The non-catalytic functions of kinases have provided novel and crucial mechanisms underlying kinase inhibitor resistance, thereby presenting new opportunities for discovery of kinase-targeted drugs. Nevertheless, only a very limited modulators targeting non-catalytic functions have been reported. As the first discovered proto-oncogene, the non-receptor tyrosine kinase SRC promotes the progression of hematologic malignancies and numerous solid tumors via complex pathways. Several SRC kinase inhibitors were approved for the treatment of hematologic malignancies, yet not for solid tumors. The rapid development of resistance, attributed to the complexity of the signaling pathway, is a key factor restricting the application of SRC kinase inhibitors. The non-kinase function of SRC is one of the primary reasons, but the non-kinase function of SRC remains unclear. In this study, it was discovered that upon abrogation of phosphorylation by SRC kinase inhibitors, non-catalytic SRC promotes the transcription of the oncogenes TRIB3 and SPC24 transcription by binding to their promoter sequence. Simultaneously, it interacts with TRIB3 and mutually prevents the proteasome-mediated degradation by the E3 ligase CHIP, ultimately inducing resistance to SRC kinase inhibitors. The peptide TS1-2 inhibits the tumor progression of pancreatic ductal adenocarcinoma (PDAC), kidney renal papillary cell carcinoma (KIRP), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), and breast invasive carcinoma (BRCA), and enhances the efficacy of SRC kinase inhibitors in solid tumors. This study elucidates the mechanism of non-catalytic functions of SRC, validates the tumor-promoting function and mechanism of nuclear accumulation of SRC/TRIB3 following SRC kinase inhibitor treatment, identifies a potential target SRC/TRIB3, and offers the candidate peptide TS1-2 as a new strategy for SRC kinase inhibitor resistance. ### Competing Interest Statement The authors have declared no competing interest.

The interactions of environmental compartments with epithelial cells are essential for mammary gland development and homeostasis. Currently, the direct crosstalk between the endothelial niche and mammary epithelial cells remains poorly understood. Here, we show that faciogenital dysplasia 5 (FGD5) is enriched in mammary basal cells (BCs) and mediates critical interactions between basal and endothelial cells (ECs) in the mammary gland. Conditional deletion of Fgd5 reduced, whereas conditional knockin of Fgd5 increased, the engraftment and expansion of BCs, regulating ductal morphogenesis in the mammary gland. Mechanistically, murine mammary BC-expressed FGD5 inhibited the transcriptional activity of activating transcription factor 3 (ATF3), leading to subsequent transcriptional activation and secretion of CXCL14. Furthermore, activation of CXCL14/CXCR4/ERK signaling in primary murine mammary stromal ECs enhanced the expression of HIF-1α-regulated hedgehog ligands, which initiated a positive feedback loop to promote the function of BCs. Collectively, these findings identify functionally important interactions between BCs and the endothelial niche that occur through the FGD5/CXCL14/hedgehog axis.

Protein kinase C α (PKCα) regulates diverse biological functions of cancer cells and is a promising therapeutic target. However, clinical trials of PKC-targeted therapies have not yielded satisfactory results. Recent studies have also indicated a tumor-suppressive role of PKCs via unclear molecular mechanisms. In this study, we found that PKCα inhibition enhances CD8+ T-cell-mediated tumor evasion and abolishes antitumor activity in immunocompetent mice. We further identified PKCα as a critical regulator of programmed cell death-ligand 1 (PD-L1) and found that it enhances T-cell-dependent antitumor immunity in breast cancer by interacting with PD-L1 and suppressing PD-L1 expression. We demonstrated that PKCα-mediated PD-L1 phosphorylation promotes PD-L1 degradation through β transducin repeat-containing protein. Notably, the efficacy of PKCα inhibitors was intensified by synergizing with anti-PD-L1 mAb therapy to boost antitumor T-cell immunity in vivo. Clinical tissue array analysis revealed that PKCα expression is positively correlated with T-cell function and the interferon-gamma signature in patients with breast cancer. This study demonstrated the antitumor capability of PKCα, identified potential therapeutic strategies to avoid tumor evasion via PKC-targeted therapies, and provided a proof of concept for targeting PKCα in combination with anti-PD-L1 mAb therapy as a potential therapeutic approach against breast cancer, especially TNBC.

Oxidative stress can attack precursor nucleotides, resulting in nucleic acid damage in cells. It remains unclear how 8-oxo-dGTP and 8-oxoGTP, oxidized forms of dGTP and GTP, respectively, could affect DNA or RNA oxidation levels and tumor development. To address this, we intravenously administered 8-oxo-dGTP and 8-oxoGTP to wild-type and MTH1-knockout mice. 8-oxoGTP administration increased frequency of tumor incidence, which is more prominent in MTH1-knockout mice. However, 8-oxo-dGTP treatment rather reduced tumor development regardless of the mouse genotype. The tumor suppressive effects of 8-oxo-dGTP were further confirmed using xenograft and C57/6J-ApcMin/Nju mouse models. Mechanistically, 8-oxo-dGTP increased the 8-oxo-dG contents in DNA and DNA strand breakage, induced cell cycle arrest in S phase and apoptosis mediated by AIF, eventually leading to reduced tumor incidence. These results suggest distinct roles of 8-oxo-dGTP and 8-oxoGTP in tumor development.

