Characterization of Hippo Pathway Components by Gene Inactivation.

The Hippo pathway is important for regulating tissue homeostasis, and its dysregulation has been implicated in human cancer. However, it is not well understood how the Hippo pathway becomes dysregulated because few mutations in core Hippo pathway components have been identified. Therefore, much work in the Hippo field has focused on identifying upstream regulators, and a complex Hippo interactome has been identified. Nevertheless, it is not always clear which components are the most physiologically relevant in regulating YAP/TAZ. To provide an overview of important Hippo pathway components, we created knockout cell lines for many of these components and compared their relative contributions to YAP/TAZ regulation in response to a wide range of physiological signals. By this approach, we provide an overview of the functional importance of many Hippo pathway components and demonstrate NF2 and RHOA as important regulators of YAP/TAZ and TAOK1/3 as direct kinases for LATS1/2.

The role of TAOK3 in cancer progression and development as a prognostic marker: A pan-cancer analysis study.

The protein kinase TAOK3, belongs to the MAP kinase family, is one of three closely related members, namely TAOK1, TAOK2, and TAOK3. We performed a pan-cancer investigation of TAOK3 across different cancer types, including uterine carcinosarcoma, adenocarcinoma of the stomach and pancreas, and endometrial carcinoma of the uterus, to better understand TAOK3's role in cancer. In at least 16 types of cancer, our findings indicate that TAOK3 expression levels differ considerably between normal and tumor tissues. In addition, our study is the first to identify the oncogenic role of TAOK3 locus S331 and S471 in renal clear cell carcinoma, Glioblastoma Multiforme, hepatocellular carcinoma, Lung adenocarcinoma, and Pancreatic adenocarcinoma, indicating their involvement in cancer progression. In addition, our data analysis indicates that copy number variation is the most prevalent form of mutation in the TAOK3 gene, and that there is a negative correlation between TAOK3 mRNA and DNA promoter methylation. Moreover, our analysis suggests that TAOK3 may serve as a prognostic marker for several kinds of cancer, including Colon adenocarcinoma, renal clear cell carcinoma, Lower Grade Glioma, Lung adenocarcinoma, Mesothelioma, and hepatocellular carcinoma. In addition, our research on signature cancer genes has uncovered a positive association between TAOK3 and SMAD2, SMAD4, and RNF168 in most of the malignancies we have examined. TAOK3 is also correlated with the frequency of mutations and microsatellite instability in four types of cancer. Numerous immune-related genes are closely associated with TAOK3 levels in numerous malignancies. TAOK3 expression is positively correlated with immune infiltrates, which include activated CD4 T cells, CD8 T cells, and type 2T helper cells. Our pan-cancer analysis of TAOK3 provides vital insight into its potential role across a variety of cancer types.

Knockout of STE20-type kinase TAOK3 does not attenuate diet-induced NAFLD development in mice.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD), the primary hepatic consequence of obesity, is affecting about 25% of the global adult population. The aim of this study was to examine the in vivo role of STE20-type protein kinase TAOK3, which has been previously reported to regulate hepatocellular lipotoxicity in vitro, in the development of NAFLD and systemic insulin resistance in the context of obesity. METHODS: Taok3 knockout mice and wild-type littermates were challenged with a high-fat diet. Various in vivo tests were performed to characterize the whole-body metabolism. NAFLD progression in the liver, and lipotoxic damage in adipose tissue, kidney, and skeletal muscle were compared between the genotypes by histological assessment, immunofluorescence microscopy, protein and gene expression profiling, and biochemical assays. Intracellular lipid accumulation and oxidative/ER stress were analyzed in cultured human and mouse hepatocytes where TAOK3 was knocked down by small interfering RNA. The expression of TAOK3-related STE20-type kinases was quantified in different organs from high-fat diet-fed Taok3-/- and wild-type mice. RESULTS: TAOK3 deficiency had no impact on body weight or composition, food consumption, locomotor activity, or systemic glucose or insulin homeostasis in obese mice. Consistently, Taok3-/- mice and wild-type littermates developed a similar degree of high-fat diet-induced liver steatosis, inflammation, and fibrosis, and we detected no difference in lipotoxic damage of adipose tissue, kidney, or skeletal muscle when comparing the two genotypes. In contrast, the silencing of TAOK3 in vitro markedly suppressed ectopic lipid accumulation and metabolic stress in mouse and human hepatocytes. Interestingly, the hepatic mRNA abundance of several TAOK3-related kinases, which have been previously implicated to increase the risk of NAFLD susceptibility, was significantly elevated in Taok3-/- vs. wild-type mice. CONCLUSIONS: In contrast to the in vitro observations, genetic deficiency of TAOK3 in mice failed to mitigate the detrimental metabolic consequences of chronic exposure to dietary lipids, which may be partly attributable to the activation of liver-specific compensation response for the genetic loss of TAOK3 by related STE20-type kinases.

