Induction of chronic lymphocytic leukemia-like disease in STYK1/NOK transgenic mice.

STYK1/NOK functions in a ligand independent and constitutive fashion to provoke tumor formation and to be up-regulated in many types of cancer cells. However, how STYK1/NOK functions at the whole animal level is completely unknown. Here, we found that STYK1/NOK-transgenic (tg) mice spontaneously developed immunosuppressive B-CLL-like disease with generally shorter life spans. The phenotype of STYK1/NOK-induced B-CLL was typically heterogeneous, and most often, presented lymphadenectasis accompanied with hepatomegaly and/or splenomegaly. STYK1/NOK-tg mice also suffered reduced immune responses. The expanded CD5+CD19+ (B1) lymphocyte pool was detected within peripheral lymphoid organs. Analysis on GEO profile revealed that expression of STYK1/NOK were significantly up-regulated in primary human B-CLL. Inoculation of blood cells from sick STYK1/NOK-tg mice into immune-deficient recipients recaptured the B1 malignant phenotype. Our study demonstrated that STYK1/NOK transgenic mouse may serve as a useful model system for the developments of novel diagnosis and treatment of B-CLL.

Lnc-STYK1-2 regulates bladder cancer cell proliferation, migration, and invasion by targeting miR-146b-5p expression and AKT/STAT3/NF-kB signaling.

BACKGROUND: Epigenetic modulation by noncoding RNAs substantially contributes to human cancer development, but noncoding RNAs involvement in bladder cancer remains poorly understood. This study investigated the role of long noncoding RNA (lncRNA) lnc-STYK1-2 in tumorigenesis in cancerous bladder cells. METHODS: Differential lncRNA and mRNA profiles were characterized by high-throughput RNA sequencing combined with validation via quantitative PCR. Bladder cancer cell proliferation was assessed through MTS, and bladder cancer cell migration and invasion were assessed through a Transwell system. The in vivo tumorigenesis of bladder cancer cells was evaluated using the cancer cell line-based xenograft model. The dual-luciferase reporter assay verified the association of miR-146b-5p with lnc-STYK1-2 and the target gene. Protein abundances and phosphorylation were detected by Western blotting. RESULTS: Alterations in lncRNA profiles, including decreased lnc-STYK1-2 expression, were detected in bladder cancer tissues compared with adjacent noncancerous tissues. lnc-STYK1-2 silencing effectively promoted proliferation, migration, and invasion in two bladder cancer cell lines, 5637 and T24, and their tumorigenesis in nude mice. lnc-STYK1-2 siRNA promoted miR-146b-5p and reduced ITGA2 expression in bladder cancer cells. Moreover, miR-146b-5p suppressed ITGA2 expression in bladder cancer cells through direct association. Also, lnc-STYK1-2 directly associated with miR-146b-5p. Finally, miR-146b-5p inhibitors abrogated the alterations in bladder cell functions, ITGA2 expression, and phosphorylation of AKT, STAT3, and P65 proteins in 5637 and T24 cells induced by lnc-STYK1-2 silencing. CONCLUSION: lnc-STYK1-2 inhibited bladder cancer cell proliferation, migration, and tumorigenesis by targeting miR-146b-5p to regulate ITGA2 expression and AKT/STAT3/NF-kB signaling.

Styk1 is specifically expressed in NK1.1+ lymphocytes including NK, γδ T, and iNKT cells in mice, but is dispensable for their ontogeny and function.

Innate T cells, NK cells, and innate-like lymphocytes (ILCs) share transcriptional signatures that translate into overlapping developmental and functional programs. A prominent example for genes that are highly expressed in NK cells but not in ILCs is serine-threonine-tyrosine kinase 1 (Styk1 encoded by Styk1). We found Styk1 to be specifically expressed in lymphocytes positive for Killer cell lectin-like receptor subfamily B, member 1, also known as CD161 or NK1.1, i.e. in NK cell, αß iNKT, and γδ NKT cell lineages. To investigate the role of Styk1 in the development and function of NK1.1+ innate T-cell subsets, we generated and analyzed a novel Styk1null mutant mouse line. Furthermore, we validated Styk1 expression in γδ NKT cells and in thymic, but not in peripheral invariant αß iNKT cells through ex vivo analysis of a concomitantly generated transgenic Styk1 reporter mouse line. Despite the very specific expression of Styk1 in NK cells, γδ NKT cells, and thymic αß iNKT, its absence did not alter homeostasis and function of these lineages. Thus, Styk1 expression is specific for NK cells and selected NK-like innate T-cell subsets, but dispensable for their development and function.

