Brain tumors represent a group of heterogeneous neoplasms which, despite originating in the same anatomical region, differ in morphology, etiology, molecular biology, and especially in clinical behavior. It has been estimated that brain tumors affect about 200,000 people worldwide every year, representing approximately 2% of cancer deaths. Among all primary human brain tumors, glioblastoma (GBM) is the most malignant and frequent (~70%), with a median survival of about 14 months and a 5-year survival rate at 5%, thus representing an extreme therapeutic challenge. GBM is characterized by sustained proliferation and survival, immune system escape, intense angiogenesis, invasion, cell infiltration, rapid progression, resistance to radio- and chemotherapies, with high frequency of relapse. In cancer pathogenesis, particularly in high grade tumors as GBM, aberrant neo-angiogenesis is a vital process for the mass growth: it is driven by neoplastic cells in order to respond to the tumoral hypoxic environment in the necrotic core, which increases the demand for oxygen and nutrients by neoplastic cells, and is, therefore, essential to carry out the metabolic functions on which their survival is based. Several observations led to the knowledge that tumoral neo-angiogenesis gives rise to ultra-structurally abnormal vessels, larger than their normal counterparts, dilated, convoluted, irregularly branched and exceptionally permeable due to the presence of fenestrations and the lack of a complete basal membrane. Due to these destructive features, despite aggressive therapeutic treatment, consisting in surgical resection followed by chemotherapy with temozolomide (TMZ), most patients experience tumor recurrence in less than one year, suggesting the urgent need to implement clinical practice with novel prognostic and therapeutic strategies. To this aim, this research project has been focused on: i) aberrant angiogenesis mediated by endothelial cells (ECs), which promote tumor infiltration into surrounding tissues, with consequent compromission of cognitive skills, and ii) genetic instability, which characterize GBM with a high intra- and intertumoral heterogeneity. In particular, the underlying hypothesis is that a specific molecular signature characterizes “resistant” and “sensitive” GBM, and it may be responsible for patient overall survival. Indeed, although GBMs from short- and long-term survivors (STS and LTS) are histologically the same, their biological and molecular characteristics are remarkably different, suggesting that factors that contribute to patients’ longevity are important for precise diagnosis and correct clinical management of the disease. The genetic profiling obtained by array CGH on STS and LTS revealed a high number of copy number variation (CNVs) across chromosomes 1 to 22, among which several novel potential prognostic and predictive biomarkers have been described and discussed. In particular, aCGH highlighted the presence of an altered chromosomic pattern relative to calpain family genes. Calpains are a conserved family of cysteine proteinases that catalyze the controlled proteolysis of many specific substrates. Calpain activity is implicated in several 5 fundamental physiological processes, including cytoskeletal remodeling, cellular signalling, apoptosis and cell survival. Alterations of the calpain activity balance has been observed in numerous cancer types, as they can reduce apoptosis, increase cell proliferation and stimulate cell migration and invasiveness. The characterization of calpain expression in primary GBM endothelial cells (GECs) showed an upregulation of calpains and a positive correlation between expression level and patient survival. The blockade of calpain activation with natural and synthetic inhibitors resulted in the inhibition of GEC viability and proliferation, as well as in the promotion of caspase-induced cell apoptosis. Further, calpain inhibition led to the arrest of tumor angiogenesis and migration in vitro. Finally, the molecular investigation revealed a downregulation of proliferative signalling as MAPK and anti-apoptotic regulators as Bcl-2, as well as an upregulation of proapoptotic mediators, as Caspases and Bax-family. From this evidence, calpains may be considered as novel potential therapeutic targets to treat cancer and to limit its progression. Overall, the discovery of novel potential prognostic and predictive biomarkers, by the “omics” approach, will allow to optimize patient’s management, as early prediction of those patients who are likely to be STS or LTS, and a full understanding of their clinical course can thus assist clinicians in providing tailored treatments and support patients and their families. However, beyond prognosis, what will really impact clinical management of GBM is if genomic analyses can lead to customized treatment and ultimately improved survival, following the concept of precision medicine.

DEVELOPMENT AND VALIDATION OF TARGET THERAPIES FOR PATIENTS WITH BRAIN CANCER, THROUGH THE MODULATION OF ANGIOGENESIS, INVASIVENESS, AND PHARMACOLOGICAL SENSITIVITY/RESISTANCE, IN THE ERA OF PRECISION MEDICINE / L. Guarnaccia ; supervisor: G. Mantovani ; co-supervisor: G. Marfia, S. E. Navone ; PhD coordinator: C. Sforza. - : . Dipartimento di Scienze Cliniche e di Comunità, 2022 Jan 14. ((34. ciclo, Anno Accademico 2021.

