Aberrant hyperexpression of the RNA binding protein FMRP in tumors mediates immune evasion

[James]

Aberrant hyperexpression of the RNA binding protein FMRP in tumors mediates immune evasion

https://www.science.org/doi/10.1126/science.abl7207

Many human cancers manifest the capability to circumvent attack by the adaptive immune system. In this work, we identified a component of immune evasion that involves frequent up-regulation of fragile X mental retardation protein (FMRP) in solid tumors. FMRP represses immune attack, as revealed by cancer cells engineered to lack its expression. FMRP-deficient tumors were infiltrated by activated T cells that impaired tumor growth and enhanced survival in mice. Mechanistically, FMRP’s immunosuppression was multifactorial, involving repression of the chemoattractant C-C motif chemokine ligand 7 (CCL7) concomitant with up-regulation of three immunomodulators—interleukin-33 (IL-33), tumor-secreted protein S (PROS1), and extracellular vesicles. Gene signatures associate FMRP’s cancer network with poor prognosis and response to therapy in cancer patients. Collectively, FMRP is implicated as a regulator that orchestrates a multifaceted barrier to antitumor immune responses.

INTRODUCTION
Cancer biology and therapy have been transformed by knowledge about immunoregulatory mechanisms that govern adaptive immunity. Although some forms of treatment resistance are related to the intentionally transitory operations of the adaptive immune system, others reflect more subtle requirements to modulate the immune system in different contexts. In this work, we identified an immunoregulatory mechanism involving the neuronal RNA binding protein fragile X mental retardation protein (FMRP), which broadly regulates protein translation and mRNA stability and is aberrantly up-regulated in multiple forms of cancer.

RATIONALE
This study was motivated by reports that cancer cells naturally overexpressing FMRP, whose loss of expression in developing neurons causes cognitive defects, were invasive and metastatic. We investigated the expression of FMRP in human tumors, further assessed its tumor-promoting functions in mouse models of cancer, and evaluated its association with prognosis for human cancer patients.

RESULTS
When human tumor tissue microarrays were immunostained for expression of FMRP, a majority of tumors expressed FMRP, whereas cognate normal tissues did not. To investigate the functional significance of this broad up-regulation, the FMR1 gene was ablated through CRISPR-Cas9 gene editing (FMRP-KO, where KO indicates knockout) in mouse cancer cell lines that were inoculated into both immunodeficient and syngeneic immunocompetent mice to establish tumors in parallel with wild-type (WT) FMRP-expressing cell lines. Mice bearing FMRP-KO tumors had similar survival compared with isogenic WT tumors in immunodeficient hosts, indicating that FMRP was not involved in stimulating tumor growth per se. By contrast, tumor growth was impaired and survival extended in immunocompetent hosts, implicating the adaptive immune system. Indeed, FMRP-expressing WT tumors were largely devoid of T cells, whereas FMRP-KO tumors were highly inflamed. Depletion of CD8 and CD4 T cells restored tumor growth and reduced survival, implicating FMRP in immune evasion in WT tumors. WT and FMRP-KO tumors were profiled by single-cell RNA sequencing, revealing marked differences in genome-wide transcription and abundance of cancer cells, macrophages, and T cells. To elucidate the effects of this multifaceted regulatory protein, we performed several functional perturbations, revealing that: FMRP-expressing cancer cells produce the chemokine interleukin-33 (IL-33), which induces regulatory T cells, as well as tumor-secreted protein S (PROS1) ligand and exosomes that elicit tumor-promoting (M2) macrophages. Both cell types are immunosuppressive, collectively contributing to the barrier against T cell attack. By contrast, FMRP-KO cancer cells down-regulate all three factors and up-regulate C-C motif chemokine ligand 7 (CCL7), which helps recruit and activate T cells. Additionally, immunostimulatory macrophages develop in this context that express three proinflammatory chemokines—CCL5, CXCL9, and CXCL10—which cooperate with CCL7 in recruiting T cells. Finally, neither FMR1 mRNA nor FMRP protein levels were sufficient to predict outcomes in cohorts of cancer patients. Recognizing FMRP’s function as an RNA binding protein that modulates mRNA stability and hence levels in transcriptome datasets, a gene signature reflecting FMRP’s cancer regulatory activity (involving 156 genes) was developed by comparing FMRP-expressing versus FMRP-deficient cancer cells, both in culture and within tumors. Our FMRP cancer activity signature was prognostic for survival across multiple human cancers; anticorrelated with the intensity of T cell infiltration in different tumor types, consistent with FMRP’s immunosuppressive effects; and was associated with comparatively poor responses to immune checkpoint inhibitors and immune-dependent chemotherapy in selected cohorts.

