Epithelial de-differentiation triggered by co-ordinate epigenetic inactivation of the EHF and CDX1 transcription factors drives colorectal cancer progression

Epigenetics; Tumour-suppressor proteinsEpigenética; Proteínas supresoras de tumoresEpigenètica; Proteïnes supresores de tumorsColorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC.This project was supported by NHMRC project grant (1107831), a Cancer Council Victoria Grant (1164674) and the Operational Infrastructure Support Programme, Victorian Government, Australia. JMM was supported by a NHMRC Senior Research Fellowship (1046092). IYL was supported by F J Fletcher Research Scholarship and Randal and Louisa Alcock Scholarship from the University of Melbourne. LJJ was supported by La Trobe University Australian Postgraduate Awards. IN was supported by La Trobe University Postgraduate Research Scholarship. JWTT was supported by the University of Melbourne Australian Postgraduate Awards. OMS is a National Health and Medical Research Council (NHMRC) Senior Research Fellow (APP1136119). Open Access funding enabled and organized by CAUL and its Member Institutions

Investigating the mechanisms by which histone deacetylase inhibitors induce apoptosis in cancer cells

© 2017 Dr. Janson W. T. TseCancer develops as a multi-step process through the accumulation of abnormal genetic alterations in tumour suppressor genes and oncogenes. Superimposed upon these genetic changes are changes in the epigenome which work together to induce the hallmarks of cancer. Histone deacetylase inhibitors (HDACi) are a class of epigenetic therapeutics approved for the treatment of cutaneous T-cell lymphoma (CTCL). These agents induce anti-tumour activity in a variety of ways, including inhibition of cell proliferation and induction of autophagy, differentiation and apoptosis. The mechanism by which HDACi induce apoptosis has been extensively investigated and shown to involve induction of a pro-apoptotic gene signature encompassing the up-regulation of pro-apoptotic genes such as BIM, BAX and BAK and the repression of anti-apoptotic genes such as BCL-2 and BCL-XL. However, the specific molecular mechanisms by which this pro-apoptotic signature is induced have yet to be clearly identified. Recent reports have demonstrated that HDACi-induced apoptosis is associated with upregulation of the AP-1 complex genes c-FOS, c-JUN and ATF3. The objective of this thesis was to determine whether induction of c-FOS, c-JUN or ATF3 is directly required for HDACi-induced apoptosis, and elucidate whether these changes in turn drive altered expression of the pro-apoptotic gene signature. The expression of cFOS, c-JUN and ATF3 expression was found to be robustly and selectively induced upon HDACi treatment in HDACi-sensitive tumour cell lines. These effects transcended tumour type and included melanoma, colorectal, breast, lung, gastric and haematological cell lines. Through systematic knockdown experiments, induction of ATF3 but not c-FOS or cJUN was found to be a functional driver of HDACi-induced apoptosis. This was demonstrated in HDACi-sensitive lung, colorectal and gastric cancer cell lines. These results were further confirmed using ATF3-/- MEFs which were significantly less sensitive to HDACi-induced apoptosis compared to wild-type MEFs. As HDACi treatment alters the expression of pro- and anti-apoptotic genes, we also determined the role of ATF3-induction in mediating these changes. Correlation of HDACi-induced ATF3 expression with the altered expression of intrinsic apoptotic members across 15 different cancer cell lines, revealed an inverse correlation between the magnitude of ATF3 induction and the repression of expression of the pro-survival gene BCL-XL. Furthermore, we demonstrated that ATF3 induction is directly required for HDACi-mediated repression of BCL-XL. A central role for repression of BCL-XL in HDACi-induced apoptosis was demonstrated in knockdown studies whereby siRNA-mediated silencing of BCL-XL was able to re-sensitise refractory cell lines to HDACi-induced apoptosis. Similarly, BH3 mimetics and BCL-XL-specific inhibitors could also re-sensitise refractory cell lines to HDACi-induced apoptosis both in vitro and in xenograft models in vivo. In addition to our finding that ATF3 induction is required for HDACI-induced apoptosis, it has previously been reported that proteasome inhibitor treatment can also induce ATF3 expression. Furthermore, the combination of HDACi and proteasome inhibitors has recently been approved for the treatment of multiple myeloma, although the mechanistic basis for this effect is unclear. We therefore postulated that additive or synergistic induction of ATF3 may underpin this effect. This thesis demonstrates that proteasome inhibitors robustly induce ATF3 in both colorectal cancer and multiple myeloma cell lines, and ATF3 induction is further enhanced by combination treatment with HDACi. We also demonstrate that these agents induce ATF3 through independent mechanisms, and that the combination treatment synergistically enhances apoptosis in these cell lines. Notably, knockdown of ATF3 attenuated the apoptotic response induced by the combination establishing ATF3 as a central component of the apoptotic response. Collectively, these findings demonstrate that HDACi-induced apoptosis is driven by ATF3 induction and subsequent repression of BCL-XL. We also demonstrate that combination treatment with a BCL-XL inhibitor can overcome inherent resistance to HDACi. Additionally, we demonstrate combination treatment with HDACi and proteasome inhibitors synergistically enhances apoptosis through additive induction of ATF3. These studies provide novel insight into the basis for differential response of cell lines to single agent HDACi therapy, and identify avenues for enhancing the activity of HDACi through rationally developed drug combinations