Colorectal cancer is the third leading cause of death in developed countries. Although the huge progresses reached in therapeutical approach, in the presence of metastasis colorectal cancer remains still uncurable. Ion channels have a prominent role in the acquirement of aggressive phenotype, and they are considered the ideal target to design an anticancer therapy. The main problem is that most of them are involved in normal physiological processes and treatments against their activity could result in high toxicity. From several years, our laboratory has focused its attention on the study of CLIC1 protein, considered a promisingcandidatefor a targeted anticancer adjuvant therapy. CLIC1 is a ubiquitous protein belonging to a partially unexplored protein family, the CLICs, that have the peculiar characteristic to be metamorphic. In normal conditions, CLIC1 protein is expressed by all cell types as a cytosolic monomer. Upon stress stimuli, monomers undergo a conformational change and translocate to the plasma membrane where increase chloride permeability. CLIC1 protein was found to be overexpressed in different pathological states, including cancer, but the mechanism in tumorigenesis is still unclear. Our laboratory has demonstrated that in glioblastoma stem cells tmCLIC1 is chronically expressed and here has a pivotal role in tumorigenesis and cancer development. CLIC1 mRNA overexpression was identified in colorectal cancer cells and is related to a poor prognosis. In this scenario, the main aim of the present work is to understand the role of tmCLIC1 in tumorigenesis, invasion, and migration potential of colorectal cancer. To this purpose, were chosen different human immortalized colorectal cancer cells at different stage of aggressiveness according to the Dukes’ classification; a method that categorizes tumor development based of the invasion of tissues and mucosa. Here, was investigated the level of expression of CLIC1 protein and the activity of its transmembrane form as an ion channel. Our results have demonstrated that tmCLIC1protein activity is proportional to the level of aggressiveness of cells. We evaluated the effect on cellular proliferation, migration and invasion of the specific inhibitor of tmCLIC1, IAA94, and a monoclonal antibody, designed in our laboratory and made to specifically recognized the NH2 terminal domain of CLIC1, exposed to extracellular space when the protein is docked in membrane. IAA94 and our antibody, now under license and named tmCLIC1omab, have demonstrated the same inhibitory proprieties. tmCLIC1omab could represent a good alternative for an adjuvant therapy to impair tmCLIC1 activity, considering the high toxicity in vivo of IAA94. Our results have demonstrated that the inhibition of tmCLIC1 activity results in a downregulation of proliferation in the most aggressive cell lines in 2D model, as well as in 3D models. Moreover, the migration and invasion potential of colorectal cancer cells is mainly abolished when tmCLIC1 is impaired. To demonstrate that the effects observed in the presence of tmCLIC1 inhibitors are due to a specific action of the drugs on the protein, and not for the presence of other off targets, we established stable cell lines in which CLIC1 was knocked down after a lentiviral infection. Results obtained in silenced cells have demonstrated the same phenotype of cells treated with IAA94 and tmCLIC1omab. The role of tmCLIC1 activity in tumorigenesis and cancer development was confirmed also in in vivo models. Experiments performed in Zebrafish embryos and immunodeficient mice have demonstrated that the silencing of CLIC1 results in the impairment of primary tumor development and formation of distal metastases. Experiments were also replicated in immunocompetent mice injected with murine colorectal cancer cells that have shown the same phenotype of human colorectal cancer cells used for all the previous trials. Our preliminary experiments have demonstrated that murine colorectal cancer cell line in which CLIC1 protein was silenced were not able to form tumors in the major part of the cases. We tried to investigate the alteration of metastatic pathways after CLIC1 silencing. We obtained that many different genes are downregulated when CLIC1 protein is absent; in particular, genes regulated by Reactive Species of Oxygen (ROS). In this scenario, we tried to define a mechanism of action in which CLIC1 is involved. Altogether, all the results obtained in the present work could be considered a starting point to design a new therapeutic strategy to counteract against metastatic colorectal cancer. Moreover, if our hypothesis of tmCLIC1 mechanism of action will be confirmed and conserved in solid tumors, could represent a new milestone in anticancer research.

TRANSMEMBRANE CHLORIDE INTRACELLULAR CHANNEL 1 (TMCLIC1) PROTEIN IS A KEY REGULATOR OF COLORECTAL CANCER¿S PROLIFERATION AND METASTASIS / F. Cianci ; tutor: M. Mazzanti. - : . Dipartimento di Bioscienze, 2022 Mar 18. ((34. ciclo, Anno Accademico 2021.

