Microtubule associated serine/threonine kinase-3 inhibits the malignant phenotype of breast cancer by promoting phosphorylation-mediated ubiquitination degradation of yes-associated protein

Abstract

Background

Microtubule associated series/threonine kinase-3 (MAST3) is a member of microtubule associated serine/threonine kinase family (MAST1-4, MAST-like), and the expression and underlying molecular mechanism of MAST3 in human tumors, including breast cancer, is not yet elucidated.

Methods

We employed immunohistochemistry to assess the significant expression of MAST3 in breast cancer tissue samples. Additionally, we utilized an overexpression vector and shRNA to bi-directionally regulate MAST3 expression, aiming to observe the impact of MAST3 on the proliferation, migration, and invasion capabilities of breast cancer cells. Furthermore, we employed immunoprecipitation, immunoblotting, luciferase reporter genes and real-time quantitative PCR to investigate the interaction between MAST3 and YAP, as well as the regulatory effects on the expression of Hippo pathway-related target genes.

Results

Low MAST3 expression was observed both in breast cancer cells and tissues, which was significantly associated with advanced tumor T stage, lymph node metastasis, and poor patient prognosis. Functional experiments found that overexpression of MAST3 can gradually inhibit the proliferation and invasion of breast cancer cells, knocking-out MAST3 showed the opposite functional effect. Immunoprecipitation showed that MAST3 interacts with the key effector factor, yes-associated protein (YAP), in the Hippo pathway. The combination of MAST3-YAP promoted the phosphorylation of YAP, which led to its degradation through the ubiquitin-proteasome pathway and reduced nuclear translocation.

Conclusions

MAST3 was identified as a novel tumor suppressor protein in breast cancer, which directly regulates the expression of YAP through the non-dependent mammalian sterile-20-like (MST)-large tumor suppressor (LATS) classical signaling pathway, providing a theoretical and experimental basis for the development of small-molecule tumor inhibitors in breast cancer.

Background

Breast cancer is the most common malignant tumor among women and poses a serious health threat. Although considerable progress has been made in the treatment of breast cancer (radiotherapy and chemotherapy, endocrine therapy, human epidermal growth factor receptor-2 (HER2) targeted therapy, and immunotherapy), some patients still respond poorly to treatment [1,2,3,4,5,6]. Key aspects of breast cancer research include identifying specific biomarkers involved in the occurrence and development of breast cancer, exploring their underlying molecular mechanisms, and developing targeted therapeutic drugs.

The Hippo/YAP pathway is a highly conserved signaling pathway during species evolution that is involved in cell proliferation and differentiation and has important functions in controlling organ volume size [7,8,9]. Recently, the inactivation of the Hippo/YAP pathway and enhancement of the malignant phenotype of breast cancer have attracted considerable attention. The occurrence and development of breast cancer, drug resistance, and other malignant biological processes are closely related to the upregulation of YAP expression and degree of nuclear penetration [10,11,12,13]. The classical Hippo/YAP signaling pathway mainly consists of upstream signaling molecules (such as Merlin and KIBRA), the central cascade kinase complex (MST-SAV-LATS-MOB), and the downstream key effector factor YAP and its homologous transcriptional co-activator with PDZ-binding motif (TAZ). When the classical Hippo pathway is activated, upstream molecules trigger a cascade of phosphorylation reactions in the central kinase complex, thereby promoting YAP phosphorylation. Phosphorylated YAP binds to 14-3-3 proteins in the cytoplasm and is degraded through the ubiquitin proteasome pathway. By contrast, when the Hippo pathway is inhibited, nonphosphorylated YAP/TAZ cannot be degraded and accumulates in large quantities in the cytoplasm and enters the nucleus. YAP/TAZ in the nucleus interacts with the transcription factor TEA domain family (TEADs), promoting their transcriptional activity and activating the transcription of downstream target genes such as CTGF, CYR61, and CCNE1 [14,15,16,17]. In addition to the classical signal transduction processes mentioned above, YAP expression can also be regulated through non-classical pathways. Yang et al. found that the E3 ubiquitin ligase SKP2 interacts with YAP through ubiquitination pathways to promote YAP entry into the nucleus [18]; similarly, CK1α kinases can directly phosphorylate YAP to promote its ubiquitin-proteasome degradation process [19]. This indicates that the regulation of the YAP oncoprotein expression is complex and can be precisely regulated by other protein kinases and ubiquitin systems.

Microtubule associated serine/threonine kinase-3 (MAST3) is a member of the MAST kinase family, of 53.9 kb, and encoded by 27 exons. MAST3 is structurally highly conserved during evolution, and the alignment of the human amino acid sequence of MAST3 with mouse homologous sequences shows approximately 91% consistency [20]. In terms of the molecular structure, the MAST3 protein is composed of four different structural domains α-spiral bundles that mainly include protein kinase domains and PDZ domains. To date, there are no published studies investigating the relationship between MAST3 and tumors. The expression mode of MAST3 in human malignant tumors (including breast cancer) and its underlying mechanism of action remain unclear.

In this study, we first explored the expression pattern of MAST3 in breast cancer by immunohistochemical detection and observed changes in the biological behavior of proliferation, invasion, and metastasis of breast cancer cells by bi-directional regulation of MAST3 expression, construction and transfection of various plasmids and short-hairpin RNA sequences, bidirectional regulation of the expression of MAST3 and YAP, detection of the effect of MAST3 on YAP activity by immunoprecipitation, reverse-transcription quantitative PCR (RT-qPCR) of luciferase reporter gene, and clarification of the biological role and mechanism of MAST3, providing a new molecular site for the regulation of the nonclassical Hippo pathway activity and a new experimental basis for the prevention and treatment of breast cancer.

