Large Tumor Suppressor Kinase Research Articles

Toxoplasma gondii causes changes in the host's expression of cancer-associated miRNAs.

Throughout the world, numerous individuals are infected with Toxoplasma gondii, which may improve immunity against cancer. Furthermore, microRNAs (miRs) may be differentially expressed in the host upon infection with T. gondii. In the present study, RNA-sequencing analysis and reverse transcription-quantitative PCR revealed that miR-429-3p, miR-145a-5p, miR-211-5p, miR-31-3p and miR-135a-5p were determined to be downregulated, while miR-21a-3p, miR-135b-5p, miR-210-5p and miR-146-3p were upregulated in mice post-infection with T. gondii. Antitumor genes [TNF receptor superfamily member 11b, large tumor suppressor kinase (Lats)2 and Lats1] were identified as targets of miR-429-3p, miR-145a-5p, miR-211-5p, miR-31-3p and miR-135a-5p with a luciferase reporter assay. In addition, the protein levels of Lats2 and Lats1 were detected to be higher in T. gondii-infected mice than in the control group. Therefore, these results provide favorable evidence for the suppression of cancer upon T. gondii infection and may give novel ideas for the treatment of tumors.

HnRNPL-activated circANKRD42 back-splicing and circANKRD42-mediated crosstalk of mechanical stiffness and biochemical signal in lung fibrosis

Increasing circular RNAs (circRNAs) are involved in the progression of idiopathic pulmonary fibrosis (IPF). However, circRNA biogenesis and circRNA-mediated crosstalk between mechanical stiffness and biochemical signals in IPF remain obscure. In this study, a novel circRNA-ankyrin repeat domain 42 (ANKRD42) from peripheral blood of patients with IPF, which participated in pulmonary fibrosis through the close communication of mechanical stiffness and biochemical signals, was identified. Mechanistic studies revealed that theheterogeneous nuclear ribonucleoprotein L (hnRNP L) activated the circANKRD42 reverse splicing biogenesis. The biogenetic circANKRD42 sponged miR-324-5p to promote the AJUBA expression, which blocked the binding between phosphorylated yes-associated protein 1 (YAP1) and large tumor suppressor kinase 1/2 (LATS1/2), leading to increased YAP1 entering the nucleus. circANKRD42 also sponged miR-136-5p to promote the YAP1 translation. Accumulating YAP1 in nucleus bound to TEAD, which initiated the transcription of genes related to mechanical stiffness. Finally,the therapeutic effect of circANKRD42 was evaluated in mice and the association between circANKRD42 and clinicopathological features was analyzed in IPF patients. Our findings supported that circANKRD42 is a promising biomarker and a potential therapeutic target related to cytoskeleton tension for IPF treatment.

LINC00174 Suppresses Non-Small Cell Lung Cancer Progression by Up-Regulating LATS2 via Sponging miR-31-5p.

Objective Dysregulation of long non-coding RNAs (lncRNAs) is associated with the progression of non-small cell lung cancer (NSCLC). This study aimed to investigate the role of long intergenic non-protein coding RNA 174 (LINC00174) in NSCLC. Materials and Methods In this experimental study, LINC00174 expression in NSCLC tissues and cell lines was investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Besides, cell counting kit-8 (CCK-8), 5-bromo-2'-deoxyuridine (BrdU). Transwell and Flow Cytometry assays were applied to detect the regulatory function of LINC00174 on the growth, migration and apoptosis of NSCLC cells. Bioinformatics analysis, dual luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay predicted and verified the targeting relationship between LINC00174 and miR-31-5p, and between miR-31-5p and the 3´-untranslated region (3´UTR) of large tumor suppressor kinase 2 (LATS2), respectively. Western blotting was performed to detect the regulatory function of LINC00174 and miR-31-5p on LATS2 protein expression. Results Compared with that in normal lung tissues, LINC00174 expression in NSCLC tissues and cell lines was reduced. LINC00174 expression was negatively associated with the TNM stage of the patients. Functional experiments showed that LINC00174 overexpression inhibited NSCLC cell multiplication and migration, and induced apoptosis. Furthermore, LINC00174 targeted miR-31-5p and repressed its expression. Additionally, LINC00174 upregulated LATS2 expression through competitively binding to miR-31-5p. ConclusionLINC00174, as a competitive endogenous RNA, elevates LATS2 expression by adsorbing miR-31-5p, thereby inhibiting the viability and migration of NSCLC cells, and promoting apoptosis.

