To understand cancer, it is important to interpret, analyze and understand the patient’s genomic background, and its profoundly influence on treatment outcomes. However, all the necessary informations are hidden inside the 3 billion bases of the human genome. Hence, new technologies were designed to analyze genomic variation and to develop bioinformatic tools for analyzing sequence data, to identify new biomarkers capable to predict the individual patient’s response to specific cancer treatments and correlated toxicity profiles. As a result of the introduction of next-generation sequencing (NGS), it has become much easier to establish correlations between sequence variants in the human genome and illness risk. It is the goal of the Moli-sani study to uncover genetic and environmental risk factors for cancer and chronic degenerative illnesses in a Mediterranean-derived population. In this study, a large-scale sample (26,000 participants) was recruited and followed up for an average of 10 years after the start of the study [3-5]. The final goal of this project, which involves 1.500 subjects, is the definition of cancer disease risk factors through Next Generation Sequencing (NGS) approaches performed on Mediterranean populations, taking into consideration information such as information on lifestyle and life environment. The mutational landscape, in specific the presence and frequency of sequence mutations (coding and non-coding sections) and changes in the number of copies of the gene, obtained by NGS provide the basis to study multi-factorial interactions. The genetic variants that are identified as associated with cancer disease risk are validated by molecular and cellular analyses. Among them, cancer-related changes in the activation profile of the Trop-1 and Trop-2 genes were identified as cancer risk factors in humans, and as benchmarks for genetic risk factor genomic, transcriptomic, and functional analysis. Studies in this thesis take advantage of these technologies and explore the possibility to identify broad genomic factors behind cancer development. The studies here presented have taken advantage of genomic-transcriptomic technologies to explore the functional role of Trop-2 in cancer progression. This was investigated in particular through the expression of designer mutants of the Trop-2 molecule, somatic knock-out and/or chemical inhibition of survival mechanisms in cancer cell systems (Article I, Article II). In the work presented in Article I [1] we revealed that Trop-2 is cleaved at the first thyroglobulin domain loop of its extracellular region, between R87 and T88, as shown by antibody targeting and N-terminal Edman degradation. Molecular modeling revealed that this cleavage alters the Trop-2 structure and biological function. We have demonstrated that Trop-2 recruits ADAM10 and is activated by ADAM10-mediated cleavage to trigger molecular pathways for tumor growth and metastasis. We have then demonstrated that Trop-2 activation by post-translational processing promotes tumor growth and metastasis. Our findings that the ADAM10-mediated cleavage of Trop-2 at the R87-T88 site is necessary to activate the transformed growth stimulatory activity of Trop-2 and promote its metastasis driver function may pave the way for the development of new therapeutic precision strategies. In the work presented in Article II [2] we identified, through transcriptomic analysis by NGS of colon cancer cells (metastasis models), that Trop-2 is a unique upregulated gene. Transcriptomic analysis of distinct cases of primary tumors and metastases, showed no down-regulation of CDH1 by transcription factors for epithelial-to-mesenchymal transition, thus suggesting that the pro-metastatic activity of Trop-2 operates through alternative mechanisms. In addition, and for the first time, we showed that the binding of Trop-2 to E-cadherin stimulated the proteolytic cleavage of the intracellular domain of E-cadherin by ADAM10. Detachment of E-cadherin from β-actin, loss of cell-cell adhesion, acquisition of invasive capability, and membrane-driven activation of β-catenin signaling, were further enhanced by the tail-less ΔcytoTrop-2 mutant. The Trop-2/E-cadherin/ β-catenin program led to anti-apoptotic signaling, increased cell migration, and enhanced cancer-cell survival.

Genomic risk for cancer activation profiles of Trop-1 and Trop-2

BOUJNAH, KHOULOUD
2022

Abstract

To understand cancer, it is important to interpret, analyze and understand the patient’s genomic background, and its profoundly influence on treatment outcomes. However, all the necessary informations are hidden inside the 3 billion bases of the human genome. Hence, new technologies were designed to analyze genomic variation and to develop bioinformatic tools for analyzing sequence data, to identify new biomarkers capable to predict the individual patient’s response to specific cancer treatments and correlated toxicity profiles. As a result of the introduction of next-generation sequencing (NGS), it has become much easier to establish correlations between sequence variants in the human genome and illness risk. It is the goal of the Moli-sani study to uncover genetic and environmental risk factors for cancer and chronic degenerative illnesses in a Mediterranean-derived population. In this study, a large-scale sample (26,000 participants) was recruited and followed up for an average of 10 years after the start of the study [3-5]. The final goal of this project, which involves 1.500 subjects, is the definition of cancer disease risk factors through Next Generation Sequencing (NGS) approaches performed on Mediterranean populations, taking into consideration information such as information on lifestyle and life environment. The mutational landscape, in specific the presence and frequency of sequence mutations (coding and non-coding sections) and changes in the number of copies of the gene, obtained by NGS provide the basis to study multi-factorial interactions. The genetic variants that are identified as associated with cancer disease risk are validated by molecular and cellular analyses. Among them, cancer-related changes in the activation profile of the Trop-1 and Trop-2 genes were identified as cancer risk factors in humans, and as benchmarks for genetic risk factor genomic, transcriptomic, and functional analysis. Studies in this thesis take advantage of these technologies and explore the possibility to identify broad genomic factors behind cancer development. The studies here presented have taken advantage of genomic-transcriptomic technologies to explore the functional role of Trop-2 in cancer progression. This was investigated in particular through the expression of designer mutants of the Trop-2 molecule, somatic knock-out and/or chemical inhibition of survival mechanisms in cancer cell systems (Article I, Article II). In the work presented in Article I [1] we revealed that Trop-2 is cleaved at the first thyroglobulin domain loop of its extracellular region, between R87 and T88, as shown by antibody targeting and N-terminal Edman degradation. Molecular modeling revealed that this cleavage alters the Trop-2 structure and biological function. We have demonstrated that Trop-2 recruits ADAM10 and is activated by ADAM10-mediated cleavage to trigger molecular pathways for tumor growth and metastasis. We have then demonstrated that Trop-2 activation by post-translational processing promotes tumor growth and metastasis. Our findings that the ADAM10-mediated cleavage of Trop-2 at the R87-T88 site is necessary to activate the transformed growth stimulatory activity of Trop-2 and promote its metastasis driver function may pave the way for the development of new therapeutic precision strategies. In the work presented in Article II [2] we identified, through transcriptomic analysis by NGS of colon cancer cells (metastasis models), that Trop-2 is a unique upregulated gene. Transcriptomic analysis of distinct cases of primary tumors and metastases, showed no down-regulation of CDH1 by transcription factors for epithelial-to-mesenchymal transition, thus suggesting that the pro-metastatic activity of Trop-2 operates through alternative mechanisms. In addition, and for the first time, we showed that the binding of Trop-2 to E-cadherin stimulated the proteolytic cleavage of the intracellular domain of E-cadherin by ADAM10. Detachment of E-cadherin from β-actin, loss of cell-cell adhesion, acquisition of invasive capability, and membrane-driven activation of β-catenin signaling, were further enhanced by the tail-less ΔcytoTrop-2 mutant. The Trop-2/E-cadherin/ β-catenin program led to anti-apoptotic signaling, increased cell migration, and enhanced cancer-cell survival.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11570/3228216
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