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Cited by 11Fibroblast Growth Factor ResearchHepatocellular Carcinoma Treatment and PrognosisLiver physiology and pathology

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FGF19 and its analog Aldafermin cooperate with MYC to induce aggressive hepatocarcinogenesis

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Ursic-Bedoya, José, Desandré, Guillaume, Chavey, Carine, Marie, Pauline, Polizzi, Arnaud, Rivière, Benjamin, Guillou, Hervé, Assenat, Eric, Hibner, Urszula, Gregoire, Damien·2024·EMBO Molecular Medicine
DOI 10.1038/s44321-023-00021-xPMC PMC10897482

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Abstract

FGF19 hormone has pleiotropic metabolic functions, including the modulation of insulin sensitivity, glucose/lipid metabolism and energy homeostasis. On top of its physiological metabolic role, FGF19 has been identified as a potentially targetable oncogenic driver, notably in hepatocellular carcinoma (HCC). Nevertheless, FGF19 remained an attractive candidate for treatment of metabolic disease, prompting the development of analogs uncoupling its metabolic and tumor-promoting activities. Using pre-clinical mice models of somatic mutation driven HCC, we assessed the oncogenicity of FGF19 in combination with frequent HCC tumorigenic alterations: p53 inactivation, CTNNB1 mutation, CCND1 or MYC overexpression. Our data revealed a strong oncogenic cooperation between FGF19 and MYC. Most importantly, we show that this oncogenic synergy is conserved with a FGF19-analog Aldafermin (NGM282), designed to solely mimic the hormone’s metabolic functions. In particular, even a short systemic treatment with recombinant proteins triggered rapid appearance of proliferative foci of MYC-expressing hepatocytes. The fact that FGF19 analog Aldafermin is not fully devoid of the hormone’s oncogenic properties raises concerns in the context of its potential use for patients with damaged, mutation-prone liver.

