Science: The "accomplice" behind immunotherapy resistance

Immunotherapy is a cutting-edge treatment for cancer that fights tumors
by enabling the patient's own immune system.
Our growing knowledge of the body's mechanisms that regulate immune responses is revolutionizing our fight against cancer
.

But despite the high success rate of immunotherapy, it has run into a stubborn obstacle time and time again: Tumor cells tend to evade the "radar"
of immune cells that try to destroy them.
This, in turn, leads to the treatment of drug resistance, which in many cases benefits if the mechanisms that help avoid resistance are better understood
.

A new study led by scientists at EPFL has now identified a protein that plays a key role
in helping tumors evade immune destruction.
The protein, named Fragile X Intellectual Disability Protein (FMRP), regulates gene and cellular networks in the tumor microenvironment, giving it the ability to "hide" immune cells
.
Under normal circumstances, FMRP is involved in regulating protein translation and mRNA stability
in neurons.
But the researchers found that it was abnormally upregulated
in a variety of cancers.

The study, published in the journal Science, was led by researchers from Douglas Hanahan's team at the Swiss Institute for Experimental Cancer Research (ISREC) and the Ludwig Cancer Institute Lausanne Branch, as well as colleagues from the University Hospital de Lausanne (CHUV) and other Swiss institutions
.
The discovery also led to a branch of EPFL, Opna Bio, whose employees were also involved in the study
.

The idea comes from previous studies that have shown that cancer cells that naturally overexpress FMRP are aggressive and metastatic
.
Other studies have shown that, conversely, if FMRP is not expressed in developing neurons, it can lead to cognitive deficits (hence the "retarded" part of the protein's name).

Armed with this evidence, the researchers set out to study FMRP expression
in human tumors.
They then evaluated its tumor-promoting function in mouse cancer models and finally investigated its relationship
to the prognosis of human cancer patients.
The study included several data collection steps
.
First, the scientists performed FMRP immunostaining
on human tumor tissue.
Most tumors test positive, while corresponding normal tissue does not
.
This means that FMRP is highly
expressed specifically in cancer cells.

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

FMRP is associated with the immune system

To explore this, scientists developed so-called "knockout" cancer cell lines
.
Gene knockout cells or organisms are genetically engineered to lose – "knock out" – a specific gene in order to find clues
about its function.
Basically, whatever changes have occurred in the cells where the gene was knocked out compared to the cells that retained the gene (called "wild type"), the missing gene
can usually be traced.

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

The researchers first evaluated survival rates
between tumor mice containing FMRP knockout cancer cells and tumor mice containing FMRP wild-type cells in mice with compromised immune systems.
The comparison showed similar survival rates
.
Remarkably, when they compared the knocked out tumors to wild-type tumors grown in mice with functioning immune systems, they found that tumors without FMRP grew more slowly and the animals survived longer
.
This part of the study suggests that FMRP itself has nothing to do with stimulating tumor growth, but rather with the adaptive immune system (the part of the immune system that we "train" with vaccines
).

This is further confirmed by the observation that wild-type tumors have very few infiltrating T lymphocytes, while knockout tumors are highly inflammatory
.
Depleting T cells from tumors knocked out by FMRP caused them to start growing faster, reducing survival in mice, meaning that FMRP was somehow involved in tumors evading the immune system
.

How tumors with FMRP defend against immune cells

The team continues molecular genetic analysis
of knockout and wild-type tumors.
This revealed significant differences in gene transcription throughout the genome, indicating that FMRP interacts
with multiple genes.
In addition, tumors showed significant differences in the abundance of cancer cells, macrophages, and T cells, further suggesting the role of FMRP in regulating components of
the immune system.

The next phase of the study looked at the production of specific factors associated with unique immune responses — evasion or aggression
.
The study found that tumors expressing FMRP produced interleukin-33, a protein that induces the production of regulatory T cells, a subset
of T cells that suppress immune responses.
They also produce protein S, a glycoprotein
known to promote tumor growth.
Finally, tumors produce exosomes, an organelle that has been shown to trigger the production of a type of macrophage that normally contributes to wound healing and tissue repair
.
Collectively, all three factors have immunosuppressive effects that help form a tumor barrier against attack by T lymphocytes
.

In contrast, FMRP knockout tumor cells actually downregulated all three factors (interleukin-33, protein S, and exosomes) and upregulated a different chemokine called "C-C motif chemokine ligand 7" (CCL7), which helps recruit and activate T cells
.
This process is further aided
by the induction of immunostimulated (rather than immunosuppressive) macrophages.
These cells produce three other pro-inflammatory proteins that recruit T cells
along with CCL7.

Predicting immunotherapy outcomes in human patients

In a clinical context, the question is whether FMRP levels contribute to shaping the prognosis
of patients receiving immunotherapy.
Counterintuitively, neither the mRNA nor FMRP protein levels of the FMR1 gene were sufficient to predict the prognosis
of the cancer patient cohort.

To solve this problem, the researchers built on the fact that in cells, FMRP upregulates or down-regulates its stability
by binding directly to mRNA.
This means that FMRP may alter RNA levels in transcriptome datasets that can be collected to define "gene signatures" to help track their functional activity
.
The approach worked, allowing scientists to track the genetic signature
of FMRP's cancer-regulating activity through a network of 156 genes.

The FMRP cancer network activity profile has been shown to be a prognostic factor for poor survival of multiple human cancers, consistent with the immunosuppressive effects of FMRP and, in some patients, it is associated with
adverse effects of immunotherapy.
Studies have shown that FMRP regulates gene and cellular networks in the tumor microenvironment, all of which help tumors evade immune destruction
.

Douglas Hanahan said: "After decades of studying the complex cellular composition of solid tumors, I was personally surprised by our discovery that an additional neuronal regulatory protein – FMRP – can coordinate to form a multifaceted protective barrier against an attack by the immune system, thereby limiting the benefits of immunotherapy, thus making FMRP a new therapeutic target for cancer
.
"

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