Integrated screens uncover a cell surface tumor suppressor gene KIRREL involved in Hippo pathway.

Cell surface proteins play essential roles in various biological processes and are highly related to cancer development. They also serve as important markers for cell identity and targets for pharmacological intervention. Despite their great potentials in biomedical research, comprehensive functional analysis of cell surface proteins remains scarce. Here, with a de novo designed library targeting cell surface proteins, we performed in vivo CRISPR screens to evaluate the effects of cell surface proteins on tumor survival and proliferation. We found that Kirrel1 loss markedly promoted tumor growth in vivo. Moreover, KIRREL was significantly enriched in a separate CRISPR screen based on a specific Hippo pathway reporter. Further studies revealed that KIRREL binds directly to SAV1 to activate the Hippo tumor suppressor pathway. Together, our integrated screens reveal a cell surface tumor suppressor involved in the Hippo pathway and highlight the potential of these approaches in biomedical research.

Protein Phosphatase 2A with B' specificity subunits regulates the Hippo-Yorkie signaling axis in the Drosophila eye disc.

Hippo-Yorkie (Hpo-Yki) signaling is central to diverse developmental processes. Although its redeployment has been amply demonstrated, its context-specific regulation remains poorly understood. The Drosophila eye disc is a continuous epithelium folded into two layers, the peripodial epithelium (PE) and the retinal progenitor epithelium. Here, Yki acts in the PE, first to promote PE identity by suppressing retina fate, and subsequently to maintain proper disc morphology. In the latter process, loss of Yki results in the displacement of a portion of the differentiating retinal epithelium onto the PE side. We show that Protein Phosphatase 2A (PP2A) complexes comprising different substrate-specificity B-type subunits govern the Hpo-Yki axis in this context. These include holoenzymes containing the B‴ subunit Cka and those containing the B' subunits Wdb or Wrd. Whereas PP2A(Cka), as part of the STRIPAK complex, is known to regulate Hpo directly, PP2A(Wdb) acts genetically upstream of the antagonistic activities of the Hpo regulators Sav and Rassf. These in vivo data provide the first evidence of PP2A(B') heterotrimer function in Hpo pathway regulation and reveal pathway diversification at distinct developmental times in the same tissue.

Core Hippo pathway components act as a brake on Yap and Taz in the development and maintenance of the biliary network.

The development of the biliary system is a complex yet poorly understood process, with relevance to multiple diseases, including biliary atresia, choledochal cysts and gallbladder agenesis. We present here a crucial role for Hippo-Yap/Taz signaling in this context. Analysis of sav1 mutant zebrafish revealed dysplastic morphology and expansion of both intrahepatic and extrahepatic biliary cells, and ultimately larval lethality. Biliary dysgenesis, but not larval lethality, is driven primarily by Yap signaling. Re-expression of Sav1 protein in sav1-/- hepatocytes is able to overcome these initial deficits and allows sav1-/- fish to survive, suggesting cell non-autonomous signaling from hepatocytes. Examination of sav1-/- rescued adults reveals loss of gallbladder and formation of dysplastic cell masses expressing biliary markers, suggesting roles for Hippo signaling in extrahepatic biliary carcinomas. Deletion of stk3 revealed that the phenotypes observed in sav1 mutant fish function primarily through canonical Hippo signaling and supports a role for phosphatase PP2A, but also suggests Sav1 has functions in addition to facilitating Stk3 activity. Overall, this study defines a role for Hippo-Yap signaling in the maintenance of both intra- and extrahepatic biliary ducts.

Molecular profiling of nonalcoholic fatty liver disease-associated hepatocellular carcinoma using SB transposon mutagenesis.

