Int J Clin Exp Pathol 2018;11(4):2053-2063 www.ijcep.com /ISSN:1936-2625/IJCEP0073054 Original Article LATS1 inhibits metastasis and epithelial-mesenchymal transition in head and neck squamous cell carcinoma Jicheng Wu1, Zhijuan Zhao2, Huiling Zhang1, Fanshuang Kong1, Huamao Jiang3, Keqiang Huang4, Huachuan Zheng1 1Department of Experimental Oncology, Shengjing Hospital of China Medical University, Shenyang, China; Depart- ments of 2Pathology, 3Urology, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China; 4Depart- ment of Stomatology, The Second Affiliated Hospital of Jinzhou Medical University, Jinzhou, China Received January 19, 2018; Accepted February 22, 2018; Epub April 1, 2018; Published April 15, 2018 Abstract: LATS1 is a serine/threonine kinase of the Hippo signaling pathway that phosphorylates and inactivates transcriptional co-activators YAP1 and WWTR1. To investigate roles of LATS1 expression in head and neck squamous cell carcinomas (HNSCCs), we transfected LATS1-expressing plasmid into B88 cells and examined the phenotypes and their relevant molecules. LATS1 expression was analyzed using immunohistochemistry on tissue microarray, Oncomine, and TCGA databases. LATS1 overexpression was found to suppress growth, migration and invasion, and induce apoptosis, G2 arrest, and mesenchymal to epithelial transition (MET) (P < 0.05). Both increased expression of P21, Bax, and E-cadherin and decreased expression of Cyclin B1, D1, Bcl-2, and MMPs. Twist and N-cadherin were detected in B88 transfectants, in comparison to mock and control by Western blot. Nuclear LATS1 expression was weaker in primary cancers than in normal squamous tissue and dysplasia (P < 0.05) but versa for cytoplasmic counterpart (P < 0.05). Cytoplasmic LATS1 expression was positively correlated with lymph node metastasis (P < 0.05). Survival analysis showed that differentiation degree was an independent factor of long overall and relapse- free survival of HNSCC patients (P < 0.05). According to bioinformatics analysis, we found upregulated LATS1 mRNA expression in HNSCCs (P < 0.05). Cox proportional hazards model indicated that perineural invasion and distant metastasis were independent prognostic factors for overall survival of HNSCC (P < 0.05). These findings suggest nu- cleocytoplasmic translocation of LATS1 protein and upregulated expression of LATS1 mRNA during tumorigenesis of HNSCC. LATS1 mRNA overexpression may reverse aggressive phenotypes of HNSCC cells, as a gene therapy target. Keywords: HNSCC, LATS1, aggressiveness, prognosis, gene therapy Introduction LATS1 (large tumor suppressor 1) serine/threo- nine kinase is a core member of Hippo signal- Head and neck squamous cell carcinoma ing, which inactivates and phosphorylates tran- (HNSCC) is the 5th most common cancer and scriptional co-activators YAP1 and WWTR1. It 8th in cancer deaths worldwide. Although early- belongs to the Ndr/LATS subfamily of AGC (pro- stage head and neck cancers (especially laryn- tein kinase A/PKG/PKC) kinases and is encod- geal and oral cavity) have high cure rates, up ed by LATS1 gene [2]. The interaction of pro- to 50% of head and neck cancer patients pres- tein’s N-terminal domain with CDC2 to form a ent with advanced disease because it frequent- complex attenuates H1 histone kinase activity, ly transfers to neck lymph nodes earlier as indicating a characteristic as negative regula- the first symptom and sometimes only [1]. tor of CDC2/cyclin A. However, its C-terminal Therefore, recognition of original biomarkers kinase region binds to its own N-terminal do- and gene therapy targets could be conducive to main to negatively interfere with complex for- increase survival rate and quality of HNSCC mation. LATS1 protein is localized to the mitotic patients, in spite of the development of radio organ and interacts with CDC2 kinase in early imaging, surgery, radiotherapy, chemotherapy, stage of mitosis [3, 4]. Further investigation has and immunotherapy. indicated that RASSF1A-LATS1 signaling stabi- LATS1 in head and neck squamous cell carcinoma lized replication forks by restricting CDK2- mary gland development, infertility and growth mediated phosphorylation of BRCA2 [5]. CHO1 retardation, development of soft-tissue sarco- phosphorylation by LATS1 regulated centro- mas, ovarian stromal cell tumors, pituitary somal activation of LIMK1 during cytokinesis dysfunction, and a high sensitivity to carcino- [6], but LATS1 suppressed centrosome overdu- genic treatments [28]. Yabuta et al. [29] fo- plication by modulating stability of Cdc25B [7]. und that mouse embryonic fibroblasts (MEFs) from LATS1 knockout mice displayed mitotic A great number of evidences indicate that bio- defects, centrosomal overduplication, chromo- logical functions of LATS1 