Expression Of LATS1 Research Articles

Micronuclei in Cord Blood Lymphocytes and Associations with Biomarkers of Exposure to Carcinogens and Hormonally Active Factors, Gene Polymorphisms, and Gene Expression: The NewGeneris Cohort

Background: Leukemia incidence has increased in recent decades among European children, suggesting that early-life environmental exposures play an important role in disease development.Objectives: We investigated the hypothesis that childhood susceptibility may increase as a result of in utero exposure to carcinogens and hormonally acting factors. Using cord blood samples from the NewGeneris cohort, we examined associations between a range of biomarkers of carcinogen exposure and hormonally acting factors with micronuclei (MN) frequency as a proxy measure of cancer risk. Associations with gene expression and genotype were also explored.Methods: DNA and protein adducts, gene expression profiles, circulating hormonally acting factors, and GWAS (genome-wide association study) data were investigated in relation to genomic damage measured by MN frequency in lymphocytes from 623 newborns enrolled between 2006 and 2010 across Europe.Results: Malondialdehyde DNA adducts (M1dG) were associated with increased MN frequency in binucleated lymphocytes (MNBN), and exposure to androgenic, estrogenic, and dioxin-like compounds was associated with MN frequency in mononucleated lymphocytes (MNMONO), although no monotonic exposure–outcome relationship was observed. Lower frequencies of MNBN were associated with a 1-unit increase expression of PDCD11, LATS2, TRIM13, CD28, SMC1A, IL7R, and NIPBL genes. Gene expression was significantly higher in association with the highest versus lowest category of bulky and M1dG–DNA adducts for five and six genes, respectively. Gene expression levels were significantly lower for 11 genes in association with the highest versus lowest category of plasma AR CALUX® (chemically activated luciferase expression for androgens) (8 genes), ERα CALUX® (for estrogens) (2 genes), and DR CALUX® (for dioxins). Several SNPs (single-nucleotide polymorphisms) on chromosome 11 near FOLH1 significantly modified associations between androgen activity and MNBN frequency. Polymorphisms in EPHX1/2 and CYP2E1 were associated with MNBN.Conclusion: We measured in utero exposure to selected environmental carcinogens and circulating hormonally acting factors and detected associations with MN frequency in newborns circulating T lymphocytes. The results highlight mechanisms that may contribute to carcinogen-induced leukemia and require further research.Citation: Merlo DF, Agramunt S, Anna L, Besselink H, Botsivali M, Brady NJ, Ceppi M, Chatzi L, Chen B, Decordier I, Farmer PB, Fleming S, Fontana V, Försti A, Fthenou E, Gallo F, Georgiadis P, Gmuender H, Godschalk RW, Granum B, Hardie LJ, Hemminki K, Hochstenbach K, Knudsen LE, Kogevinas M, Kovács K, Kyrtopoulos SA, Løvik M, Nielsen JK, Nygaard UC, Pedersen M, Rydberg P, Schoket B, Segerbäck D, Singh R, Sunyer J, Törnqvist M, van Loveren H, van Schooten FJ, Vande Loock K, von Stedingk H, Wright J, Kleinjans JC, Kirsch-Volders M, van Delft JHM, NewGeneris Consortium. 2014. Micronuclei in cord blood lymphocytes and associations with biomarkers of exposure to carcinogens and hormonally active factors, gene polymorphisms, and gene expression: The NewGeneris Cohort. Environ Health Perspect 122:193–200; http://dx.doi.org/10.1289/ehp.1206324

