Characterization of a Novel MAP4K4-SASH1 Kinase Cascade Regulating&nbsp;Breast Cancer Tumorigenesis and Metastasis

doi:10.21203/rs.3.rs-101160/v1

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Characterization of a Novel MAP4K4-SASH1 Kinase Cascade Regulating Breast Cancer Tumorigenesis and Metastasis

https://doi.org/10.21203/rs.3.rs-101160/v1

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Background: The SAM and SH3 domain containing protein 1(SASH1) was previously described as a candidate tumor-suppressor gene in breast cancer and colon cancer to mediate tumor metastasis and tumor growth. However，the underlying mechanisms by which SASH1 implements breast cancer tumorigenesis and the question why SASH1 is downregulated in most solid cancers remain unexplored.

Methods: The expression and clinical relevance of SASH1 and mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) were analyzed by immunohistochemistry(IHC). The bindings of SASH1 and MAP4K4 were investigated by pull down assay, nano-flow LC-MS/MS , immunoprecipitation- western blot(IP-WB) and immunofluorescence(IF). The knockdown of SASH1- and/or MAP4K4-induced cell proliferation, apoptosis, migration and invasion were investigated by flow cytometry and transwell assays. The cell proliferation and apoptosis treated by GNE-495 were assessed by flow cytometry. The in vitro regulation relationship between SASH1 and MAP4K4 were identified by in vitro kinase assay. The functional effects of the silencing of SASH1 and/or MAP4K4 on tumorigenesis and metastasis of T47D cells-xenograft tumors were assessed by HE staining and IHC .

Results: SASH1 and MAP4K4 was significantly downregulated in hormone- dependent subtypes of breast cancers and SASH1 was significantly correlated with MAP4K4 in hormone-dependent subtypes. SASH1 is identified to be a novel serine/threonine protein kinase that binds to MAP4K4, and is phosphorylated by MAP4K4. SASH1 and MAP4K4 synergistically regulate cell proliferation，migration and invasion of hormone-dependent breast cancer cells and mediate liver and lung metastasis of T47D cells-xenograft tumors.Ser801 in MAP4K4 might be the serine site for MAP4K4 to phosphorylate SASH1. GNE-495， a MAP4K4-specific inhibitors by upregulating the expression of MAP4K4 and SASH1 might be a potential reagent in treating hormone-dependent breast cancers.

Conclusion: Our findings characterize a novel MAP4K4-SASH1 kinase cascade to mediate the tumorigenesis and metastasis of breast cancer, which can be of targeted intervention by GNE-495.

Cancer Biology

Oncology

SASH1

MAP4K4

Tumorigenesis

Metastasis

Hormone-dependent breast cancers

SASH1 was originally identified as a candidate tumor-suppressor gene in breast cancer and colon cancer, regulating tumorigenesis of breast and other solid cancers and the adhesive and migratory behavior of cancer cells in tumor formation[1, 2]. A growing number of reports have confirmed the tumor suppressor roles of SASH1 in breast and other solid cancers[3–8]. Treatment of breast cancer cells treated with chloropyramine results in higher SASH1 protein levels [9]. SASH1 was recently suggested to be potential therapeutic target for cancer [10]. SASH1 was confirmed to bind to 14-3-3 proteins and phospho-Ser90 of SASH1 provides the 14-3-3-binding sites to promote SASH1 phosphorylation by phosphatidylinositol 3-kinase and MAPK/p90RSK signaling[11]. And the most recent study has indicated that SASH1 was phosphorylated by LATS1 [12].

MAP4K4, is also known as a hepatocyte progenitor kinase-like/germinal center kinase-like kinase (HGK).HGK is a member of the human STE20/ mitogen-activated protein kinase kinase kinase kinase family of serine/threonine kinases and is the ortholog of mouse NIK (Nck-interacting kinase). Strikingly increasing rates of invasion and morphogenesis are induced by enhanced HGK kinase activity through overexpression[13]. MAP4K4 overexpresses in many types of human cancer [14–16]. MAP4K4 is commonly overexpressed in TNBC(triple negative breast cancer)- breast tumor-initiating cells(BTICs) and suggested to be a common regulator in TNBC [17]. The present study indicates that SASH1 is identified to be a novel protein kinase interacts with MAP4K4. MAP4K4 and SASH1 coordinately mediate tumorigenesis and metastasis of breast cancer in hormone-dependent breast cancer cell lines and xenograft tumors. SASH1 is phosphorylated and regulated by MAP4K4, which forms a novel MAP4K4-SASH1 phosphokinase cascade and this cascade can be inhibited by GNE-495 .

Collection of breast cancer tissues and human breast carcinoma tissue arrays.

Fresh primary breast cancer tissues from breast carcinoma patients undergoing resection and normal tissues were collected between July 2015 and May 2020 at the Chongqing Cancer Hospital. A set of human breast cancer tissue arrays and a set of matched adjacent tissue arrays of human breast cancer were provided by Shanghai Biochip(Shanghai, China).

Antibodies, cell lines, recombinant DNA or shRNA and siRNAs, and primers for gene cloning and site-directed mutagenesis，and siRNA construction.

The detailed information on chemical reagents, antibodies, cell lines, BALB/c-nude mice, recombinant DNA or shRNA and siRNAs, and primers for gene cloning, shRNA silencing of lentivirus, site-directed mutagenesis , siRNA construction used in this study is indicated in Table S1.

