NCK-interacting Protein Kinase Research Articles

TNIK inhibition sensitizes TNIK-overexpressing lung squamous cell carcinoma to radiotherapy.

Most patients with lung squamous cell carcinoma (LSCC) undergo chemotherapy, radiotherapy, and adjuvant immunotherapy for locally advanced disease. The efficacy of these treatments is still limited due to dose-limiting toxicity or locoregional recurrence. New combination approaches and targets such as actionable oncogenic drivers are needed to advance treatment options for LSCC patients. Moreover, other options for chemotherapy-ineligible patients are also limited. As such there is a critical need for the development of selective and potent chemoradiosensitizers for locally advanced LSCC. Here, we investigated inhibiting TRAF2 and NCK-interacting protein kinase (TNIK), which is amplified in 40% of LSCC patients, as a strategy to sensitize LSCC tumors to chemo- and radiotherapy. Employing a range of human LSCC cell lines and the TNIK inhibitor NCB-0846, we investigated the potential of TNIK as a chemo- and radiosensitizing target with in vitro and in vivo preclinical models. The combination of NCB-0846 with cisplatin or etoposide was at best additive. Interestingly, pre-treating LSCC cells with NCB-0846 prior to ionizing radiation (IR) potentiated the cytotoxicity of IR in a TNIK-specific fashion. Characterization of the radiosensitization mechanism suggested that TNIK inhibition may impair the DNA damage response and promote mitotic catastrophe in irradiated cells. In a subcutaneous xenograft in vivo model, pretreatment with NCB-0846 significantly enhanced the efficacy of IR and caused elevated necrosis in TNIKhigh LK2 tumors but not TNIKlow KNS62 tumors. Overall, these results indicate that TNIK inhibition may be a promising strategy to increase the efficacy of radiotherapy in LSCC patients with high TNIK expression.

Abstract 6799: The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy with rapidly acquired chemotherapy resistance. While the inflamed SCLC subtype is associated with a significantly improved response to immunotherapy, the non-inflamed SCLC subtypes, encompassing most SCLC patients, experience only modest survival gains with chemo-immunotherapy. Wnt signaling pathway activation is associated with resistance to chemotherapy and an immune checkpoint inhibitor in several tumors. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers, but the efficacy of TNIK inhibition in SCLC has not previously been researched. We previously demonstrated that single agent TNIK inhibitors are effective in a subset of SCLC. Here, we hypothesize that targeting TNIK will show an anti-tumor effect based on specific biomarkers and also modify the tumor immune environment in SCLC. Methods: We evaluated susceptibility to a small molecule TNIK inhibitor, NCB-0846, in 29 human-derived SCLC cell lines using 96-hour proliferation assays. To investigate efficacy biomarkers, we correlated NCB-0846 IC50 values with proteomic expression (profiled using reverse phase protein array). cMYC and FOXK1 expression was reduced by siRNA. Chemokine concentrations were determined by ELISA. Results: SCLC cell lines with high cMYC levels, were more sensitive to the TNIK inhibitor, NCB-0846 (F.C.=-2.07, p=0.01), while SCLC cell lines with high expression of TTF-1 were more resistant (F.C.=2.48, p<0.01). Knockdown of cMYC conferred resistance to NCB-0846 in cMYC-high cells, while NCB-0846 treatment resulted in decreased cMYC expression in a concentration-dependent manner. Additionally, NCB-0846 treatment decreased the immunosuppressive chemokine CCL2 levels in the supernatant of human-derived SCLC cell lines and GEMM-derived cell lines. The expression of FOXK1, a transcription factor of CCL2, was also reduced in cell lines treated with NCB-0846. Conclusions: These findings show that TNIK inhibition is more effective in cMYC-high SCLC, acting through downregulating cMYC levels. TNIK inhibition also decreases the production of CCL2, an immunosuppressive chemokine implicated in resistance to anti-PD-L1 immunotherapy. Together, these findings highlight the promising potential of TNIK inhibitors in cMYC-high SCLC. Additionally, suppressing CCL2 by TNIK inhibition supports the rationale for combining the TNIK inhibitor and an anti-PD-L1 antibody in the non-inflamed SCLC subtypes. Citation Format: Azusa Tanimoto, Kavya Ramkumar, Allison Stewart, Bingnan Zhang, Robert Cardnell, Shen Li, Qi Wang, Jing Wang, Carl Gay, Lauren Averett Byers. The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6799.