Small molecule LATS kinase inhibitors block the Hippo signaling pathway and promote cell growth under 3D culture conditions

Although 3D cell culture models are considered to reflect the physiological microenvironment and exhibit high concordance with in vivo conditions, one disadvantage has been that cell proliferation is slower in 3D culture as compared to 2D culture. However, the signaling differences that lead to this slower proliferation are unclear. Here, we conducted a cell-based high-throughput screening study and identified novel small molecules that promote cell proliferation, particularly under 3D conditions. We found that one of these molecules, designated GA-017, increases the number and size of spheroids of various cell-types in both scaffold-based and scaffold-independent cultures. In addition, GA-017 also enhances the ex vivo formation of mouse intestinal organoids. Importantly, we demonstrate that GA-017 inhibits the serine/threonine protein kinases large tumor suppressor kinase 1/2, which phosphorylate Yes-associated protein and transcriptional coactivator with PDZ-binding motif , key effectors of the growth- and proliferation-regulating Hippo signaling pathway. We showed that GA-017 facilitates the growth of spheroids and organoids by stabilizing and translocating Yes-associated protein and transcriptional coactivator with PDZ-binding motif into the cell nucleus. Another chemical analog of GA-017 obtained in this screening also exhibited similar activities and functions. We conclude that experiments with these small molecule large tumor suppressor kinase inhibitors will contribute to further development of efficient 3D culture systems for the ex vivo expansion of spheroids and organoids.

The E3 ubiquitin ligase, FBXW5, promotes the migration and invasion of gastric cancer through the dysregulation of the Hippo pathway

F-box and WD repeat domain-containing 5 (FBXW5), with WD40 repeats, can bind to the PPxY sequence of the large tumor suppressor kinases 1/2 (LATS1/2) kinase domain, resulting in ubiquitination. Ubiquitination and the subsequent degradation of LATS1/2 abrogate the Hippo pathway and worsen gastric cancer (GC). However, the effects and molecular mechanisms of FBXW5 in GC remain unexplored. To elucidate the clinical significance of FBXW5, immunohistochemistry was conducted to reveal the positive correlation between FBXW5 expression and lymph node metastasis (p < 0.001) and TNM stage (training cohort: p = 0.018; validation cohort: p = 0.001). Further, patients with high FBXW5 expression were found to have poor prognosis (training cohort: log-rank p = 0.020; validation cohort: log-rank p = 0.025). Cell experiments revealed the promoting effects of FBXW5 on the proliferation, invasion, metastasis, and chemoresistance of GC cells. Blocking LATS1-YAP1 leads to the loss of FBXW5-mediated regulation of the Hippo pathway and partial functions. Further, co-immunoprecipitation and in vivo ubiquitination assays revealed the interaction between FBXW5 and LATS1, which promoted the ubiquitination and degradation of LATS1. Based on mouse xenograft assays, FBXW5 silencing attenuated the growth of subcutaneous tumor xenografts. Altogether, FBXW5 was found to inactivate the Hippo signaling pathway by enhancing LATS1 ubiquitination and degradation, which promoted the invasion, metastasis, and drug resistance of GC cells.

Protein kinase Cα activation switches YAP1 from TEAD-mediated signaling to p73-mediated signaling.