Gene cloning

SASH1-Pegfp-C3 vectors were constructed as described previously[18]. MAP4K4 cDNA was cloned into Pbabe-Flag-puro and Pegfp-C3 using KpnI and XhoI restriction sites. SASH1 mutant plasmids were constructed as described previously [18]. Full- length cDNA of SASH1 was cloned into the pSec Tag vector using XhoI and HindIII restriction sites to construct His-SASH1- pSec Tag vector.

Cell Culture, RNA Interference (RNAi), Transfection and lentivirus infection.

Human breast cancer cell lines and HEK-293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai，China). After passages, cells were transfected recombined vectors. Cells were transfected with two unique and efficient SASH1-shRNA or MAP4K4-shRNA constructs in a GFP vector (Origene) using PEI(prepared by us) according to our previous reports[18, 19]. The sequences used for silencing of SASH1-shRNA vector, silencing of MAP4K4-shRNA vector, silencing of MAP4K4–shRNA lentivirus and silencing of SASH1–shRNA lentivirus were shown in Table S1.

Pull down assay, Nano-flow LC-MS/MS and Database search

The Pull down assay, nano-flow LC-MS/MS， and the database search were constructed as previously described. The binding partners of SASH1 and the phosphorylation sites on SASH1 in SK-BR-3 cells were identified with nano-LC MS/MS analysis on an HPLC system as previously described [20].

Immunoprecipitation and immunoblotting

The immunoprecipitation-western blot(IP-WB) assays were performed as previously described[18, 19]. Most of western blot were mainly performed as our previous reports [18, 19].The primary antibodies used for western blotting were as listed in Table S1.

Cell cycle and apoptosis assays

Transfected cells were harvested , fixed , stained and analyzed by flow cytometry (Navious, Beckman Coulter) for cell cycle profile determination [21]. Apoptosis was measured by staining with 7AAD/APC. The results were analyzed using Flowjo software (CT, USA).

Transwell migration and Invasion assay

Transwell migration assay and invasion assays were performed as previously described[22].

Immunofluorescence

Immunofluorescence (IF) was performed as previously described by us[20].

In vitro Kinase assay

For the SASH1 kinase assay, HEK-293T cells were transfected with GFP-MAP4K4 and treated with 150 nM GNE-495 for 24hr and MAP4K4 was immunoprecipitated. His-SASH1 was also introduced into HEK-293T cells and at 36hr after transfection, His-SASH1 was immunoprecipitated with His Ab.The purified His-SASH1 was used as the substrate for the immunoprecipitated MAP4K4. The protocol of this study were mainly referred to the previous report[23] and the detailed protocol of In vitro kinase assay was indicated in the Supplementary Materials and Methods .

Immunohistochemical staining and quantification

Tissue sections (5μm) were dehydrated and subjected to peroxidase blocking. of human breast cancer tissues, normal breast tissues, corresponding adjacent tissues，and T47D cells-xenograft tumors were immunohistochemically stained with anti-SASH1, anti-MAP4K4, anti-phospho-MAP4K4, anti-Ki67, anti-MMP2 ，anti-caspase9 and anti-MMP9. Detailed information on these antibodies can be found in Table S2. The IHC analyses results of p53 and the breast cancer patients’ information including age, tumor size, tumor location, tumor stage, node status and clinical stage were provided by the manufacturer of the breast cancer tissue arrays. IHC were mainly performed as previously described[18]. The staining intensity of SASH1, MAP4K4, phospho-MAP4K4, Ki67, MMP2 and MMP9 of the positive cells, and the positive areas’ percentage of the positive cells , and total scores of each visual field were calculated as previously described [18].

Site directed mutagenesis

The wild type SASH1-Pegfp-C3 vector was used to construct SASH1- Pegfp-C3 vectors with S90A，S355A , S359A, S814A and S819A mutations by site-directed mutagenesis using the KOD-Plus-Mutagenesis kit(TOYOBO,Janpan). The wild type MAP4K4- Flag-Pcdna 3.1 vector was also used to construct MAP4K4- Pegfp-C3 vectors with S801A,S629A and S631A mutations by site-directed mutagenesis.

Xenograft

Animals were housed and maintained under specific pathogen-free experimental animal center. T47D stable cells containing SASH1-shRNA or/and were suspended in Matrigel (BD)-DMEM (5 ×10⁶) and injected subcutaneously into the right side fossa axillaris of each 5- to 6-week-old female BALB/c nude mice in each group. Tumor volume was calculated and tumor diameter was measured. All mice were sacrificed 3 weeks after observation, and tumor weights were measured.

Statistical analyses

Data are presented as mean ± SD. Pearson’s chi-squared Tests was used to analyze the relationship between IHC scores of the relevant proteins and between clinicopathological parameters and expression levels of SASH1 and p53. The statistical significance of differences was assessed using SPSS16.0 software. The densitometry values of protein bands on western blot were analyzed using a homogeneity of variance test and one-way ANOVA was used for multiple comparisons using the least significant difference in SPSS16.0 to generate the required P-values. P-values of less than 0.05 were considered statistically significant. The cartograms were made and plotted using GraphPad Prism 5 (GraphPad, Inc, CA, USA). Additional methods can be found in the Supplementary Materials and Methods.

SASH1 is down-regulated in four breast cancer subtypes and its expression is associated with the pathological diagnosis index and receptor expression of breast cancer.