Nonalcoholic fatty liver disease (NAFLD) is the fastest rising cause of hepatocellular carcinoma (HCC) in Western countries; however, the molecular mechanisms that cause NAFLD-HCC remain elusive. To identify molecular drivers of NAFLD-HCC, we performed Sleeping Beauty (SB) transposon mutagenesis screens in liver-specific Pten knockout and in high-fat diet-fed mice, which are murine models of NAFLD-HCC. SB mutagenesis accelerated liver tumor formation in both models and identified 588 and 376 candidate cancer genes (CCGs), respectively; 257 CCGs were common to both screens and were enriched in signaling pathways known to be important for human HCC. Comparison of these CCGs with those identified in a previous SB screen of hepatitis B virus-induced HCC identified a core set of 141 CCGs that were mutated in all screens. Forty-one CCGs appeared specific for NAFLD-HCC, including Sav1, a component of the Hippo signaling pathway and the most frequently mutated gene identified in both NAFLD-HCC screens. Liver-specific deletion of Sav1 was found to promote hepatic lipid accumulation, apoptosis, and fibrogenesis, leading to the acceleration of hepatocarcinogenesis in liver-specific Pten mutant mice. Sav1/Pten double-mutant livers also showed a striking up-regulation of markers of liver progenitor cells (LPCs), along with synergistic activation of Yap, which is a major downstream effector of Hippo signaling. Lastly, Yap activation, in combination with Pten inactivation, was found to accelerate cell growth and sphere formation of LPCs in vitro and induce their malignant transformation in allografts. Our forward genetic screens in mice have thus identified pathways and genes driving the development of NAFLD-HCC.

Angiomotins stimulate LATS kinase autophosphorylation and act as scaffolds that promote Hippo signaling.

The Hippo pathway controls cell proliferation, differentiation, and survival by regulating the Yes-associated protein (YAP) transcriptional coactivator in response to various stimuli, including the mechanical environment. The major YAP regulators are the LATS1/2 kinases, which phosphorylate and inhibit YAP. LATS1/2 are activated by phosphorylation on a hydrophobic motif (HM) outside of the kinase domain by MST1/2 and other kinases. Phosphorylation of the HM motif then triggers autophosphorylation of the kinase in the activation loop to fully activate the kinase, a process facilitated by MOB1. The angiomotin family of proteins (AMOT, AMOTL1, and AMOTL2) bind LATS1/2 and promote its kinase activity and YAP phosphorylation through an unknown mechanism. Here we show that angiomotins increase Hippo signaling through multiple mechanisms. We found that, by binding LATS1/2, SAV1, and YAP, angiomotins function as a scaffold that connects LATS1/2 to both its activator SAV1-MST1 and its target YAP. Deletion of all three angiomotins reduced the association of LATS1 with SAV1-MST1 and decreased MST1/2-mediated LATS1/2-HM phosphorylation. Angiomotin deletion also reduced LATS1/2's ability to associate with and phosphorylate YAP. In addition, we found that angiomotins have an unexpected function along with MOB1 to promote autophosphorylation of LATS1/2 on the activation loop motif independent of HM phosphorylation. These results indicate that angiomotins enhance Hippo signaling by stimulating LATS1/2 autophosphorylation and by connecting LATS1/2 with both its activator SAV1-MST1/2 and its substrate YAP.

SAV1, regulated by microRNA-21, suppresses tumor growth in colorectal cancer.

This study investigated the role and action of the Salvador 1 protein (SAV1, also called WW45) in colorectal cancer (CRC). For this, CRC SW480 and HCT116 cells were infected with lentiviruses of SAV1 overexpression vector (lenti-SAV1) and SAV1 short hairpin RNA (sh-SAV1) to overexpress and silence SAV1 respectively, or transfected with microRNA-21 (miR-21) mimic to overexpress miR-21. Relative mRNA levels of SAV1 and relative miR-21 levels in CRC tissues or cells were detected. The effects of SAV1 and miR-21 on cell proliferation and apoptosis were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and annexin V - fluorescein isothiocyanate (FITC) - propidium iodide (PI) flow cytometry, respectively. Our results revealed that SAV1 was downregulated in CRC tissues compared with the adjacent noncancerous tissues. Furthermore, SAV1 overexpression inhibited proliferation and promoted apoptosis in SW480 and HCT116 cells, whereas knockdown of SAV1 exerted the opposite effect. Additionally, the tumorigenesis of SW480 cells in xenografted mice was significantly inhibited by SAV1 overexpression but promoted by SAV1 knockdown. MiR-21 levels significantly and negatively correlated with SAV1 expression in CRC tissues. More importantly, miR-21 overexpression significantly abolished the SAV1-mediated inhibition of proliferation and stimulation of apoptosis of SW480. In conclusion, SAV1 suppresses tumor growth in CRC and is regulated by miR-21.

Mammalian Hippo kinase pathway is downregulated by BCL-2 via protein degradation.

