PTK6 Inhibition Suppresses Metastases of Triple-Negative Breast Cancer Via SNAIL-Dependent E-Cadherin Regulation Koichi Ito1, Sun Hee Park1, Anupma Nayak2, Jessica H

Published OnlineFirst June 14, 2016; DOI: 10.1158/0008-5472.CAN-15-3445

Cancer Molecular and Cellular Pathobiology Research

PTK6 Inhibition Suppresses Metastases of Triple-Negative Breast Cancer via SNAIL-Dependent E-Cadherin Regulation Koichi Ito1, Sun Hee Park1, Anupma Nayak2, Jessica H. Byerly1, and Hanna Y. Irie1,3

Abstract

Patients with triple-negative breast cancers (TNBC) are at high E-cadherin expression, promoted cell migration, and increased risk for recurrent or metastatic disease despite standard treatment, levels of mesenchymal markers in nontransformed MCF10A underscoring the need for novel therapeutic targets and strategies. breast epithelial cells, consistent with a role in promoting an Here we report that protein tyrosine kinase 6 (PTK6) is expressed epithelial–mesenchymal transition (EMT). SNAIL downregula- in approximately 70% of TNBCs where it acts to promote survival tion and E-cadherin upregulation mediated by PTK6 inhibition and metastatic lung colonization. PTK6 downregulation in mes- induced anoikis, leading to impaired metastatic lung colonization enchymal TNBC cells suppressed migration and three-dimension- in vivo. Finally, effects of PTK6 downregulation were phenocopied al culture growth, and enhanced anoikis, resistance to which is by treatment with a recently developed PTK6 kinase inhibitor, considered a prerequisite for metastasis. PTK6 downregulation further implicating kinase activity in regulation of EMT and meta- restored E-cadherin levels via proteasome-dependent degradation stases. Our ﬁndings illustrate the clinical potential for PTK6 of the E-cadherin repressor SNAIL. Beyond being functionally inhibition to improve treatment of patients with high-risk TNBC. required in TNBC cells, kinase-active PTK6 also suppressed Cancer Res; 76(15); 4406–17. 2016 AACR.

Introduction undergone an EMT exhibit enhanced cellular migration, invasion, and survival, which are required for tumor cell dissem- Triple-negative breast cancers (TNBC) represent approximately ination and metastasis formation. In many preclinical models, 15% of all diagnosed breast cancers, and are a genomically suppression or reversal of EMT impairs metastatic potential of heterogeneous subtype for which current systemic treatments are many tumor cell types (8, 9). limited (1, 2). Although chemotherapy is the mainstay of systemic EMT is also characterized by loss of tight junction and treatment for these patients, many patients develop metastatic adherens junction proteins (e.g., E-cadherin) and increased recurrences due to the heterogeneity within this subtype. The lack expression of mesenchymal markers (e.g., N-cadherin, vimen- of targeted therapies, intrinsic biologic aggressiveness, and che- tin) via both transcriptional and posttranscriptional mechan- motherapy resistance contribute to adverse outcomes for a subset isms (reviewed in refs. 5, 6, 10). Transcriptional repressors, of patients diagnosed with TNBC. Insights into the processes that such as TWIST, SNAIL, SLUG, or ZEB1, suppress transcription of regulate TNBC growth and metastases, as well as drivers of these the epithelial marker E-cadherin (CDH1). Many of the known processes, could lead to novel therapeutic targets and approaches EMT-inducing factors and stimuli, including cytokines such as for high-risk patients. TGFb, EGF, and hepatocyte growth factor (HGF; ref. 10), hyp- A subset of TNBC exhibits a mesenchymal gene expression oxia (11) as well as epigenetic alterations (12, 13), promote profile, which is associated with high histologic grade and EMT via regulation of these E-cadherin transcriptional repres- relative resistance to chemotherapy (3, 4). Epithelial–mesen- sors. Furthermore, the expression of these repressors in tumors chymal transition (EMT) is a developmental process often co- of patients with early-stage TNBC may be associated with early opted by cancer cells and can be initiated by distinct oncogenic relapse and worse prognoses (14–17). signaling pathways (reviewed in refs. 5–7). Cells that have In this article, we investigated the function of protein tyrosine kinase 6 (PTK6) in mesenchymal TNBC cells. PTK6 is an intracellular nonreceptor kinase, distantly related to the Src 1 Division of Hematology and Medical Oncology, Department of Med- family kinases, and is expressed in normal, differentiated epi- icine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York. 2Department of Pathology, Icahn School of thelial cells of the skin and gastrointestinal tract (18) as well as Medicine at Mount Sinai, New York, New York. 3Department of Onco- in a variety of tumor types, including breast, ovary, prostate, logical Sciences, Tisch Cancer Institute, Icahn School of Medicine at lung, and pancreas (19–21). Although among breast cancer Mount Sinai, New York, New York. subtypes, TNBCs express the lowest level of PTK6 transcript Note: Supplementary data for this article are available at Cancer Research (22), PTK6 protein is expressed in patients with TNBCs and Online (http://cancerres.aacrjournals.org/). TNBC cell lines (20, 23). Both PTK6 transcript and protein Corresponding Author: Hanna Y. Irie, Icahn School of Medicine at Mount Sinai, levels have prognostic significance within this subtype, with fl Annenberg 24th oor, Room 04D, 1468 Madison Avenue, New York, NY 10029. higher levels associated with worse prognosis (20, 23, 24). Phone: 212-241-3720; Fax: 646-537-8698; E-mail: [email protected] Furthermore, PTK6 protein expression in TNBC cells can also be doi: 10.1158/0008-5472.CAN-15-3445 induced by known oncogenic stimuli; for example, hypoxia- 2016 American Association for Cancer Research. inducible factor 1a/2a activity promotes PTK6 expression (20).

