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The INPP4B Tumor Suppressor Modulates EGFR Trafficking and Promotes Triple-Negative Breast Cancer

Hui Liu, Marcia N. Paddock, Haibin Wang, Charles J. Murphy, Renee C. Geck, Adrija J. Navarro, Gerburg M. Wulf, Olivier Elemento, Volker Haucke, Lewis C. Cantley and Alex Toker
Hui Liu
1Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.
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  • For correspondence: atoker@bidmc.harvard.edu LCantley@med.cornell.edu hliu5@bidmc.harvard.edu
Marcia N. Paddock
2Meyer Cancer Center, Weill Cornell Medicine, New York, New York.
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Haibin Wang
3Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.
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Charles J. Murphy
2Meyer Cancer Center, Weill Cornell Medicine, New York, New York.
4Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, New York.
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Renee C. Geck
1Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.
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  • ORCID record for Renee C. Geck
Adrija J. Navarro
1Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.
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Gerburg M. Wulf
5Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.
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Olivier Elemento
4Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, New York.
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Volker Haucke
3Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.
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Lewis C. Cantley
2Meyer Cancer Center, Weill Cornell Medicine, New York, New York.
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  • For correspondence: atoker@bidmc.harvard.edu LCantley@med.cornell.edu hliu5@bidmc.harvard.edu
Alex Toker
1Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.
6Ludwig Center at Harvard, Boston, Massachusetts.
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  • For correspondence: atoker@bidmc.harvard.edu LCantley@med.cornell.edu hliu5@bidmc.harvard.edu
DOI: 10.1158/2159-8290.CD-19-1262 Published August 2020
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    Figure 1.

    Genetic ablation of Inpp4b promotes TNBC formation in a K14cre; Trp53flox/flox; Brca1flox/flox mouse model. A, Mammary tumor incidence in INPP4B wild-type (WT), heterozygous (HET), or knockout (KO) mice. B, Survival in Inpp4b WT (n = 28), HET (n = 53), or KO (n = 43) mice. C, Life span of mammary tumor–bearing mice in Inpp4b WT, HET, or KO mice. D, Representative images of different histologies from K14cre; Trp53flox/flox; Brca1flox/flox; Inpp4B-KO background, including adenocarcinomas (a), ductal carcinomas (b), mixed adenocarcinomas with focal squamous cell carcinomas (adeno+focal SCC; c), and cystic carcinoma (d). E, AIMS analysis was performed using RNA-sequencing data generated from tumors developed in K14cre; Trp53flox/flox; Brca1flox/flox; Inpp4B WT, HET, and KO mice. PR, progesterone receptor.

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    Figure 2.

    Inpp4b loss does not significantly affect genomic instability. A, Number of chromosome breaks in Inpp4b WT, HET, and KO in the K14cre; Trp53flox/flox; Brca1flox/flox background. B, Number of chromosome translocations in same cohort as A. C, Number of insertions/deletions (indels) in same cohort as A. D, Number of point mutations in same cohort as A. n.s., not significant. Statistical analysis was performed using one-way ANOVA.

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    Figure 3.

    INPP4B loss enhances both PI3K and ERK signaling pathway activation in vivo and in vitro. A, GSEA of tumors developed from Inpp4b HET (left) or KO (right) backgrounds for AKT pathway activation compared with Inpp4b WT background. B, GSEA of tumors developed from Inpp4b HET (left) or KO (right) backgrounds for MEK pathway activation compared with Inpp4b WT background. C, MCF10A cells infected with virus harboring either pLK0.1 vector or pLK01 Inpp4b shRNA were washed, trypsinized, and pelleted. Total cell lysates were immunoblotted with the indicated antibodies. D, MCF10A cells transfected with nontarget control siRNA (NT-si), or INPP4B smart-pool siRNA pool (si-pool-1; Dharmacon) or siRNA (si-2; Cell Signaling Technology), serum starved and stimulated with 50 ng/mL EGF, and immunoblotted with the indicated antibodies (n = 5, representative images are shown). E, Proliferation of MCF10A-pLK0.1 cells or MCF10A-pLK-shINPP4B cells in serum-free medium containing 50 ng/mL of EGF; figure is representative of three independent experiments, error bars represent SEM, statistical analysis was performed using two-way ANOVA.

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    Figure 4.

    INPP4B loss increases sensitivity to PI3K and MEK inhibitors. A, MCF10A cells harboring control guide RNA (gRNA) or INPP4B gRNA1 treated with increasing concentrations of GDC0068 for 72 hours in serum-free medium containing 50 ng/mL EGF. Live cells were measured using CellTiter-Glo and IC50 was calculated (n = 4, Student t test, error bars represent SEM). B, MCF10A cells transiently transfected with control siRNA or INPP4B smart-pool siRNA and treated with increasing concentrations of BKM120 for 72 hours in serum-free medium containing 50 ng/mL EGF. Live cells were measured as in A (n = 3, Student t test, error bars represent SEM). C, Dose and frequency of in vivo drug treatment regimen. D, Nude mice implanted with GEMM tumors treated with BKM120 or BYL719. Statistical analysis of overall survival was performed using log-rank (Mantel–Cox) test. E, Nude mice bearing GEMM tumors were treated with trametinib, and tumor volume was measured. Tumor sizes on day 9 were divided to those on day 1; statistical analysis was performed using Student t test. F, Overall survival of nude mice implanted with GEMM tumors and treated with BKM120 or BYL719 was analyzed on the basis of INPP4B genotypes, WT and INPP4B loss (LOSS = HET + KO). Statistical analysis was performed using log-rank (Mantel–Cox) test. G, Mice bearing GEMM tumors were treated with trametinib and percent change in tumor volume on day 9 of trametinib was normalized to control treatment. Statistical analysis was performed using Student t test; n.s., not significant.

