Integration of Distinct Shca Signaling Complexes Promotes Breast Tumor Growth and Tyrosine Kinase Inhibitor Resistance Jacqueline R

Published OnlineFirst February 16, 2018; DOI: 10.1158/1541-7786.MCR-17-0623

Signal Transduction Molecular Cancer Research Integration of Distinct ShcA Signaling Complexes Promotes Breast Tumor Growth and Tyrosine Kinase Inhibitor Resistance Jacqueline R. Ha1,2, Ryuhjin Ahn1,2, Harvey W. Smith3,4, Valerie Sabourin1, Steven Hebert 1, Eduardo Cepeda Canedo~ 1,2, Young Kyuen Im1,2, Claudia L. Kleinman1,5, William J. Muller3,5, and Josie Ursini-Siegel1,2,3,4,6

Abstract

The commonality between most phospho-tyrosine signaling breast cancers. Using genetic and pharmacologic approaches, networks is their shared use of adaptor proteins to transduce we show that PTB-independent ShcA complexes augment mitogenic signals. ShcA (SHC1)isonesuchadaptorprotein mammary tumorigenesis by increasing the activity of the Src that employs two phospho-tyrosine binding domains (PTB and Fyn tyrosine kinases in an SH2-dependent manner. This and SH2) and key phospho-tyrosine residues to promote bifurcation of signaling complexes from distinct ShcA pools mammary tumorigenesis. Receptor tyrosine kinases (RTK), transduces non-redundant signals that integrate the AKT/ such as ErbB2, bind the ShcA PTB domain to promote breast mTOR and SFK pathways to cooperatively increase breast tumorigenesis by engaging Grb2 downstream of the ShcA tumor growth and resistance to tyrosine kinase inhibitors, tyrosine phosphorylation sites to activate AKT/mTOR signal- including lapatinib and PP2. This study mechanistically dis- ing. However, breast tumors also rely on the ShcA PTB domain sects how the interplay between diverse intracellular ShcA to bind numerous negative regulators that limit activation of complexes impacts the tyrosine kinome to affect breast secondary mitogenic signaling networks. This study examines tumorigenesis. the role of PTB-independent ShcA pools in controlling breast tumor growth and resistance to tyrosine kinase inhibitors. We Implications: The ShcA adaptor, within distinct signaling com- demonstrate that PTB-independent ShcA complexes predom- plexes, impacts tyrosine kinase signaling, breast tumor growth, inately rely on the ShcA SH2 domain to activate multiple Src and resistance to tyrosine kinase inhibitors. Mol Cancer Res; 1–15. family kinases (SFK), including Src and Fyn, in ErbB2-positive 2018 AACR.

Introduction targeted therapy, into clinical practice has significantly improved patient outcome. Unfortunately, numerous tyrosine kinases, Aberrant phospho-tyrosine signaling networks are essential including Met, EGFR, and Lyn (4–6), contribute to the emergence drivers of breast cancer progression (1). With the knowledge that of basal-like breast cancer that has confounded the identification breast cancer is a heterogenous disease, intense research efforts of effective targeted therapies against this poor outcome subtype. have reinforced the understanding that tyrosine kinases contrib- þ In this regard, HER2 breast cancer cells reprogram their tyrosine ute to the emergence of specific breast cancer subtypes. For þ kinome through the aberrant activation of numerous receptor and example, HER2 breast cancer is an aggressive disease driven by nonreceptor tyrosine kinases, including but not limited to ErbB3, the dysregulated activation of the HER2/ErbB2 receptor tyrosine Met, and Src, to confer trastuzumab resistance (7–10). Thus, kinase (RTK; refs. 2, 3). Introduction of trastuzumab, a HER2- tyrosine kinases serve an essential role in establishing breast cancer heterogeneity and therapeutic sensitivity. 1Lady Davis Institute for Medical Research, Montreal, Quebec, Canada. 2Division Adaptor proteins serve an equally important role in phospho- of Experimental Medicine, McGill University, Montreal, Quebec, Canada. 3Depart- tyrosine signaling. Although many adaptor proteins lack intrinsic ment of Biochemistry, McIntyre Medical Building, McGill University, Montreal, catalytic activity, they are critical intermediates that transduce Quebec, Canada. 4Goodman Cancer Research Centre, Montreal, Quebec, signals downstream of tyrosine kinases by integrating multimeric Canada. 5Department of Human Genetics, Strathcona Anatomy & Dentistry signaling complexes. The ShcA adaptor protein is an essential 6 Building, McGill University, Montreal, Quebec, Canada. Gerald Bronfman signaling node downstream of ErbB2 to promote breast cancer Department of Oncology, McGill University, Montreal, Quebec, Canada. initiation (11). Specifically, ShcA possesses two phospho-tyrosine Note: Supplementary data for this article are available at Molecular Cancer binding domains (PTB, SH2) and three tyrosine phosphorylation Research Online (http://mcr.aacrjournals.org/). sites (Y239/Y240/Y313) to transduce phospho-tyrosine– þ Corresponding Author: Josie Ursini-Siegel, Lady Davis Institute for Medical dependent signals (12). Key RTKs that are activated in HER2 Research, 3755 Cote St. Catherine Road, Montreal, Quebec H3T 1E2, Canada. breast tumors, including ErbB2, EGFR, and ErbB3, bind the ShcA Phone: 514-340-8222, ext. 26557; Fax: 514-340-7502; E-mail: PTB domain through a conserved NPXpY motif (13, 14) and [email protected] require a functional PTB domain to promote breast tumor initi- doi: 10.1158/1541-7786.MCR-17-0623 ation and progression (15). Once activated, RTKs bind the PTB 2018 American Association for Cancer Research. domain of ShcA, leading to phosphorylation of tyrosine residues

