LATS1 Promotes B-ALL Tumorigenesis by Regulating YAP1 Phosphorylation and Subcellular Localization.

OBJECTIVE: YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors; differentiating between these roles may depend on the YAP1 phosphorylation pattern. The specific function of YAP1 in B cell acute lymphoblastic leukemia (B-ALL), however, is currently unclear. Thus, in the present study, the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets. METHODS: The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting, quantitative real-time polymerase chain reaction, flow cytometry, immunostaining, and nude mouse subcutaneous tumorigenesis experiments. Gene expression levels of Hippo pathway-related molecules before and after verteporfin (VP) treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells. RESULTS: Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels. YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells; YAP1 was distributed in the nuclei in NALM6 cells. Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase. Before and after VP treatment, the expression of the upstream gene LATS1 was upregulated; its overexpression promoted YAP1-Ser127 phosphorylation. Further, YAP1 was distributed in the plasma. CONCLUSION: LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function; thus, VP, which targets this axis, may serve as a new therapeutic method for improving the outcomes for B-ALL patients.

LncRNA surfactant associated 1 activates large tumor suppressor kinase 1/Yes-associated protein pathway via modulating hypoxic exosome-delivered miR-4766-5p to inhibit lung adenocarcinoma metastasis.

LncRNA surfactant associated 1 (SFTA1P) exhibits low expression in non-small cell lung cancer (NSCLC) tissues as compared with that in adjacent tissues, and may play a suppressing role in NSCLC. However, the effect and mechanism of SFTA1P on the metastasis of lung adenocarcinoma (LUAD) remain undefined, which are thus investigated in this research. Herein, potential impacts of SFTA1P on LUAD were determined through the Cancer Genome Atlas (TCGA) database and Gene Expression Profiling Interactive Analysis (GEPIA). After knockdown/overexpression of SFTA1P, the metastatic ability of LUAD cells was evaluated by molecular biology experiments (cell counting kit-8 assay, scratch test, Transwell assay and Western blot). The effect of SFTA1P on Yes-associated protein (YAP) nuclear translocation was assessed by Western blot. Hypoxia-induced exosomes were extracted for LUAD metastasis analysis. The targeting relationship of SFTA1P/miR-4766-5p/large tumor suppressor kinase 1 (LATS1) was verified by dual-luciferase reporter assay and molecular biology experiments. Xenograft and lung metastasis models were constructed for in vivo validation. SFTA1P was lowly expressed in LUAD, which was associated with the poor prognosis of patients with LUAD. Up-regulated SFTA1P prevented the metastasis of LUAD cells and the nuclear translocation of YAP. Hypoxia-induced exosomes stimulated LUAD cell metastasis, but inhibited the SFTA1P and LATS1/YAP axes. MiR-4766-5p acted as an intermediate "bridge" for SFTA1P to regulate LATS1. SFTA1P repressed xenograft growth and LUAD cell metastasis. To sum up, SFTA1P activates hypoxic exosome-delivered miR-4766-5p through modulating LATS1/YAP pathway, thereby suppressing LUAD cell metastasis, which may serve as a suitable target for the LUAD therapy.

lncRNA TINCR attenuates the proliferation and invasion, and enhances the apoptosis of cutaneous malignant melanoma cells by regulating the miR­424­5p/LATS1 axis.