Effects of the Hippo Signaling Pathway in Human Gastric Cancer

The Hippo signaling pathway is a newly discovered and conserved signaling cascade, which regulates organ size control by governing cell proliferation and apoptosis. This study aimed to investigate its effects in human gastric cancer. Tumor tissues (n=60), adjacent non-tumor tissues (n=60) and normal tissues (n=60) were obtained from the same patients with primary gastric cancer (GC). In addition, 70 samples of chronic atrophic gastritis (CAG) tissues were obtained from patients with intestinal metaplasia (IM) by endoscopic biopsy. Hippo signaling molecules, including Mst1, Lats1, YAP1, TAZ, TEAD1, Oct4 and CDX2, were determined by quantitative polymerase chain reaction (qPCR). Protein expression of Mst1, Lats1, YAP1, TEAD1 and CDX2 was assessed by immunohistochemistry and Western blotting. Mst1, Lats1 and Oct4 mRNA expression showed an increasing tendency from GC tissues to normal gastric tissues, while the mRNA expression of YAP1, TAZ and TEAD1 was up-regulated (all P<0.01). Mst1 and Lats1 protein expression presented a similar trend with their mRNA expression. In addition, YAP1 and TEAD1 protein expression in GC was significantly higher than in the other groups (all P<0.01). CDX2 mRNA and protein expression in the CAG group were higher than in the other groups (all P<0.01). In GC, mRNA expression of Mst1, Lats1, Oct4, YAP1, TAZ, TEAD1 and CDX2 had a close correlation with lymphatic metastasis and tumor TNM stage (all P<0.01). Furthermore, protein expression of Mst1, Lats1 ,YAP1, TAZ, TEAD1 and CDX2 had a close correlation between each other (P<0.05). The Hippo signaling pathway is involved in the development, progression and metastasis of human gastric cancer. Therefore, manipulation of Hippo signaling molecules may be a potential therapeutic strategy for gastric cancer.

MicroRNA-135b promotes lung cancer metastasis by regulating multiple targets in the Hippo pathway and LZTS1

Dysregulation of microRNAs has a critical role in cancer progression. Here we identify an intronic microRNA, miR-135b that is upregulated in highly invasive non-small-cell lung cancer cells. Expression of miR-135b enhances cancer cell invasive and migratory abilities in vitro and promotes cancer metastasis in vivo, while specific inhibition of miR-135b by a miR-135b-specific molecular sponge and antagomirs suppresses cancer cell invasion, orthotopic lung tumour growth and metastasis in a mouse model. miR-135b targets multiple key components in the Hippo pathway, including LATS2, β-TrCP and NDR2, as well as LZTS1. Expression of miR-135b, LZTS1, LATS2 and nuclear TAZ predicts poor outcomes of non-small-cell lung cancer. We find that miR-135b is dually regulated by DNA demethylation and nuclear factor-kappaB signalling, implying that abnormal expression of miR-135b in cancer may result from inflammatory and epigenetic modulations. We conclude that miR-135b is an oncogenic microRNA and a potential therapeutic target for non-small-cell lung cancer.

L3MBTL1: A Polycomb Protein At the Node of Crosstalk Between the BMP4 and Hippo Signaling Pathways in Erythropoiesis.