SMAD3 inducing the transcription of STYK1 to promote the EMT process and improve the tolerance of ovarian carcinoma cells to paclitaxel.

OBJECTIVE: To figure out the relationship between SMAD3 and serine-threonine tyrosine kinase (STYK1) in ovarian carcinoma cell's paclitaxel resistance. METHODS: The quantitative reverse transcription-polymerase chain reactpostion and Western blot analysis were used to analyze RNA and protein content of SMAD3 and STYK1, respectively. The chromatin immunoprecipitation assay was used to confirm the binding site of SMAD3 to the STYK1 promoter region. Transwell assay was used to detect cell invasion and migration, and Western Blot was used to detect the marker proteins (vimentin and E-cadherin) of epithelial-mesenchymal transition (EMT) process. MTT and apoptosis assay were used to, respectively, measure cell vitality and apoptosis. In vivo experiments, rats were subcutaneously implanted with A2780 cells to establish an animal model of ovarian cancer and the survival curve was drawn. RESULTS: Upregulating SMAD3 induced the expression of STYK1 in ovarian cancer cell lines. STYK1 is a direct transcriptional target of SMAD3. Upregulating STYK1 improved the paclitaxel resistance of ovarian carcinoma cells. Upregulating STYK1 promoted cell invasion, migration, and the EMT process, and SMAD3 had the same effect with STYK1 on cell invasion, cell migration, and the EMT process. The animal assay showed that downregulating STYK1 inhibited the EMT process and the paclitaxel resistance, further promoting the treatment of cervical cancer. CONCLUSION: SMAD3 combined with the promoter region of STYK1 to promote the transcription process of STYK1, thereby promoting the EMT process and paclitaxel resistance of ovarian cancer cells.

NOK/STYK1 promotes the genesis and remodeling of blood and lymphatic vessels during tumor progression.

Previous studies have indicated that the overexpression of NOK, also named STYK1, led to tumorigenesis and metastasis. Here, we provide evidence that increased expression of NOK/STYK1 caused marked alterations in the overall and inner structures of tumors and substantially facilitates the genesis and remodeling of the blood and lymphatic vessels during tumor progression. In particular, NOK-expressed HeLa stable cells (HeLa-K) significantly enhanced tumor growth and metastasis in xenografted nude mice. Hematoxylin and eosin (HE) staining demonstrated that the tumor tissues generated by HeLa-K cells were much more ichorous and had more interspaces than those generated by control HeLa cells (HeLa-C). The fluorescent areas stained with cluster of differentiation 31 (CD31), a marker protein for blood vessels, appeared to be in different patterns. The total blood vessels, especially the ring patterns, within the tumors of the HeLa-K group were highly enriched compared with those in the HeLa-C group. NOK-HA was demonstrated to be well colocalized with CD31 in the wall of the tubular structures within tumor tissues. Interestingly, antibody staining of the lymphatic vessel endothelial hyaluronan receptor (LYVE-1) further revealed the increase in ring (oratretic strip-like) lymphatic vessels in either the peritumoral or intratumoral areas in the HeLa-K group compared with the HeLa-C group. Consistently, the analysis of human cancerous tissue also showed that NOK was highly expressed in the walls of tubular structures. Thus, our results reveal a novel tumorigenic function of NOK to mediate the genesis and remodeling of blood and lymphatic vessels during tumor progression.

Depletion of STYK1 inhibits intrahepatic cholangiocarcinoma development both in vitro and in vivo.

