Expression Of LATS1 Research Articles

Abstract 534: Large Tumor Suppressor 2 (Lats2), a Downstream Target of Mammalian Sterile 20 Like Kinase 1 (Mst1), Negatively Regulates Ventricular Mass.

Mst1 is an evolutionarily conserved serine threonine kinase which is a critical regulator of growth and death in cardiac myocytes. Mst1 forms a complex with hWW45 and Lats2, thereby activating Lats2. Lats2 partially mediates the proapoptotic effect of Mst1 in cardiac myocytes in vitro . In order to elucidate the cardiac function of Lats2 in vivo , we generated transgenic mice with cardiac specific expression of Lats2 (Tg-Lats2, line #8) and dominant negative Lats2 (Tg-DN-Lats2). At 5– 6 months of age, Tg-Lats2 exhibited a greater left ventricular (LV) end-diastolic dimension (4.3 vs 3.7 mm, n = 8, n = 7, p < 0.05) and lower LV ejection fraction (EF, 63 vs 72 %, p < 0.05) than wild type (WT) mice. In addition, many Tg-Lats2 exhibited a remarkably enlarged right atrium with mural thrombus. Although LV weight /body weight (LVW/BW) in Tg-Lats2 was not significantly different from that in WT, right ventricular weight (RVW)/BW in Tg-Lats2 was significantly smaller than that in WT (0.65 vs 0.96, p < 0.05). The LV myocyte cross sectional area (CSA) was significantly smaller in Tg-Lats2 than in WT (82 vs 100%, p < 0.05) and the RV myocyte CSA showed a similar tendency. Significantly reduced RVW/BW and LVEF were also observed in Tg-Lats2 line #20 (milder expression). Tg-Lats2 exhibited greater levels of interstitial fibrosis (13.0 vs 3.4%, p < 0.05) at baseline. Although the number of TUNEL positive myocytes was not different between Tg-Lats2 and WT at baseline, it was significantly greater in Tg-Lats2 after transverse aortic constriction for 4 weeks (0.12 vs 0.09%, p < 0.05). In contrast, Tg-DN-Lats2 exhibited significantly greater LVW/BW (3.88 vs 3.27, n = 10, n = 10, p < 0.05) and RVW/BW (0.96 vs 0.73, p < 0.05), and normal LV function. Furthermore, adenovirally expressed Lats2 reduced both the relative cell size and protein/DNA content in cultured neonatal rat cardiac myocytes (53.5%, 63.2% vs LacZ control virus, n = 3, p < 0.05). Conversely, DN-Lats2 virus transduction significantly increased the cell size and protein/DNA content of cardiac myocytes (125.0%, 121.1% vs LacZ control virus, n = 3, p < 0.05). These results suggest that Lats2 is a critical negative regulator of ventricular mass and that stimulation of Lats2 leads to increases in cell death, fibrosis and cardiac dysfunction.

Promoter hypermethylation-mediated down-regulation of LATS1 and LATS2 in human astrocytoma

LATS1 and LATS2 are tumor suppressor genes implicated in the regulation of cell cycle, but their methylation statuses are still unknown in human astrocytoma. Here, we found that the promoter hypermethylation frequencies of LATS1 and LATS1 were 63.66% (56/88) and 71.5% (63/88) in 88 astrocytomas by methylation-specific PCR. But no methylation of LATS1 and LATS2 promoter was detected in the 10 normal brain tissues. There was an increased methylation frequency of LATS1 and LATS2 with the malignant development of astrcytoma. By real-time PCR, the mRNA expression of LATS1 or LATS2 was detected significantly decreased in different pathological grade astrocytomas ( P < 0.05). And the mRNA levels of LATS1 and LATS2 in astrocytomas with hypermethylation were both significantly ( P < 0.01) lower than those without methylation. The methylation of LATS1 and LATS2 was detected in U251 and SHG-44 cell lines, and 5-aza-deoxycytidine could restore their expression to induce cell apoptosis. Our results suggested that LATS1 and LATS2 mRNA was down-regulated in astrocytoma by hypermethylation of the promoter. The methylation and mRNA expression of LATS1 and LATS2 may provide useful clues to the development of the diagnostic assays for astrocytoma. Our results also suggested that LATS1 and LATS2 may be a useful target for astrocytoma therapy.