TAOK3 limits age-associated inflammation by negatively modulating macrophage differentiation and their production of TNFα.

BACKGROUND: Human aging is characterized by a state of chronic inflammation, termed inflammaging, for which the causes are incompletely understood. It is known, however, that macrophages play a driving role in establishing inflammaging by promoting pro-inflammatory rather than anti-inflammatory responses. Numerous genetic and environmental risk factors have been implicated with inflammaging, most of which are directly linked to pro-inflammatory mediators IL-6, IL1Ra, and TNFα. Genes involved in the signaling and production of those molecules have also been highlighted as essential contributors. TAOK3 is a serine/threonine kinase of the STE-20 kinase family that has been associated with an increased risk of developing auto-immune conditions in several genome-wide association studies (GWAS). Yet, the functional role of TAOK3 in inflammation has remained unexplored. RESULTS: We found that mice deficient in the serine/Threonine kinase Taok3 developed severe inflammatory disorders with age, which was more pronounced in female animals. Further analyses revealed a drastic shift from lymphoid to myeloid cells in the spleens of those aged mice. This shift was accompanied by hematopoietic progenitor cells skewing in Taok3-/- mice that favored myeloid lineage commitment. Finally, we identified that the kinase activity of the enzyme plays a vital role in limiting the establishment of proinflammatory responses in macrophages. CONCLUSIONS: Essentially, Taok3 deficiency promotes the accumulation of monocytes in the periphery and their adoption of a pro-inflammatory phenotype. These findings illustrate the role of Taok3 in age-related inflammation and highlight the importance of genetic risk factors in this condition.

The STE20 kinase TAOK3 controls the development of house dust mite-induced asthma in mice.

BACKGROUND: The most common endotype of asthma is type 2-high asthma, which is sometimes driven by adaptive allergen-specific TH2 lymphocytes that react to allergens presented by dendritic cells (DCs), or sometimes by an innate immune response dominated by type 2 innate lymphocytes (ILC2s). Understanding the underlying pathophysiology of asthma is essential to improve patient-tailored therapy. The STE20 kinase thousand-and-one kinase 3 (TAOK3) controls key features in the biology of DCs and lymphocytes, but to our knowledge, its potential usefulness as a target for asthma therapy has not yet been addressed. OBJECTIVE: We examined if and how loss of Taok3 affects the development of house dust mite (HDM)-driven allergic asthma in an in vivo mouse model. METHODS: Wild-type Taok3+/+ and gene-deficient Taok3-/- mice were sensitized and challenged with HDM, and bronchoalveolar lavage fluid composition, mediastinal lymph node cytokine production, lung histology, and bronchial hyperreactivity measured. Conditional Taok3fl/fl mice were crossed to tissue- and cell-specific specific deletor Cre mice to understand how Taok3 acted on asthma susceptibility. Kinase-dead (KD) Taok3KD mice were generated to probe for the druggability of this pathway. Activation of HDM-specific T cells was measured in adoptively transferred HDM-specific T-cell receptor-transgenic CD4+ T cells. ILC2 biology was assessed by in vivo and in vitro IL-33 stimulation assays in Taok3-/- and Taok3+/+, Taok3KD, and Red5-Cre Taok3fl/fl mice. RESULTS: Taok3-/- mice failed to mount salient features of asthma, including airway eosinophilia, TH2 cytokine production, IgE secretion, airway goblet cell metaplasia, and bronchial hyperreactivity compared to controls. This was due to intrinsic loss of Taok3 in hematopoietic and not epithelial cells. Loss of Taok3 resulted in hampered HDM-induced lung DC migration to the draining lymph nodes and defective priming of HDM-specific TH2 cells. Strikingly, HDM and IL-33-induced ILC2 proliferation and function were also severely affected in Taok3-deficient and Taok3KD mice. CONCLUSIONS: Absence of Taok3 or loss of its kinase activity protects from HDM-driven allergic asthma as a result of defects in both adaptive DC-mediated TH2 activation and innate ILC2 function. This identifies Taok3 as an interesting drug target, justifying further testing as a new treatment for type 2-high asthma.

TAOK3 is a MAP3K contributing to osteoblast differentiation and skeletal mineralization.

