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The INPP4B Tumor Suppressor Modulates EGFR Trafficking and Promotes Triple

Negative Breast Cancer

Hui Liu1*, Marcia N. Paddock2, Haibin Wang3, Charles J. Murphy2,4, Renee C. Geck1,

Adrija J. Navarro1, Gerburg M. Wulf5, Olivier Elemento4, Volker Haucke3, Lewis C.

Cantley2* and Alex Toker1,6*

1 Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center,

Harvard Medical School, Boston, MA 02115, USA

2 Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA

3 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Roessle-.

Strasse 10, 13125 Berlin, Germany

4 Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY

10021, USA

5 Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess

Medical Center, Harvard Medical School, Boston, MA 02115, USA

6 Ludwig Center at Harvard, Boston, MA 02115, USA

*Corresponding Authors:

Alex Toker: Department of Pathology, Beth Israel Deaconess Medical Center, 330

Brookline Avenue, EC/CLS-633A, Boston MA 02215. Phone: (617) 735-2482; FAX:

(617) 735-2480; email: [email protected]

Research. on December 14, 2020. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 8, 2020; DOI: 10.1158/2159-8290.CD-19-1262

mailto:[email protected]

http://cancerdiscovery.aacrjournals.org/

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Lewis C. Cantley: The Sandra and Edward Meyer Cancer Center, Weill Cornell

Medicine, 413 East 69th St., 13th Floor, Box 50, New York, NY 10021. Phone: (646)

962-6132; Fax: (646) 962-0575; email: [email protected]

Hui Liu: Department of Pathology, Beth Israel Deaconess Medical Center, 330

Brookline Avenue, CLS-628, Boston, MA 02215. Phone: (617) 275-3922; Email:

[email protected]

Running Title: INPP4B Inactivation Drives Triple Negative Breast Cancer

Keywords: INPP4B, triple negative breast cancer, EGFR, PI 3-kinase,

phosphoinositide.

Conflict of Interest Statement:

A.T. is a consultant for Oncologie, Inc. and Bertis, Inc. L.C.C. is a founder and member

of the SAB of Agios Pharmaceuticals and is a founder and member of SAB of Petra

Pharmaceuticals and receives research support from Petra Pharmaceuticals. These

companies are developing novel therapies for cancer.






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Abstract

Inactivation of the tumor suppressor lipid phosphatase INPP4B is common in triple

negative breast cancer (TNBC). We generated a genetically-engineered TNBC mouse

model deficient in INPP4B. We found a dose-dependent increase in tumor incidence in

INPP4B homozygous and heterozygous knockout mice compared to wild-type,

supporting a role for INPP4B as a tumor suppressor in TNBC. Tumors derived from

INPP4B knockout mice are enriched for AKT and MEK gene signatures. Consequently,

mice with INPP4B deficiency are more sensitive to PI3K or MEK inhibitors, compared to

wild-type mice. Mechanistically, we found that INPP4B deficiency increases PI(3,4)P2

levels in endocytic vesicles but not at the plasma membrane. Moreover, INPP4B loss

delays degradation of EGFR and MET, while promoting recycling of RTKs, thus

enhancing the duration and amplitude of signaling output upon growth factor

stimulation. Therefore, INPP4B inactivation in TNBC promotes tumorigenesis by

modulating RTK recycling and signaling duration.




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Significance

Inactivation of the lipid phosphatase INPP4B is frequent in triple negative breast cancer.

Using a genetically engineered mouse model, we show that INPP4B functions as a

tumor suppressor in TNBC. INPP4B regulates receptor tyrosine kinase trafficking and

degradation, such that loss of INPP4B prolongs both PI3K and ERK activation.




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Introduction

The phosphoinositide 3-kinase (PI3K) pathway is one of the most frequently

altered signaling pathways in human cancer, and genetic gain oncogenes or loss of

tumor suppressors that regulate or transduce the PI3K signal leads to tumorigenesis

(1). In growth factor signaling, activation of class I PI3K results in production of the

second messenger PI(3,4,5)P3 (PIP3), which recruits effector proteins such as the

protein kinase AKT to promote cell growth, survival, migration and metabolic

reprogramming (2). Termination of PI3K signaling is achieved by dephosphorylation of

PIP3 by phosphatase and tensin homolog (PTEN). Alternatively, PIP3 can be removed

by the sequential action of the SH2-domain containing inositol phosphatases 1 and 2

(SHIP1/2), to generate PI(3,4)P2, followed by inositol polyphosphate 4-phosphatases A

and B (INPP4A/B) and PTEN, ultimately generating the precursor phosphoinositides

PI(3)P and PI(4)P (3-5).

Gain-of-function, oncogenic mutations in PIK3CA, the gene that encodes the

p110 catalytic subunit of class Ia PI3K, occur with high frequency in estrogen receptor

positive (ER+ve) breast cancers (6). TNBC is a subtype of breast cancer that lacks

targeted therapy options due to lack of expression of ER or HER2, and exhibits a high

degree of molecular heterogeneity (7). In contrast to ER+ve breast cancers, PIC3CA is

not frequently altered in TNBC, instead inactivating mutations or deletion of PTEN and

heterozygous deletion of INPP4B are frequent (8-10). While PTEN has been

established as a bona fide tumor suppressor in many cancer types and loss of PTEN

sensitizes tumors to PI3K inhibitors (11), the function and mechanistic basis of INPP4B

as a tumor suppressor is less clear. INPP4B was originally identified as a tumor




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suppressor in a genetic RNAi screen (12), and in cell-based and xenograft experiments,

INPP4B inactivation leads to elevated PI(3,4)P2 levels, AKT activation, and increased

tumor growth (13,14). By contrast, in ER+ve cells and tumors, INPP4B can actually

function as an oncogene through activation of the serum and glucocorticoid-regulated

kinase 3 (SGK3) pathway (15,16). Although loss of INPP4B protein expression is

observed in 70%-80% of TNBC patient samples (13,17), to the extent that INPP4B-

negativity is identified as the most specific biomarker in basal-like breast cancer (18,19),

whether INPP4B loss functionally mediates TNBC development has not been evaluated

in vivo.

