WNK Lysine Deficient Protein Kinase Research Articles

Downregulation of WNK4 expression facilitates the proliferation of gastric cancer cells via activation of the STAT3 signaling pathway.

WNK lysine deficient protein kinase 4 (WNK4) has been shown to be significantly associated with cancer progression. Nevertheless, its involvement in gastric cancer (GC) is unclear. The objective of this work was to investigate the WNK4's regulatory mechanism in GC. Quantitative RT-PCR and immunoblots revealed that WNK4 expression was downregulated in GC and that low expression of WNK4 was strongly linked to poor prognosis. Functional assays including cell counting kit-8 assay and colony formation assay demonstrated that overexpression of WNK4 led to limited tumor proliferation both in vitro and in vivo, while the WNK4 reduction yielded to the opposite results. Gene Set Enrichment Analysis (GSEA) indicated a potential association between WNK4 and the signal transducer and activator of transcription (STAT3). WNK4 suppressed the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) in GC cells. The inhibition of the STAT3 pathway with Stattic reversed growth and proliferation induced by WNK4 knockdown in GC cells. These findings provide new insights for identifying key therapeutic targets for GC in the future.

Abstract 1757 Investigating the effect of WNK lysine deficient protein kinase inhibition in breast cancer

Abstract 1757 Investigating the effect of WNK lysine deficient protein kinase inhibition in breast cancer

Profiling Kinase Activities for Precision Oncology in Diffuse Gastric Cancer.

Gastric cancer (GC) remains a leading cause of cancer-related mortality globally. This is due to the fact that majority of the cases of GC are diagnosed at an advanced stage when the treatment options are limited and prognosis is poor. The diffuse subtype of gastric cancer (DGC) under Lauren's classification is more aggressive and usually occurs in younger patients than the intestinal subtype. The concept of personalized medicine is leading to the identification of multiple biomarkers in a large variety of cancers using different combinations of omics technologies. Proteomic changes including post-translational modifications are crucial in oncogenesis. We analyzed the phosphoproteome of DGC by using paired fresh frozen tumor and adjacent normal tissue from five patients diagnosed with DGC. We found proteins involved in the epithelial-to-mesenchymal transition (EMT), c-MYC pathway, and semaphorin pathways to be differentially phosphorylated in DGC tissues. We identified three kinases, namely, bromodomain adjacent to the zinc finger domain 1B (BAZ1B), WNK lysine-deficient protein kinase 1 (WNK1), and myosin light-chain kinase (MLCK) to be hyperphosphorylated, and one kinase, AP2-associated protein kinase 1 (AAK1), to be hypophosphorylated. LMNA hyperphosphorylation at serine 392 (S392) was demonstrated in DGC using immunohistochemistry. Importantly, we have detected heparin-binding growth factor (HDGF), heat shock protein 90 (HSP90), and FTH1 as potential therapeutic targets in DGC, as drugs targeting these proteins are currently under investigation in clinical trials. Although these new findings need to be replicated in larger study samples, they advance our understanding of signaling alterations in DGC, which could lead to potentially novel actionable targets in GC.

Lateral Diffusion of NKCC1 Contributes to Chloride Homeostasis in Neurons and Is Rapidly Regulated by the WNK Signaling Pathway.

An upregulation of the Na+-K+-2Cl- cotransporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABA type A receptors (GABAARs) and, thus, to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cell mechanisms regulating NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK lysine deficient protein kinase 1 (WNK1) and the downstream Ste20 Pro-line Asparagine Rich Kinase (SPAK) kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl- concentration, but it participates in the elevation of intraneuronal Cl- concentration in hyperactivity conditions associated with an up-regulation of NKCC1. The fact that the main chloride exporter, the K+-Cl- cotransporter KCC2, is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.

Loss of WNK1 Suppressed the Malignant Behaviors of Hepatocellular Carcinoma Cells by Promoting Autophagy and Activating AMPK Pathway.

