Search for genetic polymorphisms and molecular markers associated with colorectal cancer for diagnostic and prognostic purposes

Il carcinoma del colon-retto (CCR) comprende tutte le neoplasie con sede tra il cieco ed il retto. E’ il tumore più frequente nella popolazione italiana. Tra le cause che ne influenzano il rischio sono noti sia fattori esogeni che endogeni. La componente genetica riveste un ruolo chiave, infatti le cellule tumorali presentano mutazioni ereditate o acquisite a carico di numerosi geni coinvolti nelle principali vie di segnalazione, replicazione e riparazione. I nostri studi si sono rivolti all’identificazione di modificazioni nell’ambito degli acidi nucleici, mediante analisi di un semplice campione di sangue periferico. Abbiamo voluto indagare sia sul rischio genetico di contrarre la malattia e dunque sulla predittività, che sul riscontro della patologia e dunque sulla diagnosi e sulla prognosi. Studi molecolari hanno difatti evidenziato che alcuni polimorfismi a carico di determinati geni possono contribuire allo sviluppo di patologie tumorali. Il mio lavoro ha mirato, da una parte, a verificare se alcune varianti alleliche dei geni CDH1, MDR1 (ABCB1), ROCK1 e ROCK2 potessero essere considerate fattori di suscettibilità alla patologia, per un possibile impiego in qualità di marcatori predittivi e, dall'altra, ad identificare mRNA marcatori specifici, selezionati tramite un programma bioinformatico utilizzando, per la validazione, campioni di sangue provenienti da pazienti affetti da cancro del colon-retto e controlli sani. L’indagine condotta sui polimorfismi scelti per i geni indagati, ha evidenziato, per alcuni, un’associazione significativa, che mi ha permesso di raccogliere dati interessanti sull’eziologia del CCR. Lo studio di mRNA candidati marcatori, mi ha permesso di identificare TSPAN8, LGALS4 e COL1A2 quali promettenti strumenti di diagnosi e prognosi per il carcinoma del colon-retto. Ulteriori studi saranno necessari per testare i candidati biomarcatori in test di predittività, screening e di follow-up.The colorectal cancer (CCR) includes all cancers located between the caecum and the rectum. It is the most common cancer in the Italian population. Among the causes that influence the risk they are known both exogenous and endogenous factors. The genetic component plays a key role, in fact, cancer cells have inherited or acquired mutations of many genes involved in major signaling, replication and repair pathways. Our studies were directed to the identification of modifications in the context of nucleic acids, by analyzing a simple peripheral blood sample. We investigated both the genetic risk of contracting the disease and thus the prediction, and on the detection of the disease and thus on diagnosis and prognosis. Molecular studies have indeed shown that some polymorphisms of certain genes may contribute to the development of cancer. My work has focused, on the one hand, to see if some allelic variants of CDH1, MDR1 (ABCB1), ROCK1 and ROCK2 genes could be regarded as factors of susceptibility to disease, for possible use as predictive markers and, on the other hand, to identify specific mRNA markers, selected through a bioinformatics program using, for the validation, blood samples from patients with colorectal cancer and healthy controls. The investigation conducted on polymorphisms chosen for the investigated genes, showed, for some of them, a significant association, which allowed me to gather interesting data on the etiology of the CRC. The study of candidated mRNA markers, allowed me to identify TSPAN8, LGALS4 and COL1A2 such promising diagnostic and prognosis tools for colorectal cancer. Further studies will be needed to test the candidated biomarkers for predictive testing, screening and follow-up

EGFR positive feedback loops and βeta Catenin driven miR-17-92 cluster converge to regulate EMT and drug resistance

Epidermal growth factor receptor (EGFR)-targeted cancer drug represents a mile- stone in oncology. Nevertheless the responses are invariably limited by the emer- gence of secondary drug-resistance (Misale, Di Nicolantonio et al. 2014). We found that drug-treated ‘‘EGFR-addicted’’ cancer cells engage a positive feedback loop lead- ing to NF-KB/βCatenin axis activation (Lauriola, Enuka et al. 2014), consequently promoting cell survival and limiting overall drug response. Specifically, secondary activation of βCatenin drives the production of an oncogenic cluster of microRNAs 17-92 (Lauriola, Donghwa et al. 2015) implicated in EMT transformation and resist- ance in colon clones. Hence βCatenin and EGFR combination pharmacological inhi- bition overcome the colon spheres growth and enhance tumor regression. These findings suggest that inhibition of EGFR feedback loop along with NF-kB/βCatenin axis may increase the response to a broad spectrum of drugs that target pathways of oncogene addiction

A module of inflammatory cytokines defines resistance of colorectal cancer to EGFR inhibitors