AbstractAbstract 2296The Hippo signaling pathway, first discovered in Drosophila, is emerging as an important regulator of stem cell behavior. Upon still-unclear upstream stimuli, the hippo pathway kinase cascade phosphorylates and inhibits the function of YAP, a transcription coactivator, by inducing its cytoplasmic retention. While recent evidences indicate that inhibition of YAP affects cell fate decisions, and proliferation, in many tissues, little is known about the relevance of this pathway in hematopoiesis. However, the interaction of YAP with Smad1, identified in flies and human cells (Alarcon C. et al. Cell 2009), prevents smurf-mediated Smad1 degradation, potentially enhancing BMP signaling.Our ongoing studies have indentified crosstalk between the BMP4 and the Hippo pathways in hematopoietic cells, and in induced-pluripotent stem (iPS) cells that we differentiated towards the erythroid lineage. This crosstalk involves the chromatin-binding, Polycomb protein L3MBTL1, which clearly regulate the effects of BMP on the erythroid differentiation of hematopoietic stem/progenitor cells and on fetal globin gene expression.We find that the Lats2 kinase, a core component of the Hippo pathway, physically interacts with L3MBTL1 and that treatment with BMP4 or Erythropoietin decreases the expression of both proteins in various hematopoietic cells, including primary human cord blood-derived CD34+ cells. By altering L3MBTL1 levels in K562 cells, we were able to show that the L3MBTL1-Lats2 interaction enhances Lats-mediated phosphorylation and the cytoplasmic retention of YAP. Furthermore, L3MBTL1-depleted iPS cells have an enhanced smad-mediated transcriptional response; by analyzing the gene expression profile of these cells, we found increased expression of several BMP target genes (such as HHEX and ID genes), suggesting that L3MBTL1 negatively titrates the BMP4 signaling pathway at least in part by affecting YAP phosphorylation and localization. Gene Set Enrichment Analysis confirmed enrichment of many smad-related genes, and yet, these cells presented enhanced smad1/5/8 phosphorylation by WB analysis, indicating that BMP4 signaling is triggered by L3MBTL1 depletion. We also found that hematopoietic differentiation of L3MBTL1-KD iPS cells generates high-fetal globin gene expressing erythroid progeny, suggesting a role for the BMP4 signaling pathway and the targeting of L3MBTL1 in the treatment of hemoglobinopathies.To further evaluate the effect of BMP4 signaling on hematopoietic cells that lack L3MBTL1, we analyzed the stress erythroid response of L3MBTL1 KO mice: while no difference was observed at baseline in the null mice compared to wt littermates, the L3mbtl1 null mice had a more severe anemia, with increased leukocytosis, and thrombocytosis post-hydrazine (PHZ) or Epo. We found a significant increase in the colony-forming ability of the l3mbtl1 null spleen and bone marrow cells, compared to controls, as well as increased spleen size and an expansion of the spleen erythroid compartment. Thus, l3mbtl1 null hematopoietic stem cells are more sensitive to the PHZ-mediated cytokine storm, which includes BMP4. Interestingly, the L3mbtl1 null BM and spleen cells showed diminished expression of Lats2 and phospho-YAP, consistent with our in vitro findings.In conclusion, these investigations have shown that L3MBTL1 not only negatively titrates the BMP4 signaling pathway, but also provides a nodal point for crosstalk between the BMP4 and Hippo signaling pathways in erythropoiesis. Thus, these data provide insights into possible novel treatments for genetic red cell disorders (such as β-thalassemia) and for acquired bone marrow failure syndromes such as Epo-resistant anemia. Disclosures:Levine:Agios Pharmaceuticals: Research Funding.

Decreased expression of LATS1 is correlated with the progression and prognosis of glioma

BackgroundLATS1 is a tumor suppressor genes implicated in the pathogenesis of certain types of tumors, but its role is not known in human glioma.MethodsUsing real-time PCR and immunohistochemistry, we detected the mRNA and protein expression of LATS1 in glioma. The effect of LATS1 on cell growth and invasion were investigated.ResultsWe found that mRNA and protein of LATS1 expression is significantly downregulated in glioma compared with normal control brain tissues. Furthermore, reduced LATS1 expression was markedly negatively correlated with WHO grade and KPS (p<0.001 and p<0.001) in glioma patients. Patients with lower LATS1 expression had a significantly shorter overall survival time than did patients with higher LATS1 expression. Multivariate analysis suggested that the level of LATS1 expression was an independent prognostic indicator (p<0.001) for the survival of patients with glioma. Forced expression of LATS1 in glioma U251 cells not only significantly suppressed cell growth, migration and invasion, but retarded cell cycle progression from G2/M to G1 in vitro. Finally, we found that overexpressed LATS1 markedly inhibited the expression level of cell cycle factor CCNA1.ConclusionThese results indicate that LATS1 is an important candidate tumor suppressor and its downregulated expression may contribute to glioma progression.

An indirect role for ASPP1 in limiting p53-dependent p21 expression and cellular senescence

In addition to acting as a transcriptional cofactor for p53, ASPP1 has been shown to function in the cytoplasm to regulate the nuclear localization and activity of YAP/TAZ. We show here that the ability of ASPP1 to activate YAP results in the decreased expression of LATS2, which lowers the ability of p53 to induce p21, cell-cycle arrest and senescence. ASPP1 expression peaks in S-phase, and down-regulation of ASPP1 leads to a reduction in DNA synthesis and enhanced senescence in response to drugs that impede DNA replication. These activities of cytoplasmic ASPP1 in opposing p53-mediated p21 expression are in contrast to the role of nuclear ASPP1 in cooperating with p53 to induce the expression of apoptotic target genes, and may help to dampen p53 activity in normal cells.