Cutaneous malignant melanoma (CMM) is responsible for ≥1/2 of skin cancer­related mortalities. The aberrant expression of long non­coding RNAs (lncRNAs) has been associated with the development of CMM. However, to the best of our knowledge, the role of the lncRNA TINCR ubiquitin domain containing (TINCR) in CMM has not been previously investigated, and thus, the current study aimed to evaluate this in vitro and in vivo. Reverse transcription­quantitative PCR (RT­qPCR) was used to analyze microRNA (miR)­424­5p expression, and RT­qPCR and western blotting were used to measure TINCR, large tumor suppressor kinase 1 (LATS1), cellular communication network factor 2 (CTGF), cellular communication network factor 1 (CCN1) and AXL receptor tyrosine kinase (AXL) mRNA and protein expression levels, respectively. Cell Counting Kit­8, flow cytometry and Transwell assays were used to detect the proliferation, apoptosis and invasion of CMM cell lines, respectively. The binding sites between TINCR and miR­424­5p were predicted using the miRDB database. A dual luciferase reporter assay and RT­qPCR were used to identify the relationship between TINCR and miR­424­5p in CMM cell lines. The bioinformatics analysis revealed that TINCR was one of the most significantly downregulated lncRNAs in CMM, and advanced stage CMM tissues showed the greatest decrease in TINCR expression. Moreover, in the collected CMM tissues and tested cell lines of the current study, TINCR expression was found to be downregulated compared with the respective controls. Notably, TINCR overexpression inhibited the expression levels of CTGF, CCN1 and AXL, decreased the proliferation and invasion, and induced the apoptosis of CMM cell lines. In addition, a mutual binding association was identified between miR­424­5p and TINCR in CMM cells. LATS1, a target of miR­424­5p, was found to be positively regulated by TINCR. TINCR activated Hippo signaling and repressed the activity of Yes 1 associated transcriptional regulator by regulating LATS1 expression, while LATS1 knockdown reversed the effect of TINCR overexpression on CMM cells. Collectively, the findings of the present study suggested that TINCR may attenuate the progression of CMM by regulating the miR­424­5p/LATS1 signaling axis. These results indicated that TINCR may play a tumor suppressive role in CMM.

Identification of the tumor­suppressive role of circular RNA­FOXO3 in colorectal cancer via regulation of miR­543/LATS1 axis.

Colorectal cancer (CRC) is a common malignancy with significant prevalence and mortality rates. Circular RNA FOXO3 (circ­FOXO3; hsa_circ_0006404) has been reported to be involved in cancer regulation; however, its role in CRC is yet to be fully elucidated. Therefore, the aim of the present study was to investigate the effect of circ­FOXO3 on CRC progression and identify its underlying mechanism. In the present study, the expression of circ­FOXO3 was investigated in CRC tissues and cells via reverse transcription­quantitative PCR. A Cell Counting Kit­8 and colony formation assays were used to assess cell proliferation. The cell migratory and invasive abilities were detected using the Transwell migration and invasion assays. The luciferase assay and RNA pull­down assay were conducted to verify the relationship of circ­FOXO3, microRNA (miR)­543 and Large tumor suppressor kinase 1 (LATS1). The results demonstrated that circ­FOXO3 expression was downregulated in CRC tissues and cells, and was associated with poor overall survival of patients with CRC. Moreover, circ­FOXO3 was associated with tumor size, distant metastasis, differentiation, lymph node metastasis and TMN stages of patients with CRC. circ­FOXO3 overexpression suppressed CRC cell proliferation, migration and invasion. Luciferase assay and RNA pull­down assay results indicated that circ­FOXO3 functioned as a sponge for miR­543. In addition, circ­FOXO3 increased the expression of LATS1 via sponging miR­543, thus inhibiting CRC cell aggressive features. Collectively, the present results suggested that circ­FOXO3 inhibited CRC metastasis and progression via elevated LATS1 expression by sponging miR­543. Therefore, circ­FOXO3 may be a promising target for CRC therapy.

Deubiquitinase JOSD2 stabilizes YAP/TAZ to promote cholangiocarcinoma progression.

Cholangiocarcinoma (CCA) has emerged as an intractable cancer with scanty therapeutic regimens. The aberrant activation of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are reported to be common in CCA patients. However, the underpinning mechanism remains poorly understood. Deubiquitinase (DUB) is regarded as a main orchestrator in maintaining protein homeostasis. Here, we identified Josephin domain-containing protein 2 (JOSD2) as an essential DUB of YAP/TAZ that sustained the protein level through cleavage of polyubiquitin chains in a deubiquitinase activity-dependent manner. The depletion of JOSD2 promoted YAP/TAZ proteasomal degradation and significantly impeded CCA proliferation in vitro and in vivo. Further analysis has highlighted the positive correlation between JOSD2 and YAP abundance in CCA patient samples. Collectively, this study uncovers the regulatory effects of JOSD2 on YAP/TAZ protein stabilities and profiles its contribution in CCA malignant progression, which may provide a potential intervention target for YAP/TAZ-related CCA patients.

