Hippo cooperates with p53 to maintain foregut homeostasis and suppress the malignant transformation of foregut basal progenitor cells.

Basal progenitor cells serve as a stem cell pool to maintain the homeostasis of the epithelium of the foregut, including the esophagus and the forestomach. Aberrant genetic regulation in these cells can lead to carcinogenesis, such as squamous cell carcinoma (SCC). However, the underlying molecular mechanisms regulating the function of basal progenitor cells remain largely unknown. Here, we use mouse models to reveal that Hippo signaling is required for maintaining the homeostasis of the foregut epithelium and cooperates with p53 to repress the initiation of foregut SCC. Deletion of Mst1/2 in mice leads to epithelial overgrowth in both the esophagus and forestomach. Further molecular studies find that Mst1/2-deficiency promotes epithelial growth by enhancing basal cell proliferation in a Yes-associated protein (Yap)-dependent manner. Moreover, Mst1/2 deficiency accelerates the onset of foregut SCC in a carcinogen-induced foregut SCC mouse model, depending on Yap. Significantly, a combined deletion of Mst1/2 and p53 in basal progenitor cells sufficiently drives the initiation of foregut SCC. Therefore, our studies shed light on the collaborative role of Hippo signaling and p53 in maintaining squamous epithelial homeostasis while suppressing malignant transformation of basal stem cells within the foregut.

LHCGR and ALMS1 defects likely cooperate in the development of polycystic ovary syndrome indicated by double-mutant mice.

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder with evidence of polygenetic components, and obesity may be a risk factor for hyperandrogenism. Previous studies have shown that LHCGR is enriched in the ovary and LHCGR deficiency causes infertility without typical PCOS phenotypes. ALMS1 is implicated in obesity and hyperandrogenism, the common phenotypes among PCOS patients. Through whole-exome sequencing of 22 PCOS families and targeted candidate gene sequencing of additional 65 sporadic PCOS patients, we identified potential causative mutations in LHCGR and ALMS1 in a sibling-pair PCOS family and three sporadic PCOS patients. The expression of LHCGRL638P in granulosa-like tumor cell line (KGN) cells promoted cyclic adenosine monophosphate production and granulosa cell proliferation, indicating that LHCGRL638P is an activating mutation. LhcgrL642P/L642P mice showed an irregular estrous cycle, reduced follicles with dynamic folliculogenesis, and increased testosterone (T), estradiol (E2), and dehydroepiandrosterone. Lhcgr+/L642PAlms1+/PB mice displayed increased T and E2 but decreased late secondary and preovulatory follicles. We showed that activating mutation of LHCGR likely plays important roles in the pathophysiology of PCOS involving abnormal reproductive physiology, whereas ALMS1 deficiency may promote anovulatory infertility via elevated androgens, suggesting that the disturbed LHCGR and ALMS1 cooperatively induce PCOS phenotypes, characterized as anovulation and hyperandrogenemia frequently observed in PCOS patients with obesity.

Exosomal miR-199a-5p promotes hepatic lipid accumulation by modulating MST1 expression and fatty acid metabolism.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) and its complications has become an expanding health problem worldwide with limited therapeutic approaches. The current study was aiming to identify novel microRNA in the regulation of hepatic lipid metabolism in NAFLD. APPROCHES AND RESULTS: Systematic screening of microRNA expression by high-throughput small RNA sequencing demonstrated that microRNA 199a-5p (miR-199a-5p) was significantly upregulated in high fat diet-induced steatosis mouse model, with the most abundant expression in adipose tissue. MST1 was further identified as the target gene for miR-199a with specific recognition at the 3' untranslated region with dural luciferase reporter assay. Delivery of miR-199a-5p with exosomes into mice aggravated liver lipid accumulation in hepatocytes, accompanied by down-regulation of hepatic MST1 expression and modulation of hepatic lipogenesis and lipolysis, including SREBP-1c, AMPK signaling cascades and the down-stream CPT1α and FASN. Conversely, administration of exosome containing anti-miR-199a-5p resulted in attenuated steotosis in mice fed on high fat diet. Importanly, miR-199a-5p-induced abnormal cellular lipid accumulation could be markedly reversed by overexpression of MST1. CONCLUSION: miR-199a-5p might be an essentail regulator for hepatic lipid metabolism, possibly through its interction with MST1 and the subsequent signaling cascade. Thus, miR-199a-5p may serve as an important therapeutic target in the treatment of NAFLD.