The substrate of INPP4B, PI(3,4)P2, belongs to the family of phosphoinositides

where the head group of the inositol ring can be reversibly phosphorylated and de-

phosphorylated to generate seven distinct species, with preferential localization on

distinct subcellular membrane compartments (20,21). By binding and recruiting effector

proteins, phosphoinositides regulate a multitude of cellular functions including

endocytosis and intracellular vesicle trafficking, cytoskeletal remodeling and signal

transduction (20-22). Importantly, the mechanisms that regulate endocytosis,

degradation or recycling of receptor tyrosine kinases (RTKs) including epidermal growth

factor receptor (EGFR), MET and fibroblast growth factor receptor (FGFR), profoundly

affect the amplitude and duration of signaling output and tumorigenesis (23-28).

PI(3,4)P2 recruits specific effector proteins such as lamellipodin (29) and Tapp1/2 (30),

as well as effectors with more promiscuous phosphoinositide binding such as AKT (31),

Bam32 (32) and sorting nexins (33-35). In addition to promoting AKT signaling,

regulating actin cytoskeletal rearrangements and facilitating clathrin-mediated




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endocytosis (36,37), localized production of PI(3,4)P2 at late endosome/ lysosomes can

suppress mTORC1 activation under specific nutrient-deprived conditions (38,39).

However, the precise mechanism(s) by which INPP4B-loss contributing to TNBCs has

not been determined. Here, we have generated a mouse model of INPP4B deletion in

the context of TNBC, and have used it to decipher the role of PI(3,4)P2 and RTK

trafficking in tumorigenesis.




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Results

Tumor Penetrance Increases Upon Genetic Ablation of INPP4B in a TNBC Mouse

Model

To determine whether genetic loss of INPP4B plays a functional role in the

etiology of TNBC, we generated an INPP4B-deficient model by crossing INPP4B-

phosphatase knockout mice (40) with a TNBC model in which Tp53 and Brca1 are

deleted upon K14-driven Cre-expression in mammary epithelial cells (41)

(Supplementary Figure S1A for breeding schematics). We confirmed deletion of exon

22 by genotyping (Supplementary Figure S1B) as described (42). As previously

reported (40,43), INPP4B phosphatase deletion alone did not result in any appreciable

phenotype, although we did observe age- and sex-dependent weight gain resulting in

increased body weight in female mice over 8 months of age with regular chow

(Supplementary Figure S1C). Similarly, modest alterations in glucose clearance when

challenged with glucose, but not with insulin, were observed in INPP4B heterozygous

(HET) and knockout (KO) mice compared to wild-type (WT) littermates (Supplementary

Figure S1D). When crossed into the TNBC mouse model, INPP4B phosphatase loss

resulted in a dose-dependent increase in mammary tumor penetrance. While mammary

tumors developed in 17.2% of WT mice, 38% of mice developed mammary tumors in

INPP4B HET mice, which increased to 53.7% in INPP4B in KO mice (Figure 1A). This

significant increase in mammary tumor penetrance was also manifested as increased

mammary tumor-related death (Figure 1B). In addition, we observed a slightly

shortened lifespan for mammary tumor-bearing mice. The mean life span due to

mammary tumor development for INPP4B WT mice was 290.8 days, while that for




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INPP4B HET mice was 232.9 days (p=0.006, one-way ANOVA), and similarly 239 days

for INPP4B KO mice (p=0.01, one-way ANOVA) (Figure 1C).

To understand the nature of mammary tumors developed from distinct INPP4B

genetic backgrounds, we analyzed tumor histology by immunohistochemistry (IHC).

The majority (76.19%) of tumors scored as mammary adenocarcinomas (Figure 1D:a),

although ductular carcinomas, mammary adenocarcinoma mixed with focal squamous

cell carcinoma and mammary cystic differentiated adenocarcinomas were also noted

(Figure 1D:b-d). The triple negative nature of these mammary tumors was confirmed

by estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth

factor receptor 2 (HER2) IHC as previously reported using this TNBC model (42), as

well as RNAseq analyses using AIMS classifiers (Figure 1E), PAM50 classifiers

(Supplementary Figures S1E), and unsupervised hierarchical clustering

(Supplementary Figures S1F).

Gross Genome Instability is not Significantly Affected in Murine Tumors Upon

INPP4B Deletion

In addition to its role in PI3K pathway signaling, loss of INPP4B elicits DNA

repair defects in ovarian cancer which can result in chromosomal instability and

increased tumor incidence (44). Therefore, we performed whole exome sequencing

and evaluated markers for genome instability, including the number of chromosome

breaks (Figure 2A), chromosomal translocations (Figure 2B), and small insertions

and deletions (INDELs) (Figure 2C), but did not find any statistically significant

differences in these parameters. By contrast, an increase in the number of point




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mutations was observed in INPP4B KO tumors compared to WT (Figure 2D). Among

the different point mutations, we observed a significant increase in C to T mutations

when comparing INPP4B HET or KO to WT tumors (Supplementary Figures S2A

and S2B), but no differences in T to other nucleotide mutations (Supplementary

Figure S2C). Overall, our data demonstrate that INPP4B loss does not affect gross

chromosomal instability during tumor development in this model, although the number

of single nucleotide point mutations is increased.