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in the CH1 domain (Y239/Y240/Y313). This creates docking 450-141-WL). NIC/Src / cell lines were established from mam- sites for Grb2/SOS and Grb2/Gab1 complexes, which activate mary tumor virus (MMTV)/Neu-internal ribosome entry site the Ras/ERK and PI3K/AKT pathways, respectively (16, 17). (IRES)-Cre (NIC) mammary tumors, in which both c-Src alleles In addition to its protumorigenic properties, the ShcA PTB have been deleted by Cre-mediated excision (25–27). Culture domain is also paradoxically important for signal termination. conditions for these cells have also been described previously This property of ShcA ensures that the strength and duration of (25–27). NIC/Src / cells were stably transfected with a pQCXIB protumorigenic responses is tightly controlled. Indeed, the ShcA expression vector (Addgene catalog no. 22266) subcloned with PTB domain binds numerous negative regulators, including Flag-tagged wild-type ShcA or the various ShcA mutants described SHIP2, PTPe, and ERK. While some of these interactions require above. Transfected cells were selected with 8 mg/mL blasticidin the phospho-tyrosine binding pocket of the ShcA PTB domain (Wisent Bio Products catalog no. 400-190-EM). Cell lines repre- (PTP-PEST, SHIP2), others are phospho-tyrosine independent sent pools of six expressing clones. All cell lines were routinely (PTPe, ERK; refs. 18–20). Several studies have established the screened for mycoplasma contamination using a Mycoprobe ShcA PTB domain as a temporal switch to control EGFR signaling Mycoplasma Detection Kit (R&D Systems catalog no. CUL001B), networks by facilitating the delayed recruitment of PTP-PEST at least once per month or one day prior to any mammary fat pad to terminate ShcA-coupled mitogenic responses (21–23). This injection. corroborates in vivo studies, which demonstrated that the loss of PTB-driven ShcA signaling delayed breast tumor initiation but Cell line authentication paradoxically potentiated subsequent tumor growth (15). NMUMG cells were purchased from ATCC and NMuMG- We previously demonstrated that PTB-dependent signaling NeuNT cell lines (ErbB2 transformed) were generated from tumor complexes promote breast cancer growth by increasing explants and cultured as described previously (11). NIC/Src / AKT/mTOR signaling downstream of the ShcA tyrosine phos- and MT cell lines were generated and cultured as described phorylation sites (24). In addition, we showed that the ShcA PTB previously (25, 27–29). Experiments performed on cell lines were domain dynamically controls signaling networks in breast cancer passaged no more than 8 times per month. cells, not only to transduce tumorigenic signals downstream of RTKs including ErbB2 (PTB-dependent), but also to create a Pharmacologic inhibitors negative feedback loop that prevents secondary activation of Cell lines were treated with media containing DMSO (BioShop ShcA-SH2–driven complexes (PTB-independent). Given the catalog no. DMS666), PP2 pan-Src Family kinase inhibitor þ absolute requirement for ShcA signaling in ErbB2 breast cancer (2 mmol/L; Sigma-Aldrich catalog no. P0042), lapatinib ditosylate progression, whether and how unique PTB-dependent and EGFR and HER2/ErbB2 inhibitor (500 nmol/L; Selleck Chemicals -independent signaling complexes converge to promote mam- catalog no. S1028), or Torin1 mTOR inhibitor (50 nmol/L; Tocris mary tumorigenesis is unknown. In this study, we show that catalog no. 4247). PTB-independent complexes augment mammary tumorigenesis by increasing the activity of the Src and Fyn tyrosine kinases in an Clonogenic assay SH2-dependent manner. Surprisingly, the ShcA tyrosine phos- For each cell line, 50 cells were seeded into 12-well plates and phorylation sites are dispensable for the ability of these treated with various inhibitors 24 hours later. Inhibitors were PTB-independent ShcA pools to amplify tumor growth. Finally, replenished every two days over a 10-day period. Cells were fixed we establish that increased Src activation downstream of with 10% buffered formalin (VWR catalog no. 16004-128) for PTB-independent ShcA signaling complexes increases resistance 20 minutes at room temperature. Subsequently, each well was to lapatinib, a dual EGFR/ErbB2 inhibitor. These results show washed with distilled water and then stained with 0.1% Crystal that perturbation of discrete ShcA-dependent signaling complexes Violet/20% Methanol solution for 30 minutes. Plates were then significantly impacts breast tumor growth and therapeutic rinsed with water and dried overnight. Plates were scanned using responsiveness. the Oxford Optronix GelCount system. Quantification of the number of colonies and size of foci were analyzed with Aperio ImageScope software. Materials and Methods Cell culture Soft agar assay NMuMG-NeuNT, an ErbB2-transformed mammary tumor cell NMuMG-NeuNT. A total of 1.5 104 cells were plated into 1.5 mL line, was generated and cultured as described previously (11). of 0.4% Agar (Bioshop catalog no. AGR001.500)/20% NMuMG-NeuNT cell lines were stably transfected with a pMSCV/ FBS (Wisent Bio Products catalog no. 080-150) DMEM (Wisent Hygromycin expression vector (Clontech catalog no. 634401) Bio Products catalog no. 319-005-CL) over a layer of 2 mL 0.6% WT subcloned with cDNAs expressing: (i) wild-type ShcA (ShcA ), Agar/20% FBS DMEM in 6-well plates. Cells were treated with (ii) a ShcA mutant harboring an arginine to glutamine substitu- either full culture media or media containing inhibitor (DMSO, tion at amino acid 175 in the phospho-tyrosine binding pocket of PP2, or Torin1). To account for the volume of agar, inhibitor MUT MUT the PTB domain of ShcA (PTB ), (iii) a PTB harboring concentrations were adjusted to 6 mmol/L PP2 and 150 nmol/L tyrosine to phenylalanine substitution at amino acids 239, 240, Torin1 to obtain an approximate final concentration of 2 mmol/L MUT MUT and 313 (PTB /3F), or (iv) a PTB with an arginine to lysine and 50 nmol/L, respectively. For inhibitor studies, media contain- substitution at amino acid 397 in the phospho-tyrosine binding ing inhibitors or DMSO was replenished every 3 days and mon- MUT pocket of the SH2 domain (PTB/SH2 ). All ShcA constructs itored over a 10-day period. are C-terminally tagged with a 3-FLAG epitope. Four ShcA- / expressing clones were pooled for each cell line, which were NIC/Src . A total of 1.5 104 cells were plated into 1.5 mL of maintained in 0.5 mg/mL Hygromycin (Wisent Bio Products Cat: 0.4% Agar/10% FBS DMEM supplemented with mammary

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epithelial growth supplement (MEGS; 3 ng/mL human epidermal either mutated all three ShcA tyrosine phosphorylation sites to growth factor (Invitrogen catalog no. PHG0311), 0.5 mg/mL phenylalanine residues (PTBMUT/3F) or introduced a loss of Hydrocortisone (Wisent Bio Products catalog no. 511-002-UG), function mutation (R397K) in the phospho-tyrosine binding 5 mg/mL insulin, 0.4% v/v bovine pituitary extract (Wisent Bio pocket of the SH2 domain (PTB/SH2MUT), in the context of Products catalog no. 002-011-IL) over a layer of 2 mL 0.6% Agar/ PTB mutation (Fig. 1A). FLAG-tagged ShcA mutants were 10% FBS DMEM supplemented with MEGS in 6-well plates and stably expressed in ErbB2-expressing breast cancer cells monitored for 10 days. Images of four fields per well were (Fig. 1B). FLAG-tagged ShcA alleles harboring a wild-type PTB acquired by an inverted light microscope using Infinity Capture domain (ShcAWT; PTB-dependent) or the loss-of-function PTB software. The number of colonies and size of foci were analyzed mutant (PTBMUT; PTB-independent) alone, were also stably using Aperio ImageScope software. expressed (Fig. 1B). We first confirmed that an intact PTB domain is required for ShcA to engage in PTB-dependent Mammary fat pad injection interactions, including ErbB2, PTP-PEST, and SHIP2 (refs. 18, A total of 5 104 NMuMG-NeuNT or 2.5 105 NIC/Src / 21–23, 31; Fig. 1C). Second, SH2-dependent interactions wild-type or mutant ShcA-expressing breast cancer cells were with ShcBP1 (32) were selectively abrogated in PTB/ injected into the fourth mammary fat pad of 8- to 10-week-old SH2MUT-expressing cells (Fig. 1C). Finally, mutation of the SCID-Beige female mice. Animals were monitored for tumor ShcA tyrosine phosphorylation sites ablated Grb2 binding initiation and tumor outgrowth every two days using caliper (PTBMUT vs. PTBMUT/3F;Fig.1D;ref.17).Theseresultsconfirm measurements. Breast tumors were fixed in 10% buffered forma- the specificity of the mutants generated. lin, embedded in optimal cutting temperature medium Our previous studies demonstrated that loss of PTB-dependent (OCT; VWR catalog no. CA95057-838), or frozen in liquid inhibitory signals augments ErbB2-driven mammary tumorigen- nitrogen. All animal studies were approved by the Animal esis (15). To mechanistically define how these PTB-independent Resources Council at McGill University and comply with guide- ShcA pools (PTBMUT) promote breast tumor growth, we first lines set by the Canadian Council of Animal Care. tested the requirement of either phospho-tyrosine- and/or SH2-mediated ShcA signaling for tumor growth in soft agar. Statistical analyses PTBMUT-expressing breast cancer cells had a reduced capacity to Unless otherwiseindicated,allin vitro studies were carried form colonies in soft agar relative to ShcAWT controls (Fig. 1E). out with three biological replicates and with six technical This corroborated previous observations, which defined an replicates per experimental group. Data were normalized to important role for the ShcA PTB domain in ErbB2-driven tumor the control groups as appropriate. For the in vivo studies, growth (15, 22, 33). However, once formed, PTBMUT-expressing mammary fat pad injections were performed with 4–5age- foci were significantly larger than ShcAWT controls (Fig. 1F), matched mice inoculated with breast cancer cells in both validating several studies demonstrating that the ShcA PTB mammary fat pads (n ¼ 8–10 breast tumors per cohort). The domain restricts tumor growth when ShcA is uncoupled from following statistical analyses were used throughout the study. oncogenes such as ErbB2 (15, 18, 19, 31). Interestingly, the Two-tailed, unpaired Student t test (Figs. 3B–Gand4B–F, tumorigenic potential from these PTB-independent ShcA and 6A, B, and H; Supplementary Figs. S2A, S3A, S3B, S5A pools was significantly debilitated in both PTBMUT/3F and PTB/ and S7). One-way ANOVA with a Tukey multiple comparisons SH2MUT-expressing cells (Fig. 1F). This indicated that both phos- test (Figs. 1E–I, 2A–E, 5C–F, and 6E, F, I, and J; Supplementary pho-tyrosine and SH2-dependent ShcA signals are important for Figs.S1C,S4A,andS5B).Two-wayANOVAwithaTukey breast cancer cell growth in vitro. multiple comparison test (Figs. 2F, 4G, 5G–J, and 6G, K–L). We next investigated whether PTB-uncoupled ShcA pools Additional information regarding the experimental procedures (PTBMUT) relied on the tyrosine phosphorylation sites and/or employed for the Crispr/Cas9 genomic editing, immunoblot SH2 domain to amplify ErbB2-driven mammary tumor growth analysis, immunoprecipitation, IHC, BioID, and bioinformatics in vivo. As expected, unrestrained ShcA signaling from approaches can be found in the Supplementary Methods section. PTB-independent complexes (PTBMUT) significantly accelerated tumor growth relative to ShcAWT controls (Fig. 1G). Surprisingly, the ShcA tyrosine phosphorylation sites were dispensable for the Results enhanced tumor growth observed from PTB-independent PTB-independent ShcA pools require an intact SH2 domain to ShcA complexes (PTBMUT vs. PTBMUT/3F). Rather, loss of potentiate mammary tumorigenesis SH2-dependent signaling from these ShcA pools (PTB/SH2MUT) To model the consequence of deregulated ShcA signaling profoundly delayed breast tumor onset (Fig. 1G). To better from PTB-independent ShcA pools on breast tumorigenesis, understand the mechanism underlying this phenotype, we per- we employed a loss-of-function mutant (R175Q) in the phos- formed Ki67 and cleaved caspase-3 IHC staining on these tumors pho-tyrosine binding pocket of the ShcA PTB domain as markers of cell proliferation and apoptosis, respectively. At the (PTBMUT; refs. 15, 30). Ectopic expression of a ShcA-PTBMUT experimental endpoint, we did not observe appreciable differ- allele in breast cancer cells that endogenously express wild- ences in the degree of cell proliferation or apoptosis in ShcAWT, type ShcA creates distinct intracellular ShcA pools that can PTBMUT, and PTBMUT/3F–expressing tumors. However, we did independently transduce oncogenic signals downstream from observe that PTB/SH2MUT–expressing tumors had the greatest RTKs, including ErbB2, (ShcA; PTB-dependent) and that also proliferative potential, which was counteracted by a significantly lack the ability to bind negative regulators, permitting ampli- increased apoptotic rate (Fig. 1H and I). This suggests that loss of fication of protumorigenic signals from PTB-independent SH2-dependent signaling from PTB-uncoupled ShcA complexes ShcA complexes (PTBMUT; Fig. 1A). To determine how these initiates an apoptotic response, leading to a compensatory PTB-independent ShcA pools transduce oncogenic signals, we increase in mitogenic responses to permit tumor growth.