Mammalian ste20-like kinase (MST) signaling pathway plays a significant part in control of cell death and cell cycle. It was originally found as Hippo pathway in Drosophila and composed of MST kinase and Salvador-1 (SAV1), a scaffold protein. In mammalian cells, MST pathway induces cell-cycle exit and apoptosis in response to various signals. BCL-2, an anti-apoptotic protein, inhibits cell death and plays an important part in tumorigenesis. In the present report, we present evidence showing that BCL-2 is a new regulator of MST pathway. First, protein levels of MST2 and SAV1 were reduced significantly by co-expression of BCL-2. Physical interaction of BCL-2 with SAV1 was correlated with proteasomal degradation of SAV1 and MST2 proteins. In SH-SY5Y neuroblastoma cell line expressing a high level of BCL-2 but low levels of MST2 and SAV1, siRNA-induced knockdown of BCL-2 restored the expression of MST2 and SAV1. Inhibition of BCL-2 with BAD or ABT-737, a BCL-2 inhibitor, reversed its effect on MST2 and SAV1 proteins. ABT737 increased HEK293 cell death significantly when both MST2 and SAV1 were co-expressed. These results suggest that cancer cells may avoid cell death through enhanced expression of BCL-2 which down-regulates the pro-apoptotic MST pathway.

MST2 kinase regulates osteoblast differentiation by phosphorylating and inhibiting Runx2 in C2C12 cells.

The mammalian Ste20-like kinase (MST) pathway or Hippo pathway plays essential roles in cell proliferation, apoptosis, organ size control, and development. Runx2 is a key transcription factor in osteoblast differentiation. The objective of this study was to investigate whether the MST pathway could modulate Runx2 and osteoblast differentiation. First, we found that Runx2 interacted with MST2 and SAV1 via the WW domain of SAV1 and amino acid 292-445 of Runx2 containing a PY motif. Results of OSE luciferase reporter assay revealed that co-expression of MST2 and SAV1 inhibited the transcriptional activity of Runx2 whereas siRNA-mediated down-regulation of Mst1 and Mst2 increased its activity. MST2 and SAV1 significantly reduced mRNA levels of osteoblast differentiation marker genes such as alkaline phosphatase and osteocalcin in differentiating C2C12 cells. MST2 and SAV1 also hampered osteoblast differentiation of C2C12 cells induced by Runx2 as shown by alkaline phosphatase activity assay and Alizarin Red staining. Mass spectrometric analysis of immunoprecipitated Runx2 protein from HEK293 cells overexpressing MST2 and SAV1 revealed two novel phosphorylation sites at Ser-339 and Ser-370 residues of mouse Runx2 protein. Mutation of both serine residues to alanine interfered with the inhibitory effect of MST2 and SAV1 on the transcriptional activity of Runx2 and osteoblast differentiation induced by Runx2. Our results suggest that the MST kinase pathway can directly regulate osteoblast differentiation by modulating Runx2 activity through phosphorylation.

Kidney-specific knockout of Sav1 in the mouse promotes hyperproliferation of renal tubular epithelium through suppression of the Hippo pathway.

We have previously reported that Salvador homologue 1 (SAV1), a component of the Hippo pathway, is significantly down-regulated in high-grade clear cell renal cell carcinoma (ccRCC) due to 14q copy number loss, and that this down-regulation contributes to the proliferation and survival of renal tubular epithelial cells through activation of Yes-associated protein 1 (YAP1), a downstream target of the Hippo pathway. However, the impact of SAV1 loss on the proliferation and survival of kidney cells in vivo remained to be determined. To address this issue, we generated kidney-specific Sav1-knockout (Cdh16-Cre;Sav1(fl/fl) ) mice. Sav1 deficiency enhanced the proliferation of renal tubular epithelial cells in Cdh16-Cre;Sav1(fl/fl) mice, accompanied by nuclear localization of Yap1, suggesting suppression of the Hippo pathway. Sav1 deficiency in renal tubules also caused structural and cellular abnormalities of the epithelial cells, including significant enlargement of their nuclei. Furthermore, Cdh16-Cre;Sav1(fl/fl) mice developed both glomerular and tubular cysts. Although lining cells of the glomerular cysts showed no atypia, those of the tubular cysts showed variations in cell size and nuclear shape, which became more severe as the mice aged. In aged Cdh16-Cre;Sav1(fl/fl) mice, we observed focal disruption of proximal tubules and perivascular lymphocytic infiltration. In conclusion, Sav1 is required for the maintenance of growth, nuclear size and structure of renal tubules under physiological conditions, and its deficiency leads to the acquisition of enhanced proliferation of renal epithelial cells through suppression of Hippo signalling.