TRANSMEMBRANE CHLORIDE INTRACELLULAR CHANNEL 1 (TMCLIC1) PROTEIN IS A KEY REGULATOR OF COLORECTAL CANCER¿S PROLIFERATION AND METASTASIS

F. Cianci
2022-03-18

Abstract

Colorectal cancer is the third leading cause of death in developed countries. Although the huge progresses reached in therapeutical approach, in the presence of metastasis colorectal cancer remains still uncurable. Ion channels have a prominent role in the acquirement of aggressive phenotype, and they are considered the ideal target to design an anticancer therapy. The main problem is that most of them are involved in normal physiological processes and treatments against their activity could result in high toxicity. From several years, our laboratory has focused its attention on the study of CLIC1 protein, considered a promisingcandidatefor a targeted anticancer adjuvant therapy. CLIC1 is a ubiquitous protein belonging to a partially unexplored protein family, the CLICs, that have the peculiar characteristic to be metamorphic. In normal conditions, CLIC1 protein is expressed by all cell types as a cytosolic monomer. Upon stress stimuli, monomers undergo a conformational change and translocate to the plasma membrane where increase chloride permeability. CLIC1 protein was found to be overexpressed in different pathological states, including cancer, but the mechanism in tumorigenesis is still unclear. Our laboratory has demonstrated that in glioblastoma stem cells tmCLIC1 is chronically expressed and here has a pivotal role in tumorigenesis and cancer development. CLIC1 mRNA overexpression was identified in colorectal cancer cells and is related to a poor prognosis. In this scenario, the main aim of the present work is to understand the role of tmCLIC1 in tumorigenesis, invasion, and migration potential of colorectal cancer. To this purpose, were chosen different human immortalized colorectal cancer cells at different stage of aggressiveness according to the Dukes’ classification; a method that categorizes tumor development based of the invasion of tissues and mucosa. Here, was investigated the level of expression of CLIC1 protein and the activity of its transmembrane form as an ion channel. Our results have demonstrated that tmCLIC1protein activity is proportional to the level of aggressiveness of cells. We evaluated the effect on cellular proliferation, migration and invasion of the specific inhibitor of tmCLIC1, IAA94, and a monoclonal antibody, designed in our laboratory and made to specifically recognized the NH2 terminal domain of CLIC1, exposed to extracellular space when the protein is docked in membrane. IAA94 and our antibody, now under license and named tmCLIC1omab, have demonstrated the same inhibitory proprieties. tmCLIC1omab could represent a good alternative for an adjuvant therapy to impair tmCLIC1 activity, considering the high toxicity in vivo of IAA94. Our results have demonstrated that the inhibition of tmCLIC1 activity results in a downregulation of proliferation in the most aggressive cell lines in 2D model, as well as in 3D models. Moreover, the migration and invasion potential of colorectal cancer cells is mainly abolished when tmCLIC1 is impaired. To demonstrate that the effects observed in the presence of tmCLIC1 inhibitors are due to a specific action of the drugs on the protein, and not for the presence of other off targets, we established stable cell lines in which CLIC1 was knocked down after a lentiviral infection. Results obtained in silenced cells have demonstrated the same phenotype of cells treated with IAA94 and tmCLIC1omab. The role of tmCLIC1 activity in tumorigenesis and cancer development was confirmed also in in vivo models. Experiments performed in Zebrafish embryos and immunodeficient mice have demonstrated that the silencing of CLIC1 results in the impairment of primary tumor development and formation of distal metastases. Experiments were also replicated in immunocompetent mice injected with murine colorectal cancer cells that have shown the same phenotype of human colorectal cancer cells used for all the previous trials. Our preliminary experiments have demonstrated that murine colorectal cancer cell line in which CLIC1 protein was silenced were not able to form tumors in the major part of the cases. We tried to investigate the alteration of metastatic pathways after CLIC1 silencing. We obtained that many different genes are downregulated when CLIC1 protein is absent; in particular, genes regulated by Reactive Species of Oxygen (ROS). In this scenario, we tried to define a mechanism of action in which CLIC1 is involved. Altogether, all the results obtained in the present work could be considered a starting point to design a new therapeutic strategy to counteract against metastatic colorectal cancer. Moreover, if our hypothesis of tmCLIC1 mechanism of action will be confirmed and conserved in solid tumors, could represent a new milestone in anticancer research.
MAZZANTI, MICHELE
Settore BIO/11 - Biologia Molecolare
Settore BIO/09 - Fisiologia
Settore MED/06 - Oncologia Medica
TRANSMEMBRANE CHLORIDE INTRACELLULAR CHANNEL 1 (TMCLIC1) PROTEIN IS A KEY REGULATOR OF COLORECTAL CANCER¿S PROLIFERATION AND METASTASIS / F. Cianci ; tutor: M. Mazzanti. - : . Dipartimento di Bioscienze, 2022 Mar 18. ((34. ciclo, Anno Accademico 2021.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/909390
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