CONCLUSION
FMRP is revealed as a regulator of a network of genes and cells in the tumor microenvironment that contribute to the capability of tumors to evade immune destruction.

[James]

FMRP – the protein behind immunotherapy resistance
By Jim Cornall

https://www.labiotech.eu/trends-news/fmrp-protein-behind-immunotherapy-resistance/

Immunotherapy is a cutting-edge approach to treating cancer by turning the patient’s own immune system against their tumor.

Despite success rates, immunotherapy has time and again met with a stubborn obstacle: tumor cells often evade 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 mechanisms that can help circumvent it.

A new study led by scientists at Swiss university EPFL has now uncovered a protein that plays a key role in helping tumors evade immune destruction. The protein, 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 regulating protein translation and the stability of mRNA in neurons. But the researchers found it is up-regulated in multiple forms of cancer.

The study, published in Science, was led by researchers in the group of Douglas Hanahan at the Swiss Institute for Experimental Cancer Research (ISREC) and the Lausanne Branch of the Ludwig Institute for Cancer Research, along with colleagues from the University Hospital of Lausanne (CHUV) and other Swiss institutions.

The discovery has also led to an EPFL spin-off, Opna Bio, whose staff were also involved in the research.
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 if, in contrast, FMRP fails to be 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 assessed its tumor-promoting functions in mouse models of cancer, and finally studied its association with prognosis for human cancer patients.

The study involved several data-gathering steps. First, the scientists performed immunostaining for FMRP on tissue from human tumors. The majority of the tumors tested positive, while corresponding normal tissue did not. This showed FMRP is specifically and highly expressed in cancer cells.

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

FMRP and the immune system

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

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

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

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

This was further confirmed by the observation that wild-type tumors had very few infiltrating T lymphocytes, whereas knockout tumors were highly inflamed. Depleting T cells from the FMRP-knockout tumors caused them to start growing more rapidly and reduced the survival rates of the mice, meaning 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 entire genome, suggesting that FMRP interacts with multiple genes. In addition, 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 the distinctive immune responses – evasion versus attack. The tumors expressing FMRP were 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, the tumors produce exosomes – cell organelles that triggered the production of a type of macrophage cell that normally helps with wound healing and tissue repair. All three factors are immunosuppressive and contribute to the tumor’s barrier against attacks from T lymphocytes.

In contrast, the FMRP knockout tumors cells actually downregulated all three factors (interleukin-33, protein S, and exosomes) while they up-regulated a different chemokine, C-C motif chemokine ligand 7 (CCL7), which helps recruit and activate T cells. This process is further aided by inducing immunostimulatory (and not immunosuppressive) macrophages. These cells produce three other proinflammatory proteins that work with CCL7 in recruiting T cells.

Predicting immunotherapy outcomes in human patients

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

To address this, the researchers built on the fact that, in the cell, FMRP up- and down-modulates the stability of mRNA by binding it directly. This means FMRP might change 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 the scientists to track a gene signature of FMRP’s cancer regulatory activity with a network of 156 genes.

The FMRP cancer network activity signature proved to be prognostic for poor survival across multiple human cancers, consistent with the immunosuppressive effects of FMRP, and, in some patients, it was linked to poor responses to immunotherapy treatments.

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

Hanahan said: “Having studied the complex cellular composition of solid tumors for decades, I am personally astonished by our discovery that a co-opted neuronal regulatory protein – FMRP – can orchestrate the formation of a multi-faceted protective barrier against attack by the immune system that consequently limits the benefit of immunotherapies, thereby presenting FMRP as a new therapeutic target for cancer.”