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PTK6 localizes to both the cytoplasm and nucleus, and can also performed with RUO Discovery Multimer Ultra instrument be recruited to the cell membrane via interactions with activated (Roche). Anti-PTK6 antibody [HPA036070] (Sigma) was used at growth factor receptors such as IGF-1R and HER2 (22, 25, 26). 1:250 dilution. Nuclei were counterstained with hematoxylin. PTK6 enhances cell proliferation, migration, growth, and survival PrEST Antigen PTK6 (Sigma, APREST78020) was used for the via a growing number of interacting proteins and substrates (e.g., peptide competition assay. The slides were scanned using Pan- Paxillin, STAT3/5, FAK, p130Cas, BKS) and synergy with other oramic Scanner 250 (Perkin Elmer) at 40 magnification and oncogenic signaling pathways such as ERK/MAPK and PI3K/AKT images were analyzed by CaseViewer 1.4 (3DHISTECH). (20, 22, 27–29). However, the role of PTK6 in metastasis formation of triple-negative cancers has not been studied previously. In RNAi knockdown the current study, we show that PTK6 promotes EMT and regulates For all siRNAs and short hairpin RNAs (shRNA), the specific metastases of TNBC cells by modulating E-cadherin expression via sequence is identified in the figures by the last two digits of the fi SNAIL protein degradation. For the rst time, we also show the TRC or catalog number (provided in Supplementary Tables S1 biologic activity of a novel small-molecule inhibitor of PTK6 and S2). siRNAs targeting PTK6 and GSK3B were obtained from fi kinase activity and its ef cacy in preventing TNBC metastases. GE Healthcare Dharmacon. The targeting sequences are shown in Supplementary Table S1. Transfection was performed using Materials and Methods Oligofectamine (Life Technologies), following the manufacturer's PTK6, SNAI1 CDH1 Antibodies and reagents protocol. shRNAs targeting ,or were pur- Antibodies purchased from Cell Signaling Technology are: chased from Sigma-Aldrich. The TRC number and targeting GAPDH (14C10), b-tubulin (9F3), SNAIL (C15D3), SNAIL sequence are shown in Supplementary Table S2. Lentivirus was (L70G2), SLUG (C19G7), E-Cadherin (24E10), Claudin-1 generated by cotransfecting 293T cells with shRNA vector and (D5H1D), and GSK3b (27C10). Antibodies purchased from BD packaging plasmids (Delta 8.9 and pCMV-VSV-G) using Lipofec- Transduction Laboratories are: N-Cadherin (clone 32), vimentin tamine 2000 and Plus reagent (Life Technologies; ref. 22). The (RV202), and GSK3b (pY216, 13A). Phospho-PTK6 (Tyr342) Ab viral supernatants were collected and stored at 80 C. Cells were was purchased from Millipore. Antibodies obtained from Santa infected with viral supernatants. Cruz Biotechnology are: BRK antibodies (C-18 and D-7), ZEB1 (H-102), goat anti-rabbit IgG-HRP, and goat anti-mouse 3D cell growth assays IgG-HRP. PTK6 inhibitor (Pyrazin-21d, P21d) was provided by MDA-MB-231 or MMTV-myc cells (3,000 cells) were added to Merck Research Laboratories (30). a-Tubulin Ab [DM1D] and 8-well chamber slides (BD Biosciences) coated with 50 mLof MG132 were purchased from Sigma-Aldrich. TWS119 was growth factor–reduced Matrigel (Corning). Media were replaced purchased from Santa Cruz Biotechnology. PTK6 cDNA every 3 days. Cells were imaged using the Axiovert 25 inverted constructs [myristoylated (MF), constitutively active Y447F (CA), microscope (Carl Zeiss AB). CellTiter-Glo 3D Cell Viability Assay or kinase dead K219M (KD)] were reported previously (22). (Promega) was used to quantitate viability of cells grown in Matrigel in 96-well plates. Cell culture and protein overexpression MCF10A cells (ATCC) were cultured as reported previously Confocal microscopy (22). MDA-MB-231 and BT549 cells (ATCC) were cultured in Cells were cultured on coverslips and fixed with 4% para- RPMI1640 supplemented with 10% FBS. Hs578t cells (ATCC) formaldehyde/PBS for 20 minutes, followed by permeabilization were cultured in DMEM supplemented with 10% FBS. MMTV- with ice-cold 95% methanol for 30 minutes. Cells were washed myc cells obtained from Dr. Eduardo Farias (Icahn School of with PBS, blocked with 3% BSA/PBS/0.1% TritonX for 1 hour at Medicine at Mount Sinai, New York, NY) and were cultured in room temperature, and incubated with phospho-PTK6 Ab (1:100 DMEM/F12 supplemented with 5% FBS and 4 mg/mL insulin dilution). After overnight incubation at 4C, cells were washed, (31). MMTV-myc cells were validated as a triple-negative cell line incubated with AlexaFluor 568 conjugated anti-rabbit IgG (1:200, by Western blot and qRT-PCR analyses. Cell circularity was Life Technologies) for 1 hour at room temperature, and mounted calculated using ImageJ software [circularity ¼ 4 p (area/peri- with VECTASHELD anti-fade media with DAPI (Vector). Images meter2)]. Generation of PTK6-overexpressing cell lines was were captured by SP5 confocal microscopy (Leica Microsystems). performed by infecting cells with retrovirus generated using The gain and offset setting were fixed across all samples. GPG-293T cells, as described previously (32). SNAIL-overexpressing MDA-MB-231 cells were generated using lentiviral construct Lung colonization assay pLEX-SNAIL-F-Luc (obtained from Dr. Yibin Kang, Princeton MDA-MB-231 or MMTV-myc cells were transduced with PTK6 – University, Princeton, NJ; ref. 33). Human E-cadherin overex- shRNA virus for 72 hours. For experiments with the PTK6 inhib- pressing MCF10A cells were generated using lentiviral construct itor, MMTV-myc cells were treated with P21d for 48 hours in vitro. pEX-M0954-Lv151 (control: pEX-NEG-Lv151; GeneCopoeia). shRNA-infected or inhibitor-treated cells were washed with PBS three times. MDA-MB-231 cells (2 106 per mouse) or Tissue microarray analysis MMTV-myc cells (5 104 per mouse) in 100 mL of PBS were Triple-negative breast tumor samples (paraffin-embedded injected into the tail veins of 6-week-old NOD-SCID or FVB tumor tissue blocks; n ¼ 60) were obtained from the Archives female mice (Charles River Laboratories), respectively. Lung of Pathology Department at Icahn School of Medicine at Mount tissues were harvested after 4 weeks for MDA-MB-231 and 3 weeks Sinai. Tissue microarrays (TMA) were constructed with Chemicon for MMTV-myc, and fixed in Bouin solution. The number of surface International ATA-100 using two representative 2-mm cores from lung metastases (size greater than 0.5 mm diameter for MDA-MB- each sample. Automated immunohistochemical staining was 231 and 1 mm for MMTV-myc cells) was counted. All animal

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PTK6 Inhibition Suppresses TNBC Metastases

procedures were conducted in compliance with the guidelines of and MMTV-myc cells were increased, as assessed by qRT-PCR the IACUC Committees of Mount Sinai School of Medicine. (Fig. 1H; primer sequences provided in Supplementary Table S3). Mesenchymal markers (N-cadherin and vimentin) were Statistical analysis not reproducibly affected by PTK6 downregulation in the cell All values were expressed as mean SEM, and the number of lines evaluated (Fig. 1G). Therefore, PTK6 downregulation n fi samples ( ) was indicated. The statistical signi cance of differ- impairs growth and migration of TNBC cells, and enhances ences between control and experimental groups was determined E-cadherin expression, partially reversing mesenchymal fea- t P < fi P < by Student test with 0.05 considered signi cant ( , 0.05; tures of these cells. P < P < , 0.01; , 0.001). All statistical analyses were performed Our data support a role for PTK6 in EMT. We wanted to using GraphPad Prism v6.0d. determine whether PTK6 is sufficient to induce EMT. In our For more information, see Supplementary Materials and previous studies, we generated MCF10A breast epithelial cells Methods. overexpressing a CA-mutant PTK6 (CA-PTK6, Y447F; ref. 22). PTK6 activity was confirmed by detection of enhanced autopho- Results sphorylation (tyrosine 342), as well as increased phosphorylation of STAT3 (tyrosine 705), an established substrate of PTK6 (Fig. 2A PTK6 is expressed in patients with triple-negative cancers and and B). We observed that with expression of CA-PTK6, but not regulates EMT kinase-dead PTK6 (KD-PTK6, K219M), MCF10A cells assumed a We sought to elucidate the role of PTK6 in TNBC growth and more spindle-shaped morphology, which we quantitated using metastases. As reported previously by others, we found that PTK6 measures of cell circularity (Fig. 2C). In addition to morphologic protein is expressed in patient triple-negative tumors (20, 23). In changes, increased expression of kinase-active, but not catalyti- our tumor microarrays of 60 patients with triple-negative cancers cally inactive, PTK6 enhanced migration of MCF10A cells in (TNBC), the majority expressed PTK6 protein (74%) with 13% wound-healing assays (Fig. 2D). PTK6 activation suppressed þ fi exhibiting strong (3 ) staining (Fig. 1A). This signal is speci cas expression of epithelial markers, E-cadherin and Claudin-1, and coincubation with blocking (competition) peptide completely increased levels of the mesenchymal marker, N-cadherin (Fig. 2E). abrogated staining with this PTK6 antibody (Supplementary Fig. E-cadherin regulation directly and functionally contributes to S1). PTK6 protein is also expressed in several TNBC cell lines PTK6-associated changes in migration; overexpression of exoge- (human and mouse; Fig. 1B). We chose two PTK6-expressing nous E-cadherin partially suppresses cell migration induced by mesenchymal breast cancer cell lines (human MDA-MB-231 and active PTK6 in wound-healing assays (Fig. 2F). These findings murine MMTV-myc) for further study. support the ability of PTK6 to drive EMT in a kinase activity– To determine the roles of PTK6 in TNBC cell growth and dependent manner metastases, we downregulated expression of PTK6 using siRNA or shRNA vectors (sequences provided in Supplementary PTK6 downregulation enhances E-cadherin expression Tables S1 and S2). We confirmed expression downregulation in TNBC cells via proteasome-dependent SNAIL by Western blot analysis using two different antibodies (C-18 protein regulation and D-7, Santa Cruz Biotechnology; Fig. 1C). PTK6 down- In PTK6-downregulated TNBC cells, we observed an increase regulation suppressed cell proliferation of MDA-MB-231 and in E-cadherin transcript levels (Fig. 1G), supporting a transcrip- MMTV-myc cells in two-dimensional (2D) culture (Fig. 1D) as tional mechanism of E-cadherin regulation by PTK6. well as growth in three-dimensional (3D) Matrigel cultures E-cadherin transcription is regulated by several known CDH1 (Fig. 1E). PTK6 downregulation also significantly impaired transcriptional repressors, such as SNAIL, SLUG, ZEB, and migration of MDA-MB-231 and MMTV-myc cells in transwell TWIST (6). We examined the levels of these known transcrip- Boyden chamber assays (Fig. 1F). This impaired migration is tional repressors in PTK6-downregulated cells and consistently not due to differences in proliferation; migration of mitomycin observed changes only in SNAIL; transfection with two inde- C–treated, nonproliferating cells (34) was also inhibited by pendent PTK6 siRNAs or infection with two distinct PTK6 PTK6 downregulation (Supplementary Fig. S2). shRNA viruses resulted in decreased SNAIL protein levels in Given these effects on migration, we wondered whether both MDA-MB-231 and MMTV-myc cells (Fig. 3A). The levels PTK6 regulates epithelial and mesenchymal adhesion molecule of SLUG, ZEB, and TWIST were not consistently affected. profiles. siRNA- or shRNA-mediated downregulation of PTK6 Decrease in the level of SNAIL protein was sufficient to enhance increased levels of epithelial E-cadherin in both MDA-MB-231 E-cadherin levels in both MDA-MB-231 and MMTV-myc cell and MMTV-myc cells (Fig. 1G). The increase in E-cadherin lines (Fig. 3B). Moreover, overexpression of CA PTK6 in protein levels is at least partially due to increased transcription; MCF10A, MDA-MB-231 or BT549 cells increased levels of hCDH1 transcript levels in PTK6-downregulated MDA-MB-231 SNAIL, whereas overexpression of kinase-inactive PTK6 in

