FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted

[EN] Background & Aims: Cholangiocarcinoma (CCA) is a neoplasia of the biliary tract driven by genetic, epigenetic and transcriptional mechanisms. Herein, we investigated the role of the transcription factor FOSL1, as well as its downstream transcriptional effectors, in the development and progression of CCA. Methods: FOSL1 was investigated in human CCA clinical samples. Genetic inhibition of FOSL1 in human and mouse CCA cell lines was performed in in vitro and in vivo models using constitutive and inducible short-hairpin RNAs. Conditional FOSL1 ablation was done using a genetically engineered mouse (GEM) model of CCA (mutant KRAS and Trp53 knockout). Followup RNA and chromatin immunoprecipitation (ChIP) sequencing analyses were carried out and downstream targets were validated using genetic and pharmacological inhibition. Results: An inter-species analysis of FOSL1 in CCA was conducted. First, FOSL1 was found to be highly upregulated in human and mouse CCA, and associated with poor patient survival. Pharmacological inhibition of different signalling pathways in CCA cells converged on the regulation of FOSL1 expression. Functional experiments showed that FOSL1 is required for cell proliferation and cell cycle progression in vitro, and for tumour growth and tumour maintenance in both orthotopic and subcutaneous xenograft models. Likewise, FOSL1 genetic abrogation in a GEM model of CCA extended mouse survival by decreasing the oncogenic potential of transformed cholangiocytes. RNA and ChIP sequencing studies identified direct and indirect transcriptional effectors such as HMGCS1 and AURKA, whose genetic and pharmacological inhibition phenocopied FOSL1 loss. Conclusions: Our data illustrate the functional and clinical relevance of FOSL1 in CCA and unveil potential targets amenable to pharmacological inhibition that could enable the implementation of novel therapeutic strategies. Lay summary: Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention.A.V. was supported by ADA of the University of Navarra, Spain, O.E. by FSE; MINECO; FJCI-2017-34233, Spain, R.E. by a donation from Mauge Burgos de la Iglesia’s family, Spain, and P. Olaizola by the Basque Government (PRE_2016_1_0269), Basque Country, Spain. M.J.P. was funded by ISCIII [FIS PI14; 00399, PI17; 00022] cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain; Spanish Ministry of Economy and Competitiveness (MINECO: “Ramón y Cajal” Program RYC-2015-17755), Spain. M.A.A was funded by La Caixa Foundation, HEPACARE project, Spain, ISCIII FIS PI16/01126 cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain, and “Fundación Científica de la Asociación Española Contra el Cáncer’’ (AECC Scientific Foundation) Rare Cancers 2017, Spain. J.M.B. was funded by the Spanish Carlos III Health Institute (ISCIII) (FIS PI15; 01132, PI18; 01075 and Miguel Servet Program CON14; 00129 and CPII19; 00008), Spain, co-financed by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain; “Euskadi RIS3” (2019222054) and BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15; CA; 016; BD), Basque Country, Spain; “Fundación Científica de la Asociación Española Contra el Cáncer” (AECC Scientific Foundation) Rare Cancers 2017, Spain. S.V. was supported by FEDER; MINECO (SAF2017-89944-R), Spain, by the Government of Navarra-Health Research Department (58; 2018), Navarra, Spain, by La Caixa and Caja Navarra Foundation-CIMA agreement, Spain. None of the funding sources were involved in the decision to submit the article for publication. This article is based upon work from COST Action CA18122 European Cholangiocarcinoma Network, supported by COST (European Cooperation in Science and Technology). COST (European Cooperation in Science and Technology) is a funding agency for research and innovation networks (www.cost.eu)

FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted

Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention

Neoadjuvant Chemotherapy Is Associated with Altered Immune Cell Infiltration and an Anti-Tumorigenic Microenvironment in Resected Pancreatic Cancer

