Targeting the FOXM1 signature for early diagnosis and treatment in cholangiocarcinoma

Lead Research Organisation: Imperial College London
Department Name: Dept of Surgery and Cancer

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Alasiri G (2018) ER stress and cancer: The FOXO forkhead transcription factor link. in Molecular and cellular endocrinology

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Gomes AR (2016) Sirtuin1 (SIRT1) in the Acetylation of Downstream Target Proteins. in Methods in molecular biology (Clifton, N.J.)

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Gong C (2018) BQ323636.1, a Novel Splice Variant to 2, as a Predictor for Tamoxifen-Resistant Breast Cancer. in Clinical cancer research : an official journal of the American Association for Cancer Research

 
Description Fluorouracil (5-FU) is the first-line chemotherapeutic drug for cholangiocarcinoma (CCA), but its efficacy has been compromised by the development of resistance.
Development of 5-FU resistance is associated with elevated expression of its cellular target, thymidylate synthase (TYMS).
Our findings suggest that the FOXM1-TYMS axis can be a novel diagnostic, predictive and prognostic marker as well as a therapeutic target for cholangiocarcinoma (CCA).
Exploitation Route We have developed strong and long-lasting collaboration with KKU and other Thai research groups, whom we will export future finding opportunities.
Sectors Education,Environment,Healthcare

 
Description CRUK Centre MultiDisciplinary Grants
Amount £29,900 (GBP)
Organisation Cancer Research UK 
Department CRUK Centre for Cancer Therapeutics
Sector Private
Country United Kingdom
Start 12/2016 
End 04/2017
 
Description Institute for Molecular Science & Engineering Seed Funding
Amount £24,837 (GBP)
Funding ID EP/P51116X/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 03/2017
 
Description PhD Studentship Grant (Hibah Alsadah)
Amount £75,000 (GBP)
Organisation Royal Embassy of Saudi Arabia 
Sector Public
Country United States
Start 03/2017 
End 02/2020
 
Description PhD studentship Grant (Glowi Alasiri)
Amount £84,000 (GBP)
Organisation Royal Embassy of Saudi Arabia 
Sector Public
Country United States
Start 01/2016 
 
Title Study the role of FOXM1 in CCA angiogenesis and metastasis 
Description Established a CCA xenograft system in zebrafish embryo model. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2017 
Provided To Others? Yes  
Impact Hitherto, the work on cancer metastasis and angiogenesis has relied primarily on in vivo animal models. 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). This approach avoids the use of animals defined as 'protected' under the Animals (Scientific Procedures) Act 1986, amended 2012 (ASPA) in research and also improves animal welfare by minimizing pain, and in this way, addresses societal concerns about animal research. The '3Rs' is one of the strategic aims of MRC and other research councils. The information on the zebrafish work will be disseminated through publications in high impact Journals, MRC and the public media (after liasing with ICL and MRC), scientific meetings and public engagement workshops. This will help to change future research models for angiogenesis and metastasis. 
 