Yes‐associated protein 1 (YAP1) interacts with TEAD transcription factor in the nucleus and upregulates TEAD‐target genes. YAP1 is phosphorylated by large tumor suppressor (LATS) kinases, the core kinases of the Hippo pathway, at 5 serine residues and is sequestered and degraded in the cytoplasm. In human cancers with the dysfunction of the Hippo pathway, YAP1 becomes hyperactive and confers malignant properties to cancer cells. We have observed that cold shock induces protein kinase C (PKC)‐mediated phosphorylation of YAP1. PKC phosphorylates YAP1 at 3 serine residues among LATS‐mediate phosphorylation sites. Importantly, PKC activation recruits YAP1 to the cytoplasm even in LATS‐depleted cancer cells and reduces the cooperation with TEAD. PKC activation induces promyelocytic leukemia protein‐mediated SUMOylation of YAP1. SUMOylated YAP1 remains in the nucleus, binds to p73, and promotes p73‐target gene transcription. Bryostatin, a natural anti‐neoplastic reagent that activates PKC, induces YAP1/p73‐mediated apoptosis in cancer cells. Bryostatin reverses malignant transformation caused by the depletion of LATS kinases. Therefore, bryostatin and other reagents that activate PKC are expected to control cancers with the dysfunction of the Hippo pathway.

Cigarette smoke extract induces migration of rat vascular smooth muscle cells through regulation of expressions of MOB2 and LATS1

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To study the impact of cigarette smoke extract (CSE) on the migratory capacity of rat vascular smooth muscle cells (VSMCs), and the likely mechanism of action involved.&#x0D; Methods: Rat VSMCs were exposed to varying concentrations of CSE. The migration capacity of the cells was determined by Transwell migration assay and scratch test. Protein and RNA expression levels of LATS1 (large tumor suppressor kinase 1) and MOB2 (monopolar spindle-one-binder protein 2) were assayed using immunoblotting and quantitative real-time-polymerase chain reaction (qRT-PCR), respectively.&#x0D; Results: The results showed that within a certain concentration range, the migratory capacity of VSMCs increased as the concentration of CSE increased (p &lt; 0.05). In contrast, the protein and RNA expressions of LATS1 and MOB2 were negatively correlated with CSE concentrations (p &lt; 0.05).&#x0D; Conclusion: CSE promotes abnormal migration of rat VSMCs by inhibiting the expressions of LATS1 and MOB2 associated with Hippo signaling pathway. Thus, smoking may induce vascular remodeling and cardiovascular events via this mechanism.&#x0D; &#x0D; &#x0D; &#x0D;

Hsa_circ_0067514 suppresses gastric cancer progression and glycolysis via miR-654-3p/LATS2 axis.

Gastric cancer is the third most frequent cancer with high prevalence and mortality globally. Circular RNAs (circRNAs) play a key role in cancer regulation, including gastric cancer. Nevertheless, only a few circRNAs have been well elucidated in gastric cancer. Hence, we investigated the action of circ_0067514 on gastric cancer and clarified the underlying mechanism. Here, we found that circ_0067514 was decreased in gastric cancer patients and cancer cells. The circ_0067514 expression was correlated with gastric cancer overall survival, lymph node metastasis, tumor, node, metastasis (TNM) stage, and histological differentiation. Overexpression of circ_0067514 blocked proliferation, invasion, and glycolysis of gastric cancer cells. Besides, circ_0067514 regulated large tumor suppressor kinase 2 (LATS2) expression by absorbing microRNA (miR)-654-3p. Furthermore, circ_0067514 modulated gastric cancer aggressive behaviors and glycolysis via miR-654-3p/LATS2 axis. Moreover, circ_0067514 constrained tumor growth in vivo. Together, this study showed that circ_0067514 suppressed gastric cancer aggressive development and glycolysis via miR-654-3p/LATS2 axis, making circ_0067514 a valuable target for preventing gastric cancer progression.