PURPOSE: Neoadjuvant chemotherapy is increasingly administered to patients with resectable or borderline resectable pancreatic ductal adenocarcinoma (PDAC), yet its impact on the tumor immune microenvironment is incompletely understood. DESIGN: We employed quantitative, spatially resolved multiplex immunofluorescence and digital image analysis to identify T-cell subpopulations, macrophage polarization states, and myeloid cell subpopulations in a multi-institution cohort of up-front resected primary tumors (n = 299) and in a comparative set of resected tumors after FOLFIRINOX-based neoadjuvant therapy (n = 36) or up-front surgery (n = 30). Multivariable-adjusted Cox proportional hazards models were used to evaluate associations between the immune microenvironment and patient outcomes. RESULTS: In the multi-institutional resection cohort, immune cells exhibited substantial heterogeneity across patient tumors and were located predominantly in stromal regions. Unsupervised clustering using immune cell densities identified four main patterns of immune cell infiltration. One pattern, seen in 20% of tumors and characterized by abundant T cells (T cell-rich) and a paucity of immunosuppressive granulocytes and macrophages, was associated with improved patient survival. Neoadjuvant chemotherapy was associated with a higher CD8:CD4 ratio, greater M1:M2-polarized macrophage ratio, and reduced CD15+ARG1+ immunosuppressive granulocyte density. Within neoadjuvant-treated tumors, 72% showed a T cell-rich pattern with low immunosuppressive granulocytes and macrophages. M1-polarized macrophages were located closer to tumor cells after neoadjuvant chemotherapy, and colocalization of M1-polarized macrophages and tumor cells was associated with greater tumor pathologic response and improved patient survival. CONCLUSIONS: Neoadjuvant chemotherapy with FOLFIRINOX shifts the PDAC immune microenvironment toward an anti-tumorigenic state associated with improved patient survival

Spatially Resolved Single-Cell Assessment of Pancreatic Cancer Expression Subtypes Reveals Co-expressor Phenotypes and Extensive Intratumoral Heterogeneity

Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in \u3e 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell-cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC

Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer1, 2, 3, 4, 5. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation6, 7, 8, 9, 10. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs4, 5, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy

The Origin of Intrahepatic Cholangiocarcinoma and the Importance of MCL-1 for Tumor Development

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Genetics, 2017.Intrahepatic cholangiocarcinoma (iCCA) is an especially lethal liver cancer, with poor prognosis and no targeted therapies. Historically the cell of origin for this disease was assumed to be the biliary epithelial cell (BEC), however recent studies have shown that hepatocytes can transdifferentiate into BECs. Moreover, two studies have shown that in the context of AKT and Notch co-mutations, as well as extreme carcinogenic injury, mutated hepatocytes can give rise to iCCA. While Notch mutations are entirely undescribed in human iCCA, we find that this pathway is activated in the setting of two of the most common mutations in iCCA, Kras and Tp53, when directed to mature hepatocytes in mice. Furthermore, targeting these mutations to hepatocytes can result in iCCA. In a related, but separate line of inquiry, we investigated the functional importance of MCL-1 over-expression in iCCA. MCL-1 is the most commonly amplified gene in iCCA and is over-expressed even when not amplified, likely related to its position downstream of multiple inflammatory signals and KRAS. MCL-1 is most commonly described as an anti-apoptotic molecule, but it has also been implicated in autophagy and ATP production. We show that MCL-1 is important for xenograft tumor growth in a Kras G12D;Tp53f/+ murine iCCA cell line. We found that knockdown of Mcl-1 in vitro does not affect baseline levels of apoptosis, autophagy, ATP levels, or TCA-cycle intermediates. In vivo we observe that MCL-1 status affects the liver's response to injury. We further observe found that human IDH1 mutant iCCA cell lines cannot tolerate knockdown of MCL-1. Thus, our work shows not only that the hepatocyte may serve as a cell of origin in this disease, but that MCL-1 is important for iCCA and that its function to support cancer growth, as not yet fully defined, is distinct from the anti-apoptotic and energy production functions ascribed to the protein in other systems