DEVELOPMENT AND VALIDATION OF TARGET THERAPIES FOR PATIENTS WITH BRAIN CANCER, THROUGH THE MODULATION OF ANGIOGENESIS, INVASIVENESS, AND PHARMACOLOGICAL SENSITIVITY/RESISTANCE, IN THE ERA OF PRECISION MEDICINE

L. Guarnaccia
2022-01-14

Abstract

Brain tumors represent a group of heterogeneous neoplasms which, despite originating in the same anatomical region, differ in morphology, etiology, molecular biology, and especially in clinical behavior. It has been estimated that brain tumors affect about 200,000 people worldwide every year, representing approximately 2% of cancer deaths. Among all primary human brain tumors, glioblastoma (GBM) is the most malignant and frequent (~70%), with a median survival of about 14 months and a 5-year survival rate at 5%, thus representing an extreme therapeutic challenge. GBM is characterized by sustained proliferation and survival, immune system escape, intense angiogenesis, invasion, cell infiltration, rapid progression, resistance to radio- and chemotherapies, with high frequency of relapse. In cancer pathogenesis, particularly in high grade tumors as GBM, aberrant neo-angiogenesis is a vital process for the mass growth: it is driven by neoplastic cells in order to respond to the tumoral hypoxic environment in the necrotic core, which increases the demand for oxygen and nutrients by neoplastic cells, and is, therefore, essential to carry out the metabolic functions on which their survival is based. Several observations led to the knowledge that tumoral neo-angiogenesis gives rise to ultra-structurally abnormal vessels, larger than their normal counterparts, dilated, convoluted, irregularly branched and exceptionally permeable due to the presence of fenestrations and the lack of a complete basal membrane. Due to these destructive features, despite aggressive therapeutic treatment, consisting in surgical resection followed by chemotherapy with temozolomide (TMZ), most patients experience tumor recurrence in less than one year, suggesting the urgent need to implement clinical practice with novel prognostic and therapeutic strategies. To this aim, this research project has been focused on: i) aberrant angiogenesis mediated by endothelial cells (ECs), which promote tumor infiltration into surrounding tissues, with consequent compromission of cognitive skills, and ii) genetic instability, which characterize GBM with a high intra- and intertumoral heterogeneity. In particular, the underlying hypothesis is that a specific molecular signature characterizes “resistant” and “sensitive” GBM, and it may be responsible for patient overall survival. Indeed, although GBMs from short- and long-term survivors (STS and LTS) are histologically the same, their biological and molecular characteristics are remarkably different, suggesting that factors that contribute to patients’ longevity are important for precise diagnosis and correct clinical management of the disease. The genetic profiling obtained by array CGH on STS and LTS revealed a high number of copy number variation (CNVs) across chromosomes 1 to 22, among which several novel potential prognostic and predictive biomarkers have been described and discussed. In particular, aCGH highlighted the presence of an altered chromosomic pattern relative to calpain family genes. Calpains are a conserved family of cysteine proteinases that catalyze the controlled proteolysis of many specific substrates. Calpain activity is implicated in several 5 fundamental physiological processes, including cytoskeletal remodeling, cellular signalling, apoptosis and cell survival. Alterations of the calpain activity balance has been observed in numerous cancer types, as they can reduce apoptosis, increase cell proliferation and stimulate cell migration and invasiveness. The characterization of calpain expression in primary GBM endothelial cells (GECs) showed an upregulation of calpains and a positive correlation between expression level and patient survival. The blockade of calpain activation with natural and synthetic inhibitors resulted in the inhibition of GEC viability and proliferation, as well as in the promotion of caspase-induced cell apoptosis. Further, calpain inhibition led to the arrest of tumor angiogenesis and migration in vitro. Finally, the molecular investigation revealed a downregulation of proliferative signalling as MAPK and anti-apoptotic regulators as Bcl-2, as well as an upregulation of proapoptotic mediators, as Caspases and Bax-family. From this evidence, calpains may be considered as novel potential therapeutic targets to treat cancer and to limit its progression. Overall, the discovery of novel potential prognostic and predictive biomarkers, by the “omics” approach, will allow to optimize patient’s management, as early prediction of those patients who are likely to be STS or LTS, and a full understanding of their clinical course can thus assist clinicians in providing tailored treatments and support patients and their families. However, beyond prognosis, what will really impact clinical management of GBM is if genomic analyses can lead to customized treatment and ultimately improved survival, following the concept of precision medicine.
MANTOVANI, GIOVANNA
MARFIA, GIOVANNI
NAVONE, STEFANIA
SFORZA, CHIARELLA
brain tumors ; glioblastoma; angiogenesis; personalized medicine; array-CGH; calpains
Settore BIO/11 - Biologia Molecolare
DEVELOPMENT AND VALIDATION OF TARGET THERAPIES FOR PATIENTS WITH BRAIN CANCER, THROUGH THE MODULATION OF ANGIOGENESIS, INVASIVENESS, AND PHARMACOLOGICAL SENSITIVITY/RESISTANCE, IN THE ERA OF PRECISION MEDICINE / L. Guarnaccia ; supervisor: G. Mantovani ; co-supervisor: G. Marfia, S. E. Navone ; PhD coordinator: C. Sforza. - : . Dipartimento di Scienze Cliniche e di Comunità, 2022 Jan 14. ((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/2434/888881
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