Fragment growth-based discovery of novel TNIK inhibitors for the treatment of colorectal cancer

Traf2-and Nck-interacting protein kinase (TNIK) plays an important role in regulating signal transduction of the Wnt/β-catenin pathway and is considered an important target for the treatment of colorectal cancer. Inhibiting TNIK has potential to block abnormal Wnt/β-catenin signal transduction caused by colorectal cancer mutations. We discovered a series of 6-(1-methyl-1H-imidazole-5-yl) quinoline derivatives as TNIK inhibitors through Deep Fragment Growth and virtual screening. Among them, 35b exhibited excellent TNIK kinase and HCT116 cell inhibitory activity with IC50 values of 6 nM and 2.11 μM, respectively. 35b also shown excellent kinase selectivity, PK profiles, and oral bioavailability (84.64%). At a p. o. dosage of 50 mg/kg twice daily 35b suppressed tumor growth on the HCT116 xenograft model. Taken together, 35b is a promising lead compound of TNIK inhibitors, which merits further investigation.

LKB1 loss promotes colorectal cancer cell metastasis through regulating TNIK expression and actin cytoskeleton remodeling.

Colorectal cancer (CRC) is one of the most common malignant tumors. Approximately 5%-6% of CRC cases are associated with hereditary CRC syndromes, including the Peutz-Jeghers syndrome (PJS). Liver kinase B1 (LKB1), also known as STK11, is the major gene responsible for PJS. LKB1 heterozygotic deficiency is involved in intestinal polyps in mice, while the mechanism of LKB1 in CRC remains elusive. In this study, we generated LKB1 knockout (KO) CRC cell lines by using CRISPR-Cas9. LKB1 KO promoted CRC cell motility in vitro and tumor metastases in vivo. LKB1 attenuated expression of TRAF2 and NCK-interacting protein kinase (TNIK) as accessed by RNA-seq and western blots, and similar suppression was also detected in the tumor tissues of azoxymethane/dextran sodium sulfate-induced intestinal-specific LKB1-KO mice. LKB1 repressed TNIK expression through its kinase activity. Moreover, attenuating TNIK by shRNA inhibited cell migration and invasion of CRC cells. LKB1 loss-induced high metastatic potential of CRC cells was depended on TNIK upregulation. Furthermore, TNIK interacted with ARHGAP29 and further affected actin cytoskeleton remodeling. Taken together, LKB1 deficiency promoted CRC cell metastasis via TNIK upregulation and subsequently mediated cytoskeleton remodeling. These results suggest that LKB1-TNIK axis may play a crucial role in CRC progression.

Abstract 3854: TNIK inhibition as a novel therapeutic in cMyc high/TTF1 low SCLC

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy, with rapidly acquired chemotherapy resistance. Some studies have reported that Wnt signaling pathway activation promoted cell proliferation and was correlated with chemo-resistance in SCLC. None of the therapies targeting Wnt pathway components of the transmembrane and the cytoplasm have been successful in a clinical application due to toxicity and insufficient efficacy. However, targeting Wnt signaling inside the nucleus has been drawing increasing attention as cancer therapeutics. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers for cell proliferation, thus TNIK is expected as a novel druggable target. On the other hand, the question remains whether TNIK is a critical target in SCLC. We hypothesize that a TNIK inhibitor has potent anti-tumor effects in SCLC and its promising biomarkers exist. Methods: We used 29 SCLC cell lines including all four subtypes defined by differential expression of transcription factors ASCL1, NEUROD1, POU2F3, and inflamed gene signature (SCLC-A, N, P, and I, respectively) to evaluate the effect of a TNIK inhibitor, NCB-0846 in vitro. We correlated NCB-0846 IC50 values with proteomic profiling (Reverse Phase Protein Array, RPPA) data. Protein expression was examined by western blotting. Results: NCB-0846 markedly reduced cell proliferation in SCLC-N and P cell lines. There was a strong positive correlation between cMyc and the efficacy of NCB-0846 (r=-0.484, P<0.01), while a negative correlation between TTF1 and the efficacy (r=0.569, P<0.01). Additionally, NCB0846 decreased the expression of cMyc in cMyc-high/TTF-1 low SCLC cells. Conclusions: These findings indicate that TNIK inhibitors may be a new personalized molecular-targeted therapy in cMyc-high/TTF-1-low SCLC. Citation Format: Azusa Tanimoto, Robert J. Cardnell, Benjamin B. Morris, Kavya Ramkumar, Shen Li, Qi Wang, Allison C. Stewart, Carl Michael Gay, Jing Wang, Lauren Averett Byers. TNIK inhibition as a novel therapeutic in cMyc high/TTF1 low SCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3854.

