SIP1/ZEB2 induced epthelial to mesenchymal transition promotes metastasis and chemoresistance in colorectal cancer

Sreekumar, Rahul (2017) SIP1/ZEB2 induced epthelial to mesenchymal transition promotes metastasis and chemoresistance in colorectal cancer. University of Southampton, Doctoral Thesis, 228pp.

Record type: Thesis (Doctoral)

Abstract

Colorectal cancer (CRC) is the second commonest cause of cancer-associated mortality in Europe, and a key public health issue. Cancer metastasis is the principle cause of death and occurs in up to 30% at presentation, and subsequently develops in 50% after curative surgery. The majority of patients with metastases are incurable, and can expect a median survival of only up to 2 years, even with the latest chemotherapeutic and biological agents. Additionally, not all patients respond and side effects are frequent and at times life threatening. These findings highlight the pressing need for identification of new markers of metastatic capability and chemotherapy response, to improve precision with which therapy can be tailor to patients. Although development of primary CRC has served as a paradigm for understanding multistage carcinogenesis, the mechanisms influencing metastasis and chemoresistance are still poorly understood.

Epithelial-Mesenchymal Transition (EMT) is an embryologically conserved genetic program by which cancer cells down regulate epithelial junctions, express mesenchymal markers, and manifest a migratory phenotype. While the significance of EMT during development and embryogenesis is well established, an emerging role is its involvement in metastasis and chemo/radio resistance in cancer. EMT is activated by TGFβ, FGF, EGF, WNT and Notch signalling pathways, which converge to activate transcription factors that subsequently repress the expression of critical epithelial genes. Key transcription factors in this process include members of the SNAIL, Twist, and ZEB families, which promote cellular phenotypic switch. In addition to enhanced migration, metastatic cells also acquire apoptosis resistance to chemo/radio therapy through currently poorly understood mechanism. Despite growing evidence that EMT promotes apoptosis resistance to DNA damaging agents, ZEB family of transcription factors have been sparsely studied in gastrointestinal malignancies and the molecular mechanism mediating apoptosis resistance poorly understood.
Based on these observations the following hypothesis was formulated:

• SMAD interacting protein (SIP1/ZEB2) induced EMT promotes metastasis and apoptosis resistance in colorectal cancer (CRC).

The primary objectives of the study are: -

1. Assess if SIP1/ZEB2 induces EMT in CRC.

2. Investigate whether expression of SIP1/ZEB2 could serve as a biomarker to detect patients at high risk or recurrence after surgical resection in CRC.

3. Study the molecular mechanisms that promote SIP1/ZEB2 induced apoptosis resistance to chemotherapeutic and radiotherapeutic treatment regimens.

4. Validate in-vitro findings in a murine model SIP1/ZEB2 expression resulted in the acquisition of all the cardinal features of EMT, namely E-cadherin down regulation, increased metastatic capacity and apoptosis resistance to chemotherapeutic agents commonly using in clinical practice.

SIP/ZEB2 expression in primary CRC, exhibited a statistically significant association with increased risk of distant recurrence in two independent patient cohorts. Addition of SIP1/ZEB2 expression status to the TNM staging system improved precision in the ability to identify patients at high risk of disease recurrence after curative surgery. Further studied also highlighted an important association between SIP1/ZEB2 expression and chemoresistance to cytotoxic drugs used to in the FOLFOX regimen. A qPCR array, with a focus on DNA damage response highlighted SIP1/ZEB2 induced EMT associated with increased expression of multiple components of the nucleotide excision repair (NER) pathway, in particular excision repair cross complementation group 1 (ERCC1).

ERCC1 hetero-dimerises with excision repair cross complementation group 4, which functions as an exonuclease during repair of DNA crosslinks generated by platinum based chemotherapeutic agents such as oxaliplatin. Stable over expression of ERCC1; lead to attenuate DNA damage, apoptosis resistance and enhanced viability. Whilst siRNA mediated knockdown (KD) sensitise cells to oxaliplatin treatment. Assessment of DNA repair kinetics, as a mechanism of repair kinetics revealed higher expression levels of ERCC1 associated with faster kinetics of DNA cross-link clearance. The influence of ERCC1 over expression in vivo was demonstrated by impaired tumour regression in ERCC1 over-expressing cells in an orthotopic murine model of primary CRC.

ZEB proteins have also been implicated with the enhanced ability to repair DNA DSB’s and consequently promote resistance to ionising radiation. For many decades, models of DNA DSB repair have highlighted the critical influence of the histone architecture in accessing damaged DNA and subsequently undertaking DNA repair. Heterochromatin rich DNA domains are known to be prone to accruing mutations, due to attenuated DNA repair. Recent studies have suggested EMT leads to epigenetic reprograming, which results in genome wide loss of heterochromatin rich domains. SIP1/ZEB2 expression in DLD-SIP1 cells enhanced apoptosis resistance secondary to faster repair of DNA DSB’s. ChIP-Seq analysis of SIP1/ZEB2 expressing mesenchymal cells highlighted genome wide loss of heterochromatin mark H3K27me3. The mechanism responsible for this epigenetic change was found to be direct transcriptional repression of the methyltransferase EZH2, by SIP1/ZEB2. Inhibition of EZH2 by small molecule inhibitor GSK126 in uninduced DLDSIP1 cells enhanced apoptosis resistance and viability in response to IR. The above results suggest the epigenetic architecture of mesenchymal cancer can influence DNA repair kinetics and consequently resistance to IR. The above body of work clearly demonstrates SIP1/ZEB2 plays a central role in promoting metastasis and treatment resistance in CRC. Further in vitro studies and clinical trials to dissect the impact of SIP1/ZEB2 expression in CRC will facilitate clinical translation in future years.