Recent Developments in Cancer Systems Biology

This ebook includes original research articles and reviews to update readers on the state of the art systems approach to not only discover novel diagnostic and prognostic biomarkers for several cancer types, but also evaluate methodologies to map out important genomic signatures. In addition, therapeutic targets and drug repurposing have been emphasized for a variety of cancer types. In particular, new and established researchers who desire to learn about cancer systems biology and why it is possibly the leading front to a personalized medicine approach will enjoy reading this book

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

New Prognostic and Predictive Markers in Cancer Progression

Biomarkers are of critical medical importance for oncologists, allowing them to predict and detect disease and to determine the best course of action for cancer patient care. Prognostic markers are used to evaluate a patient’s outcome and cancer recurrence probability after initial interventions such as surgery or drug treatments and, hence, to select follow-up and further treatment strategies. On the other hand, predictive markers are increasingly being used to evaluate the probability of benefit from clinical intervention(s), driving personalized medicine. Evolving technologies and the increasing availability of “multiomics” data are leading to the selection of numerous potential biomarkers, based on DNA, RNA, miRNA, protein, and metabolic alterations within cancer cells or tumor microenvironment, that may be combined with clinical and pathological data to greatly improve the prediction of both cancer progression and therapeutic treatment responses. However, in recent years, few biomarkers have progressed from discovery to become validated tools to be used in clinical practice. This Special Issue comprises eight review articles and five original studies on novel potential prognostic and predictive markers for different cancer types

Identification of new genetic alterations and potential biomarkers in papillary thyroid carcinoma

