Pleckstrin‐2‐promoted PPM1B degradation plays an important role in transforming growth factor‐β‐induced breast cancer cell invasion and metastasis

Abstract Transforming growth factor‐β (TGF‐β) is known to promote breast cancer cell migration, invasion, and dissemination; however, the underlying molecular mechanisms are not yet well characterized. Here, we report that TGF‐β induces pleckstrin‐2 (PLEK2) expression by Smad3 and signal transducer and activator of transcription 3 (STAT3) activating PLEK2 promoter activity. Higher PLEK2 expression is associated with poor prognosis in breast cancer patients. Overexpression and knockout experiments in MDA‐MB‐231 and MCF‐7 breast cancer cells revealed that PLEK2 promotes cell migration, invasion, and dissemination in 2D and 3D cell culture. Moreover, PLEK2 promotes metastasis of breast cancer cells in vivo. Pleckstrin‐2 localizes to the cell membrane and cell protrusions following TGF‐β treatment. Furthermore, inhibition of PI3K phosphorylation abolishes TGF‐β‐ and PLEK2‐induced cell invasion. The carboxyl‐terminal PH domain of PLEK2 is critical for TGF‐β‐ and PLEK2‐induced Akt activation and plays an important role in cell invasion. Pleckstrin‐2 interacts with PPM1B and promotes its ubiquitin‐dependent degradation. The PLEK2‐PPM1B axis utilizes nuclear factor‐κB signaling to promote cell migration and invasion. Our data implicate the TGF‐β‐STAT3/Smad3‐PLEK2‐PPM1B signaling cascade in TGF‐β‐induced breast cancer cell migration and invasion. These findings suggest that PLEK2/PPM1B could represent novel targets for the intervention of breast cancer metastasis.


| INTRODUC TI ON
Breast cancer is the most commonly diagnosed cancer among women, 1 and metastasis is the leading cause of breast cancerassociated death and poor prognosis. 2 The invasion-metastasis cascade is a multistep process through which cancer cells from the primary tumor disseminate by migration and invasion, colonizing at distant organs. 2,3 The vast majority of patients with advanced metastatic disease are incurable with current therapeutic regimens.
Transforming growth factorβ can function as a tumor suppressor or tumor promoter at different stages of cancer progression. 4 During the early stages, TGFβ inhibits cancer cell proliferation and induces cell apoptosis. By contrast, during the late stages, TGFβ induces EMT 5 and stem cell phenotypes to promote cancer cell migration, invasion, and metastasis. 6 A better understanding of the molecular mechanisms through which TGFβ promotes invasion and migration of breast cancer cells could allow the development of improved therapeutic strategies for cancer patients.
In the present study, we found that TGFβ induced the expression of several genes including PLEK2. A high expression level of PLEK2 was related to poor prognosis in breast cancer patients.
Pleckstrin-2 is a member of the Pleckstrin family 7 and contains two PH domains at the N-and C-terminus, as well as a Disheveled, Egl-10, and Pleckstrin (DEP) domain in the center. 8 Recent studies have shown that PLEK2 plays an important role in the metastasis and progression of many cancer types. [9][10][11][12][13][14] Pleckstrin-2 promotes cell migration and invasion in vitro and cancer metastasis in vivo through the EMT program in non-small-cell lung cancer and gallbladder cancer. 14,15 Evidence suggests that PLEK2 exerts its pro-oncogenic function through the PI3K/Akt signaling pathway. 16,17 Although it has been reported that high expression levels of PLEK2 are related to the dissemination of luminal type A breast cancer cells, 18 the exact function and molecular mechanism of PLEK2 in breast cancer progression has not yet been well characterized.
Here, we focus on the molecular mechanisms underlying the TGFβ-induced expression of PLEK2 and its regulation of cell invasion and migration in breast cancer. We show that Smad3/ STAT3 signaling is involved in TGFβ-induced PLEK2 expression.
Pleckstrin-2 plays an important role in TGFβ-induced cell migration and invasion in 2D cell culture, tumor organoid invasion and cell dissemination in 3D cell culture, and lung metastasis in a mouse xenograft model of breast cancer. The C-terminal PH domain of PLEK2 is required for PLEK2-induced breast cancer cell invasion, which involves the PI3K/Akt signaling pathway. Furthermore, PLEK2 interacts with PPM1B to promote its ubiquitin-dependent degradation. PPM1B suppresses the TGFβ-and PLEK2-mediated phosphorylation of p65. Together, our data reveal that PLEK2 plays an essential role in TGFβ-induced breast cancer metastasis, suggesting that PLEK2/PPM1B could represent a novel target for the intervention of breast cancer metastasis.

