Targeting the FOXM1 signature for early diagnosis and treatment in cholangiocarcinoma

Cholangiocarcinoma (CCA) is rare cancer of the intrahepatic and extra hepatic bile ducts. High incidences of CCA are clustered in the Greater Mekong sub-region, particularly in the northeast of Thailand. Relatively little is known about the cause of CCA, and the treatment options are limited because of late diagnosis. Researching for early diagnosis and effective treatment is an urgent need to prevent the loss from this cancer. Accumulating evidence indicates that the Forkhead box M1 (FOXM1) transcription factor has a key role in tumour initiation, progression, metastasis, angiogenesis and drug sensitivity. A previous gene array study in Thai CCA patient tissues has revealed that FOXM1 and its downstream targets as genes upregulated in almost all CCA cases. We hypothesise that FOXM1 plays a key role in CCA initiation, development and drug resistance. We aim to identify the critical FOXM1 gene transcription signatures involved in CCA tumorigenesis, progression and drug sensitivity. This will not only help to identify reliable biomarkers for early diagnosis and for predicting disease relapse but also aid the design of targeted therapies and strategies to overcome drug resistance in CCA. This joint project will also strengthen the collaborations between ICL and KKU, and promote the career development of junior researchers from both institutes.

We will study the regulation and role of FOXM1 and its downstream targets in the cholagiocarcinoma (CCA) initiation, progression and drug resistance using the zebrafish, mouse and human in vivo models as well as CCA cell lines. This study will identify key FOXM1 expression signatures essential for CCA tumorigenesis, metastasis, angiogenesis and the development of drug resistance. The proposed project consists of 4 overlapping objectives (1-4).
In objective 1, we will identify and characterize the putative FOXM1 signature genes in CCA cell lines using proliferation, clonogenic, Western blot and RT-qPCR analyses. We will then confirm these putative genes as FOXM1 downstream targets in CCA and in response to chemotherapy treatment using siRNA to selectively silence FOXM1 in drug sensitive and resistant CCA lines cells. We will also confirm these results and uncover novel FOXM1 targets by studying the mRNA and miRNA expression using microarrays.
In objective 2, the role and regulation of FOXM1 and its targets during cholangiocarcinogenesis will be confirmed in an in vivo model of liver fluke infection by immunohistochemistry staining. The role and expression of the FOXM1 signatures in CCA angiogenesis and metastasis will be examined in in vivo systems, including a novel zebrafish embryo model. The function of the FOXM1 signature in drug resistance will also be tested in CCA cell lines.
In objective 3, we will attempt to identify the components of the FOXM1 gene signature/signalling network that can be targeted to enhance cell death and senescence and to overcome drug resistance using CCA cell lines.
In objective 4, the findings from the in vitro and in vivo studies will then be validated samples from CCA patients.
It is anticipated these studies will identify key FOXM1 gene expression signatures that are important for CCA initiation, progression and drug resistance and help to uncover crucial early diagnostic markers and therapeutic targets for CCA.

FOXM1 has been implicated in cholagiocarcinoma (CCA) initiation, progression and the development of chemotherapy resistance. This study will unveil FOXM1 downstream expression signatures and help to identify crucial prognostic markers and therapeutic targets for CCA. This project will directly benefit the CCA patients and the communities that are at risk of contracting liver fluke and CCA. This project will improve CCA survival and help to relieve the health, economic and societal burden of the CCA affected communities as well as the Thai population. In addition, this project will also generate useful hypothesis, reagents, and techniques relevant for CCA researchers. A wider circle of researchers who are investigating the function and regulation of forkhead transcription factors and the potent oncogenic transcription factor FOXM1 will gain from the knowledge and information generated from this project.
The proposed work on CCA metastasis and angiogenesis using < 5 day zebrafish embryos will aid the development of new 3Rs approaches and technologies that facilitates the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). Particularly for scientists researching into angiogenesis and metastasis, this project will help to establish novel in vivo models, which are more economical and ethical, with a very short generation time.
This study and its finding will benefit the KKU Co-Is, who are integral members of the Liver Fluke and CCA Research Centre, whose aims are: 1) to research into the prevention, diagnosis and treatment of CAA; 2) to facilitate local authority in district and local communities in education for prevention of liver fluke infection and CCA; 3) to build CCA research capability. As part of this programme, junior researchers from KKU will also gain from research visits and placements at ICL. They will be exposed to cutting edge technology and stimulating research environment at ICL. They will develop expertise in cancer and molecular biology as well as communication skills and research networks, which will aid their career development. High impact 'open access' publications from the joint project will also help to attract and recruit future scientists and collaborators to KKU and the Liver Fluke and CCA Research Centre to ensure that their future sustainable research capability.