Intrahepatic cholangiocarcinoma (ICC) has been reported to be the second most common primary hepatic carcinoma worldwide, and very limited therapies are currently available. Serine threonine tyrosine kinase (STYK1), a member of the receptor tyrosine kinase family, exhibits tumorigenicity in many types of cancers and is a potential therapeutic target for ICC. In this study, STYK1 was knocked down in the ICC cell lines HCCC-9810 and RBE via a lentivirus-mediated system using short hairpin RNA (shRNA). Next, cell proliferation, colony formation, cell cycle progression, tumor formation in nude mice, migration and invasion, and the expression levels of cell cycle proteins in Lv-sh STYK1- or Lv-sh Con-infected cells were analyzed by CCK-8 assay, colony formation evaluation, flow cytometry, tumor formation evaluation, wound scratch assay, transwell assay, and western blotting. The results indicated that depletion of STYK1 inhibits ICC development both in vitro and in vivo.

The malignant transformation potential of the oncogene STYK1/NOK at early lymphocyte development in transgenic mice.

B-cell Chronic Lymphocytic Leukemia (B-CLL) is a malignancy caused by the clonal expansion of mature B lymphocytes bearing a CD5+CD19+ (B1) phenotype. However, the origin of B-CLL remains controversial. We showed previously that STYK1/NOK transgenic mice develop a CLL-like disease. Using this model system in this study, we attempt to define the stage of CLL initiation. Here, we show that the phenotype of STYK1/NOK-induced B-CLL is heterogeneous. The expanded B1 lymphocyte pool was detected within peripheral lymphoid organs and was frequently associated with the expansions of memory B cells. Despite this immunophenotypic heterogeneity, suppression of B cell development at an early stage consistently occurred within the bone marrow (BM) of STYK1/NOK-tg mice. Overall, we suggest that enforced expression of STYK1/NOK in transgenic mice might significantly predispose BM hematopoietic stem cells (HSCs) towards the development of B-CLL.

Phosphorylated STYK1 restrains the inhibitory role of EGFR in autophagy initiation and EGFR-TKIs sensitivity.

Epidermal growth factor receptor (EGFR) plays critical roles in cell proliferation and tumorigenesis. Autophagy has emerged as a potential mechanism involved in the acquired resistance to anti-EGFR treatments, however, the molecular mechanisms has not been fully addressed. In this study, we identified EGFR interacts with STYK1, a positive autophagy regulator, in EGFR kinase activity dependent manner. We found that EGFR phosphorylates STYK1 at Y356 site and STYK1 inhibits activated EGFR mediated Beclin1 tyrosine phosphorylation and interaction between Bcl2 and Beclin1, thus enhances PtdIns3K-C1 complex assembly and autophagy initiation. We also demonstrated that STYK1 depletion increased the sensitivity of NSCLC cells to EGFR-TKIs in vitro and in vivo. Moreover, EGFR-TKIs induced activation of AMPK phosphorylates STYK1 at S304 site. STYK1 S304 collaborated with Y356 phosphorylation to enhance the EGFR-STYK1 interaction and reverse the inhibitory effects of EGFR to autophagy flux. Collectively, these data revealed new roles and cross-talk between STYK1 and EGFR in autophagy regulation and EGFR-TKIs sensitivity in NSCLC.

RBM15 condensates modulate m6A modification of STYK1 to promote tumorigenesis.

RBM15 expression is recurrently upregulated in several types of malignant tissues, and its high expression level is typically associated with poor prognosis. However, whether and how RBM15 is involved in the tumor progression remains unclear. In this study, we found that overexpressing RBM15 in NIH3T3 cells was able to enhance proliferation rate in vitro and induced subcutaneous tumor formation in vivo. Moreover, we imaged the subcellular localization of RBM15 with our home-built structured illumination super-resolution microscopy, and revealed that RBM15 formed substantial condensates dispersed in the nucleus, undergoing dynamic fusion and fission activities. These condensates were partially colocalized with m6A-modified transcripts in the nucleus. In addition, we confirmed that RBM15 formed "liquid-like" droplets in a protein/salt concentration-dependent manner in vitro, and the addition of RNA further enhanced its phase-separation propensity. To identify downstream targets of RBM15, we performed meRIP-seq and RNA-seq, revealing that RBM15 preferentially bound to and promoted the m6A modification on the mRNA of Serine/threonine/tyrosine kinase 1 (STYK1), thereby enhancing its stability. The upregulated STYK1 expression caused MAPK hyperactivation, thereby leading to oncogenic transformation of NIH3T3 cells.

STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1.

The novel oncogene STYK1/NOK plays critical roles in cancer development. However, its regulation during cell division is less defined. In this paper, we show that over-expression of STYK1/NOK caused mitotic arrest and cytokinesis defects. The protein level of STYK/NOK fluctuated during the cell cycle, with a peak at mitosis and a quick reduction upon mitotic exit. The cell cycle-related expression pattern of STYK1/NOK resembled the one of aurora kinases and polo-like kinase 1. Depletion of APC3 led to accumulation of STYK1/NOK and to the G2/M arrest. Co-immunoprecipitation experiment demonstrated the direct interaction of STYK1/NOK with CDH1. Overexpression of CDH1 shortened the half-life of STYK1/NOK. The kinase domain, but not the five D boxes, of STYK1/NOK was responsible for the interaction with CDH1. Altogether, our data demonstrated for the first time that STYK1/NOK could affect cell division, probably by directly targeting key components of APC/C such as CDH1 at late mitosis. Current study may provide a vital mechanistic clue for understanding the roles of STYK1/NOK in mitosis and cytokinesis during STYK1NOK mediated genomic instability and oncogenesis.

STYK1/NOK Promotes Metastasis and Epithelial-Mesenchymal Transition in Non-small Cell Lung Cancer by Suppressing FoxO1 Signaling.

AIMS: Serine/threonine/tyrosine kinase 1 (STYK1) has been previously shown to have oncogenic properties, and emerging evidence suggests that STYK1 expression correlates with epithelial-mesenchymal transition (EMT). However, the mechanism of STYK1 involvement in oncogenesis remains unknown. The present study aimed to elucidate how STYK1 expression level relates to the metastasis, migration, invasion, and EMT in non-small cell lung cancer (NSCLC) and to determine the molecular mechanism of STYK1 effects. METHODS: Serine/threonine/tyrosine kinase 1 (STYK1) expression level and its relationship with the prognosis of NSCLC were determined using the ONCOMINE database and clinical cases. Non-small cell lung cancer cell lines with the overexpression or knockdown of STYK1 were established to determine whether STYK1 promotes cell migration, invasion, and EMT in vitro and in vivo. In addition, a constitutively active FoxO1 mutant (FoxO1AAA) was used to examine the role of FoxO1 in the STYK1-mediated upregulation of metastasis and EMT in NSCLC. RESULTS: Serine/threonine/tyrosine kinase 1 (STYK1) was upregulated in NSCLC tissues and cell lines, and its overexpression correlated with poor prognosis in patients with NSCLC after surgery. Enhanced expression of STYK1 potentiated the migration, invasion, and EMT in SW900 cells, thereby promoting metastasis, whereas knockdown of STYK1 inhibited these cellular phenomena in Calu-1 cells. Furthermore, STYK1 expression was positively related to the level of phosphorylated-FoxO1, whereas the constitutively active FoxO1 mutant protected against the positive effect of STYK1 overexpression on cell migration, invasion, and EMT. CONCLUSION: Serine/threonine/tyrosine kinase 1 (STYK1) was upregulated in NSCLC and correlated with poor clinical outcomes. In addition, STYK1 suppressed FoxO1 functions, thereby promoting metastasis and EMT in NSCLC.

STYK1 promotes autophagy through enhancing the assembly of autophagy-specific class III phosphatidylinositol 3-kinase complex I.