DVL mutations identified from human neural tube defects and Dandy-Walker malformation obstruct the Wnt signaling pathway.

Wnt signaling pathways, including the canonical Wnt/ß-catenin pathway, planar cell polarity pathway, and Wnt/Ca2+ signaling pathway, play important roles in neural development during embryonic stages. The DVL genes encode the hub proteins for Wnt signaling pathways. The mutations in DVL2 and DVL3 were identified from patients with neural tube defects (NTDs), but their functions in the pathogenesis of human neural diseases remain elusive. Here, we sequenced the coding regions of three DVL genes in 176 stillborn or miscarried fetuses with NTDs or Dandy-Walker malformation (DWM) and 480 adult controls from a Han Chinese population. Four rare mutations were identified: DVL1 p.R558H, DVL1 p.R606C, DVL2 p.R633W, and DVL3 p.R222Q. To assess the effect of these mutations on NTDs and DWM, various functional analyses such as luciferase reporter assay, stress fiber formation, and in vivo teratogenic assay were performed. The results showed that the DVL2 p.R633W mutation destabilized DVL2 protein and upregulated activities for all three Wnt signalings (Wnt/ß-catenin signaling, Wnt/planar cell polarity signaling, and Wnt/Ca2+ signaling) in mammalian cells. In contrast, DVL1 mutants (DVL1 p.R558H and DVL1 p.R606C) decreased canonical Wnt/ß-catenin signaling but increased the activity of Wnt/Ca2+ signaling, and DVL3 p.R222Q only decreased the activity of Wnt/Ca2+ signaling. We also found that only the DVL2 p.R633W mutant displayed more severe teratogenicity in zebrafish embryos than wild-type DVL2. Our study demonstrates that these four rare DVL mutations, especially DVL2 p.R633W, may contribute to human neural diseases such as NTDs and DWM by obstructing Wnt signaling pathways.

Hippo Kinases Mst1 and Mst2 Sense and Amplify IL-2R-STAT5 Signaling in Regulatory T Cells to Establish Stable Regulatory Activity.

Interleukin-2 (IL-2) and downstream transcription factor STAT5 are important for maintaining regulatory T (Treg) cell homeostasis and function. Treg cells can respond to low IL-2 levels, but the mechanisms of STAT5 activation during partial IL-2 deficiency remain uncertain. We identified the serine-threonine kinase Mst1 as a signal-dependent amplifier of IL-2-STAT5 activity in Treg cells. High Mst1 and Mst2 (Mst1-Mst2) activity in Treg cells was crucial to prevent tumor resistance and autoimmunity. Mechanistically, Mst1-Mst2 sensed IL-2 signals to promote the STAT5 activation necessary for Treg cell homeostasis and lineage stability and to maintain the highly suppressive phosphorylated-STAT5+ Treg cell subpopulation. Unbiased quantitative proteomics revealed association of Mst1 with the cytoskeletal DOCK8-LRCHs module. Mst1 deficiency limited Treg cell migration and access to IL-2 and activity of the small GTPase Rac, which mediated downstream STAT5 activation. Collectively, IL-2-STAT5 signaling depends upon Mst1-Mst2 functions to maintain a stable Treg cell pool and immune tolerance.

Hdac7 promotes lung tumorigenesis by inhibiting Stat3 activation.

BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. However, the molecular mechanisms underlying lung cancer development have not been fully understood. The functions of histone deacetylases (HDACs), a class of total eighteen proteins (HDAC1-11 and SIRT1-7 in mammals) that deacetylate histones and non-histone proteins, in cancers are largely unknown. METHODS: Hdac7 +/-/K-Ras mice and HDAC7-depleted human lung cancer cell lines were used as models for studying the function of Hdac7 gene in lung cancer. Kaplan-Meier survival analysis was performed to explore the relationship between HDAC7 expression and prognosis of human lung cancers. Recombinant lentivirus-mediated in vivo gene expression or knockdown, Western blotting, and pull-down assay were applied to investigate the underlying molecular mechanism by which Hdac7 promotes lung tumorigenesis. RESULTS: The number and burden of lung tumor were dramatically reduced in Hdac7 +/-/K-Ras mice compared to control K-Ras mice. Also, in Hdac7 +/-/K-Ras mice, cell proliferation was significantly inhibited and apoptosis in lung tumors was greatly enhanced. Similarly, cell proliferation and anchorage-independent growth of human lung cancer cell lines expressing shHDAC7 were also significantly suppressed and apoptosis was dramatically elevated respectively. Mechanistic study revealed that Hdac7 mutation in mouse lung tumors or HDAC7 depletion in human tumor cell lines resulted in significantly enhanced acetylation and tyrosine-phosphorylation of Stat3 and HDAC7 protein directly interacted with and deacetylateed STAT3. The Hdac7 mutant-mediated inhibitory effects on lung tumorigenesis in mice and cell proliferation/soft agar colony formation of human lung cancer cell lines were respectively reversed by expressing dnStat3. Finally, the high HDAC7 mRNA level was found to be correlated with poor prognosis of human lung cancer patients. CONCLUSION: Our study suggests that Hdac7 promotes lung tumorigenesis by inhibiting Stat3 activation via deacetylating Stat3 and may shed a light on the design of new therapeutic strategies for human lung cancer.

