Role of interferon-induced PKR as predictive biomarker and anti-cancer stem cells properties of interferon

Abstract

Colon cancer and malignant melanoma figure among the leading causes of morbidity and mortality whose incidence is increasing worldwide. Currently, available cytotoxic treatment options show low response rates, significant side effects and low/medium impact on survival, thus emphasizing the need for more effective therapies and new predictive biomarkers that could be achieved by a personalized treatment, based on the individual tumours molecular profile. The correct and early diagnosis of cancer is essential for adequate and effective treatment because every cancer patient requires a specific treatment regimen, which encompasses one or more modalities such as surgery, radiotherapy and/or chemotherapy. Despite the success of new treatments, most patients progress due to the presence of intraand intertumoral heterogeneity conferring resistance and thus disease relapse. Factors contributing to heterogeneity include interactions with the microenvironment, inflammatory response, genetic mutations and the presence of cancer stem cells subpopulations (CSCs). This small cell subpopulation is characterized, among others, by relative quiescence, longevity, slow cell cycle progression and accordingly high resistance towards radiation and cytotoxic drugs, which finally accounts for metastasis formation and local relapse after long periods of dormancy. Therefore, to prevent cancer relapse, new therapeutic effective strategies against CSCs are required; and the search for specific drugs with low-toxicity and new predictive and pronostic biomarkers are still a very attractive approach. In this way, with the aim of searching new predictive biomarkers for cancer patients, our group has identified the protein kinase, PKR. The protein kinase R (PKR, also called EIF2AK2) is an interferon-inducible double-stranded RNA protein kinase with multiple effects on cells that plays an active part in the cellular response to numerous types of stress. PKR has been extensively studied and documented for its relevance as an antiviral agent and a cell growth regulator. PKR has been previously identified by our group as a therapeutic target of the chemotherapeutic drug 5-Fluorouracil (5-FU), inducing tumor cell death by apoptosis in a complementary and independent manner to the tumor suppressor p53. In addition, PKR is regulated by a non-coding pre-mir886 (nc886), which has been identified in serum of patients with metastases having high potential to be a detectable biomarker in liquid biopsies. Our data show the great variability in PKR expression levels and localization between tumor samples and healthy tissues in patients with metastatic colon cancer (plasma and colon epithelium). The expression levels and localization of PKR together with the expression of nc886 have allowed the identification of groups of patients with different responses to treatment based on the use of 5-FU, supporting the great potential of this protein and the pre-microRNA that regulates it as possible predictive biomarkers in different types of cancer. In addition, PKR is induced by Interferon type I (IFN), a cytokine widely known for their antiviral and antitumor activity. In fact, non metastatic high-risk melanoma is still treated with high dose of Interferon alpha (IFN-α) with a significant improvement in Disease Free Survival in patients through a mechanism that is not yet fully understood. In this thesis, we have analyzed the effect of low and high dose of IFN-α-treatment over melanospheres enriched in CSCs subpopulations, demonstrating that this cytokine has a potent antitumor action against these subpopulations both in vitro and in vivo, which contributes to deep in the mechanism of action of these cytokines and to devise new therapies based on effective combinations against these resistant populations. Since new immunotherapies are being imposed in melanoma and other solid tumors, as well as different combinations are under clinical trial to avoid resistances, the efficacy of interferons over CSCs even at low doses with fewer side effects, should be considered as a potentially important combination treatment against the relapse of the disease in oncology. Recently, cellular communication by extracellular vesicles has gained great importance for its likely involvement in cancer. Tumor-derived exosomes are abundant in the body fluids of cancer patients, including those with malignant melanoma, and are involved in several processes such as tumor initiation, progression and tumoral metastasis. In this work, we have demostrated the IFN- α modulation on specific traffic vesicles and exosomes production from melanoma stem cell, suggesting their role of potential biomarkers in novel cancer therapies. Moreover, for the first time, we have found significant differences on the metabolomic profile of exosomes derived from melanoma CSCs as compared with the population of differentiated IFN- α - treated cells. The metabolomic characterization of exosomes opens the door for the discovery of prognosis and diagnosis biomarkers of malignant melanoma with a clear translational and clinical application.