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Figure 1. PTB-independent ShcA pools require a functional SH2 domain, but not the tyrosine phosphorylation sites to promote mammary tumorigenesis. A, Schematic diagram illustrating how the ShcA PTB domain coordinates distinct intracellular signaling complexes that initiate and amplify mammary tumorigenesis in ErbB2-driven breast cancers. Schematic representation of ShcA alleles employed in this study is also shown. B, Immunoblot analysis characterizing the expression of FLAG-tagged PTB domain mutant ShcA alleles in ErbB2-driven mammary epithelial cell lines (NMuMG-NeuNT). C, MMTV Middle T antigen mammary epithelial cells expressing indicated Myc-BirA fusion proteins were subjected to BioID analysis. Biotinylated proteins were probed using the indicated antibodies by immunoblot analysis. D, Grb2 interactions assessed by the immunoprecipitation of FLAG-tagged PTB domain mutant ShcA alleles expressed in NMuMG-NeuNT cells. Number (E) and average area (F) of foci formed in a soft agar assay from the indicated cell lines. The data is representative of three independent experiments (means SEM). ShcAWT vs. PTBMUT: ,P< 0.01; ,P< 0.0001; PTBMUT versus PTBMUT/3F or PTB/SH2MUT: d, P < 0.05; dd, P < 0.01; ddd, P < 0.001. G, Mammary fat pad injection of the indicated cell lines into immunodeﬁcient mice. The data are shown as average tumor volume (mm3) SEM and is representative of 14 tumors per group. ShcAWT versus PTBMUT: ,P< 0.001; PTBMUT versus PTBMUT/3F or PTB/SH2MUT: dd, P < 0.01; ddd, P < 0.001. IHC staining of mammary tumors using Ki67 (H) and cleaved Casp3 (I). The data depicts the average positively stained cells or pixels SEM and is representative of 6–8 tumors per group. ,P< 0.01.

PTB-independent ShcA pools simultaneously increase mTOR (Fig. 2A). Thus, mTOR and Src activation bifurcates downstream signaling and Src activity of PTB-independent ShcA pools through the ShcA tyrosine phos- We previously demonstrated that PTB-uncoupled ShcA signal- phorylation sites and SH2 domain, respectively. ing complexes (PTBMUT) activate Src (15). We conﬁrmed this To further examine how perturbation of ShcA-driven signaling observation and further show that these PTB-independent ShcA complexes impacted breast tumor growth in vivo, we performed pools required an intact SH2 domain, and not the tyrosine IHC analyses using pS240/244-ribosomal S6 (pS240/244-rS6) phosphorylation sites, to activate Src (Fig. 2A). Moreover, and pY416-Src speciﬁc antibodies. We show that pS240/244-rS6 PTBMUT-expressing cells upregulated mTOR signaling, as evi- levels are selectively reduced in PTBMUT/3F tumors compared with denced by elevated pS65-4EBP1 and pT389-S6K levels, compared PTBMUT controls (Fig. 2B), corroborating an important role for with ShcAWT controls (Fig. 2A). This increase in mTOR signaling ShcA tyrosine phosphorylation in promoting mTOR signaling. required the ShcA tyrosine phosphorylation sites. Interestingly, Given that PTBMUT- and PTBMUT/3F-expressing tumors display AKT activation was relatively unchanged, irrespective of the muta- comparable growth rates in vivo (Fig. 1G), these data suggest that tional status of these PTB-uncoupled ShcA signaling complexes mTOR activation is not primarily responsible for the increased

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Figure 2. PTB-independent ShcA pools employ distinct domains to hyperactivate mTOR and Src signaling. A, Immunoblot analysis with the indicated antibodies in ErbB2-driven breast cancer cells expressing specified FLAG-tagged ShcA alleles. The data are representative of three independent experiments. ,P< 0.05. B and C, IHC staining of tumors using pS240/244-rS6 and pY416-Src–specific antibodies, respectively. The data depict the average positively stained cells or pixels SEM is representative of 6–8 tumors per group. ,P< 0.05; ,P< 0.001. D, FLAG immunoprecipitates were probed with pY239/240-ShcA or ShcA-specific antibodies by immunoblot analysis. Both DMSO and PP2 (2 mmol/L)-treated cells were assayed. The barplot represents densitometric quantification of three independent experiments using ImageJ software. One-way ANOVA (DMSO or PP2)—ShcAWT versus PTBMUT: ,P< 0.01; ,P< 0.0001; PTBMUT vs. PTBMUT/3F: dd, P < 0.01. We also compared the differences in ShcA tyrosine phosphorylation in PTBMUT (DMSO vs. PP2) and PTB/SH2MUT (DMSO vs. PP2) using a two-tailed, paired t test (#, P < 0.05). E, FLAG immunoprecipitates from indicated cell lines were probed with pY416-Src, Src, Fyn, Lyn, and FLAG-specific antibodies via immunoblot analysis. The barplot represents densitometric quantification of three independent experiments using ImageJ software. PTBMUT versus PTB/SH2MUT: d, P < 0.05. F, Clonogenic assays of specified cell lines treated with DMSO and lapatinib (0.5 mmol/L). The data is depicted as a fold change in viability relative to DMSO control and is representative of three independent experiments (means SEM). ShcAWT versus PTBMUT: ,P< 0.001; PTBMUT vs. PTBMUT/3F or PTB/SH2MUT: d, P < 0.05; dd, P < 0.01. tumorigenic potential of PTB-uncoupled ShcA pools (Fig. 2B). On Once activated, ErbB2 recruits and phosphorylates ShcA in the other hand, pY416-Src levels were profoundly elevated in a PTB-dependent manner (34, 35). We now demonstrate that PTBMUT and PTBMUT/3F–expressing tumors relative to ShcAWT PTB-independent ShcA pools (PTBMUT) are also tyrosine phos- controls (Fig. 2C). This is consistent with our in vitro studies phorylated, albeit to a significantly lower extent (3 fold) relative showing that increased tumor growth was associated with an to PTB-dependent ShcA complexes (ShcAWT; Fig. 2D). Given that SH2-dependent increase in Src activity (Fig. 2A). At endpoint, Src numerous Src family kinases (SFK) bind ShcA, both in a consti- signaling was restored in PTB/SH2MUT tumors (Fig. 2C) even tutive and phospho-tyrosine inducible manner (36–39), we though their growth potential was severely impaired in vivo employed a pan SFK inhibitor (PP2) at lower doses (2 mmol/L) (Fig. 2C). These results demonstrate that the ability of to test whether PTB-independent ShcA pools are more reliant on PTB-uncoupled ShcA pools to accelerate mammary tumorigenesis SFKs to phosphorylate ShcA (Supplementary Fig. S1A). Pharma- was strictly dependent on a functional SH2 domain. Moreover, cologic SFK inhibition (2 mmol/L PP2) did not impact ShcA when faced with loss of SH2-mediated signaling, there were tyrosine phosphorylation from PTB-coupled complexes (ShcAWT) significant selective pressures to reacquire Src activity to permit but attenuated pY-ShcA levels from PTB-uncoupled ShcA pools eventual tumor growth. (PTBMUT) relative to their respective DMSO controls (Fig. 2D). In