Epidermal Growth Factor Receptor (EGFR) activates a robust signalling network to which colon cancer tumours often become addicted. Cetuximab, one of the monoclonal antibodies targeting this pathway, is employed to treat patients with colorectal cancer. However, many patients are intrinsically refractory to this treatment, and those who respond develop secondary resistance along time. Mechanisms of cancer cell resistance include either acquisition of new mutations or non genomic activation of alternative signalling routes. In this study, we employed a colon cancer model to assess potential mechanisms driving resistance to cetuximab. Resistant cells displayed increased ability to grow in suspension as colonspheres and this phenotype was associated with poorly organized structures. Factors secreted from resistant cells were causally involved in sustaining resistance, indeed administration to parental cells of conditioned medium collected from resistant cells was sufficient to reduce cetuximab efficacy. Among secreted factors, we report herein that a signature of inflammatory cytokines, including IL1A, IL1B and IL8, which are produced following EGFR pathway activation, was associated with the acquisition of an unresponsive phenotype to cetuximab in vitro. This signature correlated with lack of response to EGFR targeting also in patient-derived tumour xenografts. Collectively, these results highlight the contribution of inflammatory cytokines to reduced sensitivity to EGFR blockade and suggest that inhibition of this panel of cytokines in combination with cetuximab might yield an effective treatment strategy for CRC patients refractory to anti-EGFR targeting

Human Multidrug Resistance 1 gene polymorphisms and Idiopathic Pulmonary Fibrosis

Background: For the first time we tested an association between the human multidrug resistance gene 1 (MDR1) polymorphisms (SNPs) and idiopathic pulmonary fibrosis (IPF). Several MDR1 polymorphisms are associated with pathologies in which they modify the drug susceptibility and pharmacokinetics. Materials and Methods: We genotyped three MDR1 polymorphisms of 48 IPF patients and 100 control subjects with Italian origins. Results: No evidence of association was detected. Conclusion: There are 50 known MDR1 SNPs, and their role is explored in terms of the effectiveness of drug therapy. We consider our small-scale preliminary study as a starting point for further research

Transcriptomic and morphological alterations in hTNPO3 MUT-microinjected Zebrafish embryos modelling Limb Girdle Muscular Dystrophy D2 in vivo

Transportin-3, encoded by the TNPO3 gene, is physiologically involved in the translocation to the nucleus of many different proteins, such as SR proteins, which are associated with RNA metabolism. Mutations in the termination codon of the TNPO3 gene have been proved to cause Limb Girdle Muscular Dystrophy D2 (LGMD D2). More specifically, a single nucleotide deletion leads to the production of a protein with a 15 amino acid extension of its C-terminal domain. This project aimed to investigate the cause of LGMD D2, utilizing an in vivo approach, focusing on the role of TNPO3 on muscle development and to identify any potential molecular pathways linked to the disorder. This procedure involved the microinjection of mRNAs encoding the mutated form of human TNPO3 in Zebrafish embryos, to follow its effects on the myogenic processes during development. The subsequent analysis revealed changes in the gene expression profiles of Myogenic Regulatory Factors (MRFs) and muscle-specific proteins. The transcriptomic alterations are reflected on a phenotypical level by an aberrant organization of muscle fibres, as demonstrated by immunofluorescence staining. The methods we employed enabled us to gain an initial understanding of the role of Transportin-3 in muscle development and the pathogenic mechanism of its mutated form underlying LGMD D2, which has yet to be determined

Electrospun Poly(L-lactide-co-ε-caprolactone) Scaffold Potentiates C2C12 Myoblast Bioactivity and Acts as a Stimulus for Cell Commitment in Skeletal Muscle Myogenesis

Tissue engineering combines a scaffold, cells and regulatory signals, reproducing a biomimetic extracellular matrix capable of supporting cell attachment and proliferation. We examined the role of an electrospun scaffold made of a biocompatible polymer during the myogenesis of skeletal muscle (SKM) as an alternative approach to tissue regeneration. The engineered nanostructure was obtained by electrospinning poly(L-lactide-co-ε-caprolactone) (PLCL) in the form of a 3D porous nanofibrous scaffold further coated with collagen. C2C12 were cultured on the PLCL scaffold, and cell morphology and differentiation pathways were thoroughly investigated. The functionalized PLCL scaffold recreated the SKM nanostructure and performed its biological functions, guiding myoblast morphogenesis and promoting cell differentiation until tissue formation. The scaffold enabled cell–cell interactions through the development of cellular adhesions that were fundamental during myoblast fusion and myotube formation. Expression of myogenic regulatory markers and muscle-specific proteins at different stages of myogenesis suggested that the PLCL scaffold enhanced myoblast differentiation within a shorter time frame. The functionalized PLCL scaffold impacts myoblast bioactivity and acts as a stimulus for cell commitment, surpassing traditional 2D cell culture techniques. We developed a screening model for tissue development and a device for tissue restoration

