Fig 2: PTK7 regulates EGFR-PI3K-Akt pathway in breast cancer. (A) PTK7 co-expression analysis in invasive breast carcinoma using breast cancer datasets including 1,904 patients with Agilent microarray data (http://www.cbioportal.org/) and 374 PTK7 positively- (Spearman’s correlation > 0.3, P < 0.01) and 289 PTK7 negative- (Spearman’s correlation < -0.3, P < 0.01) correlated genes were selected and used as candidate genes in the following analysis. (B) PTK7 positively-correlated genes were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) by DAVID: Functional Annotation Tools (https://david.ncifcrf.gov/tools.jsp). PTK7 positively correlated genes enriched in PI3K-Akt signaling (hsa04151) and Regulation of actin cytoskeleton (hsa04810) pathways are listed in the frames, respectively. (C) PTK7 and EGFR pair-wise gene correlation in breast invasive carcinoma were analyzed using GEPIA Correlation Analysis tools (http://gepia.cancer-pku.cn/detail.php?clicktag=correlation). (D, E) MDA-MB-468 (D) and MDA-MB-231 (E) cells were transduced with a non-targeting control shRNA (shNC) or two different PTK7-specific shRNAs (shPTK71 and shPTK7#2). Cells were stimulated with EGF (500 ng/ml) for 0, 20 or 40 minutes and phospho-EGFR and phosphor-Akt levels were evaluated using immunoblotting assay.

Fig 3: Potential autocrine RTK signaling in MIBC patient samples and BCa cell lines.(A) Heat map showing relative mRNA expression levels of ctRTK-cognate ligand pairs (RNA Seq V2) among 408 tumor samples from patients with MIBC [22] (left), and among 15 BCa-derived cell lines (absolute mRNA copy number, right). (B) Immunoblot detection of ctRTKs and their cognate ligands, CSF1/CSF1R, GAS6/AXL, MST1/MST1R, and evidence of DDR1, DDR2 and PTK7 proteins in 9 BCa-derived cell lines. GAPDH (bottom panel) serves as a loading control. (C) Left: heat map showing relative mRNA expression levels of 9 EMT-associated transcriptional activators (listed at right) among 408 tumor samples from patients with MIBC [22]. Samples are grouped on the horizontal axis by mRNA subtype classifications defined previously [22] indicated at the top (from left to right): neuronal (N, green); basal squamous (BS, red); luminal (L, gray); luminal infiltrating (LI, gold); luminal papillary (LP, purple), and clustered hierarchically on the vertical axis. Right: heat map showing absolute mRNA copy levels of 9 EMT-associated transcriptional activators among 15 BCa-derived cell lines (identified at bottom).

Fig 4: PTK7 associates with extracellular matrix organization and migration in breast cancer cells. (A) Gene Ontology (GO) was performed using DAVID: Functional Annotation Tools (https://david.ncifcrf.gov/tools.jsp) and Biological Process (BP) of PTK7 positively- and negatively-correlated genes were shown. (B) Pair-wise gene correlation of PTK7 with COL1A1, FN1, WNT5B, MMP11, MMP14 or SDC1 in breast invasive carcinoma were analyzed using GEPIA Correlation Analysis tools (http://gepia.cancer-pku.cn/detail.php?clicktag=correlation). (C) MDA-MB-231 and MDA-MB-468 cells were transduced with shNC, shPTK71 or shPTK72. Cells were stained for F-actin with TRITC-phalloidin. Pictures show the TRITC-tagged Phalloidin (red) and DAPI (purple). Presentative images are shown. Magnification, 400×; scale bars, 50 μm.

Fig 5: PTK7 participates in cell proliferation and migration in TNBC cell lines. (A, B) TNBC cell lines MDA-MB-436 (A) and MDA-MB-468 (B) were transfected with control vector pcDNA3 or PTK7 expression vector pcDNA3-PTK7 for 48 h and proliferation was evaluated using MTT assay. Data are shown as mean ± s.d. ****P < 0.0001. (C, D) MDA-MB-436 (C) and MDA-MB-468 (D) were transduced with a non-targeting control shRNA (shNC) or two different PTK7-specific shRNAs (shPTK71 and shPTK7#2). Cell proliferation was evaluated using MTT assay. Data are shown as mean ± s.d. ****P < 0.0001. (E) Colony formation assay was performed to determine proliferation in shNC-, shPTK7#1- or shPTK7#2-transduced MDA-MB-468 cells. Presentative images are shown (left) and colony numbers and colony diameters were shown as mean ± s.d. Magnification, ×100; ****P < 0.0001. (F, G) Transwell migration assay using Boyden chamber in shNC-, shPTK7#1- or shPTK7#2-transduced MDA-MB-436 (F) and MDA-MB-468 (G) cells was performed and photographed under a light microscope. Presentative images are shown (left) and migrated cells were counted and shown as mean ± s.d. Magnification, ×100; ****P < 0.0001.