WNK lysine deficient protein kinase 1 (WNK1) has been shown to be highly expressed in hepatocellular carcinoma (HCC) samples and related to poor prognosis of HCC patients based on bioinformatics analysis. However, the specific function of WNK1 in HCC has not been analyzed. This study is aimed at exploring the function of WNK1 in HCC progression as well as its related molecular mechanism. After knockdown of WNK1 by small interference RNA, cell counting kit-8, colony formation, western blot, Transwell, and wound healing assays were employed to evaluate the biological behaviors of HCC cells. Immunofluorescent staining was applied to detect the effect of WNK1 on LC3 II. GSK690693 or si-AMPK was applied to block AMPK pathway. The expression of autophagy and AMPK pathway related molecules was examined by western blot assay. WNK1 was highly expressed in HCC cell lines and loss of WNK1 inhibited HCC cell proliferation, cell cycle, migration, and invasion. Additionally, we demonstrated that loss of WNK1 promoted the autophagy and activated AMPK pathway in HCC cells. While, GSK690693 treatment or si-AMPK transfection suppressed the autophagy and promoted HCC cells proliferation. However, WNK1 knockdown counteracted the effect of GSK690693 or si-AMPK in regulating HCC cell proliferation. Finally, we demonstrated that WNK1 regulated the malignant behaviors of HCC cells by modulating autophagy and AMPK pathway. The above results indicated that WNK1 may be a worthwhile target to be considered for therapy of HCC.

STEM-04. CONNEXIN 43 DRIVES GLIOBLASTOMA CANCER STEM CELL PHENOTYPES THROUGH A WNK1 SIGNALING AXIS

Abstract Tumors represent a highly dynamic system that relies on coordinated signaling to drive growth and adapt to selective pressures, including those induced by anti-cancer therapies. Given the need for tumors to engage in rapid and coordinated cell-cell communication, mechanisms including gap junction intracellular communication would be essential. Surprisingly, the connexin proteins that make up gap junctions have traditionally been considered tumor suppressors. This hypothesis was based on the loss of GJIC in cancer cells and, frequently, lower connexin expression in tumor cells compared to non-neoplastic tissue, including in glioblastoma (GBM), where connexin 43 (Cx43) was shown to be decreased. However, recent data suggest that this tumor-suppressive effect is context dependent and that connexins can function in a tumor-promoting role. We previously found that GBM cancer stem cells (CSCs) rely on gap junctional intercellular communication through connexin 46 (Cx46) for survival. We now identify a subset of GBM patient-derived xenograft (PDX) CSC models that also rely on Cx43 in addition to Cx46. This dependence on Cx43 is independent of Cx43 expression level. Knockdown of Cx43 using shRNA in these PDX models compromises their survival and self-renewal while inducing apoptosis. We leveraged a protein kinase phospho array to identify signaling changes associated with Cx43 knockdown and observed reductions in phosphorylation of WNK lysine-deficient protein kinase 1 (WNK1) in addition to ERK1/2, AKT, and p38MAPK. Immunoblotting validated that WNK1 phosphorylation is lost in the absence of Cx43. Treatment with a WNK1 inhibitor was toxic to CSCs. Current work is focused on interrogating the link among Cx43, WNK1 phosphorylation, and downstream signaling events and the therapeutic window of WNK1 inhibition. This work identifies a potential new signaling axis downstream of Cx43 in glioblastoma CSCs and further strengthens the hypothesis that connexins may act as both tumor suppressors and tumor drivers depending on context.