Knockout of LATS1 induces neoplastic phenotype in hepatic oval cells.

BACKGROUND: Primary liver cancer (PLC) is the second leading cause of cancer-related death worldwide. It has been reported that PLC can be originated from malignant transformed adult hepatic progenitor cells. Mammalian large tumor suppressor kinase 1 (LATS1) is one of the core components of the Hippo pathway and it has been implicated in regulating invasion and metastasis of different cancer cell. However, the underlying connections between hepatic progenitor cells and LATS1 in the pathogenesis of PLC are still elusive. METHODS: LATS1 gene knockout (LATS1-KO) hepatic oval cells (HOCs) were constructed by the CRISPR/Cas9 system. Cell viability was evaluated by the CCK-8 assay. Cell migration was measured by scrape assay. Cell invasion was examined by Transwell assay. Cell apoptosis was evaluated by flow cytometry. The expression of LATS1 and Yes-associated protein (YAP) in HOCs was determined by Q-PCR and Western blot analysis. RESULTS: Here, we found that knockout of LATS1 significantly induced the migration and invasion of WB-F344 cells. Knockdown of YAP suppressed the neoplastic phenotype of LATS1-KO WB-F344 cells. Furthermore, overexpression of YAP promoted the migration and invasion of LATS1-KO WB-F344 cells. CONCLUSIONS: In summary, the current study demonstrated that LATS1 is required for inhibiting the neoplastic phenotype of normal hepatic progenitor cell via downregulating YAP.

Expressions of large tumor suppressor kinase 1 and large tumor suppressor kinase 2 in gastric cancer and their significances / 肿瘤研究与临床

Objective To investigate the expressions of large tumor suppressor kinase 1 (LATS1) and large tumor suppressor kinase 2 (LATS2) proteins in gastric cancer tissues, and to explore the correlation between expressions of LATS1 and LATS2 proteins and the occurrence and development of gastric cancer. Methods A total of 93 gastric cancer paraffin tissues and the corresponding adjacent gastric normal mucosa in the Department of Pathology in Baotou Cancer Hospital from September 2008 to June 2010 were collected. The immunohistochemistry was used to detect the expressions of LATS1 and LATS2 proteins in gastric cancer and adjacent normal tissues. The differences of the expressions of LATS1 and LATS2 proteins in gastric cancer and adjacent normal tissues were compared by usingχ2 test. The relationship between the expressions of LATS1 and LATS2 proteins and the clinicopathological features was also analyzed. Results In gastric cancer tissues, LATS1 was negatively expressed in 54 cases (58.1％), weakly positive expressed in 15 cases (16.1％), moderately positive expressed in 16 cases (17.2％), and strongly positive expressed in 8 cases (8.6％);in adjacent normal tissues, LATS1 was negatively expressed in 17 cases (18.3％), weakly positive expressed in 16 cases (17.2％), moderately positive expressed in 31 cases (33.3％), and strongly positive expressed in 29 cases (31.2％). The positive expression rate of LATS1 in gastric cancer tissues was lower than that in adjacent normal tissues, and the difference was statistically significant (χ2=37.460, P<0.01). In gastric cancer tissues, LATS2 was negatively expressed in 28 cases (30.1％), weakly positive expressed in 17 cases (18.3％), moderately positive expressed in 33 cases (35.5％), strongly positive expressed in 15 cases (16.1％);in adjacent normal tissues, LATS2 was negatively expressed in 5 cases (5.4％), weakly positive expressed in 7 cases (7.5％), moderately positive expressed in 32 cases (34.4％), strongly positive expressed in 49 cases (52.7％). The positive expression rate of LATS2 in gastric cancer tissues was lower than that in adjacent normal tissues, and the difference was statistically significant (χ2=38.275, P<0.01). The expressions of LATS1 and LATS2 were not related to patients'age, gender, lymph node metastasis, degree of differentiation and tumor diameter (all P>0.05). Conclusion LATS1 and LATS2 proteins may be involved in the occurrence of gastric cancer and have the inhibiting effect on the occurrence and development of gastric cancer.