Itchy E3 ubiquitin-protein ligase promotes neuroblastoma progression in vitro and in vivo

[Objective] Neuroblastoma is the most common pediatric neuroendocrine tumor. Patients with high-risk neuroblastoma have poor clinical outcomes. Understanding the mechanisms underlying neuroblastoma progression could help identify potential therapeutic targets. This study aimed to explore the roles of itchy E3 ubiquitin-protein ligase (ITCH) in neuroblastoma progression using neuroblastoma cell lines and xenograft models of neuroblastoma. [Methods] ITCH-silencing or overexpressing neuroblastoma cells were established using two different human neuroblastoma cell lines, SK-N-AS and SH-SY5Y. In vitro and in vivo experiments were carried out to determine the effects of ITCH on neuroblastoma cell behaviors. The dual-luciferase reporter assay and co-transfection experiments were applied to determine the interaction of ITCH and miR-145-5p during neuroblastoma progression. [Results] In both cell lines, ITCH overexpression significantly promotes the proliferation, migration, and invasion capacities of neuroblastoma cells, while ITCH silencing with ShITCH suppressed neuroblastoma cell proliferation and induced apoptosis. Moreover, overexpression of ITCH decreased 51% and 54% the protein expressions of large tumor suppressor kinase 1 (LATS1), and inhibited 59% and 66% the phosphorylation of Yes-associated protein (YAP), concomitant with 2.02-fold and 2.56-fold increased expressions of cell proliferation marker Ki67 and 2.51-fold and 2.26-fold elevated levels of anti-apoptosis marker Bcl2 in SK-N-AS and SH-SY5Y cells, respectively. The dual-luciferase reporter assay demonstrated that ITCH interacted with miR-145-5p. Further in vitro and xenograft experiments showed that ITCH negatively affected the tumor-suppressive effect of miR-145-5p. [Conclusion] ITCH promotes neuroblastoma cell proliferation and metastasis by inhibiting LATS1 and promoting YAP nuclear translocation.

MicroRNA miR-4709-3p targets Large Tumor Suppressor Kinase 2 (LATS2) and induces obstructive renal fibrosis through Hippo signaling

ABSTRACT Obstructive renal fibrosis is the consequence of abnormal extracellular matrix assembly, which eventually results in renal failure, acute, and end‑stage renal infection. MicroRNAs (miRNAs), a particular category of small RNAs, modulate the expression of genes post-transcriptionally and regulate biological activities, including fibrogenesis. The study probed to estimate the key functions of miR-4709-3p in obstructive renal fibrosis. This investigation used TGF-β1 stimulated HK-2 in-vitro model, unilateral ureteral occlusion (UUO) mice model, and human Diabetic nephropathy (DN) and Renal interstitial fibrosis (RIF) specimens to depict the abundance of the miR-4709-3p level using FISH and RT-qPCR. MiR-4709-3p mimics and inhibitors were utilized to evaluate the functions of miR-4709-3p in-vitro. Luciferase assay was exploited to verify miR-4709-3p and LATS2 3ʹUTR binding. Finally, to depict the functions of miR-4709-3p in-vivo, the UUO model was injected with miR-4709-3p inhibitors. Results exhibited the upregulation of miR-4709-3p in UUO-induced in-vivo model, TGF-β1 stimulated HK-2, and human RIF and DN samples. Moreover, it was determined that modulating miR-4709-3p regulated the level of fibrosis markers. Luciferase assay miR-4709-3p modulates renal fibrosis by targeting LATS2. Finally, it was found that miR-4709-3p regulates obstructive renal fibrosis through the Hippo signaling pathway. Overall, the study concludes that aberrant miR-4709-3p expression plays an essential function in the renal fibrosis progression, and miR-4709-3p overexpression could advance obstructive renal fibrosis via LATS2 targeting in Hippo signaling pathway. Therefore, miR-4709-3p inhibition may be a potential renal fibrosis therapy target.