To examine the expression of SASH1 in different breast cancer subtypes, 284 carcinoma specimens of four breast cancer subtypes including 92 luminal A, 105 luminal B, 45 HER2 positive and 42 TNBC specimens, and 79 normal breast tissues including tissues of breast fibroadenoma and hyperplasia of mammary glands were collected to compare SASH1 expression in normal breast tissues and breast cancer tissues. Positive SASH1 staining was found in both ductal and lobular epithelial cells. SASH1 expression was observed predominantly in the cytoplasm (Fig. 1A). SASH1 was significantly downregulated in all four subtypes as compared with normal breast tissues(Fig. 1A and Fig. 1B). SASH1 staining of human breast carcinoma tissue arrays indicated that SASH1 was downregulated in four subtypes as compared with the matched breast tissues (Fig. 1C and Fig. 1D). Immunoblot analyses indicated that in 18 benign breast tissues and 18 cancer tissues, SASH was downregulated in different subtypes of breast cancer (Fig. 1G). Statistical analyses based on IHC staining scores of SASH1 and Ki67 in human breast carcinoma tissue arrays suggested that SASH1 expression was significantly related to that of Ki67 in subtypes of luminal and Her2 positive, however, not associated with that of Ki67 in TNBC subtype(Fig. 1E). The relationship between SASH1 expression and p53 expression obtained by the human breast cancer tissue array was assessed by Pearson’s chi-squared test. SASH1 expression was significantly related to p53 in subtypes of luminal and Her2 positive, however, not related to Ki67 in TNBC subtype (Fig. 1F). SASH1 downregulation was significantly associated with early tumor stages,early lymph node metastasis, and early clinical stage; however, it was not correlated with tumor size(Table S2). We also analyzed the relationship of SASH1 expression with ER, PR, EGFR, p53 and CK5/6 in human breast carcinoma tissue arrays. Pearson’s χ2 test based their IHC scores and their positive rate indicated that SASH1 expression was inversely correlated with the ER of nucleus and cytoplasm(Fig.S1A and S1B). SASH1 expression was negatively correlated with the nucleic and cytoplasmic PR (Fig.S1C and S1D).However, SASH1 expression was positively correlated with the nucleic HER2 (Fig.S1E). SASH1 expression was positively correlated with the nucleic and cytoplasmic p53 ae well as Ki67(Fig.S1F, Fig.S1G and Fig.S1H).However, SASH1 expression was not correlated with the EGFR of cytoplasm and cytoplasm membrane(Fig.S1I and S1J) and CK5/6(Fig.S1K).

SASH1 is associated with MAP4K4.

To investigate the molecular network in which SASH1 involved, pull down assays and LC-MS/MS spectrometry analysis were performed. Our results indicated that SASH1 may interact with MAP4K4 in stable SK-BR-3 cells. MAP4K4 was shown to have a high possibility to bind to SASH1 (Fig. 2A), and the interacting peptide sequences of SASH1 and MAP4K4 were identified (Fig. 2B). IP-WB analysis further confirmed that endogenous SASH1 was associated with endogenous MAP4K4 (Fig. 2C). We also found exogenous SASH1 binds to endogenous MAP4K4 using IP with anti GFP antibody. As demonstrated in Fig. 2D, exogenous SASH1 was shown to immunoprecipitate with endogenous MAP4K4 in SK-BR-3 cells. We further identified the binding domains of SASH1 to MAP4K4. Deleted SASH1 constructs were created, transfected into HEK-293T cells and immunoprecipitated with anti-GFP antibody. The associated endogenous MAP4K4 were identified by IP-WB. Compared with the binding of full- length SASH1 to endogenous MAP4K4, the N-terminal domain of SASH1 (SASH1-ΔC terminal) was shown to bind to endogenous MAP4K4 (Fig. 2F and Fig. 2G). In addition, we observed co-localization between endogenous SASH1 and endogenous MAP4K4 in T47D and MCF-7 cells (Fig. 2H and Fig. 2I).

SASH1 expression is positively correlated with MAP4K4 expression and downregulation of SASH1 is positively associated with increased phospho-MAP4K4 levels in breast cancer.

Since SASH1 was shown to bind to MAP4K4, the expression of MAP4K4 and the relationship between SASH1 and MAP4K4 levels in breast cancers were investigated. A total of 284 carcinoma specimens of breast cancer and 79 normal breast tissues including tissues of breast fibroadenoma and hyperplasia of mammary glands were collected to analyze the relationship between SASH1 and MAP4K4 in normal breast tissues and breast cancer tissues. MAP4K4 expression was detected predominantly in the cytoplasm and on the plasma membrane in some cases. In four breast cancer subtypes especially the luminal and HER2 positive subtypes as well as normal breast tissues,SASH1 was significantly associated with MAP4K4. Statistical analysis of IHC scores of SASH1 and MAP4K4 revealed that SASH1 was obviously correlated with MAP4K4 in normal breast tissues (Fig. 3A). In the subtypes of luminal A, luminal B and HER2 positive (Fig. 3B, Fig. 3C and Fig. 3D) SASH1 was obviously correlated with MAP4K4. However,SASH1 was also correlated with MAP4K4 in TNBC subtype (Fig. 3E). Although MAP4K4 mRNA levels in TNBC cells were higher than in non-TNBC cells༌MAP4K4 protein levels were not higher than in non-TNBC cells[17]. Statistical analysis of IHC scores indicated that MAP4K4 expression of 284 breast carcinoma specimens was lower than that of 79 normal breast tissues(Fig. 3G and Fig. 3H). Western blot analysis indicated that in 10 pairs of breast cancer tissues and normal breast tissues, in most breast cancer specimens༌ downregulation of MAP4K4 was accompanied by decreased SASH1 levels (Fig. 3I). In addition, statistical analysis of IHC scores indicated that SASH1 expression was correlated with phospho-MAP4K4 levels in 75 breast cancer tissues (Fig. 3J). These IHC results revealed that in breast cancer tissues༌ SASH1 expression is correlated with MAP4K4 expression༌and increased phospho-MAP4K4 levels are correlated with SASH1 expression.