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Figure 3. PTB-independent ShcA pools cannot accelerate tumor growth in the absence of Src. A, NIC/Src/ mammary tumor cells were engineered to express FLAG-tagged ShcAWT or PTBMUT alleles. B, FLAG immunoprecipitates from indicated cell lines were probed with pY239/240-ShcA- or ShcA-specific antibodies via immunoblot analysis. The barplot represents densitometric quantification of three independent experiments using ImageJ software. ,P< 0.0001. C, Mammary fat pad injection of indicated cell lines into immunodeficient mice. The data are shown as average tumor volume (mm3) SEM and are representative of 10 tumors per group. IHC staining of mammary tumors using Ki67 (D), cleaved Casp3 (E), pY416-SFK (F), and pS240/244-rS6–specific antibodies (G). The data depict the average positively stained cells or pixels SEM and is representative of 7–9 tumors per group. ,P< 0.05.

addition, PP2 inhibition of SFKs at a higher dose (20 mmol/L) cells to pharmacologic inhibition of ErbB2 and EGFR (lapatinib), completely abrogated ShcA tyrosine phosphorylation, specifically both of which bind the ShcA PTB domain. Recall that PTBMUT- from PTB-independent ShcA complexes (Supplementary expressing cells retain the ability to engage both PTB-independent Fig. S1B). As expected, a ShcA mutant lacking the tyrosine phos- ShcA complexes alongside PTB-coupled interactions from phorylation sites (PTBMUT/3F) was not phosphorylated in control endogenous ShcA alleles. We demonstrate that increased or PP2-treated cells. Remarkably, ShcA pools that cannot engage PTB-independent ShcA signaling promoted lapatinib resistance in PTB- or SH2-driven interactions (PTB/SH2MUT) were residually in ErbB2-transformed breast cancer cells (Fig. 2F). Indeed, the ERK tyrosine phosphorylated in an SFK-dependent manner (Fig. 2D; and AKT pathways remained elevated in lapatinib-treated Supplementary Fig. S1B). This highlights an accessory role PTBMUT-expressing cells compared with ShcAWT controls for constitutive ShcA/SFK interactions in transducing phospho- (Supplementary Fig. S1C). Loss of either phospho-tyrosine tyrosine driven ShcA signals. These results suggest that (PTBMUT/3F)- or SH2 (PTB/SH2MUT)-driven signaling from these PTB-uncoupled ShcA pools are more reliant on SFKs to phos- PTB-independent ShcA pools reversed lapatinib resistance phorylate the ShcA tyrosine phosphorylation sites. (Fig. 2F) and impaired ERK and AKT/mTOR signaling (Supple- Given that the ShcA SH2 domain is required to activate Src from mentary Fig. S1C). Significant inhibition of ErbB2 signaling was PTB-independent signaling complexes, we next interrogated confirmed upon treatment with lapatinib (500 nmol/L) (Supple- whether they rely on an intact SH2 domain to bind SFKs. Unex- mentary Fig. S1C). Moreover, ErbB2 inhibition had no impact on pectedly, the ShcA SH2 domain is dispensable for Src, Fyn, or Src activation in any cell line tested (Supplementary Fig. S1C). Lyn recruitment to PTB-uncoupled ShcA pools (PTBMUT vs. Combined, these data suggest that increased signaling from PTB- PTB/SH2MUT; Fig. 2E). Paradoxically, PTB-independent ShcA independent ShcA pools can induce lapatinib resistance by pools with an intact SH2 domain (PTBMUT) exhibited the lowest increasing ERK and AKT activation both through the SH2 domain level of SFK binding, relative to ShcAWT controls (Fig. 2E), despite and tyrosine phosphorylation sites. displaying a 2-fold increase in Src activation (Fig. 2A). Collec- tively, these results suggest that the ability of PTB-uncoupled ShcA The increased tumorigenic potential of PTB-independent ShcA pools to activate Src required a functional SH2 domain but signaling complexes requires the Src tyrosine kinase was uncoupled from direct SFK recruitment to ShcA signaling Although these studies demonstrate an essential role for the complexes. ShcA-SH2 domain in promoting mammary tumorigenesis PTB-independent ShcA signaling complexes coordinately acti- from PTB-independent complexes, they do not identify a causal vate SFKs in ErbB2-positive breast cancer cells. Thus, we assessed role for Src in this phenotype. To test this, we ectopically whether their hyperactivation altered sensitivity of breast cancer expressed ShcAWT and PTBMUT alleles in ErbB2 transformed

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Figure 4. PTB-independent ShcA pools require Src to promote mammary tumorigenesis. A, Src was deleted from ErbB2 mammary epithelial cell line (NMuMG-NeuNT) from the indicated ShcA-expressing cell lines using Crispr/Cas9 genomic editing. Immunoblot analysis of vector control and Src-Crispr (CR) cell lines using Src- and Tubulin-specific antibodies. Vector control and Src-CR of NMuMG-NeuNT ShcAWT (B) and PTBMUT (C) cell lines were injected into the mammary fat pads of immunodeficient mice. The data depict the average treumor volumes (mm3) SEM and are representative of 9–10 tumors per group. ,P< 0.05; ,P< 0.01; ,P< 0.0001. IHC staining of pY416-Src (D) and pS240/244-rS6 (E) in ShcAWTand PTBMUT-expressing NMuMG-NeuNT mammary tumors. The data depict the average positively stained cells or pixels SEM is representative of 8–10 tumors per group. ,P< 0.01. F, FLAG immunoprecipitates from indicated cell lines were probed with pY239/240-ShcA or ShcA-specific antibodies via immunoblot analysis. The graph represents densitometric quantification of three independent experiments using ImageJ software. ,P< 0.01. G, Clonogenic assay of specified cell lines treated with DMSO, lapatinib (0.5 mmol/L), PP2 (2 mmol/L) alone, or in combination. The data is shown as fold change in viability relative to DMSO control and is representative of three independent experiments (means SEM). ,P< 0.05; ,P< 0.001. mammary tumors established from transgenic mice lacking Src signaling is likely not a major driver of mitogenic signals (NIC/Src / ) in the mammary epithelial compartment (Fig. 3A; downstream of PTB-independent ShcA complexes. ref. 27). Consistent with our previous studies (Fig. 2D), Given that mammary tumors derived from NIC/Src / mice PTB-independent ShcA pools were significantly hypopho- emerged in the absence of Src, it is possible that these tumors sphorylated relative to ShcAWT controls in Src-deficient cells significantly rewired their kinome in response to Src loss. To (Fig. 3B). In the absence of Src, mammary tumor growth was confirm that adaptive mechanisms did not account for the inabil- comparable between ShcAWT-andPTBMUT-expressing cells, ity of PTB-uncoupled ShcA complexes to augment mammary both in vitro and in vivo (Fig. 3C; Supplementary Fig. S2A). tumor growth in a Src-deficient background, we used Crispr/Cas9 IHC staining further revealed that both Src-deficient ShcAWT genome editing to delete Src (Src-CR) from ShcAWT- and PTBMUT- þ þ and PTBMUT breast tumors displayed comparable rates of expressing, ErbB2-transformed (ErbB2/Src / ) breast cancer proliferation and apoptosis (Fig. 3D and E; Supplementary cells (Fig. 4A). While Src was dispensable for the growth of ShcAWT Fig. S2B). Moreover, pY416-SFK levels were comparable mammary tumors (Fig. 4B), Src deletion impaired the ability between ShcAWT-andPTBMUT-expressing mammary tumors of PTB-independent ShcA pools to promote mammary tumori- (Fig. 3F; Supplementary Fig. S2B), suggesting that the ability genesis (Fig. 4C). At the experimental endpoint, proliferation and of PTB-independent ShcA pools to promote mammary tumor- apoptosis were comparable in all groups, irrespective of the igenesis required Src. In addition, reduced pS240/244-rS6 presence or absence of Src (Supplementary Fig. S3A–S3D). IHC levels in ShcA-PTBMUT-expressing mammary tumors analysis confirmed that both ShcAWT/Src-CR and PTBMUT/Src-CR (Fig. 3G) did not correlate with their tumorigenic potential tumors were debilitated in SFK activation relative to their respec- (Fig. 3C). This confirms our previous observation that mTOR tive vector controls (Fig. 4D; Supplementary Fig. S3E). However,