INPP4B Loss Enhances PI3K and ERK Pathway Activation

We next compared the transcriptional profile of tumors developed from the

INPP4B mouse models. Using gene set enrichment analysis (GSEA), we found an

enhanced AKT pathway gene signature in INPP4B HET or KO mice compared to WT

mice (Figure 3A). Surprisingly, tumors developed from INPP4B HET or KO mice also

showed an increased mitogen-activated protein kinase kinase (MEK) pathway gene

signature (Figure 3B). To confirm this observation in vitro, we knocked down

INPP4B in breast epithelial MCF10A cells using shRNA, and this also resulted in

enhanced PI3K pathway activation as reported by increased pAKT1, pAKT2 and

pPRAS40 as well as increased extracellular-signal-regulated kinase (ERK)

phosphorylation and pS6 (pS235/p236) (Figure 3C). Moreover, both the duration and

magnitude of pAKT and pERK were enhanced in EGF-stimulated cells in which

INPP4B was transiently downregulated using siRNA (Figure 3D and Supplementary

Figure S3A and S3B). This was also observed in primary mammary epithelial cells

stimulated with EGF over a time course (Supplementary Figure S3C).




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Phenotypically, INPP4B reduction promoted MCF10A cell proliferation under serum-

deprived conditions when supplemented with EGF (Figure 3E), but not in cells grown

with complete media (Supplementary Figure S3D). Finally, over-expression of

INPP4B in the TNBC cell line MDA-MB-231 resulted in significantly reduced spheroid

growth in 3D (Supplementary Figure S3E). In summary, the in vivo and in vitro

results above show INPP4B loss promotes both PI3K and ERK pathway activation,

which may contribute to mammary tumor development in TNBC.

Endogenous Tumors with INPP4B Ablation are More Sensitive to PI3K and MEK

Inhibition

We reasoned that if TNBC cells with reduced INPP4B levels become more

dependent on PI3K and ERK signaling for tumor initiation and/or maintenance, then

pathway inhibition may show a more pronounced effect compared to cells that retain

INPP4B. We first tested this hypothesis in vitro, and generated INPP4B knockdown

(using si/shRNA and CRISPR-Cas9) (Supplementary Figure S4A) and treated cells

with PI3K pathway inhibitors, including the pan class I PI3K inhibitor BKM-120 and the

catalytic AKT inhibitor GDC0068. We found that in both cases, INPP4B loss

increased sensitivity to inhibitor treatment, resulting in statistically significant

decreases in IC50 to both BKM120 (Figure 4A, Supplementary Figures S4B and

S4C, n=3) and GDC0068 (Figure 4B, Supplementary Figure S4D, n=3). We also

observed a trend showing decreased IC50 for the MEK inhibitor trametinib, however

this decrease was not statistically significant (Supplementary Figure S4E). To test

this hypothesis in vivo, we implanted tumors developed from INPP4B WT, HET, or




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KO backgrounds into the mammary glands of recipient nude mice(45), and carried out

in vivo drug treatment using pre-determined doses once tumors reached 7-8 mm in

diameter (42) (Figure 4C). We confirmed the efficacy of BKM120, the p110-specific

PI3K inhibitor BYL719 and the MEK inhibitor trametinib in pathway inhibition by

immunoblotting tumor lysates for pAKT and pERK, respectively (Supplementary

Figures S4F and S4G). We found that both BKM120 and BYL719 improved overall

survival (Figure 4D), while trametinib delayed tumor growth (Figure 4E) without

affecting overall survival (Supplementary Figure S4H). However, tumors developed

from the INPP4B loss (HET and KO) background were more sensitive to either

BKM120 or BYL719 (Figure 4F) or trametinib (Figure 4G), when compared to tumors

developed from the INPP4B WT background. Therefore, INPP4B loss confers

sensitivity to both PI3K and ERK pathway inhibition.

INPP4B Loss Results in Increased PI(3,4)P2 in Intracellular Vesicles

INPP4B is a lipid phosphatase that dephosphorylates the 4’ position of

PI(3,4)P2 to generate PI(3)P, and therefore loss or inactivation of INPP4B leads to

increased pools of PI(3,4)P2. Consistent with this model, CRISPR-Cas9-mediated

INPP4B reduction in MCF10A cells resulted in increased EGF-stimulated PI(3,4)P2 as

measured by 3H-inositol labelling, whilst the levels of other phosphoinositides were

unaffected (Figure 5A and Supplementary Figure S5A). PI(3,4)P2 has been shown

to be localized to the plasma membrane where it regulates clathrin-mediated

endocytosis, as well as at intracellular vesicles where it stimulates AKT signaling

(46,47). Consistent with previous studies, immunofluorescence staining using anti-




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PI(3,4)P2 antibodies in INPP4B-downregulated cells revealed an increase in PI(3,4)P2

in intracellular vesicles following EGF stimulation (Figure 5B and 5C). By contrast,

the PI(3,4)P2 plasma membrane pool only transiently increased at 3min and remained

largely unaffected at other time points upon INPP4B downregulation (Supplementary

Figures S5B and S5C). These data are consistent with the model that INPP4B

contributes to the intracellular endomembrane pool of PI(3,4)P2 biosynthesis upon

growth factor stimulation, leading to downstream pathway activation.

INPP4B Depletion Results in Delayed EGFR Degradation

In initial INPP4B knockdown experiments we noticed that in addition to

enhanced pAKT and pERK, total EGFR protein levels were consistently elevated

compared to control cells (Figure 3D and Figure 6A). Increased EGFR expression

was confirmed by siRNA-mediated downregulation of INPP4B, with no alterations in

EGFR and slight change in MET mRNA transcript levels (Figure 6B and

Supplementary Figure S6A, S6B and S6C). Given the established functions of

phosphoinositides, including PI(3,4)P2 in RTK trafficking (36), we investigated whether

INPP4B loss affects RTK trafficking and signaling. We found that whilst total EGFR

protein levels decreased in control siRNA-treated cells upon a time course of EGF

stimulation, degradation of EGFR was delayed in INPP4B-siRNA treated cells (Figure

6C and 6D). This was confirmed using INPP4B CRISPR-Cas9 cells stimulated with

EGF (Figure 6E and Supplementary Figures S6D and S6E). However, INPP4B

reduction did not alter sensitivity to the EGFR inhibitor Erlotinib (Supplementary

Figure S6F). In TNBC, EGFR overexpression has been shown to play an important




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role during tumor development, although other RTKs including MET also contribute

(48). Consistent with this, total MET protein levels are increased in response to HGF

stimulation in INPP4B knockdown cells compared to control (Figure 6F). In addition,

pAKT and pERK were enhanced and prolonged in response to HGF (Figure 6F),

similar to that observed in EGF-stimulated cells.