WNK3 inhibition elicits antitumor immunity by suppressing PD-L1 expression on tumor cells and activating T-cell function

Immune checkpoint therapies, such as programmed cell death ligand 1 (PD-L1) blockade, have shown remarkable clinical benefit in many cancers by restoring the function of exhausted T cells. Hence, the identification of novel PD-L1 regulators and the development of their inhibition strategies have significant therapeutic advantages. Here, we conducted pooled shRNA screening to identify regulators of membrane PD-L1 levels in lung cancer cells targeting druggable genes and cancer drivers. We identified WNK lysine deficient protein kinase 3 (WNK3) as a novel positive regulator of PD-L1 expression. The kinase-dead WNK3 mutant failed to elevate PD-L1 levels, indicating the involvement of its kinase domain in this function. WNK3 perturbation increased cancer cell death in cancer cell–immune cell coculture conditions and boosted the secretion of cytokines and cytolytic enzymes, promoting antitumor activities in CD4+ and CD8+ T cells. WNK463, a pan-WNK inhibitor, enhanced CD8+ T-cell-mediated antitumor activity and suppressed tumor growth as a monotherapy as well as in combination with a low-dose anti-PD-1 antibody in the MC38 syngeneic mouse model. Furthermore, we demonstrated that the c-JUN N-terminal kinase (JNK)/c-JUN pathway underlies WNK3-mediated transcriptional regulation of PD-L1. Our findings highlight that WNK3 inhibition might serve as a potential therapeutic strategy for cancer immunotherapy through its concurrent impact on cancer cells and immune cells.

Identification of Differential Expression Genes between Volume and Pressure Overloaded Hearts Based on Bioinformatics Analysis.

Volume overload (VO) and pressure overload (PO) are two common pathophysiological conditions associated with cardiac disease. VO, in particular, often occurs in a number of diseases, and no clinically meaningful molecular marker has yet been established. We intend to find the main differential gene expression using bioinformatics analysis. GSE97363 and GSE52796 are the two gene expression array datasets related with VO and PO, respectively. The LIMMA algorithm was used to identify differentially expressed genes (DEGs) of VO and PO. The DEGs were divided into three groups and subjected to functional enrichment analysis, which comprised GO analysis, KEGG analysis, and the protein–protein interaction (PPI) network. To validate the sequencing data, cardiomyocytes from AR and TAC mouse models were used to extract RNA for qRT-PCR. The three genes with random absolute values of LogFC and indicators of heart failure (natriuretic peptide B, NPPB) were detected: carboxylesterase 1D (CES1D), whirlin (WHRN), and WNK lysine deficient protein kinase 2 (WNK2). The DEGs in VO and PO were determined to be 2761 and 1093, respectively, in this study. Following the intersection, 305 genes were obtained, 255 of which expressed the opposing regulation and 50 of which expressed the same regulation. According to the GO and pathway enrichment studies, DEGs with opposing regulation are mostly common in fatty acid degradation, propanoate metabolism, and other signaling pathways. Finally, we used Cytoscape’s three techniques to identify six hub genes by intersecting 255 with the opposite expression and constructing a PPI network. Peroxisome proliferator-activated receptor (PPARα), acyl-CoA dehydrogenase medium chain (ACADM), patatin-like phospholipase domain containing 2 (PNPLA2), isocitrate dehydrogenase 3 (IDH3), heat shock protein family D member 1 (HSPD1), and dihydrolipoamide S-acetyltransferase (DLAT) were identified as six potential genes. Furthermore, we predict that the hub genes PPARα, ACADM, and PNPLA2 regulate VO myocardial changes via fatty acid metabolism and acyl-Coa dehydrogenase activity, and that these genes could be employed as basic biomarkers for VO diagnosis and treatment.

Abstract 1121: Identification and in vivo validation of unique anti-oncogenic properties and mechanisms involving protein kinase signalling and autophagy mediated by the investigational new agent PV-10

