Effects of fermented wheat germ extract on oral cancer cells and research of biomarkers for diagnosis and prognosis of oral cancer

Oral squamous cell carcinoma (OSCC) represents one of the most aggressive types of cancer. The disease occurs when the accumulation of multiple genetic mutations in the oral epithelial cells leads to an irreversible damage of DNA and the cells lose their normal life cycle. The prognosis correlates to several factors and an early diagnosis, certainly, improves the outcome. The treatment strategy for OSCC incorporates both the surgical and oncologic approaches. There are two main challenges of the current research in cancer treatment: the first one is the development of more personalized and effective therapies, since not all tumors of the same stage respond to the therapy in the same way, and the second one is the setup of a more targeted therapy, that can affect only the cancer cells, without destroying healthy ones. Many efforts are made to find compounds that can support and improve the cancer therapy, and great attention is focused on some of natural products, known to have beneficial properties on the human organism. The aim of this thesis is to present results deriving from a research directed to investigate a possible use of a natural compound, Fermented Wheat Germ Extract (FWGE), for the treatment of Oral squamous cell carcinoma (OSCC). In order to summarize the scientific evidence of the use of FWGE for treatment of cancer cells, a systematic review of the literature was performed. Sixteen articles were included in the final qualitative analysis. Various types of cancer cells treated with FWGE have been analyzed, showing mainly cytotoxic effects, alteration of the cell cycle, antiproliferative effects, and induction of apoptosis. After that, a series of in vitro experiments, including MTT assay, invasion and migration assays were performed to investigate the effects of the treatment of OSCC cells (HSC-3, SAS and SCC-25) with different concentrations of FWGE. The inhibitory effect on viability 2 of OSCC cells, exerted by chemotherapeutic drugs (cisplatin and 5-fluorouracil) and the combination of these with FWGE, was also evaluated. The results showed a significant reduction of cells viability after treatment with FWGE. Regarding migration and invasion capacity, the HSC-3 cells resulted to be the most sensitive to the treatment with FWGE. The combination of chemotherapeutic drugs and FWGE at 10mg/ml led to a significantly higher decrease in cell viability. A secondary purpose of this thesis regarded the investigation of prognostic meaning of certain mutations and expression of proteins characterizing OSCC. Firstly, a histologic and bioinformatic analysis of Musashi 2 (MSI2) expression was performed and its correlation with clinic-pathologic and prognostic features of OSCC evaluated. Musashi-2 is an RNA-binding protein, playing a fundamental role in the oncogenesis of several cancers. A bioinformatic analysis was performed on data downloaded from The Cancer Genome Atlas (TCGA) database. The MSI2 expression data were analysed for their correlation with clinic-pathological and prognostic features. In addition, an immunohistochemical evaluation of MSI2 expression on 108 OSCC samples included in a tissue microarray and 13 healthy mucosae samples was performed. 241 patients’ data from TCGA were included in the final analysis. No DNA mutations were detected for the MSI2 gene, but a hyper methylated condition of the gene emerged. MSI2 mRNA expression correlated with Grading (p = 0.009) and overall survival (p = 0.045), but not with disease free survival (p = 0.549). Males presented a higher MSI2 mRNA expression than females. The immunohistochemical evaluation revealed a weak expression of MSI2 in both OSCC samples and in healthy oral mucosae. In addition, MSI2 expression directly correlated with Cyclin-D1 expression (p = 0.022). However, no correlation has been detected with prognostic outcomes (overall and disease free survival). The role of MSI2 expression in OSCC seems to be not so closely correlated with prognosis, as in other human neoplasms. 3 The correlation with Cyclin-D1 expression suggests an indirect role that MSI2 might have in the proliferation of OSCC cells, but further studies are needed to confirm such results. Secondly, the role of programmed death ligand 1 (PD‐L1) in the tumour immunity and its potential function as a marker for OSCC prognosis were investigated through a metaanalysis. The studies were identified by searching PubMed, SCOPUS, Web of Science and were assessed by two of the authors. After the selection process, 11 articles met eligibility criteria and were included in the meta‐analysis. Quality assessment of studies was performed according to the REMARK guidelines, and the risk of biases across studies was investigated through Q and I2 tests. Meta‐analysis was performed to investigate the association between the PD‐L1 expression either overall survival (OS), disease‐free survival (DFS), diseasespecific survival (DSS), gender and lymph node metastasis. A total of 1060 patients were analysed in the 11 studies included in the meta‐analysis. Pooled analysis revealed that the expression of PD‐L1 did not correlate with poor OS (HR, 0.60; 95% CI: [0.33, 1.10]; P = 0.10), DFS (HR, 0.62; 95% CI: [0.21, 1.88]; P = 0.40), DSS (HR, 2.05; 95% CI: [0.53, 7.86]; P = 0.29 and lymph node metastasis (HR, 1.15; 95% CI: [0.74, 1.81]; P = 0.53). Furthermore, results of the meta‐analysis showed that high expression of PD‐L1 is two times more frequent in female patients (OR, 0.5; 95% CI: [0.36, 0.69]; P < 0.0001) compared to males. For all the three outcomes analysed, a high rate of heterogeneity was detected (I2 > 50%). High PD‐L1 expression did not correlate with poor prognosis of patients suffering for oral squamous cell carcinoma. Studies published on the topic showed a significant variation in results, limiting the use of PD‐L1 expression by immunohistochemistry as prognostic biomarker in clinical practice. Lastly, the role of the tumour-suppressor gene TP53 was evaluated in different head and neck squamous cell carcinoma (HNSCC). A systematic bioinformatics appraisal of TP53 mutations was performed on 415 HNSCC cases available on The Cancer Genome Atlas (TCGA). The following features were analysed and correlated with known 4 clinicopathological variables: mutational profile of TP53, location (within secondary structure and predicted domains of p53 protein) and well-known hotspot mutations. Interactome–genome–transcriptome network analysis highlighted different gene networks. An algorithm was generated to develop a new prognostic classification system based on patients’ overall survival. TP53 mutations in HNSCCs exhibited distinct differences in different anatomical sites. The mutational profile of TP53 was an independent prognostic factor in HNSCC. High risk of death mutations, identified by our novel classification algorithm, was an independent prognostic factor in TCGA HNSCC database. Finally, network analysis suggested that distinct p53 molecular pathways exist in a site- and mutation-specific manner. The mutational profile of TP53 may serve as an independent prognostic factor in HNSCC patients, and is associated with distinctive site-specific biological networks.