A Genetic Screen Implicates miRNA-372 and miRNA-373 As Oncogenes in Testicular Germ Cell Tumors

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumor-suppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.

Down-Regulation of LATS1 and LATS2 mRNA Expression by Promoter Hypermethylation and Its Association with Biologically Aggressive Phenotype in Human Breast Cancers

LATS1 and LATS2 are tumor suppressor genes implicated in the regulation of cell cycle. Methylation status of the promoter regions of these genes as well as its correlation with their mRNA levels were studied in human breast cancers. Correlation of LATS1 and LATS2 mRNA levels with clinicopathologic characteristics of breast tumors were also studied. Methylation status of promoter regions of LATS1 and LATS2 was studied by a methylation-specific PCR and mRNA expression levels of LATS1 and LATS2 were determined by a real-time PCR assay in 30 breast cancers. In addition, correlation of LATS1 and LATS2 mRNA levels with clinicopathologic characteristics was studied in 117 breast cancers. Methylation-specific PCR showed that of 30 tumors, LATS1 promoter region was hypermethylated in 17 tumors (56.7%) and LATS2 promoter region was hypermethylated in 15 (50.0%) tumors. LATS1 mRNA levels in breast tumors with hypermethylation (2.15 +/- 0.37, mean +/- SE) were significantly (P < 0.01) lower than those without hypermethylation (6.09 +/- 1.38), and LATS2 mRNA levels in breast tumors with hypermethylation (1.42 +/- 0.66) were also significantly (P < 0.01) lower than those without hypermethylation (3.10 +/- 1.00). The decreased expression of LATS1 or LATS2 mRNA was significantly associated with a large tumor size, high lymph node metastasis, and estrogen receptor and progesterone receptor negativity. Furthermore, the decreased expression of LATS1 mRNA, but not LATS2 mRNA, was significantly (P < 0.05) associated with a poor prognosis. Hypermethylation of the promoter regions of LATS1 and LATS2 likely plays an important role in the down-regulation of their mRNA levels in breast cancers, and breast cancers with a decreased expression of LATS1 or LATS2 mRNA levels have a biologically aggressive phenotype.

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.

LATS1 tumor suppressor regulates G2/M transition and apoptosis.

The LATS1 gene is a mammalian member of the novel lats tumor suppressor family. Both lats mosaic flies and LATS1 deficient mice spontaneously develop tumors. Our previous studies have shown that inactivation of Drosophila lats leads to up-regulation of cyclin A in the fly, and the human LATS1 protein associates with CDC2 in early mitosis in HeLa cells, suggesting that the lats gene family may negatively regulate cell proliferation by modulating CDC2/Cyclin A activity. We demonstrate here that transduction of the human breast cancer cell MCF-7 with recombinant LATS1 adenovirus (Ad-LATS1), but not with EGFP adenovirus (Ad-EGFP), inhibits in vitro cell proliferation. Ectopic expression of LATS1 in MCF-7 cells specifically down-regulates Cyclin A and Cyclin B protein levels and dramatically reduces CDC2 kinase activity, leading to a G2/M blockade. Furthermore, Ad-LATS1 suppresses anchorage-independent growth of MCF-7 cells in soft agar and tumor formation in athymic nude mice. We also demonstrate that ectopic expression of LATS1 in MCF-7 cells and human lung cancer cell H460 up-regulates the level of BAX proteins and induces apoptosis. Finally, we show that LATS1 kinase activity is required for its ability to inhibit cell growth and induce apoptosis. The results indicate that the LATS1 tumor suppressor may play an important role in the control of human tumor development and that LATS1 suppresses tumorigenesis by negatively regulating cell proliferation and modulating cell survival.

Structure, Expression, and Chromosome Mapping of LATS2, a Mammalian Homologue of the Drosophila Tumor Suppressor Gene lats/warts

We have cloned and characterized LATS2, a novel mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2) mRNA, whereas expression of human LATS2 (HsLATS2) mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2 gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2 gene (by fluorescence in situ hybridization) to the 13q11–q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RB and BRCA2 have also been mapped.