A Breakdown of the Hippo Pathway in Gastric Cancer

The Hippo pathway is a newly discovered signaling pathway. In mammals, the Hippo pathway was reported to be a tumor-suppressor signaling that inhibited cell proliferation and promoted cell apoptosis. Inactivation of the Hippo pathway resulted in massive cell growth, enlargement in organ size and eventually cancer formation. The aim of this study was to investigate the expression of core component proteins of the Hippo pathway, Mst1/2,S Sav1 and Lats1, in normal gastric mucosa, intestinal metaplasia and gastric cancer, and to explore the role of the Hippo pathway in gastric cancer. Two-step immunohistochemical staining was used to detect the expression of Mst1/2, Sav1 and Lats1 in 46 cases of normal gastric mucosa, 37 cases of intestinal metaplasia and 78 cases of gastric cancer. The expression of Mst1/2 and Lats1 was down-regulated in gastric cancer compared with that in normal gastric epithelium and adenoma. The Sav1 expression showed an increased trend from normal mucosa through intestinal metaplasia to gastric cancer, though there was no statistically significant difference. The expression of Sav1 and Lats1 in gastric cancer with lymph node metastasis was significantly lower than that without lymph node metastasis. A breakdown of the Hippo pathway may play a role in tumorigenesis and metastasis of gastric cancer.

CTLA-4 activates the hippo pathway to regulate terminal differentiation of the CD8+ T cell. (46.17)

Abstract Replicating, antigen-specific CD8+ T cells must not commit to terminal differentiation until there has been sufficient clonal expansion. The Hippo pathway of organ size control mediates this requirement by linking expression of the differentiation-inducing transcription factor, Blimp-1, to contact between replicating cells, which would be dependent on their frequency. TCR and IL-2R stimulation assemble the Hippo pathway in the CD8+ T cell by inducing expression of WW45, Mob1, Lats1, and YAP, the transcriptional co-activator that mediates organ growth. Contact between activated CD8+ T cells triggers the Hippo pathway, causing serine phosphorylation and degradation of YAP. This is suppressed by addition of naïve CD8+ T cells, indicating that the ligand-receptor pair triggering the Hippo pathway is expressed only by activated cells. The ligand was identified by suppressing YAP degradation with blocking CD80/86 antibody, and the receptor was defined by inducing YAP degradation by crosslinking CTLA-4. That YAP regulates differentiation was shown by ectopically expressing a non-phosphorylatable, stable form of YAP in activated CD8+ T cells, which suppressed Blimp-1 expression in vitro, and the differentiation/senescence marker, KLRG1, in vivo. This role for CTLA-4 was confirmed by the presence of YAP in T cells from CTLA-4-/-, but not CTLA-4+/-, mice. Therefore, in a process that resembles quorum sensing, the Hippo pathway regulates terminal differentiation of the CD8+ T cell.

Identification of a Tumor Suppressor Relay between the FOXP3 and the Hippo Pathways in Breast and Prostate Cancers

Defective expression of LATS2, a negative regulator of YAP oncoprotein, has been reported in cancer of prostate, breast, liver, brain, and blood origins. However, no transcriptional regulators for the LATS2 gene have been identified. Here we report that spontaneous mutation of the transcription factor FOXP3 reduces expression of the LATS2 gene in mammary epithelial cells. shRNA-mediated silencing of FOXP3 in normal or malignant mammary epithelial cells of mouse and human origin repressed LATS2 expression and increased YAP protein levels. LATS2 induction required binding of FOXP3 to a specific sequence in the LATS2 promoter, and this interaction contributed to FOXP3-mediated growth inhibition of tumor cells. In support of these results, reduced expression and somatic mutations of FOXP3 correlated strongly with defective LATS2 expression in microdissected prostate cancer tissues. Thus, defective expression of LATS2 is attributable to FOXP3 defects and may be a major independent determinant of YAP protein elevation in cancer. Our findings identify a novel mechanism of LATS2 downregulation in cancer and reveal an important tumor suppressor relay between the FOXP3 and HIPPO pathways which are widely implicated in human cancer.

