Fig 2: BCAR3 was identified as a substrate of PTPN14. A) The results of IP‐MS, following CompPASS analysis, yielded high‐confidence candidate PTPN14‐interacting proteins. B) Exogenous IP‐Western Blot analysis validated the interaction of PTPN14 and BCAR3. Three independent experiments were performed. C) Endogenous IP‐Western Blot analysis validated the interaction of PTPN14 and BCAR3. D) BCAR3 was knockdown in control or PTPN14‐KO MDA‐MB‐231 cells and cell viability under ULA conditions was assessed every 24 h for 5 days using the CCK8 assay (left); validation of protein expression levels in each cell group through Western Blotting (right). Three independent experiments were performed. E) After the knockdown of BCAR3 in PTPN14‐KO MDA‐MB‐231 cells, the phosphorylation levels of AKT and ERK were assessed. Three independent experiments were performed. F) Tyrosine phosphorylated protein IP‐Western Blot analysis was conducted in both control MDA‐MB‐231 cells and PTPN14‐KO MDA‐MB‐231 cells. Three independent experiments were performed. G) Tyrosine phosphorylated protein IP‐Western Blot analysis was conducted in both PTPN14‐HA MDA‐MB‐231 cells and PTPN14‐D1079A‐HA MDA‐MB‐231 cells.

Fig 3: PTPN14 PTP domain and HPV18 E7 constitute a 2:2 complex.(A) C-terminal domain of HPV18 (green) forms a homodimer together with the crystallographic symmetry-associated molecule (navy). Residues involved in the intermolecular hydrophobic interaction are shown in sticks and labeled. (B) Two views of 2:2 dimeric structure. Two PTPN14–HPV18 E7 complexes are crystallographically associated with each other as shown in S4B Fig. HPV, human papillomavirus; PTPN14, nonreceptor-type protein tyrosine phosphatase 14.

Fig 4: A pooled genome‐wide CRISPR screen in a TNBC anoikis model. A) Genes related to anoikis resistance identified through a genome‐wide CRISPR/Cas9 positive selection screen were ranked based on MAGeCK analysis results, with the top ten genes listed. B) The overlap of the indicated datasets. C) PTPN14, APOLD1, FGL2, and LDLRAD2 mRNA expression levels in 113 breast cancer adjacent samples and 115 TNBC samples from TCGA database. Student's t‐test, *** p < 0.001. D) The mRNA expression levels of PTPN14, APOLD1, FGL2, and LDLRAD2 were assessed in 13 primary breast tumor samples and seven distant metastasis tumor samples from dataset GSE191230 obtained from the GEO database. Student's t‐test, * p < 0.05, ** p < 0.01. E) Representative IHC images (above) and semiquantitative evaluation of PTPN14 IHC expression (below) in paired primary tumors and metastatic tumors (n = 53), and normal breast tissue (n = 11). Dunn's multiple comparisons test following Kruskal–Wallis test, *** p < 0.001. Scale bar: 100 µm. (F) OS (n = 201, low expression: 140; high expression: 61) and RFS (n = 417, low expression: 234; high expression: 183) were summarized in the high and low PTPN14 expression groups by using Kaplan–Meier survival curves in TNBC. Log‐rank test, with p < 0.05 indicating statistical significance.

Fig 5: Analysis of the binding specificity of HPV18 E7 to PTP proteins.(A) Sequence alignment. Sequence of the HPV18 E7–binding interface of PTPN14 is aligned with those of the corresponding regions of 16 other nonreceptor-type PTP proteins. The secondary structures of PTPN14 are shown at the top. Conserved residues are shaded in orange. Four hydrophobic residues in PTPN14 that play a key role in the intermolecular interaction with HPV18 E7 are marked by red arrows. (B) Structural alignment. The PTP domains of two representative nonreceptor-type PTP proteins, PTP1B and PTPN3, are superimposed onto that of PTPN14 bound to HPV18 E7. Shown in sticks and labeled are PTPN14 residues interacting with Arg84 or Leu91 of HPV18 E7 and PTP1B and PTPN3 residues corresponding to the represented PTPN14 residues. (C) ITC measurements. The KD values between the C-terminal domain of HPV18 E7 and a series of PTP domains were calculated, as in Fig 1B. Deducible dissociation constants were shown only when PTPN14 or PTPN21 was subjected to the experiment. (D) SEC-MALS analysis. The complex formation was shown between the dimeric C-terminal domain of HPV18 E7 and the monomeric PTP domain of human PTPN21. Solid lines, molar masses in kg/mol; dotted lines, refractive indexes. Constructs are listed in S1 Table. The numerical data are included in S1 Data. a.a., amino acids; HPV, human papillomavirus; ITC, isothermal titration calorimetry; MBP, maltose binding protein; Mn, number-average molar mass; Mw, weight-average molar mass; PTP, protein tyrosine phosphatase; PTPN14, nonreceptor-type PTP 14; SEC-MALS, size-exclusion chromatography–multiangle light scattering.