressive) macrophages. These cells produce three other pro-inflammatory proteins that work with CCL7 to recruit T cells.

Predicting Immunotherapy Outcomes in Human Patients

In a clinical setting, the question is whether FMRP levels can help form a prognosis for patients undergoing immunotherapy. Counterintuitively, mRNA levels of the FMR1 gene and FMRP protein were insufficient to predict outcomes in cancer patient cohorts.

To solve this problem, the researchers relied on the fact that, in the cell, FMRP modulates up and down the stability of mRNA by directly binding it. This means that FMRP could alter RNA levels that could be picked up in transcriptome datasets, which could be collected to define a “gene signature” to help track its functional activity. The approach worked, allowing scientists to track a genetic signature of FMRP’s cancer-regulating activity with a network of 156 genes.

The FMRP cancer network activity signature has been shown to prognosticate poor survival in several human cancers, consistent with the immunosuppressive effects of FMRP and, in some patients, has been linked to poor responses to cancer treatments. immunotherapy.

The work shows that FMRP regulates a network of genes and cells in the tumor microenvironment, all of which help tumors evade immune destruction.

Douglas Hanahan says, “Having studied the complex cellular makeup of solid tumors for decades, I am personally amazed by our discovery that a co-opted neural regulatory protein – FMRP – can orchestrate the formation of a multi-faceted protective barrier against attack. by the immune system, which consequently limits the benefit of immunotherapies, thus presenting FMRP as a new therapeutic target for cancer.

Other contributors

  • Opna Bio SA
  • Agora Cancer Research Center
  • Swiss Institute of Bioinformatics (SIB)
  • University of Bern
  • EPFL Institute of Bioengineering
  • University of Lausanne (UNIL)
  • National Center for Children’s Health (Beijing)
  • Swiss Cancer Center Leman (SCCL)

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