The Hippo Tumor Pathway Promotes TAZ Degradation by Phosphorylating a Phosphodegron and Recruiting the SCFβ-TrCP E3 Ligase

The TAZ transcription co-activator promotes cell proliferation and epithelial-mesenchymal transition. TAZ is inhibited by the Hippo tumor suppressor pathway, which promotes TAZ cytoplasmic localization by phosphorylation. We report here that TAZ protein stability is controlled by a phosphodegron recognized by the F-box protein β-TrCP and ubiquitylated by the SCF/CRL1(β-TrCP) E3 ligase. The interaction between TAZ and β-TrCP is regulated by the Hippo pathway. Phosphorylation of a phosphodegron in TAZ by LATS primes it for further phosphorylation by CK1ε and subsequent binding by β-TrCP. Therefore, the Hippo pathway negatively regulates TAZ function by both limiting its nuclear accumulation and promoting its degradation. The phosphodegron-mediated TAZ degradation plays an important role in negatively regulating TAZ biological functions.

LATS2 is De-methylated and Overexpressed in Nasopharyngeal Carcinoma and Predicts Poor Prognosis

BackgroundLATS2, which encodes a novel serine/threonine kinase, is known to be important in centrosome duplication and in the maintenance of genomic stability. Recently, a potential role for LATS2 in cancer has been reported. In breast cancer and acute lymphoblastic leukemia (ALL), LATS2 mRNA is downregulated and has been suggested to be a tumor suppressor. However, the role of LATS2 in nasopharyngeal carcinoma has not been investigated. In this study, we aimed to investigate the expression pattern of LATS2 and its clinicopathological involvement in nasopharyngeal carcinoma to understand its effect on cell survival.MethodsUsing quantitative real time PCR and immunoblotting, the expression of LATS2 was detected in nasopharyngeal carcinoma cell lines and in the immortalized nasopharyngeal epithelial cell line NP69. Using immunohistochemistry, we analyzed LATS2 protein expression in 220 nasopharyngeal carcinoma cases. The association of LATS2 protein expression with the clinicopathological characteristics and the prognosis of nasopharyngeal carcinoma were subsequently assessed. Using methylation specific PCR, we detected the methylation status of the LATS2 promoter. RNA interference was performed by transfecting siRNA to specifically knock down LATS2 expression in 5-8F and CNE2.ResultsLATS2 protein was detected in 178 of 220 (80.91%) cases of nasopharyngeal carcinoma. LATS2 overexpression was a significant, independent prognosis predictor (P = 0.037) in nasopharyngeal carcinoma patients. Methylation specific PCR revealed that 36.7% (11/30) of nasopharyngeal carcinoma tissues and all of the chronic nasopharyngeal inflammation samples were methylated. Functional studies showed that the suppression of LATS2 expression in nasopharyngeal carcinoma (5-8F and CNE2) cell lines by using specific small interfering (siRNA) resulted in the inhibition of growth, induction of apoptosis and S-phase cell cycle increase. Overexpression of LATS2 in NP69 stimulated cell proliferation.ConclusionsOur results indicate that LATS2 might play a role in the tumorigenesis of nasopharyngeal carcinoma by promoting the growth of nasopharyngeal carcinoma cells. Transfection with specific siRNA might be feasible for the inhibition of growth, induction of apoptosis and S phase increase in nasopharyngeal carcinoma.

Regulation of ploidy and senescence by the AMPK-related kinase NUAK1

Senescence is an irreversible cell-cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss-of-function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK-related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant-negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.

