Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase

Abstract

Among breast cancers, triple-negative breast cancer (TNBC) is the most poorly understood and is refractory to current targeted therapies. Using a genetic screen, we identify the PTPN12 tyrosine phosphatase as a tumor suppressor in TNBC. PTPN12 potently suppresses mammary epithelial cell proliferation and transformation. PTPN12 is frequently compromised in human TNBCs, and we identify an upstream tumor-suppressor network that posttranscriptionally controls PTPN12. PTPN12 suppresses transformation by interacting with and inhibiting multiple oncogenic tyrosine kinases, including HER2 and EGFR. The tumorigenic and metastatic potential of PTPN12-deficient TNBC cells is severely impaired upon restoration of PTPN12 function or combined inhibition of PTPN12-regulated tyrosine kinases, suggesting that TNBCs are dependent on the proto-oncogenic tyrosine kinases constrained by PTPN12. Collectively, these data identify PTPN12 as a commonly inactivated tumor suppressor and provide a rationale for combinatorially targeting proto-oncogenic tyrosine kinases in TNBC and other cancers based on their profile of tyrosine-phosphatase activity.

Figure 1. A Genetic Screen for Tumor Suppressors Identifies PTPN12

(A) Schematic of genetic screen for suppressors of HMEC transformation. A pool of retroviral shRNAs was transduced into TLM-HMECs in duplicate, and assessed for anchorage-independent proliferation. shRNAs were PCR amplified and sequenced from macroscopic colonies. (B) Depletion of PTPN12. PTPN12 protein expression in TLM-HMECs transduced with vectors expressing the indicated shRNAs with quantification below. (C) PTPN12 loss of function transforms TLM-HMECs. Anchorage-independent proliferation in TLM-HMECs transduced with the indicated shRNAs. (D) Restoring PTPN12 expression suppresses transformation by PTPN12 shRNA. Anchorage-independent proliferation in TLM-HMECs transduced with control or PTPN12-shRNA in combination with PTPN12 cDNA as indicated. (E) PTPN12 regulates acinar morphogenesis. MCF10A cells expressing the indicated shRNAs were analyzed for 3D acinar morphogenesis in vitro (day 15 after seeding) and quantified for the number of aberrant mammary acini. (F) The enzymatic activity of PTPN12 is required for transformation suppression. TLM-HMECs expressing a PTPN12 shRNA were transduced with lentivirus-encoding control or shRNA-resistant PTPN12-C231S mutant cDNA and assessed for PTPN12 expression by western (top) and anchorage-independent proliferation (bottom). Error bars represent standard error.

Figure 2. PTPN12 Interacts with and Inhibits the HER2/EGFR Signaling Axis

(A and B) Tyrosine phosphoproteins regulated by PTPN12. HMECs expressing an inducible PTPN12-shRNA were quantified in the presence and absence of PTPN12 for tyrosine-phosphorylated peptides using a quantitative proteomic approach (described in Experimental Procedures). Interactions between the 69 PTPN12-regulated phosphoproteins were analyzed via (A) Ingenuity and (B) the HPRD. (C) HER2 and EGFR RTKs interact with PTPN12 in HMECs. The left panel shows the experimental design of the BiFC system. PTPN12 was fused with the N terminus of YFP, and RTKs were fused with the C terminus of YFP. HMECs were transduced with retroviruses expressing PTPN12-N-YFP and individual RTK-C-YFP cDNAs. In the right panel the interaction between PTPN12 and HER family RTKs was assessed by cellular fluorescence. Asterisk indicates p < 0.01. (D) Substrate-trapping PTPN12 mutant displays increased interaction with HER2. Breast cancer cells expressing PTPN12-WT-N-YFP or mutant PTPN12-C231S-N-YFP in combination with HER2-C-YFP were analyzed for cellular fluorescence. (E) PTPN12 loss of function elicits hyperactivation of HER2, EGFR, and a MAPK-signaling cascade. HMECs engineered with an inducible PTPN12-shRNA were cultured ± dox for 3 days, starved of growth factors, and analyzed for levels of the indicated total and phosphorylated proteins by western. (F) PTPN12 suppresses HER2, EGFR, and MAPK signaling. HMECs engineered with an inducible PTPN12-cDNA were cultured and analyzed as in (E). Error bars represent standard error.

Figure 3. PTPN12 Suppresses Transformation by Inhibiting HER2/EGFR Signaling

(A) EGFR and HER2 depletion in TLM-HMECs. TLM-HMECs expressing control, EGFR, or HER2-targeting shRNAs were analyzed by western blotting for EGFR, HER2, and vinculin (loading control) as indicated. (B) EGFR and HER2 RTKs are required for cellular transformation upon PTPN12 depletion. TLM-HMECs encoding an inducible PTPN12-shRNA were transduced with the indicated shRNAs and assessed for anchorage-independent proliferation ± dox. (C) SHC depletion in HMECs. TLM-HMECs expressing control or SHC-targeting shRNAs were analyzed by western for SHC and vinculin as indicated. (D) SHC is required for cellular transformation upon PTPN12 depletion. TLM-HMECs encoding an inducible PTPN12-shRNA were transduced with the indicated shRNAs and assessed for anchorage-independent proliferation ± dox. (E) HER2/EGFR inhibitors block PTPN12 depletion-induced transformation. TLM-HMECs expressing the indicated shRNAs were assessed for anchorage-independent growth ± a HER2/EGFR inhibitor (lapatinib). (F) Transformation by PTPN12 inactivation requires MAPK signaling. TLM-HMECs expressing the indicated shRNAs were assessed for anchorage-independent proliferation ± a MEK inhibitor (U0126). Error bars represent standard error.