In Vitro, Molecular Docking and In Silico ADME/Tox Studies of Emodin and Chrysophanol against Human Colorectal and Cervical Carcinoma.

Anthraquinones (AQs) are present in foods, dietary supplements, pharmaceuticals, and traditional treatments and have a wide spectrum of pharmacological activities. In the search for anti-cancer drugs, AQ derivatives are an important class. In this study, anthraquinone aglycons chrysophanol (Chr), emodin (EM) and FDA-approved anticancer drug fluorouracil were analyzed by molecular docking studies against receptor molecules caspase-3, apoptosis regulator Bcl-2, TRAF2 and NCK-interacting protein kinase (TNIK) and cyclin-dependent protein kinase 2 (CDK2) as novel candidates for future anticancer therapeutic development. The ADMET SAR database was used to predict the toxicity profile and pharmacokinetics of the Chr and EM. Furthermore, in silico results were validated by the in vitro anticancer activity against HCT-116 and HeLa cell lines to determine the anticancer effect. According to the docking studies simulated by the docking program AutoDock Vina 4.0, Chr and EM had good binding energies against the target proteins. It has been observed that Chr and EM show stronger molecular interaction than that of the FDA-approved anticancer drug fluorouracil. In the in vitro results, Chr and EM demonstrated promising anticancer activity in HCT-116 and HeLa cells. These findings lay the groundwork for the potential use of Chr and EM in the treatment of human colorectal and cervical carcinomas.

TNIK Inhibition Has Dual Synergistic Effects on Tumor and Associated Immune Cells.

Treatment with checkpoint inhibitors can be extraordinarily effective in a fraction of patients, particularly those whose tumors are pre-infiltrated by T cells. In others, efficacy is considerably lower, which has led to interest in developing strategies for sensitization to immunotherapy. Using various colorectal cancer mouse models, it is shown that the use of Traf2 and Nck-interacting protein kinase inhibitors (TNIKi) unexpectedly increases tumor infiltration by PD-1+ CD8+ T cells, thus contributing to tumor control. This appears to happen by two independent mechanisms, by inducing immunogenic cell death and separately by directly activating CD8. The use of TNIKi achieves complete tumor control in 50% of mice when combined with checkpoint inhibitor targeting PD-1. These findings reveal immunogenic properties of TNIKi and indicate that the proportion of colorectal cancers responding to checkpoint therapy can be increased by combining it with immunogenic kinase inhibitors.

Determination of the Apoptotic Effect of Raf2 and Nck-Interacting Protein Kinase Inhibitor on Metastatic Canine Mammary Gland Tumor Cells

Objective: Canine mammary gland tumors (CMGTs) are the most common tumors in female dogs and the main treatment options used in CMGTs are surgery caused some complications. Therefore, new treatment options are needed for the CMGTs. Traf2 and Nck-interacting serine protein kinase (TNIK) as a transcriptional coregulator of Wnt targeted genes is highly expressed in breast cancer development. The inhibition of TNIK may be a new therapeutic target in cancers with abnormal WNT signaling. Therefore we aimed to investigate the potential therapeutic effect of NCB-0846 on metastatic sarcoma CMGTs subtype, for the first time. Materials and Methods: The cytotoxic and apoptotic effects of NCB-0846 on CMGT cells were analyzed by WST‑1, Annexin V, cell cycle, acridine orange (AO) and DAPI staining. Results: NCB-0846 significantly inhibited cell viability in a dose and time dependent manner (p<0.05) and induced nuclear damage in CMGT cells. Furthermore, NCB-0846 caused apoptotic cell death through the accumulation of cells in the G0/G1 phase. Conclusion: Our findings demonstrated that NCB-0846 could be potentially a new therapeutic anti-cancer agent in the treatment of CMGTs. However, further investigations need to be performed in order to elucidate the inhibitory activity of TNIK and Wnt signaling by NCB-0846 on CMGT cells. 