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Figure 5. Fyn cooperates with Src to increase the tumorigenic potential of PTB-independent ShcA signaling complexes. Immunoblot blot analysis of whole-cell lysates from ShcAWT (A) and PTBMUT (B) cell lines upon deletion of Src, Fyn, or Lyn using Crispr/Cas9 genomic editing. Number (C and E) and average area (D and F) of foci formed in a soft agar assay from speciﬁed cell lines. The data are representative of three independent experiments (means SEM). , P < 0.05; , P < 0.01. The fold change in number (G and I) and average area (H and J) of foci formed in soft agar of indicated cell lines relative to DMSO controls in the absence or presence of PP2 (2 mmol/L) or Torin1 (50 nmol/L). The data is representative of three independent experiments (means SEM). DMSO versus PP2 or Torin1 treatment: ,P< 0.05; ,P< 0.01; ,P< 0.0001. VC versus Src-CR: d, P < 0.05; dd, P < 0.01; ddd, P < 0.001; dddd, P < 0.0001.

pS240/244-rS6 levels were selectively reduced in Src-deficient mitogenic signals in ErbB2-transformed breast cancer cells in vitro. tumors compared with PTBMUT controls (Fig. 4E; Supplementary On the other hand, while the loss of Src resulted in a 3-fold Fig. S3F). Thus, using two independent model systems, we show increase in the sensitivity of PTBMUT-expressing cells to lapatinib that loss of Src signaling from PTB-independent ShcA pools treatment, coincubation with lapatinib and PP2 resulted in a debilitated mTOR signaling (Figs. 3G and 4E). While SFK activity 22-fold decrease in cell viability. This was contrasted by ShcAWT- was required to phosphorylate ShcA residing within PTB-inde- expressing breast cancer cells in which cell viability upon com- pendent ShcA pools (Fig. 2D), Src deletion severely reduced, but bined lapatinib/PP2 treatment was comparable, irrespective did not ablate, ShcA tyrosine phosphorylation from these com- of Src status (Fig. 4G). These results suggest that in the absence plexes (PTBMUT vs. PTBMUT/Src-CR; Fig. 4F). In contrast, tyrosine of Src, breast tumors that engage PTB-independent signaling phosphorylation of PTB-dependent ShcA signaling complexes complexes become codependent on mitogenic signals emanating remained unaffected by Src deletion (ShcAWT vs. ShcAWT/ from both ErbB2 and other SFKs. Src-CR; Fig. 4F). These data further confirm our observation that Src, and potentially other SFKs, selectively promote ShcA tyrosine Fyn cooperates with Src to increase the tumorigenic potential of phosphorylation in PTB-independent signaling complexes. PTB-independent ShcA signaling complexes We previously established that increased SH2 signaling from To assess the importance of other SFKs downstream of PTB-independent ShcA complexes contributes to lapatinib resis- PTB-independent ShcA complexes, we used Crisper/Cas9 geno- tance, presumably through increased Src activation (Fig. 2F). We mic editing to delete Fyn (Fyn-CR) or Lyn (Lyn-CR) from ErbB2- next examined whether the loss of Src impacted lapatinib sensi- transformed breast cancer cells (Fig. 5A and B; Supplementary tivity in mammary tumors that can (PTBMUT) or cannot (ShcAWT) Fig. S4A). We did not observe a compensatory increase in Src activate ShcA from PTB-independent complexes. As expected, Src levels when either Fyn or Lyn were deleted. By the same token, Src deficiency had no impact on the sensitivity of ShcAWT tumors to deletion did not appreciably alter Fyn or Lyn expression levels lapatinib but profoundly sensitized PTBMUT-expressing tumors to (Fig. 5A and B; Supplementary Fig. S4A). Moreover, while this ErbB2 inhibitor (Fig. 4G). This suggests that in the absence of pY416-SFK and pY576/7-FAK levels (Src specific phosphorylation Src, tumors lose their ability to engage PTB-independent ShcA site) were significantly reduced in ShcAWT/Src-CR and PTBMUT/ complexes and instead are more reliant on ErbB2/ShcA signaling. Src-CR–expressing cells, SFK activation was unaltered by Fyn or On the other hand, Src deficiency minimally impacted sensitivity Lyn loss in either cell line (Fig. 5A and B; Supplementary Fig. S4A). to PP2, in both ShcAWT- and PTBMUT-expressing cells (Fig. 4G). This suggests that Fyn and Lyn may play secondary roles in This indicated that Src is likely the primary SFK to transduce supporting the ability of PTB-independent ShcA pools to promote

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Figure 6. PTB-independent ShcA complexes hyperactivate mTOR signaling to maintain mammary tumorigenesis in the absence of Src. A, Mammary fat pad injection of NIC/Src/ PTB/SH2MUT–expressing cell lines into immunodeficient mice. The data is shown as an average tumor volume (mm3) SEM and is representative of 10 tumors per group. B, Vector control and Src-CR of NMuMG-NeuNT PTB/SH2MUT–expressing cells lines injected into the mammary fat pads of immunodeficient mice. The data depicts the average tumor volumes (mm3) SEM and is representative of 9–10 tumors per group. ,P< 0.05; ,P< 0.01; ,P< 0.001. C, Immunoblot analysis of the indicated NIC/Src/ cell lines using Src, Fyn, Lyn, and Tubulin-specific antibodies. The positive control represents lysates from NMuMG-NeuNT cells. D, Immunoblot blot analysis of whole-cell lysates of NMuMG-NeuNT PTB/SH2MUT cells upon Crispr/Cas9 deletion of Src, Fyn, or Lyn with specified antibodies. IHC staining of NIC/Src/ PTB/SH2MUT (E) and vector control (F) and Src-CR NMuMG-NeuNT PTB/SH2MUT mammary tumors using pY416-SFK, Ki67, cleaved Casp3 and pS240/244-rS6 specific antibodies. The data depict the average positively stained cells or pixels SEM and is representative of 7–9 tumors per group. ,P< 0.05; ,P< 0.001; ,P< 0.0001. G, Clonogenic assays of specified cell lines treated with DMSO, lapatinib (0.5 mmol/L), PP2 (2 mmol/L) alone, or in combination. The data is shown as fold change in viability relative to DMSO control and is representative of three independent experiments (means SEM). ,P< 0.05. H, FLAG immunoprecipitates from NMuMG-NeuNT PTB/SH2MUT cell line probed with pY239/240-ShcA or ShcA-specific antibodies by immunoblot analysis. The barplot represents densitometric quantification of three independent experiments using ImageJ software. Number (I) and average area (J) of foci formed in a soft agar assay from the specified cell lines. The data are representative of three independent experiments (means SEM). ,P< 0.05. The fold change in the number (K) and average area (L) of foci formed in soft agar of indicated cell lines relative to DMSO controls in the absence or presence of PP2 (2 mmol/L) or Torin1 (50 nmol/L). The data is representative of three independent experiments (means SEM). DMSO versus PP2 or Torin1 treatment: ,P< 0.05; ,P< 0.01. tumor growth. To test this, we examined the transformation both the number and size of foci formed (Fig. 5E and F). This potential of Src-, Fyn-, or Lyn-deficient breast cancer cells in a validated our in vivo observations where Src was necessary for the soft agar assay. In support of our in vivo study, Src deletion ability of these PTB-independent ShcA pools to augment mam- impaired tumor initiation (number of foci) in breast cancer cells mary tumor growth (Fig. 4C). Fyn deletion in PTBMUT-expressing that retained a functional PTB domain (ShcAWT) but was dis- breast cancer cells did not impact foci formation but reduced their pensable for tumor growth (average area of foci; Fig. 5C and D). In growth potential (2 fold) relative to vector controls (Fig. 5E and contrast, neither Fyn nor Lyn deletion appreciably altered the F). Lyn, on the other hand, was dispensable for the transforming transforming potential of ShcAWT breast cancer cells (Fig. 5C and potential of ErbB2-driven breast cancer cells, irrespective of D). In contrast, Src loss in breast cancer cells that hyperactivate whether they had engaged PTB-independent ShcA complexes PTB-independent ShcA complexes (PTBMUT) significantly reduced (Fig. 5E and F). These data suggest that Src cooperates with Fyn