MAP4K4 and SASH1 co-regulate proliferation, migration and invasion of luminal subtype and HER2 positive subtype cell lines.

Since SASH1 expression is correlated with MAP4K4 expression in the luminal subtype and the HER2 positive subtype, we further assessed their roles in breast cancer tumorigenesis. We first performed loss-of-function studies using lentiviruses expressing two distinct short hairpin RNAs (shRNAs) against SASH1 to knockdown SASH1 in two luminal subtype cell lines, T47D and MCF-7. The distribution of G1, G2/M, and S-phases of T47D cells were determined by flow cytometry and SASH1 silencing increased the proportion of S-phase cells and decreased the proportion of G1-phase cells(Fig. 4A). Moreover, flow cytometry analyses indicated that SASH1 knockdown induced less proportion of early apoptotic cells(Fig. 4B). We further analyzed the effects of loss-of-function of SASH1 on the migration and invasion of T47D cells. Transwell assays revealed that migration and invasion of T47D cells were induced by SASH1 deletion (Fig. 4C and Fig. 4D). Flow cytometry analyses suggested that MAP4K4 knockdown increased the proportion of G2/M-phase cells and induced more proportion of early apoptotic cells (Fig. 4F and Fig. 4G). Transwell assays indicated that MAP4K4 knockdown inhibited the cell numbers of migrating and invasive cells when T47D cells were silenced by MAP4K4-shRNA lentivirus (Fig. 4H and Fig. 4I). We further co-silenced SASH1 and MAP4K4 and evaluated the effects of co-silencing of SASH1 and MAP4K4 on cell growth༌ migration and invasion of T47D. The cell cycle profiles indicated that although MAP4K4 deletion did not significantly change the inducement of SASH1 silencing to the increased proportion of S-phase cells༌MAP4K4 knockdown could significantly increase the proportion of early apoptotic cells(Fig. 4K and Fig. 4L). Meanwhile, MAP4K4 deletion significantly abolished the inducement of SASH1 silencing to enhance the migration and invasion ability of T47D cells (Fig. 4N and Fig. 4O). In the other luminal subtype cell line, MCF-7, SASH1 silencing also increased the proportion of S-phase cells and decreased the proportion of G1-phase cells(Fig.S2A)༌and SASH1 knockdown also induced decreased proportion of apoptotic cells of MCF-7 cells(Fig.S2B). The cell cycle profiles indicated that MAP4K4 deletion decreased the proportion of S-phase cells (Fig.S2C) and induced increased proportion of apoptotic cells of MCF-7 cells (Fig.S2D). Similarly, in the co-silenced MCF-7 cells, although MAP4K4 deletion did not significantly change the inducement of SASH1 silencing to the increased proportion of S-phase cells, MAP4K4 silencing significantly increased the proportion of early apoptotic cells (Fig.S2E and Fig.S2F).

In the HER2⁺ subtype cell line,SK-BR-3 cells, SASH1 was silenced by two pairs of specific SASH1-shRNA, and the distribution of G1,G2/M༌ and S-phases was determined by flow cytometry. SASH1 silencing increased the proportion of S-phase cells and decreased the proportion of G2/M-phase cells (Fig. S3A and Fig.S3C). Moreover༌ flow cytometry analysis revealed after SASH1 silencing༌a significantly smaller proportion of early apoptotic cells was observed (Fig. S3B and Fig.S3C). Specific MAP4K4-shRNAs were also introduced into SK-BR-3 cells; cell cycle analyses showed that the interference with MAP4K4 expression increased proportion of G2/M-phase cells and decreased the proportion of S-phase cells༌which suggested MAP4K4 silencing induced cell cycle arrest in the G2/M-phase (Fig. S3D and S3F). Flow cytometry analyses also demonstrated that a significantly higher proportion of early apoptotic cells were observed after MAP4K4 silencing (Fig. S3E and S3F). Co-silencing of SASH1 and MAP4K4 induced cell cycle arrest in the G2-phase and a decrease in the proportion of S-phase cells, which suggested that the increase in S-phase cells upon SASH1 silencing in SK-BR-3 cells was prevented by MAP4K4 silencing༌and MAP4K4 silencing resulted in G2/M-phase cell cycle arrest(Fig. S3G and S3I). In addition, increased proportion of apoptotic cells was induced by co-silencing of SASH1 and MAP4K4 (Fig. S3H and S3I).

Loss-of-function of MAP4K4 impairs the ability of SASH1 silencing to promote cell proliferation and metastasis of the xenograft of ER + breast cancer cells.