Expression profile and prognostic value of SAV1 in patients with pancreatic ductal adenocarcinoma.

SAV1 is a human homolog of salvador that contains two protein-protein interaction modules known as WW domains and acts as a scaffolding protein for Hpo and Warts. SAV1 is known to be a tumor suppressor, but its clinical and prognostic implications remain elusive. This study aimed at evaluating the prognostic significance and associated expression of SAV1 in pancreatic ductal adenocarcinoma (PDAC) patients. The expression of SAV1 in tissue specimens of PDAC patients were assayed with immunohistochemistry on a tissue microarray. The correlations between SAV1 expression and clinicopathological characteristics were analyzed by Pearson's chi-square test, Fisher's exact test, and Spearman's rank. The prognostic factors for overall survival were analyzed by univariate and multivariate Cox regression. The percentage of SAV1 expression in PDAC (50.6 %) was significantly lower than those in paratumor tissues (69.9 %) (P = 0.017). Expression of SAV1 was only significantly correlated with histological differentiation (P = 0.025) and N classification (P = 0.009). On multivariate analysis, elevated expression of SAV1 and N0 was a significant favorable prognostic factor of OS. Our study demonstrated for the first time that lower expression of SAV1 might be involved in the progression of PDAC, suggesting that SAV1 may be a potential prognostic marker and target for PDAC therapy.

Arsenic-induced cutaneous hyperplastic lesions are associated with the dysregulation of Yap, a Hippo signaling-related protein.

Arsenic exposure in humans causes a number of toxic manifestations in the skin including cutaneous neoplasm. However, the mechanism of these alterations remains elusive. Here, we provide novel observations that arsenic induced Hippo signaling pathway in the murine skin. This pathway plays crucial roles in determining organ size during the embryonic development and if aberrantly activated in adults, contributes to the pathogenesis of epithelial neoplasm. Arsenic treatment enhanced phosphorylation-dependent activation of LATS1 kinase and other Hippo signaling regulatory proteins Sav1 and MOB1. Phospho-LATS kinase is known to catalyze the inactivation of a transcriptional co-activator, Yap. However, in arsenic-treated epidermis, we did not observed its inactivation. Thus, as expected, unphosphorylated-Yap was translocated to the nucleus in arsenic-treated epidermis. Yap by binding to the transcription factors TEADs induces transcription of its target genes. Consistently, an up-regulation of Yap-dependent target genes Cyr61, Gli2, Ankrd1 and Ctgf was observed in the skin of arsenic-treated mice. Phosphorylated Yap is important in regulating tight and adherens junctions through its binding to αCatenin. We found disruption of these junctions in the arsenic-treated mouse skin despite an increase in αCatenin. These data provide evidence that arsenic-induced canonical Hippo signaling pathway and Yap-mediated disruption of tight and adherens junctions are independently regulated. These effects together may contribute to the carcinogenic effects of arsenic in the skin.

Profiles of Expression of SAV1 in Normoxia or Hypoxia Microenviroment are Associated with Breast Cancer Prognosis.

BACKGROUND: In breast cancer (BC), hypoxia is associated with poor prognosis. Protein Salvador homolog 1 (SAV1) acts as a tumor suppressor and is downregulated in the cancer cells. However, there is limited data on the expression profile of SAV1 and its importance in BC. It has not been studied to evaluate this phenomenon in a hypoxic microenvironment yet. AIM: This study aimed to investigate SAV1 expression profiles under normoxia and hypoxia, and the potential of SAV1 in BC prognosis. METHODS: Gene and protein expression analyses were performed using Real-Time quantitative PCR (RT-qPCR) and immunocytochemistry (ICC), respectively, and in silico analyses were performed using The Cancer Genome Atlas (TCGA). The survival curves were constructed using KMplotter. RESULTS: SAV1 expression was lower in BC samples and tumor cell lines than in normal samples. The SAV1 mRNA levels were reduced in hypoxic estrogen receptor positive (ER+) tumors, which were associated with a lower survival probability as compared to normoxic ER+ tumors. Furthermore, lower levels of SAV1 were found in advanced cancer stage samples, which are associated with worse survival curves and can be a risk factor for BC. CONCLUSIONS: These data suggest a potential prognostic role of SAV1 in BC, with lower expressions associated with worse prognosis.