Progress of large tumor suppressor kinase in tumors / 肿瘤研究与临床

Large tumor suppressor kinase 1 (LATS1) and LATS2 are the kinases found in recent years which can inhibit the tumor development. They are highly homologous and overlap in function. LATS1 and LATS2 proteins have been confirmed to be associated with the occurrence of multiple malignancies, including soft tissue sarcoma, leukemia, astrocytoma, breast cancer, prostate cancer, lung cancer, liver cancer, esophageal cancer, etc. Studies have shown that LATS has more extensive biological functions, such as regulating gene transcription and cell cycle checkpoint, inducing apoptosis, inhibiting cell migration, maintaining genetic stability and regulating organ size, and loss of any one can cause a variety of biological changes. So insighting into the structure and mechanism of LATS1 and LATS2 is the key to understanding the occurrence and development of tumors. The discovery of LATS gene and the structure, expression, target and function of LATS protein are summarized in this paper.

Role of Hippo pathway in autosomal dominant polycystic kidney disease / 中华肾脏病杂志

Objective To explore the role of Hippo pathway in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD),and find potential targets for drug therapy.Methods By means of immunofluorescence staining,Western blotting,Real-time PCR,the differences of sublocalization,expression and phosphorylation level about Hippo pathway molecules in Han:SPRD (cy/+) and ADPKD patients compared with the control were observed.Knockdown Yes kinaseassociated protein (YAP),transcriptional coactivator with PDZ binding motif (TAZ) and large tumor suppressor kinase1 (LATS1) in cystic lining epithelium cell line WT9-12 were took by siRNA interference,and then their effects on cell proliferation,apoptosis and cell cycle were assessed.Results In cystic lining epithelium of Han:SPRD(cy/+),decreased expression of LATS1 and increased expression of YAP were found compared with the control,and the immunofluorescence of YAP was distributed both in cytoplasm and nucleus,while distribution and expression level of TAZ were without significant variance.Abnormal mRNA expressions of Hippo pathway components in ADPKD patients were found (P ＜ 0.05).Down-regulation of LATS1 in WT9-12 cells could prohibit phosphorylation of YAP,and prompted proliferation and cell division.Knockdown YAP in WT9-12 cells could inhibited cell proliferation by arresting cell cycle in G0/G1 phase,but down-regulating TAZ showed no significant differences in proliferation and cell cycle.Conclusions Altered Hippo signaling exists in ADPKD,and YAP activation may be one leading cause of autosomal dominant polycystic kidney disease onset.In vitro,knockdown YAP in WT9-12 cells can inhibit cell proliferation by arresting cell cycle and depressing cell division,suggesting the expression level and activity of YAP are potential targets for ADPKD treatment.

Arsenic-induced cutaneous hyperplastic lesions are associated with the dysregulation of Yap, a Hippo signaling-related protein.

Arsenic exposure in humans causes a number of toxic manifestations in the skin including cutaneous neoplasm. However, the mechanism of these alterations remains elusive. Here, we provide novel observations that arsenic induced Hippo signaling pathway in the murine skin. This pathway plays crucial roles in determining organ size during the embryonic development and if aberrantly activated in adults, contributes to the pathogenesis of epithelial neoplasm. Arsenic treatment enhanced phosphorylation-dependent activation of LATS1 kinase and other Hippo signaling regulatory proteins Sav1 and MOB1. Phospho-LATS kinase is known to catalyze the inactivation of a transcriptional co-activator, Yap. However, in arsenic-treated epidermis, we did not observed its inactivation. Thus, as expected, unphosphorylated-Yap was translocated to the nucleus in arsenic-treated epidermis. Yap by binding to the transcription factors TEADs induces transcription of its target genes. Consistently, an up-regulation of Yap-dependent target genes Cyr61, Gli2, Ankrd1 and Ctgf was observed in the skin of arsenic-treated mice. Phosphorylated Yap is important in regulating tight and adherens junctions through its binding to αCatenin. We found disruption of these junctions in the arsenic-treated mouse skin despite an increase in αCatenin. These data provide evidence that arsenic-induced canonical Hippo signaling pathway and Yap-mediated disruption of tight and adherens junctions are independently regulated. These effects together may contribute to the carcinogenic effects of arsenic in the skin.