Methods

Cell lines

The immortalized human breast duct epithelial cell line MCF-10 A (#GNHu50, RRID: CVCL_0598) and breast cancer cell lines MCF-7 (#SCSP-531, RRID: CVCL_0031), T-47D (#SCSP-564, RRID: CVCL_0553), MDA-MB-231 (#TCHu227, RRID: CVCL_0062), MDA-MB-468 (#TCHu136, RRID: CVCL_0419), SK-BR-3 (#TCHu225, RRID: CVCL_0033), MDA-MB-453 (#TCHu233, RRID: CVCL_0418), and ZR-75-1 (#TCHu126, RRID: CVCL_0588) were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). MCF-10 A, SK-BR-3, MDA-MB-231, MDA-MB-468, and MDA-MB-453 cells were cultured in Dulbecco Modified Eagle Medium (DMEM) medium; T-47D and ZR-75-1 cells were cultured in Roswell Park Memorial Institute (RPMI-1640) medium; and MCF-7 cells were cultured in MEM medium. All culture media contained 10% fetal bovine serum and 1% ampicillin and kanamycin; all cells were cultured in an environment with 5% CO₂ at 37 ℃. All cell lines have been authenticated by short tandem repeat (STR) DNA profiling in the past three years, and all experiments were performed with mycoplasma-free cells.

Patient information and samples

This study was approved by the Ethics Committee of Liaoning Cancer Hospital (Approval number: KY20231013) and conducted in accordance with the principles of the Declaration of Helsinki. All patients with breast cancer who participated in this study signed an informed consent form. A total of 137 breast cancer specimens were obtained from wax blocks filed by the Pathology Department of Liaoning Cancer Hospital (from January 2021 to December 2022). Of these, 108 cases of breast cancer were classified as Grade I and Grade II, and 29 cases were classified as Grade III according to the 2019 World Health Organization classification of tumors of the breast [21]. No chemotherapy or radiation therapy was administered before surgery.

Immunohistochemistry

All tissues were fixed with 10% neutral buffered formalin, embedded in paraffin, and sliced into 4 μm-thick sections. S-P immunohistochemistry was used for MAST3 staining. The first antibody was incubated overnight at 4 ℃ using polyclonal rabbit-derived MAST3 antibody (# HPA035061; Sigma Aldrich, St. Louis, MO, USA) and Ki-67 antibody (MaiXin, Fuzhou, China). Phosphate-buffered saline (PBS) was used, instead of primary antibody, as a negative control. Biotin-labeled secondary antibody (MaiXin) was incubated at 37 ℃ for 30 min and 3,3-diaminobenzidine (DAB) staining was performed.

Five fields of view were randomly selected from each section and 100 tumor cells were counted in each field. Based on the percentage of counted cell staining, the expression of MAST3 was divided into five levels: 0 (no staining), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (> 75%). Based on the intensity of cell staining, the expression of MAST3 was further divided into three levels: 0 (no staining), 1 (light yellow particles), and 2 (deep yellow or yellow-brown particles). Each tissue section was assigned a percentage and coloring score. Multiplying these two values yielded the final score for each section. We evaluated the expression of MAST3 in normal breast tissue adjacent to tumors using this scoring standard and found that the expression intensity in most cases was ≥ 4 points. Therefore, we defined a score < 4 points as negative expression (-) and a score ≥ 4 points as positive expression (+). For YAP, nuclear staining (≥ 4 points) was recognized as positive expression.

Western blot and Immunoprecipitation

The cells were lysed using RIPA lysis buffer and total proteins were extracted separately. Proteins were quantified using the bicinchoninic acid (BCA) method (Beyotime, Shanghai, China). Forty micrograms of total protein were subjected to protein electrophoresis using 8% and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% skim milk powder (BD company, Danvers, USA) and incubated for 1 h. The primary antibodies used: MAST3 (# SAB4500930; Sigma Aldrich, St. Louis, IB:1:500). Myc Tag (# 2276, IB/1:1000, IP/1:50), HA tag (# 3724, IB/1:1000), p-YAP (Ser127) (# 13008, IB/1:1000), p-YAP (Ser109) (# 53749, IB/1:1000), p-YAP (Ser397) (# 13619, IB/1:1000), p-YAP (Ser61) (# 75784, IB/1:1000), YAP (# 14074, IB/1:1000), p-LATS1 (Thr1079, # 8654), LATS1 (# 3477, IB/1:1000), MST (# 14946, IB/1:1000), p-MST1 (Thr183)/MST2 (Thr180) (# 49332, IB/1:1000), CTGF (# 86641, IB/1:1000), and CYR61 (# 14479, IB/1:1000), were purchased from Cell Signaling Technology (Danvers, MA, USA). GAPDH (# sc-293335, IB/1:1000) was purchased from Santa Cruz Biotechnology Inc (CA, USA). GFP tag was purchased from Clontech (# JL-8, TaKaRa, Beijing, China) and incubated overnight at 4 ℃. The membranes were then incubated with goat anti-rabbit secondary antibody (IB/1:5000; Cell Signaling Technology) at 37 ℃ for 1 h. After ECL, ImageJ was used to perform band grayscale value measurements using the ratio to GAPDH as the relative expression level. The experiment was repeated three times, and the mean was calculated. Immunoprecipitation assay was conducted according to manufacturer’s kit protocol (Beyotime).