Impaired Expression of the Salvador Homolog-1 Gene Is Associated with the Development and Progression of Colorectal Cancer.

Salvador homolog-1 (SAV1) is a component of the Hippo pathway that regulates tissue growth and homeostasis by affecting diverse cell processes, including apoptosis, cell division, and differentiation. The aberrant expression of Hippo pathway components has been observed in various human cancers. This study aimed to examine the expression level of the SAV1 gene in colorectal cancer (CRC) and its prognostic value and associations with tumor progression. We obtained matched pairs of tumor tissue and non-cancerous mucosa of the large intestine from 94 CRC patients as well as 40 colon biopsies of healthy subjects collected during screening colonoscopy. The tissue samples and CRC cell lines were quantified for SAV1 mRNA levels using the quantitative polymerase chain reaction method, while SAV1 protein expression was estimated in the paired tissues of CRC patients using immunohistochemistry. The average level of SAV1 mRNA was decreased in 93.6% of the tumor tissues compared to the corresponding non-cancerous tissues and biopsies of healthy colon mucosa. A downregulated expression of SAV1 mRNA was also noted in the CRC cell lines. Although the average SAV1 immunoreactivity was increased in the CRC samples compared to the non-cancerous tissues, a decreased immunoreactivity of the SAV1 protein in the tumor specimens was associated with lymph node involvement and higher TNM disease stage and histological grade. The results of our study suggest that the impaired expression of SAV1 is involved in CRC progression.

lncRNA-FMR6 directly binds SAV1 to increase apoptosis of granulosa cells in premature ovarian failure.

BACKGROUND: A regulatory mechanism of lncRNA binding to protein has been detected in premature ovarian failure (POF). Therefore, this study was expected to illustrate the mechanism of lncRNA-FMR6 and SAV1 regulating POF. METHODS: Follicular fluid and ovarian granulosa cells (OGCs) from POF patients and healthy volunteers were collected. Using RT-qPCR and western blotting, lncRNA-FMR6 and SAV1 expression were detected. KGN cells were cultured, and the subcellular localization analysis of lncRNA-FMR6 was carried out. In addition, KGN cells were treated with lncRNA-FMR6 knockdown/overexpression or SAV1 knockdown. Then, cell optical density (proliferation), apoptosis rate, Bax and Bcl-2 mRNA expression were explored by CCK-8, caspase-3 activity, flow cytometry and RT-qPCR analysis. By performing RIP and RNA pull-down experiments, the interactions among lncRNA-FMR6 and SAV1 was investigated. RESULTS: Up-regulation of lncRNA-FMR6 was shown in follicular fluid and OGCs of POF patients, and ectopic overexpression of lncRNA-FMR6 promoted KGN cells apoptosis and inhibited proliferation. lncRNA-FMR6 was localized in the cytoplasm of KGN cells. SAV1 bounding to lncRNA-FMR6 was negatively regulated by lncRNA-FMR6, and was down-regulated in POF. SAV1 knockdown promoted KGN cells proliferation and inhibited apoptosis, and partially eliminated the effect of lncRNA-FMR6 low expression on KGN cells. CONCLUSION: Overall, lncRNA-FMR6 accelerates POF progression by binding to SAV1.

Smoke-induced SAV1 Gene Promoter Hypermethylation Disrupts YAP Negative Feedback and Promotes Malignant Progression of Non-small Cell Lung Cancer.

