Identification of PTPN12 Phosphatase as a Novel Negative Regulator of Hippo Pathway Effectors YAP/TAZ in Breast Cancer

Abstract

The Hippo pathway plays crucial roles in governing various biological processes during tumorigenesis and metastasis. Within this pathway, upstream signaling stimuli activate a core kinase cascade, involving MST1/2 and LATS1/2, that subsequently phosphorylates and inhibits the transcriptional co-activators YAP and its paralog TAZ. This inhibition modulates the transcriptional regulation of downstream target genes, impacting cell proliferation, migration, and death. Despite the acknowledged significance of protein kinases in the Hippo pathway, the regulatory influence of protein phosphatases remains largely unexplored. In this study, we conducted the first gain-of-functional screen for protein tyrosine phosphatases (PTPs) regulating the Hippo pathway. Utilizing a LATS kinase biosensor (LATS-BS), a YAP/TAZ activity reporter (STBS-Luc), and a comprehensive PTP library, we identified numerous novel PTPs that play regulatory roles in the Hippo pathway. Subsequent experiments validated PTPN12, a master regulator of oncogenic receptor tyrosine kinases (RTKs), as a previously unrecognized negative regulator of the Hippo pathway effectors, oncogenic YAP/TAZ, influencing breast cancer cell proliferation and migration. In summary, our findings offer valuable insights into the roles of PTPs in the Hippo signaling pathway, significantly contributing to our understanding of breast cancer biology and potential therapeutic strategies.

Keywords: Hippo pathway; PTPN12; TAZ; YAP; breast cancer; cell migration; cell proliferation; phosphatase; tumor suppressor.

Figure 1

The mammalian Hippo pathway and its regulators. LPA, lysophosphatidic acid; GPCR, G protein-coupled receptor; ECM, extracellular matrix.

Figure 2

Parallel screens for PTPs regulating the Hippo pathway by the LATS-BS and STBS-Luc (YAP/TAZ reporter). The ratio of the LATS-BS (A) and STBS-Luc reporter (B) signals after cotransfection with phosphatases to that of LATS-BS or STBS-Luc alone was calculated based on fold changes. A fold change greater than 2 was considered significant and indicated as “*”.

Figure 3

Validation of candidate PTPs regulating the Hippo pathway. A triplicate test was performed to validate the result of the first screen. The fold changes in LATS-BS and STBS-Luc reporters were calculated after cotransfection with each phosphatase. “*” represents a fold change greater than 2.

Figure 4

PTP candidates stimulate phosphorylation of YAP-pS127 endogenously. Western blot analysis of YAP-pS127 after induction of PTP candidate expression by Dox in HEK293T stable cell line. Without Dox, HEK293T-PTP stable cell lines do not express PTPs, while Dox treatment induces expression of FLAG-tagged PTPs. Band intensity of YAP and phosphorylated YAP-pS127 was quantified via ImageJ 1.54g. Expression of each of PTP candidates stimulates phosphorylation of YAP at S127, which is indicated by intensity ratio of ±Dox.

Figure 5

PTPN12 overexpression downregulates the mRNA expression of CTGF and Cyr61. qRT-PCR analysis of mRNA levels of CTGF and Cyr61 in HEK293T-PTPN12 stable cell line in the absence (−) and presence (+) of Dox treatment. The relative mRNA levels were calculated based on the ratio of mRNA levels under ± conditions. The mean ± S.D. of the triplicate samples are shown. **, p ≤ 0.01 (statistical significance).

Figure 6

Loss of PTPN12 reduces phosphorylation of YAP at S127 and increases YAP nuclear localization. (A) Western blot analysis. Reduced phosphorylation of YAP at S127 by PTPN12 in MCF10A-iCas9-gPTPN12-KO mammary cells. MCF10A-iCas9 cells stably expressing gPTPN12-2 (1) or gPTPN12-3 (2) were left untreated (−) or treated (+) with Dox for 5 days, followed by Western blot analysis of PTPN12, YAP, and S127-phosphorylated YAP (YAP-pS127) using anti-PTPN12, anti-YAP, and anti-pS127-YAP antibodies. Band intensity of YAP and pYAP was quantified using ImageJ. Relative intensity of phosphorylated YAP (pYAP) and YAP (pYAP/YAP ratio) is shown. (B,C) Immunostaining analysis of YAP subcellular localization. YAP immunostaining in MCF10A cells with wild-type (WT) or knockout (KO) PTPN12. DAPI was used to stain nucleus. For each cell line, approximately 100 cells were counted for YAP nuclear-only (red arrow in (B)) or cytoplasmic/cytoplasmic + nuclear (white arrow in (B)) staining cells. Scale bar, 200 μm. Quantitative analysis of YAP staining showed that in more than 50% of MCF10A-PTPN12-KO cell lines, YAP was localized in nucleus, whereas less than 25% of WT cell lines demonstrated YAP nuclear localization (C). *, p ≤ 0.05 (statistical significance).

Figure 7

PTPN12 regulates cell proliferation, which is mediated by the Hippo signaling pathway. (A) Western blot analysis of YAP and TAZ after the siYAP/siTAZ transfection of short interference RNAs targeting YAP/TAZ (siYAP/siTAZ) into MCF10A-gPTPN12 cells. (B) Cell proliferation assay. Cell proliferation was measured in MCF10A-PTPN12-WT (−Dox, −siYAP/siTAZ), MCF10A-PTPN12-KO (+Dox, −siYAP/siTAZ), and MCF10A-PTPN12-KO-siYAP/siTAZ (+Dox, +siYAP/siTAZ) cells. Cell numbers were counted on days 2, 4, and 6 after seeding.

Figure 8

The overexpression of PTPN12 suppresses YAP/TAZ-induced increased mammary cell proliferation. (A) Inducible expression of PTPN in MCF10A-YAP or -TAZ cells. Dox was added to induce PTPN12 in MCF10A-YAP-PTPN12/pTRIPZ or MCF10A-TAZ-PTPN12/pTRIPZ cells. MCF10A cells expressing a WPI lentiviral vector expressing GFP were used as a control. Forty-eight hours after Dox induction, proteins were extracted from cells and subjected to Western blot analysis of PTPN12, YAP, and TAZ. β-actin was used as an internal control. (B) Increased levels of phosphorylated YAP after PTPN12 overexpression were noted. Protein lysates from A were subjected to Western blot analysis of PTPN12, YAP-pS217, and YAP. (C) The suppression of YAP-induced increased cell proliferation by PTPN12. MCF10A-GFP (WPI vector) and MCF10A-YAP-PTPN12 in the absence or presence of Dox were subjected to cell proliferation analysis. (D) The suppression of TAZ-induced increased cell proliferation by PTPN12. MCF10A-GFP (WPI vector) and MCF10A-TAZ-PTPN12 in the absence or presence of Dox were subjected to cell proliferation analysis. The mean and standard deviation (S.D.) of the cell numbers in triplicate samples at each day were shown.

Figure 9

Inhibition of YAP/TAZ-induced increased cell migration by PTPN12 in MCF10A mammary cells. (A,C) Wound healing analysis using Incucyte Zoom. MCF10A-GFP (WPI vector) and MCF10A-YAP/TAZ expressing Dox-inducible PTPN12 cells in the absence or presence of Dox (+PTPN12) were seeded into 96-wells. After making the wounds, cell migration was monitored as a percentage of wound closure using Incucyte Zoom for 72 h. (B,D) Images from (A) showing wound closure at 0 h and 48 h. MCF10A-GFP was used as a control for both MCF10A-YAP/PTPN12 and MCF10A-TAZ/PTPN12. Scale bar, 300 μm.

Figure 10

Inhibition of TAZ-induced increased cell proliferation and anchorage-independent growth on soft agar in NMuMG cells. (A) Western blot analysis. Protein was extracted from NMuMG [Control (Ctrl)] cells and NMuMG cells expressing TAZ in the absence (−Dox) or presence (+Dox) of PTPN12 in pTRIPZ. About 10 μg of protein lysates was subjected to Western blot analysis. Band intensity was quantified using ImageJ. The ratios of the intensity of TAZ and β-actin was calculated. (B) Cell proliferation analysis. (C,D) Soft agar colony formation analysis. Triplicates of 2 × 10⁴ cells for each cell line were mixed with top agarose and overlaid onto base agarose in each well of 6-well plates, followed by incubation at 37 °C for 3 weeks. Colonies were stained with crystal violet (C) and quantified (D) using ImageJ. **, p ≤ 0.01 (statistical significance).

Figure 11

Inhibition of TAZ-induced increased cell migration by PTPN12 in NMuMG mammary cells. (A) Wound healing analysis using Incucyte Zoom. NMuMG (WPI vector) and NMuMG-TAZ expressing Dox-inducible PTPN12 cells in the absence or presence of Dox (TAZ-PTPN12) were seeded into 96-wells. After making the wounds, cell migration was monitored as a percentage of wound closure using Incucyte Zoom for 24 h. (B) Images from (A) showing wound closure at 0 h and 24 h. Scale bar, 300 μm.