| Cell lines, reagents, and Abs
All cell lines were obtained from the ATCC and cultured as described previously. 19 Detailed information regarding the reagents and Abs is provided in Table S2.

| Statistical analysis
All experiments were repeated at least three times. Student's t-test was used to analyze differences between two groups. Statistical analysis of more than two groups was carried out by ANOVA test.
Statistical analysis was undertaken using SigmaPlot (version 9.0, Systat Software Inc.) or GraphPad Prism (version 8.4.0, Graphpad Software Inc.). The threshold for statistical significance was p < 0.05.
Results in the bar graphs are expressed as the mean ± SD.

| Supplementary materials and methods
Detailed materials and methods are provided in Data S1.  Figure S1D). The fold changes in gene expression seen by RNA sequencing were confirmed by RT-qPCR ( Figure S1E). Pleckstrin-2 was mainly expressed in triple-negative and luminal A-type breast cancer cells ( Figure 1B). Furthermore, TGFβ induced PLEK2 expression in MDA-MB-231 and MCF-7 cells at both the mRNA and protein levels ( Figure 1C,D). Transforming growth factorβ also induced the expression of PLEK2 mRNA in SUM-159 and T47D breast cancer cells ( Figure S1F). These findings suggest that PLEK2 gene expression is induced by TGFβ in breast cancer cells.

| Pleckstrin-2 is highly expressed in human breast cancer and is associated with poor prognosis
Cells migrate by extending plasma membrane protrusions, such as lamellipodia and invadopodia, at the leading edge. 20 Pleckstrin-2 was enriched in the plasma membrane region, cell leading edge, and leading edge membrane GO terms as our GO analysis results (http://www.webge stalt.org/; Table S1). Pleckstrin-2 expression was significantly upregulated in primary tumors and different subtypes of breast cancer tissues in comparison with normal tissues (http:// ualcan.path.uab.edu/; 21 Figure 1E, F). A high expression level of PLEK2 was associated with metastasis and decreased overall and metastasis-free survival (http://www.compb io.iupui.edu/progg ene/; 22,23 Figure 1G,H).   Figure 2I). Protein expression levels of TGFBR1 and TGFBR2 were similar in both cell lines ( Figure S10A). Furthermore, the TGFBR1/2 inhibitor LY2109761 eliminated TGFβ-induced cell migration and invasion but did not completely block cell migration or invasion induced by PLEK2 ( Figure S3A,B). These findings suggest that PLEK2 is necessary for TGFβ-induced cell migration and invasion.

| Pleckstrin-2 is essential for formation of TGFβ-induced invasive protrusions and cell dissemination of organoids in 3D cell culture
A 3D cell culture system was used to mimic the in vivo tumor microenvironment as previously reported. [24][25][26] The doubling time of the indicated cells was analyzed using Trypan blue staining and a hemocytometer ( Figure S4A

| Pleckstrin-2 promotes metastasis of breast cancer cells in vivo
The effect of PLEK2 on lung metastasis in xenograft models of breast cancer was evaluated. Control, PLEK2-KO, and OE-PLEK2 MDA-MB-231 cells were injected into the tail vein of BALB/c nude mice as previously described. 27,28 There was no obvious difference in bodyweight or lung weight among the three groups ( Figure 4A,B).
Staining with H&E showed that PLEK2 KO decreased metastatic lung foci, whereas these foci were increased following PLEK2 overexpression ( Figure 4C-E). These results indicate that PLEK2 promotes metastasis of breast cancer cells to the lung.

