CJ14939, a Novel JAK Inhibitor, Increases Oxaliplatin-induced Cell Death Through JAK/STAT Pathway in Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer is reported to have the highest mortality rate among human malignancies. Although many research results for the treatment of colorectal cancer have been reported, there is no suitable treatment when resistance has developed. Therefore, it is necessary to develop new therapeutic agents. Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling plays an essential role in cell differentiation, proliferation, and survival. Abnormal activation of the JAK/STAT signaling pathway, by gene mutation or amplification, may induce cancer development, and sustained JAK/STAT activation is involved in chemoresistance. While many therapeutic agents have been developed to treat colon cancer, there remains no drug to overcome resistance to chemotherapies. The purpose of this study was to determine the potential of CJ14939 as a novel JAK inhibitor for the treatment of colorectal cancer. MATERIALS AND METHODS: In this study, cell culture, cell death assay, 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, colony formation assay, immunoblot analysis and tumor xenograft were applied. RESULTS: CJ14939 induced cell death, and inhibited phosphorylation of JAK1 and STAT3 in colorectal cancer cells. Furthermore, CJ14939 also promoted oxaliplatin-induced cell death, up-regulated expression of cleaved caspase-3, and down-regulated expression of phospho-JAK1 and phospho-STAT3. In vivo, co-treatment with CJ14939 and oxaliplatin notably reduced tumor growth when compared with CJ14939 or oxaliplatin treatment alone. CONCLUSION: This study identifies the important potential of CJ14939 in colorectal cancer treatment and suggests that combining CJ14939 with oxaliplatin might be a novel therapeutic strategy for patients with colorectal cancer.

Inhibition of JAK1/2 can overcome EGFR-TKI resistance in human NSCLC.

Non-small lung cancer (NSCLC) is the most common cancer in the world. The epidermal growth factor receptor (EGFR) gene is mutated in approximately 10% of lung cancer cases in the US and 50% of lung cancer in Asia. The representative target therapeutic agent, erlotinib (EGFR tyrosine kinase inhibitor; EGFR TKI), is effective in inactivating EGFR in lung cancer patients. However, approximately 50-60% of patients are resistant to EGFR TKI. These populations are associated with the EGFR mutation. To overcome resistance to EGFR TKI, we discovered a JAK1 inhibitor, CJ14939. We investigated the efficacy of CJ14939 in human NSCLC cell lines in vitro and in vivo. Our results showed that CJ14939 induced the inhibition of cell growth. Moreover, we demonstrated that combination treatment with erlotinib and CJ14939 induced cell death in vitro and inhibited tumor growth in vivo. In addition, we confirmed the suppression of phosphorylated EGFR, JAK1, and Stat3 expression in erlotinib and CJ14939-treated human NSCLC cell lines. Our results provide evidence that JAK inhibition overcomes resistance to EGFR TKI in human NSCLCs.

Human breast cancer cells display different sensitivities to ABT-263 based on the level of survivin.

ABT-263 (navitoclax), a Bcl-2 family protein inhibitor, was clinically tested as an anti-cancer agent. However, the clinical trials were limited given the occurrence of resistance to monotherapy in breast cancer cells. Our study investigates the mechanisms for overcoming navitoclax resistance by combining it with an mTOR inhibitor to indirectly target survivin. The apoptotic effects of navitoclax occurred in MDA-MB-231 breast cancer cells in a time- and dose-dependent fashion, but MCF-7 cells were resistant to navitoclax treatment. The expression of Bcl-2 family genes was not altered by navitoclax, but the expression of survivin, a member of the inhibitors of apoptosis proteins (IAP) family, was downregulated, which increased death signaling in MDA-MB-231 cells. In MCF-7 cells, a navitoclax-resistant cell line, combined treatment with navitoclax and everolimus synergistically reduced survivin expression and induced cell death. These data indicate that navitoclax induces cell death in MDA-MB-231 cells but not in MCF-7 cells. Decreased survivin expression in MDA-MB-231 cells may be a primary pathway for death signaling. Combined navitoclax and everolimus treatment induces cell death by reducing the stability of survivin in MCF-7 cells. Given that survivin-targeted therapy overcomes resistance to navitoclax, this strategy could be used to treat breast cancer patients.

GSK-3ß-mediated fatty acid synthesis enhances epithelial to mesenchymal transition of TLR4-activated colorectal cancer cells through regulation of TAp63.

