Author Manuscript Published OnlineFirst on December 16, 2019; DOI: 10.1158/0008-5472.CAN-19-0942 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 2 A runaway PRH/HHEX-Notch3 positive feedback loop 3 drives cholangiocarcinoma and determines response to 4 CDK4/6 inhibition. 5 Philip Kitchen1, Ka Ying Lee1, Danielle Clark2, Nikki Lau1, Jomnarong Lertsuwan3, 6 Anyaporn Sawasdichai4, Jutamaad Satayavivad4, Sebastian Oltean5, Simon Afford6, Kevin 7 Gaston7*# and Padma-Sheela Jayaraman1*# 8 9 1. Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, 10 University of Birmingham, UK. 2.Department of Biochemistry, Medical School, University 11 of Bristol, UK 3.Laboratory of Chemical Carcinogenesis, Chulabhorn Research Institute, 12 Bangkok, Thailand; 4.Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok, 13 Thailand; 5.Institute of Biomedical & Clinical Sciences, University of Exeter Medical 14 School, Exeter, UK. 6.Institute of Immunology and Immunotherapy, University of 15 Birmingham, Birmingham UK. 7.Division of Cancer and Stem Cells, School of Medicine, 16 University of Nottingham, Nottingham, UK. 17 # joint senior author and *joint corresponding author 18 Running title: PRH drives cholangiocarcinoma and chemotherapeutic response 19 7 figures 20 Email: [email protected] tel. no. +44( 0)121-414-4383 1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 16, 2019; DOI: 10.1158/0008-5472.CAN-19-0942 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 21 Email: [email protected] tel. no. +44(0)115-8231395 22 23 Declaration of Interests 24 The authors declare no competing interests. 25 26 27 2 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 16, 2019; DOI: 10.1158/0008-5472.CAN-19-0942 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 28 Abstract 29 Aberrant Notch and Wnt signalling are known drivers of cholangiocarcinoma (CCA) but the 30 underlying factors that initiate and maintain these pathways are not known. Here we show 31 that the PRH/HHEX transcription factor forms a positive transcriptional feedback loop with 32 Notch3 that is critical in CCA. PRH/HHEX expression was elevated in CCA and depletion of 33 PRH reduced CCA tumour growth in a xenograft model. Overexpression of PRH in primary 34 human biliary epithelial cells was sufficient to increase cell proliferation and produce an 35 invasive phenotype. Interrogation of the gene networks regulated by PRH and Notch3 36 revealed that unlike Notch3, PRH directly activated canonical Wnt signalling. These data 37 indicate that hyperactivation of Notch and Wnt signalling is independent of the underlying 38 mutational landscape and has a common origin in dysregulation of PRH. Moreover, they 39 suggest new therapeutic options based on the dependence of specific Wnt, Notch, and 40 CDK4/6 inhibitors on PRH activity. 41 Significance 42 The PRH/HHEX transcription factor is an oncogenic driver in cholangiocarcinoma that 43 confers sensitivity to CDK4/6 inhibitors. 44 45 Keywords: cholangiocarcinoma, bile duct, biliary epithelial cells, cholangiocyte, HHEX, 46 PRH, Notch, Wnt, epithelial-mesenchymal transition 47 3 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 16, 2019; DOI: 10.1158/0008-5472.CAN-19-0942 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 48 Introduction 49 Cholangiocarcinoma (CCA) is a tumour of the bile duct epithelium with an unmet clinical 50 need as this disease is usually advanced at diagnosis. For non-resectable disease median 51 survival is less than 12 months and 5-year survival is around 2% (1). New treatment options 52 and markers that allow early detection and/or inform personalised cancer treatment are 53 therefore urgently needed. Risk factors for CCA include viral infection (Hepatitis B and C), 54 liver fluke infection, primary sclerosing cholangitis (PSC), cholestasis (loss of bile flow), and 55 exposure to dietary toxins or metabolites, all of which lead to inflammation in and around the 56 biliary tree. Chronic inflammation can provoke uncontrolled wound-healing responses 57 involving the generation of DNA-damaging reactive oxygen and nitrogen species, activation 58 of immune cells (particularly macrophages) and stroma, and aberrant activation of autocrine 59 signalling, as well as activation of signalling pathways that promote epithelial-mesenchymal 60 transition (EMT) and angiogenesis. All these events ultimately lead to cancer development 61 and progression (2). Many of the dysregulated pathways involved in CCA are also involved 62 in embryonic liver morphogenesis, such as the Wnt, TGFβ, Notch, Hedgehog and Hippo 63 pathways. Several of these pathways (including Wnt and Notch) can transiently promote 64 EMT or the acquisition of mesenchymal features, such as increased cell migration and matrix 65 invasion or loss of the epithelial cell-cell adhesion molecule E-cadherin (3, 4). In many 66 cancers including mammary cancers, EMT is associated with an increased proportion of 67 tumour initiating cells (the so-called cancer stem cells or CSC) that show increased self- 68 renewal properties and increased chemoresistance (3). EMT and the CSC phenotype can be 69 induced by any one of a common set of aberrantly expressed EMT transcription factors (e.g. 70 Snail, Twist, Zeb) that, amongst many other things, directly repress the expression of E- 71 cadherin (reviewed (5)). CCA patient samples exhibit decreased E-cadherin expression and 72 contain CSCs (6) suggesting that EMT may also be a feature of CCA. 4 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 16, 2019; DOI: 10.1158/0008-5472.CAN-19-0942 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 73 Canonical Wnt signalling is critical for cell proliferation and contributes to the development 74 of CCA in a mouse CCA carcinogenesis model (7, 8). In canonical Wnt signaling, Wnt 75 ligands activate Frizzled family receptors and stabilise the β-catenin protein which then enters 76 the nucleus and activates transcription of Wnt target genes through interaction with TCF/LEF 77 family transcription factors and co-activators (4). β-catenin, by acting as both a co-activator 78 for TCF/LEF transcription factors and a structural component of adherens junctions via 79 interaction with E-cadherin, facilitates cross-talk between canonical Wnt signalling and cell- 80 cell adhesion. Although there are few mutations in Wnt signalling components in CCA there 81 are high levels of nuclear -catenin in the majority of CCA (7, 9). Importantly, 82 pharmacological inhibition of Wnt signalling decreases CCA formation in mouse models (7). 83 NOTCH signalling is also of central importance to the development of CCA (10). Notch 84 signalling involves four transmembrane NOTCH receptors and two families of ligands, 85 Serrate/Jagged (JAG-1, -2) and Delta-like (DLL-1, -3, -4), along with other proteins that 86 transduce and regulate the signal. Upon ligand binding, Notch receptors are sequentially 87 cleaved by an ADAM family protease and the γ-secretase protease complex. Cleavage 88 releases the NOTCH intracellular domain (ICD) which interacts with the DNA binding 89 protein RBP-J/CSL and MAML1 co-activator resulting in transcriptional activation of 90 NOTCH target genes, including the HES and HEY family of genes encoding bHLH 91 transcriptional repressors (reviewed in (11)). In hepatic regeneration models, Wnt and Notch 92 signalling promote different cell fates; Notch signalling promotes biliary fate in Hepatic 93 Progenitor Cells and Wnt signalling promotes hepatic specification (12). Dysregulation of 94 Notch signalling in mouse hepatocytes through constitutive Notch1 ICD or Notch2 ICD 95 expression can result in either hepatocellular carcinoma (HCC) or CCA depending on the co- 96 operating oncogene. NOTCH3 is not expressed in adult liver but Notch3 expression is 97 elevated in CCA patient samples and knockout of Notch3 in a mouse model of CCA 5 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 16, 2019; DOI: 10.1158/0008-5472.CAN-19-0942 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 98 abrogates tumour growth (10). Notch3 ICD is known to be a key driver of CCA in part 99 through activation of PI3K/Akt signalling (10). However, it is not known which genes the 100 Notch3 ICD regulates nor is the interplay between Notch3 ICD signalling and Wnt signalling 101 understood. 102 The Proline-Rich Homeodomain protein/Haematopoietically Expressed Homeobox 103 (PRH/HHEX) protein is a transcription factor encoded by the HHEX gene that is required in 104 the development of many tissue types including the liver and bile duct (reviewed (13)), where 105 PRH regulates hepatic (HNF4a) and biliary (Onecut1)