8 research outputs found
An integrative approach unveils FOSL1 as an oncogene vulnerability in KRAS-driven lung and pancreatic cancer
KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers
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)
Criteria for preclinical models of cholangiocarcinoma:scientific and medical relevance
Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA
p65BTK is a novel potential actionable target in KRAS-mutated/EGFR-wild type lung adenocarcinoma
BACKGROUND: Lung cancer is still the main cause of cancer death worldwide despite the availability of targeted therapies and immune-checkpoint inhibitors combined with chemotherapy. Cancer cell heterogeneity and primary or acquired resistance mechanisms cause the elusive behaviour of this cancer and new biomarkers and active drugs are urgently needed to overcome these limitations. p65BTK, a novel isoform of the Bruton Tyrosine Kinase may represent a new actionable target in non-small cell lung cancer (NSCLC).
METHODS: p65BTK expression was evaluated by immunohistochemistry in 382 NSCLC patients with complete clinico-pathological records including smoking habit, ALK and EGFR status, and in metastatic lymph nodes of 30 NSCLC patients. NSCLC cell lines mutated for p53 and/or a component of the RAS/MAPK pathway and primary lung cancer-derived cells from Kras/Trp53 null mice were used as a preclinical model. The effects of p65BTK inhibition by BTK Tyrosine Kinase Inhibitors (TKIs) (Ibrutinib, AVL-292, RN486) and first-generation EGFR-TKIs (Gefitinib, Erlotinib) on cell viability were evaluated by MTT. The effects of BTK-TKIs on cell growth and clonogenicity were assessed by crystal violet and colony assays, respectively. Cell toxicity assays were performed to study the effect of the combination of non-toxic concentrations of BTK-TKIs with EGFR-TKIs and standard-of-care (SOC) chemotherapy (Cisplatin, Gemcitabine, Pemetrexed).
RESULTS: p65BTK was significantly over-expressed in EGFR-wild type (wt) adenocarcinomas (AdC) from non-smoker patients and its expression was also preserved at the metastatic site. p65BTK was also over-expressed in cell lines mutated for KRAS or for a component of the RAS/MAPK pathway and in tumors from Kras/Trp53 null mice. BTK-TKIs were more effective than EGFR-TKIs in decreasing cancer cell viability and significantly impaired cell proliferation and clonogenicity. Moreover, non-toxic doses of BTK-TKIs re-sensitized drug-resistant NSCLC cell lines to both target- and SOC therapy, independently from EGFR/KRAS status.
CONCLUSIONS: p65BTK results as an emerging actionable target in non-smoking EGFR-wt AdC, also at advanced stages of disease. Notably, these patients are not eligible for EGFR-TKIs-based therapy due to a lack of EGFR mutation. The combination of BTK-TKIs with EGFR-TKIs is cytotoxic for EGFR-wt/KRAS-mutant/p53-null tumors and BTK-TKIs re-sensitizes drug-resistant NSCLC to SOC chemotherapy. Therefore, our data suggest that adding BTK-TKIs to SOC chemotherapy and EGFR-targeted therapy may open new avenues for clinical trials in currently untreatable NSCLC
The phospholipid transporter PITPNC1 links KRAS to MYC to prevent autophagy in lung and pancreatic cancer
Abstract Background The discovery of functionally relevant KRAS effectors in lung and pancreatic ductal adenocarcinoma (LUAD and PDAC) may yield novel molecular targets or mechanisms amenable to inhibition strategies. Phospholipids availability has been appreciated as a mechanism to modulate KRAS oncogenic potential. Thus, phospholipid transporters may play a functional role in KRAS-driven oncogenesis. Here, we identified and systematically studied the phospholipid transporter PITPNC1 and its controlled network in LUAD and PDAC. Methods Genetic modulation of KRAS expression as well as pharmacological inhibition of canonical effectors was completed. PITPNC1 genetic depletion was performed in in vitro and in vivo LUAD and PDAC models. PITPNC1-deficient cells were RNA sequenced, and Gene Ontology and enrichment analyses were applied to the output data. Protein-based biochemical and subcellular localization assays were run to investigate PITPNC1-regulated pathways. A drug repurposing approach was used to predict surrogate PITPNC1 inhibitors that were tested in combination with KRASG12C inhibitors in 2D, 3D, and in vivo models. Results PITPNC1 was increased in human LUAD and PDAC, and associated with poor patients’ survival. PITPNC1 was regulated by KRAS through MEK1/2 and JNK1/2. Functional experiments showed PITPNC1 requirement for cell proliferation, cell cycle progression and tumour growth. Furthermore, PITPNC1 overexpression enhanced lung colonization and liver metastasis. PITPNC1 regulated a transcriptional signature which highly overlapped with that of KRAS, and controlled mTOR localization via enhanced MYC protein stability to prevent autophagy. JAK2 inhibitors were predicted as putative PITPNC1 inhibitors with antiproliferative effect and their combination with KRASG12C inhibitors elicited a substantial anti-tumour effect in LUAD and PDAC. Conclusions Our data highlight the functional and clinical relevance of PITPNC1 in LUAD and PDAC. Moreover, PITPNC1 constitutes a new mechanism linking KRAS to MYC, and controls a druggable transcriptional network for combinatorial treatments
New molecular mechanisms in cholangiocarcinoma: signals triggering interleukin-6 production in tumor cells and KRAS co-opted epigenetic mediators driving metabolic reprogramming
Background: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. Methods: Cholangiocarcinogenesis was induced in rats (TAA) and mice (JnkΔhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. Results: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. Conclusions: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA
Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance
Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA