Breast Tumor Kinase (Brk/PTK6) Mediates Advanced Cancer Phenotypes via SH2-Domain Dependent Activation of RhoA and Aryl Hydrocarbon Receptor (AhR) Signaling

Abstract

Protein tyrosine kinase 6 (PTK6; also called Brk) is overexpressed in 86% of patients with breast cancer; high PTK6 expression predicts poor outcome. We reported PTK6 induction by HIF/GR complexes in response to either cellular or host stress. However, PTK6-driven signaling events in the context of triple-negative breast cancer (TNBC) remain undefined. In a mouse model of TNBC, manipulation of PTK6 levels (i.e., via knock-out or add-back) had little effect on primary tumor volume, but altered lung metastasis. To delineate the mechanisms of PTK6 downstream signaling, we created kinase-dead (KM) and kinase-intact domain structure mutants of PTK6 via in-frame deletions of the N-terminal SH3 or SH2 domains. While the PTK6 kinase domain contributed to soft-agar colony formation, PTK6 kinase activity was entirely dispensable for cell migration. Specifically, TNBC models expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were unresponsive to growth factor-stimulated cell motility relative to SH3-del, KM, or wild-type PTK6 controls. Reverse-phase protein array revealed that while intact PTK6 mediates spheroid formation via p38 MAPK signaling, the SH2 domain of PTK6 limits this biology, and instead mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Inhibition of RhoA and/or AhR blocked TNBC cell migration as well as the branching/invasive morphology of PTK6⁺/AhR⁺ primary breast tumor tissue organoids. Inhibition of RhoA also enhanced paclitaxel cytotoxicity in TNBC cells, including in a taxane-refractory TNBC model. IMPLICATIONS: The SH2-domain of PTK6 is a potent effector of advanced cancer phenotypes in TNBC via RhoA and AhR, identified herein as novel therapeutic targets in PTK6⁺ breast tumors.

Figure 1:. Elevated PTK6 expression is correlated with lower overall survival of breast cancer patients and promotes cell motility, invasion and lung metastasis.

(A) SurvExpress was used to examine the prognostic index of patients in the TCGA Invasive Breast Cancer cohort (n = 502 samples) based on PTK6 expression. (B) Overall survival of ER-negative patients stratified on PTK6 mRNA expression using KM plotter (TCGA database, n = 1214 cases). (C) A high-throughput scratch wound assay was used to measure wound width over time (n=4 wells/genotype; data representative of three replicate experiments). (D) Transwell assays were used to chemoattract serum-starved HIF-1/HIF-2 double knockout (DKO) MDA-MB-231 cells to 10% FBS that were stably transfected with vector only (+Vector) or re-expressed PTK6 (+PTK6) to compare migration or cell invasion; bar graphs represent the grand mean ± SEM of three biological experiments; NS = not significant. (E) MDA-MB-231 HIF DKO + vector or HIF DKO + PTK6 cells were injected into the tail vein of 8-10-week old female NSG mice (n=6 DKO+ Vec; n=5 DKO +PTK6) and metastatic burden measured by densitometry analysis following immunostaining for an anti-human marker for pan-mitochondrial proteins (AbCam ab92824) with a hematoxylin counterstain. Whole immunostained lung sections representative of the mean percent positive area of metastasis are included. (F) A high-throughput scratch wound assay was used to compare the time elapsed to wound filling when a mixture of Cas9-deleted PTK6 KO cells are compared to parent or non-targeting control (NTC) gRNA transfected cells (n=4 wells/genotype; data representative of three replicate experiments). (G) Parent, NTC or PTK6 KO cells were bi-laterally injected in the mammary fat pad (n=8-9 mice/genotype) and tumor volume ± SEM compared over time. At experiment endpoint, tumors were excised and the tumor volumes ± SEM compared ex vivo (H). (I-J) MDA-MB-231 NTC or PTK6 KO (I) (n=10 mice/genotype) or parent + Vector or +PTK6 (J) (n=8 mice/genotype) cells were injected into the tail vein of 8-10-week old female NSG mice and the extent of metastatic colonization measured by densitometry analysis as described in (E). All metastasis data are expressed as ± SD; at least 5 animals/genotype were compared.

Figure 2:. PTK6 SH2 domain is critical for TNBC motility.

(A) PTK6 domain structure schematic. (B) Western blot of MDA-MB-231 PTK6 KO cells re-expressing FLAG-tagged PTK6 domain mutant constructs. (C) Anchorage-independent growth was assessed in soft agar supplemented with EGF. (D) Transwell migration of MDA-MB-231 PTK6 domain mutants was examined in response to an HGF gradient (50 ng/mL). Data is shown as grand mean ± SD of n = 3 experiments with 3 replicate transwells each. Tumorsphere-forming assays were performed in domain mutant and the number (E) and size (F) were quantified. (G) PTK6 knockout models. Inset confirms knockout of PTK6 using CRISPR/Cas9n. Graphed data were analyzed by one-way ANOVA with multiple comparisons tests and are representative of n = 4 independent experimental repeats; *** p < 0.001.

Figure 3:. SH2-dependent pathways in MDA-MB-231 cells.