We further identified the effects of loss-of-function of SASH1 and/or MAP4K4 on T47D cells xenograft. T47D cells with stable SASH1 knockdown, MAP4K4 knockdown and co-silencing of SASH1 and MAP4K4 were cultured and injected subcutaneously into the left side fossa axillaris of BALB/c female nude mice. MAP4K4 deletion inhibited the tumor growth including tumor size and tumor weight in the xenograft mouse model with MAP4K4 silencing and co-silencing of SASH1 and MAP4K4 (Fig. 5A, 5B, 5C and 5D). We also evaluated the influence of silencing of SASH1 and/or MAP4K4 on the metastasis of T47D cells xenograft. SASH1 silencing induced more liver metastatic lesions in the xenograft mouse models with SASH1 silencing compared with those in the xenograft mouse models with negative control(NC), MAP4K4 silencing and co-silencing of SASH1 and MAP4K4(Fig. 5E). We further identified the micro-metastases of liver using H&E staining by counting the number of metastatic lesions in the liver tissues of each mouse. H&E staining revealed that knockdown of SASH1 substantially enhanced the invasiveness of the T47D derived xenograft tumors(Fig. 5F and Fig. 5G). MAP4K4 deletion did not significantly reduce the number of liver metastatic lesions, however, MAP4K4 deletion could significantly reduce the increased numbers of liver metastatic lesions induced by SASH1 silencing (Fig. 5F and Fig. 5G). Furthermore, pulmonary micro-metastases were examined by H&E staining and the total number of pulmonary metastatic lesions in each mouse were counted. The results showed that more and larger pulmonary micro-metastases were detected in the mice model with SASH1-depleted T47D cells than in the control mice. Although MAP4K4 deletion did not significantly reduce the number of pulmonary metastatic lesions, MAP4K4 deletion could significantly reduce the increased numbers of liver metastatic lesions induced by SASH1 silencing (Fig. 5H and Fig. 5I). We further evaluated the influence of SASH1 and/or MAP4K4 silencing on the molecular index of proliferation and metastasis. MAP4K4 knockdown in vivo reversed the SASH1 downregulation induced by SASH1 silencing in the T47D cells-xenograft tumors co-silenced with SASH1 and MAP4K4-shRNA lentivirus, and MAP4K4 silencing upregulated SASH1 expression in the xenograft tumors silenced with MAP4K4-shRNA lentivirus(Fig. 6A). SASH1 silencing induced upregulated expression of Ki67 and increased numbers of Ki67-positive cells in the xenograft tumors silenced with SASH1- shRNA lentivirus. MAP4K4 knockdown abolished the inducement of Ki67 expression and increased Ki67-positive cells by SASH1 silencing in the xenograft tumors co-silenced with the lentivirus of SASH1-shRNA and MAP4K4-shRNA(Fig. 6C). However ,SASH1 silencing did not change the expression of caspase 9༌MMP2 and MMP9 in the T47D cells-xenograft tumors silenced with SASH1-shRNA lentivirus(data not shown).

SASH1 acts as a protein kinase and is phosphorylated by MAP4K4.

To identify the reason that SASH1 is downregulated in most of tumors and tumor cells, The phosphorylation modification sites on SASH1 were identified by LC- MS/MS analysis. Ser355, Ser359, Ser914 and Ser918 are potential phosphorylation modification sites (Fig. S4A and S4B). Actually, previous report has suggested that Ser90 of SASH1 is the phosphorylation modification site of SASH1[11]. We further investigated whether SASH1 is phosphorylated by MAP4K4 and which serine sites of SASH1 were the key phosphorylation sites. SASH1 was identified to be a very unstable protein and exogenous SASH1 and endogenous MAP4K4 began to degrade after cycloheximide (CHX) treatment (Fig. 7A). Decreased exogenous SASH1 expression and a backward shift of the SASH1 protein bands in GFP-SASH1-transfected SK-BR-3 cells were observed after treatment with the serine/threonine phosphatase inhibitor calyculin A (Fig. 7B), which indicates that SASH1 is a novel serine protein kinase. Calyculin A-induced SASH1 degradation and calyculin A-induced MAP4K4 degradation were blocked upon the proteasome inhibitor MG132(Fig. 7C), which indicated that MAP4K4 and SASH1 are both phosphorylated. GNE-495 is a potent and selective MAP4K4 inhibitor and inhibit MAP4K4 function in vivo[24]. To identify the inhibitory effects of GNE-495 to MAP4K4 in breast cancer cells and investigate whether GNE-495 can block the phosphorylation of MAP4K4. SK-BR-3 cells were treated with different concentrations of GNE-495 and western blot showed that 100 nM GNE-495 could efficiently inhibit phospho-MAP4K4 expression and upregulated the protein levels of endogenous MAP4K4 and SASH1 (Fig. 7D). Treatment with GNE-495 not only increased expression of SASH1 and MAP4K4 but also enhanced bindings of SASH1 to MAP4K4 (Fig. 7E). The calyculin A- induced decreases in phosphorylation of exogenous SASH1 and exogenous MAP4K4 were abolished by GNE-495 treatment (Fig. 7F). The SASH1 phosphorylation sites Ser355, Ser359, Ser914, and Ser918 are highly conserved, as analyzed by Clustal X2 software(Fig. 7G). Immunoblot analysis indicated that Ser359 and Ser914 are the key phosphorylation sites of SASH1 because the mutations of S359A and S914A caused little changes in SASH1 expression after GNE-495 treatment as compared with wild type SASH1 and other SASH1 mutants. Especially S914A caused a forward shift of the SASH1 protein band (Fig. 7H, 7I and 7J). IP-WB revealed that these two mutations increased binding of SASH1 to MAP4K4༌ and GNE-495 not only promoted the binding of wild type SASH1 but also of mutated SASH1 to MAP4K4 (Fig. 7K).