Abstract Introduction: PV-10 (10% Rose Bengal) is a small molecule agent previously shown to have potent immunotherapeutic and anti-tumor activities against a number of tumors including metastatic melanoma and refractory neuroblastoma, and is currently undergoing clinical testing as a single-agent for refractory metastatic neuroendocrine cancer (NCT02693067) and in combination with checkpoint inhibitors for metastatic melanoma (NCT02557321) and metastatic uveal melanoma (NCT00986661). We have previously determined that PV-10 induces cell death at pharmacologically relevant concentrations in a panel of phenotypically diverse adult solid tumor cell lines. However, the molecular consequences of this phenomenon have not yet been fully elucidated. In this study, we investigated the target validation and modulation of PV-10 on protein kinase signalling and their associated impact on specific oncogenic pathways in these tumor cells. Methods: A panel of human tumor cell lines derived from breast (MCF-7, T-47D, MDA-MB-231), colorectal (LoVo, T-84), head and neck (CAL-27, Detroit-562, FaDu, UM-SCC-1), and testicular (NCC-IT, NTERA-2, TCAM-2) tissues were treated with PV-10 and their cytotoxic and pro-apoptotic effects were described. Protein kinase profiling was performed using the human phospho-kinase antibody array (#ARY003C, R&D Systems). Western blotting was used to investigate autophagic markers and protein kinase activity. Cell migration inhibition was determined by wound healing assay following treatment with a sublethal dose of PV-10 or pan-WNK inhibitor WNK463. Tumor xenograft studies were carried out according to established protocols. Results: Treatment with PV-10 leads to consistent inhibition of WNK lysine deficient protein kinase 1 (WNK1) phosphorylation based on protein kinase profiling of drug-treated cancer cells when compared with vehicle-treated cells. WNK1 has been implicated as an inhibitor of autophagy and a promoter of cell migration and invasion in several cancers. Western blot analysis showed that PV-10 leads to the downregulation of SQSTM1/p62, upregulation of Beclin-1, and conversion of LC3B-I into LC3B-II, indicating activation of autophagy. PV-10 was also found to significantly inhibit the migration of cancer cells similar to the inhibitor WNK463. Significant tumor regression and target modulation was noted in tumor-bearing animals treated with PV-10. Conclusion: In addition to the known activity of PV-10 to mediate tumor-specific immune responses and cytotoxic effects in neoplasms, we have identified novel therapeutic targets for PV-10, such as WNK1, and provide new insights into its effect on autophagy and metastasis. Our data provide essential mechanism-based evidence and biomarkers of activity to formulate effective PV-10 backbone clinical studies in the future. Citation Format: Son Tran, Satbir Thakur, Mohit Jain, Chunfen Zhang, Aru Narendran. Identification and in vivo validation of unique anti-oncogenic properties and mechanisms involving protein kinase signalling and autophagy mediated by the investigational new agent PV-10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1121.

Clinicopathologic and molecular characteristics of EGFR-mutant lung adenocarcinomas that transform to small cell lung cancer after TKI therapy.

BackgroundSmall cell lung cancer (SCLC) transformation is one of the mechanisms of drug resistance to tyrosine kinase inhibitors (TKIs) in advanced epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) and represents an increasingly recognized clinical dilemma.MethodsWe performed a retrospective review of 964 cases at the University of California, San Diego of patients with EGFR sensitizing mutations. Nine patients had a biopsy-confirmed small cell transformation. The unique gene alterations and clinicopathologic features were collected and analyzed.ResultsNine cases (9/964, 0.9%) were identified, all with stage IV adenocarcinoma (ADC) at diagnosis, 7 were poorly differentiated, and 7 had an EGFR exon 19 deletion. All nine patients had tumor protein p53 (TP53) mutation. Among seven cases that had next-generation sequencing (NGS), 5 harbored retinoblastoma 1 (RB1) loss. WNK lysine deficient protein kinase 1 (WNK1) mutation was found in two patients that had longer survival. The median time from the initial diagnosis to transformation was 22.7 months (IQR: 15.1–25.1). After small cell transformation on EGFR inhibition, all patients were treated with etoposide/platinum, conferring a median progression-free survival (PFS) of 3.2 months (IQR, 2.2–6.5 months) and post-chemotherapy survival of 8.6 months (IQR, 4.0–19.0 months). Six patients, as they retained the initial EGFR mutations, resumed (did so after terminating chemotherapy)/continued (did so concomitantly with chemotherapy) TKIs with a median duration of 13.8 months (IQR, 3.8–27.7 months). Two patients received immunotherapy but had no benefit.ConclusionsIn our series, most patients with small cell transformation had poorly differentiated adenocarcinomas at baseline. RB1 loss was not universal in transformed patients in this series, though TP53 mutation was present in all tumor samples. WNK1 mutation may be a new resistance mechanism to TKIs that may be associated with improved survival.