LncRNA HCG11 mediated by METTL14 inhibits the growth of lung adenocarcinoma via IGF2BP2/LATS1

Lung adenocarcinoma (LUAD) is a common malignancy the pathogenesis of which is terribly complicated and remains largely unclear. Long non-coding RNAs (lncRNAs) are a group of endogenous RNA molecules that are involved in various malignant processes. In this study, we explored the roles of lncRNA Human leukocyte antigen complex group 11 (HCG11) in LUAD. Our data revealed that lncRNA HCG11 expression was downregulated in LUAD, which was modulated by the hypermethylation of HCG11 promoter and Methyltransferase Like 14 (METTL14) mediated N6-methyladenosine (m6A) modification. The m6A modification of HCG11 promoted its nuclear exportation and binding by Insulin Like Growth Factor 2 MRNA Binding Protein 2 (IGF2BP2), resulting in increased stability. HCG11 could recruit IGF2BP2 to target Large Tumor Suppressor Kinase 1 (LATS1) mRNA to enhance the stability and promote the expression of LATS1. HCG11 served as a tumor suppressor to restrain tumor growth in LUAD by regulating LATS1. In summary, this study demonstrated that HCG11 mediated by METTL14 inhibited the growth of lung adenocarcinoma via IGF2BP2/LATS1.

LncRNA TINCR attenuates the proliferation and invasion, and enhances the apoptosis of cutaneous malignant melanoma cells by regulating the miR‑424‑5p/LATS1 axis

Cutaneous malignant melanoma (CMM) is responsible for ≥1/2 of skin cancer-related mortalities. The aberrant expression of long non-coding 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 transcription-quantitative PCR (RT-qPCR) was used to analyze microRNA (miR)-424-5p expression, and RT-qPCR 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 Kit-8, 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 miR-424-5p were predicted using the miRDB database. A dual luciferase reporter assay and RT-qPCR were used to identify the relationship between TINCR and miR-424-5p 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 miR-424-5p and TINCR in CMM cells. LATS1, a target of miR-424-5p, 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 miR-424-5p/LATS1 signaling axis. These results indicated that TINCR may play a tumor suppressive role in CMM.

Identification of the tumor‑suppressive role of circular RNA‑FOXO3 in colorectal cancer via regulation of miR‑543/LATS1 axis.

Colorectal cancer (CRC) is a common malignancy with significant prevalence and mortality rates. Circular RNA FOXO3 (circ-FOXO3; 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 circ-FOXO3 on CRC progression and identify its underlying mechanism. In the present study, the expression of circ-FOXO3 was investigated in CRC tissues and cells via reverse transcription-quantitative PCR. A Cell Counting Kit-8 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 pull-down assay were conducted to verify the relationship of circ-FOXO3, microRNA (miR)-543 and Large tumor suppressor kinase 1 (LATS1). The results demonstrated that circ-FOXO3 expression was downregulated in CRC tissues and cells, and was associated with poor overall survival of patients with CRC. Moreover, circ-FOXO3 was associated with tumor size, distant metastasis, differentiation, lymph node metastasis and TMN stages of patients with CRC. circ-FOXO3 overexpression suppressed CRC cell proliferation, migration and invasion. Luciferase assay and RNA pull-down assay results indicated that circ-FOXO3 functioned as a sponge for miR-543. In addition, circ-FOXO3 increased the expression of LATS1 via sponging miR-543, thus inhibiting CRC cell aggressive features. Collectively, the present results suggested that circ-FOXO3 inhibited CRC metastasis and progression via elevated LATS1 expression by sponging miR-543. Therefore, circ-FOXO3 may be a promising target for CRC therapy.

Hypoxia-Inducible Exosomes Facilitate Liver-Tropic Premetastatic Niche in Colorectal Cancer.