Next, we tracked EGFR subcellular localization dynamics upon EGF

stimulation using immunofluorescence (IF). At early time points, accumulation of

EGFR in intracellular vesicles was not affected by INPP4B reduction in response to

EGF (Supplementary Figure S6G). By contrast, by 60 and 90 min EGFR trafficked

to a perinuclear region in control siRNA-treated cells, but remained scattered in

INPP4B siRNA-treated cells. By 180 min, EGFR staining was significantly diminished

in control cells, whereas in INPP4B knockdown cells EGFR was still detectable with a

perinuclear staining pattern (Figure 6G and Supplementary Figure S6H).

The prediction from the in vitro INPP4B knockdown experiments would be that

in the setting of INPP4B loss in vivo, EGFR protein levels would be elevated.

Immunohistochemistry staining of EGFR on the INPP4B mouse tumors revealed that

while both tumors derived from the INPP4B WT cohort showed low levels of EGFR

expression, 5/6 of tumors from the INPP4B HET or 7/10 tumors from the KO

backgrounds showed medium to high level expression of total EGFR (Figure 6H).

INPP4B Affects Trafficking of EGFR to Late Endosome/Lysosomes and RTK

Recycling




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To further investigate trafficking of RTKs in the context of INPP4B loss, we

performed IF to measure co-localization of EGFR with endosomal markers. At all

time points tested, the intensity of staining and vesicle size of the early endosome

antigen 1 (EEA1) marker was not affected upon INPPB reduction with siRNA

(Supplementary Figure S7A and S7B). Similarly, the co-localization of EEA1 with

EGFR in INPP4B siRNA-treated cells was unaffected at early time points, but

persisted at later time points (60, 90 and 180min) (Figure 7A and Supplementary

Figure 7C). At early time points (10 and 30 min), we also did not observe significant

differences in the staining intensity of the late endosome/lysosome marker CD63 or

CD63-positive vesicle size (Figure 7B). By contrast, at later time points when EGFR

traffics to the late endosome/lysosome for degradation, CD63 intensity decreased

with a concomitant increase in vesicle size in control cells, however, there was no

increase in INPP4B knockdown cells (Figure 7B). Consistent with this observation,

the dynamics of EGFR-CD63 co-localization was significantly altered: In control cells

internalized EGFR progressively accumulated in CD63-positive late endosomes

before being degraded, while a much less pronounced late endosomal EGFR

accumulation was observed in INPP4B knockdown cells (Figure 7C and

Supplementary Figure S7D). These data suggest a defect in EGFR trafficking from

early endosomes to late endosomes/lysosomes. Furthermore, we found increased

recycling of EGFR to the plasma membrane in INPP4B-siRNA treated cells (Figure

7D), explaining its increased surface levels (Figure 7E). Our data indicate that loss of

INPP4B delays trafficking of the EGFR from early endosomes to the late




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endosomes/lysosomes, thus delaying receptor degradation, and thereby sustaining

downstream signaling.




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Discussion

In this study, we developed a genetically-engineered mouse model to provide

evidence that INPP4B inactivation drives TNBC. Mechanistically, we uncovered a

function for INPP4B in regulating the trafficking and degradation of EGFR and MET.

INPP4B reduction results in increased PI(3,4)P2 accumulation in intracellular vesicles,

delaying trafficking of EGFR from early endosomes to late endosomes/lysosomes upon

EGF stimulation, whilst promoting recycling of EGFR to the cell surface. As a result,

INPP4B loss delays EGFR degradation with a concomitant prolonged duration and

amplitude of both AKT and ERK signaling, thereby promoting tumorigenesis.

Consequently, INPP4B inactivation sensitizes TNBC cells to both PI3K and MEK

inhibitors in vitro and in vivo.

Transcriptional profiling has revealed the extensive genetic heterogeneous nature of

TNBC, with multiple distinct subgroups classified according to unique expression

profiles with important clinical implications (49). Similarly, large-scale sequencing

studies have detected diverse but low-frequency oncogenic mutations in numerous

genes in TNBC, many of which contribute to PI3K and ERK pathway (8,50-52).

Approximately 40% of ER+ve breast cancer patients harbor activating PIK3CA

mutations, and the p110-specific inhibitor Piqray (Alpelisib) was recently approved for

the treatment of PIK3CA-mutant, ER+ve breast cancer in combination with fulvestrant in

post-menopausal women with advanced or metastatic disease (53). By contrast, less

than 10% of TNBC patients possess oncogenic PIK3CA mutations and to date there is

no approved molecular targeted therapy for TNBC. Given the frequency of PI3K




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pathway hyperactivation in TNBC, targeting aberrant PI3K pathway activation remains a

promising option. In our GEMM model, mammary tumors resulting from INPP4B

heterozygous or homozygous deletion mice are more sensitive to PI3K pathway

inhibition (Figure 4), suggesting that loss of INPP4B may be a predictive marker for

sensitivity to PI3K inhibition, consistent with previous studies in cell lines (54).

Since AKT functions to modulate phenotypes associated with malignancy including

proliferation, survival, migration, and metabolism, and is frequently hyperactivated in

many cancers, numerous small molecule inhibitors have been developed for clinical use

(2). As single agents, AKT inhibitors have shown minimal efficacy in clinical trials (55).