Transcriptomic and morphological characterization of a first Zebrafish model of Limb Girdle Muscular Dystrophy D2

Limb Girdle Muscular Dystrophy D2 (LGMD D2) is a rare neuromuscular disorder caused by a heterozygous mutation in the termination codon of the TNPO3 gene. This gene encodes for Transportin-3 (TNPO3), an importin which normally mediates the translocation to the nucleus of SR proteins, a family of splicing factors and other proteins related to RNA metabolism. LGMD D2 is characterized by high variability in the onset and progression of the disorder, and the main clinical features are progressive muscle weakness and marked atrophy. Several mutations have been described as causative of LGMD D2, all resulting in a mutated protein that is 15-aminoacids longer in its C-terminal domain. However, the pathogenetic mechanism of this disorder remains unknown. This project aims at investigating the pathogenesis of LGMD D2 utilizing an in vivo approach based on the creation of a Zebrafish model of disease. We focused on the role of TNPO3 on muscle development to identify any potential molecular pathways linked to the disorder. Experimentally we proceeded with the microinjection of mRNAs encoding the wild type or mutated form of human TNPO3 into Zebrafish embryos, to follow their effects on the myogenic processes during development up to 48 hpf. The subsequent analysis showed abnormalities in the gene expression profiles of Myogenic Regulatory Factors (MRFs) and muscle-specific proteins, suggesting an unbalance in the normal myogenic process. Next, we analysed how the transcriptomic alterations were reflected at a morphological level by transmission electron microscopy studies. At ultrastructural level we observed a random organization of myofibrils limited to the embryos microinjected with the mutant form of human TNPO3. These results suggest that our approach could be effective in establishing a Zebrafish model of LGMD D2 and enabled us to gain an initial understanding of the role of TNPO3 in muscle development and the pathogenic mechanism of LGMD D2

Mechanisms of acquired resistance to cetuximab: role of interleukin-1

Cetuximab (CX) is a monoclonal antibody targeting the Epidermal Growth Factor Receptor (EGFR), which is commonly utilized to treat patients with metastatic colorectal cancer (mCRC). Unfortunately, clinicians often observe a residual disease, with a population of cells surviving the treatment and eventually enabling CX resistance. Our previous studies, performed with a cohort of 150 CRC xenopatients, associated poor response to CX with increased abundance of a set of inflammatory cytokines, including IL1A, B and IL8. Stemming from these observations, our working hypothesis assumes that, resistance to CX is acquired, in a subset of CRC patients, through cell plasticity and consequent rewiring of signalling networks, which confer to tumors dependency on the IL1 pathway. In order to assess the effect of IL1 activity, we employed a colon cancer model unresponsive to cetuximab, as previously characterized in our laboratory. To inhibit activation of the IL1 pathway we used anakinra, an IL1-receptor antagonist and parthenolide, which modulates the activity of NF-kB, the transcription factor involved in the feed-forward loop of inflammation mediators. Furthermore, we employed a recombinant decoy (IL1R-Fc), namely a soluble protein combining the human immunoglobulin Fc portion linked to the extracellular region of IL1-receptor, with the ability to sequester IL1 directly from the medium. We generated stable clones of CX-resistant cells expressing IL1R-Fc. Our preliminary results show that inhibition of IL1R leads to a proliferation decrease of colorectal cancer cells. These findings support the hypothesis of a compensatory activation of the IL1-receptor pathway in cetuximab-resistant CRC cells. Hence, modulating IL1 signalling might represent a new therapeutic strategy suitable for patients who acquired refractoriness to monoclonal antibody therapy

Possible Gender-Related Modulation by the ROCK1 Gene in Colorectal Cancer Susceptibility

AIM: In view of accumulating evidence supporting a pivotal role of the Rho/ROCK pathway in cancer, we investigated Rho-kinase polymorphisms as potential susceptibility factors in colorectal cancer (CRC) in a representative sample of the Italian population. METHODS: DNA obtained from the peripheral blood samples of 137 CRC patients and 141 healthy controls was genotyped for four ROCK1 (rs35996865; rs73963110; rs2127958; rs288980) and five ROCK2 (rs12692437; rs7563468; rs35768389; rs17463896; rs16857265) selected single nucleotide polymorphisms. RESULTS: None of the allelic variants of the nine selected markers was associated with the occurrence of CRC or with the development of regional lymph node metastasis. By contrast, the ROCK1 rs35996865 G variant allele was significantly more frequent in male patients (p = 0.028) than in the control group. CONCLUSION: This finding is, at present, the first that points to a possible gender-related modulation by the ROCK1 gene in CRC susceptibility