Aberrant expression of WNK lysine deficient protein kinase 1 is associated with poor prognosis of colon adenocarcinoma.

WNK1 (WNK lysine deficient protein kinase 1) is a kind of protein kinase and participates in angiogenesis, having a potent tumor promoting role. WNK1 is ubiquitously expressed, and its upregulated expression has been reported in several tumor types. Here, we aimed to investigate the correlation between WNK1 expression and colon adenocarcinoma (COAD) progression. In the current study, WNK1 expression was evaluated by immunohistochemically in adjacent normal colonic mucosae and primary adenocarcinomas. The effect of WNK1 on overall survival (OS) and its associations with the clinicopathological parameters were analyzed in a retrospective cohort of COAD patients (n = 185). The tumor-related effects of WNK1 in COAD were further tested via cellular and mice experiments. According to our cohort, higher WNK1 expression was significantly associated with unfavorable prognostic factors, such as high pT stage, pN stage, as well as shorter OS. Moreover, WNK1 exhibited tumor promoting role in COAD cancer cell lines as well as in nude mice. Silencing WNK1 can significantly inhibit the proliferation of COAD both in vitro and in vivo. In all, WNK1 acts as a tumor promoter and may be used as a COAD prognostic biomarker.

Long non-coding RNA LINC00858 inhibits colon cancer cell apoptosis, autophagy, and senescence by activating WNK2 promoter methylation

Accumulating evidence shows the involvement of long non-coding RNAs (lncRNAs) in tumorigenesis of many types of human cancers. However, the role of LINC00858 in colon cancer has not been fully elucidated. Therefore, we investigated the involvement of LINC00858 in the progression of colon cancer and identified its downstream targets. After examining the expression of LINC00858 in colon cancer tissues and cell lines, we then identified the possible interaction between LINC00858 and WNK lysine deficient protein kinase 2 (WNK2) by fluorescence in situ hybridization, RNA immunoprecipitation, chromatin immunoprecipitation, and RNA pull-down assays. Next, the role of the LINC00858/WNK2 axis was explored by evaluating the apoptosis, autophagy, and senescence of colon cancer cells in vitro after ectopic expression and depletion experiments in HCT116 cells. Moreover, a mouse xenograft model of HCT116 cells was established to verify the function of the LINC00858/WNK2 axis in vivo. There was high expression of LINC00858 and low expression of WNK2 in colon cancer tissues and cell lines. Silencing of LINC00858 promoted apoptosis, senescence, and autophagy in colon cancer cells. Additionally, the enrichment of WNK2 was promoted when LINC00858 bound to DNA methyltransferases. Furthermore, in vivo assays demonstrated that silencing of LINC00858 resulted in inhibited tumor growth by upregulating WNK2. In summary, LINC00858 acts as a tumor-promoting lncRNA in colon cancer by downregulating WNK2. Our results may provide novel targets for the treatment for colon cancer.

Prognostic and Clinical Implications of WNK Lysine Deficient Protein Kinase 1 Expression in Patients With Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a particularly malignant form of cancer prevalent throughout the world; however, there is a pressing need for HCC biomarkers to facilitate prognosis and risk assessment. This paper reports on the potential prognostic value of WNK lysine deficient protein kinase 1 (WNK1) in cases of HCC. We analyzed the expression of WNK1 at the mRNA level using omics data from the UALCAN database. We then verified our findings through the immunohistochemical (IHC) staining of various human cancer tissue as well as 59 HCC samples paired with corresponding normal tissues. The prognostic value of mRNA or protein expression by WNK1 was evaluated using the Kaplan-Meier method. Initial screening results revealed significantly higher WNK1 expression levels in HCC tissue compared to normal tissue. Verification using the paired HCC samples confirmed that the expression of WNK1 was indeed significantly higher in HCC tissue samples than in adjacent normal tissues. High WNK1 expression levels were significantly correlated with clinicopathological variables, including gender and histologic grade. Kaplan-Meier survival analysis revealed that high WNK1 expression levels were associated with poor HCC prognosis. Finally, univariate and multivariate analysis identified WNK1 as a prognostic factor for TNM stage in cases of HCC. In summary, WNK1 is overexpressed at the mRNA and protein levels, and correlated with poor prognosis. Thus, WNK1 expression could potentially be used as a biomarker in HCC prognosis.