MiR-372 Regulates Cell Cycle and Apoptosis of AGS Human Gastric Cancer Cell Line through Direct Regulation of LATS2

Previously, we have reported tissue- and stage-specific expression of miR-372 in human embryonic stem cells and so far, not many reports speculate the function of this microRNA (miRNA). In this study, we screened various human cancer cell lines including gastric cancer cell lines and found first time that miR-372 is expressed only in AGS human gastric adenocarcinoma cell line. Inhibition of miR-372 using antisense miR-372 oligonucleotide (AS-miR-372) suppressed proliferation, arrested the cell cycle at G2/M phase, and increased apoptosis of AGS cells. Furthermore, AS-miR-372 treatment increased expression of LATS2, while over-expression of miR-372 decreased luciferase reporter activity driven by the 3' untranslated region (3' UTR) of LATS2 mRNA. Over-expression of LATS2 induced changes in AGS cells similar to those in AGS cells treated with AS-miR-372. Taken together, these findings demonstrate an oncogenic role for miR-372 in controlling cell growth, cell cycle, and apoptosis through down-regulation of a tumor suppressor gene, LATS2.

Transcriptional activation of the Lats1 tumor suppressor gene in tumors of CUX1 transgenic mice

BackgroundLats1 (large tumor suppressor 1) codes for a serine/threonine kinase that plays a role in the progression through mitosis. Genetic studies demonstrated that the loss of LATS1 in mouse, and of its ortholog wts (warts) in Drosophila, is associated with increased cancer incidence. There are conflicting reports, however, as to whether overexpression of Lats1 inhibits cell proliferation. CUX1 is a transcription factor that exists in different isoforms as a result of proteolytic processing or alternative transcription initiation. Expression of p110 and p75 CUX1 in transgenic mice increases the susceptibility to cancer in various organs and tissues. In tissue culture, p110 CUX1 was shown to accelerate entry into S phase and stimulate cell proliferation.ResultsGenome-wide location arrays in cell lines of various cell types revealed that Lats1 was a transcriptional target of CUX1. Scanning ChIP analysis confirmed that CUX1 binds to the immediate promoter of Lats1. Expression of Lats1 was reduced in cux1-/- MEFs, whereas it was increased in cells stably or transiently expressing p110 or p75 CUX1. Reporter assays confirmed that the immediate promoter of Lats1 was sufficient to confer transcriptional activation by CUX1. Lats1 was found to be overexpressed in tumors from the mammary gland, uterus and spleen that arise in p110 or p75 CUX1 transgenic mice. In tissue culture, such elevated LATS1 expression did not hinder cell cycle progression in cells overexpressing p110 CUX1.ConclusionWhile inactivation of Lats1/wts in mouse and Drosophila can increase cancer incidence, results from the present study demonstrate that Lats1 is a transcriptional target of CUX1 that can be overexpressed in tumors of various tissue-types. Interestingly, two other studies documented the overexpression of LATS1 in human cervical cancers and basal-like breast cancers. We conclude that, similarly to other genes involved in mitotic checkpoint, cancer can be associated with either loss-of-function or overexpression of Lats1.

Lats1 differential expression in selected human cancers

Lats1 is a tumor suppressor gene that was studied extensively in Drosophila melangaster. Several studies were done to address the molecular pathway of Lats 1, yet several roles in carcinogenesis are not elucidated. Lats1 is a serine/threonine kinase similar to human myotonic dystrophy kinase (Justice et al., 1995). Lats 1 is phosphorylated in a cell cycle dependent manner and was shown to modulate CDC2 and cyclin A activity (Tao et al., 1999). Transgenic Lats1-/mice develop soft-tissue sarcomas and ovarian stromal cell tumours and sensitivity to carcinogenic treatments (St John et al., 1999). Lats1 was shown to play a crucial role in controlling mitosis progression by forming a Lats1/zyxin complex on mitotic apparatus (Hirota et al., 2000). Overexpression of LATS1 significantly suppressed the human tumor cell growth in vitro and tumorigenicity in vivo by either G2-M arrest or apoptosis (Yang et al., 2001). The kinase inactive Lats1 impaired the G1 tetraploidy checkpoint due absence of p53 induction (Iida et al., 2004). Few clinical studies correlated the expression of Lats1, promoter methylation and tumor progression. We previously correlated Lats 1 overexpression with the transcription regulator CDP/Cux in selected tumors (Siam et al., unpublished results). We are currently correlating these findings in clinical samples to investigate the differential expression of Lats1 in many human carcinomas. The hypermethylation of the Lats1 promoter and how this correlates with regulation of Lats1 expression by CDP/Cux p110 and/or p75 isoforms is under investigation. We have preliminary evidence suggesting that Lats1 expression and methylation status of the Lats1 promoter are distinct in selected cancers. Additionally, we are suggesting the use of Lats1 expression and the methylation status as prognostic marker for selected tumors. from 2009 American University in Cairo Research Conference Cairo, Egypt. 5 April 2009