Liver metastasis is a frequent occurrence in patients with colorectal cancer (CRC), with 15%-25% of CRC patients having liver metastases at the time of initial diagnosis. Specifically, some regional-stage patients with mild symptoms (stage 1 or 2) will also advance to liver metastases rapidly, even if the CRC lesion in situ is resected in time. Nevertheless, the precisemechanism of liver metastasis isstill unclear. Fresh tumor tissues from patients with CRC, adjacent noncancerous tissues, and colorectal adenoma tissues were subjected to microarray analysis to identify differentially expressed microRNA. Exosomes from human serum and cell culture medium were separated, quantitated, and verified by transmission electronic microscopy and Zetasizer Nano. Luciferase reporter assay, real-time quantitative PCR, western blot, immunoprecipitation, chromatin and re-chromatin immunoprecipitation, migration and invasion assay, PDX mouse model, flow cytometry, immunohistochemistry, and immunofluorescence staining were employed to explore the regulation among CRC liver metastases, immunosuppression, and cell adhesion. In this study, we demonstrated that the hypoxic microenvironment in primary CRC lesions boosted exosome release, selectively initiated favorable premetastatic niche formation in the liver but not in other organs. Mechanistically, Kupffer cells (KCs) can phagocytose exosomes containing highly expressed miR-135a-5p from the blood circulation into the liver. Exosomal miR-135a-5p initiated the large tumor suppressor kinase 2-yes-associated protein-matrix metalloproteinase 7 axis to promote the occurrence of CRC liver metastasis, and cluster of differentiation 30-TNF receptor-associated factor 2-p65-mediated immunosuppression signaling also contributed to this process. Hypoxia-induced exosomal miR-135a-5p correlates with the development, clinical severity, and prognosis of CRC liver metastases through the premetastatic niche; and our findings revealed that miR-135a-5p might be a promising target in halting CRC liver metastases.

Loss of Caenorhabditis elegans homologue of human MOB4 compromises life span, health life span and thermotolerance.

Mob1/phocein family proteins are conserved from yeast to mammals. Human has four MOB genes, MOB1, 2, 3 and 4. Human MOB1 protein, which is a component of the Hippo pathway, is involved in the inhibition of yes-associated protein (YAP1) through large tumor suppressor (LATS) kinases and plays a tumor suppressive role. In contrast, MOB4 activates YAP1. Caernorhabditis elegans (C. elegans) also has four MOB genes. Moreover, C. elegans has homologues of YAP1 (Ce_YAP-1) and LATS kinases (WTS-1). Nevertheless, our previous study revealed that the Hippo pathway is not conserved in C. elegans and that heat shock activates Ce_YAP-1. We also reported that Ce_YAP-1 is involved in the regulation of life span, healthy lifespan and thermotolerance. In this study, we raised a question whether and how C. elegans homologue of MOB4 (Ce_MOB-4) is involved in the regulation of Ce_YAP-1. Ce_MOB-4 is ubiquitously expressed in adult worms. This expression pattern is similar to that of Ce_YAP-1. mob-4 loss-of-function mutants show short life span, short health life span and compromise thermotolerance. However, heat shock activates Ce_YAP-1 in mob-4 mutant. In conclusion, the role of MOB4 in the activation of YAP1 is not conserved in C. elegans.

A phosphatidic acid-binding lncRNA SNHG9 facilitates LATS1 liquid-liquid phase separation to promote oncogenic YAP signaling.

Long noncoding RNAs (lncRNAs) are emerging as a new class of important regulators of signal transduction in tissue homeostasis and cancer development. Liquid-liquid phase separation (LLPS) occurs in a wide range of biological processes, while its role in signal transduction remains largely undeciphered. In this study, we uncovered a lipid-associated lncRNA, small nucleolar RNA host gene 9 (SNHG9) as a tumor-promoting lncRNA driving liquid droplet formation of Large Tumor Suppressor Kinase 1 (LATS1) and inhibiting the Hippo pathway. Mechanistically, SNHG9 and its associated phosphatidic acids (PA) interact with the C-terminal domain of LATS1, promoting LATS1 phase separation and inhibiting LATS1-mediated YAP phosphorylation. Loss of SNHG9 suppresses xenograft breast tumor growth. Clinically, expression of SNHG9 positively correlates with YAP activity and breast cancer progression. Taken together, our results uncover a novel regulatory role of a tumor-promoting lncRNA (i.e., SNHG9) in signal transduction and cancer development by facilitating the LLPS of a signaling kinase (i.e., LATS1).

Regulatory effect of microRNA-223-3p on breast cancer cell processes via the Hippo/Yap signaling pathway.