Figure 1. Downregulation of PTK6 partially reverses EMT in TNBC cells. A, TMA of TNBC patient tissues (total n ¼ 60). The slides were stained with anti-PTK6 antibody. B, Western blot analyses for PTK6 and EMT-related markers in TNBC cell lines. C, validation of siRNA- or shRNA-mediated downregulation of PTK6 in MDA-MB-231 and MMTV-myc cells. D, 2D proliferation assays (CellTiter-Glo) of MDA-MB-231 or MMTV-myc expressing pLKO.1 vector control (VC) or PTK6 shRNA (day 3). E, 3D Matrigel cultures of MDA-MB-231 (day 12) or MMTV-myc (day 8) expressing pLKO.1 vector control or PTK6 shRNA. Scale bar, 30 mm. Viability was assessed by 3D CellTiter-Glo. F, transwell migration assay using MDA-MB-231 cells transfected with siLuciferase control or PTK6 siRNA, or MMTV-myc expressing VC or PTK6 shRNA. Representative figures from three independent experiments are shown. The quantification of migrated cells per field is shown in the bar graphs; mean SEM (three individual experiments). G, Western blot analysis for EMT markers in MDA-MB-231 and MMTV-myc cells expressing VC or PTK6 siRNA/shRNA. H, qRT-PCR analysis of CDH1 mRNA transcripts in PTK6-downregulated MDA-MB-231 and MMTV-myc cells. All bar graphs are shown as mean SEM.

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Figure 2. PTK6 overexpression in MCF10A cells promotes EMT. A, whole cell lysates of MCF10A cells expressing vector control (pBabe), constitutively active (CA) PTK6 (Y447F), or kinase-dead (KD PTK6 (K219M) were probed with the indicated antibodies. B, ﬂow cytometric analysis of MCF10A cells expressing pBabe, CA-, or KD-PTK6 using phospho-PTK6 (Y342) antibody. Relative change in mean ﬂuorescence intensity (MFI; vs. pBabe) is shown in the bar graph; mean SEM (three individual experiments). C, cell circularity of pBabe-, CA-PTK6-, or KD-PTK6–expressing MCF10A cells was analyzed (n ¼ 50). Representative phase images of each group of MCF10A cells grown in monolayer cultures are shown (scale bar, 30 mm). D, wound-healing assay with MCF10A cells expressing pBabe, CA-, or KD-PTK6. Images were captured 4 and 12 hours after wound was created. Relative wound width is shown in the graph; mean SEM (three individual experiments). E, Western blot analysis of EMT markers using whole cell lysates of MCF10A cells expressing pBabe, CA-, or KD-PTK6. F, wound-healing assay with MCF10A cells expressing pBabe or CA-PTK6 pEx-CDH1. Relative wound width is shown; mean SEM (three individual experiments).

MCF10A cells did not (Fig. 3C). The reduction in SNAIL protein dation. In cells serially transfected with GSK3b siRNA followed by caused by PTK6 downregulation is due to a nontranscriptional PTK6 siRNA, downregulation of SNAIL was still observed (Fig. mechanism, as we did not observe any signiﬁcant changes in 4B). Furthermore, treatment with TWS119, a GSK3b-selective SNAI1 mRNA levels in PTK6-downregulated MDA-MB-231 or inhibitor, did not prevent the decrease in SNAIL expression MMTV-myc cells (Fig. 3D). Therefore, we hypothesized that following PTK6 downregulation, whereas the level of b-catenin, PTK6 regulates the posttranscriptional stability of SNAIL protein. another known target of GSK3b-dependent degradation, was As SNAIL is known to be degraded via ubiquitination and increased in a dose-dependent manner (Fig. 4C). Therefore, PTK6 proteasome activity, we wondered if PTK6 downregulation pro- downregulation enhances degradation of SNAIL protein via a motes proteasome-dependent degradation of SNAIL. Treatment mechanism that is independent of GSK3b activity. of PTK6 siRNA-transfected cells with MG132, an inhibitor of proteasome activity, restored SNAIL protein levels to basal levels PTK6 small-molecule inhibitor treatment downregulates SNAIL (Fig. 4A). We next determined whether PTK6 downregulation– protein and restores E-cadherin expression in TNBC cells induced SNAIL degradation is dependent on GSK3b activity. Zheng and colleagues previously described the synthesis of GSK3b is known to target SNAIL for proteasome-dependent imidazo[1,2-a]pyrazin-8-amines that inhibit PTK6 kinase activity degradation by phosphorylating residues in the serine-rich with relative speciﬁcity (30). We evaluated the ability of these domain of SNAIL (35). This phosphorylation is required for compounds to inhibit PTK6 kinase activity in breast cells. recognition and ubiquitination by the E3 ligases, We selected P21d for our initial studies given its potency with b-TrCP1/FBXW1 SCF , and subsequent proteasome-dependent degra- in vitro IC50 ¼30 nmol/L. Utilizing MCF10A cells overexpressing