The expression of COX-2, hTERT, MDM2, LATS2 and S100A2 in different types of non-small cell lung cancer (NSCLC)

Several studies have reported different expression levels of certain genes in NSCLC, mostly related to the stage and advancement of the tumours. We investigated 65 stage I-III NSCLC tumours: 32 adenocarcinomas (ADC), 26 squamous cell carcinomas (SCC) and 7 large cell carcinomas (LCC). Using the real-time reverse transcription polymerase chain reaction (RT-PCR), we analysed the expression of the COX-2, hTERT, MDM2, LATS2 and S100A2 genes and researched the relationships between the NSCLC types and the differences in expression levels. The differences in the expression levels of the LATS2, S100A2 and hTERT genes in different types of NSCLC are significant. hTERT and COX-2 were over-expressed and LATS2 under-expressed in all NSCLC. We also detected significant relative differences in the expression of LATS2 and MDM2, hTERT and MDM2 in different types of NSCLC. There was a significant difference in the average expression levels in S100A2 for ADC and SCC. Our study shows differences in the expression patterns within the NSCLC group, which may mimic the expression of the individual NSCLC type, and also new relationships in the expression levels for different NSCLC types.

Abstract 484: Large Tumor Suppressor 2, A Downstream Target of Mammalian Sterile 20 Like Kinase 1, Is an Endogenous Regulator of Myocyte Size and Apoptosis in the Heart

Mammalian sterile 20-like kinase 1 (Mst1) plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Mst1 forms a complex with hWW45 and Large tumor suppressor 2 (Lats2), thereby activating Lats2. Lats2 dose dependently increased apoptosis in cultured cardiac myocytes and transgenic mice with cardiac specific expression of Lats2 exhibited a cardiac dysfunction, suggesting that stimulation of Lats2 leads to increased cell death and cardiac dysfunction. In order to elucidate the function of endogenous Lats2 in the heart, we generated adenovirus harboring dominant-negative Lats2 (Ad-DN-Lats2) and transgenic mice with cardiac specific expression of dominant-negative Lats2 (Tg-DN-Lats2). Ad-DN-Lats2 prevented induction of apoptosis and inhibition of hypertrophy by Mst1 in cultured cardiac myocytes, suggesting that Lats2 mediates the function of Mst1. In Tg-DN-Lats2, both LV weight/tibia length (LVW/TL; 4.34 vs 3.68, N=9, N=7, p&lt;0.01) and RV weight/tibia length (1.15 vs 0.88, N=9, N=7, p&lt;0.01) were greater than in NTg. Echocardiographically determined LV function was normal in Tg-DN-Lats2 at baseline except that LV wall thickness was greater in Tg-DN-Lats2 than in NTg (LV posterior wall thickness; 0.97 vs 0.79 mm, N=10, N=7, p&lt;0.05). Since expression of Lats2 is increased in response to pressure overload (8 fold), we examined whether upregulation of endogenous Lats2 during pressure overload works as a negative feedback mechanism for cardiac hypertrophy. Tg-DN-Lats2 mice exhibited greater LVW/TL than NTg after transverse aortic constriction (TAC) (5.9 vs 4.7 mm, N=9, N=7, p&lt;0.05). Increases in LVW/TL (+37% vs 27%, N=9, N=7, p&lt;0.05) and LV cross sectional area (+25% vs +16%, N=5, N=5, p&lt;0.05) after TAC were greater in Tg-DN-Lats2 than those in NTg. Moreover, Tg-DN-Lats2 exhibited significantly lower cardiac myocyte apoptosis than NTg after TAC. These results suggest that Lats2 is a negative regulator of cardiac hypertrophy at baseline and in response to pressure overload, and a positive mediator of cardiac myocyte apoptosis under pressure overload. In conclusion, Lats2 is an evolutionarily conserved negative regulator of organ size: namely inhibiting hypertrophy while stimulating apoptosis in the heart. This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).