According to the 2018 global cancer statistics, the incidence and mortality rates of breast cancer are increasing gradually, which seriously threatens the health of women. MicroRNA-223-3p (miR-223-3p) can promote the proliferation and invasion of breast cancer cells. Hippo/Yes-related protein (Yap) signaling pathway activation has been found in a variety of tumors. The present study aimed to investigate the potential mechanism of miR-223-3p in breast cancer. The Cell Counting Kit-8 assay was used to detect cell viability and flow cytometry was used to detect apoptosis. The abilities of cell migration and invasion were detected using scratch and Transwell assays, as well as reverse transcription-quantitative PCR and western blotting to detect gene and protein expression, respectively. The current results demonstrated that miR-223-3p transcription levels were increased in breast cancer cells, and inhibition of miR-223-3p gene expression decreased cell proliferation, migration and invasion. Additionally, inhibition of miR-223-3p expression inhibited epithelial-mesenchymal transition (EMT) in breast cancer cells. miR-223-3p promoted cell proliferation, migration, invasion and EMT, and the western blotting results demonstrated that miR-223-3p inhibition increased the phosphorylation of Yap1 and the protein expression levels of large tumor suppressor kinase 1. In conclusion, results from the present results suggested that miR-223-3p may promote cell proliferation, migration, invasion and EMT through the Hippo/Yap signaling pathway. Therefore, miR-223-3p may be a potential biomarker for breast cancer.

JAM‐A signals through the Hippo pathway to regulate intestinal epithelial proliferation

The intestinal mucosa is lined by a columnar epithelial monolayer that serves as a dynamic interface, regulating barrier function and homeostasis. Perturbation of homeostasis, including changes in intestinal epithelial cell (IEC) proliferation and migration, can give rise to pathological states including ulcerative colitis and cancer. Regulation of proliferation and migration is a complex process coordinated, in part, by signaling events emanating from tight junction proteins at cell-cell contacts. One tight junction protein that has been linked to regulation of cellular proliferation is Junctional Adhesion Molecule A (JAM-A). JAM-A forms homodimers in cis and trans through interactions between its extracellular immunoglobulin-like domains. Loss of JAM-A results in enhanced IEC proliferation that has been linked to loss of homophilic interactions and Akt-dependent, beta-catenin signaling. However, JAM-A contributions to other signaling pathways that regulate cell proliferation are poorly understood. Since the Hippo signaling pathway is a well-known regulator of cellular proliferation, differentiation, and apoptosis, we examined whether loss of JAM-A alters Hippo pathway activity. We assessed activity of the Hippo transcriptional coactivator, Yes-1 Associated Protein (YAP), in JAM-A deficient IEC by gene-reporter activity assay and immunoblotting and observed significant increases, suggesting that JAM-A may interact with Hippo pathway constituents upstream of YAP. Immunolabeling studies with monolayers of IEC revealed association of JAM-A with two Hippo pathway constituents, Large Tumor Suppressor Kinase (LATS1) and Merlin. Co-immunoprecipitation experiments confirmed LATS1 and Merlin association with JAM-A. Moreover, phosphoprotein specific immunoblotting studies on JAM-A deficient IEC revealed decreased Hippo pathway activity characterized by decreased LATS1 activity and subsequent increased YAP activity. Such findings are consistent with JAM-A signaling upstream of Merlin and LATS1 and coordinating downstream Hippo signaling. Since there is evidence suggesting that dimerization of JAM-A is a required signal that suppresses IEC proliferation, we performed experiments using an exogenous overexpression system to study effects of a dimerization deficient JAM-A mutant on Hippo signaling. Phosphoprotein specific immunoblotting and EdU incorporation revealed that overexpression of full-length JAM-A resulted in activation of Hippo signaling, suppressing cell proliferation compared to empty vector control cells. However, overexpression of a dimerization defective JAM-A mutant protein lacking the distal immunoglobulin loop (DL1) was unable to activate Hippo signaling and suppress cell proliferation. Based on these observations, we propose that homodimerization of JAM-A functions as a cell-cell contact sensor, initiating downstream Hippo signaling to suppress IEC proliferation. These findings have important implications for understanding effects of altered proliferation on mucosal wound repair, as well as in morbidity associated with certain cancers which have been reported to express decreased levels of JAM-A protein.