YAP (gene symbol YAP1) as a potential oncoprotein, is positively correlated with the malignancy of various tumors. However, overexpression of YAP alone in multiple normal tissue cells has failed to induce tumor formation and the underlying mechanism is poorly understood. Herein, we show that YAP activation directly induces transcription of its negative regulator, SAV1, to constitute a negative feedback loop, which plays a vital role in maintaining lung epithelial cell homeostasis and was dysregulated in non-small cell lung cancer (NSCLC). Notably, smoking promotes the hypermethylation of the SAV1 promoter region, which disrupts YAP negative feedback by inactivating the Hippo pathway. Besides, exogenous overexpression of SAV1 can act as a traffic protein, activating the Hippo signaling and concurrently inhibiting the WNT pathway to decrease cancer cell growth. Furthermore, using the lung cancer organoids, we found that lentivirus-mediated SAV1 gene transfer combined with methylation inhibitor and YAP-TEAD inhibitor is a potential feasible clinical medication regimen for the lung cancer patient, especially among the smoking population. Thus, this SAV1 mediated feedback loop provides an efficient mechanism to establish the robustness and homeostasis of YAP regulation and as a potential target of gene therapy for the smoking NSCLC population.

Transmembrane protein KIRREL1 regulates Hippo signaling via a feedback loop and represents a therapeutic target in YAP/TAZ-active cancers.

The Hippo tumor-suppressor pathway is frequently dysregulated in human cancers and represents a therapeutic target. However, strategies targeting the mammalian Hippo pathway are limited because of the lack of a well-established cell-surface regulator. Here, we show that transmembrane protein KIRREL1, by interacting with both SAV1 and LATS1/2, promotes LATS1/2 activation by MST1/2 (Hippo kinases), and LATS1/2 activation, in turn, inhibits activity of YAP/TAZ oncoproteins. Conversely, YAP/TAZ directly induce the expression of KIRREL1 in a TEAD1-4-dependent manner. Indeed, KIRREL1 expression positively correlates with canonical YAP/TAZ target gene expression in clinical tumor specimens and predicts poor prognosis. Moreover, transgenic expression of KIRREL1 effectively blocks tumorigenesis in a mouse intrahepatic cholangiocarcinoma model, indicating a tumor-suppressor role of KIRREL1. Hence, KIRREL1 constitutes a negative feedback mechanism regulating the Hippo pathway and serves as a cell-surface marker and potential drug target in cancers with YAP/TAZ dependency.

The tumor suppressor role of salvador family WW domain-containing protein 1 (SAV1): one of the key pieces of the tumor puzzle.

PURPOSE: In the complex tumor scenario, understanding the function of proteins with protumor or antitumor roles is essential to support advances in the cancer clinical area. Among them, the salvador family WW domain-containing protein 1 (SAV1) is highlighted. This protein plays a fundamental role in the tumor suppressor face of the Hippo pathway, which are responsible for controlling cell proliferation, organ size, development and tissue homeostasis. However, the functional dysregulation of this pathway may contribute to tumorigenesis and tumor progression. As SAV1 is a tumor suppressor scaffold protein, we explored the functions performed by SAV1 with its partners, the regulation of its expression, and its antitumor role in various types of cancer. METHODS: We selected and analyzed 80 original articles and reviews from Pubmed that focuses on the study of SAV1 in cancer. RESULTS: SAV1 interacts with several proteins, has different functions and acts as tumor suppressor by other mechanisms besides Hippo pathway. SAV1 expression regulation seems to occur by microRNAs and rarely by mutation or promoter methylation. It is downregulated in different types of cancer, which leads to cancer promotion and progression and is associated with poor prognosis. In vivo models have shown that the loss of SAV1 contributes to tumorigenesis. CONCLUSION: SAV1 plays a relevant role as tumor suppressor in several types of cancer, highlighting SAV1 and the Hippo pathway's importance to cancer. Thus, encouraging further studies to include the SAV1 as a molecular key piece in cancer biology and in clinical approaches to cancer.

Non-Lethal Sequential Individual Monitoring of Viremia in Relation to DNA Vaccination in Fish-Example Using a Salmon Alphavirus DNA Vaccine in Atlantic Salmon Salmo salar.