| Smad3/STAT3 signaling is involved in TGFβinduced PLEK2 expression
Considering that TGFβ induced PLEK2 expression at both the mRNA and protein levels, we hypothesized that TGFβ regulates

| Transforming growth factorβ significantly increases PLEK2 localization at cell membrane and protrusions
Previous studies have shown that PLEK2 colocalizes with F-actin, has a high affinity for the PI3K products PIP2 and PIP3, and interacts with Coffilin, Rac1, EGFR, and SHIP2. 14,15,[29][30][31] Surprisingly, many of the proteins and lipids known to interact or colocalize with PLEK2 are associated with invadopodia 32 ; therefore, we hypothesized that the intracellular localization of PLEK2 affects the regulation of invadopodia formation and function. Immunofluorescence staining shows that PLEK2 localization at the cell membrane and F I G U R E 3 Pleckstrin-2 (PLEK2) is essential for cell protrusion extension and cell dissemination of organoids in 3D culture. (A,C,E) Representative invasion images of the indicated cells in 3D culture. Scale bar, 30 μm. White arrows indicate cell invasion. The inset of (C) is an enlarged view of the boxed region. Statistical analysis of (A,C,E) is shown. Invasive ability was normalized to the control group. Each group was quantitated using 100-300 cells. (B,D,F) Representative dissemination images of the indicated cells in 3D culture. Scale bar, 20 μm. Red arrows indicate cell dissemination. Statistical analysis of (B,D,F) is shown. The cell dissemination ability was normalized to the control group. Each sample was quantitated using 50-100 cells. Three independent experiments were performed. *p < 0.05, **p < 0.01, ***p < 0.001. OE, overexpression; TGFβ, transforming growth factorβ.

| The C-terminal PH domain of PLEK2 is important for activation of the PI3K/Akt signaling pathway and is required for breast cancer cell invasion
Inhibiting PI3K with Wortmannin suppresses the membrane association of PLEK2 and PLEK2-induced cell spreading in Jurkat cells. 29 Our data indicates that inhibition of PI3K phosphorylation with Wortmannin abolished TGFβ-and PLEK2-induced phosphorylation of Akt and cell invasion ( Figure 7A Figures 8E,F and S8H). These results indicate that the interaction between PLEK2 and PPM1B likely plays an important role in TGFβ-induced breast cancer progression.

| PLEK2-PPM1B axis utilizes the NFκ B signaling pathway to exert its pro-metastatic function
It has been reported that PPM1B suppresses cancer progression and negatively regulates NF-κB signaling by dephosphorylating IKKβ. 35 Gain-of-function and loss-of-function experiments were carried out to investigate the role of PPM1B in breast cancer cells. The protein expression level of PPM1B was significantly increased in PPM1Boverexpressing MDA-MB-231 and MCF-7 cells ( Figure S9A) and was decreased following PPM1B knockdown ( Figure S9D