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    Figure 5.

    INPP4B reduction results in increased PI(3,4)P2 in intracellular vesicles. A, MCF10A cells with control gRNA or INPP4B gRNA1, labeled with 3H-inositol, serum starved, and stimulated with 50 ng/mL of EGF for 0, 5, and 30 minutes, and phosphoinositide levels were measured. N = 4, statistical analysis was performed using Student t test. B, MCF10A cells transiently transfected with nontarget control siRNA (Ctrl-si) or smart-pool INPP4B siRNA (INPP4B-si) were serum starved and stimulated with 50 ng/mL of EGF, and stained for intracellular PI(3,4)P2, n = 3. C, Quantification of intracellular PI(3,4)P2 levels upon EGF stimulation. **, P = 0.003; ***, P ≤ 0.0001, Student t test; n.s., not significant.

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    Figure 6.

    INPP4B downregulation results in RTK degradation defects. A, MCF10A cell transiently transfected with control (Control-si) or INPP4B siRNA (INPP4B-si) and immunoblotted with the indicated antibodies (left) and quantitated (right). B, qRT-PCR using mRNA from the same samples as in A (n = 4, Student t test). C, MCF10A cells transfected with control or INPP4B siRNA, serum starved, and treated with cycloheximide for 1 hour prior to stimulation with 50 ng/mL of EGF for the indicated times. D, Quantification of C, n = 3, two-way ANOVA. E, MCF10A cells infected with Cas9/control gRNA or distinct INPP4B gRNAs, serum starved, and treated with cycloheximide for 1 hour and stimulated with 50 ng/mL EGF for the indicated times. F, MCF10A cells transfected with control or INPP4B siRNA, serum starved, and stimulated with 50 ng/mL of EGF for the indicated times and immunoblotted with the indicated antibodies. G, INPP4B siRNA–transfected MCF10A cells were serum starved and stimulated with EGF for the indicated times. IF was performed with primary anti-EGFR antibody followed with Alexa Fluor-488–conjugated secondary antibody. H, Tumors developed from K14cre; Trp53flox/flox; BRCA1flox/flox;INPP4B WT/HET/KO mice were sectioned and IHC was carried out using anti-mouse EGFR antibody. Quantitation is shown on the right (two-way ANOVA).

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    Figure 7.

    INPP4B knockdown increases EGFR recycling and delayed trafficking to the lysosome. A, INPP4B siRNA–transfected MCF10A cells were serum starved, treated with cycloheximide, and stimulated with 50 ng/mL EGF for the indicated times and stained with anti-EGFR and anti-EEA1. Images were analyzed and quantitated using Volocity. For each siRNA condition, percent colocalization at 3 minutes was set as 100% and was normalized against other timepoints to evaluate the dynamic change in colocalization. Error bars represent SEM, and statistical analysis was carried out using two-way ANOVA. B, Similar to A, cells were stained with anti-EGFR and anti-CD63 and images were analyzed using Volocity (error bars: SEM; statistical analysis: two-way ANOVA). C, Using the same conditions described in B, percent colocalization of CD63–EGFR was analyzed using Volocity. For each siRNA condition, percent colocalization at time 0 was set as 100% and normalized against other timepoints to evaluate the dynamics of colocalization over time (error bars: SEM; statistical analysis: two-way ANOVA). D, After overnight starvation, cells were chilled to 4°C, loaded with 50 ng/mL EGF for 30 minutes, shifted to 37°C for 15 minutes to allow internalization, acid washed, and chased for the indicated times. Cells were fixed without permeabilization and stained for surface EGFR. Images were analyzed using Volocity (error bars: SEM; statistical analysis: two-way ANOVA). E, After overnight starvation, cells were trypsinized, fixed, and surface expression of EGFR was stained for FACS analysis (error bars: SEM; statistical analysis: Student t test, n = 5). MFI, mean fluorescence intensity.

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Cancer Discovery: 10 (8)
August 2020
Volume 10, Issue 8
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The INPP4B Tumor Suppressor Modulates EGFR Trafficking and Promotes Triple-Negative Breast Cancer
Hui Liu, Marcia N. Paddock, Haibin Wang, Charles J. Murphy, Renee C. Geck, Adrija J. Navarro, Gerburg M. Wulf, Olivier Elemento, Volker Haucke, Lewis C. Cantley and Alex Toker
Cancer Discov August 1 2020 (10) (8) 1226-1239; DOI: 10.1158/2159-8290.CD-19-1262

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The INPP4B Tumor Suppressor Modulates EGFR Trafficking and Promotes Triple-Negative Breast Cancer
Hui Liu, Marcia N. Paddock, Haibin Wang, Charles J. Murphy, Renee C. Geck, Adrija J. Navarro, Gerburg M. Wulf, Olivier Elemento, Volker Haucke, Lewis C. Cantley and Alex Toker
Cancer Discov August 1 2020 (10) (8) 1226-1239; DOI: 10.1158/2159-8290.CD-19-1262
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