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Fucosylated Clusterin: a novel mechanism of tumor escape from immune response

Periodic Reporting for period 1 - FUCTURE (Fucosylated Clusterin: a novel mechanism of tumor escape from immune response)

Reporting period: 2018-06-15 to 2020-06-14

Cancers represent a group of heterogeneous diseases characterized by the transformation of a normal cell into a malignant cell following a multistage process involving genome instability, mutations and tumor-promoting inflammation. Cancer is a significant public health problem worldwide and one of the major causes of mortality around the world. Among women, breast cancer is the most common type and the leading cause of death by cancer.
Even if checkpoint-based therapies represent a true revolution in the treatment of metastatic cancer, a large proportion of patients do not respond to checkpoint inhibitors. Combination therapies aim at enhancing immune responses to tumors, giving checkpoint therapies a better chance to succeed.

During malignant transformation that drives cancer development, tumor-associated antigens arise and they can be recognized by the immune system, generating adaptive immune responses that can lead to tumor rejection. Nevertheless, tumors generally escape immune attacks due to cancer induced immunosuppression. Unraveling the mechanisms involved in this process is a major goal to improve cancer immunotherapies.
A key feature of cancer is the acquisition of glycan motifs different than those expressed by healthy tissues, a phenomenon often referred as “aberrant glycosylation”. Whereas neo expression of Sialyl Lewis X-type motifs renders neoplastic cells with the ability to bind endothelium selectins promoting metastasis, many tumors present an increased expression of non-sialylated fucosylated Lewis-type glycans, unable to bind selectins. The contribution of this glycosylation pattern to tumor growth has not been clearly characterized.
Clusterin (CLU) is a multifunctional glycoprotein present in almost all tissues and body fluids. It was shown to be involved in a number of physiological and pathological processes. Indeed, its expression is dysregulated in many types of tumors, including colon, gastric, prostate, ovarian, bladder, lung and breast cancer.
Our understanding of the diversity functions shared by CLU has been limited to the exitance of two isoforms: nuclear and secretory form. Different works show that CLU production is increased in different malignant tumors and even more, its overexpression is associated to an increased tumor aggressiveness and bad prognosis. However, the nature of this association is still unclear.
Previous data has revealed in the glycosylation patter of seminal and serum CLU, showing that the seminal isoform bears highly fucosylated glycans, which provides it with the ability to bind to DC-SIGN, an endocytic receptor express by myeloid cells such as macrophages and dendritic cells. Moreover, binding of seminal CLU to DC-SIGN might result in the modulation of cellular function promoting a tolerogenic profile.

We hypothesise that tumor secreted CLU express Lewis-type fucosylated glycans and interacts with DC-SIGN expressed by tumor infiltrating myeloid cells, favouring immunosuppressive microenvironment, thus promoting immune tolerance to tumor antigens. In this sense, fucosylated tumor CLU could represent a new mechanism of tumor escape from immune response.

We propose to study the presence of fucosylated clusterin in the tumor microenvironment and unravel its immune modulatory functions.
In the present study we show that human breast tumors express fucosylated clusterin conferring clusterin the ability to interact with DC-SIGN, a C-type lectin receptor expressed by myeloid cells. Interestingly, fucosylated clusterin was absent or diminished in juxtatumor tissues suggesting that it might represent a cancer associated glycoform. Furthermore, by analysing single cell data sets we found that tumoral and stromal cells might represent the major source of fucosylated clusterin. Moreover, we have seen the expression of DC-SIGN by intratumoral macrophages, which represent the most prominent myeloid cell population infiltrating breast tumors. Using an in vitro model, we show that fucosylated clusterin turns macrophages into a pro-angiogenic profile, suggesting a novel pathway linking clusterin and tumor growth. In summary, we have found an unexpected complexity on the structure of secretory clusterin produced by tumors suggesting that, fucosylated clusterin might play a role in tumor progression by promoting the release of pro-angiogenic factors by intratumoral macrophages.
These results have been disseminated in an international conference on Glyocbiology and published in a peer-reviewed paper.
Fucosylated Clusterin could represent a more general mechanism to generate tolerance against self-antigens. This proposal could therefore generate tools to extend the analyses to autoimmune and auto-inflammatory diseases. The highly conserved structure of Clusterin among mammals at the amino-acid level and its presence in almost all body fluids at high concentrations, suggest an important and conserved physiological function. Furthermore, understanding and manipulating this pathway could lead to the development of new therapeutic approaches in the clinics, promoting tolerance to specific antigens in, for example, organ transplantation.
We expected to develope a specific antibody against Fucosylated Clusterin, representing a fundamental tool to expand the study of Fucosylated Clusterin expression. Facilitating the evaluation of its potential use as a predictive and/or biomarker, either in serum from cancer patients or in biopsies. Moreover, the possibility to couple blocking anti-Fucosylated-Clusterin antibodies with checkpoint inhibition therapy could be exploited to enhance anti-tumor immunity.
Graphical Abstract of proposed model