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Figure 3. PTK6 downregulation suppresses SNAIL and increases E-cadherin expression. A, Western blot analysis for SNAIL, SLUG, ZEB1, and TWIST in PTK6-downregulated MDA-MB-231 and MMTV-myc cells. B, Western blot analysis for E-cadherin in MDA-MB-231 and MMTV-myc cells transfected with SNAIL siRNA or SNAIL shRNA. C, Western blot analysis for SNAIL in MCF10A, MDA-MB-231, or BT549 cells overexpressing pBabe vector control, CA-PTK6, or KD-PTK6. For BT549 cells, nuclear lysate was used. D, qRT-PCR analysis for SNAI1 mRNA transcripts in PTK6-downregulated MDA-MB-231 and MMTV-myc cells. Bar graphs are shown as mean SEM. All experiments were repeated at least three times.

myristoylated PTK6 (MF-PTK6), we observed a dose-dependent reduction in phosphorylation of tyrosine 342 (an autophosphor- ylation site) by Western blot analysis, thereby confirming the ability of P21d to inhibit PTK6 kinase activity (Fig. 5A). In addition, flow cytometric analysis demonstrated that P21d treatment significantly decreased mean fluorescence intensity (MFI) levels of phospho-PTK6 (Y342) in CA-PTK6 expressing MCF10A cells (Fig. 5B). Confocal microscopy analysis confirmed membrane-associated phospho-PTK6 in MCF10A cells expressing MF-PTK6; these phospho-PTK6 signals were suppressed by 2-hour treatment with P21d (Fig. 5C). Endogenous autophosphorylated (activated) PTK6 in TNBC cells was difficult to detect by Western blot analysis or flow cytometry. We therefore examined levels and patterns of endogenous auto-phosphorylated, active PTK6 in TNBC cells by immu- nofluorescence staining and confocal microscopy. In MDA-MB- 231 and MMTV-myc cells, we observed autophosphorylated PTK6 at the cell membrane, as well as in the nucleus (Fig. 5D and E). Two-hour treatment of MDA-MB-231 or MMTV-myc cells with the PTK6 inhibitor suppressed the phospho-PTK6 signal (Fig. 5D and E). Interestingly, activated PTK6 was previously reported to be mostly localized at the cell membrane of patient breast tumors (23). In nontransformed MCF10A cells, there was no detectable autophosphorylated PTK6 (Supplementary Fig. S3). PTK6 inhibitor treatment of TNBC cells phenocopies the effects of PTK6 expression downregulation. Treatment with PTK6 inhibitor suppressed migration of MDA-MB-231 and MMTV-myc cells (Fig. 6A). Inhibitor treatment increased E-cadherin expression and downregulated SNAIL expression in TNBC cells in a dose- dependent manner (Fig. 6B). Similar effects on E-cadherin and SNAIL were observed with in vivo MMTV-myc tumors treated with PTK6 inhibitor [daily intraperitoneal administration of P21d Figure 4. (10 mg/kg) for 3 days; Fig. 6C and D]. SNAIL and E-cadherin PTK6 downregulation decreases SNAIL protein levels by a GSK3b-independent, are critical mediators of PTK6 inhibitor–induced biologic effects; proteasome-dependent mechanism. A, PTK6-downregulated MDA-MB-231 overexpression of SNAIL or downregulation of E-cadherin pre- cells were treated with DMSO or MG132 (10 mmol/L) for 30 or 180 minutes. Levels vents the full inhibitory effects of P21d treatment on migration of of SNAIL were assessed. B, levels of SNAIL in MDA-MB-231 cells serially transfected with GSK3b siRNA followed by PTK6 siRNA were assessed. C, TNBC cells (Fig. 6E and F). levels of SNAIL were assessed in PTK6 siRNA-transfected MDA-MB-231 cells The mechanisms by which PTK6 inhibitor treatment alters treated with GSK3b inhibitor TWS119 for 8 hours. All experiments were repeated SNAIL and E-cadherin levels also parallel that responsible for at least three times and representative blots are shown.

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Figure 5. Treatment with PTK6 small-molecule inhibitor P21d inhibits PTK6 activity. A, levels of autophosphorylated PTK6 (pY342) in MCF10A cells expressing myristoylated (MF), FLAG tagged PTK6 (MF-PTK6) treated with PTK6 small-molecule inhibitor, P21d, were assessed by Western blot analysis. Cells were treated with P21d at the indicated doses for 1 hour. B, ﬂow cytometric analysis of MCF10A cells expressing pBabe, CA-PTK6, or KD-PTK6 treated with P21d (5 mmol/L) for 2 hours in suspension cultures. Levels of autophosphorylated PTK6 were assessed. Relative changes in MFI (vs. pBabe DMSO) are shown in the bar graph (mean SEM). C, confocal microscopy was used to detect autophosphorylated PTK6 (red) and a-tubulin (green) in MF-PTK6 overexpressing MCF10A cells. D and E, autophosphorylated, endogenous PTK6 in MDA-MB-231 and MMTV-myc cells treated with DMSO or P21d was assessed by confocal microscopy. Autophosphorylated PTK6 and nuclei are shown in red and blue, respectively (scale bar, 15 mm).