GNE-495 may reverse MAP4K4-mediated phosphorylation of SASH1 and upregulates both protein levels of MAP4K4 and SASH1.

MAP4K4 over-expression downregulated endogenous SASH1 expression in hormone- dependent breast cancer cell lines,SK-BR-3 and MCF-7 cells(Fig. 8A). Exogenous SASH1 was upregulated upon MAP4K4 knockdown (Fig. 8B). Bioinformatics analysis by Clustal X2 software indicated that Ser629, Ser631 and Ser801 of MAP4K4 are highly conserved phosphorylation sites (Fig. 8C). Western blot analysis suggested that the S801A mutation of MAP4K4 induced a forward shift of the MAP4K4 protein bands༌ and unlike exogenous SASH1 was upregulated after the phosphorylation inhibition of GNE-495 treatment to MAP4K4༌S801A mutation of MAP4K4 could not upregulate or attenuated the expression of exogenous SASH1 in SK-BR-3 and HEK-293T cells (Fig. 8D and Fig. 8E). In addition, the S801A mutation of MAP4K4 not only induced a forward shift of MAP4K4 main bands but also caused the appearance of multiple SASH1protein (Fig. 8D and Fig. 8E). Similarly, the S801A mutation in MAP4K4 attenuated the GNE-495-induced-endogenous SASH1 upregulation (Fig. 8F and Fig. 8G). Further IP-WB analyses revealed that S801A mutation in MAP4K4 attenuated the bindings of MAP4K4 to SASH1 compared with wild type MAP4K4 (Fig. 6H) in vivo. We performed in vitro kinase assay to identify the phosphorylation of MAP4K4 to SASH1. GNE-495 not only upregulates the expression of wild type MAP4K4 but also that of S801A-MAP4K4. Being similar to the in vivo results indicated above, GNE-495 promoted the bindings of wild type MAP4K4 to his-SASH1, however, attenuated the association S801A- MAP4K4 tohis-SASH1 in vitro (Fig. 6I). In addition༌ GNE-495 treatment caused a significant apoptosis in hormone-dependent breast cancer cells and MAP4K4 deletion can impair the induced apoptosis by GNE-495(supplementary Fig.S5).

Although growing evidence suggests that SASH1 is a tumor suppressor in breast cancer[25] [2, 26, 27], the mechanism by which SASH1 regulates tumor behaviors including tumorigenesis,adhesion and migration on cancer cells in cancer development and progression remain completely unclear[25]. Recent evidence reveal that SASH1 might be phosphorylated by LATS1 and LATS2[12, 28]. In this study, we firstly reveal that SASH1 acting as a novel protein kinase is phosphorylated by MAP4K4, to constitute a novel MAP4K4-SASH1 phosphokinase cascade to mediate tumorigenesis and metastasis of hormone-dependent breast cancer. Taken the previous findings into consideration, a MAP4K4-LATS1/LATS2-SASH1 signal cascade may mediate the tumorigenesis and metastasis of breast cancer. Functionally༌MAP4K4 existence is necessary to SASH1. MAP4K4 deletion will abolish the SASH1-induced tumorigenesis and metastases in vitro and in vivo. Importantly, GNE-495 specifically inhibits MAP4K4 phosphorylation and not only upregulate MAP4K4 protein level but also that of SASH1. GNE-495 by preventing the phosphorylation of the MAP4K4-SASH1 phosphorylation cascade might be a novel effective inhibitor, inhibits tumorigenesis of hormone-dependent breast cancer. Therefore, our findings firstly reveal a novel kinase cascade that can be targeted by small molecular compounds to treat hormone-dependent breast cancers. Previous reports have revealed that Ser90 in SASH1 serves as the 14-3-3-binding sites to induce SASH1 phosphorylation by phosphatidylinositol 3-kinase and MAPK/p90RSK signaling [17] and Ser407 in SASH1 is the key serine site for LATS1-mediated phosphorylation of SASH1[12]. Ser 914 in SASH1 in the present study is identified to be another serine site for MAP4K4-mediated phosphorylation of SASH1.

The serine/threonine kinase MAP4K4 is a member of the Ste20p (sterile 20 protein) family and increasing evidence suggests that MAP4K4 also plays an important role in cancer[29]. Current evidence suggests that MAP4K4 potentially serves as a negative prognostic indicator in patients with colorectal cancer[30], hepatocellular carcinoma [31], pancreatic ductal adenocarcinoma [32], lung adenocarcinoma [33] and prostate cancer [34]. MAP4K2, MAP4K4 and MAPK1 have been identified as kinases that mediate paracrine growth signaling in ER-negative breast cancers[35]. In this study, our findings indicate that in breast cancer, especially the luminal A and TNBC subtypes, MAP4K4 is downregulated compared to normal breast tissues. Generally, cell apoptosis[31, 36–39], cell cycle arrest[31, 38, 39], and migration and invasion [40] [38, 39, 41, 42] are induced by MAP4K4 downregulation in cancer cells. In this study, MAP4K4 silencing in induces cell cycle arrest, apoptosis and block migration and invasion in hormone-dependent breast cancer cell lines and T47D cells-xenograft tumors. MAP4K4, in a kinase activity-dependent manner, positively regulate cell transformation and invasion and negatively regulates cell spreading and adhesion[13]. MAP4K4 physically interacts with both LATS1 and LATS2 and phosphorylates LATS1 and LATS2 [28] and SASH1 was phosphorylated by LAST1[12]. Our findings indicated that MAP4K4 physically interacts with SASH1 and MAP4K4 may indirectly phosphorylate SASH1. However, MAP4K4 deletion will impair the inducement of loss-of-function of SASH1 to tumorigenesis,adhesion and migration of hormone-dependent breast cancer in breast cancer cell lines and xenograft tumors. So functionally, the existence of MAP4K4 is necessary to SASH1 and there may be a balance of expression levels between MAP4K4 and SASH1. Ser801 in MAP4K4 are identified might to be the key sites for MAP4K4-mediated phosphorylation of SASH1.