Current anabolic drugs to treat osteoporosis and other disorders of low bone mass all have important limitations in terms of toxicity, contraindications, or poor efficacy in certain contexts. Addressing these limitations will require a better understanding of the molecular pathways, such as the mitogen activated protein kinase (MAPK) pathways, that govern osteoblast differentiation and, thereby, skeletal mineralization. Whereas MAP3Ks functioning in the extracellular signal-regulated kinases (ERK) and p38 pathways have been identified in osteoblasts, MAP3Ks mediating proximal activation of the c-Jun N-terminal kinase (JNK) pathway have yet to be identified. Here, we demonstrate that thousand-and-one kinase 3 (TAOK3, MAP3K18) functions as an upstream activator of the JNK pathway in osteoblasts both in vitro and in vivo. Taok3-deficient osteoblasts displayed defective JNK pathway activation and a marked decrease in osteoblast differentiation markers and defective mineralization, which was also confirmed using TAOK3 deficient osteoblasts derived from human MSCs. Additionally, reduced expression of Taok3 in a murine model resulted in osteopenia that phenocopies aspects of the Jnk1-associated skeletal phenotype such as occipital hypomineralization. Thus, in vitro and in vivo evidence supports TAOK3 as a proximal activator of the JNK pathway in osteoblasts that plays a critical role in skeletal mineralization.

Kinase shRNA screening reveals that TAOK3 enhances microtubule-targeted drug resistance of breast cancer cells via the NF-κB signaling pathway.

BACKGROUND: Chemotherapy is currently one of the most effective treatments for advanced breast cancer. Anti-microtubule agents, including taxanes, eribulin and vinca-alkaloids are one of the primary major anti-breast cancer chemotherapies; however, chemoresistance remains a problem that is difficult to solve. We aimed to discover novel candidate protein targets to combat chemoresistance in breast cancer. METHODS: A lentiviral shRNA-based high-throughput screening platform was designed and developed to screen the global kinome to find new therapeutic targets in paclitaxel-resistant breast cancer cells. The phenotypes were confirmed with alternative expression in vitro and in vivo. Molecular mechanisms were investigated using global phosphoprotein arrays and expression microarrays. Global microarray analysis was performed to determine TAOK3 and genes that induced paclitaxel resistance. RESULTS: A serine/threonine kinase gene, TAOK3, was identified from 724 screened kinase genes. TAOK3 shRNA exhibited the most significant reduction in IC50 values in response to paclitaxel treatment. Ectopic downregulation of TAOK3 resulted in paclitaxel-resistant breast cancer cells sensitize to paclitaxel treatment in vitro and in vivo. The expression of TAOK3 also was correlated to sensitivity to two other anti-microtubule drugs, eribulin and vinorelbine. Our TAOK3-modulated microarray analysis indicated that NF-κB signaling played a major upstream regulation role. TAOK3 inhibitor, CP43, and shRNA of NF-κB both reduced the paclitaxel resistance in TAOK3 overexpressed cells. In clinical microarray databases, high TAOK3 expressed breast cancer patients had poorer prognoses after adjuvant chemotherapy. CONCLUSIONS: Here we identified TAOK3 overexpression increased anti-microtubule drug resistance through upregulation of NF-κB signaling, which reduced cell death in breast cancer. Therefore, inhibition of the interaction between TAOK3 and NF-κB signaling may have therapeutic implications for breast cancer patients treated with anti-microtubule drugs. Video abstract.

TAOK3 Facilitates Esophageal Squamous Cell Carcinoma Progression and Cisplatin Resistance Through Augmenting Autophagy Mediated by IRGM.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers because of its robust aggressive phenotype and chemoresistance. TAO kinase belongs to mitogen-activated protein kinases, which mediate drug resistance in multiple cancers. However, the role of TAO kinase in ESCC progression and chemoresistance has never been explored. Here, it is reported that TAOK3 augments cell autophagy and further promotes ESCC progression and chemoresistance. Mechanistically, TAOK3 phosphorylates KMT2C at S4588 and strengthens the interaction between KMT2C and ETV5. Consequently, the nuclear translocation of KMT2C is increased, and the transcription of autophagy-relevant gene IRGM is further upregulated. Additionally, the inhibitor SBI-581 can significantly suppress cell autophagy mediated by TAOK3 and synergizes with cisplatin to treat ESCC in vitro and in vivo.