Traditionally, commercial testing for vaccine efficacy has relied on the mass infection of vaccinated and unvaccinated animals and the comparison of mortality prevalence and incidence. For some infection models where disease does not cause mortality this approach to testing vaccine efficacy is not useful. Additionally, in fish experimental studies on vaccine efficacy and immune response the norm is that several individuals are lethally sampled at sequential timepoints, and results are extrapolated to represent the kinetics of immune and disease parameters of an individual fish over the entire experimental infection period. In the present study we developed a new approach to vaccine testing for viremic viruses in fish by following the same individuals over the course of a DNA vaccination and experimental infection through repeated blood collection and analyses. Injectable DNA vaccines are particularly efficient against viral disease in fish. To date, two DNA vaccines have been authorised for use in fish farming, one in Canada against Infectious Haemorrhagic Necrotic virus and more recently one in Europe against Salmon Pancreatic Disease virus (SPDv) subtype 3. In the current study we engineered and used an experimental DNA vaccine against SPDv subtype 1. We measured viremia using a reporter cell line system and demonstrated that the viremia phase was completely extinguished following DNA vaccination. Differences in viremia infection kinetics between fish in the placebo group could be related to subsequent antibody levels in the individual fish, with higher antibody levels at terminal sampling in fish showing earlier viremia peaks. The results indicate that sequential non-lethal sampling can highlight associations between infection traits and immune responses measured at asynchronous timepoints and, can provide biological explanations for variation in data. Similar to results observed for the SPDv subtype 3 DNA vaccine, the SPDv subtype 1 DNA vaccine also induced an interferon type 1 response after vaccination and provided high protection against SPDv under laboratory conditions when fish were challenged at 7 weeks post-vaccination.

SAV1, regulated by HERC4, inhibits the proliferation, migration, and invasion of hepatocellular carcinoma.

BACKGROUND: Hepatic carcinoma is one of the most malignant cancers worldwide. Salvador 1 (SAV1) plays a key role in a variety of human carcinogenesis. This study investigated the role of SAV1 and HERC4 in hepatocellular carcinoma (HCC). METHODS: SAV1 and HERC4 expressions in HCC tissues were examined using RT-qPCR assay. The regulatory effect of HERC4 on SAV1 was verified by co-immunoprecipitation (Co-IP), RT-qPCR, Western blot, and immunofluorescent assays in HEP3B and Huh 7 cell lines. In addition, functional experimental verification was performed through Edu staining, colony formation, and Transwell assay. Finally, Xenograft tumor model was finally used in nude mice. RESULTS: Clinical features showed significant difference with SAV1 and HERC4 expression. HERC4 was found to be upregulated, while SAV1 was downregulated in HCC. Patients with high HERC4 or low SAV1 had a worse prognosis. Results showed that HERC4 could notably decreased the expression level of SAV1 in HCC cells. Our results showed that overexpression HERC4 could reverse the inhibitory effects of SAV1 on HCC cell proliferation, migration, and invasion. SAV1 overexpression repressed tumor growth and enhance caspase 3 expression. CONCLUSION: SAV1 can be directly downregulated by HERC4, indicating that the HERC4/SAV1 axis might have great promise for targeted therapies of HCC.

RNA-binding protein Musashi2 regulates Hippo signaling via SAV1 and MOB1 in pancreatic cancer.

Musashi 2 (MSI2), a member of the Musashi RNA-binding family, is reported to be an oncoprotein in pancreatic ductal adenocarcinoma (PDAC), but the mechanisms of MSI2 in the development and progression of PDAC have not been fully demonstrated. In this research, we studied the clinical significance, biologic effects and the underlying mechanism of MSI2 in the progression of PDAC. The expression of MSI2, Mps-binding protein 1 (MOB1) and Salvador family WW domain-containing protein 1 (SAV1) in PDAC tissues were analyzed immunohistochemically. The biologic effects of MSI2 regarding PDAC cell proliferation, migration and invasion were studied using gain- and loss-of-function assays. MSI2 regulated Hippo signaling pathway via SAV1 and MOB1 was tested in several PDAC cell lines, and the mechanisms were studied using molecular biologic methods. The expression of MSI2 was significantly increased in PDAC cell lines and tissues, and positively associated with tumor poorer differentiation, lymph nodes metastasis and TNM stages. Overexpression of MSI2 promoted PDAC cells proliferation, migration and invasion. Further studies demonstrated that MSI2 regulated the Hippo signaling pathway via directly binding to the mRNAs of SAV1 and MOB1, and controlled the translation and stability of SAV1 and the translation of MOB1. This study demonstrated that MSI2 regulated the Hippo signaling pathway via suppressing SAV1 and MOB1 at post-transcriptional level and promoted PDAC progression.