PTK6 shRNA/siRNA-induced changes; P21d treatment downregulation (Fig. 6I). Furthermore, SNAIL regulation by enhances E-cadherin transcript levels without signiﬁcantly PTK6 is independent of other previously reported mechanisms altering SNAIL transcript levels (Fig. 6G). MG132 treatment that promote SNAIL ubiquitination and degradation; down- restored SNAIL protein levels in P21d-treated cells (Fig. 6H). regulation of BRTC, FBXL5, FBXO11, MDM2, and CBL all P21d treatment enhances proteasome-dependent degradation failed to fully rescue SNAIL protein levels in P21d-treated cells of SNAIL by a GSK3b-independent mechanism as downregula- (Supplementary Fig. S4). These data again support a novel tion of GSK3b by siRNA did not prevent P21d-induced SNAIL mechanism for SNAIL protein regulation by PTK6.

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Figure 6. PTK6 small-molecule inhibitor treatment phenocopies the effects of PTK6 downregulation on EMT. A, transwell migration assay of MDA-MB-231 and MMTV-myc cells treated with DMSO or P21d (5 mmol/L) for 72 hours. B, Western blot analysis for E-cadherin and SNAIL in MDA-MB-231 and MMTV-myc cells treated with DMSO or P21d for 24 or 72 hours. Normalized band intensities are shown below. C, Western blot analysis for SNAIL in MMTV-myc tumor tissues. FVB mice bearing MMTV-myc tumors were treated with P21d 10 mg/kg for 3 days (i.p.). The tumor tissues were harvested and lysed. D, immunohistochemical analysis of E-cadherin expression in MMTV-myc primary tumor allografts treated with P21d at 10 mg/kg daily for 3 days (green, E-cadherin; blue, nuclei stained with DAPI; scale bar, 30 mm). E and F, transwell migration assay of MDA-MB-231 cells expressing exogenous SNAIL or E-cadherin shRNA with or without P21d (5 mmol/L) treatment. The number of migrated cells per ﬁeld is shown in the bar graphs; mean SEM (three individual experiments). G, qRT-PCR analysis of CDH1 and SNAI1 mRNA transcripts in DMSO- or P21d (5 mmol/L)-treated MDA-MB-231 cells. Fold changes in relative expression (vs. DMSO) are shown as mean SEM (three individual experiments). H, MDA-MB-231 cells were treated with DMSO or P21d for 72 hours, followed by treatment with MG132 (10 mmol/L) for 30 or 180 minutes. Levels of SNAIL were assessed by Western blot analysis. I, MDA-MB-231 cells were transfected with GSK3b siRNA and then treated with P21d for 48 hours. Levels of SNAIL were assessed by Western blot analysis.

In sum, PTK6 inhibitor treatment phenocopies the effects PTK6 inhibition induces anoikis and prevents observed with PTK6 siRNA/shRNA expression, speciﬁcally metastases of TNBC cells impaired migration, SNAIL downregulation, and increased EMT of cancer cells has been associated with increased E-cadherin expression, thereby supporting a critical role for dissemination and metastasis formation. We therefore hypoth- PTK6 kinase activity in EMT of TNBC cells. esized that PTK6 inhibition could impact TNBC metastasis