Allelically and Differentially Expressed Genes After Infection of Edwardsiella ictaluri in Channel Catfish as Determined by Bulk Segregant RNA-Seq.

Identification of genetic markers associated with resistance against enteric septicemia of catfish (ESC) is of great interest for genetic enhancement programs of catfish. In the present study, bulk segregant RNA-Seq analysis was applied to determine differentially expressed genes and alleles after ESC infection. Here we report three genomic regions on LG1, LG12, and LG26, containing significant single-nucleotide polymorphisms (SNPs). These genomic regions aligned well with quantitative trait loci (QTL) previously identified. Within the QTL regions, eleven genes were found to be differentially regulated between phenotypic bulks. Importantly, the QTL on linkage group 1 (LG1) were found to be expressed in the liver, whereas the QTL on LG12 and LG26 were expressed in the intestine, suggesting multiple mechanisms of ESC resistance. It is apparent that apolipoproteins may be important for ESC resistance as the QTL on LG1 included the 14-kDa apolipoprotein genes that are both allelically expressed and differentially expressed between the resistant and susceptible bulks. Traf2 and NCK-interacting protein kinase (TNIK) were found in the QTL on LG12, and it was downregulated in resistant fish, suggesting the importance of NCK downregulation in ESC resistance, as previously reported. In addition, we observed divergent gene expression patterns between the liver and intestine after infection. Immune/inflammatory-related processes were overrepresented from liver DEGs, while those DEGs identified from intestine were enriched for proteolysis and wounding processes. Taken together, the BSR-Seq analysis presented here advanced the knowledge of ESC resistance, providing information of not only positions of QTL but also genes and their differential expression between resistant and susceptible fish, making it one step closer to the identification of the causal genes for ESC resistance.

Discovery of 3,4-Dihydrobenzo[f][1,4]oxazepin-5(2H)-one Derivatives as a New Class of Selective TNIK Inhibitors and Evaluation of Their Anti-Colorectal Cancer Effects.

The Traf2- and Nck-interacting protein kinase (TNIK) is a downstream signal protein of the Wnt/β-catenin pathway and has been thought of as a potential target for the treatment of colorectal cancer (CRC) that is often associated with dysregulation of Wnt/β-catenin signaling pathway. Herein, we report the discovery of a series of 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one derivatives as a new class of TNIK inhibitors. Structure-activity relationship (SAR) analyses led to the identification of a number of potent TNIK inhibitors with compound 21k being the most active one (IC50: 0.026 ± 0.008 μM). This compound also displayed excellent selectivity for TNIK against 406 other kinases. Compound 21k could efficiently suppress CRC cell proliferation and migration in in vitro assays and exhibited considerable antitumor activity in the HCT116 xenograft mouse model. It also showed favorable pharmacokinetic properties. Overall, 21k could be a promising lead compound for drug discovery targeting TNIK and deserves further studies.

Identification of TNIK as a novel potential drug target in thyroid cancer based on protein druggability prediction.

Thyroid cancer is a common endocrine malignancy; however, surgery remains its primary treatment option. A novel targeted drug for the development and application of targeted therapy in thyroid cancer treatment remain underexplored.We obtained RNA sequence data of thyroid cancer from The Cancer Genome Atlas database and identified differentially expressed genes (DEGs). Then, we constructed co-expression network with DEGs and combined it with differentially methylation analysis to screen the key genes in thyroid cancer. PockDrug-Server, an online tool, was applied to predict the druggability of the key genes. Finally, we constructed protein-protein interaction (PPI) network to observe potential targeted drugs for thyroid cancer.We identified 3 genes correlated with altered DNA methylation level and oncogenesis of thyroid cancer. According to the druggable analysis and PPI network, we predicted TRAF2 and NCK-interacting protein kinase (TNIK) sever as the drug targeted for thyroid cancer. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that genes in protein-protein interaction network of TNIK enriched in mitogen-activated protein kinase signaling pathway. For drug repositioning, we identified a targeted drug of genes in PPI network.Our study provides a bioinformatics method for screening drug targets and provides a theoretical basis for thyroid cancer targeted therapy.

Direct conversion of osteosarcoma to adipocytes by targeting TNIK.