Plasmids, short-hairpin RNA and reagents

pCMV6 empty vector (#PS-100001) and Myc/DDK-pCMV6-MAST3 (#RC-219098) were purchased from Origene (Rockville, MD, USA). Myc/DDK-pCMV6-MAST3-△PDZ, Myc/DDK-pCMV6-MAST3-△kinase, pEGFP-N1 empty vector, pEGFP-N1-YAP, pEGFP-N1-YAP-△PDZ-BD, pEGFP-N1-YAP-S127A, and the lentivirus envelope shMAST3 and MAST3 were purchased and constructed by Genechem company (Shanghai, China). pGL3b 8xGTIIC luciferase plasmid (# 34615) was purchased from Addgene (Cambridge, MA, USA). The pRL-TK vector (# E2241) was purchased from Promega (Madison, Wisconsin, USA). shYAP (# sc-38637-SH) was purchased from Santa Cruz Biotechnology, Inc. HA-Ub plasmid was a gift kindly provided by Dr. Yao Zhang (China Medical University). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) transfection reagent was used for plasmid transfection. Puromycin (Sigma-Aldrich, St. Louis, MO, USA) was used to select stably transfected cells. Cycloheximide (CHX, #239763) and MG132 (#M8699) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Dual-luciferase assay

Assays were performed according to the manufacturer’s protocol. Cells were transfected to express the indicated proteins and with Renilla luciferase as a control for signal normalization. Three independent transfections were carried out for each experiment.

Immunofluorescence assay

The cells were fixed with 4% paraformaldehyde and sealed with 3% bovine serum albumin (BSA) for 1 h. MAST3 and YAP antibody (1:50) were incubated overnight at 4 ℃, followed by goat anti-rabbit/mouse secondary antibody and Rhodamine secondary antibody incubation. Nuclei were stained with DAPI and confocal scanning was performed using a Radiance 2000 laser confocal microscope (Carl Zeiss, Thornwood, NY, USA).

3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) experiment

Approximately 3000 MDA-MB-468 and MCF-7 cells were cultured in a 96-well plate containing 10% FBS medium after 24 h of transfection and 72 h post knockdown. Next, 20 µL of 5 mg/mL MTT (thiazolyl blue) solution was added to each well and incubated at 37 °C for 4 h. The solution was then removed, the MTT crystals that had formed were dissolved in 150 µL of dimethyl sulfoxide (DMSO), and the absorption peak at 490 nm was measured using a spectrophotometer.

Matrigel invasion experiment

The Matrigel (BD Biosciences) was diluted at a ratio of 1:3 using DMEM medium with dual absence in a 24-well plate and applied to an 8 μm pore size upper chamber (Costa, USA). MDA-MB-468 cells were stably transfected with MAST3 plasmid, MCF-7 cells stably knocked down by shRNA-MAST3 were treated with 100 µL of 5 × 10⁵ cells with a density of 2% calf serum in the upper chamber. Culture medium containing 10% calf serum was placed in the lower chamber. After seeding, the cells were cultivated for 24 h, fixed in ice-cold methanol for 15 min, and stained with hematoxylin. Ten fields of view were randomly selected, and the number of cells invading the subventricular space was counted. The experiment was repeated three times, and the mean value was calculated.

Colony formation experiment

The MDA-MB-468 and MCF-7 cells were overexpressed for 48 h or underwent interference experiments for 72 h. The cells were then inoculated into a 6-cm cell culture dish (1000 cells/dish) and incubated for 12 d. Next, the cells were washed three times with PBS every 5 min and stained with hematoxylin for 10 min. The experiment was repeated three times, and the mean value was calculated.

Wound healing assay

When the cultured cells achieved a density of < 90% confluence, wounds were inflicted on the cells using the tip of a 200 mL pipette. The cells were then washed to remove debris before subculturing in 2% serum medium. Wound healing was observed within the scratch lines at various time points and representative scratch lines were photographed for each cell line. For each experiment, the replicate wells were examined for each condition, and the experiment was repeated three times. ImageJ software was used to perform optical measurements of the wound distance.

RNA extraction and RT-qPCR

Total RNA was extracted from cells using RNeasy Plus Mini Kit (Qiao, Hilden, Germany), and RT-qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster, CA, USA); 20 µL of the cells was added to a 7900 RT-qPCR system with the following conditions: 50 ℃ for 2 min, 95 ℃ for 10 min, 95 ℃ for 40 s, followed by 60 ℃ for 60 s. GAPDH was used as an internal reference, with the relative expression level of the gene represented by Δ Ct (Ct _{value of the gene} - Ct _{value of the internal reference)}. Multiple changes in gene expression were calculated as 2^{− ΔΔCt} using the Ct method, and all experiments were conducted in triplicate. The RT-qPCR primer sequences are listed in Table 1.

Table 1 Primers for real-time reverse transcriptase polymerase chain reaction

Animal experiment

The nude mice used in this study were treated by following the experimental animal ethics guidelines issued at China Medical University. The study was approved by Institutional Animal Research Committee of China Medical University (No. CMU20231358; Approval Date: October 13, 2023). The maximal tumor size permitted by animal ethics committee was 2 × 10³ mm³, and the maximal tumor size in our assay was not exceeded. The nude mice (BALB/c, SPF grade, 16–18 g, 4 weeks old, and female) were purchased from Charles River (Beijing, China), then housed in the pathogen-free room with a 12-hour l dark/light cycle and fed with standard chow. The following assays have been described previously [22]. the axilla or tail vein of each mouse was subcutaneously or intravenously inoculated with 5 × 10⁶ or 2 × 10⁵ tumor cells, respectively, in 0.2 mL of sterile PBS. After six weeks, the mice were euthanized and autopsied to examine tumor volume and weight. The mice lungs were fixed in 4% formaldehyde and embedded in paraffin and stained with hematoxylin-eosin (H&E).

Statistical analysis and gene set enrichment analysis (GSEA)

All data were analyzed using SPSS 26.0 (Chicago, USA). The chi-square test was used to test the association between MAST3 expression and clinicopathological factors. The t-test was used to analyze the differences between the groups. A P-value < 0.05 was considered significant. The relationship between MAST3 and ubiquitin-proteasome pathway was analyzed using the Gene Set Enrichment Analysis (GSEA) online database (http://sangerbox.com/).