Establishment and characterization of a novel cell line (cc‑006cpm8) of moderately/poorly differentiated colorectal adenocarcinoma derived from a primary tumor of a patient.

In the present study, the cc‑006cpm8 novel colon cell line was established from a sample of right colorectal adenocarcinoma obtained from a woman with liver metastasis. It was possible to culture this cell line for ≥100passages invitro with vigorous growth. Morphologically, the cells grew as several layers with tight adhesion to the surface of the culture plate. The morphological, immunological and ultrastructural features of these cells suggested their epithelial origin. The characterization of this cell line indicated a doubling time of 27h, a colony forming efficiency of 73.2% in semisolid media and a plate efficiency of 66.5% in liquid culture. The modal number of chromosomes was50. Invivo, the cc‑006cpm8 cells underwent tumorigenesis in all nude mice used. Immunohistochemical analysis demonstrated that mutS homolog2(MSH2) and MSH6 were expressed; however, mutL homolog1 and postmeiotic segregation2 were downregulated in cc‑006cpm8 cells. To determine the mutation profile of the cell line analyzed, exome capture DNA sequencing was performed. The results revealed 20hypermutated exons comprising single nucleotide polymorphisms, and insertion and deletions(InDels), including single nucleotide variants of mucin(MUC)19, MUC16, MUC12, filaggrin and AHNAK nucleoprotein2, and InDels of βdefensin‑126, microRNA‑3665, WNK lysine deficient protein kinase1 and SLAIN motif‑containing protein1. In addition, commonly mutated genes in colorectal cancer and exon mutations of genes in cc‑006cpm8 cells were analyzed, including adenomatous polyposis coli, tumor protein p53, Drosophila mothers against decapentaplegic4, phosphatidylinositol‑4,5‑bisphosphate3‑kinase catalytic subunitα and Kirsten rat sarcoma, and genes associated with the DNA mismatch repair pathway were investigated.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na+/Cl- cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4-/- mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2+-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Integrated analysis of gene expression and copy number variations in MET proto‑oncogene‑transformed human primary osteoblasts.

The aim of the present study was to screen the potential osteosarcoma (OS)‑associated genes and to obtain additional insight into the pathogenesis of OS. Transcriptional profile (ID: GSE28256) and copy number variations (CNV) profile were downloaded from Gene Expression Omnibus database. Differentially expressed genes (DEGs) between MET proto‑oncogene‑transformed human primary osteoblast (MET‑HOB) samples and the control samples were identified using the Linear Models for Microarray Data package. Subsequently, CNV areas and CNVs were identified using cut‑off criterion of >30%‑overlap within the cases using detect_cnv.pl in PennCNV. Genes shared in DEGs and CNVs were obtained and discussed. Additionally, the Database for Annotation, Visualization and Integrated Discovery was used to identify significant Gene Ontology (GO) functions and pathways in DEGs with P<0.05. A total of 1,601 DEGs were screened out in MET‑HOBs and compared with control samples, including 784 upregulated genes, such as E2F transcription factor 1 (E2F1) and 2 (E2F2) and 817 downregulated genes, such as retinoblastoma 1 (RB1) and cyclin D1 (CCND1). DEGs were enriched in 344 GO terms, such as extracellular region part and extracellular matrix and 14 pathways, including pathways in cancer and extracellular matrix‑receptor interaction. Additionally, 239 duplications and 439 deletions in 678 genes from 1,313 chromosome regions were detected. A total of 12 genes were identified to be CNV‑driven genes, including cadherin 18, laminin subunit α 1, spectrin β, erythrocytic, ciliary rootlet coiled‑coil, rootletin pseudogene2, β‑1,4-N-acetyl-galactosaminyltransferase 1, G protein regulated inducer of neurite outgrowth 1, EH domain binding protein 1‑like 1, growth factor independent 1, cathepsin Z, WNK lysine deficient protein kinase 1, glutathione S‑transferase mu 2 and microsomal glutathione S‑transferase1. Therefore, cell cycle‑associated genes including E2F1, E2F2, RB1 and CCND1, and cell adhesion‑associated genes, such as CDH18 and LAMA1 may be used as diagnosis and/or therapeutic markers for patients with OS.

