Lorenzi, Federica
(2017)
Cellular & molecular mechanisms of drug resistance in cancer cells mutated for FBXW7 tumour suppressor gene.
PhD thesis, University of Nottingham.
Abstract
Common anticancer strategies often fail resulting in tumour recurrences and metastasis that cause the majority of cancer-related deaths. Only about 6% of colorectal cancer (CRC) metastatic patients live more than 5 years after diagnosis. Thus, elucidating the crucial mechanisms underlying drug resistance and metastasis is an urgent need in cancer biology and therapy.
The tumour suppressor gene FBXW7 (also called hCDC4, FBW7, Sel10, Ago) is one of the most frequently mutated genes in CRC. FBXW7, as a receptor subunit of the SCF-E3 ligase complex, drives the ubiquitination and degradation of many oncogenic proteins influencing cell proliferation, differentiation, senescence and other crucial pathways. Our group and others recently demonstrated that murine intestinal Fbxw7 loss of function induced tumorigenic activity, however its exact position in the multistep model of colorectal carcinogenesis remains to be clarified.
More recent data from Dr Nateri’s laboratory (Li et al., under review) suggested that ZEB2, transcriptional inducer of epithelial-mesenchymal transition (EMT), represents a specific target of FBXW7 and FBXW7 loss induces an EMT-like phenotype. We have therefore proposed that FBXW7 suppression may confer resistance to chemotherapy, stem-like cell properties and invasive and metastatic activity via EMT, following accumulation of ZEB2. To test our hypothesis, we investigated FBXW7 role in the occurrence of drug resistance in human CRC cells and three-dimensional murine organoids, after treatment with 5-fluorouracil (5-FU) and oxaliplatin, DNA damaging chemotherapeutic agents. We explored EMT activation due to loss of FBXW7 by examining epithelial and mesenchymal markers in response to drug treatment as well as stem-like cell properties by colonosphere formation assay. EMT-associated properties were also analysed following stable down-regulation of ZEB2 in CRC cells and organoids. Moreover, to explore a putative involvement of FBXW7 through accumulation of ZEB2 in invasive and metastatic potential, we performed in vivo xenograft analyses by intra-splenic (for liver and lymph node metastasis) and intravenous (migration/invasion in lungs) injection of human CRC cells deleted for FBXW7 and stably down-regulating ZEB2.
Herein, we show that CRC cell lines and murine organoids carrying deletion of FBXW7 are significantly less sensitive to 5-FU and oxaliplatin treatments. FBXW7 loss of function by accumulation of ZEB2 confers EMT phenotype to CRC cells including reduction of E-cadherin, up-regulation of Vimentin, stem-like properties and induction of invasive and metastatic activity. This suggest that FBXW7-deleted CRC cells may represent cancer stem cells.
Moreover, this study also aimed to employ and establish an innovative in vitro model to evaluate the cell type-specific responses of gut epithelium to chemotherapeutic agents and how these responses are influenced by FBXW7. Chemotherapeutic sensitivity of the different cell types in normal gut and tumour tissues is poorly understood, impeding the development of improved targeted therapies. We have therefore established the organoid culture which mimics the structure of both gut and colorectal tumours comprising all the cell types likewise the human in vivo situation. To further investigate the mechanism(s) of action of Fbxw7, we grew organoids from isolated small intestinal crypts of mice in which fbxw7 gene is conditionally knocked-out in the intestine (fbxw7∆G), in parallel with wildtype floxed-fbxw7 (fbxw7fl/fl) organoids. The fbxw7∆G organoids exhibited rapid budding events in the crypt region. To test organoids for drug response, we exposed organoids from fbxw7fl/fl and fbxw7∆G mice to various concentrations of 5-FU for 72 hours. 5-FU triggers phenotypic differences in organoids including changing shape, survival, resistance, and death. 5-FU, however, rescues the drug resistance phenotype of fbxw7∆G through the induction of terminal differentiation. Our results support the hypothesis that a differentiating therapy successfully may target FBXW7-mutated CRC cells.
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