Results

Low expression of MAST3 in clinical resected specimens of human breast cancer is associated with poor patient prognosis

We collected 137 breast cancer tissue specimens to investigate the expression of MAST3 in human breast cancer. Through immunohistochemical staining, we found that MAST3 was located in the cytoplasm of breast cancer cells and was strongly expressed in the normal ductal epithelium adjacent to cancer cells (26/36, 75%). Ductal carcinoma in situ and invasive carcinoma (non-special types) were weakly positive-to-negative (42.3%, 58/137, P < 0.05; Fig. 1-A, B). Low expression of MAST3 was significantly associated with positive lymph node metastasis (P = 0.005) and tumor T-stage (P = 0.002) in patients with breast cancer (Table 2). Meanwhile, we accessed The Cancer Genome Atlas (TCGA, http://timer.cistrome.org/) database, which showed that the expression level of MAST3 in breast cancer was significantly lower than that in normal breast tissue (Fig. 1-C), consistent with our conclusion. In addition, the online breast cancer database (https://bcgenex.ico.unicancer.fr/) indicates that, compared to other molecular subtypes of breast cancer (such as Luminal A, Luminal B, and HER2-amplified), MAST3 expression exerts the lowest in the Basal-like subtype (i.e., triple-negative breast cancer) (Figure-D, E). Finally, Kaplan–Meier survival online analysis (https://bcgenex.ico.unicancer.fr/ and http://kmplot.com/analysis/) revealed that patients with high MAST3 expression had significantly higher disease-free and overall survival than those with low MAST3 expression (P < 0.01, Fig. 1-F, G, H).

Fig. 1

Low expression of MAST3 in breast cancer is associated with poor prognosis. (A-B). Immunohistochemical testing (A) revealed that MAST3 is highly expressed in normal mammary ductal epithelium and acinar cells (a-200×, b-400×). In ductal carcinoma in situ (DCIS, c-200×, d-400×) and invasive breast cancer (IBC, non-specific type) (grade 2, e-200×, f-400×; grade 3, g-200×, h-400×) MAST3 expression is negative or weakly positive. The areas (200×, scale bar: 50 μm) using black rectangle box labels were enlarged at the right side (400×, scale bar: 20 μm). Statistical percentage chart (B) of MAST3 expression in breast cancer tissues (n = 137) and normal Adjacent Tissues (n = 36) tested by IHC. (C): In the network database (http://timer.cistrome.org/), MAST3 expression was significantly lower in breast cancer than that in normal breast tissue, using paired Student t-test, * * *: P < 0.001. (D, E). The online breast cancer database (https://bcgenex.ico.unicancer.fr/) indicates that, compared to other molecular subtypes of breast cancer (such as Luminal A, Luminal B, and HER2-amplified), MAST3 expression exerts the lowest in the Basal-like subtype. P < 0.0001. (F-H). Multiple networks of Kaplan–Meier analysis (https://bcgenex.ico.unicancer.fr/ & http://kmplot.com/analysis/) indicated that low MAST3 expression positively correlated with poor progression-free survival and overall survival. P < 0.01

Table 2 Association of MAST3 expression with the clinical and pathological characteristics in breast cancer

Overexpression of MAST3 can inhibit the malignant phenotype of breast cancer cells

To further explore whether MAST3 affects the proliferation and migration of breast cancer cells, we detected MAST3 protein levels in normal breast duct epithelial cells, MCF-10 A, and a panel of breast cancer cell lines (n = 7). Western blot analysis showed that the expression of MAST3 in breast cancer cell lines (7/7) was significantly lower than that in MCF-10 A cells (Fig. 2-A). The MDA-MB-468 and T-47D cell lines, which have low MAST3 expression, were selected for the overexpression experiments. After gradient transfection of the lentivirus-coated MAST3 plasmid into the MDA-MB-468 and T-47D cell lines (Fig. 2-B, Supplementary Figure S1-A), the MTT experiments showed that compared with the control group, the proliferation ability of cells overexpressing MAST3 gradually weakened with the gradual upregulation of MAST3 expression (Fig. 2-C). Colony formation experiments showed that the number of clones formed in cells overexpressing MAST3 gradually decreased with the upregulation of MAST3 expression (Fig. 2-D, E, Supplementary Figure S1-C, E). Additionally, the Transwell and wound healing assays showed that the degree of cell migration in cells overexpressing MAST3 gradually decreased as the expression level of MAST3 increased (Fig. 2-F-I, Supplementary Figure S1-G, I, K, M). Subsequently, in vivo experiments showed that overexpression of MAST3 significantly reduced the volume, weight, and Ki-67 index of transplanted tumors in nude mice (Fig. 2-J-L, Supplementary Figure S2-A, B). The tail-vein lung metastasis experiment showed that overexpression of MAST3 can significantly reduce the formation of lung metastases (Fig. 2-M-N).