In our GEMM model, INPP4B loss increases sensitivity to the AKT inhibitor GDC0068

(Ipatasertib) in vitro. In clinical trials, the AKT inhibitor AZD5363 did not significantly

improve progression-free survival compared to paclitaxel alone in ER+ve breast cancer

harboring PIK3CA mutations (NCT01625286, (56)). By contrast, addition of GDC0068

to paclitaxel as neoadjuvant therapy in early-stage TNBC patients harboring

PIK3CA/AKT/PTEN alternations showed a favorable response (complete response 39%

for GDC0068+Paclitaxel vs 9% Paclitaxel alone; NCT02301988 (57)). Given that

INPP4B loss is common in TNBC (13,14,18,19) and promotes hyperactivation of AKT, it

would therefore be interesting to evaluate the response of INPP4B-deficient TNBC to

AKT inhibitors in vivo and in the clinic.

Compared to ER+ve or ERBB2- (HER2)-amplified breast cancer, EGFR overexpression

is a frequent event in TNBC. Depending on the patient population and IHC test used,




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13%-76% of TNBC tumors overexpress EGFR at the protein level (58). Yet, analyses

of METABRIC and TCGA reveal that only approximately 5% of patients harbor ERBB1

(EGFR) gene amplification (59). This indicates that additional mechanisms must exist

to account for increased EGFR protein in TNBC. Several mechanisms have been

shown, including loss of PTEN (60), loss of BRCA1 (61) and increased expression of

tissue transglutaminase (62). Our data are indicative of an additional mechanism

whereby loss of INPP4B increases EGFR stability contributing to enhanced EGFR

downstream signaling.

EGFR overexpression in TNBC indicates that it could serve as a potential therapeutic

vulnerability for this subtype of breast cancer. However, numerous anti-EGFR

therapeutics used as single agents or in combination with chemotherapy in TNBC have

not shown durable therapeutic responses (63-66). One can speculate that more

accurate patient stratification could improve treatment outcomes, for example by

accruing patients expressing elevated levels of EGFR. In our studies, drug sensitivity

assays with Erlotinib administered in cells with INPP4B reduction did not appreciably

shift the IC50. These data are consistent with previous observations which showed that

EGFR activation is required for initial endocytosis (27,67). Since Erlotinib inhibits EGFR

phosphorylation, and stabilization of EGFR upon INPP4B reduction occurs after ligand-

induced receptor endocytosis, EGFR inhibition would be predicted to be ineffective in

INPP4B-null tumors.




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The mechanism(s) by which loss of INPP4B and increased vesicular PI(3,4)P2 affect

EGFR degradation and recycling, and as a result total EGFR expression, is presently

unknown. Upon ligand-induced clathrin-mediated endocytosis, ubiquitinated EGFR and

endosomal PI(3)P recruit ESCRT (endosomal sorting complexes required for transport)

complexes for sorting into intraluminal vesicles for degradation (68-70). The INPP4B

interactome has not been deciphered and it is not yet clear whether INPP4B can directly

affect the ubiquitin ligase activity of c-Cbl docked onto the EGFR, thereby affecting

EGFR/Hrs interaction, as has been shown for SHIP2 (71). Alternatively, INPP4B could

directly or indirectly interact with ESCRT complexes, since INPP4A/B also localizes to

endosomes (43,72,73). Our results show that INPP4B depletion results in increased

PI(3,4)P2 in intracellular vesicles, consistent with previous studies (46,74). Although a

subset of PI(3,4)P2-positive vesicles colocalize with EEA1, our preliminary studies show

that only a small percentage (1.7%-6.1% depending on the time point) colocalize with

internalized EGFR. By contrast, up to 50% of EGFR-positive vesicles are also positive

for EEA1. Within this small subset of EGFR-positive vesicles, PI(3,4)P2 intensities were

comparable between control siRNA- and INPP4B siRNA-treated cells. Several potential

mechanisms may contribute to defects in RTK degradation and enhanced recycling.

First, a block in PI(3,4)P2 degradation due to INPP4B loss may result in the reduction of

a local endosomal pool of PI(3)P derived from PI(3,4)P2 (i.e. a minor pool not separable

from the total cellular PI(3)P pool in our HPLC analysis), upstream of PI(3,5)P2

production via PIKFYVE and EGFR degradation. In addition, failure to dephosphorylate

PI(3,4)P2 via INPP4B may promote funneling of PI(3,4)P2 into an alternative PTEN-

mediated degradation pathway towards PI(4)P (3), a lipid that promotes recycling to the




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cell surface (75,76). Alternatively, PI(3,4)P2-rich endomembranes may recruit effectors

to promote recycling. One possibility is that SNX18, a PX-BAR-containing sorting nexin

that belongs to the SNX9/18/30 family may contribute to this mechanism. SNX18 binds

to PI(3,4)P2 as well as other phosphoinositides (33,47) and colocalizes with Rab11 but

not EEA1, and has been shown to promote tubulated recycling endomembranes (77-

79).

Traditionally viewed as a breakdown product of PI(3,4,5)P3, recent studies have shown

that PI(3,4)P2 has important signaling roles in its own right (36,80). Different enzymes

contribute to the localized production of this signaling lipid, exerting seemingly different

biological activities. For example, class II PI3K C2 at clathrin-coated pits is important

for the synthesis of PI(3,4)P2 to mediate constriction of late-stage clathrin-coated pits

(47,81,82). Class II PI3K C2 at late endosomes/lysosomes during growth factor

starvation suppresses mTORC1 activity (39). Alternatively, class I PI3K activation by

RTKs such as EGFR generates PI(3,4,5)P3, which is dephosphorylated by 5’

phosphatases to give rise to PI(3,4)P2, contributing to endosomal PI(3,4)P2 (5).

Although the relative quantitative contribution from each of the routes during EGFR

intracellular trafficking is not precisely understood, it is generally accepted that

degradation from PI(3,4,5)P3 contributes to a significant fraction of PI(3,4)P2 in

intracelllular vesicles upon RTK activation (36,37). Additional studies are required to

determine whether class II PI3Ks can function in an analogous manner to INPP4B in

TNBC etiology, especially given their importance in physiology and disease (83).




