Colorectal cancer (CRC) is one of the most prevalent and incident cancers worldwide and it has become soon an important public health issue. Although surgery represents the principal curative treatment, chemotherapy is one of the most important tools currently available for the treatment of CRC. 5-fluorouracil (5FU) is the chemotherapeutic agent of election for CRC treatment. Two main problems affect the outcome of cancer chemotherapy: the use of poorly specific drugs and, in a high percentage of patients, the lack of response due to drug resistance, seen as a major obstacle to improve the overall response and survival of cancer patients, limiting the effectiveness of chemotherapy. GSK-3β and TGF-β are known to be master regulators of a plethora of signalling pathways in various mechanisms involved in cancer development, resistance and dissemination. In this thesis is shown that inhibition of GSK-3β (mediated by shRNA or Lithium) is able to revert chemoresistance to 5-FU in HCT116 P53-/- cells (a colon cancer cell line resistant to chemotherapy because of P53 deletion), both in vitro and in vivo through a heterotopic xenograft model. We started from this model of restoration of chemosensitivity to analyze some major features of the TGF-β pathway. Microvasculature analysis on xenografted tumor sections, revealed a dramatic increase of tumor vascularization in consequence of 5FU administration whereas Lithium and 5FU combination was able to significantly decrease the vasculature density, restoring the basal value. Moreover, 5FU is able to stimulate nuclear translocation of SMAD3 and the transcription of specific genes such as ACVRL1,FN1 and TGFB1. Contrarily, the specific inhibition of TGF-βRI, not only is able to inhibit the 5FU-induced genes transcription, but also restores the sensitivity of chemoresistant cells to the action of chemotherapeutic, causing the repression of BCL2L1 and ID1 genes. Chemoresistant cells behavior was consistent to a sort of autocrine protective loop acted by TGFB1 in consequence of 5FU administration. Moreover, in this work is presented a bioinformatics study on colorectal cancer patients. Patients enrolled in the study underwent preoperative chemoradiotherapy, followed by surgical excision. By determining gene expression profiles, responders and non-responders showed significantly different expression levels for 19 genes (P < 0.001). We fitted a logistic model selected with a stepwise procedure optimizing the Akaike Information Criterion (AIC) and then validated by means of leave one out cross validation (LOOCV, accuracy D 95%). Four genes were retained in the achieved model: ZNF160, XRCC3, HFM1 and ASXL2. Real time PCR confirmed that XRCC3 is overexpressed in responders group. In vitro test on colon cancer resistant/susceptible to chemoradioterapy cells, finally prove that XRCC3 deregulation is extensively involved in the chemoresistance mechanisms. Protein-protein interactions (PPI) analysis involving the predictive classifier revealed a network of 45 interacting nodes (proteins) with TRAF6 gene playing a keystone role in the network. In this thesis is also presented a work on Glioblastoma multiforme (GBM) drug resistance. GBM is one of the most fatal and least successfully treated solid tumors: current therapies provide a median survival of 12-15 months after diagnosis, due to the high recurrence rate. Glioma Stem Cells (GSCs) are believed to be the real driving force of tumor initiation, progression and relapse. Therefore, better therapeutic strategies GSC-targeted are needed. Resveratrol (RSV) is a polyphenolic phytoalexin found in fruits and vegetables displaying pleiotropic health benefits. Results showed that response to RSV exposure was highly heterogeneous among GSC lines, but generally it was able to inhibit cell proliferation, increasing cell mortality, and strongly decrease cell motility, modulating the Wnt signaling pathway.

(2015). Molecular mechanisms of cancer drug resistance. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

Molecular mechanisms of cancer drug resistance

ROMANO, GABRIELE
2015

Abstract

Colorectal cancer (CRC) is one of the most prevalent and incident cancers worldwide and it has become soon an important public health issue. Although surgery represents the principal curative treatment, chemotherapy is one of the most important tools currently available for the treatment of CRC. 5-fluorouracil (5FU) is the chemotherapeutic agent of election for CRC treatment. Two main problems affect the outcome of cancer chemotherapy: the use of poorly specific drugs and, in a high percentage of patients, the lack of response due to drug resistance, seen as a major obstacle to improve the overall response and survival of cancer patients, limiting the effectiveness of chemotherapy. GSK-3β and TGF-β are known to be master regulators of a plethora of signalling pathways in various mechanisms involved in cancer development, resistance and dissemination. In this thesis is shown that inhibition of GSK-3β (mediated by shRNA or Lithium) is able to revert chemoresistance to 5-FU in HCT116 P53-/- cells (a colon cancer cell line resistant to chemotherapy because of P53 deletion), both in vitro and in vivo through a heterotopic xenograft model. We started from this model of restoration of chemosensitivity to analyze some major features of the TGF-β pathway. Microvasculature analysis on xenografted tumor sections, revealed a dramatic increase of tumor vascularization in consequence of 5FU administration whereas Lithium and 5FU combination was able to significantly decrease the vasculature density, restoring the basal value. Moreover, 5FU is able to stimulate nuclear translocation of SMAD3 and the transcription of specific genes such as ACVRL1,FN1 and TGFB1. Contrarily, the specific inhibition of TGF-βRI, not only is able to inhibit the 5FU-induced genes transcription, but also restores the sensitivity of chemoresistant cells to the action of chemotherapeutic, causing the repression of BCL2L1 and ID1 genes. Chemoresistant cells behavior was consistent to a sort of autocrine protective loop acted by TGFB1 in consequence of 5FU administration. Moreover, in this work is presented a bioinformatics study on colorectal cancer patients. Patients enrolled in the study underwent preoperative chemoradiotherapy, followed by surgical excision. By determining gene expression profiles, responders and non-responders showed significantly different expression levels for 19 genes (P < 0.001). We fitted a logistic model selected with a stepwise procedure optimizing the Akaike Information Criterion (AIC) and then validated by means of leave one out cross validation (LOOCV, accuracy D 95%). Four genes were retained in the achieved model: ZNF160, XRCC3, HFM1 and ASXL2. Real time PCR confirmed that XRCC3 is overexpressed in responders group. In vitro test on colon cancer resistant/susceptible to chemoradioterapy cells, finally prove that XRCC3 deregulation is extensively involved in the chemoresistance mechanisms. Protein-protein interactions (PPI) analysis involving the predictive classifier revealed a network of 45 interacting nodes (proteins) with TRAF6 gene playing a keystone role in the network. In this thesis is also presented a work on Glioblastoma multiforme (GBM) drug resistance. GBM is one of the most fatal and least successfully treated solid tumors: current therapies provide a median survival of 12-15 months after diagnosis, due to the high recurrence rate. Glioma Stem Cells (GSCs) are believed to be the real driving force of tumor initiation, progression and relapse. Therefore, better therapeutic strategies GSC-targeted are needed. Resveratrol (RSV) is a polyphenolic phytoalexin found in fruits and vegetables displaying pleiotropic health benefits. Results showed that response to RSV exposure was highly heterogeneous among GSC lines, but generally it was able to inhibit cell proliferation, increasing cell mortality, and strongly decrease cell motility, modulating the Wnt signaling pathway.
GIOVANNONI, ROBERTO
Chemoresistance; colon cancer; glioblastoma
MED/04 - PATOLOGIA GENERALE
English
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
28
2014/2015
(2015). Molecular mechanisms of cancer drug resistance. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/93577
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