Fig. 2

Overexpression of MAST3 significantly inhibits the proliferation, invasion, and metastasis of breast cancer cells. (A). Western blot was used to detect the expression of MAST3 in different breast cancer cell lines (n = 7) and normal breast cell lines (MCF-10 A). GAPDH was used as an internal reference. (B). Gradient transfection of MAST3 lentivirus-coated plasmid in MDA-MB-468. Western blot detection of MAST3 transfection efficiency. (C–I): MTT assay (C), colony formation assay (D, E), matrix gel invasion assay (F, G) and wound healing assay (H, I) revealed that the proliferation, clone formation, invasion and migration abilities of tumor cells gradually weakened with increasing MAST3 expression. Scale bar: 50 μm. (J–L): After overexpression of MAST3, the volume and weight of subcutaneous transplanted tumors in nude mice decreased significantly compared to those in the control group. Scale bar: 1 cm. (M-N): Overexpression of MAST3 can significantly reduce the number of lung metastases formed by tumor cells through the tail-vein. (Magnification 20×, Scale Bar: 1000 μm; Magnification 400×, Scale Bar: 30 μm). Paired Student t-test, Columns: mean numbers, Bars: SD, each experiment was performed triplicate. *: P < 0.05; * *: P < 0.01; * * *: P < 0.001

Silencing MAST3 can promote the proliferation and migration of breast cancer cells

We selected MCF-7 and MDA-MB-231 cell lines with relatively high MAST3 expression and used shRNA to silence the MAST3 expression to observe the effect of MAST3 on the malignant phenotype of breast cancer cells. We used two independent MAST3 shRNAs to knockdown MAST3 and then overexpress MAST3 for the recovery experiment (Fig. 3-A, Supplementary Figure S1-B). The MTT experiment results showed that compared with the control group, in the silenced MAST3 group, the proliferation ability of cells was significantly enhanced, whereas that in the recovery group was significantly weakened (Fig. 3-B). The colony formation experiment showed that after silencing MAST3, the number of cell clones formed significantly increased compared to that in the control group, whereas that in the recovery group significantly decreased (Fig. 3-C, D, Supplementary Figure S1-D, F). Transwell and wound healing assays revealed that the degree of cell migration and invasion in the silenced MAST3 group significantly increased compared to that in the control group, whereas that in the recovery group significantly decreased (Fig. 3-E-H), Supplementary Figure S1-H, J, L, N). Similarly, in vivo experiments on nude mouse xenografts showed that knocking down MAST3 significantly increased the volume, weight, and Ki-67 index of the transplanted tumor (Fig. 3-I-K, Supplementary Figure S2-C, D), while restoring MAST3 expression inhibited this phenomenon. The tail-vein lung metastasis experiment showed that MAST3 knockdown significantly increased the formation of lung metastases (Fig. 3-L, M), whereas metastases formation in the recovery group was inhibited. Based on the experimental results of cell function after the overexpression and silencing of MAST3, we concluded that MAST3 plays a biological role as a tumor suppressor protein in the occurrence and development of breast cancer.

Fig. 3

Knockdown of MAST3 significantly promotes the malignant phenotype of breast cancer. (A). Western blot detection of MAST3 transfection efficiency. Recovery of MAST3 expression owing to the table transfection of shMAST3 plasmid in MCF-7. (B–H): MTT (B), colony formation (C, D), matrix gel invasion (E, F), and wound healing (G, H) assays revealed that the proliferation, clone formation, invasion, and migration abilities of tumor cells increased with the decrease in MAST3 expression, MAST3 expression recovery induced the opposite effect. Scale bar: 100 μm. (I–K): In vivo experiments showing that knocking down MAST3 significantly increased the volume and weight of subcutaneous transplanted tumors in nude mice compared to those in the control group, whereas restoring the expression of MAST3 resulted in the opposite effect. Scale bar: 1 cm. (L–M): Knockdown of MAST3 significantly increased the number of tumor cells forming metastatic foci in the tail-vein lung metastasis experiment, whereas restoring the expression of MAST3 resulted in the opposite effect. (Magnification 20×, Scale Bar: 1000 μm; Magnification 400×, Scale Bar: 30 μm). Paired Student t-test, Columns: mean numbers, Bars: SD, each experiment was performed triplicate. *: P < 0.05; * *: P < 0.01; * * *: P < 0.001

MAST3 is a positive regulatory factor for the non-classical Hippo pathway

The Hippo pathway plays an important role in the occurrence and development of breast cancer; however, the relationship between MAST3 and the Hippo pathway has not been previously reported. Therefore, we first transfected the lentivirus-coated MAST3 plasmid into the MDA-MB-468 cell line. Using luciferase reporter genes, we found that the overexpression of MAST3 significantly inhibited YAP-induced TEAD transcriptional activity (that is, YAP activity was inhibited), whereas knocking down MAST3 using lentivirus-coated shRNA resulted in the opposite effect (Fig. 4-A, B). Western blot analysis showed that after overexpression of MAST3 in MDA-MB-468 and T-47D cell lines, the total YAP level was significantly downregulated, whereas the knockdown of MAST3 in MCF-7 and MDA-MB-231 cell lines resulted in the opposite effect (Fig. 4-C, Supplementary Figure S3-A, B). The total amount and phosphorylation level of the intermediate kinase complex MST-LATS in the classical Hippo signaling pathway remained the same despite the changes in MAST3 expression. We postulated that MAST3 affects the expression and activity of YAP through the independent MST-LATS pathway. Furthermore, we examined the mRNA expression levels of the target genes CTGF, CYR61, and CCNE1 in the Hippo pathway after the bidirectional regulation of MAST3. The results showed that overexpression of MAST3 significantly downregulated the transcription levels of the three downstream genes induced by YAP, whereas the knockdown of MAST3 resulted in the opposite effect (Fig. 4-D, E). The changes in MAST3 expression did not cause significant changes in YAP mRNA levels (P < 0.05). YAP nuclear entry is essential for the inhibition of the Hippo pathway; thus, we further investigated the effect of MAST3 on YAP nuclear entry levels. Immunofluorescence results showed that after overexpression of MAST3 in MDA-MB-468 cells under sparse cell culture conditions, the nuclear uptake of YAP was significantly decreased compared to that in the control group, whereas MAST3 knockdown in MCF-7 cells showed the opposite result (Fig. 4-F). Therefore, we conclude that MAST3 promotes the degradation of YAP and inhibits its nuclear entry via another pathway that is not dependent on the MST-LATS pathway, which leads to the activation of the non-classical Hippo pathway activity.