Given the diverse roles of MAP4K4 in many cell processes, the therapeutic potential of MAP4K4 inhibition will be appealing and it's worth identifying the MAP4K4 inhibitor that would function in vivo[24]. Some of the inhibitors show promise in treating pathological angiogenesis in mice[24, 43]. MAP4K4 may represent a novel actionable cancer therapeutic target. Whether these inhibitors possess potent antitumor properties remains to be determined[29]. In this study, GNE-495 not only inhibits phospho-MAP4K4 protein level and upregulates MAP4K4 but also increases SASH1 protein level, which eventually induce the apoptosis of hormone-dependent breast cancer cells.

Therefore, our findings firstly reveal a novel SASH1-MAP4K4 phosphoprotein kinase cascade that may serve as a therapeutic intervention to treat hormone-dependent breast cancers.

BTICs: Breast tumor-initiating cells; CHX: Cycloheximide; DMEM: Dulbecco's modified eagle medium; HER2: Human epidermal growth factor receptor 2;

HGK: Hepatocyte progenitor kinase-like/germinal center kinase-like kinase; IF: Immunofluorescence; IP-WB: Immunoprecipitation- western blot; LATS1: Large tumor suppressor kinase 1; LATS2: Large tumor suppressor kinase 2; MAPK : Mitogen-activated protein kinase kinase; MAP4K4: Mitogen-activated protein kinase kinase kinase kinase 4; NIK: Nck-interacting kinase; SASH1: SAM and SH3 domain containing protein 1; TNBC: Triple negative breast cancer.

Acknowledgements

Not applicable.

Authors' Contributions

Conception and design: D.A. Zhou, X.H. Zeng; Development of methodology: D.A. Zhou, Y.D. Li; Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y.D. Li，D.Q. Wu, J. Hou，X. Zeng, L. Chen, X. Wan, J. Zhang, Z.X Wu， K. Wang, Y. Wang , D. Yang, H.Y Chen，Z.X. Xu，L. Jia，Q.F. Liu, J. Wang，Z.S Kuang， G.L Jiang, H. Zhang, J. Luo, W. Li, X. Zou, X.H Zeng; Analysis and interpretation of data (e.g., statistical analysis, biostatistics,

computational analysis): D.A. Zhou, Y.D. Li; Writing, review, and/or revision of the manuscript: D.A. Zhou, X.H. Zeng; Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.A. Zhou, X.H. Zeng, J. Hou.

Funding

This work was supported by the National Natural Science Foundation Project of China (grant numbers: 31860319 and 32060165 to D.A. Zhou，and grant number: 81702640 and 81660439 to J. Hou) and the Guizhou Provincial Natural Science Foundation (grant numbers: Qian Ke He Zhicheng [2020]4Y125 to D.A. Zhou) , the Science and technology bureau of Chongqing (grant number:cstc2018jxjl130044) to X.H. Zeng ) , and the Guizhou Province Education Department Youth Science and Technology Talent Growth Project (No. [2016]143) to X.Zou).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

This study was approved by the Institutional Ethics Committee of the Affiliated Hospital of Guizhou Medical University and Chongqing Cancer Hospital (authorization number: 2018 lunshen No.059).

Consent for publication

Informed consent was obtained from all individual participants included in

the study.

Competing interests

The authors declare that they have no conflicts of interest.

Author details

¹Clinical Research Center, the Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, 550004,P.R. China. ² Clinical College, Guizhou Medical University, Guiyang, Guizhou, 550004, P.R. China. ³School of Clinical Laboratory Science, Guizhou Medical University, Guiyang,Guizhou, 550004, P.R. China. ⁴ Breast Cancer Surgery Department, Guizhou Provincial People’s Hospital. Guiyang,Guizhou, 550001, P.R. China. ⁵ The Fifth Clinical College, Chongqing Medical University, Chongqing, 400010, China. ⁶School of Biology and Engineering, Guizhou Medical University, Guiyang,Guizhou, 550004, P.R. China. ⁷Department of Laboratory Medicine, Yongchuan District Hospital, Chongqing, 402160, P.R. China. ⁸ Zhongshan Hospital, Fudan University, Shanghai, 200032, China. ⁹Chongqing University Cancer Hosiptal & ChongqingCancer Institute & Chongqing cancer hospital, Chongqing, 400030, P.R. China. # These authors contribute equally to these work.