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Recent studies have found that PTEN can also degrade PI(3,4)P2, and that combined

depletion of INPP4B and PTEN results in synergistic accumulation of PI(3,4)P2 and AKT

activation (3). Since PTEN inactivation is also a frequent event in TNBC and combined

PTEN and INPP4B loss leads to AKT hyperactivation, this provides additional rationale

for preclinical and clinical studies for AKT inhibitors in this setting. At the same time, it is

important to note that while INPP4B functions as a bona fide tumor suppressor in TNBC

and other cancers, studies in cell lines and mice have shown that in ER+ve breast

cancer and colorectal cancer, INPP4B actually functions as an oncogene, potentially

due to copy number gain or overexpression in these tumor types (15,84).

In summary, we have developed a mouse model of TNBC in which INPP4B functions as

a tumor suppressor and regulates receptor tyrosine kinase trafficking and degradation.

We propose a model whereby INPP4B inactivation results in PI(3,4)P2 accumulation in

intracellular vesicles, delaying degradation of RTKs, prolonging both PI3K and ERK

signaling and tumorigenesis and leading to sensitization to pathway inhibitors.




23

Materials and Methods

Mice and histology: Animal experiments were conducted in accordance with

Institutional Animal Care and Use Committee (IACUC) approved protocols (# 099-2015)

at Beth Israel Deaconess Medical Center. K14cre; Brca1flox/flox; Trp53flox/flox mice were

obtained from the Dr. Jos Jonkers (Netherlands Cancer Institute), and INPP4B del/del

mice were obtained from Dr. Takehiko Sasaki (Tokyo Medical and Dental University).

Mouse breeding was carried out as shown in Figure S1A, and genotyping was carried

out as described (40,41)

Orthotopic tumor implantation: Tumor pieces were cut into 2 mm in diameter and

inserted into the 4th mammary fat pad of 8-10-week-old recipient mice via a 0.5 cm2

incision in the skin. The skin was closed with VetBond.

Tumor treatment and tumor measurement: Once tumors reached approximately 8

mm in diameter as measured by electrical caliper (Fisher Scientific), mice were treated

with indicated drugs obtained from MedChemExpress, LLC (BKM120, BYL719 and

Tremetinib). For oral gavage, 100l of drug suspension was administrated daily for six

consecutive days, followed by one drug holiday. Tumor sizes were measured twice a

week (length and width), and tumor volume was calculated as

(3.14*length*width*width/6).

Genomic DNA and library preparation: Genomic DNA from tumor or liver samples

was prepared following the protocol for Promega ReliaPrep Tissue DNA Miniprep




24

System (A2051). SureSelect or NimbleGen Mouse exome capture kits were used to

generate DNA library according to manufacturer’s instructions. Sequencing was carried

out using HiSeq4000 (Illumina) using paired end clustering and 51x2 cycles

sequencing.

Mutation and copy number analysis: See the supplementary methods for details.

Somatic mutations were identified upon removing any mutations found in any tail, liver

or normal mammary control samples, in mouse dbSNP, or with insufficient coverage in

the control samples. Mutations were annotated with SnpEff. Copy number variants were

called using CNVkit after removing low-quality reads. Sample-specific thresholds were

computed to call amplifications and deletions.

RNA preparation and library preparation: Total RNA was prepared following the

protocol for Promega ReliaPrep RNA Tissue Miniprep System (Z6111), and RNA

integrity and concentration were measured using the Agilent 2100 Bioanalyzer (Agilent

Technologies). cDNA libraries were prepared from 15–35 ng RNA starting material (RIN

values >6.0), using the TruSeq RNA Sample Preparation Kit (Illumina) according to the

manufacturer’s instructions, and quality was checked on an Agilent 2100 Bioanalyzer

(Agilent Technologies). Sequencing was carried out on the HiSeq 2500 (Illumina) using

paired end clustering and 51x2 cycles sequencing.

Immunoblotting: Tumors and cells were lysed in RIPA lysis buffer with protease

inhibitors (Roche) and phosphatase inhibitors (Sigma). Equal amount of total protein




25

lysates were used for immunoblotting. The following antibodies were from Cell Signaling

Technology (Beverly, MA) and were used at 1:1000 dilution: pAKT (#3787), AKT

(#4691), pERK (#4370), ERK (#4695), pMET (#3077), MET (#3127), pEGFR (#3777),

EGFR (#4267), pPRAS40 (#2997), PRAS40 (#2691), Vinculin (#13901). Other

antibodies used in this paper are as follows: INPP4B (Abcam, Ab81269), beta-actin

(Sigma, A2228).

Cell lines: MCF10A and MDA-MB-231cells were obtained from the American Type

Culture Collection (ATCC) and authenticated using short tandem repeat (STR) profiling.

Primary human mammary epithelial cells (HMECs) were obtained as described (42).

MCF10A cells and HMECs were maintained in DMEM/Ham’s F12 (CellGro)

supplemented with 5% equine serum (CellGro), 10 mg/mL insulin (Life Technologies),

500 ng/mL hydrocortisone (Sigma-Aldrich), 20 ng/mL EGF (R&D Systems), and 100

ng/mL cholera toxin (Sigma-Aldrich). MDA-MB-231 cells were maintained in DMEM

(CellGro) supplemented with 10% fetal bovine serum (FBS) (Gemini). Cells were

passaged for no more than 2 months and routinely assayed for mycoplasma

contamination (MycoAlert, Lonza).

Lentivirus Production and Cell Infection: Lentivirus preparation and infection were

carried out as described using Lipofectamine 2000 (ThermoFisher) (42).

Cell Proliferation: 1500 cells were plated in 96-well plates in 200l of cell culture

medium and measured with CellTiter-Glo (Promega, G7572). For 3D culture, each well




26

of the 8 well chamber slides were coated with 50l growth-factor reduced Matrigel

(Corning), followed by seeding 3000 cells in growth medium containing 2% Matrigel.