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formation. We assessed rates of lung metastases following tail findings highlight the possibility of PTK6 inhibition as an attrac- vein injection of MDA-MB-231 or MMTV-myc cells expressing tive strategy to inhibit growth and prevent metastasis of TNBCs. vector control or PTK6 shRNA. PTK6 downregulation signifi- EMT is induced by oncogenes and transcription factors (TWIST, cantly decreased the number of surface metastatic foci in both SNAIL, SLUG), cytokines (TGFb, EGF, HGF, FGF) as well as models (Fig. 7A and B). Pretreatment with PTK6 small-mole- stressors such as hypoxia (36). In in vivo models, induction of cule inhibitor P21d for 48 hours prior to tail vein injection also EMT promotes dissemination of tumor cells, which is a prereq- significantly decreased the number of lung metastases, when uisite for metastasis formation (reviewed in ref. 37). Furthermore, compared with DMSO-treated control cells (Fig. 7C). The mesenchymal gene expression profiles correlate with relative observed difference in metastatic lung colonization is not likely resistance to chemotherapy, which is also associated with due to a difference in proliferation between tail vein-injected increased likelihood for metastases and adverse outcomes for control and PTK6 inhibitor or shRNA-treated cells; using cells patients (37, 38). cultured in suspension as a model for TNBC cells in circulation PTK6, a nonreceptor tyrosine kinase, was previously implicated þ following tail vein injection, we did not observe any differences in regulation of EMT of prostate cancer cells and HER2 breast in cell-cycle profiles between DMSO- and PTK6 inhibitor– cancers via distinct mechanisms. Overexpression of PTK6 in treated cells (Supplementary Fig. S5). prostate cancer cells promoted EMT via Akt activation (39). In As we reported previously that PTK6 regulates anoikis sensi- MCF7 breast cancer cells overexpressing HER2 that were selected þ þ tivity of HER2 and ER breast cancer cells (22), we wanted to in vivo for increased invasive potential, downregulation of PTK6 determine whether regulation of anoikis sensitivity by PTK6 could suppressed migration and expression of mesenchymal markers, be responsible for the decreased lung colonization of PTK6 shRNA and restored E-cadherin expression; these effects were prevented or inhibitor-treated TNBC cells. Downregulation of PTK6 expres- by restoration of active Stat3, a known substrate of PTK6 (40). sion using either of two independent shRNA vectors enhanced However, the mechanisms by which PTK6 regulates core tran- anoikis of MDA-MB-231 and MMTV-myc cells grown in suspen- scriptional factors and repressors directly responsible for EMT sion cultures, as assessed by AnnexinV/PI staining (Fig. 7D and E). were not elucidated by these studies. Our study is the first to Similarly, P21d treatment for 72 hours enhanced anoikis of identify PTK6-dependent regulation of transcriptional repressors MMTV-myc cells (Fig. 7F). responsible for changes in epithelial adhesion markers, and is also We wanted to determine whether regulation of EMT, specif- the first to address the mechanistic links between PTK6-dependent ically SNAIL/E-cadherin, by PTK6 directly contributes to anoi- SNAIL/E-cadherin regulation, anoikis resistance and metastasis kis resistance and metastasis formation. Overexpression of formation. In addition, we demonstrate the kinase dependency of SNAIL suppressed anoikis of P21d-treated or PTK6 shRNA- EMT regulation by PTK6 and highlight the ability of a small- expressing MDA-MB-231 cells (Fig. 7G and H). Suppression molecule inhibitor to suppress metastasis formation. of P21d treatment–induced E-cadherin expression was also SNAIL is a member of the family of transcriptional repressors of sufficient to suppress anoikis of MMTV-myc cells in suspension the E-cadherin promoter. PTK6 specifically regulates the expres- cultures (Fig. 7I). Finally, suppression of E-cadherin abrogated sion of SNAIL, and not other transcriptional repressors. SNAIL has effects of P21d treatment on lung metastases rates; the number been linked to generation of cancer stem cell properties (41), drug of lung metastases was restored to levels observed in control resistance (37), cancer recurrence and metastasis, all contributors mice (vector control shRNA/DMSO-treated; Fig. 7J). These to poor prognosis for patients (16, 42). Although SNAIL is often results support SNAIL/E-cadherin regulation as a critical mech- transcriptionally regulated by EMT-inducing stimuli, here we anistic mediator of PTK6-associated anoikis resistance and show that PTK6 regulates the proteasome-dependent degradation metastatic colonization (Fig. 7K). of SNAIL protein. Although GSK3b-dependent phosphorylation of SNAIL is a major mechanism by which SNAIL is targeted for proteasome-dependent degradation (35), our results show that Discussion PTK6 inhibition-dependent SNAIL protein regulation is indepen- There is increasing evidence supporting a critical role for PTK6 dent of GSK3b expression or activity. SNAIL regulation by PTK6 is in a variety of tumor cell types. Although its roles in proliferation also independent of other known regulators, such as F-box and survival of cancer cells have been well documented, less is proteins (FBXO1 FBX11), MDM2, and CBL. Our ongoing studies known about PTK6's role in EMT, a process likely associated with will identify the specific mediators of this GSK3b-independent dissemination, chemotherapy resistance, and enhanced metastat- regulation of SNAIL protein degradation. ic potential of tumor cells. In the current study, we investigate the Our studies highlight the requirement for PTK6 kinase activ- functions of PTK6 in TNBC cells, a subtype of breast cancer that ity in the regulation of EMT. Overexpression only of a kinase includes tumors with mesenchymal gene expression profiles active, but not catalytically inactive PTK6, in MCF10A breast (3, 4). We show that inhibition of PTK6, either via expression epithelial cells induced EMT (e.g., suppression of E-cadherin, downregulation or by inhibition of kinase activity, partially increase N-cadherin, increase migration). Furthermore, treat- reverses mesenchymal properties of TNBC cells, specifically restor- ment of mesenchymal TNBC cells with P21d, a recently devel- ing E-cadherin expression and impairing migration and growth. oped small-molecule inhibitor of PTK6, inhibited migration We identified a novel mechanism by which PTK6 regulates and restored E-cadherin expression, phenocopying the effects E-cadherin and EMT; PTK6 regulates the stability of SNAIL of PTK6 expression downregulation on EMT. PTK6 inhibitor protein, a transcriptional repressor of E-cadherin, via pathways treatment of mesenchymal TNBC cells suppressed metastasis independent of known mechanisms. Finally, we establish that the formation, again phenocopying the effects of PTK6 shRNA reversal of mesenchymal properties and restoration of expression. Although there are a growing number of PTK6 E-cadherin expression by PTK6 inhibition sensitizes tumor cells substrates, such as AKT, b-catenin, STAT3/5, Paxillin, SAM68 to anoikis, and suppresses metastasis formation in vivo. Our (29, 43–48), none has been specifically linked to regulation of

4414 Cancer Res; 76(15) August 1, 2016 Cancer Research

PTK6 Inhibition Suppresses TNBC Metastases

Figure 7. P21d treatment sensitizes TNBC cells to anoikis and suppresses metastasis formation. A, MDA-MB-231 cells expressing vector control (VC) or PTK6 shRNA were injected into tail veins of NOD/SCID female mice. After 4 weeks, the lungs were harvested and fixed. The number of metastases was counted. B, MMTV-myc cells expressing VC or PTK6 shRNA were injected into tail veins of FVB female mice. Lungs were harvested after 3 weeks and metastatic foci were counted. C, MMTV- myc cells pretreated with DMSO or P21d (5 mmol/L) for 2 days were injected into tail veins of FVB female mice. After 3 weeks, the lungs were harvested and fixed, and the number of metastases counted. Graphs show mean number of metastases SEM. D and E, anoikis of MDA-MB-231 or MMTV-myc cells expressing VC or PTK6 shRNA was assessed. Cells were cultured in suspension for the indicated number of hours, stained with AnnexinV/PI, and analyzed by flow cytometry. Representative plots from 8-hour time points are shown. F, anoikis of MMTV-myc cells was assessed. Cells were pretreated with DMSO or P21d (5 mmol/L) for 72 hours and then cultured in suspension for 24 hours. Anoikis was assessed by cell death ELISA assay. G, anoikis of MDA-MB-231 cells expressing VC or PTK6 shRNA (SNAIL overexpression) was assessed by AnnexinV/PI staining. H, MDA-MB-231 cells (SNAIL overexpression) were treated with DMSO or P21d (5 mmol/L) for 72 hours in suspension cultures. Cell death was assessed by AnnexinV/PI staining. I, MMTV-myc cells expressing E-cadherin shRNA were treated with DMSO or P21d (5 mmol/L) for 24 hours in suspension cultures. Cells were stained with AnnexinV/PI. Relative changes of AnnexinVþ/PIþ (vs. DMSO-treated vector control) are shown in the bar graph as mean SEM. J, MMTV-myc cells expressing vector control or E-cadherin shRNA were treated with DMSO or P21d (5 mmol/L) for 2 days in vitro. Cells were injected into tail veins of FVB female mice. After 3 weeks, lung surface metastases were counted. Graphs show mean number of metastases SEM. K, model of E-cadherin and metastasis regulation by PTK6.