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downstream of PTB-independent ShcA pools to increase the (Fig. 5). Moreover, Src deficiency in two independent models þ tumorigenic potential of ErbB2 breast cancers. To test this, we (NIC/Src / and ErbB2/Src-CR) significantly upregulated Fyn first assessed how Src deletion, in combination with, SFK inhi- levels when PTB-independent ShcA pools could no longer signal bition (2 mmol/L PP2) impacted the tumorigenic potential of through the SH2 domain (Fig. 6C and D, Supplementary Fig. S5A ShcAWT- and PTBMUT-expressing cells. As expected, pharmacolog- and S5B). Lyn expression levels, on the other hand, were mini- ic SFK inhibition did not appreciably impact the transforming mally impacted in these cells (Fig. 6C and D, Supplementary potential of ShcAWT-expressing cells (Fig. 5G and H; Supplemen- Fig. S5A and S5B). Moreover, we did not observe increased pY416- tary Fig. S4B). In addition, genetic loss of Src in ShcAWT-expressing SFK levels in Src-deficient PTB/SH2MUT cancer cells, both in vitro cells impacted the number but not the size of foci formed in a soft and in vivo (Fig. 6C–F; Supplementary Fig. S5A and S5B). This is agar assay. Similarly, even in the absence of Src, pharmacologic consistent with the observation that the growth potential of SFK inhibition moderately reduced the number of foci but did not these cells was insensitive to SFK inhibition (PP2) in a clonogenic impact the size of the foci (Fig. 5G and H). These data further assay (Fig. 6G; Supplementary Fig. S5C). Rather, Src deficiency support the observation that ShcAWT breast tumors are less coupled with the lack of SH2-driven signaling from PTB-inde- dependent on SFKs. In this regard, we previously established pendent ShcA pools (PTB/SH2MUT), significantly upregulated that the ErbB2/ShcA signaling axis predominately activates pS240/244-rS6 levels in mammary tumors (Fig. 6E and F). This AKT/mTOR signaling to promote mammary tumorigenesis was coincident with an increased proliferative index compared (24). Consistent with this observation, ShcAWT-expressing breast with SH2-proficient controls (Fig. 6E and F; Supplementary cancer cells were exquisitely sensitive to suboptimal doses of Fig. S6A and S6B). Thus, in the absence of Src and a functional mTOR inhibitor, Torin1 (50 nmol/L) in a soft agar assay, showing SH2 domain, PTB-independent ShcA pools elicit a compensatory a significant reduction in the number and size of foci formed increase in mTOR signaling to facilitate eventual tumor growth. (Fig. 5G and H; Supplementary Fig. S4B). Moreover, the ability of However, the hyperactivation of mTOR signaling in these Torin1 to impair focus formation (# foci) was independent of Src cells was not associated with increased pY-ShcA levels in ShcAWT-expressing cells. Alternatively, the tumorigenic poten- (Fig. 6H), suggesting that increased mTOR signaling was inde- tial of breast cancer cells engaging PTB-independent ShcA pendent of the ShcA tyrosine phosphorylation sites. These signaling complexes (PTBMUT) were similarly sensitive to Torin1 observations reinforce the importance of mTOR signaling in (Fig. 5I and J; Supplementary Fig. S4B). These results highlighted ErbB2-driven breast cancer, particularly in response to Src inhi- þ an important role for mTOR signaling in ErbB2 breast bition, and demonstrate the ability of mammary tumors to cancers, irrespective of whether they transduce signals from reprogram their kinomes. PTB-independent ShcA pools. Suboptimal doses of SFK inhibitor, We next tested whether Src-null breast tumors that engage PP2, had no impact on the tumor-forming potential of Src PTB-independent ShcA signaling, become more reliant on ErbB2. proficient PTBMUT-expressing cells but did attenuate the growth As expected, Src deletion sensitized PTB/SH2MUT-expressing cells of these foci (Fig. 5I and J; Supplementary Fig. S4B). In contrast, to lapatinib, demonstrating an increased reliance on ErbB2/ShcA Src deletion was sufficient to decrease both the number and size signaling complexes (Fig. 6G). Moreover, simultaneous ErbB2 of foci formed by PTBMUT-expressing cells (Fig. 5I and J). The and SFK inhibition (lapatinib and PP2) completely ablated the average area of PTBMUT-expressing foci was further and signifi- viability of PTB/SH2MUT breast cancer cells, suggesting a potential cantly diminished upon SFK inhibition by PP2 (Fig. 5I and J). accessory role for Fyn or Lyn in the absence of Src. To test this, Fyn These data suggest that Src is principally responsible for the and Lyn were also stably deleted from PTB/SH2MUT-expressing increased tumorigenic potential of PTB-independent ShcA com- cells using Crispr/Cas9 genomic editing (Fig. 6D). Fyn deletion plexes but that other SFKs, including Fyn play a cooperative role in reciprocally increased Src expression levels in PTB/SH2MUT- this process. expressing cells, again highlighting the adaptive responses trig- gered in ErbB2-driven breast tumors to retain SFK function Loss of c-Src signaling downstream of PTB-independent ShcA (Fig. 6D; Supplementary Fig. S5B). To test whether these pertur- complexes increases the dependency of ErbB2-driven breast bations in SFK levels were biologically relevant, we assessed the tumors on mTOR signaling impact of Src, Fyn or Lyn deficiency on the transforming potential Our data suggest that breast tumors require a functional ShcA of these cells in a soft agar assay. As observed in our in vivo study SH2 domain to activate Src downstream from PTB-independent (Fig. 6B), deletion of Src in PTB/SH2MUT-expressing cells ShcA complexes. Given that Src deficiency reduced the tumori- increased the average size but not number of foci formed, relative genic potential of PTB-uncoupled ShcA pools (Figs. 3C and 4C), to Src-proficient controls (Fig. 6I and J). These data suggest that we interrogated the impact of simultaneous Src loss and impaired compensatory activation of mTOR signaling likely sustained their SH2-driven signaling from tumor-amplifying ShcA complexes growth in soft agar. To test this, we examined the sensitivity of (PTB/SH2MUT). Surprisingly, upon ectopic expression of both Src-proficient and Src-deficient PTB/SH2MUT-expressing cells PTB/SH2MUT alleles in NIC/Src / breast cancer cells, loss of to mTOR inhibition (Torin1) in a soft agar assay. As expected, the SH2-driven ShcA signaling did not ablate breast tumor formation transforming potential of these cells was insensitive to pharma- in vivo (Fig. 6A). In addition, Src deficiency (by Crispr/Cas9) cologic (PP2) or genetic (Src-CR) Src inhibition (Fig. 6K and L). paradoxically increased the growth potential of tumors that lack Rather, the loss of a functional ShcA-SH2 domain from the ability to transduce SH2-driven signals from PTB-independent PTB-independent ShcA complexes was sufficient to render these ShcA complexes (Fig. 6B). This indicated that, in the absence of Src cancer cells exquisitely sensitive to Torin1, independently and SH2 signaling from these PTB-uncoupled ShcA pools, sec- of Src (Fig. 6K and L). These data support the notion that ondary adaptive responses likely facilitate breast tumor growth. PTB-independent ShcA signaling complexes rely on the ShcA SH2 We already showed that Fyn cooperates with Src to amplify the domain to activate Src signaling to augment mammary tumor tumorigenic potential of PTB-independent signaling complexes growth; however, loss of the ShcA/Src signaling axis from these