IC50 Measurement: 5000 cells were plated in 96 well plates and changed to medium

(growth media or serum-free media/50ng/ml EGF) containing inhibitors at different

concentrations. The following inhibitors were used: BKM-120 and BYL719

(MedChemExpress, LLC), GDC0068 (Selleck), Trametinib (Selleck), Erlotinib (Selleck).

After 72 hours, the relative numbers of remaining cells were measured with CellTiter-

Glo (Promega, G7572).

Immunofluorescence: Cells were seeded on serum pre-coated glass cover slips

overnight, serum-starved for 16-18 hours, and stimulated with 50ng/ml EGF. At

indicated time points, cells were fixed in 4% paraformaldehyde, followed by 0.1% Triton

X-100 in PBS (5min, RT), blocked at 37°C with 10% normal goat serum in PBS for

30min, and incubated with the following primary antibodies at 4°C overnight. After

washing, cells were incubated with Alexa Fluor 488 or 568 conjugated secondary

antibodies (Molecular Probes). Primary antibodies are as following: EGFR (CST #4267

for intracellular staining), EGFR-AF488 (Biolegend #352908 for surface staining); EEA1

(BD bioscience#610457), CD63 (BioLegend #353013). Images were acquired with

Zeiss LSM 880 upright confocal system and analyzed with Volocity imaging software

(Improvision, Perkin Elmer).

For PI(3,4)P2 staining, after fixation, cells were permeabilized and blocked with PBS

containing 0.5% Saponin, 1% BSA and 10% normal goat serum for 30min for plasma




27

membrane (PM) staining, or with 20µM Digitonin in buffer A (20mM Pipes,PH6.8,

137mM NaCl and 2.7mM KCl) followed by 30min in buffer A containing 5% normal goat

serum and 50mM NH4Cl for intracellular vesicle (IV) staining. Anti-PI(3,4)P2 antibody

(Echelon Biosciences Z-P034b) was diluted 1:150 in PBS or Buffer A (for PM or IV,

respectively) containing 5% normal goat serum, incubated for 1 hour at room

temperature, washed before incubating with Alexa Fluor 568 conjugated anti-mouse

secondary antibody. Images were acquired using a spinning disk confocal microscope

(Ultraview ERS, Perkin Elmer), analyzed with Volocity imaging software (Improvision,

Perkin Elmer). PI(3,4)P2 levels were quantified using custom written ImageJ macros as

described (47).

Immunohistochemistry (IHC): Slides were deparaffinized, rehydrated and antigen

retrieval was performed using SignalStain® Citrate Unmasking Solution (CST #14746).

Slides were incubated with freshly prepared 3% H2O2 for 10min, washed twice with

ddH2O, once with TBST, blocked in TBST/5% normal goat serum at R.T. for 1 hour.

After incubating with anti-mouse-EGFR antibody (CST #71655) diluted in TBST/5%

goat serum overnight at 40C, the slides were washed 3 x TBST and incubated with

goat-anti-rabbit-biotin secondary antibody at R.T. for 30min. Color development was

carried out following instruction (Vectastain Elite ABC HRP Kit). Slides were

counterstained hematoxylin (#14166), dehydrated and mounted.

Phosphoinositide measurements: as previously reported (73). Briefly, cells were

labeled for 48 hours in inositol-free DMEM with glutamine, 10% dialyzed FBS, and 20



https://www.cellsignal.com/product/productDetail.jsp?productId=14166


28

mCi/ mL 3H myo-inositol. At the indicated times, cells were washed and harvested by

scraping in 1.5 mL ice-cold aqueous solution (1M HCl, 5 mM Tetrabutylammonium

bisulfate, 25 mM EDTA) before adding 2 mL of MeOH and 4mL of CHCl3, vortexed and

centrifuged. The aqueous layer was extracted 3 times using theoretical lower reagent

(CHCl3:MeOH:aqueous solution in 8:4:3 v/v), and organic phase was collected and

dried, deacylated at 55 degrees for 1 hour and dried. To the dried vials, 1 mL of

theoretical upper and 1.5 mL of theoretical lower were added, vortexed, centrifuged and

the aqueous phase was collected and dried. Samples were resuspended in 150 uL

Buffer A (1mM EDTA), injected in anion-exchange HPLC using Partisphere SAX column

and eluted with Buffer B (1mM EDTA, 1M NaH2PO4), detected using an on-line

continuous flow scintillation detector (detailed gradient is provided in the supplementary

methods).




29

Acknowledgements

We thank Drs. Jos Jonkers and Takehiko Sasaki for providing mouse strains, Junyan

Zhang and Kangkang Yang for technical support, members of the Toker and Cantley

laboratories for advice and discussion, Roderick Bronson at the DH/FCC Rodent

Histopathology Core, Lay-Hong Ang and Aniket Gad at BIDMC Confocal Image Core,

Suzanne L. White and Lena Liu for histology work and Eva Csizmadia for

immunohistochemistry, and Luke Dow for plasmid constructs. This work was supported

by: Susan G. Komen postdoctoral fellowship (H.L.); the Ludwig Center at Harvard

(A.T.); the Breast Cancer Alliance (A.T); the Deutsche Forschungsgemeinschaft

TRR186/ A08 (V.H.); NIH R35 CA197588 (L.C.C.), NIH R01 CA226776 (G.M.W.),

Breast Cancer Research Foundation (L.C.C. and G.M.W); a gift from the Jon and Mindy

Gray Foundation (L.C.C.); NIH U54CA210184 (L.C.C.) and NCI F31 CA213460

(R.C.G.).

Author Contributions

Conception and design: H. Liu, L.C. Cantley, A. Toker.

Development of methodology: H. Liu, M.N. Paddock, H. Wang.

Acquisition of data (provided animals, acquired and managed patients, provided

facilities, etc.): H. Liu, M.N. Paddock, H. Wang, R.C. Geck, A.J. Navarro.

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,

computational analysis): H. Liu, M.N. Paddock, H. Wang, C.J. Murphy, R.C. Geck, O.