depend on phosphoryla- somal misalignment, multipolar spindle forma- tion and ubiquitination [8-21]. hMOB1 was tion, chromosomal bridging, and cytokinesis reported to recruit LATS1 to the plasma mem- failure. They also showed anchorage-indepen- brane for multi-site phosphorylation and activa- dent growth and continued cell cycles and tion [8]. Although Han et al. [9] found that dis- cell growth, by passing cell-cell contact inhibi- sociation of WWC3 from LATS1 reduced LATS1 tion similar to tumor cells. In our study, the phosphorylation to suppress lung cancer inva- effects of ectopic LATS1 overexpression on sion and metastasis, the interaction between aggressive phenotypes of HNSCC cells were TNFAIP8 and LATS1 decreased phosphoryla- found and relevant mechanisms were analyzed. tion of the latter, finally promoting aggressive- Moreover, LATS1 expression in squamous epi- ness of hepatocellular carcinoma cells [10]. On thelium, cancer of head and neck, and dyspla- the other hand, LATS1-mediated YAP phosphor- sia was examined. We also examined the cor- ylation at HX(R/H/K)XX(S/T) sequence facili- relation of LATS1 expression with clinicopa- tated dissociation of the YAP-TEAD4 complex thological and prognostic parameters of can- and association of YAP-RUNX3 complex [11, cers by immunohistochemistry and bioinfor- 12], controlled nephron progenitor epitheliali- matics analysis. zation, and inhibited myofibroblast formation [13]. LATS1 phosphorylated CDC26 to modu- Materials and methods late assembly of the tetratricopeptide repeat subcomplex of APC/C [14], Angiomotin to inhibit Cell culture and transfection YAP transcription and cell growth due to serum deprivation [15], and FOXL2 to repress StAR Tongue cancer, B88, was bought from ATCC mRNA expression [16]. LATS1 ubiquitination by and cultured in MEM (HyClone, Logan, UT, USA) NEDD4, E3 ubiquitin ligase CRL4, and WWP1 along with fetal bovine serum (10%) (FBS; E3 ligase leads to proteasomal degradation HyClone), penicillin (100 U/ml; Sigma, St-Louis, and promotes nuclear localization and tran- MO, USA), and streptomycin (100 μg/ml; Sigma) scriptic activity of YAP [17-19]. Ni et al. [20] in a humidified atmosphere of 5% CO2 at 37°C. found that a novel lncRNA uc.134 repressed B88 cells were transfected with pcDNA3.1- hepatocellular carcinoma progression by inhib- LATS1 at 24 hours using Attractene Transfec- iting CUL4A-mediated ubiquitination of LATS1 tion Reagent (QIAGEN, USA) and subjected to and increasing YAP (S127) phosphorylation to monocloning selection with pcDNA3.1 as a silence target genes. Additionally, LATS1 abla- mock. Cells were collected by centrifugation, tion promotes the luminal phenotype and washed twice with phosphate buffered saline increases number of bipotent and luminal pro- (PBS), and finally used for RNA and protein genitors, the proposed cells-of-origin of most extraction. human breast cancers, by targeting ubiquitina- tion and DCAF1-dependent proteasomal degra- Proliferation assay dation of ERα [21]. Cells were seeded in triplicate in 96-well plates LATS1 is downregulated in various human can- at a density of 3 × 103 cells/well. Every 24 cers such as breast cancer, lung cancer, colo- hours after cell adhesion, cell counting Kit-8 rectal cancer, astrocytoma, and glioma [22-26]. (CCK-8; Dojindo, Kumamoto, Japan) was em- Sun et al. [27] found that LATS1 deletion ployed to determine number of viable cells by increased germ cell apoptosis and follicular detecting absorbance at 450 nm using a cysts in mouse ovaries. LATS1 knockout mice FL600 fluorescence plate reader (Bio-Rad, showed low neonate survival, a lack of mam- Hercules, CA, USA). 2054 Int J Clin Exp Pathol 2018;11(4):2053-2063 LATS1 in head and neck squamous cell carcinoma Table 1. Primary antibodies used in the present study the invasion assay, 2.5 × 105 cells Name Source Company Dilution in 100 µl serum-free culture medi- um were seeded into the top cham- N-cadherin Rabbit Cell Signaling Technology 1:1000 ber of matrigel-coated Transwell E-cadherin Rabbit Cell Signaling Technology 1:1000 inserts (BD Bioscience). The lower Twist Rabbit Abcam 1:1000 compartment of the chamber was ZEB2 (E-11) Mouse Santa Cruz Biotechnology 1:500 filled up with corresponding media LATS1 Mouse Santa Cruz Biotechnology 1:500 containing 10% v/v FBS as a che- CyclinB 1 Mouse Santa Cruz Biotechnology 1:500 moattractant. After incubation for CyclinD 1 Rabbit Santa Cruz Biotechnology 1:500 24 hours, non-invading cells on the p21 Rabbit Santa Cruz Biotechnology 1:500 upper surface of the membrane Bcl-2 Mouse Santa Cruz Biotechnology 1:500 were wiped away and invading cells MMP-9 Mouse Santa Cruz Biotechnology 1:500 on the bottom were washed with PBS, fixed in 100% methanol, and MMP-2 Rabbit Wanleibio 1:1000