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.

Negative Regulation of YAP by LATS1 Underscores Evolutionary Conservation of the Drosophila Hippo Pathway

The Hippo pathway defines a novel signaling cascade regulating cell proliferation and survival in Drosophila, which involves the negative regulation of the transcriptional coactivator Yorkie by the kinases Hippo and Warts. We have recently shown that the human ortholog of Yorkie, YAP, maps to a minimal amplification locus in mouse and human cancers, and that it mediates dramatic transforming activity in MCF10A primary mammary epithelial cells. Here, we show that LATS proteins (mammalian orthologs of Warts) interact directly with YAP in mammalian cells and that ectopic expression of LATS1, but not LATS2, effectively suppresses the YAP phenotypes. Furthermore, shRNA-mediated knockdown of LATS1 phenocopies YAP overexpression. Because this effect can be suppressed by simultaneous YAP knockdown, it suggests that YAP is the primary target of LATS1 in mammalian cells. Expression profiling of genes induced by ectopic expression of YAP or by knockdown of LATS1 reveals a subset of potential Hippo pathway targets implicated in epithelial-to-mesenchymal transition, suggesting that this is a key feature of YAP signaling in mammalian cells.

Integrated Genomic Profiling of Chronic Lymphocytic Leukemia Identifies Subtypes of Deletion 13q14 with Differential Expression of LATS2.

Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous illness with a variable clinical course. Loss of chromosomal material on chromosome 13 at cytoband 13q14 is the most frequent genetic abnormality in CLL but the molecular aberrations underlying del13q14 in CLL remain incompletely characterized. We analyzed 171 CLL cases for LOH and sub-chromosomal copy loss on chromosome 13 in DNA from FACS-sorted CD19+ cells and paired buccal cells using the Affymetrix XbaI 50K SNP array platform. Comparative analysis of SNP-array-derived detection of del13q14 using copy number analysis compared with clinical FISH results for del13q14 demonstrated strong agreement between both methods: FISH detected 91/171=53% overall del13q14 incidence and 77/171=45% del13q14 cases with greater than 25% of cells involved. SNP-arrays detected 82/171=48% del13q14 incidence. SNP-arrays detected 74/77=96% of all CLL cases that were greater than 25% FISH positive and 82/91=90% of all CLL cases that were FISH positive (% positive range for FISH was 2–97%). Finally, SNP-arrays detected 2 CLL cases not previously known to be del13q14 by FISH. The resulting high-resolution genomic maps, together with array-based measurements of expression levels of RNA in CLL cases with and without del13q14 and Q-PCR-based expression analysis of selected genes, support the following conclusions:i.del13q14 is heterogeneous and composed of multiple subtypes (types Ia, Ib and II) with deletion of Rb or the miR15a/16 loci serving as anatomic landmarks, respectively;ii.del13q14 type Ia deletions are relatively uniform in length and extend from breakpoints close to the miR15a/16 cluster to a newly identified telomeric breakpoint cluster at ∼50.2–50.5 Mb physical position;iii.LATS2 RNA levels are ∼2.6–2.8 fold lower in cases with del13q14 type I that do not delete Rb, as opposed to del13q14 type II lesions or all other CLL cases;iv.∼15% of CLL cases display marked reductions in miR15a/16 expression that are often but not invariably associated with bi-allelic miR15a/16 loss; andv.Bcl-2 levels and miR16/15a locus loss or miR16/15a expression are not correlated.This data should aid future investigations into biological differences imparted on CLL by different del13q14 subtypes, including investigations into LATS2 as one of the genes found deregulated as part of del13q14 type I and investigations into additional miR16/15a targets.