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PTB-independent pools enable the reprogramming of signal PTB-driven interactions result in parallel activation of distinct networks to increase their reliance on mTOR signaling. ShcA pools that amplify breast tumor growth (15). This approach enabled us to specifically dissect the molecular mechanisms by Increased Src and mTOR signaling correlates with a PTBMUT which PTB-independent ShcA complexes augment mammary gene signature in human breast cancers tumorigenesis without altering the strength or duration of sig- We previously generated a gene signature that distinguished naling responses from PTB-coupled ShcA pools. In this study, we tumors that did (PTBMUT) or did not (ShcAWT) augment mitogenic identified a significant divergence in the signaling properties of signals from PTB-independent ShcA complexes (15). We used this PTB-dependent versus PTB-independent signaling complexes. gene signature (>2.5-fold differentially expressed; Supplementary One of the most striking differences between these two ShcA Table S1); to stratified 1,218 human breast cancers using the pools lies in their use of the ShcA tyrosine phosphorylation sites to publicly available TCGA RNA-seq dataset. We performed single promote tumor growth. PTB-coupled ShcA complexes require sample gene set enrichment analysis (ssGSEA), a computational phosphorylation of the tyrosine residues in the CH1 domain to method that calculates the absolute degree of enrichment of a gene activate AKT/mTOR signaling (16, 24). Interestingly, while ShcA set in individual samples (40). Breast cancer patients were strat- tyrosine phosphorylation activates AKT/mTOR signaling from ified into four quartiles based on the degree to which they most PTB-independent ShcA complexes, these sites are surprisingly resemble a PTBMUT gene signature (quartile 1: most ShcAWT-like/ dispensable for mammary tumor growth in vivo. Instead, PTB-dependent vs. quartile 4: most PTBMUT-like/PTB-indepen- PTB-independent ShcA complexes primarily relied on the SH2 dent). These signatures represented gene expression changes that domain to activate SFKs and amplify tumor growth (Fig. 7). are correlated with increased signaling from PTB-independent Previous studies have identified a phosphorylation-dependent ShcA pools. We first examined relative Src, Fyn, and Lyn transcript gating mechanism whereby tyrosine phosphorylation of ShcA levels within each quartile (Supplementary Fig. S7A). We show induces a conformational change, which opens the SH2 domain that the acquisition of a PTBMUT-like gene signature is associated to increase its ability to bind ligands (42). This may explain why with a modest but significant increase in Src mRNA levels (1.14- ShcA-PTBMUT/3F–expressing cells are debilitated in their trans- fold increase between 1st and 4th quartiles). In contrast Fyn, and forming potential in vitro. On the other hand, we show that a Lyn mRNA levels are much more significantly increased in PTBMUT- mutant ShcA allele lacking both a functional PTB domain and the like human breast tumors compared with those that are most like tyrosine phosphorylation sites (PTBMUT/3F) can still engage ShcAWT-like breast tumors (1.37-fold and 1.47-fold increase ShcBP1, an SH2-specific ShcA interactor, and robustly potentiate respectively between 1st and 4th quartiles). We also took advan- tumor growth in vivo. These data suggest that cross talk with the tage of the fact that the TCGA database contains RPPA data for 747 tumor microenvironment may transduce signals that permit SH2- of these patients (n ¼ 171:1st quartile; n ¼ 164: 2nd quartile; n ¼ driven signaling independently of ShcA tyrosine phosphoryla- 196: 3rd quartile; n ¼ 216: 4th quartile). We demonstrate that tion. As the previous study was in the context of a wild-type ShcA PTBMUT-like breast tumors display significantly reduced pY317- allele (42), the impact of this gating mechanism on the ability ShcA levels coincident with elevated pY416-Src and pS235/6-rS6 of PTB-independent ShcA pools to activate the SH2 domain has levels compared with ShcAWT-like breast tumors (Supplementary yet to be determined. Fig. S7B). These results correlate with our preclinical studies, Although the ShcA SH2 domain can serve an accessory role to demonstrating that PTB-independent ShcA complexes hyperacti- augment AKT/mTOR signaling downstream of activated RTKs vate Src and mTOR signaling, independently of the ShcA tyrosine (43), the very fact that the SH2 domain is dispensable for AKT/ phosphorylation sites. Finally, as described previously, several mTOR signaling from PTB-independent ShcA pools, highlights negative regulators, particularly PTP-PEST and SHIP2, have been the inherent plasticity of these breast cancers to adapt to the loss of shown to bind the PTB domain of ShcA to dampen ShcA-driven extracellular stimuli required for tumor initiation. However, this mitogenic signaling (18, 21–23, 31). Copy number analyses does not suggest that the hyperactivation of PTB-independent recently showed that the SHC1 (ShcA) gene, residing within the ShcA signaling complexes renders breast tumors insensitive to the genomic region 1q21-1q23, is amplified in a subset of human mTOR pathway. Indeed, we show that breast tumors are exqui- breast cancers (41). Thus, breast cancers could potentially hyper- sitely sensitive to pharmacologic mTOR inhibitors regardless of activate PTB-independent ShcA signaling complexes, either whether they are reliant on PTB-coupled or PTB-independent through genomic amplification of the ShcA gene and/or genomic ShcA pools (Fig. 7). Rather, these data establish that activation loss of one or more PTB-dependent negative regulators. We asked of the AKT/mTOR pathway in breast tumors is primarily through whether copy number variations of SHC1, PTPN12 (PTP-PEST), PTB-dependent ShcA signaling complexes. Despite this fact, loss INPPL1 (SHIP2) were correlated with tumors that most closely of SH2-driven ShcA signaling from PTB-independent complexes resemble a ShcAWT-like or PTBMUT-like gene signature. Interest- attenuated Src activation and consequently reduced tumor ingly, ShcA amplification was selectively enriched in ShcAWT-like growth. Altogether, these data corroborate the notion that distinct breast tumors (Supplementary Fig. S7C). In contrast, PTP-PEST, intracellular ShcA signaling complexes transduce diverse and and not SHIP2, genomic loss was specifically enriched in PTBMUT- nonredundant mitogenic signals, that activate the AKT/mTOR like breast tumors (Supplementary Fig. S7C). Although correla- (PTB-dependent) and Src (PTB-independent) pathways to tive, these data suggest a possible relationship between PTP-PEST, cooperatively promote mammary tumorigenesis. ShcA signaling, and Src activation in human breast cancers. Intense research efforts continue to focus on the inhibition of RTKs in the treatment of specific breast cancer subtypes. For example, in ErbB2-driven breast cancers, targeted therapies inhi- Discussion biting ErbB2 and/or ErbB3 signaling are either the standard of care We previously demonstrated that simultaneous expression or are actively being pursued in clinical trials. However, muta- of ShcA alleles that can (ShcAWT) or cannot (PTBMUT) partake in tional activation, amplification, or overexpression of multiple

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Figure 7. Distinct ShcA-dependent pools inﬂuence breast tumor growth, heterogeneity, and therapeutic responsiveness downstream of RTKs during mammary tumorigenesis. Schematic diagram summarizing the biological impact of PTB-dependent versus -independent ShcA signaling complexes on breast tumor growth along with sensitivity to pharmacologic and/or genetic inhibition of the ErbB2, mTOR, and Src family kinase pathways.