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.

Figure 5. PTPN12 Is Regulated by the REST Tumor Suppressor via miR-124

(A) Loss of REST expression in human breast cancer. Primary human breast cancers (n = 185) were analyzed by immunohistochemistry for REST expression. Representative panels exhibiting negative and positive REST expression in invasive breast cancers. (B) Loss of REST expression strongly correlates with loss of PTPN12 expression. Primary human breast cancers (n = 185) were analyzed by immunohisto-chemistry for REST and PTPN12 expression. The level of PTPN12 (y axis) is plotted for tumors with absent, intermediate, or high REST levels (x axis). The median and mean PTPN12 values for each group are represented by a solid red line and plus symbol, respectively. The boxes represent the 25th to 75th percentiles. Association between PTPN12 and REST expression was tested by Fisher’s exact test. Error bars represent maximum and minimum observations within inner fences. (C) Ectopic REST expression increases PTPN12 protein levels in REST-deficient TNBC cells. HCC70 TNBC cells were transduced with control or REST cDNA, cultured for 9 days, and analyzed for expression of REST and PTPN12 by western. (D) Model for REST regulation of PTPN12 expression. REST regulates transcription of the neuronal microRNA miR-124. The PTPN12 3′UTR contains three conserved binding sites for miR-124. The sequences surrounding the three miR-124 binding sites are shown for human and five other vertebrate species. (E) Ectopic miR-124 expression decreases PTPN12 protein levels in HMECs. HMECs were transduced with control or miR-124-containing plasmid, cultured for 7 days, and analyzed for PTPN12 expression by western. (F) Ectopic miR-124 expression transforms TLM-HMECs. Cells from (E) were assessed for anchorage-independent proliferation. Error bars represent standard error.

Figure 6. PTPN12 Suppresses Growth and Metastasis of TNBC Cells

(A) PTPN12 expression is reduced in TNBC cell lines. PTPN12 protein levels were quantified by western in HMECs, TNBC cells, and HER2-amplified breast cancer cells as indicated. (B) Reconstituting PTPN12 expression suppresses proliferation in PTPN12-deficient breast cancer cells. TNBC cells expressing low endogenous PTPN12 were transduced with equivalent multiplicity of infection (moi) of retrovirus encoding eGFP (control) or PTPN12 cDNAs and analyzed for macroscopic colony formation in vitro. (C) Reconstituting PTPN12 expression suppresses proliferation in PTPN12-deficient breast cancer cells. Quantification of colony number from cells in (B). (D) PTPN12 expression is reduced in aggressive lung metastatic subpopulation of TNBC MDA-MB231 cells. PTPN12 protein expression was assessed in MDA-MB231 breast cancer cells (231-parent) and in MDA-MB231-LM2 subpopulation that exhibits enhanced primary and lung metastatic tumor growth. MDA-MB231-LM2 cells were engineered with an inducible PTPN12-cDNA (LM2-IP cells) that expresses similar PTPN12 levels as parental MDA-MB231 cells upon addition of dox. (E) Restoring PTPN12 expression suppresses primary tumor growth in aggressive TNBC cells. Cells from (D) were transplanted in the mouse mammary gland and monitored for primary tumor growth in the presence or absence of dox (n = 12 for each group). (F) Restoring PTPN12 expression suppresses lung metastatic growth in aggressive TNBC cells. Cells from (D) were tail vein injected and monitored for lung metastatic growth (via luminescence detection) in the presence or absence of dox (n = 6 for each group). Representative +dox and −dox images and quantification are shown in upper and lower panels, respectively. (G) Restoring PTPN12 expression suppresses lung metastatic growth in aggressive TNBC cells. Cells from (D) were tail vein injected (as in F) in the presence or absence of dox (n = 7) and analyzed for lung metastatic lesions via standard H&E. Error bars represent standard error.

Figure 7. PTPN12 Inhibits Proliferation and Survival of TNBCs by Inhibiting Multiple RTKs

(A) HER2 and PDGFR-β RTKs interact with PTPN12 in TNBC cells. HCC1937 cells expressing PTPN12-N-YFP and individual RTK-C-YFP cDNAs (as indicated) were analyzed for cellular fluorescence using flow cytometry. (B) Ectopic PTPN12 expression inhibits HER2 and PDGFR-β RTK signaling in TNBC cells. HCC1937 cells engineered with control or PTPN12-cDNA were assessed for PTPN12 expression and levels of phosphorylated HER2 and PDGFR-β by western. (C) Combined HER family and PDGFR inhibitors suppress proliferation of PTPN12-deficient TNBC cells. HCC1937 cells were cultured ± HER2/EGFR inhibitor lapatinib (1 μM) ± PDGFR inhibitor sunitinib (5 μM) for 8 days. Cell numbers were determined by DAPI cell counting. (D and E) Combined HER family and PDGFR inhibitors suppress tumorigenicity of PTPN12-deficient TNBC cells. MDA-MB231-LM2 cells were transplanted in the mouse mammary gland and monitored for primary tumor growth in the presence or absence of HER2/EGFR inhibitor (lapatinib) and PDGFR inhibitor (sunitinib) as indicated (n = 10 for each group). Tumor volumes on day 26 postinjection are shown in (D). Tumor growth curves are shown in (E). (F) Combined HER family and PDGFR inhibitors extend event-free survival of animals harboring PTPN12-deficient TNBC tumors. Animals transplanted with MDA-MB231-LM2 cells (as above) were treated with the indicated inhibitor and monitored for tumor volume. Events are denoted as animals with tumors greater than 1000 mm³. Error bars represent standard error.