Description Collaboration between Prof Lam's and Prof Quentin Liu's Groups on FOXM1, Cancer Stem Cells and drug resistance 
Organisation Dalian Medical University
Country China 
Sector Hospitals 
PI Contribution Prof Lam's group supply expertise, reagents, and researchers in studying FOXM1, Cancer Stem Cells and drug resistance
Collaborator Contribution Prof Quentin Liu's group supply expertise, reagents, and researchers in studying FCancer Stem Cells and drug resistance
Impact Results in publication of over 12 high impact papers1: Peng F, Wang JH, Fan WJ, Meng YT, Li MM, Li TT, Cui B, Wang HF, Zhao Y, An F, Guo T, Liu XF, Zhang L, Lv L, Lv DK, Xu LZ, Xie JJ, Lin WX, Lam EW, Xu J, Liu Q. Glycolysis gatekeeper PDK1 reprograms breast cancer stem cells under hypoxia. Oncogene. 2018 Feb 22;37(8):1119. doi: 10.1038/onc.2017.407. Epub 2017 Dec 18. PubMed PMID: 29251717. 2: Peng F, Wang JH, Fan WJ, Meng YT, Li MM, Li TT, Cui B, Wang HF, Zhao Y, An F, Guo T, Liu XF, Zhang L, Lv L, Lv DK, Xu LZ, Xie JJ, Lin WX, Lam EW, Xu J, Liu Q. Glycolysis gatekeeper PDK1 reprograms breast cancer stem cells under hypoxia. Oncogene. 2018 Feb 22;37(8):1062-1074. doi: 10.1038/onc.2017.368. Epub 2017 Nov 6. Erratum in: Oncogene. 2017 Dec 18;:. PubMed PMID: 29106390. 3: Li SS, Xu LZ, Zhou W, Yao S, Wang CL, Xia JL, Wang HF, Kamran M, Xue XY, Dong L, Wang J, Ding XD, Bella L, Bugeon L, Xu J, Zheng FM, Dallman MJ, Lam EWF, Liu Q. p62/SQSTM1 interacts with vimentin to enhance breast cancer metastasis. Carcinogenesis. 2017 Oct 26;38(11):1092-1103. doi: 10.1093/carcin/bgx099. PubMed PMID: 28968743. 4: Lyu Y, Lou J, Yang Y, Feng J, Hao Y, Huang S, Yin L, Xu J, Huang D, Ma B, Zou D, Wang Y, Zhang Y, Zhang B, Chen P, Yu K, Lam EW, Wang X, Liu Q, Yan J, Jin B. Dysfunction of the WT1-MEG3 signaling promotes AML leukemogenesis via p53-dependent and -independent pathways. Leukemia. 2017 Dec;31(12):2543-2551. doi: 10.1038/leu.2017.116. Epub 2017 Apr 12. PubMed PMID: 28400619; PubMed Central PMCID: PMC5729340. 5: Kamran M, Long ZJ, Xu D, Lv SS, Liu B, Wang CL, Xu J, Lam EW, Liu Q. Aurora kinase A regulates Survivin stability through targeting FBXL7 in gastric cancer drug resistance and prognosis. Oncogenesis. 2017 Feb 20;6(2):e298. doi: 10.1038/oncsis.2016.80. PubMed PMID: 28218735; PubMed Central PMCID: PMC5337621. 6: Yang N, Wang C, Wang Z, Zona S, Lin SX, Wang X, Yan M, Zheng FM, Li SS, Xu B, Bella L, Yong JS, Lam EW, Liu Q. FOXM1 recruits nuclear Aurora kinase A to participate in a positive feedback loop essential for the self-renewal of breast cancer stem cells. Oncogene. 2017 Jun 15;36(24):3428-3440. doi: 10.1038/onc.2016.490. Epub 2017 Jan 23. PubMed PMID: 28114286; PubMed Central PMCID: PMC5485180. 7: Peng F, Li TT, Wang KL, Xiao GQ, Wang JH, Zhao HD, Kang ZJ, Fan WJ, Zhu LL, Li M, Cui B, Zheng FM, Wang HJ, Lam EW, Wang B, Xu J, Liu Q. H19/let-7/LIN28 reciprocal negative regulatory circuit promotes breast cancer stem cell maintenance. Cell Death Dis. 2017 Jan 19;8(1):e2569. doi: 10.1038/cddis.2016.438. PubMed PMID: 28102845; PubMed Central PMCID: PMC5386357. 8: Yan M, Wang C, He B, Yang M, Tong M, Long Z, Liu B, Peng F, Xu L, Zhang Y, Liang D, Lei H, Subrata S, Kelley KW, Lam EW, Jin B, Liu Q. Aurora-A Kinase: A Potent Oncogene and Target for Cancer Therapy. Med Res Rev. 2016 Nov;36(6):1036-1079. doi: 10.1002/med.21399. Epub 2016 Jul 13. Review. PubMed PMID: 27406026. 9: Xu LZ, Li SS, Zhou W, Kang ZJ, Zhang QX, Kamran M, Xu J, Liang DP, Wang CL, Hou ZJ, Wan XB, Wang HJ, Lam EW, Zhao ZW, Liu Q. p62/SQSTM1 enhances breast cancer stem-like properties by stabilizing MYC mRNA. Oncogene. 2017 Jan 19;36(3):304-317. doi: 10.1038/onc.2016.202. Epub 2016 Jun 27. PubMed PMID: 27345399; PubMed Central PMCID: PMC5269535. 10: Zheng F, Yue C, Li G, He B, Cheng W, Wang X, Yan M, Long Z, Qiu W, Yuan Z, Xu J, Liu B, Shi Q, Lam EW, Hung MC, Liu Q. Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype. Nat Commun. 2016 Jan 19;7:10180. doi: 10.1038/ncomms10180. PubMed PMID: 26782714; PubMed Central PMCID: PMC4735655. 11: Zheng FM, Long ZJ, Hou ZJ, Luo Y, Xu LZ, Xia JL, Lai XJ, Liu JW, Wang X, Kamran M, Yan M, Shao SJ, Lam EW, Wang SW, Lu G, Liu Q. A novel small molecule aurora kinase inhibitor attenuates breast tumor-initiating cells and overcomes drug resistance. Mol Cancer Ther. 2014 Aug;13(8):1991-2003. doi: 10.1158/1535-7163.MCT-13-1029. Epub 2014 Jun 4. PubMed PMID: 24899685. 12: Yan M, Zhang Y, He B, Xiang J, Wang ZF, Zheng FM, Xu J, Chen MY, Zhu YL, Wen HJ, Wan XB, Yue CF, Yang N, Zhang W, Zhang JL, Wang J, Wang Y, Li LH, Zeng YX, Lam EW, Hung MC, Liu Q. IKKa restoration via EZH2 suppression induces nasopharyngeal carcinoma differentiation. Nat Commun. 2014 Apr 17;5:3661. doi: 10.1038/ncomms4661. PubMed PMID: 24739462.
Start Year 2006
 
