Figure 4. PTPN12 Is Functionally Inactivated in Human TNBC via Multiple Mechanisms.

(A) PTPN12 mutations occur more frequently in human TNBC. Frequency of mutations observed in TNBCs (75 primary tumors and eight cell lines) and 202 primary breast cancers positive for ER and/or HER2.

(B) Schematic of PTPN12 mutations in TNBCs. Red stars indicate altered amino acids. The sequence surrounding the catalytic cysteine residue is expanded. The preceding histidine, H230, is conserved among all tyrosine phosphatases. The phosphatase domain (Phos Domain) and Proline rich regions (Pro1-5) are shown.

(C) H230Y mutant PTPN12 fails to suppress transformation. TLM-HMECs expressing the PTPN12-shRNA were engineered with the indicated dox-inducible cDNAs. Cells were assessed for anchorage-independent growth.

(D) E690 and W699 PTPN12 mutants fail to suppress transformation. TLM-HMECs expressing the PTPN12-shRNA were transduced with lentiviral PTPN12 cDNAs (as indicated) and assessed for anchorage-independent growth.

(E) The PTPN12-T573A SNP is a partial loss-of-function allele for suppressing transformation. TLM-HMECs were transduced with PTPN12-shRNA in combination with lentiviral cDNAs encoding PTPN12-WT (threonine at residue 573), or PTPN12-T573A (alanine at residue 573). Cells were measured for anchorage-independent growth.

(F) PTPN12-H230Y mutation disrupts proper acinar formation. MCF10A cells expressing control or PTPN12 shRNA in combination with wild-type or H230Y mutant PTPN12 were analyzed for 3D acinar formation (day 15 after seeding).

(G) PTPN12-H230Y mutation disrupts proper acinar formation. Quantification of aberrant mammary acini from (F).

(H) The PTPN12-T573A SNP allele disrupts acinar formation. MCF10A cells transduced with lentiviral cDNAs encoding control, PTPN12-WT (threonine at residue 573), or PTPN12-T573A (alanine at residue 573) as indicated were analyzed for 3D acinar morphogenesis in vitro (day 15 after seeding).

(I) Loss of PTPN12 expression occurs more frequently in human TNBC. Primary human breast cancers (n = 185) were analyzed by immunohistochemistry for PTPN12 expression. Representative panels exhibiting positive PTPN12 expression in HER2-amplified breast cancer (left panel) and lack of expression in TNBC (right panel).

(J) Loss of PTPN12 expression occurs predominantly in TNBC. Primary human breast cancers (n = 185) were analyzed by immunohistochemistry for PTPN12 expression. The number of samples showing no detectable PTPN12 expression (red area of bars) was quantified in HER2-positive, ER-positive, and triple-negative subtypes. Association between PTPN12 expression and breast cancer subtypes was tested by Fisher’s exact test.

Error bars represent standard error.