STE20-type kinase TAOK3 regulates hepatic lipid partitioning.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD), defined by excessive lipid storage in hepatocytes, has recently emerged as a leading global cause of chronic liver disease. The aim of this study was to examine the role of STE20-type protein kinase TAOK3, which has previously been shown to associate with hepatic lipid droplets, in the initiation and aggravation of human NAFLD. METHODS: The correlation between TAOK3 mRNA expression and the severity of NAFLD was investigated in liver biopsies from 62 individuals. In immortalized human hepatocytes, intracellular fat deposition, lipid metabolism, and oxidative and endoplasmic reticulum stress were analyzed when TAOK3 was overexpressed or knocked down by small interfering RNA. Subcellular localization of TAOK3 was characterized in human and mouse hepatocytes by immunofluorescence microscopy. RESULTS: We found that the TAOK3 transcript levels in human liver biopsies were positively correlated with the key lesions of NAFLD (i.e., hepatic steatosis, inflammation, and ballooning). Overexpression of TAOK3 in cultured human hepatocytes exacerbated lipid storage by inhibiting ß-oxidation and triacylglycerol secretion while enhancing lipid synthesis. Conversely, silencing of TAOK3 attenuated lipid deposition in human hepatocytes by stimulating mitochondrial fatty acid oxidation and triacylglycerol efflux while suppressing lipogenesis. We also found aggravated or decreased oxidative/endoplasmic reticulum stress in human hepatocytes with increased or reduced TAOK3 levels, respectively. The subcellular localization of TAOK3 in human and mouse hepatocytes was confined to intracellular lipid droplets. CONCLUSIONS: This study provides the first evidence that hepatic lipid droplet-coating kinase TAOK3 is a critical regulatory node controlling liver lipotoxicity and susceptibility to NAFLD.

Single-Nucleotide Polymorphisms in TAOK3 Are Associated With High Opioid Requirement for Pain Management in Patients With Advanced Cancer Admitted to a Tertiary Palliative Care Unit.

PURPOSE: Different amounts of opioid are required for the relief of cancer pain in different individuals, raising the possibility that genetic factors play a role. We tested the hypothesis that genetic variations in the TAOK3 (TAO kinase 3, encoding serine/threonine-protein kinase) explain some of the interindividual variations related to the morphine-equivalent daily dose (MEDD) in patients with cancer. EXPERIMENTAL DESIGN: We selected two single-nucleotide polymorphisms (SNPs) in the TAOK3, reported earlier to associate with higher MEDD in postoperative pain based on genome-wide association study. We investigated their association with MEDD in Canadian patients with cancer (n = 110) admitted to a tertiary palliative care unit. SNPs analyzed were rs1277441 (C/T, C = minor allele) and rs795484 (A/G, A = minor allele). RESULTS: Minor allele frequencies in our population were 0.29 (rs1277441) and 0.28 (rs795484). These SNPs were in perfect linkage disequilibrium (r2 = 0.97). SNPs in TAOK3 showed a significant association with mean MEDD ≥800 mg. For rs795484, MEDD values ≥800 mg occurred in patients who were GG (7%), GA (18%), and AA (57%) (P = 0.004; Fisher's exact test); similar results were obtained for rs1277441. Homozygous variants for either SNP had received higher numbers of different opioids (P = 0.021). CONCLUSION: In this cohort of patients with advanced cancer pain, TAOK3 SNPs were associated with opioid doses. This result supports the original findings from a GWAS in postoperative patients. The proportions of variant homozygotes (8.2% of patients) and their requirement for higher doses of opioids would appear potentially clinically important and should be validated in further studies.

TAOK3 phosphorylates the methylenecyclopropane nucleoside MBX 2168 to its monophosphate.

Monohydroxymethyl methylenecyclopropane nucleosides (MCPNs) with ether or thioether substituents at the 6-position show promise as broad-spectrum herpes virus inhibitors. Their proposed mechanism of action involves sequential phosphorylation to a triphosphate, which can then inhibit viral DNA polymerase. The inhibition of herpes simplex virus (HSV) by these compounds is not dependent on the viral thymidine kinase (TK), which is known to phosphorylate acyclovir (ACV), a standard treatment for HSV infections. Previous studies on the mechanism of action of these compounds against human cytomegalovirus (HCMV) implicated a host kinase in addition to HCMV UL97 kinase in performing the initial phosphorylation. After first eliminating other candidate HSV-1 encoded kinases (UL13 and US3) as well as potential host nucleoside kinases, using activity-based fractionation, we have now identified the host serine-threonine protein kinase TAOK3 as the kinase responsible for transforming the representative monohydroxymethyl MCPN analog MBX 2168 to its monophosphate.