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Figures

Figure 1 FGF19 and FGF15 cooperate with MYC to trigger liver carcinogenesis. (A) Experimental strategy to test FGF19/15 cooperation with other oncogenic events. Female C57BL/6J mice are subjected to hydrodynamic gene transfer (HGT) of a combination of oncogenes and the Sleeping-Beauty transposase 100 (SB100). (B) Representative livers and hematoxylin & eosin & Saffron (HES) stained sections following HGT with empty vector (RFP) or FGF19 plasmids, as indicated. (C) Kaplan–Meier curve of tumor-free survival for distinct oncogenic combinations, as indicated. Statistical significance was determined using Mantel-Cox (Log-rank) test; p value is indicated. (D) Representative livers and HES stained sections following hydrodynamic injection with RFP + MYC, FGF19 + MYC and FGF15 + MYC. (E) Liver/body weight ratio and tumor number per liver following HGT with the indicated combinations of oncogenes. (F) RT-qPCR expression analysis of transfected FGF 19/15 and HCC markers Glypican 3 and Alpha fetoprotein in RFP parenchyma (NT) and FGF19 + MYC and FGF15 + MYC tumors (T). Data information: Scale-bars: 1 cm (macroscopic liver), 100 µm (HES staining). Data are represented as mean ± SD. Number of mice per group and tumor incidence indicated are indicated in the figure. The data are representative of at least two independent experiments and all replicates shown are biological replicates. All tumoral samples come from independent mice. Statistical significance was evaluated using Mann–Whitney test performed on liver/body weight (E) or mRNA levels (F). Significant p values are indicated: ns (non significant) p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. RFP red fluorescent protein, T tumor, NT non-tumor parenchyma. Source data are available online for this figure.
Figure 1 FGF19 and FGF15 cooperate with MYC to trigger liver carcinogenesis. (A) Experimental strategy to test FGF19/15 cooperation with other oncogenic events. Female C57BL/6J mice are subjected to hydrodynamic gene transfer (HGT) of a combination of oncogenes and the Sleeping-Beauty transposase 100 (SB100). (B) Representative livers and hematoxylin & eosin & Saffron (HES) stained sections following HGT with empty vector (RFP) or FGF19 plasmids, as indicated. (C) Kaplan–Meier curve of tumor-free survival for distinct oncogenic combinations, as indicated. Statistical significance was determined using Mantel-Cox (Log-rank) test; p value is indicated. (D) Representative livers and HES stained sections following hydrodynamic injection with RFP + MYC, FGF19 + MYC and FGF15 + MYC. (E) Liver/body weight ratio and tumor number per liver following HGT with the indicated combinations of oncogenes. (F) RT-qPCR expression analysis of transfected FGF 19/15 and HCC markers Glypican 3 and Alpha fetoprotein in RFP parenchyma (NT) and FGF19 + MYC and FGF15 + MYC tumors (T). Data information: Scale-bars: 1 cm (macroscopic liver), 100 µm (HES staining). Data are represented as mean ± SD. Number of mice per group and tumor incidence indicated are indicated in the figure. The data are representative of at least two independent experiments and all replicates shown are biological replicates. All tumoral samples come from independent mice. Statistical significance was evaluated using Mann–Whitney test performed on liver/body weight (E) or mRNA levels (F). Significant p values are indicated: ns (non significant) p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. RFP red fluorescent protein, T tumor, NT non-tumor parenchyma. Source data are available online for this figure.CC BY · PubMed Central
Figure EV1 FGF19 cooperates with β-cateninS33Y to induce hepatic carcinogenesis. Representative livers and HES-stained sections from mice following hydrodynamic gene transfer with either FGF19, β-cateninS33Y or both. Tumor incidence is indicated. Liver/body weight ratio for each mouse, with dot sizes proportional to tumor burden. Scale bar for zoom: 200 μm. Scale bar for large view: 1 cm. Mann–Whitney test statistical significance is indicated. Data are represented as mean ± SD. ns (non significant) p > 0.05, *p < 0.05, **p < 0.01.
Figure EV1 FGF19 cooperates with β-cateninS33Y to induce hepatic carcinogenesis. Representative livers and HES-stained sections from mice following hydrodynamic gene transfer with either FGF19, β-cateninS33Y or both. Tumor incidence is indicated. Liver/body weight ratio for each mouse, with dot sizes proportional to tumor burden. Scale bar for zoom: 200 μm. Scale bar for large view: 1 cm. Mann–Whitney test statistical significance is indicated. Data are represented as mean ± SD. ns (non significant) p > 0.05, *p < 0.05, **p < 0.01.CC BY · PubMed Central
Figure 2 Aldafermin and FGF19 are indistinguishable in cooperating with MYC to promote liver carcinogenesis. (A) Sequence alignment of FGF19 and Aldafermin proteins. Amino-acid sequences of N-terminal part of the proteins are indicated. 3D AlphaFold prediction of proteins’ structure and folding are shown. (B) Representative livers, HES stained sections, liver/body weight ratio and tumor number per liver following HGT with MYC + RFP, Aldafermin or Aldafermin + MYC, 2–4 weeks after injection. Of note, quantifications for MYC + RFP tumors were already presented in Fig. 1E. (C) Principal Component Analysis of RNAseq gene expression profiles of tumors obtained after HGT with FGF19 + MYC, Aldafermin + MYC, sg-Trp53 + MYC or liver (HGT with RFP plasmid) as control. (D) Correlation plot of the mean log10(CPM) of the Aldafermin + MYC and FGF19 + MYC tumors. Each point represents one gene, and the black line is a linear regression over the whole dataset. Equation of the linear regression and its Pearson’s correlation coefficient are indicated. (E) Heat map showing hierarchical clustering of genes (n = 786) separating tumoral transcriptomes along dimension 2 of the PCA (contribution to Dim.2 > 0.003 and |correlation to Dim.2| > 0.5). (F) Gene set enrichment analysis (GSEA) pathways specifically deregulated in Aldafermin/FGF19 driven tumors. A selection of most significant pathways is shown. Data information: Data are presented as mean ± SD. Mann–Whitney test statistical significance is indicated. ns (non significant) p > 0.05, *p < 0.05, **p < 0.01. Scale bars: 1 cm. All tumoral samples for RNA-seq come from independent mice, all replicates shown are biological replicates. Data from at least two independent experiments. HES Hematoxylin & eosin & saffron. GSEA analysis were performed with ViSEAGO package. Source data are available online for this figure.