Macroautophagy/autophagy plays key roles in development, oncogenesis, and cardiovascular and metabolic diseases. Autophagy-specific class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) is essential for autophagosome formation. However, the regulation of this complex formation requires further investigation. Here, we discovered that STYK1 (serine/threonine/tyrosine kinase 1), a member of the receptor tyrosine kinases (RTKs) family, is a new upstream regulator of autophagy. We discovered that STYK1 facilitated autophagosome formation in human cells and zebrafish, which was characterized by elevated LC3-II and lowered SQSTM1/p62 levels and increased puncta formation by several marker proteins, such as ATG14, WIPI1, and ZFYVE1. Moreover, we observed that STYK1 directly binds to the PtdIns3K-C1 complex as a homodimer. The binding with this complex was promoted by Tyr191 phosphorylation, by means of which the kinase activity of STYK1 was elevated. We also demonstrated that STYK1 elevated the serine phosphorylation of BECN1, thereby decreasing the interaction between BECN1 and BCL2. Furthermore, we found that STYK1 preferentially facilitated the assembly of the PtdIns3K-C1 complex and was required for PtdIns3K-C1 complex kinase activity. Taken together, our findings provide new insights into autophagy induction and reveal evidence of novel crosstalk between the components of RTK signaling and autophagy. Abbreviations: AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ATG: autophagy related; ATP: adenosine triphosphate; BCL2: BCL2 apoptosis regulator; BECN1: beclin 1; Bre A: brefeldin A; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DAPI: 4',6-diamidino-2-phenylindole; EBSS: Earle's balanced salt solution; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GSEA: gene set enrichment analysis; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAPK8/JNK1: mitogen-activated protein kinase 8; mRFP: monomeric red fluorescent protein; MTOR: mechanistic target of rapamycin kinase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; qRT-PCR: quantitative reverse transcription PCR; RACK1: receptor for activated C kinase 1; RUBCN: rubicon autophagy regulator; siRNA: small interfering RNA; SQSTM1: sequestosome 1; STYK1/NOK: serine/threonine/tyrosine kinase 1; TCGA: The Cancer Genome Atlas; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; WIPI1: WD repeat domain, phosphoinositide interacting 1; ZFYVE1: zinc finger FYVE-type containing 1.

Aberrantly High Expression Of NOK/STYK1 Is Tightly Associated With The Activation Of The AKT/GSK3ß/N-Cadherin Pathway In Non-Small Cell Lung Cancer.

PURPOSE: High metastasis is a leading risk factor for the survival of non-small cell lung cancer (NSCLC) and epithelial-mesenchymal transition (EMT) is a vital step of metastasis. The expression of novel oncogene with kinase domain (NOK) has been observed in some human malignancies, including non-small cell lung cancer (NSCLC); however, the biological function of NOK in NSCLC remains unclear. In the study, we explored the function of NOK in NSCLC, with an aim to elucidate the relevant underlying mechanisms. PATIENTS AND METHODS: We investigate the expression of NOK, p-Akt, p-GSK-3ß, E-cadherin and N-cadherin expression by immunohistochemical analysis using tissue microarrays of 72 paired NSCLC samples of cancerous and adjacent normal tissues. The associations between NOK expression and clinicopathological factors, overall survival, other proteins were assessed. Immunofluorescence analysis of NSCLC tissues was performed to study the location of NOK, Akt and GSK-3ß. Up or down-regulated of NOK were conducted in two NSCLC cell lines to analyze its impact on AKT/GSK3ß pathway. RESULTS: Statistical analysis revealed NOK expression increased in NSCLC tissues compared with normal tissues (P<0.05). It also showed that low NOK expression were associated with a higher possibility of non-lymphatic metastasis, an early pN stage and clinical stage (P<0.05). Moreover, NOK expression was positively correlated with the expression of oncogene p-Akt (Thr308), p-GSK-3ß (Ser9) and N-cadherin (P<0.05). Immunofluorescence analysis of NSCLC tissues revealed that NOK is co-located with Akt and GSK-3ß. Further study in NSCLC cell lines revealed that NOK overexpression can activate the AKT/GSK3ß pathway. Conversely, knockdown of NOK can suppress the AKT/GSK3ß pathway. CONCLUSION: Our results suggest that NOK overexpression correlated significantly with lymphatic metastasis, advanced pN and clinical stage in NSCLC. And NOK may promote EMT by activating the AKT/GSK3ß/N-cadherin pathway in NSCLC.