Fig. 4

MAST3 is identified as a positive regulator of the Hippo pathway. (A–B): MAST3 overexpression vector and shRNA-MAST3 are transfected into MDA-MB-468 and MCF-7, respectively. Fluorescence reporter gene detection reveals that MAST3 significantly reduces the transcriptional activity of YAP-TEAD, with YAP acting as a signal amplifier. (C): Western blot analysis revealed that MAST3 significantly downregulates the amount of YAP protein, while the phosphorylation levels and content of upstream kinases MST-LATS did not show significant changes. GAPDH served as an internal reference. (D–E): RT-qPCR detection showed that MAST3 downregulates the mRNA levels of target genes CTGF, CYR61, and CCNE1 in the Hippo pathway but induced no significant change in the mRNA levels of YAP. (F): Immunofluorescence experiments showed that MAST3 overexpression in MDA-MB-468 weakened the ability of YAP to enter the nucleus, whereas MAST3 knockdown led to the opposite result. The areas (200×, scale bar: 50 μm) using red rectangle box labels were enlarged at the right side (400×, scale bar: 20 μm). Paired Student t-test, Columns: mean numbers, Bars: SD, each experiment was performed triplicate. *: P < 0.05; * *: P < 0.01; * * *: P < 0.001

MAST3 interacts with the PDZ binding motif of YAP through its PDZ domain and promotes Serine phosphorylation at YAP 127 site

We also investigated the mechanism underlying MAST3 inhibition of YAP protein activity. As MAST3 is a protein kinase, bidirectional regulation of MAST3 expression can cause changes in the total amount of YAP protein. Therefore, we examined whether MAST3 could bind to YAP and cause changes in its phosphorylation levels. Immunoprecipitation revealed that MAST3 interacts with endogenous levels of YAP (Fig. 5-A). We co-transfected MAST3 and YAP plasmids into MCF-7 cells and confirmed the interaction through exogenous immunoprecipitation (Fig. 5-B). Laser confocal and immunofluorescence staining showed that MAST3 and YAP were colocalized in the cytoplasm of breast cancer cells (Fig. 5-C). We further investigated which protein domains were involved in the interaction between MAST3 and YAP. We constructed the corresponding spliceosomes based on the known protein domains of MAST3 and YAP (Fig. 5-D). Immunoprecipitation experiments revealed that MAST3 lacking the PDZ domain could not bind to YAP (Fig. 5-E), whereas YAP lacking the PDZ binding motif could not bind to MAST3 (Fig. 5-F). This indicated that MAST3 binds to the PDZ binding motif of YAP through its PDZ domain. Since MAST3 is a protein kinase that causes the phosphorylation of interacting protein substrates, we explored whether protein kinase MAST3 can promote YAP phosphorylation through interaction with YAP. First, we bidirectionally regulated the expression of MAST3 in MDA-MB-468 and MCF-7 cell lines and used western blot to detect changes in YAP phosphorylation levels (Fig. 5-G, H). We preliminarily detected common YAP phosphorylation sites (S127, S109, S397, and S61) that are already commercially available. We found that MAST3 can significantly alter the phosphorylation of the YAP-S127 site. Immunofluorescence staining further revealed that MAST3 could inhibit the nuclear translocation of wild-type YAP, but it had no obvious impact on mutant YAP-S127A (Fig. 5-I). Upon transfection of MDA-MB-468 cells with MAST3 wild-type plasmid, western blot results indicated that the phosphorylation of YAP-S127 was obviously upregulated compared to that of the empty vector group, whereas the MAST3 mutant plasmids groups (delta-kinase domain and delta-PDZ domain) did not exhibit any significant changes (Fig. 5-J). It is noteworthy that we transfected the MAST3 plasmid into MDA-MB-468 cells and utilized immunoprecipitation assays to detect alterations in the affinity between LATS1 and YAP. The findings revealed that overexpressing MAST3 did not significantly alter the binding amount between LATS1 and YAP, even after the addition of cycloheximide (CHX) to inhibit the downregulation of YAP expression mediated by MAST3 (Fig. 5-K). Thus, MAST3 can interact with the PDZ binding motif of YAP through its PDZ domain and promotes serine phosphorylation at the YAP 127 site in an independent manner of LATS1-YAP pathway.

Fig. 5

The interaction between MAST3 and YAP promotes the phosphorylation of YAP (S127). (A–B): Endogenous and exogenous immune coprecipitation experiments were conducted to verify the interaction between MAST3 and YAP. (C): Immunofluorescence experiments confirmed the co-localization of MAST3 and YAP in the cytoplasm of MCF-7 cell lines. Scale bar: 25 μm. (D): Schematic diagram of the structure of MAST3 and YAP splicing bodies. (E–F): MAST3 interacts with YAP’s PDZ-BD domain through its PDZ domain. Co-transfection of GFP-YAP and Myc MAST3-WT and Myc MAST3 in MCF-7-Δ PDZ; immunoprecipitation showed that MAST3 could not interact with YAP in the absence of the PDZ domain (E); Similarly, YAP lacking the PDZ-BD domain cannot bind to MAST3 (F). (G–H): Western blot detection of changes in YAP phosphorylation levels after the bidirectional regulation of MAST3 expression in MDA-MB-468 and MCF-7. After MAST3 overexpression, the phosphorylation level of YAP-ser127 was significantly upregulated, whereas after knockdown, the results showed the opposite effect (phosphorylation levels at other sites did not show significant changes). GAPDH served as an internal reference. (I): The MDA-MB-468 cell line was co-transfected with MAST3 as well as YAP wild-type and mutant YAP-S127A plasmids, and immunofluorescence experiments were performed to detect the changes in YAP nuclear translocation. Scale bar: 25 μm. (J): MAST3 wild-type, MAST3-delta kinase, and MAST3-delta PDZ mutant plasmids were transfected into MDA-MB-468 cells, and western blot was performed to verify that MAST3 upregulates the phosphorylation levels of the YAP-ser127 site, whereas the mutants abrogated this effect. (K): MAST3 plasmids were transfected into MDA-MB-468. After 36 h, cycloheximide (CHX, 532.5 nM) was added and proteins at 0 h and 12 h were collected for immunoprecipitation analysis. GAPDH served as an internal reference protein. IgH: heavy chain