Mst-1 deficiency promotes post-traumatic spinal motor neuron survival via enhancement of autophagy flux.

The mammalian Ste20-like kinase 1 (Mst-1) is a serine-threonine kinase and a component of the Hippo tumor suppressor pathway, which reacts to pathologically relevant stress and regulates cell death. However, little is known about its role in spinal cord injury. Here, we found that p-Mst-1, the activated form of Mst-1, was induced in the post-traumatic spinal motor neurons. In vivo evidence demonstrated that Mst-1 deficiency promoted post-traumatic spinal motor neuron survival, Basso mouse scale scores, and synapse survival. Moreover, we found that autophagosome formation and autolysosome degradation enhanced by Mst-1 deficiency were crucial to attenuate the death of injured spinal motor neurons. Taken together, our findings demonstrate that Mst-1 deficiency promotes post-traumatic spinal motor neuron survival via enhancement of autophagy flux.

Dendritic cell MST1 inhibits Th17 differentiation.

Although the differentiation of CD4+T cells is widely studied, the mechanisms of antigen-presenting cell-dependent T-cell modulation are unclear. Here, we investigate the role of dendritic cell (DC)-dependent T-cell differentiation in autoimmune and antifungal inflammation and find that mammalian sterile 20-like kinase 1 (MST1) signalling from DCs negatively regulates IL-17 producing-CD4+T helper cell (Th17) differentiation. MST1 deficiency in DCs increases IL-17 production by CD4+T cells, whereas ectopic MST1 expression in DCs inhibits it. Notably, MST1-mediated DC-dependent Th17 differentiation regulates experimental autoimmune encephalomyelitis and antifungal immunity. Mechanistically, MST1-deficient DCs promote IL-6 secretion and regulate the activation of IL-6 receptor α/ß and STAT3 in CD4+T cells in the course of inducing Th17 differentiation. Activation of the p38 MAPK signal is responsible for IL-6 production in MST1-deficient DCs. Thus, our results define the DC MST1-p38MAPK signalling pathway in directing Th17 differentiation.

Mammalian Sterile 20-like Kinase 1 (Mst1) Enhances the Stability of Forkhead Box P3 (Foxp3) and the Function of Regulatory T Cells by Modulating Foxp3 Acetylation.

Regulatory T cells (Tregs) play crucial roles in maintaining immune tolerance. The transcription factor Foxp3 is a critical regulator of Treg development and function, and its expression is regulated at both transcriptional and post-translational levels. Acetylation by lysine acetyl transferases/lysine deacetylases is one of the main post-translational modifications of Foxp3, which regulate Foxp3's stability and transcriptional activity. However, the mechanism(s) by which the activities of these lysine acetyl transferases/lysine deacetylases are regulated to preserve proper Foxp3 acetylation during Treg development and maintenance of Treg function remains to be determined. Here we report that Mst1 can enhance Foxp3 stability, its transcriptional activity, and Treg function by modulating the Foxp3 protein at the post-translational level. We discovered that Mst1 could increase the acetylation of Foxp3 by inhibiting Sirt1 activity, which requires the Mst1 kinase activity. We also found that Mst1 could attenuate Sirt1-mediated deacetylation of Foxp3 through directly interacting with Foxp3 to prevent or interfere the interaction between Sirt1 and Foxp3. Therefore, Mst1 can regulate Foxp3 stability in kinase-dependent and kinase-independent manners. Finally, we showed that treatment of Mst1(-/-) Tregs with Ex-527, a Sirt1-specific inhibitor, partially restored the suppressive function of Mst1(-/-) Tregs. Our studies reveal a novel mechanism by which Mst1 enhances Foxp3 expression and Treg function at the post-translational level.