Osteosarcoma (OS) is an aggressive mesenchymal tumor for which no molecularly targeted therapies are available. We have previously identified TRAF2- and NCK-interacting protein kinase (TNIK) as an essential factor for the transactivation of Wnt signal target genes and shown that its inhibition leads to eradication of colorectal cancer stem cells. The involvement of Wnt signaling in the pathogenesis of OS has been implicated. The aim of the present study was to examine the potential of TNIK as a therapeutic target in OS. RNA interference or pharmacological inhibition of TNIK suppressed the proliferation of OS cells. Transcriptome analysis suggested that a small-molecule inhibitor of TNIK upregulated the expression of genes involved in OS cell metabolism and downregulated transcription factors essential for maintaining the stem cell phenotype. Metabolome analysis revealed that this TNIK inhibitor redirected the metabolic network from carbon flux toward lipid accumulation in OS cells. Using in vitro and in vivo OS models, we confirmed that TNIK inhibition abrogated the OS stem cell phenotype, simultaneously driving conversion of OS cells to adipocyte-like cells through induction of PPARγ. In relation to potential therapeutic targeting in clinical practice, TNIK was confirmed to be in an active state in OS cell lines and clinical specimens. From these findings, we conclude that TNIK is applicable as a potential target for treatment of OS, affecting cell fate determination.

Effect of TNIK upregulation on JQ1-resistant human colorectal cancer HCT116 cells

JQ1 disrupts the binding of bromodomain and extra-terminal (BET) family of proteins to acetylated histones, modulates the expression of various genes, and inhibits the proliferation of cancer cells. We established two JQ1-resistant sublines from human colorectal cancer HCT116 cells. These resistant cells showed an 8- to 9-fold higher resistance to JQ1, and a 2- to 4-fold higher resistance to various anti-cancer agents, such as doxorubicin, etoposide, mitoxantrone, SN-38, cisplatin, and methotrexate than the parental HCT116 cells. The JQ1-resistant cells expressed higher levels of TRAF2 and NCK-interacting protein kinase (TNIK), cyclin D1 (CCND1), cyclin E1 (CCNE1), and their corresponding mRNAs than the parental cells. TNIK is a regulator of Wnt/β-catenin signaling and is known to transactivate CCND1. Transient transfection of HCT116 cells with a TNIK expression plasmid resulted in the upregulation of cyclin D1, cyclin E1, and their corresponding mRNAs, as well as an increase in CCNE1 promoter activity. Furthermore, luciferase assay revealed that the JQ1-resistant cells showed high CCNE1 promoter activity. These results suggest that TNIK also transactivates CCNE1. Three stable TNIK transfectant clones of HEK293 cells expressed 1.5- to 2-fold higher levels of TNIK, cyclin D1, and cyclin E1 than the parental cells. The 293/TNIK-6 cells, which expressed the highest level of TNIK among the transfectants, showed a 2.3-fold higher resistance to JQ1 than the parental cells. These results suggest the possible involvement of TNIK in cellular resistance to JQ1.