www.aacrjournals.org Cancer Res; 76(15) August 1, 2016 4415

Ito et al.

EMT in TNBC. The specific substrates of PTK6 responsible for central role in the oncogenic activities of PTK6 in mesenchymal regulation of SNAIL and E-cadherin are the focus of current, TNBCs, directly impacting metastatic colonization. ongoing studies. Our data collectively support the importance In conclusion, PTK6 inhibition reverses mesenchymal proper- of PTK6 kinase activity in the regulation of EMT, as well as the ties of TNBC cells and suppresses their growth and migration in possibility of therapeutic approaches incorporating this PTK6 vitro, as well as metastasis formation in vivo. Our results with a kinase inhibitor to prevent TNBC metastasis. recently developed small-molecule inhibitor of PTK6 phenocopy Interestingly, PTK6 may play a tissue-dependent role in our findings with PTK6 downregulation and raises the exciting regulating EMT. In contrast to our results with breast epithelial possibility that PTK6 inhibition may be further developed as a cells and TNBC cells, Mathur and colleagues recently reported therapeutic approach for TNBC. that PTK6 promotes the epithelial phenotype in the context of a colon cancer cell line, and PTK6 shRNA expression increased Disclosure of Potential Conflicts of Interest migration, suppressed E-cadherin expression and increased No potential conflicts of interest were disclosed. mesenchymal marker expression (49). These seemingly con- tradictory roles of PTK6 in EMT may be due to tissue-specific Authors' Contributions contexts, as PTK6 specificallyplaysaroleinthedifferentiation Conception and design: K. Ito, S.H. Park, H.Y. Irie of colonic epithelial cells (50). The distinct genetic alterations Development of methodology: K. Ito, H.Y. Irie found in colon cancer, such as those in APC and Ras, which are Acquisition of data (provided animals, acquired and managed patients, less frequently found in breast cancers may also interact dif- provided facilities, etc.): K. Ito, A. Nayak, J.H. Byerly, H.Y. Irie fi Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ferentially with PTK6-induced signaling pathways. These nd- computational analysis): K. Ito, J.H. Byerly, H.Y. Irie ings, along with our results, underscore the importance of Writing, review, and/or revision of the manuscript: K. Ito, S.H. Park, A. Nayak, individualized evaluation of the consequences of PTK6 inhi- J.H. Byerly, H.Y. Irie bition in specificcancertypes. Administrative, technical, or material support (i.e., reporting or organizing Although EMT is associated with invasive, migratory behavior data, constructing databases): S.H. Park, A. Nayak, H.Y. Irie of cancer cells, and is likely important for tumor cell dissemina- Study supervision: A. Nayak, H.Y. Irie tion, it has been challenging to establish EMT as a causal require- Acknowledgments ment for metastasis formation, as well as to elucidate the specific The authors thank Dr. Eduardo Farias for providing 4T1 and MMTV-myc roles for EMT in the multi-step process of metastasis initiation and cells. The authors acknowledge the Tisch Cancer Institute Biorepository and formation. EMT has been associated with resistance to anoikis, a Histopathology Cores at Icahn School of Medicine at Mount Sinai (Dr. Michael form of cell death that occurs when cells are detached from their Donovan, Dr. Tin Htwe Thin, and Anastasiya Dzhun). The authors also thank surrounding extracellular matrix (reviewed in refs. 51, 52). Anoi- Dr. Tatyana Grushko and Dr. Olufunmilayo Olopade for initial work with PTK6 kis resistance contributes to the ability of cells to survive in altered antibody for IHC. matrix environments as they travel in circulation to secondary organ sites to form metastases. Loss of E-cadherin expression, a Grant Support marker of EMT, is sufficient to promote anoikis resistance; for This work was supported by Susan G Komen for the Cure Career Catalyst example, E-cadherin downregulation promoted anoikis resis- Award (CCR12225655 to H.Y. Irie). The costs of publication of this article were defrayed in part by the payment of tance of mammary carcinoma cells derived from p53-null back- page charges. This article must therefore be hereby marked advertisement in ground mice (53). Our studies demonstrate that modulation of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. EMT, via SNAIL or E-cadherin expression alone, was sufficient to fully reverse the effects of PTK6 inhibition on anoikis and metas- Received December 17, 2015; revised April 14, 2016; accepted May 12, 2016; tasis formation. Therefore, SNAIL and EMT regulation plays a published OnlineFirst June 14, 2016.

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www.aacrjournals.org Cancer Res; 76(15) August 1, 2016 4417

PTK6 Inhibition Suppresses Metastases of Triple-Negative Breast Cancer via SNAIL-Dependent E-Cadherin Regulation

Koichi Ito, Sun Hee Park, Anupma Nayak, et al.

Cancer Res 2016;76:4406-4417. Published OnlineFirst June 14, 2016.

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