STK4 regulates TLR pathways and protects against chronic inflammation-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is frequently associated with pathogen infection-induced chronic inflammation. Large numbers of innate immune cells are present in HCCs and can influence disease outcome. Here, we demonstrated that the tumor suppressor serine/threonine-protein kinase 4 (STK4) differentially regulates TLR3/4/9-mediated inflammatory responses in macrophages and thereby is protective against chronic inflammation-associated HCC. STK4 dampened TLR4/9-induced proinflammatory cytokine secretion but enhanced TLR3/4-triggered IFN-ß production via binding to and phosphorylating IL-1 receptor-associated kinase 1 (IRAK1), leading to IRAK1 degradation. Notably, macrophage-specific Stk4 deletion resulted in chronic inflammation, liver fibrosis, and HCC in mice treated with a combination of diethylnitrosamine (DEN) and CCl4, along with either LPS or E. coli infection. STK4 expression was markedly reduced in macrophages isolated from human HCC patients and was inversely associated with the levels of IRAK1, IL-6, and phospho-p65 or phospho-STAT3. Moreover, serum STK4 levels were specifically decreased in HCC patients with high levels of IL-6. In STK4-deficient mice, treatment with an IRAK1/4 inhibitor after DEN administration reduced serum IL-6 levels and liver tumor numbers to levels similar to those observed in the control mice. Together, our results suggest that STK4 has potential as a diagnostic biomarker and therapeutic target for inflammation-induced HCC.

The non-canonical Hippo/Mst pathway in lymphocyte development and functions.

The canonical Hippo/Mst pathway, originally discovered in Drosophila, is famous for its function in promoting apoptosis, inhibiting cell proliferation and tumorigenesis, and regulating tissue regeneration. However, emerging evidence shows that multiple non-canonical Hippo signaling pathways are also implicated in the regulation of various other biological processes. Recent studies have revealed that Mst1/2, the core kinases of Hippo/Mst pathway are required for T cell development, function, survival, trafficking, and homing, and also involved in regulation of autoimmunity. In this review, we discuss the roles of non-canonical Hippo/Mst signaling pathways in lymphocyte development and functions.

Mst1 and mst2 are essential regulators of trophoblast differentiation and placenta morphogenesis.

The placenta is essential for survival and growth of the fetus because it promotes the delivery of nutrients and oxygen from the maternal circulation as well as fetal waste disposal. Mst1 and Mst2 (Mst1/2), key components of the mammalian hpo/Mst signaling pathway, encode two highly conserved Ser/Thr kinases and play important roles in the prevention of tumorigenesis and autoimmunity, control of T cell development and trafficking, and embryonic development. However, their functions in placental development are not fully understood, and the underlying cellular and molecular mechanisms remain elusive. Here, we investigated the functions of Mst1/2 in mouse placental development using both conventional and conditional (endothelial) Mst1/2 double knockout mice. We found that the number of trophoblast giant cells dramatically increased while spongiotrophoblast cells almost completely disappeared in Mst1/2 deficient placentas. We showed that Mst1/2 deficiency down regulated the expression of Mash2, which is required for suppressing the differentiation of trophoblast giant cells. Furthermore, we demonstrated that endothelial-specific deletion of Mst1/2 led to impaired placental labyrinthine vasculature and embryonic lethality at E11.5, but neither affected vasculature in yolk sac and embryo proper nor endocardium development. Collectively, our findings suggest that Mst1/2 regulate placental development by control of trophoblast cell differentiation and labyrinthine vasculature at midgestation and Mst1/2 control labyrinth morphogenesis in trophoblast- and fetal endothelial-dependent manners. Thus, our studies have defined novel roles of Mst1/2 in mouse placental development.

MST1 is a key regulator of beta cell apoptosis and dysfunction in diabetes.