PKM2 regulates proliferation and apoptosis through the Hippo pathway in oral tongue squamous cell carcinoma

Oral tongue squamous cell carcinoma (OTSCC) is a highly malignant type of tumor. The 5-year survival rate of patients with advanced tongue squamous cell carcinoma is only ~50%. Pyruvate kinase M2 (PKM2) is the key rate-limiting enzyme of glycolysis, maintaining the Warburg effect in tumor cells. The present study aimed to investigate the relationship between PKM2 expression and the poor prognosis of patients with OTSCC and to determine oral squamous carcinoma tumor cell proliferation and apoptosis. Reverse transcription-quantitative (RT-q) PCR, western blotting and immunohistochemistry were used to analyze the expression levels of PKM2 in OTSCC, and the clinicopathological characteristics and prognosis of patients with OTSCC were further analyzed by statistical analysis. The results from RT-qPCR and immunohistochemistry demonstrated that PKM2 was upregulated in OTSCC tissues and highly expressed in advanced stage OTSCC tissues compared with paired adjacent tissues and lower stage OTSCC tissues. Patients with OTSCC and high PKM2 expression had shorter overall survival (OS) compared with those with low PKM2 expression. Furthermore, high expression of PKM2 was significantly associated with Tumor-Node-Metastasis (TNM) stage. TNM stage and PKM2 expression were independent predictive factors for OS in patients with OTSCC. In addition, PKM2 knockdown inhibited the proliferation and increased the apoptosis of oral squamous carcinoma tumor cells. Furthermore, PKM2 knockdown could regulate the expression of cell cycle and apoptosis-related proteins by activating Hippo signaling pathway, as confirmed by the decreased expression of yes-associated protein 1 (YAP), Bcl-2 and Ki-67 and the increased expression of large tumor suppressor kinase 1, phosphorylated YAP and Bax. Taken together, the findings from this study demonstrated that PKM2 may be considered as a potential target for the diagnosis and treatment of OTSCC.

SEPT6 drives hepatocellular carcinoma cell proliferation, migration and invasion via the Hippo/YAP signaling pathway.

Septin 6 (SEPT6) is a member of the GTP-binding protein family that is highly conserved in eukaryotes and regulates various biological functions, including filament dynamics, cytokinesis and cell migration. However, the functional importance of SEPT6 in hepatocellular carcinoma (HCC) is not completely understood. The present study aimed to investigate the expression levels and roles of SEPT6 in HCC, as well as the underlying mechanisms. The reverse transcription quantitative PCR, western blotting and immunohistochemistry staining results demonstrated that SEPT6 expression was significantly elevated in HCC tissues compared with corresponding adjacent non-tumor tissues, which indicated that SEPT6 expression may serve as a marker of poor prognosis for HCC. By performing plasmid transfection and G418 treatment, stable SEPT6-knockdown and SEPT6-overexpression cell lines were established. The Cell Counting Kit-8, flow cytometry and Transwell assay results demonstrated that SEPT6 overexpression significantly increased HCC cell proliferation, cell cycle transition, migration and invasion compared with the Vector group, whereas SEPT6 knockdown displayed significant suppressive effects on HCC cell lines in vitro compared with the control group. Mechanistically, SEPT6 might facilitate F-actin formation, which induced large tumor suppressor kinase 1 dephosphorylation, inhibited Hippo signaling, upregulated yes-associated protein (YAP) expression and nuclear translocation, and upregulated cyclin D1 and matrix metallopeptidase 2 (MMP2) expression. Furthermore, YAP overexpression significantly reversed SEPT6 knockdown-induced inhibitory effects on HCC, whereas YAP knockdown significantly inhibited the oncogenic effect of SEPT6 overexpression on HCC. Collectively, the present study demonstrated that SEPT6 may promote HCC progression by enhancing YAP activation, suggesting that targeting SEPT6 may serve as a novel therapeutic strategy for HCC.