Elemento, V. Haucke, L.C. Cantley, A. Toker.




30

Writing, review, and/or revision of the manuscript: H. Liu, M.N. Paddack, H. Wang,

C. J. Murphy, G.M. Wulf, O. Elemento, V. Haucke, L.C. Cantley, A. Toker.

Administrative, technical, or material support (i.e., reporting or organizing data,

constructing databases): H. Liu, C.J. Murphy.

Study supervision: H. Liu, O. Elemento, V. Haucke, L.C. Cantley, A. Toker.




31

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Figure Legends

Figure 1: Genetic ablation of INPP4B promotes TNBC formation in a K14cre;

Tp53flox/flox; Brca1flox/flox mouse model. A) Mammary tumor incidence in INPP4B WT,

HET or 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; Tp53flox/flox; Brca1flox/flox;

Inpp4B KO background, including adenocarcinomas, ductal carcinomas, mixed

adenocarcinomas with focal squamous cell carcinomas, and cystic. E) AIMS analysis

was performed using RNAseq data generated from tumors developed in K14cre;

Tp53flox/flox; Brca1flox/flox; Inpp4B WT, HET and KO mice.

Figure 2. INPP4B loss does not significantly affect genomic instability. A) Number of

chromosome breaks in INPP4B WT, HET and KO in the K14cre; Tp53flox/flox; Brca1flox/flox

background. B) Number of chromosome translocations in same cohort as (A). C)

number of INDELS in same cohort as (A). D) Number of point mutations in same cohort

as (A).

Figure 3. INPP4B loss enhances both PI3K and ERK signaling pathway activation in

vivo and in vitro. A) GSEA analysis of tumors developed from INPP4B HET (left) or

KO (right) backgrounds for AKT pathway activation compared to INPP4B WT

background. B) GSEA analysis of tumors developed from INPP4B HET (left) or KO

(right) backgrounds for MEK pathway activation compared to INPP4B WT

background. C) MCF10A cells infected with virus harboring either pLK0.1 vector or




39

pLK01-INPP4B-shRNA were washed, trypsinized, and pelleted. Total cell lysates

were immunoblotted with the indicated antibodies. D) MCF10A cells transfected with

non-target control siRNA (control-siRNA), or INPP4B smart-pool siRNA pool (si-pool-

1, Dharmacon) or siRNA (si-2 from CST), serum starved and stimulated 50ng/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 50ng/ml of EGF, figure is representative of 3

independent experiments, error bars represent SEM, statistics analysis was

performed using 2-way ANOVA.

Figure 4. INPP4B loss increases sensitivity to PI3K and MEK inhibitors. A) MCF10A

cells harboring control-gRNA or INPP4B-gRNA1 treated with increasing

concentrations of GDC0068 for 72 hours in serum-free medium containing 50ng/ml

EGF. Live cells measured using CellTiter Glow and IC50 was calculated (n=4,

student’s 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 50ng/ml

EGF. Live cells were measured as in (A) (n=3, student’s 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 measured.

Tumor sizes on day 9 were divided to those on day 1, statistical analysis was




40

performed using student’s t-test. F) Overall survival of nude mice implanted with

GEMM tumors and treated with BKM120 or BYL719 were analyzed based on 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. Statistics was performed using student’s t-test.

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 50ng/ml of EGF for 0, 5min and 30min and

phosphoinositide levels were measured. N=4, Statistics was performed using

student’s t-test. B) MCF10A cells transiently transfected with non-target control

siRNA or smart-pool INPP4B-siRNA were serum starved and stimulated with 50ng/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.

Figure 6. INPP4B downregulation results in RTK degradation defects. A) MCF10A

cell transiently transfected with control- or INPP4B-siRNA and immunoblotted with the

indicated antibodies (left panel) and quantitated (right panel). B) qRT-PCR using

mRNA from the same samples as in (A) (n=4, student’s t-test). C) MCF10A cells

transfected with control or INPP4B-siRNA, serum-starved and treated with

cycloheximide for 1-hour prior stimulation with 50ng/ml of EGF for the indicated times.

D) Quantification of (C), n=3, 2-way ANOVA. E) MCF10A cells infected with

Cas9/control gRNA or distinct INPP4B-gRNAs, serum starved, and treated with




41

cycloheximide for 1 hour and stimulated with 50ng/ml EGF for the indicated times. F)

MCF10A cells transfected with control or INPP4B siRNA, serum-starved and

stimulated with 50ng/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. Immunofluorescence was

performed with primary anti-EGFR antibody followed with Alexa Fluor-488 conjugated

secondary antibody. H) Tumors developed from K14cre; Tp53flox/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 (2-way ANOVA).

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 50ng/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 co-localization at 3min was set as

100% and was normalized against other time points to evaluate the dynamic change

in co-localization. Error bars represent SEM, and statistical analysis was carried out

using 2-way ANOVA. B) Similar to (A), cells were stained with anti-EGFR and anti-

CD63 and images analyzed using Volocity (Error Bars: SEM; Statistics: 2-way

ANOVA). C) Using the same conditions described in (B), percent co-localization of

CD63-EGFR was analyzed using Volocity. For each siRNA condition, percent co-

localization at time 0 was set as 100% and normalized against other time points to

evaluate the dynamics of co-localization over time (Error Bars: SEM; Statistics: 2-way




42

ANOVA). D) After overnight starvation, cells were chilled to 4°C, loaded with 50ng/ml

EGF for 30min, shifted to 37°C for 15min 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; Statistics:

2-way ANOVA). E) After overnight starvation, cells were trypsinized, fixed and

surface expression of EGFR were stained for FACS analysis. (Error Bars: SEM;

Statistics: student t test, n=5).




Published OnlineFirst June 8, 2020.Cancer Discov Hui Liu, Marcia N Paddock, Haibin Wang, et al. Promotes Triple Negative Breast CancerThe INPP4B Tumor Suppressor Modulates EGFR Trafficking and

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