2014 - 2015Papillary thyroid carcinoma (PTC) is the most frequent thyroid malignant neoplasia. Oncogene activation occurs in more than 70% of the cases. BRAF mutations occur in about 40% of PTCs, whereas RET rearrangements (RET/PTC oncogenes) are present in about 20% of cases. Finally, RAS mutations and TRK and PPARG rearrangements account for about 5% each of these malignancies. However, despite the presence of tumor-initiating driver events, cancer results from the progressive accumulation of mutations in genes that confer growth advantage over surrounding cells. A better understanding of molecular alterations of PTC will provide important insights into cancer etiology. It will also lead to advance in their diagnosis, possibly opening the way for developing novel molecular therapies. Thus, the aim of this PhD project is to deeply explore the transcriptome of PTC in order to identify new driver events in this type of cancer. In the first part of this study, we used RNA-Sequencing in a discovery cohort of 18 patients with papillary thyroid carcinoma to identify fusion transcripts and expressed mutations in cancer driver genes. Furthermore, we used targeted sequencing on the DNA of these same patients to validate identified mutations. We extended the screening to thyroids of 50 PTC patients and of 30 healthy individuals. Using this approach we identified new somatic mutations in CBL, NOTCH1, PIK3R4 and SMARCA4 genes. We also found mutations in DICER, MET and VHL genes, previously found mutated in other tumors, but not described yet in PTC. We also identified a new chimeric transcript generated by the fusion of lysine deficient protein kinase 1 (WNK1) and beta-1,4-N-acetylgalactosaminyl transferase 3 (B4GALNT3) genes and correlated with an overexpression of B4GALNT3 gene. Moreover, although protein coding genes play a leading role in cancer genetics, in recent years, many studies focused on a novel class of non coding RNAs, long non coding RNAs (lncRNAs), which regulate the expression levels of protein coding genes. Since deregulated expression of lncRNAs has been reported in many cancers, it suggests that that they may act as potential oncogene or tumorsuppressor. Thus, to assess if lncRNAs can exert a tumorigenic role in thyroid, in the second part of my PhD project I systematically quantified lncRNAs’ expression in PTC vs healthy thyroids using our RNA-Seq data. Combining ab initio reconstruction to a custom computational pipeline we found that novel and known lncRNAs are significantly altered in PTC, and some of them are possibly associated with cancer driver genes. Then we extensively focused on an unannotated lncRNA transcribed antisense to MET oncogene, named in this study MET-AS. Both genes are significantly up-regulated in a sub-class of PTCs - i.e. patients with BRAF gene mutations and RET gene rearrangements, compared to other PTCs and "non-tumor" thyroid biopsies. Preliminary data indicate that MET-AS knockdown induces down-regulation of MET, and induces a changes in cell cycle in a PTC cell line, suggesting the novel lncRNA might be a new MET regulator. Further studies should be conducted to demonstrate detailed mechanism of our findings. Finally, our data confirmed the genetic heterogeneity of papillary thyroid carcinoma revealing that gene expression correlates more with the mutation pattern than with tumor staging. Overall, this study provides new information about PTC genetic alterations, suggesting potential pharmacological adjuvant therapies in PTC. [edited by author]Il carcinoma papillare tiroideo (PTC) costituisce circa l’80% di tutti i tumori maligni della tiroide. Ad oggi, sono state identificate mutazioni a carico del gene BRAF in circa il 40% di casi, mentre riarrangiamenti che coinvolgono il gene RET (RET/PTC) sono presenti in circa il 20% dei casi. Infine, mutazioni nei geni RAS e riarrangiamenti dei geni TRK e PPARG occorrono in circa il 5% dei casi ciascuno. Tuttavia, nonostante la presenza di alterazioni genetiche che possano dare inizio al processo canceroso, il tumore è il risultato del progressivo accumulo di mutazioni in geni che conferiscono un vantaggio di crescita sulle cellule circostanti. Pertanto, una conoscenza più approfondita delle alterazioni molecolari del carcinoma papillare tiroideo è fondamentale per migliorare gli aspetti diagnostici e prognostici, e la risposta individuale ai trattamenti farmacologici. Alla luce di ciò, il mio progetto di dottorato ha avuto come obiettivo principale l’analisi del trascrittoma del PTC al fine di individuare nuovi eventi molecolari che possano essere implicati in questo tipo di cancro. La prima parte di questo progetto è stata focalizzata sul sequenziamento - mediante RNA-Seq – di 22 RNA isolati da biopsie di tiroide (18 tiroidi affette da carcinoma papillare tiroideo, 4 tiroidi non tumorali) per identificare trascritti di fusione e mutazioni somatiche in geni espressi. I risultati sono stati validati sul DNA dei medesimi pazienti mediante sequenziamento diretto di Sanger. Inoltre, l’analisi mutazionale è stata estesa ad ulteriori 50 pazienti affetti da carcinoma papillare tiroideo e 30 individui sani. Mediante quest’approccio sono state identificate nuove mutazioni puntiformi nei geni CBL, NOTCH1, PIK3R4 e SMARCA4. Inoltre, l’analisi ha rivelato la presenza di mutazioni somatiche nei geni DICER1, MET e VHL, già note nella patogenesi in altri tipi di cancro, ma ad oggi non note nel PTC. Inoltre, è stato individuato un nuovo evento di fusione intra-cromosomico generato dalla fusione tra il primo esone del gene WNK1 (lysine deficient protein kinase 1) e il secondo esone del gene B4GALNT3 (beta-1,4-N-acetyl-galactosaminyl transferase 3). I geni codificanti rivestono un ruolo di primo piano nella genetica del cancro, ma negli ultimi anni, molti studi si sono concentrati su una nuova classe di RNA non codificanti, i long non coding RNA (lncRNAs), che regolano l’espressione dei geni codificanti. I livelli di espressione dei lncRNA sono spesso alterati in diversi tipi di tumori, suggerendo che essi possano agire sia da oncogeni sia da oncosoppressori. Al fine di valutare il loro potenziale ruolo nella tumorigenesi del PTC, la seconda parte di questo progetto è stata focalizzata sull’analisi computazionale dei lncRNA, sia nuovi che annotati, nei nostri dataset di RNASeq. Attraverso l’utilizzo di approcci per la ricostruzione ab initio del trascrittoma e di una pipeline computazionale sono stati indentificati i lncRNA significativamente deregolati nei campioni tumorali. Inoltre, per individuare i lncRNA che potessero regolare l’espressione genica in cis, alcuni di essi sono stati associati - per vicinanza al TSS (transcription start site) - a geni driver in diversi tipi di cancro. Infine, mi sono focalizzata su un lncRNA non annotato nei database pubblici, associato all’oncogene MET, ma trascritto dal filamento opposto. Si tratta pertanto di un lncRNA antisenso al gene MET, chiamato in questo lavoro di tesi MET-AS. Entrambi i geni sono significativamente sovraespressi in una sotto-classe di PTC - vale a dire i pazienti con mutazioni del gene BRAF e riarrangiamenti dell’oncogene RET – chiamati BRAF-like-, rispetto ai campioni tumorali PTC, con profilo trascrizionale simile ai campioni mutati nei geni RAS – chiamati RAS-like - e campioni di tiroide "non-tumorali". Esperimenti preliminari condotti in vitro su una linea cellulare di carcinoma papillare tiroideo, TPC-1, indicano che il silenziamento del lncRNA MET-AS induce una down-regolazione dell’oncogene MET, e induce un blocco del ciclo cellulare in fase G1, suggerendo che il lncRNA potrebbe essere un nuovo regolatore dell’oncogene MET. In conclusione, i risultati ottenuti in questo lavoro di tesi confermano l'eterogeneità genetica del carcinoma papillare della tiroide rivelando che l'espressione genica correla più con il profilo mutazionale dei pazienti che con la stadiazione del tumore. Inoltre, questo studio fornisce nuove informazioni sulle alterazioni genetiche del PTC, suggerendo potenziali terapie adiuvanti farmacologiche per questo tipo di cancro. [a cura dell'autore]XIV n.s

Molecular Mechanisms and Therapies of Colorectal Cancer

Colorectal cancer (CRC) is currently the third leading cause of cancer-related mortality, with 1.9 million incidence cases and 0.9 million deaths worldwide. The global number of new CRC cases is predicted to reach 3.2 million in 2040, based on the projection of aging, population growth, and human development.In clinics, despite advances of diagnosis and surgical procedures, 20% of the patients with CRC present with metastasis at the time of diagnosis, caused by residual tumor cells that have spread to distant organs prior to surgery, affecting the patient survival rate. Standard systemic chemotherapy, alternative therapies that target mechanisms involved in cancer progression and metastasis, immunotherapy, and combination therapies are the major CRC-treatment strategies. In the advanced stage of CRC the transforming growth factor-beta (TGF-β) plays an oncogenic role by promoting cancer cell proliferation, cancer cell self-renewal, epithelial-to-mesenchymal transition, invasion, tumor progression, metastatic spread, and immune escape. Furthermore, high levels of TGF-β1 confers poor prognosis and is associated with early recurrence after surgery, resistance to chemo- or immunotherapy, and shorter survival. Based on the body of experimental evidence indicating that TGF-β signaling has the potential to be a good therapeutic target in CRC, several anti-TGF-β drugs have been investigated in cancer clinical trials. Here, we presented a comprehensive collection of manuscripts regarding studies on targeting the TGF-β signaling in CRC to improve patient’s prognosis and personalized treatments

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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