WNK lysine deficient protein kinase 1 regulates human endometrial stromal cell decidualization, proliferation, and migration in part through mitogen-activated protein kinase 7.

The differentiation of endometrial stromal cells into decidual cells, termed decidualization, is an integral step in the establishment of pregnancy. The mitogen-activated protein kinase homolog, WNK lysine deficient protein kinase 1 (WNK1), is activated downstream of epidermal growth factor receptor during decidualization. Primary human endometrial stromal cells (HESCs) were subjected to small interfering RNA knockdown of WNK1 followed by in vitro decidualization. This abrogated expression of the decidual marker genes, insulin like growth factor binding protein 1 (IGFBP1) and prolactin (PRL), and prevented adoption of decidual cell morphology. Analysis of the WNK1-dependent transcriptome by RNA-Seq demonstrated that WNK1 regulates the expression of 1858 genes during decidualization. Gene ontology and upstream regulator pathway analysis showed that WNK1 regulates cell migration, differentiation, and proliferation. WNK1 was required for many of the gene expression changes that drive decidualization, including the induction of the inflammatory cytokines, C-C motif chemokine ligand 8 (CCL8), interleukin 1 beta (IL1B), and interleukin 15 (IL15), and the repression of transforming growth factor-beta (TGF-beta) pathway genes, including early growth response 2 (EGR2), SMAD family member 3 (SMAD3), integrin subunit alpha 2 (ITGA2), integrin subunit alpha 4 (ITGA4), and integrin subunit beta 3 (ITGB3). In addition to abrogating decidualization, WNK1 knockdown decreased the migration and proliferation of HESCs. Furthermore, mitogen-activated protein kinase 7 (MAPK7), a known downstream target of WNK1, was activated during decidualization in a WNK1-dependent manner. Small interfering RNA knockdown of MAPK7 demonstrated that MAPK7 regulates a subset of WNK1-regulated genes and controls the migration and proliferation of HESCs. These results indicate that WNK1 and MAPK7 promote migration and proliferation during decidualization and regulate the expression of inflammatory cytokines and TGF-beta pathway genes in HESCs.

MiR-93 inhibits the invasive potential of triple-negative breast cancer cells in vitro via protein kinase WNK1

Despite advances in treatment, the highly metastatic nature of breast tumors has given rise to the urgent need for development of novel therapeutic and prognostic markers. miR-93 is known to regulate the epithelial to mesenchymal transition process and to influence metastatic spread in breast carcinoma, although the exact mechanism(s)/genes involved remain unknown. In the present study, we examined the role of miR-93 in MDA-MB-231 breast cancer cells. Overexpression of mature miR-93-5p in MDA-MB-231 cells decreased cell migratory capability and invasive potential, as well as increased adhesion. In contrast, inhibition of miR-93 induced the opposite effects. miRNA-mRNA target prediction (TargetScan) identified WNK lysine deficient protein kinase1 (WNK1), which is known to interact with diverse signaling pathways and regulate cell proliferation, survival, angiogenesis and metastasis, as one of the potential targets of miR-93. Furthermore, we showed by luciferase assay that WNK1 is a putative miR-93 target. siRNA mediated silencing of WNK1 also decreased the invasive ability of the cells, suggesting that the effects of miR-93 may be attributed at least in part to decreased WNK1 expression. Further invivo studies are required to ascertain the miR-93-WNK1-metastasis cascade, that has potential implications in breast cancer therapy.

