the protein behind resistance to immunotherapy

Immunotherapy is an advanced approach to cancer treatment that targets the patient’s own immune system against their tumor.

Despite its successes, immunotherapy has repeatedly faced a stubborn obstacle: tumor cells often escape the “radar” of immune cells that seek to destroy them. This in turn leads to resistance to treatment, which in many cases would benefit from a better understanding of the mechanisms that can help circumvent it.

A new study led by scientists from Swiss University EPFL discovered a protein that plays a key role in helping tumors avoid immune destruction. The protein, fragile X mental 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 increases in different forms 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, together with colleagues from the Lausanne University Hospital (CHUV) and other Swiss institutions.

The discovery also led to the separation of EPFL, Opna Biowhose employees also participated in the study.

FMRP

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, conversely, when 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, the researchers investigated the expression of FMRP in human tumors. They then evaluated its tumor-promoting functions in mouse models of cancer and finally studied its association with prognosis in human cancer patients.

The study included several stages of data collection. First, the scientists performed immunostaining for FMRP on human tumor tissues. Most of the tumors tested positive, while the corresponding normal tissue did not. This showed that FMRP is specifically and highly 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 and the immune system

To investigate this, scientists developed lines of “knockout” cancer cells. Knockout cells or organisms are genetically engineered to lose—”knock out”—a particular gene in order to find clues about its function. Essentially, any changes that occur in the knockout cells compared to cells that still have the gene — so-called “wild-type” — can usually be traced back to the missing gene.

In this case, scientists used CRISPR-Cas9 gene editing to turn off the FMR1 gene, which produces FMRP, in mouse cancer cells arising from pancreatic, colon, breast, and skin melanocytes. They then compared FMRP-knockout cancer cells with cancer cells that still had the FMR1 gene and thus expressed the FMRP protein.

The researchers evaluated the survival rate of mice with tumors containing FMRP-knockout cancer cells and mice with wild-type FMRP cells, first in mice whose immune systems were compromised. The comparison showed similar survival rates. In contrast, when they compared the knockout tumors to wild-type tumors growing in mice with a properly functioning immune system, they found that tumors lacking FMRP grew more slowly and the animals survived longer.

This showed that FMRP itself is not involved in stimulating tumor growth, and rather is involved in the adaptive immune system (the part of the immune system that is “trained” by vaccines).

This was further supported by the observation that wild-type tumors had very few infiltrating T lymphocytes, whereas knockout tumors were highly inflamed. Depleting T cells from FMRP-knockout tumors caused them to grow faster and lower the survival rate of the mice, suggesting that FMRP is somehow involved in tumors evading the immune system.

How tumors with FMRP defend against immune cells

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

The next stage of the study looked at the development of specific factors associated with distinctive immune responses – avoidance of attack. Tumors expressing FMRP have been found 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 trigger the production of a type of macrophage cell that normally aids in wound healing and tissue repair. All three factors are immunosuppressive and contribute to the tumor barrier against T-lymphocyte attacks.

In contrast, FMRP-knockout tumor cells actually downregulated all three factors (interleukin-33, protein S, and exosomes), while they upregulated another chemokine, chemokine ligand 7 (CCL7), which helps recruit and activate T cells. This process further contributes to the induction of immunostimulatory (rather than immunosuppressive) macrophages. These cells produce three other proinflammatory proteins that work with CCL7 to recruit T cells.

Prediction of the results of immunotherapy in patients

In a clinical context, the question is whether the level of FMRP can help in shaping the prognosis for patients undergoing immunotherapy. Counterintuitively, FMR1 gene mRNA and FMRP protein levels 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 the stability of mRNA up and down by directly binding to it. This means that FMRP can change the level of RNA that can be found in transcriptome data sets that can be collected to define a “gene signature” to help track its functional activity. This approach worked, allowing the scientists to track the gene signature of FMRP’s regulatory activity against cancer using a network of 156 genes.

The FMRP cancer network activity signature has been shown to be predictive of poor survival in several human cancers, consistent with the immunosuppressive effects of FMRP, and has been associated with poor response to immunotherapy in some patients.

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

Hanahan said: “Having studied the complex cellular makeup of solid tumors for decades, I was personally surprised by our discovery that a co-opted neuronal regulatory protein – FMRP – can orchestrate the formation of a multifaceted protective barrier against immune system attacks. system, which consequently limits the benefit of immunotherapy, thereby presenting FMRP as a novel therapeutic target for cancer treatment.’