Glycogen synthase kinase-3ß (GSK-3ß) in cancer cells is a critical regulatory component of both cellular metabolism and epithelial-mesenchymal transition (EMT) processes via regulation of the ß-catenin/E-cadherin and phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Lipogenesis of cancer cells also plays a critical role in survival and metastasis. We investigated the role of GSK-3ß-mediated intracellular fatty acid synthesis to control EMT in TLR4-activated colorectal cancer cells and the underlying regulatory mechanism. Engagement of TLR4 with lipopolysaccharide (LPS) in colon cancer cells promoted the induction of phosphorylated GSK-3ß and related lipogenic enzymes as well as the expression of CD74, CD44 and macrophage inhibitory factor (MIF), but decreased expression of transcriptionally active p63 (TAp63). In addition, targeted inhibition of GSK-3ß using SB216763 was accompanied by decreased intracellular fatty acid synthesis and blockage of CD74 and CD44 expression, whereas it reversed the level of TAp63. Although TAp63 overexpression had no effect on the expression of CD74 and CD44 in LPS-treated colon cancer cells, GSK-3ß-dependent fatty acid synthesis and invasive activity were significantly suppressed. Notably, inhibition of CD44 or CD74 by siRNA not only attenuated de novo lipogenesis and migratory activity but also restored the expression of TAp63 in LPS-activated colon cancer cells. These results suggest that TAp63-mediated GSK-3ß activation induced by TLR4 stimulation triggers migration and invasion of colon cancer cells through the regulation of lipid synthesis and GSK-3ß-mediated CD74/CD44 expression could be a target to control fatty acid-related EMT process through the modulation of TAp63 expression.

Biochemical characterization of evasion from peptidoglycan recognition by Staphylococcus aureus D-alanylated wall teichoic acid in insect innate immunity.

We recently reported that D-alanylation of Staphylococcus aureus wall teichoic acid (WTA) mitigates an induction of the Toll-mediated humoral response in Drosophila by interfering with peptidoglycan (PG) recognition by PG recognition protein-SA (PGRP-SA). Here, we investigated the mode of this interference by using an in vitro cell free system from larvae of the coleoptran insect Tenebrio molitor. WTA modification on PG had a potent inhibitory effect on PGRP-SA-mediated Toll proteolytic cascade activation, and the D-alanylation of WTA enhanced its inhibitory effect. Purified D-alanylated WTA released from PG lost its inhibitory action on both Toll cascade activation and PGRP-SA binding to insoluble PG. The inhibition of PGRP-SA binding to PG by D-alanylated WTA took place not only on polymeric PG but also on WTA-attached disaccharide units of monomeric PG. These results suggest that D-alanylation-mediated evasion requires the covalent bonding of D-alanylated WTA to PG, but not net-like cross-linking structure of PG.

Inhibitory role for D-alanylation of wall teichoic acid in activation of insect Toll pathway by peptidoglycan of Staphylococcus aureus.

Pathogenic bacteria mitigate host immunity to establish infections, but the mechanism of this bacterial action has not been fully elucidated. To search for cell wall components that modulate innate immune responses in host organisms, we examined Staphylococcus aureus mutants, which were deficient in components of the cell wall, for pathogenicity in Drosophila. A mutation of dltA, which is responsible for the D-alanylation of teichoic acids, brought about an increase in the survival rate of adult flies that had received a septic infection with the bacteria. The growth of dltA-deficient S. aureus in adult flies was less efficient than that of the parental strain. The level of mRNA of Toll pathway-dependent antimicrobial peptides was higher in flies infected with the dltA mutant than that observed after the infection with the parental strain. The defective phenotype associated with the mutation of dltA, reduced pathogenicity and growth, was not evident in flies lacking the Toll pathway. Finally, a fraction of peptidoglycan prepared from the dltA mutant induced the expression of mRNA of a Toll-dependent antimicrobial peptide in flies and was bound by peptidoglycan recognition protein-SA in vitro more effectively than that obtained from the parental strain, and this difference was lost after the removal of wall teichoic acid from peptidoglycan. Taken together, we conclude that D-alanylated wall teichoic acid of S. aureus mitigates a Toll-mediated humoral response in Drosophila interfering with the recognition of peptidoglycan by a pattern recognition receptor.

Three pairs of protease-serpin complexes cooperatively regulate the insect innate immune responses.

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and beta-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.

Structural basis for the recognition of lysozyme by MliC, a periplasmic lysozyme inhibitor in Gram-negative bacteria.

Lysozymes are an important component of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall constituent. Many bacteria have contrived various means of dealing with this bactericidal enzyme, one of which is to produce lysozyme inhibitors. Recently, a novel family of bacterial lysozyme inhibitors was identified in various Gram-negative bacteria, named MliC (membrane bound lysozyme inhibitor of C-type lysozyme). Here, we report the crystal structure of Pseudomonas aeruginosa MliC in complex with chicken egg white lysozyme. Combined with mutational study, the complex structure demonstrates that the invariant loop of MliC plays a crucial role in the inhibition of the lysozyme by its insertion to the active site cleft of the lysozyme, where the loop forms hydrogen and ionic bonds with the catalytic residues. Since MliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC will be of relevance to the control of bacterial growth in animal hosts.