Abstract 1123: TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells

Abstract [Introduction] Osteosarcoma (OS) is a rare malignant bone tumor and affects predominantly adolescents and young adults. There is no molecular targeted therapy for OS and breakthrough intervention has long been awaited for decades. Disturbances in differentiation towards mature osteoblast from mesenchymal stem cell are related to OS pathogenesis. Wnt signaling is a key component in managing this differentiation process. We previously reported that TNIK was a positive regulator of Wnt signaling. To develop the first molecular targeted therapy for OS patients, we focused on Wnt pathway-targeted therapy in OS and initiated this study to demonstrate TNIK as a novel therapeutic target for OS treatment. [Methods] Eleven different osteosarcoma cell lines were used in the present study. Twenty clinical samples were obtained at biopsy and surgical resection from ten OS patients. TNIK expression was assessed by western blotting, immunofluorescence and immunohistochemical staining. The generation of the novel TNIK inhibitor was previously described (Masuda et al., 2016). Cells were cultured with TNIK inhibitor or siTNIK. Cell viability was evaluated by real-time cell analysis system or ATP quantitation assay. The effect of TNIK inhibitor in vivo was also assessed by a mouse xenograft model. RNA-seq was performed using the RNA extracted from vehicle or TNIK inhibitor-treated OS cells. Cancer stem cell (CSC)-like properties were assessed by soft-agar colony-formation assay, limiting dilution assay, ALDH activities and the expression of CSC markers. Adipogenic phenotypes were qualitatively and quantitatively analyzed by Oil red O staining and the expression of adipogenesis markers. [Results] TNIK was identified in all cell lines and most of cases. Immunohistochemistry of OS tissue microarray also revealed that 52 out of 55 OS samples were positive for TNIK protein. TNIK inhibitor suppressed OS cell proliferation both in vitro and in vivo. SiTNIK also inhibited OS cell growth. Global gene expression analysis revealed a significant decrease in the expression of the genes relating to Wnt signaling pathway and maintenance of stem cell pluripotency. Soft-agar colony-formation assay and limiting dilution assay revealed that TNIK inhibitor decreased sphere formation activities in OS cells. Accordingly, TNIK inhibitor significantly suppressed ALDH activities and the expression of the proteins that are involved in the maintenance of CSCs, including NANOG, SOX2, OCT4A and MYC. Concomitantly, TNIK inhibitor drove adipogenic transdifferentiation of OS cells with PPARG activation. TNIK knock-down U2OS cells created by shRNAs showed nearly identical results. [Conclusion] We present that TNIK inhibition promotes adipogenic transdifferentiation of OS cells and eliminates CSCs. Our study thus suggests the feasibility of TNIK as a target for OS treatment. Citation Format: Toru Hirozane, Mari Masuda, Naoko Goto, Teppei Sugano, Naofumi Asano, Eisuke Kobayashi, Keisuke Horiuchi, Hideo Morioka, Akira Kawai, Masaaki Sawa, Morio Matsumoto, Masaya Nakamura, Tesshi Yamada. TRAF2 and NCK-interacting protein kinase (TNIK) regulates cancer stemness and adipogenesis of osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1123.

Therapeutic targets in the Wnt signaling pathway: Feasibility of targeting TNIK in colorectal cancer

The genetic and epigenetic alterations occurring during the course of multistage colorectal carcinogenesis have been extensively studied in the last few decades. One of the most notable findings is that the great majority of colorectal cancers (>80%) have mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. Loss of functional APC protein results in activation of canonical Wnt/β-catanin signaling and initiates intestinal carcinogenesis. Mutational inactivation of APC is the first genetic event, but colorectal cancer cells retain their dependency on constitutive Wnt signal activation even after accumulation of other genetic events. Accordingly, pharmacological blocking of Wnt signaling has been considered an attractive therapeutic approach for colorectal cancer. Several therapeutics targeting various molecular components of the Wnt signaling pathway, including porcupine, frizzled receptors and co-receptor, tankyrases, and cAMP response element binding protein (CREB)-binding protein (CBP), have been developed, and some of those are currently being evaluated in early-phase clinical trials. Traf2- and Nck-interacting protein kinase (TNIK) has been identified as a regulatory component of the T-cell factor-4 and β-catenin transcriptional complex independently by two research groups. TNIK regulates Wnt signaling in the most downstream part of the pathway, and its inhibition is expected to block the signal even in colorectal cancer cells with APC gene mutation. Here we discuss some of the TNIK inhibitors under preclinical development.

Quantitative phosphoproteomics of transforming growth factor‐β signaling in colon cancer cells

The transforming growth factor-β (TGF-β) signaling pathway progresses through a series of protein phosphorylation regulated steps. Smad4 is a key mediator of the classical TGF-β signaling pathway; however, reports suggest that TGF-β can activate other cellular pathways independent of Smad4. By investigating the TGF-β-regulated phosphoproteome, we aimed to uncover new functions controlled by TGF-β. We applied titanium dioxide to enrich phosphopeptides from stable isotope labeling with amino acids in cell culture (SILAC)-labeled SW480 cells stably expressing Smad4 and profiled them by mass spectrometry. TGF-β stimulation for 30 min resulted in the induction of 17 phosphopeptides and the repression of 8 from a total of 149 unique phosphopeptides. Proteins previously not known to be phosphorylated by TGF-β including programmed cell death protein 4, nuclear ubiquitous casein and cyclin-dependent kinases substrate, hepatoma-derived growth factor and cell division kinases amongst others were induced following TGF-β stimulation, while the phosphorylation of TRAF2 and NCK-interacting protein kinase are examples of proteins whose phosphorylation status was repressed. This phosphoproteomic screen has identified new TGF-β-modulated phosphorylation responses in colon carcinoma cells.