MAST3 promotes YAP degradation through the ubiquitin-proteasome pathway

Based on these experimental results, we verified that MAST3 can regulate changes in the total amount of YAP protein but cannot change its transcription level. This indicates that MAST3 regulates changes in YAP expression at the post-transcriptional level. Considerable evidence indicates that YAP phosphorylation is closely related to its degradation process. We postulated that MAST3 can promote YAP phosphorylation, thereby increasing the degradation process. We analyzed the Gene Set Enrichment Analysis database (http://sangerbox.com/) and found that MAST3 participates in protein degradation (Fig. 6-A). Based on the bidirectional regulation of MAST3 expression, we added a protein biosynthesis inhibitor, cycloheximide (CHX), and observed that the ability of MAST3 to downregulate YAP protein levels was significantly weakened, (Fig. 6-B-E), confirming that MAST3 regulates YAP expression through post-transcriptional levels. The ubiquitin–proteasome pathway is the primary pathway involved in protein degradation. Furthermore, after transfecting GFP-YAP and adding the protein enzyme inhibitor MG132, the degradation ability of YAP was inhibited, indicating that YAP could be degraded through the ubiquitin-proteasome pathway (Fig. 6-F). We co-transfected MAST3 and YAP plasmids in MDA-MB-468 cell line, and found that as the expression of MAST3 increased, the expression level of exogenous YAP gradually decreased (Fig. 6-G). Furthermore, bidirectional regulation of the expression of MAST3 revealed that overexpression of wild-type MAST3 in MDA-MB-468 cells significantly increased the ubiquitination levels of YAP, whereas deletion of the kinase or PDZ domain of MAST3 abrogated this effect (Fig. 6-H). On the other hand, knocking down MAST3 in MCF-7 cells resulted in the upregulation of YAP ubiquitination levels (Fig. 6-I). Besides, western blot analysis and IHC results indicated that MAST3 expression was negatively correlated with YAP expression both in breast cancer cell lines and samples (Fig. 6-J, K, Table 3). Therefore, MAST3 can promote YAP degradation process through the ubiquitin-proteasome pathway.

Fig. 6

MAST3 is involved in the YAP-degradation process mediated by ubiquitin-proteasome pathway. (A). GSEA analysis showed that MAST3 is involved in ubiquitin-mediated protein degradation process. (B, C). siRNA-MAST3 and MAST3 plasmids were transfected into MCF-7 and MDA-MB-468, respectively. After 36 h, cycloheximide (CHX, 532.5 nM) was added and proteins at different time points were collected for western blot analysis. GAPDH served as an internal reference protein. (D, E). MAST3 mediated the metabolic curve of YAP protein degradation. *: P < 0.05. (F). MDA-MB-468 cells were transfected with GFP-tagged YAP plasmid, 24 h later, the cells were added to DMSO or MG132 (concentration: 20 µM) for different time points (including 4 and 8 h, respectively). Cells were lysed and subjected to western blot. GAPDH serves as a loading control. (G): GFP-YAP plasmid and Myc-MAST3 plasmid were co-transfected into MDA-MB-468, and after 24 h, DMSO or MG132 (20 µM) was added. After 24 h of treatment, the protein collected was subjected to western blot analysis. GAPDH served as an internal reference protein. (H and I). MAST3 wild type-plasmid or its mutant plasmids (delta-kinase domain and delta-PDZ domain), along with HA-ubiquitin (Ub) plasmid were co-transfected in MDA-MB-468 cells for 48 h (H), shRNA-MAST3 and HA-Ub plasmid were co-transfected in MCF-7 cells for 72 h, respectively (I). Cells were lysed and the changes in YAP ubiquitination level was subjected to immunoprecipitation and western blot. (J). The association between MAST3 and YAP expression in multiple breast cancer cell lines was detected using western blot. GAPDH served as an internal reference protein. (K). Representative photographs of the correlation between MAST3 and YAP expression in human breast cancer specimens (magnification, 200×, scale bar: 100 μm; magnification, 400×, scale bar: 50 μm). Paired Student t-test, Columns: mean numbers, Bars: SD, each experiment was performed triplicate

Table 3 Correlation of MAST3 with the expression of YAP in 40 cases of breast cancer specimens

MAST3 weakens the proliferation and migration of breast cancer cells by inhibiting YAP

Since MAST3 serves as a positive regulator of YAP degradation process, we postulated that MAST3 weakens the malignant phenotype of breast cancer by inhibiting YAP. To verify this hypothesis, we silenced the expression of MAST3 in MCF-7 cell lines and applied a small-molecule inhibitor, verteporfin (selective inhibition of YAP-TEAD transcriptional activity). The results of colony formation and transwell experiments showed that, consistent with previous results, knocking down MAST3 can increase the degree of colony formation and cell invasion of the MCF-7 breast cancer cell line, while this increase was abrogated after verteporfin was applied (Fig. 7-A, B); conversely, we added YAP shRNA also obtained similar results (Fig. 7-C, D). RT-qPCR and western blot showed that adding verteporfin or silencing YAP could reverse the upregulation of Hippo target genes (CTGF, CYR61, and CCNE1), mRNA, and protein levels caused by silencing MAST3 (Fig. 7-E, F). Thus, MAST3 inhibits the proliferation and migration of breast cancer cells by inhibiting YAP-TEAD transcriptional activity and reducing the expression of its downstream genes.