Apoptotic cell death is a hallmark of the loss of insulin-producing beta cells in all forms of diabetes mellitus. Current treatments fail to halt the decline in functional beta cell mass, and strategies to prevent beta cell apoptosis and dysfunction are urgently needed. Here, we identified mammalian sterile 20-like kinase-1 (MST1) as a critical regulator of apoptotic beta cell death and function. Under diabetogenic conditions, MST1 was strongly activated in beta cells in human and mouse islets and specifically induced the mitochondrial-dependent pathway of apoptosis through upregulation of the BCL-2 homology-3 (BH3)-only protein BIM. MST1 directly phosphorylated the beta cell transcription factor PDX1 at T11, resulting in the latter's ubiquitination and degradation and thus in impaired insulin secretion. MST1 deficiency completely restored normoglycemia, beta cell function and survival in vitro and in vivo. We show MST1 as a proapoptotic kinase and key mediator of apoptotic signaling and beta cell dysfunction and suggest that it may serve as target for the development of new therapies for diabetes.

Mst1/Mst2 regulate development and function of regulatory T cells through modulation of Foxo1/Foxo3 stability in autoimmune disease.

Foxp3 expression and regulatory T cell (Treg) development are critical for maintaining dominant tolerance and preventing autoimmune diseases. Human MST1 deficiency causes a novel primary immunodeficiency syndrome accompanied by autoimmune manifestations. However, the mechanism by which Mst1 controls immune regulation is unknown. In this article, we report that Mst1 regulates Foxp3 expression and Treg development/function and inhibits autoimmunity through modulating Foxo1 and Foxo3 (Foxo1/3) stability. We have found that Mst1 deficiency impairs Foxp3 expression and Treg development and function in mice. Mechanistic studies reveal that Mst1 enhances Foxo1/3 stability directly by phosphorylating Foxo1/3 and indirectly by attenuating TCR-induced Akt activation in peripheral T cells. Our studies have also shown that Mst1 deficiency does not affect Foxo1/3 cellular localization in CD4 T cells. In addition, we show that Mst1(-/-) mice are prone to autoimmune disease, and mutant phenotypes, such as overactivation of naive T cells, splenomegaly, and autoimmune pathological changes, are suppressed in Mst1(-/-) bone marrow chimera by cotransplanted wt Tregs. Finally, we demonstrate that Mst1 and Mst2 play a partially redundant role in Treg development and autoimmunity. Our findings not only identify Mst kinases as the long-searched-for factors that simultaneously activate Foxo1/3 and inhibit TCR-stimulated Akt downstream of TCR signaling to promote Foxp3 expression and Treg development, but also shed new light on understanding and designing better therapeutic strategies for MST1 deficiency-mediated human immunodeficiency syndrome.

MST1 promotes apoptosis through regulating Sirt1-dependent p53 deacetylation.

Mammalian Sterile 20-like kinase 1 (MST1) protein kinase plays an important role in the apoptosis induced by a variety of stresses. The MST1 is a serine/threonine kinase that is activated upon apoptotic stimulation, which in turn activates its downstream targets, JNK/p38, histone H2B and FOXO. It has been reported that overexpression of MST1 initiates apoptosis by activating p53. However, the molecular mechanisms underlying MST1-p53 signaling during apoptosis are unclear. Here, we report that MST1 promotes genotoxic agent-induced apoptosis in a p53-dependent manner. We found that MST1 increases p53 acetylation and transactivation by inhibiting the deacetylation of Sirtuin 1 (Sirt1) and its interaction with p53 and that Sirt1 can be phosphorylated by MST1 leading to the inhibition of Sirt1 activity. Collectively, these findings define a novel regulatory mechanism involving the phosphorylation of Sirt1 by MST1 kinase which leads to p53 activation, with implications for our understanding of signaling mechanisms during DNA damage-induced apoptosis.

Mst1 is an interacting protein that mediates PHLPPs' induced apoptosis.

PHLPP1 and PHLPP2 phosphatases exert their tumor-suppressing functions by dephosphorylation and inactivation of Akt in several breast cancer and glioblastoma cells. However, Akt, or other known targets of PHLPPs that include PKC and ERK, may not fully elucidate the physiological role of the multifunctional phosphatases, especially their powerful apoptosis induction function. Here, we show that PHLPPs induce apoptosis in cancer cells independent of the known targets of PHLPPs. We identified Mst1 as a binding partner that interacts with PHLPPs both in vivo and in vitro. PHLPPs dephosphorylate Mst1 on the T387 inhibitory site, which activate Mst1 and its downstream effectors p38 and JNK to induce apoptosis. The same T387 site can be phosphorylated by Akt. Thus, PHLPP, Akt, and Mst1 constitute an autoinhibitory triangle that controls the fine balance of apoptosis and proliferation that is cell type and context dependent.