Description Collaboration with Dr Raimundo Freire 
Organisation University Hospital of the Canary Islands
Country Spain 
Sector Hospitals 
PI Contribution Study the role of OTUBs and E3 ligase in the control of FOXM1
Collaborator Contribution Binding assays with OTUBs, E3 ligases and FOXM1
Impact 4 publications 1. RNF168 cooperates with RNF8 to mediate FOXM1 ubiquitination and degradation in breast cancer epirubicin treatment. Kongsema M, Zona S, Karunarathna U, Cabrera E, Man EP, Yao S, Shibakawa A, Khoo US, Medema RH, Freire R, Lam EW. Oncogenesis. 2016 Aug 15;5(8):e252. doi: 10.1038/oncsis.2016.57. PMID: 27526106 Free PMC Article Similar articles Select item 26148240 2. OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance. Karunarathna U, Kongsema M, Zona S, Gong C, Cabrera E, Gomes AR, Man EP, Khongkow P, Tsang JW, Khoo US, Medema RH, Freire R, Lam EW. Oncogene. 2016 Mar 17;35(11):1433-44. doi: 10.1038/onc.2015.208. PMID: 26148240 Free PMC Article Similar articles Select item 24362530 3. SUMOylation inhibits FOXM1 activity and delays mitotic transition. Myatt SS, Kongsema M, Man CW, Kelly DJ, Gomes AR, Khongkow P, Karunarathna U, Zona S, Langer JK, Dunsby CW, Coombes RC, French PM, Brosens JJ, Lam EW. Oncogene. 2014 Aug 21;33(34):4316-29. doi: 10.1038/onc.2013.546. PMID: 24362530 Free PMC Article Similar articles Select item 24141789 4. FOXM1 targets NBS1 to regulate DNA damage-induced senescence and epirubicin resistance. Khongkow P, Karunarathna U, Khongkow M, Gong C, Gomes AR, Yagüe E, Monteiro LJ, Kongsema M, Zona S, Man EP, Tsang JW, Coombes RC, Wu KJ, Khoo US, Medema RH, Freire R, Lam EW. Oncogene. 2014 Aug 7;33(32):4144-55. doi: 10.1038/onc.2013.457. PMID: 24141789 Free PMC Article Similar articles
Start Year 2013
 
Description Functional Role if interaction between FOXO3 and cyclin D1 in senescence 
Organisation Siriraj Medical School
Country Thailand 
Sector Academic/University 
PI Contribution Reagents to study the interaction between FOXO3 and cyclin D1 in senescence
Collaborator Contribution PI. Interaction and functional experiments on the the interaction between FOXO3 and cyclin D1
Impact Pending publications
Start Year 2015
 
Description Role of FOXM1 in CCA drug resistance 
Organisation Khon Kaen University
Country Thailand 
Sector Academic/University 
PI Contribution Collaborative research on the role of FOXM1 in CCA drug resistance Hosting a Newton Fund/RGJ student for 1 year from 12/15-12/16.
Collaborator Contribution Collaborative research on the role of FOXM1 in CCA drug resistance
Impact Hosting a Newton Fund/RGJ student from KKU (Kitti Intuyod ) for 1 year from 12/15-12/16. Publications pending.
Start Year 2015
 
Description Single-cell FoxO3a/FoxM1 dynamics and their roles on cancer therapeutic resistance 
Organisation Siriraj Medical School
Country Thailand 
Sector Academic/University 
PI Contribution Role and Regulation of FOXO3 and FOXM1 in single cancer cells.
Collaborator Contribution Project leader: Role and Regulation of FOXO3 and FOXM1 in single cancer cells.
Impact Pending publications. Thailand TRF Grant for New Researcher Project title: Single-cell FoxO3a/FoxM1 dynamics and their roles on cancer therapeutic resistance Project duration: 2 years Total budget: 600,000 Baht Principal investigator / Department / Faculty / Institute Somponnat Sampattavanich Department of Pharmacology Faculty of Medicine Siriraj Hospital Mahidol University
Start Year 2015
 