Oncogenic Mutations and Injury in the Mouse Liver

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biomedical Genetics, 2017.Chronic injury and inflammation are among the most established determinants of cancer risk, and the myriad of factors that contribute to this association is an active area of investigation. A key aspect of injury is the consequent regenerative and proliferative response. Understanding the mechanisms which restrain these proliferative processes and how these controls can be bypassed may shed light on the processes that drive early cancer development. Focusing on the mouse liver as a model tissue, we sought to determine how common oncogenic mutations impact the ductular reaction, the liver’s response to injury and one of the earliest events in cholangiocarcinoma development. Here we identify, among the most commonly mutated genes in cholangiocarcinoma, a uniquely critical role for the tumor suppressor Smad4 in restraining proliferation of ductular reactive cells, the biliary/epithelial compartment of the ductular reaction. Experiments testing the tumor suppressive role of Smad4 in mouse liver revealed a complex role as a suppressor of hepatocellular carcinoma, cholangiocarcinoma, and biliary cyst development. Focusing on Smad4’s role in the ductular reaction, we identify an IGF activation signature in Smad4-perturbed ductular reactive cells and in SMAD4-mutant cancers that is potentially mediated by SMAD4-regulated transcription of IGF binding proteins, key inhibitors of IGF signaling. This study therefore importantly links a key tumor suppressive pathway to regulation of the injury response, thereby shedding light on the earliest events in cancer development. In a related project, we sought to understand the cellular origins of cholangiocarcinoma. Hepatocytes and biliary epithelial cells may both be potential cells of origin for cholangiocarcinoma, although the contexts in which this has been demonstrated are limited. For example, although hepatocytes can be cells of origin in the setting of active Notch signaling, a key driver of biliary differentiation, Notch pathway mutations are not common events in human cases; therefore, it remains unknown if common genetic lesions identified in human cholangiocarcinoma are sufficient to promote hepatocyte-derived cholangiocarcinoma. To shed light on these matters, we demonstrate that targeting Kras and Tp53 mutations, two of the most common mutations in human cholangiocarcinoma, to the SOX9+ biliary compartment promotes cholangiocarcinoma development. Similarly, targeting these mutations to the hepatocyte compartment can lead to cholangiocarcinoma development in the setting of injury. We further establish that Tp53 in particular has a critical function in restricting reprogramming of hepatocytes to biliary epithelial cells, a role that likely enables hepatocyte-derived iCCA when it is mutated. Finally, mirroring what has been observed in other biliary tract-derived models, we observed active Notch and Wnt signaling in our hepatocyte-derived model, suggesting hepatocyte-derived cholangiocarcinoma may have similar programming regarding these important targetable pathways. Understanding distinguishing biological features of hepatocyte-derived and biliary tract-derived cholangiocarcinoma may prove to have clinically relevant prognostic and therapeutic value

Stereotactic Body Radiation Therapy as a Bridge to Transplantation and for Recurrent Disease in the Transplanted Liver of a Patient with Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer mortality worldwide. Despite orthotopic liver transplantation (OLT), recurrent HCC is a major cause of morbidity. In this case report, we evaluate the efficacy of stereotactic body radiation therapy (SBRT) as a bridge to OLT and for recurrence in the transplanted liver of a patient with HCC. A 52-year-old male with a history of chronic hepatitis C presented with a 1.7-cm liver lesion radiographically consistent with HCC, which was subsequently treated with a course of SBRT to 50 Gy in 5 fractions followed by OLT in 2009. The patient had a 2.2-cm recurrence in the transplanted liver in 2012, which was treated with SBRT to 62.5 Gy in 5 fractions. He tolerated the course of radiotherapy well with no significant radiation-related toxicity and remains in complete remission approximately 1 year after SBRT. SBRT is a safe and effective modality for the treatment of recurrent HCC in the transplanted liver of the same patient initially treated with SBRT as a bridge to OLT