STYK1 promotes cancer cell proliferation and malignant transformation by activating PI3K-AKT pathway in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract with extremely poor prognosis. The malignant transformation of GBC is associated with cell proliferation, invasion, and epithelial-mesenchymal transition (EMT). However, the molecular mechanisms underlying GBC progression are poorly understood. We found that serine threonine tyrosine kinase 1 (STYK1) was elevated in GBC and was negatively correlated with clinical outcomes and prognosis. Overexpression of STYK1 in GBC cell lines gave rise to increased cell proliferation, colony formation, migration and invasion, thus committing cells to undergoing EMT. In contrast, silence of STYK1 led to opposite effects on cell transformation. Consistent with STYK1 gene knockdown, AKT specific inhibitor MK2206 abrogated tumor promoting action induced by STYK1, suggesting that PI3K/AKT pathway is essential for the oncogenic role of STYK1 in GBC. STYK1 shRNA in GBC cells inhibited development of xenografted tumors compared with control cells. Collectively, our findings suggest that STYK1 is a critical regulator of tumor growth and metastasis, and may serve as a potential target for GBC therapy.

Low STYK1 expression indicates poor prognosis in gastric cancer.

BACKGROUND: The expression of serine threonine tyrosine kinase 1 (STYK1), a member of the receptor protein tyrosine kinase (RPTK) family, is abnormal in several cancers. However, the molecular mechanism of STYK1 regulation of gastric cancer (GC) progression is unknown. MATERIALS AND METHODS: We evaluated STYK1 expression in GC tissues and the corresponding normal tissues. Specimens from 93 patients with GC were examined with immunohistochemical staining. The relationship between STYK1 protein expression and the patients' clinicopathological features was assessed. Kaplan-Meier and Cox proportional regression analyses were used to evaluate the association between STYK1 expression and survival. RESULTS: STYK1 expression was decreased in GC tissues. Low STYK1 expression was significantly associated with poor tumor differentiation (P=0.023), advanced clinical stage (P=0.021), and poor overall survival (OS; P=0.034). Univariate and multivariate analyses revealed that STYK1expression was an independent prognostic indicator (HR =0.53, 95% CI =0.29-0.95, P=0.039; HR =0.51, 95% CI =0.24-0.91, P=0.030, respectively). CONCLUSION: Downregulated STYK1 expression correlated significantly with poor tumor differentiation, advanced clinical stage, and poor OS in GC. STYK1 might be a diagnostic and prognostic indicator in patients with GC.

Aberrant expression of STYK1 and E-cadherin confer a poor prognosis for pancreatic cancer patients.

Previous studies showed that aberrant Serine/threonine/tyrosine kinase 1 (STYK1, also known as NOK) or/and E-cadherin were involved in the progression of some types of human cancers. However, whether they contributed to the development of pancreatic cancer was unknown. Here, we investigated the prognostic significance of aberrant STYK1 and E-cadherin in pancreatic cancer. Our results showed that STYK1 expression increased while E-cadherin decreased in pancreatic cancer tissues compared with normal pancreas tissues. STYK1 level was positively correlated with lymph node metastasis and clinical stage in pancreatic cancer patients. E-cadherin expression was inversely correlated with STYK1 expression in pancreatic cancer tissue samples. Patients with high STYK1 and low E-cadherin expression had the worst prognosis. In addition, STYK1 knockdown in pancreatic cancer cell lines inhibited cell proliferation, enhanced cell apoptosis, induced cell cycle arrest, and prohibited cell migration, while STYK1 over-expression showed the opposite effects. Silencing STYK1 also increased E-cadherin expression and inhibited epithelial-to-mesenchymal transition (EMT) and p-p38 expression in vitro. Over-expression had showed the opposite trends, and treatment with p38 inhibitor, SB203580, could reverse the trends. Thus, STYK1 repressed E-cadherin expression and promoted EMT, mediated by p38 MAPK signaling pathway, which was the possible mechanism for STYK1-mediated pancreatic cancer cell proliferation and migration. In summary, our results showed that STYK1 might be a prognostic marker for pancreatic cancer patients and might be a novel strategy for the treatment of pancreatic cancer.