(A) Heatmap by unsupervised consensus clustering of protein lysates analyzed for protein expression by RPPA. Broken line box indicates protein staining intensities unchanged between samples. Solid line box highlights RPPA intensities that appear different in SH2-del PTK6 expressing cells. (B) Supervised clustering dendrogram of samples. (C) Immunoblot analysis of p38 MAPK activation by HGF (50 ng/mL). Phospho-p38 densitometry (indicated underneath) was calculated by first normalizing to total p38 MAPK, then calculating fold change over vehicle for each cell line. Data is representative of n = 3 independent repeats. (D) Primary tumorsphere assays in MDA-MB-231 PTK6 domain mutant models. Cells were treated with DMSO (vehicle) or SB203580 (10 mM) for 7 days. Data were analyzed by one-way ANOVA with posthoc corrections (n = 3); *** p < 0.001, ** p < 0.01.

Figure 4:. PTK6 SH2 domain is required for RhoA activation.

(A) RhoA activation assay using GST-Rhotekin-RBD pull down in MDA-MB-231 cells expressing WT or SH2-del PTK6. (B) Expression of Rho and AhR was examined in a panel of TNBC models by immunoblotting. (C) Rho inhibition by CT04 (1 μg/mL) in a panel of TNBC cell lines. Transwell migration (MDA-MB-231 and Hs578T) was stimulated by HGF (50 ng/mL). Significance was determined by one-way ANOVA with multiple comparisons testing; ** p < 0.01, *** p < 0.001. (D) Soft agar colony formation of WT and SH2-del PTK6 expressing cells in the presence of Rho inhibitor CT04 (1 μg/mL). Graphed data are representative of at least n = 3 independent replicates.

Figure 5:. Pairing CT04 with Taxol is synergistic in TNBC models.

(A-D). Tumor cell growth was measured over time using the IncuCyte S3 live-cell imager as described in the materials and methods. Holding either CT04 or CH223191 constant near each cell line’s specific IC₅₀ value, increasing concentrations of Taxol (paclitaxel) were added to the cells and the change in the growth ratio relative to time after drug was added (t = 0) was plotted over time (hours). All data are shown as the mean ± SEM for technical replicates at each time point; each line graph shown is representative of at least 3 independent experiments. # indicate significant differences in the growth ratio among treatments.

Figure 6:. AhR activity requires PTK6 SH2 domain.

(A) Unsupervised heatmap of RPPA dataset identified by IPA “AhR Signaling”. (B) MDA-MB-231 cells expressing WT or SH2-del PTK6 were starved and treated with veh or HGF (50 ng/mL; 30 min). AhR-containing complexes were isolated using AhR-specific antibodies and protein G-agarose beads. Western blots were performed on immunocomplexes and whole-cell lysates (FL; full length). Representative blot of n = 3 experimental repeats are shown. (C) TNBC migration was examined with AhR inhibitor CH223191 (10 μM). Data were analyzed by Student’s t-test for significance; ** p < 0.01, *** p < 0.001 (n = 4). (D) Colony formation with AhR inhibitor CH223191 (10 μM). Data was analyzed by two-way ANOVA with post-hoc corrections; *** p < 0.001 (n = 3). (E) HGF-induced transwell migration in the presence of AhR inhibitor CH223191. Data was analyzed by two-way ANOVA with post-hoc corrections; *** p < 0.001, ** p < 0.01 (n = 3). (F) mRNA levels of AhR and canonical target genes CYP1A1 and CYP1B1 in MDA-MB-231 cells stably expressing WT or SH2-del PTK6. Data was analyzed by Student’s t-test; * p < 0.05 (n = 3). (G) Recruitment of AhR to the CYP1B1 promoter region was examined by ChIP-PCR. Data are shown as fold recruitment over vehicle control of combined n = 3 experiments. In parallel, CYP1B1 mRNA expression was analyzed by qRT-PCR. Two-way ANOVA with post-hoc corrections was used to test significance; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7:. AhR and PTK6 expression promote aggressive breast cancer phenotypes.

(A) AhR and (B) PTK6 mRNA expression was analyzed in organoids isolated from ER-negative human breast tumor samples or normal adjacent tissue. Data were analyzed by Student’s t-test; * p < 0.05. Data represents the mean ± SD (n = 2 patient samples). (C) Organoids were grown from human malignant tissue and treated with vehicle or CH223191 (10 μM) in duplicate wells. Representative images of two separate experiments are shown. Branching organoids were quantified by manual counting in ImageJ (n = >5 organoids fields/sample) and expressed as a percentage of total organoid number. Significance was determined by Student’s t-test; * p < 0.05. (D) SurvExpress was used to stratify recurrence-free survival (left) and distant metastasis free survival (right) based on median expression of PTK6, AhR, and GR (NR3C1) across all breast cancer subtypes. (E) A representative cartoon model of the oncogenic activities of PTK6 in TNBC. PTK6 expression is regulated by the AhR/GR/HIFs transcriptional complex [8, 9, 26]. In turn, PKT6 is a key signaling node of HGF-mediated oncogenic signaling in TNBC. Specifically, the SH2 domain of PTK6 is integral in the regulation of both RhoA and p38 MAPK. As shown, deletion of the SH2 domain, promotes tumorsphere formation via activation of the p38 MAPK. However, deletion of the SH2 domain decreased RhoA and AhR oncogenic activity in our TNBC models (i.e. cellular migration, cellular growth and resistance to Taxol chemotherapy).