Fig. 7

Inhibiting YAP activity or reducing YAP expression can partially counteract the effect of low MAST3. (A–D): In MCF-7 cell lines, shRNA/MAST3 was used to knockdown the expression of MAST3, YAP-TEAD binding inhibitor, verteporfin (12 h, 9.5 µM) and shRNA-YAP (72 h, 2.5 µg) were added to inhibit YAP activity or expression, respectively. Colony formation (A, C) and matrix gel invasion experiments (B, D) showed that verteporfin and YAP knockdown both counteract the enhanced malignant phenotype of tumor cells induced by knocking down MAST3. Scale bar: 100 μm. *: P < 0.05, ***: P < 0.001. E-F: Western blotting and RT-qPCR detection reveal that verteporfin and shYAP counteract the upregulation of target genes (CTGF and CYR61) induced by shMAST3, *: P < 0.05. GAPDH was used as a loading control. The yellow dashed line represents the reference line. Columns: mean numbers, Bars: SD, each experiment was performed triplicate. shRNA: short hairpin RNA; MAST3, Microtubule associated serine/threonine kinase-3; YAP: yes-associated protein; CTGF: connective tissue growth factor; CYR61: Cysteine-rich- 61; CCNE1: G1/S-specific cyclin-E1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase

Discussion

MAST3 is a member of the MAST protein family, and its role in tumors has not been extensively studied. One study suggested that array comparative genomic hybridization combined with gene expression analysis may be associated with the survival time of patients with glioma [23]. MAST3 may regulate the NF-κB signaling pathway and promote the proliferation and inflammation of FLSs [24]. Knockout of the MAST3 gene specifically reduces Toll like receptor-4 dependent NF-κB activity, demonstrating the key role of NF-κB activity modulators in the pathogenesis of IBD [25,26,27,28]. Pathogenic MAST3 variants in the STK domain are associated with epilepsy [29]. However, the expression of MAST3 in breast cancer and its possible molecular mechanism remain unknown.

Using TCGA database, immunohistochemistry, and western blot, we found that MAST3 showed low expression in both breast cancer tissues and cell lines. Furthermore, the low expression of MAST3 was closely related to the clinicopathological parameters and poor prognosis of patients with breast cancer. Notably, the analysis results from the online network database indicate that transcriptome sequencing results from large samples consistently suggest that, compared to other molecular types, MAST3 has the lowest expression level in triple-negative breast cancer. However, our statistical findings do not indicate a correlation between MAST3 expression and the hormone receptor status of breast cancer. We speculate that this may be attributed to the limited number of samples we examined. Moreover, the reason for the low expression of MAST3 (such as mutations in the MAST3 gene, promoter methylation, post-translational modifications, or regulation by other transcription factors or microRNAs) remains unclear and also should be investigated in future studies.

The inactivation of the Hippo pathway is closely associated with the occurrence and development of breast cancer and drug resistance. The classic LATS/MST kinase complex YAP target gene (CTGF/CYR61) molecular axis dominates the activity of the Hippo signaling pathway, in which the expression content and nuclear entry status of the main effector YAP/TAZ in this pathway play a key role in cellular functional changes. In addition to the classical processes mentioned above, YAP expression can be regulated through the non-dependent MST-LATS pathway, considered the non-classical Hippo signaling pathway. For example, SIRT1 can reduce YAP acetylation and enhance YAP-binding activity with the transcription factor TEAD, thereby promoting the transcription of target genes [30–31]. Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) mediates YAP phosphorylation and affects cellular energy pressure metabolism [32], while mammalian target of rapamycin complex-2 (mTORC2) directly phosphorylates YAP (Ser 463), enhancing YAP activity and promoting the malignant phenotype of glioblastoma [33]; the deubiquitinase OTUB1 reduces YAP ubiquitination levels and inhibits YAP degradation, thereby promoting the proliferation activity of gastric cancer cells [34]. The above studies demonstrate that a series of key enzymes with catalytic activities, such as protein kinases/phosphatases, deubiquitinases/ligases, and acetylases/deacetylases, can precisely regulate the expression and protein activity of YAP, by participating in a series of biological functions and tumor formation.

In this study, we found that upregulating the expression of MAST3 significantly inhibits YAP-TEAD luciferase activity in the Hippo pathway and downregulates the expression of target genes. We identified and demonstrated that MAST3 can bind to the PDZ binding motif of YAP through its PDZ domain, promoting the phosphorylation and ubiquitination degradation of YAP (Ser 127). Therefore, we consider that MAST3 kinase can directly upregulate the phosphorylation level of YAP through the independent MST-LATS pathway, promoting its ubiquitination degradation process (Fig. 8). However, in exploring the regulation of the YAP phosphorylation site by MAST3, we only detected common and commercially available YAP phosphorylation site antibodies and did not perform high-throughput mass spectrometry and gel zymography detection. Therefore, a limitation of this study is that we do not know whether MAST3 can regulate sites other than serine 127. Moreover, it remains unclear whether the phosphorylation of YAP mediated by MAST3 leads to its degradation through the MAST3-YAP direct pathway or if other kinases (such as CK1) are involved. This process still needs further elucidation.

Fig. 8

Schematic diagram of the molecular mechanism of MAST3-mediated regulation of YAP phosphorylation and ubiquitination

Conclusions

MAST3 was identified as a novel tumor suppressor protein in breast cancer, which directly downregulates the expression of YAP through its phosphorylation-mediated ubiquitination in non-classical Hippo signaling pathway, suggesting potential avenues for personalized treatments.