SH3BP5L triggers the RAB11A-regulated integrin recycling network implicated in breast cancer metastasis
Huayi Li, Maria Chiara De Santis, Francesco A. Tucci, Daniela Tosoni, Ping Zhang, Meredith L. Jenkins, Giulia Villari, Maria Grazia Filippone, Elisa Guerrera, Simone Tealdi, Luca Gozzelino, Federico Gulluni, Lorenzo Prever, Cristina Zanini, Marco Forni, Irene Franco, Miriam Martini, John E. Burke, Guido Serini, Carlo Cosimo Campa, Salvatore Pece, Jean Piero Margaria, Emilio Hirsch
Huayi Li, Maria Chiara De Santis, Francesco A. Tucci, Daniela Tosoni, Ping Zhang, Meredith L. Jenkins, Giulia Villari, Maria Grazia Filippone, Elisa Guerrera, Simone Tealdi, Luca Gozzelino, Federico Gulluni, Lorenzo Prever, Cristina Zanini, Marco Forni, Irene Franco, Miriam Martini, John E. Burke, Guido Serini, Carlo Cosimo Campa, Salvatore Pece, Jean Piero Margaria, Emilio Hirsch
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Research Article Cell biology Oncology

SH3BP5L triggers the RAB11A-regulated integrin recycling network implicated in breast cancer metastasis

Abstract

Metastatic progression in aggressive breast cancer (BC) depends on a tightly controlled vesicular recycling network regulated by RAB11, a small guanosine triphosphate enzyme (GTPase). In a cohort of more than 1,000 patients with BC, we identified SH3BP5L as the most highly expressed guanine nucleotide exchange factor (GEF) for RAB11A. High SH3BP5L expression marked an advanced tumor stage, distant metastasis, and poor prognosis, with significant associations in human epidermal growth factor receptor 2–positive (HER2+) and triple-negative breast cancer (TNBC). Using Förster resonance energy transfer (FRET) sensors and artificial intelligence– (AI-assisted) microscopy, we showed that cargo delivery to the plasma membrane required SH3BP5L-dependent activation of RAB11A and assembly of a complex with the anterograde motor KIF5B. This trafficking governed key metastatic features of TNBC, including β1 integrin recycling and α3β1 integrin surface exposure. Inhibition of SH3BP5L or its GEF activity reduced cell spreading in zebrafish and lung metastasis in mouse models, revealing a previously unidentified driver of BC dissemination and a potential therapeutic vulnerability.

Authors

Huayi Li, Maria Chiara De Santis, Francesco A. Tucci, Daniela Tosoni, Ping Zhang, Meredith L. Jenkins, Giulia Villari, Maria Grazia Filippone, Elisa Guerrera, Simone Tealdi, Luca Gozzelino, Federico Gulluni, Lorenzo Prever, Cristina Zanini, Marco Forni, Irene Franco, Miriam Martini, John E. Burke, Guido Serini, Carlo Cosimo Campa, Salvatore Pece, Jean Piero Margaria, Emilio Hirsch

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Figure 3

SH3BP5L interacts with KIF5B to recycle cargo back to the plasma membrane.

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SH3BP5L interacts with KIF5B to recycle cargo back to the plasma membran...
(A) Volcano plot of mass spectrometry analysis performed from GFP immunoprecipitation of HEK293T cells transfected with GFP or GFP-SH3BP5L in the control (serum-free) or recycling (serum-fed) condition. (B and C) Bubble plot illustrating the relationship between the average expression and Top3 metric for (B) RAB and (C) kinesin family proteins. Bubble size represents the log fold change (log FC), and bubble color corresponds to the P value. (D) Representative Western blot of GFP immunoprecipitation performed in HEK293T cells transfected with GFP, GFP-SH3BP5L(WT), GFP-SH3BP5L(AAA) , or GFP-SH3BP5L(AK) constructs in serum-starved or -fed conditions. (E and F) Representative confocal images (E) and quantification (F) of a PLA for RAB11A and KIF5B in MDA-MB-231 cells. n = 6 images for each group from 3 independent experiments. Scale bars: 5 μm. (G and H) Representative Western blots (G) and relative quantification (H) of GFP immunoprecipitation performed on HEK293T cells transfected with GFP, GFP-RAB11A(WT), GFP-RAB11AQ70L, or GFP-RAB11A(S25N) constructs. (I and J) Representative Western blots (I) and relative quantification (J) of a pull-down assay for GST-RAB11A loaded with GDP or GTPγS performed on HEK293T cells transfected with HA-KIF5B. (K) Kymograph of fluorescence intensity for mCherry-SH3BP5L (red) and GFP-KIF5B (green) along an iRFP-RAB11A-labeled (magenta) vesicle over time. Merge shows the colocalization dynamics. (L) Representative image of the vesicle positive for iRFP-RAB11A, mCherry-SH3BP5L, and GFP-KIF5B (top) showing its transport toward the plasma membrane (PM). Quantification of relative fluorescence intensity over time for iRFP-RAB11A, mCherry-SH3BP5L, and GFP-KIF5B, with shaded regions indicating standard deviation (bottom). Scale bars: 1 μm. Data represent the mean of at least 3 independent experiments ± SEM. **P < 0.01, ***P < 0.005, and ****P < 0.001, by 1-way ANOVA.