Figure 2 Aldafermin and FGF19 are indistinguishable in cooperating with MYC to promote liver carcinogenesis. (A) Sequence alignment of FGF19 and Aldafermin proteins. Amino-acid sequences of N-terminal part of the proteins are indicated. 3D AlphaFold prediction of proteins’ structure and folding are shown. (B) Representative livers, HES stained sections, liver/body weight ratio and tumor number per liver following HGT with MYC + RFP, Aldafermin or Aldafermin + MYC, 2–4 weeks after injection. Of note, quantifications for MYC + RFP tumors were already presented in Fig. 1E. (C) Principal Component Analysis of RNAseq gene expression profiles of tumors obtained after HGT with FGF19 + MYC, Aldafermin + MYC, sg-Trp53 + MYC or liver (HGT with RFP plasmid) as control. (D) Correlation plot of the mean log10(CPM) of the Aldafermin + MYC and FGF19 + MYC tumors. Each point represents one gene, and the black line is a linear regression over the whole dataset. Equation of the linear regression and its Pearson’s correlation coefficient are indicated. (E) Heat map showing hierarchical clustering of genes (n = 786) separating tumoral transcriptomes along dimension 2 of the PCA (contribution to Dim.2 > 0.003 and |correlation to Dim.2| > 0.5). (F) Gene set enrichment analysis (GSEA) pathways specifically deregulated in Aldafermin/FGF19 driven tumors. A selection of most significant pathways is shown. Data information: Data are presented as mean ± SD. Mann–Whitney test statistical significance is indicated. ns (non significant) p > 0.05, *p < 0.05, **p < 0.01. Scale bars: 1 cm. All tumoral samples for RNA-seq come from independent mice, all replicates shown are biological replicates. Data from at least two independent experiments. HES Hematoxylin & eosin & saffron. GSEA analysis were performed with ViSEAGO package. Source data are available online for this figure.CC BY · PubMed Central
Figure EV2 GSEA Pathways enriched in FGF19/Aldafermin + MYC driven tumors. (A) Upset plot depicting the number of unique and shared GSEA pathways enriched in each sample groups analyzed by RNAseq. FGF19 + MYC and Aldafermin + MYC samples were pooled for this analysis. (B) GSEA analysis of the 18 pathways specifically enriched in FGF19/Aldafermin + MYC vs sgTrp53 + MYC tumors. Kolmogorov–Smirnov statistical test significance is indicated. Data information: Threshold of significance for GSEA: p < 0.01. Complementary data are provided in Dataset EV1.
Figure EV2 GSEA Pathways enriched in FGF19/Aldafermin + MYC driven tumors. (A) Upset plot depicting the number of unique and shared GSEA pathways enriched in each sample groups analyzed by RNAseq. FGF19 + MYC and Aldafermin + MYC samples were pooled for this analysis. (B) GSEA analysis of the 18 pathways specifically enriched in FGF19/Aldafermin + MYC vs sgTrp53 + MYC tumors. Kolmogorov–Smirnov statistical test significance is indicated. Data information: Threshold of significance for GSEA: p < 0.01. Complementary data are provided in Dataset EV1.CC BY · PubMed Central
Figure 3 Systemic administration of recombinant FGF19 and Aldafermin recapitulates cooperation with MYC. (A) Schematic representation of the workflow for recombinant FGF19 and Aldafermin production and purification. (B) ELISA quantification of plasma levels of FGF19 and Aldafermin 2 h after intraperitoneal injection of 2 µg of recombinant FGF19 or Aldafermin. RT-qPCR quantification of liver Cyp7a1 mRNA levels 2 h post injection. (C) Schematic representation of the experiment. After hydrodynamic gene transfer of MYC, RFP and SB100X encoding plasmids, mice were intraperitoneally injected every 12 h with either 1.2 µg rFGF19, rAldafermin or rGST. Left panel: Immunofluorescent detection of MYC and Click-iT EdU assay performed on liver sections at day 4 post-injection. Upper right panel: Size of the MYC-positive foci. Median value is indicated with a green line. Number of detected foci on one liversection is shown for each mouse. Lower right panel: Distribution of EdU-positive cells (0–100%) in the foci analyzed. Data information: Scale bar = 100 µm. Data are presented as mean ± SD, all replicates shown are biological replicates. Mann–Whitney test statistical significance is indicated (B, C). Statistical significance of differences in % of EdU-positive foci (C, lower panel) was evaluated using the Kolmogorov–Smirnov test. ns (non significant) p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 DsBc disulfide bond C, rGST recombinant glutathione-S-transferase. Source data are available online for this figure.
Figure 3 Systemic administration of recombinant FGF19 and Aldafermin recapitulates cooperation with MYC. (A) Schematic representation of the workflow for recombinant FGF19 and Aldafermin production and purification. (B) ELISA quantification of plasma levels of FGF19 and Aldafermin 2 h after intraperitoneal injection of 2 µg of recombinant FGF19 or Aldafermin. RT-qPCR quantification of liver Cyp7a1 mRNA levels 2 h post injection. (C) Schematic representation of the experiment. After hydrodynamic gene transfer of MYC, RFP and SB100X encoding plasmids, mice were intraperitoneally injected every 12 h with either 1.2 µg rFGF19, rAldafermin or rGST. Left panel: Immunofluorescent detection of MYC and Click-iT EdU assay performed on liver sections at day 4 post-injection. Upper right panel: Size of the MYC-positive foci. Median value is indicated with a green line. Number of detected foci on one liversection is shown for each mouse. Lower right panel: Distribution of EdU-positive cells (0–100%) in the foci analyzed. Data information: Scale bar = 100 µm. Data are presented as mean ± SD, all replicates shown are biological replicates. Mann–Whitney test statistical significance is indicated (B, C). Statistical significance of differences in % of EdU-positive foci (C, lower panel) was evaluated using the Kolmogorov–Smirnov test. ns (non significant) p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 DsBc disulfide bond C, rGST recombinant glutathione-S-transferase. Source data are available online for this figure.CC BY · PubMed Central

