Folliculin impairs breast tumor growth by repressing TFE3-dependent induction of the Warburg effect and angiogenesis
Leeanna El-Houjeiri, … , Peter M. Siegel, Arnim Pause
Leeanna El-Houjeiri, … , Peter M. Siegel, Arnim Pause
Published November 15, 2021
Citation Information: J Clin Invest. 2021;131(22):e144871. https://doi.org/10.1172/JCI144871.
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Research Article Angiogenesis Metabolism

Folliculin impairs breast tumor growth by repressing TFE3-dependent induction of the Warburg effect and angiogenesis

Abstract

Growing tumors exist in metabolically compromised environments that require activation of multiple pathways to scavenge nutrients to support accelerated rates of growth. The folliculin (FLCN) tumor suppressor complex (FLCN, FNIP1, FNIP2) is implicated in the regulation of energy homeostasis via 2 metabolic master kinases: AMPK and mTORC1. Loss-of-function mutations of the FLCN tumor suppressor complex have only been reported in renal tumors in patients with the rare Birt-Hogg-Dube syndrome. Here, we revealed that FLCN, FNIP1, and FNIP2 are downregulated in many human cancers, including poor-prognosis invasive basal-like breast carcinomas where AMPK and TFE3 targets are activated compared with the luminal, less aggressive subtypes. FLCN loss in luminal breast cancer promoted tumor growth through TFE3 activation and subsequent induction of several pathways, including autophagy, lysosomal biogenesis, aerobic glycolysis, and angiogenesis. Strikingly, induction of aerobic glycolysis and angiogenesis in FLCN-deficient cells was dictated by the activation of the PGC-1α/HIF-1α pathway, which we showed to be TFE3 dependent, directly linking TFE3 to Warburg metabolic reprogramming and angiogenesis. Conversely, FLCN overexpression in invasive basal-like breast cancer models attenuated TFE3 nuclear localization, TFE3-dependent transcriptional activity, and tumor growth. These findings support a general role of a deregulated FLCN/TFE3 tumor suppressor pathway in human cancers.

Authors

Leeanna El-Houjeiri, Marco Biondini, Mathieu Paquette, Helen Kuasne, Alain Pacis, Morag Park, Peter M. Siegel, Arnim Pause

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Figure 5

Loss of FLCN in luminal breast cancer cells promotes an angiogenic profile.

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Loss of FLCN in luminal breast cancer cells promotes an angiogenic profi...
(A) Heatmap representing differential gene expression in WT and FLCN-knockout (FLCNKO) MCF7 tumors following RNA-sequencing analysis. Each column represents a different mouse from each cohort, where blue is WT and red is FLCNKO. Fold increase was normalized against EV and color coded (dark red indicates 3-fold or more increase, light green indicates 3-fold or more decrease, black indicates no change). (B) Gene enrichment scores for significantly upregulated pathways in FLCNKO compared with WT MCF7 tumors highlighting regulation of angiogenesis as a differentially induced pathway, with the heatmap specifically showing the upregulation of angiogenesis-related genes. Each column represents a different mouse from each cohort, where blue is WT and red is FLCNKO. Fold increase was normalized against EV and color coded (dark red indicates 3-fold or more increase, light green indicates 3-fold or more decrease, black indicates no change). (C) Fold increase in 17 angiogenic/growth factors detected in WT and FLCNKO MCF7 tumor lysates using human angiogenesis array. Data represent the average values of 10 mice in each of the indicated cohorts. Significance was determined using Student’s t test. **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) Representative images of immunohistochemistry (IHC) staining for human VEGF-A, mouse CD31, and mouse F4/80 of WT, FLCNKO, FLCNKO plus EV, and FLCNKO FLCN-reexpressing MCF7 tumors resected 6 weeks after injection (top). Scale bar: 50 μm. Quantification of IHC results showing percentage positive VEGF-A staining, positive CD31 staining, and positive F4/80 staining (bottom). Data represent mean quantifications ± SEM of IHC images from at least 5 different mice. Statistical significance was determined using 2-way ANOVA with Bonferroni’s multiple-comparison correction. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Relative human VEGFA mRNA levels measured by RT-qPCR in WT, FLCNKO, FLCNKO plus EV, and FLCNKO FLCN-reexpressing MCF7 tumors (top). Concentration of human VEGF-A in EV and FLCNKO cells transfected with siRNA targeting TFE3 in MCF7 cells, and FLCNKO FLCN-reexpressing MCF7 tumor lysates, as measured by ELISA (bottom). Data represent the average ± SEM of at least 5 different mice from each cohort, performed in triplicate. Statistical significance was determined using 2-way ANOVA with Bonferroni’s multiple-comparison correction. *P < 0.05; **P < 0.01; ****P < 0.0001. (F) Relative human VEGFA mRNA levels measured by RT-qPCR in EV, FLCNKO, and FLCNKO cells transfected with nontargeting (NT) control siRNA or siRNA targeting TFE3 in MCF7 cells (top). Concentration of human VEGF-A in EV and FLCNKO cells transfected with siRNA targeting TFE3 in MCF7 cells, and FLCNKO FLCN-reexpressing MCF7 tumor lysates, as measured by ELISA (bottom). Data represent the average ± SEM of at least n = 3, each performed in triplicate. Statistical significance was determined using 2-way ANOVA with Bonferroni’s multiple-comparison correction. **P < 0.01; ****P < 0.0001.