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 5

SH3BP5L enhances RAB11A-mediated invasion and metastasis.

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SH3BP5L enhances RAB11A-mediated invasion and metastasis. (A) Representa...
(A) Representative image of Transwell (upper) and single-cell tracking (bottom) assay of MDA-MB-231 cells KO for SH3BP5L cells and transfected with mCherry-SH3BP5L(WT) (koSH3BP5L/mCherry-SH3BP5L(WT)). For cell tracking, n = 25 (Ctrl), n = 36 (koSH3BP5L), n = 21 (koSH3BP5L/mCherry-SH3BP5L(WT)) cells, respectively. Scale bars: 600 μm and 300 μm (enlarged insets). (B and C) Representative images (B) and relative quantification (C) of H&E staining of lung metastases in NSG mice injected in the tail vein with koSH3BP5L in MDA-MB-231 cells and transfected with mCherry-SH3BP5L(WT) (koSH3BP5L/mCherry-SH3BP5L(WT)). Black arrowheads indicate metastatic foci. Each dot in the graph in C is representative of an injected mouse (n = 5). Scale bars: 60 μm. (D and E) Representative images (D) and relative quantification (E) of metastasized foci in zebrafish injected with MDA-MB-231 cells with koSH3BP5L and transfected with mCherry-SH3BP5L(WT) (koSH3BP5L/SH3BP5L(WT)), mCherry-SH3BP5L(AAA) (koSH3BP5L/SH3BP5L(AAA)), mCherry-SH3BP5L(AK) (koSH3BP5L/SH3BP5LAK), or GFP-RAB11A(Q70L) (koSH3BP5L/RAB11AQ70L). Arrowheads indicate metastatic foci in zebrafish tails. Each dot in the graph in E is representative of an injected zebrafish (n ≥30). Scale bars: 0.5 mm. (F and G) Representative images (F) and relative quantification (G) of H&E staining of lung metastases in NSG mice orthotopically injected with MDA-MB-231 cells with koSH3BP5L and transfected with mCherry-SH3BP5L(WT) (koSH3BP5L/SH3BP5L(WT)), mCherry-SH3BP5L(AAA) (koSH3BP5L/SH3BP5L(AAA)), mCherry-SH3BP5L(AK) (koSH3BP5L/SH3BP5L(AK)), or GFP-RAB11A(Q70L) (koSH3BP5L/RAB11A(Q70L)). Each dot in the graph in G is representative of an injected mouse (n ≥3). Scale bars: 600 μm and 300 μm (enlarged insets). (H and I) In vitro Matrigel Transwell invasion assay (left) and spheroid-forming assay (right) of cells derived from 2 patients with TNBC (patient 1, H; patient 2, I) transduced with either control or 2 independent lentiviral shRNAs targeting SH3BP5L (left; n = 12 fields per group from 3 independent experiments; right: n = 8). Data represent the mean of at least 3 independent experiments ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.005, by 1-way ANOVA.