Description The study of FOXO3a in leukaemia and cancer 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution Collaboration in the study of FOXO3a in leukaemia and cancer
Collaborator Contribution Supply of expertise and reagents for FOXO3a research
Impact 9 Publications: FOXP1 acts through a negative feedback loop to suppress FOXO-induced apoptosis. van Boxtel R, Gomez-Puerto C, Mokry M, Eijkelenboom A, van der Vos KE, Nieuwenhuis EE, Burgering BM, Lam EW, Coffer PJ. Cell Death Differ. 2013 Sep;20(9):1219-29. doi: 10.1038/cdd.2013.81. Epub 2013 Jul 5. PMID: 23832113 [PubMed - indexed for MEDLINE] Free PMC Article Related citations Select item 21533107 2. Rapid temporal control of Foxp3 protein degradation by sirtuin-1. van Loosdregt J, Brunen D, Fleskens V, Pals CE, Lam EW, Coffer PJ. PLoS One. 2011 Apr 20;6(4):e19047. doi: 10.1371/journal.pone.0019047. PMID: 21533107 [PubMed - indexed for MEDLINE] Free PMC Article Related citations Select item 17132628 3. FOXO3a induces differentiation of Bcr-Abl-transformed cells through transcriptional down-regulation of Id1. Birkenkamp KU, Essafi A, van der Vos KE, da Costa M, Hui RC, Holstege F, Koenderman L, Lam EW, Coffer PJ. J Biol Chem. 2007 Jan 26;282(4):2211-20. Epub 2006 Nov 27. PMID: 17132628 [PubMed - indexed for MEDLINE] Free Article Related citations Select item 17108112 4. Chronic protein kinase B (PKB/c-akt) activation leads to apoptosis induced by oxidative stress-mediated Foxo3a transcriptional up-regulation. van Gorp AG, Pomeranz KM, Birkenkamp KU, Hui RC, Lam EW, Coffer PJ. Cancer Res. 2006 Nov 15;66(22):10760-9. PMID: 17108112 [PubMed - indexed for MEDLINE] Free Article Related citations Select item 15509806 5. FoxO3a and BCR-ABL regulate cyclin D2 transcription through a STAT5/BCL6-dependent mechanism. Fernández de Mattos S, Essafi A, Soeiro I, Pietersen AM, Birkenkamp KU, Edwards CS, Martino A, Nelson BH, Francis JM, Jones MC, Brosens JJ, Coffer PJ, Lam EW. Mol Cell Biol. 2004 Nov;24(22):10058-71. PMID: 15509806 [PubMed - indexed for MEDLINE] Free PMC Article Related citations Select item 14527951 6. FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. Sunters A, Fernández de Mattos S, Stahl M, Brosens JJ, Zoumpoulidou G, Saunders CA, Coffer PJ, Medema RH, Coombes RC, Lam EW. J Biol Chem. 2003 Dec 12;278(50):49795-805. Epub 2003 Oct 3. PMID: 14527951 [PubMed - indexed for MEDLINE] Free Article Related citations Select item 11884591 7. Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors. Kops GJ, Medema RH, Glassford J, Essers MA, Dijkers PF, Coffer PJ, Lam EW, Burgering BM. Mol Cell Biol. 2002 Apr;22(7):2025-36. PMID: 11884591 [PubMed - indexed for MEDLINE] Free PMC Article Related citations Select item 11815629 8. FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity. Dijkers PF, Birkenkamp KU, Lam EW, Thomas NS, Lammers JW, Koenderman L, Coffer PJ. J Cell Biol. 2002 Feb 4;156(3):531-42. Epub 2002 Jan 28. PMID: 11815629 [PubMed - indexed for MEDLINE] Free PMC Article Related citations Select item 11094066 9. Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Dijkers PF, Medema RH, Pals C, Banerji L, Thomas NS, Lam EW, Burgering BM, Raaijmakers JA, Lammers JW, Koenderman L, Coffer PJ. Mol Cell Biol. 2000 Dec;20(24):9138-48. PMID: 11094066 [PubMed - indexed for MEDLINE] Free PMC Article Related citations
 
Description Meetings with Director, clinicians and staff of the Khon Kaen Liver Fluke Centre to explain and report work carried out by the MRC grant 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Study participants or study members
Results and Impact Meetings with Director, clinicians and staff of the Khon Kaen Liver Fluke Centre to explain and report work carried out by the MRC grant. This is the organisation which support CCA research and education in Thailand.
Year(s) Of Engagement Activity 2016