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20201009FigureS1.jpg
Figure S1 IHC analyses reveal that SASH1 expression is correlated with those of ER, PR, p53 and Ki67.
A Expression of SASH1 is inversely correlated with nucleus ER in human breast cancer arrays and vice versa. The 139 subjects were divided into two groups based on SASH1 expression scores in the tumors, representing low (mean scores≤6.0216) and high (scores＞6.0216). The percentage of ER positive or ER negative expression in the two groups of subjects was analyzed using Pearson’s χ2 test. B Expression of SASH1 is inversely correlated with cytoplasm ER in human tissue arrays containing 73 breast cancer specimens and vice versa. C Expression of SASH1 is negatively correlated with nucleus PR in human tissue array containing138 breast cancer specimens and vice versa. D Expression of SASH1 is inversely correlated with cytoplasm PR in 74 breast cancer specimens and vice versa. E Expression of SASH1 is not correlated with HER2 in 139 breast cancer specimens and vice versa. F Expression of nucleus p53 positively correlates with SASH1 in human tissue arrays containing 139 breast carcinoma specimens. The 139 subjects were divided into two groups based on SASH1 expression scores in the tumors, representing low (scores≤6.0216) and high (scores＞6.0216). The percentage of nucleus p53 low expression(scores≤3.018) or high (scores＞3.018)in the two groups of subjects was analyzed using Pearson’s χ2 test. G Expression of SASH1positively correlates with cytoplasm p53 in 55 human breast carcinoma tissue arrays and vice versa. H Expression of SASH1 is positively correlated with Ki67 in 138 breast cancer arrays and vice versa. I Expression of SASH1 is not correlated with cytoplasm EGFR in 117 breast cancer arrays and vice versa. J Expression of SASH1 is not correlated with cytomembrane EGFR in 73 breast cancer arrays and vice versa. K Expression of SASH1 is not correlated with CK5/6 in breast cancer arrays.
20201009FigureS2.jpg
Figure S2 MAP4K4 deletion impairs the proliferation inducement of is co-regulated by MAP4K4 and SASH1 in MCF-7 cells. A An increased proportion of S-phase cells in MCF-7 cells was induced by SASH1 silencing. MCF-7 stable cells were starved for 72hr and subjected to analyze the cell cycle distribution using flow cytometry assays . B SASH1 silencing blocked the apoptosis in MCF-7 cells. Flow cytometry assays indicated that SASH1 silencing caused a significant decrease in the proportion of apoptotic cells. C MAP4K4 knockdown caused a G1-phase arrest in MCF-7 stable cells. D MAP4K4 deletion significantly increased the proportion of apoptotic cells in MCF-7 stable cells. E The increase in S-phase cells and decreasing proportion of apoptotic cells induced by SASH1 silencing in T47D cells were abolished by MAP4K4 silencing and the subsequent G1-phase arrest and the significant proportion of apoptotic cells were observed. These data were representative results of at least 3 repeated experiments with similar trends.
20201009SupplementaryFigure3.jpg
Figure S3 The Growth of HER2 positive breast cancer cells is co-regulated by MAP4K4 and SASH1 . A and C SASH1 silencing by shRNA induced an increase in the proportion pf S- phase cells in SK-BR-3 cells. SK-BR-3 cells were starved for 24hr and transfected with two specific SASH1 shRNA vectors. The silencing efficiency of SASH1- shRNAs was examined by western blot. Flow cytometry assays were performed to analyze the cell cycle distribution of SK-BR-3 cells at appropriate time-points after transfection for 48 hr. B and C SASH1 silencing inhibited apoptosis in SK-BR-3 cells. SK-BR-3 cells were starved and transfected. Flow cytometry assays indicated that SASH1 silencing caused a significant increase in the proportion of apoptotic cells. D and F MAP4K4 knockdown shRNAs caused a G2/M-phase arrest in SK-BR-3 cells. SK-BR-3 cells were starved , transfected with two MAP4K4 shRNA vectors, and subjected to flow cytometry analysis of the cell cycle. The silencing efficiency of SASH1 shRNAs was examined by western blot. E and F MAP4K4 deletion significantly increased the proportion of apoptotic cells. G, H and I The increase in S-phase cells and decreasing proportion of apoptotic cells induced by SASH1 silencing in SK-BR-3 cells were abolished by MAP4K4 silencing and the subsequent G2/M-phase arrest and the significant proportion of apoptotic cells were observed. SK-BR-3 cells were starved for 24hr and transfected with two specific shRNA vectors of SASH1 and MAP4K4 and the silencing efficiency of shRNAs of SASH1 and MAP4K4 was examined by western blot. These data were representative results of at least 3 repeated experiments with similar trends.
20201009FigureS4.jpg
Figure S4 The phosphorylation sites of SASH1 were identified by LC-MS/MS assays. A The relevant information about two peptides containing phosphorylated serine resides in SASH1 was obtained by LC-MS/MS assays. B and C Mass spectrometric identification of Ser355, Ser359, Ser914 and Ser918 as phosphorylation sites in SASH1 protein.
20201028FigureS5.tif
Figure S5 MAP4K4 deletion impaired the induced apoptosis of hormone-dependent breast cancer cells by GNE-495 A T47D cells or T47D stably expressing MAP4K4-shRNA lentivirus cells were starved for at least 24hr and treated with 100 nM GNE-495 for 24hr. Cells were trypsinizated and subjected to apoptosis assays using flow cytometry. MAP4K4 silencing significantly abolished the apoptosis inducement by GNE-495. B Significant apoptosis of MCF-7 cells were induced by GNE-495 treatment. MCF-7 cells were starved for about 24hr and treated with 100 nM GNE-495,and were subjected to flow cytometry for apoptosis analyses.
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Characterization of a Novel MAP4K4-SASH1 Kinase Cascade Regulating Breast Cancer Tumorigenesis and Metastasis

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