components of the tyrosine kinome inevitably promote thera- highlighted that potential differences may exist for SFK depen- peutic resistance to ErbB2-targeted therapies (1). Our study dency across breast cancer subtypes. Indeed, recent studies suggests that the loss of intracellular negative feedback loops describe Lyn as an important effector of tumorigenicity in basal through the aberrant activation of distinct ShcA signaling com- breast cancers (4). Moreover, reduced Src expression or dimin- plexes can result in therapeutic resistance to tyrosine kinase ished SH2-driven Src activation reduced tumor growth, which inhibitors. Indeed, we demonstrate that the hyperactivation of imposes selective pressures on breast tumors to restore Src þ PTB-independent ShcA signaling complexes confers lapatinib signaling (Fig. 7). We propose that the ability of HER2 breast resistance by increasing SH2-driven Src signaling. Moreover, cancers to augment auxiliary ShcA signaling pathways, indepen- debilitating the ShcA SH2 domain or inactivating Src overcomes dently of the PTB domain may represent one mechanism by lapatinib resistance conferred by PTB-independent ShcA signaling which these tumors increase Src activity and acquire resistance complexes (Fig. 7). This is consistent with previous studies dem- to ErbB2 inhibitors. onstrating that Src is hyperactivated in trastuzumab-resistant While PTB-independent ShcA pools can no longer bind ErbB2, þ HER2 breast cancers and that Src inhibition is able to resensitize this does not preclude the fact that it is competent to participate in them to ErbB2-targeted therapies (10). Our observations also SH2-driven interactions to increase ShcA tyrosine phosphoryla- reinforce the importance of SFKs in enhancing tumorigenic sig- tion, including Met (44), PDGFR (45), FGFR (46), and, the SFKs, nals from PTB-independent ShcA pools through multiple exper- Src, and Lyn (36, 38). Indeed, we previously demonstrated that imental approaches. First, Src or Fyn deletion by Crispr/Cas9 mammary tumors expressing PTB-independent pools of ShcA genomic editing and/or Cre-mediated excision selectively atten- establish an autocrine loop to activate Src. Src, in turn, laterally uated transformation from PTB-independent ShcA pools. This activates Met, FGFR, and PDGFR (15). On the other hand, SFK

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recruitment to ShcA is not exclusively dependent on the SH2 observations, PTP-PEST loss only marginally impacted AKT domain and can also involve alternative phospho-tyrosine– signaling but profoundly increased mTOR activation, particularly independent interactions. For example, Src can constitutively S6K and rS6 phosphorylation. As such, PTP-PEST and/or other associate with an amino-terminal region in ShcA (37), leading negative regulators may augment mTOR signaling through a to increased Src activation (47). Moreover, Fyn interacts with a distinct mechanism that is independent of AKT (51–53). Inter- proline-rich region in the CH1 domain of ShcA through its SH3 estingly, mammary epithelial PTP-PEST deletion did not alter Src domain (39). In effect, the ShcA phospho-tyrosine residues, phosphorylation levels, even though downstream effectors of Y239/240 and Y313 can be phosphorylated by either Src and integrin signaling (Cas, Pyk2) were hyperphosphorylated (53). Fyn, respectively, leading to Grb2 recruitment and the consequent However, it is unlikely that genomic loss of PTP-PEST would transduction of mitogenic signals (39, 48). Therefore, PTB- phenocopy the observations found in our study as PTP-PEST is independent ShcA complexes require SFKs to ShcA. It is likely closely involved in actin remodeling independent of ShcA (53, then, that ShcA and Src signaling is reciprocally regulated. Indeed, 54). The effects of PTP-PEST loss on actin remodeling may we found an inverse correlation between Src, Fyn, and Lyn confound the ability to observe any ShcA-regulated effects on Src recruitment to PTB-independent ShcA pools with Src activity and activation. To address this question, the introduction of a PTP- the transforming potential of these ShcA complexes. With the PEST mutant that contains an intact phosphatase domain but knowledge that both SFK activity and an intact SH2 domain is lacks the ShcA binding motif, would be required (21). Alterna- required to enhance tumor growth from PTB-independent ShcA tively, it is possible that one or more alternative negative regula- complexes, these data suggest that the ability of ShcA to interact tors that bind the PTB domain of ShcA may contribute to with SFKs was not functionally significant for increased tumor- increased Src activation from PTB-independent ShcA signaling þ igenesis in ErbB2 breast cancers. Correspondingly, ShcA tyrosine complexes. phosphorylation, which requires Src, is dispensable for increased tumorigenesis from PTB-independent ShcA pools. These data Disclosure of Potential Conflicts of Interest imply that the ShcA SH2 domain indirectly activates Src from No potential conflicts of interest were disclosed. PTB-independent ShcA pools by recruiting a yet to be identified protein, whose signaling properties are also likely to be restrained Authors' Contributions by one or more negative regulators that normally bind the PTB Conception and design: J.R. Ha, J. Ursini-Siegel domain of ShcA. Development of methodology: J.R. Ha Acquisition of data (provided animals, acquired and managed patients, Recruitment of negative regulators to the PTB domain of provided facilities, etc.): J.R. Ha, R. Ahn, H.W. Smith, V. Sabourin, E.C. Canedo,~ ShcA is dependent, in part, on serine phosphorylation of ShcA, Y.K. Im, W.J. Muller which results in the termination of ShcA-dependent signaling Analysis and interpretation of data (e.g., statistical analysis, biostatistics, responses and the subsequent shift in the ShcA interactome to computational analysis): J.R. Ha, S. Hebert, C.L. Kleinman, J. Ursini-Siegel favor cytoskeletal reorganization (23). Specifically, the recruit- Writing, review, and/or revision of the manuscript: J.R. Ha, S. Hebert, ment of PTP-PEST depends on Serine 29 phosphorylation to C.L. Kleinman, J. Ursini-Siegel Administrative, technical, or material support (i.e., reporting or organizing stabilize ShcA/PTP-PEST interactions. Moreover, Threonine data, constructing databases): J.R. Ha 214 phosphorylation of ShcA was recently shown to increase Study supervision: J. Ursini-Siegel ERK and AKT activation (49). Thus, serine/threonine phosphorylation of ShcA dynamically regulates the signaling potential Acknowledgments of the adaptor protein, yet it is still poorly understood. This work was supported by CIHR (MOP-133670; MOP-111143) grants (to How serine/threonine phosphorylation of ShcA from PTB- J. Ursini-Siegel). J.R. Ha is supported by a CIHR Doctoral Award. J. Ursini-Siegel independent complexes may impact breast tumorigenesis is is the recipient of a Senior FRQS salary support award. C.L. Kleinman is yet to be studied and poses an interesting avenue for further supported by a Junior I FRSQ salary support award. W.J. Muller acknowledges a Canada Research Chair in Molecular Oncology. investigation. The authors thank Dr. Peter Siegel for critical reading of the manuscript. We Although there is no evidence for any point mutations in ShcA, also thank Drs. Marc Fabian and Michel Tremblay for providing reagents for our data would suggest that the loss or inactivation of genes that BioID and PTP-PEST antibody, respectively. We further acknowledge support negatively regulate ShcA, would amplify both PTB-dependent and from the small-animal research and pathology cores at the Lady Davis Institute independent ShcA signaling complexes. Indeed, several studies for Medical Research and Goodman Cancer Research Centre. have interrogated the impact of PTP-PEST loss in breast cancers. The costs of publication of this article were defrayed in part by the payment of For instance, PTP-PEST is deleted or mutated in a subset of human page charges. This article must therefore be hereby marked advertisement in primary breast cancers (50). Moreover, the ability of PTP-PEST accordance with 18 U.S.C. Section 1734 solely to indicate this fact. loss to transform mammary epithelial cells requires ShcA (51). We demonstrate that signaling from PTB-independent pools Received October 25, 2017; revised December 20, 2017; accepted January 26, increases mTOR, but not AKT signaling. In accordance with these 2018; published OnlineFirst February 16, 2018.

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Distinct ShcA Pools Cooperatively Increase Tumorigenesis

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Integration of Distinct ShcA Signaling Complexes Promotes Breast Tumor Growth and Tyrosine Kinase Inhibitor Resistance

Jacqueline R. Ha, Ryuhjin Ahn, Harvey W. Smith, et al.

Mol Cancer Res Published OnlineFirst February 16, 2018.

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