A cell-intrinsic role for Mst1 in regulating thymocyte egress.

The MST1 kinase was recently identified as playing an essential role in the promotion of lymphocyte polarization and adhesion stimulated by chemokines and TCR signaling. However, the physiological relevance of the Mst1 pathway in thymocyte development is not completely understood. In this study, we analyzed the effect of Mst1 disruption on thymocyte development and migration. Mst1-deficient (Mst1(-/-)) mice displayed an accumulation of mature thymocytes in the thymus, a dramatic reduction of lymphocytes in blood and peripheral lymphoid tissues, and a decrease of homing ability to peripheral lymph nodes. Mst1(-/-) thymocytes were impaired in chemotactic response to chemokines, such as CCL19, but not to sphingosine-1-phosphate. Further analyses of Mst1(-/-) mice revealed a severe impairment in the egress of mature T cells from the thymus. T lineage-specific knockout of the Mst1 gene demonstrates a cell-intrinsic role for Mst1 in regulating T cell development. Our study indicates that Mst1 is crucial in controlling lymphocyte chemotaxis and thymocyte emigration.

Lats2 is a negative regulator of myocyte size in the heart.

Mammalian sterile 20-like kinase (Mst)1 plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Because Hippo, a Drosophila homolog of Mst1, forms a signaling complex with Warts, a serine/threonine kinase, which in turn stimulates cell death and inhibits cell proliferation, mammalian homologs of Warts, termed Lats1 and Lats2, may mediate the function of Mst1. We here show that Lats2, but not Lats1, dose-dependently increased apoptosis in cultured cardiac myocytes. Lats2 also dose-dependently reduced [(3)H]phenylalanine incorporation and cardiac myocyte size, whereas dominant negative Lats2 (DN-Lats2) increased them, suggesting that endogenous Lats2 negatively regulates myocyte growth. DN-Lats2 significantly attenuated induction of apoptosis and inhibition of hypertrophy by Mst1, indicating that Lats2 mediates the function of Mst1 in cardiac myocytes. Cardiac specific overexpression of Lats2 in transgenic mice significantly reduced the size of left and right ventricles, whereas that of DN-Lats2 caused hypertrophy in both ventricles. Overexpression of Lats2 reduced left ventricular systolic and diastolic function without affecting baseline levels of myocardial apoptosis. Expression of endogenous Lats2 was significantly upregulated in response to transverse aortic constriction. Overexpression of DN-Lats2 significantly enhanced cardiac hypertrophy and inhibited cardiac myocyte apoptosis induced by transverse aortic constriction. These results suggest that Lats2 is necessary and sufficient for negatively regulating ventricular mass in the heart. Although Lats2 is required for cardiac myocyte apoptosis in response to pressure overload, it was not sufficient to induce apoptosis at baseline. In conclusion, Lats2 affects both growth and death of cardiac myocytes, but it primarily regulates the size of the heart and acts as an endogenous negative regulator of cardiac hypertrophy.

Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L).

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.

Lats2, a putative tumor suppressor, inhibits G1/S transition.

Lats2 is a new member of the Lats tumor suppressor family. The human LATS2 gene is located at chromosome 13q11-12, which has been shown to be a hot spot (67%) for LOH in nonsmall cell lung cancer. In order to understand the function of LATS2 in the control of tumor development, we ectopically expressed mouse Lats2 via retroviral infection in NIH3T3/v-ras cells to examine whether Lats2 plays a role in suppressing tumor development and regulating cell proliferation. We have found that ectopic expression of Lats2 in NIH3T3/v-ras cells suppresses development of tumors in athymic nude mice and inhibits proliferation of NIH3T3/v-ras cells in an in vitro assay. Cell cycle profile analysis demonstrated that ectopic expression of Lats2 inhibited the G1/S transition. Further mechanistic studies revealed that cyclin E/CDK2 kinase activity was downregulated in Lats2-transduced NIH3T3/v-ras cells, while other cell cycle regulators controlling the G1/S transition were not affected. We have also shown that LATS2 kinase activity and two LATS conserved domains (LCDs) are required for Lats2 to suppress tumorigenicity and to inhibit cell growth. In addition, the LATS2 protein is cytoplasmic during interphase in NIH3T3 cells, while it becomes localized to the mitotic apparatus during mitosis. Finally, we propose a model in which a combination of mammalian Lats2 and Lats1 control cell proliferation by negatively regulating different cell cycle check points.