Molecular initiating events of the intersex phenotype: Low-dose exposure to 17α-ethinylestradiol rapidly regulates molecular networks associated with gonad differentiation in the adult fathead minnow testis

Intersex, or the presence of oocytes in the testes, has been documented in fish following exposure to wastewater effluent and estrogenic compounds. However, the molecular networks underlying the intersex condition are not completely known. To address this, we exposed male fathead minnows to a low, environmentally-relevant concentration of 17alpha-ethinylestradiol (EE2) (15ng/L) and measured the transcriptome response in the testis after 96h to identify early molecular initiating events that may proceed the intersex condition. The short-term exposure to EE2 did not affect gonadosomatic index and proportion of gametes within the testes. However, the production of 11-ketotestosterone and testosterone from the testis in vitro was decreased relative to controls. Expression profiling using a 8×60K fathead minnow microarray identified 10 transcripts that were differentially expressed in the testes, the most dramatic change being that of coagulation factor XIII A chain (20-fold increase). Transcripts that included guanine nucleotide binding protein (Beta Polypeptide 2), peroxisome proliferator-activated receptor delta, and WNK lysine deficient protein kinase 1a, were down-regulated by EE2. Subnetwork enrichment analysis revealed that EE2 suppressed transcriptional networks associated with steroid metabolism, hormone biosynthesis, and sperm mobility. Most interesting was that gene networks associated with doublesex and mab-3 related transcription factor 1 (dmrt1) were suppressed in the adult testis, despite the fact that dmrt1 itself was not different in expression from control males. Transcriptional networks involving forkhead box L2 (foxl2) (transcript involved in ovarian follicle development) were increased in expression in the testis. Noteworthy was that a gene network associated to granulosa cell development was increased over 100%, suggesting that this transcriptome network may be important for monitoring estrogenic exposures. Other cell processes rapidly downregulated by EE2 at the transcript level included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway. Conversely, lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased. These data provide new insight into the transcriptional responses that are perturbed prior to gonadal remodeling and intersex following exposure to estrogens. These data demonstrate that low concentrations of EE2 (1) rapidly suppresses male hormone production, (2) down-regulate molecular networks related to male sex differentiation, and (3) induce transcriptional networks related to granulosa cell development in the adult testis. These responses are hypothesized to be key molecular initiating events that occur prior to the development of the intersex phenotype following estrogenic exposures.

In Primary Aldosteronism, Mineralocorticoids Influence Exosomal Sodium-Chloride Cotransporter Abundance.

Distal tubular sodium retention is a potent driver of hypertension, and the thiazide-sensitive sodium-chloride cotransporter (NCC) has a key role in this process. In humans, factors regulating NCC are unclear, but in animal models, aldosterone is a potent regulator, possibly via effects on plasma potassium. We studied the effects of the mineralocorticoid fludrocortisone on the abundance of NCC and its phosphorylated form (pNCC) as well as WNK lysine deficient protein kinase 4 (WNK4) and STE20/SPS1-related, proline alanine-rich kinase (SPAK) in human urinary exosomes. We isolated exosomes from daily urine samples in 25 patients undergoing fludrocortisone suppression testing (100 μg every 6 hours for 4 days) to diagnose or exclude primary aldosteronism. Over the course of the test, NCC levels increased 3.68-fold (P<0.01) and pNCC levels increased 2.73-fold (P<0.01) relative to baseline. The ratio of pNCC/NCC dropped by 48% (P<0.01). The abundance of WNK4 increased 3.23-fold (P<0.01), but SPAK abundance did not change significantly (P=0.14). Plasma potassium concentration strongly and negatively correlated with pNCC, NCC, and WNK4 abundance (P<0.001 for all). This study shows that, in humans, mineralocorticoid administration is associated with a rapid increase in abundance of NCC and pNCC, possibly via the WNK pathway. These effects may be driven by changes in plasma potassium.