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  1. Zhou M, Wang X, Phung V, Lindhout DA, Mondal K, Hsu JY, Yang H, Humphrey M, Ding X, Arora T et al (2014b) Separating tumorigenicity from bile acid regulatory activity for endocrine hormone FGF19. Cancer Res 74:3306–3316 · DOI · Paper page

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Metadata

Authors
Ursic-Bedoya, J., Desandré, G., Chavey, C., Marie, P., Polizzi, A., Rivière, B., Guillou, H., Assenat, E., Hibner, U., & Gregoire, D.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • Université Toulouse III - Paul Sabatier (FR)
  • Université de Montpellier (FR)
Corresponding author
Desandré, Guillaume
ORCID
Venue
EMBO Molecular Medicine
Publisher
Springer Science and Business Media LLC
Published
2024-01-16
Volume
16
Issue
2
Pages
238-250
ISSN
  • 1757-4676 (ISSN-L)
  • 1757-4684
DOI
10.1038/s44321-023-00021-x
PMC
PMC10897482
OpenAlex
W4390904460
Cited by
11
References
58
Citations by year
2026: 5 · 2025: 3 · 2024: 3
FWCI
1.50
Type
journal article
Language
en
Open access
Licence
Locations
  • EMBO Molecular Medicine (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by-nc-nd)
  • HAL (submittedVersion, cc-by-nc-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Association Française pour l'Etude du Foie (AAP_2021)
  • SIRIC Montpellier (Cancer Grant INCa_Inserm_DGOS_12553)
  • Fondation ARC pour la Recherche sur le Cancer (2019)
  • Institut National Du Cancer (2019-134)
  • ITMO Cancer (EVA-Plan cancer INSERM HTE-201610)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fibroblast Growth Factor Research
  • Health Sciences › Medicine › Hepatology › Hepatocellular Carcinoma Treatment and Prognosis
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
Keywords
FGF19, c-Myc, hepatocellular carcinoma, hepatocarcinogenesis, oncogene cooperation, p53 inactivation, CTNNB1 mutation, hepatocyte proliferation, drug safety concerns
MeSH terms
Animals, Fibroblast Growth Factors, Carcinoma, Hepatocellular, Hormones, Humans, Liver Neoplasms, Mice

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