| DISCUSS ION
Metastasis is the leading cause of breast cancer-related death, and despite the recent progress in suppressing the metastasis of breast cancer, metastatic disease still lacks effective treatment. To develop effective metastasis prevention and treatment strategies, detailed knowledge of the underlying molecular mechanisms is required.
Transforming growth factorβ is a critical regulator of EMT and TGFβ signaling plays context-dependent roles in cancer, 36,37 with the effects on CSCs and NCSCs being different. 38,39 Transforming growth factorβ signaling is only activated in CSCs in response to TGFβ treatment, regulating EMT in breast cancer 39 ; however, the aggressiveness of cancer is related to new CSCs generated from NCSCs. Transforming growth factorβ promotes the transition of NCSCs to CSCs by inducing ZEB1 expression in breast cancer. 38 The common genes expressed in CSCs and NCSCs in response to TGFβ could be the key to cancer aggressiveness. To identify metastasis-related genes induced by TGFβ in breast cancer, gene expression profiles were analyzed by RNA sequencing F I G U R E 8 Pleckstrin-2 (PLEK2)-PPM1B axis acts through the nuclear factor-κB signaling pathway to exert its pro-metastatic function. Previous studies have reported that PLEK2 interacts or colocalizes with certain proteins or lipids, such as F-actin, PIP3, Rac1, Coffilin, EGFR, and SHIP2, 14,15,[29][30][31] which play essential roles in invadopodia formation and function. [40][41][42] Transforming growth factorβ significantly increased PLEK2 localization at the cell membrane and protrusions ( Figure 6A,C). Moreover, the spot-like pattern of PLEK2 and F-actin colocalization was similar to the structure of invadopodia ( Figure 6E,F). However, PLEK2 unexpectedly did not colocalize with Tks5, a marker of mature invadopodia ( Figure S7), suggesting that PLEK2 has no effect on mature invadopodia formation and function.
The C-terminal PH domain of PLEK2 is required for lipid binding and its mutation prevents PLEK2 from binding to the cell membrane, inhibiting cell spreading. 29 We hypothesized that the C-terminal PH domain of PLEK2 affects the PI3K signaling pathway to regulate PLEK2-induced cell invasion. Inhibition of PI3K phosphorylation with Wortmannin abolished TGFβ-and PLEK2-induced cell invasion ( Figure 7B), indicating that the PI3K/Akt signaling pathway is involved in PLEK2-induced cell invasion and metastasis. These results are consistent with previous reports. [15][16][17] Furthermore, our data show for the first time that the C-terminal PH domain of PLEK2 is important for TGFβ-and PLEK2-induced Akt activation ( Figure 7D) and cell invasion ( Figure 7E).
Previous studies have shown that PLEK2 interacts with protein partners to exert its pro-metastatic function in lung and gallbladder cancer. 14,15 Pleckstrin-2 can interact with the kinase domain of EGFR to maintain a state of continuous activation by inhibiting its c-CBLmediated ubiquitination. 14 Additionally, PLEK2 interacts with SHIP2 in non-small-cell lung cancer cells to target this protein for degradation, which leads to activation of the PI3K/Akt signaling pathway. 14,15 Moreover, PLEK2 interacts with c-Myc to prevent its proteasomemediated degradation in head and neck squamous cell carcinoma. 43 Here, we determined that PLEK2 interacted with PPM1B in breast cancer cells ( Figure 8A,C,D). PPM1B inhibited PLEK2-induced cell migration and invasion ( Figure 8G-J). Furthermore, PLEK2 induced the ubiquitin-dependent degradation of PPM1B ( Figures 8E,F and   S8H), which is involved in the NF-κB signaling pathway ( Figure 8K,L).
These results suggest that the PLEK2-PPM1B axis exerts prometastatic functions through the NF-κB signaling pathway.
Considering that TGFβ plays a crucial role in cancer and other diseases, numerous efforts have been made to develop targeted therapeutics. 44 However, systemic anti-TGFβ therapies using TGFβ-neutralizing Abs or receptor kinase inhibitors may cause severe adverse effects due to the spatiotemporal activation pattern of TGFβ signaling during cancer progression. [45][46][47] More specific drugs that selectively target downstream signaling without affecting other homeostatic functions of TGFβ could be used to improve current therapeutic strategies for breast cancer patients. Here, we revealed that the TGFβ-STAT3/Smad3-PLEK2-PPM1B axis is critical for TGFβ-induced breast cancer cell migration, invasion, and dissemination.
We also showed that the NF-κB signaling pathway participates in the TGFβ-STAT3/Smad3-PLEK2-PPM1B axis in breast cancer cells.
These findings provide new insights into the function and detailed mechanism of TGFβ-induced cell invasion and metastasis.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflicts of interest