Insulin signaling is essential for glucose metabolism, and insulin decreases insulin receptor (InsR) levels in a dose-dependent and time-dependent manner. However, the regulatory mechanisms of InsR reduction upon insulin stimulation remain poorly understood. Here, we show that Eph receptor B4 (EphB4), a tyrosine kinase receptor that modulates cell adhesion and migration, can bind directly to InsR, and this interaction is markedly enhanced by insulin. Due to the adaptor protein 2 (Ap2) complex binding motif in EphB4, the interaction of EphB4 and InsR facilitates clathrin-mediated InsR endocytosis and degradation in lysosomes. Hepatic overexpression of EphB4 decreases InsR and increases hepatic and systemic insulin resistance in chow-fed mice, whereas genetic or pharmacological inhibition of EphB4 improve insulin resistance and glucose intolerance in obese mice. These observations elucidate a role for EphB4 in insulin signaling, suggesting that EphB4 might represent a therapeutic target for the treatment of insulin resistance and type 2 diabetes.

FGD5 promotes basal-like breast cancer tumorigenesis and progression by inducing and maintaining cancer stemness in an EGFR-dependent manner.

The existence of breast cancer stem cells (BCSCs) is a major reason underlying cancer metastasis and recurrence after chemotherapy and radiotherapy. Targeting BCSCs may ameliorate breast cancer relapse and therapy resistance. Here we report that expression of the pseudokinase Tribble 3 (TRIB3) positively associates with breast cancer stemness and progression. Elevated TRIB3 expression supports BCSCs by interacting with AKT to interfere with the FOXO1-AKT interaction and suppress FOXO1 phosphorylation, ubiquitination, and degradation by E3 ligases SKP2 and NEDD4L. The accumulated FOXO1 promotes transcriptional expression of SOX2, a transcriptional factor for cancer stemness, which in turn, activates FOXO1 transcription and forms a positive regulatory loop. Disturbing the TRIB3-AKT interaction suppresses BCSCs by accelerating FOXO1 degradation and reducing SOX2 expression in mouse models of breast cancer. Our study provides insights into breast cancer development and confers a potential therapeutic strategy against TRIB3-overexpressed breast cancer.

Emerging evidence suggests that microbial pathogens may induce oxidative stress in infected hosts. The aim of the present study was to investigate the relationship between changes in oxidative stress and intestinal infection with and without antibiotic treatment in animal models. Sprague-Dawley (SD) rats were divided into three groups: rats infected with Salmonella enterica serovar Enteritidis (S. enteritidis), rats infected with S. enteritidis followed by norfloxacin treatment, and the control group. To evaluate oxidative stress changes, levels of 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn), which represented oxidative damage to RNA and DNA, respectively, were analysed in urine and tissue samples. In urine, the level of 8-oxo-Gsn increased significantly after oral exposure to S. enteritidis (p <= 0.001) and returned to baseline after recovery. Notably, norfloxacin treatment decreased the level of 8-oxo-Gsn in urine significantly (p = 0.001). Changes of 8-oxo-Gsn measured in tissues from the small intestine, colon, liver and spleen were consistent with 8-oxo-Gsn measured in urine. Our study suggested that 8-oxo-Gsn in urine may serve as a highly sensitive biomarker for evaluating the severity of S. enteritidis infection and the effectiveness of antibiotic treatment against infection.

Recent studies have shown that KIF5B (conventional kinesin heavy chain) mediates glucose transporter type 4 translocation and adiponectin secretion in 3T3-L1 adipocytes, suggesting an involvement of KIF5B in the homeostasis of metabolism. However, the in vivo physiologic function of KIF5B in adipose tissue remains to be determined. In this study, adipose-specific Kif5b knockout (F-K5bKO) mice were generated using the Cre-LoxP strategy. F-K5bKO mice had similar body weights to controls fed on a standard chow diet. However, F-K5bKO mice had hyperlipidemia and significant glucose intolerance and insulin resistance. Deletion of Kif5b aggravated the deleterious impact of a high-fat diet (HFD) on body weight gain, hepatosteatosis, glucose tolerance, and systematic insulin sensitivity. These changes were accompanied by impaired insulin signaling, decreased secretion of adiponectin, and increased serum levels of leptin and proinflammatory adipokines. F-K5bKO mice fed on an HFD exhibited lower energy expenditure and thermogenic dysfunction as a result of whitening of brown adipose due to decreased mitochondria biogenesis and down-regulation of key thermogenic gene expression. In conclusion, selective deletion of Kif5b in adipose tissue exacerbates HFD-induced obesity and its associated metabolic disorders, partly through a decrease in energy expenditure, dysregulation of adipokine secretion, and insulin signaling.-Cui, J., Pang, J., Lin, Y.-J., Gong, H., Wang, Z.-H., Li, Y.-X., Li, J., Wang, Z., Jiang, P., Dai, D.-P., Li, J., Cai, J.-P., Huang, J.-D., Zhang, T.-M. Adipose-specific deletion of Kif5b exacerbates obesity and insulin resistance in a mouse model of diet-induced obesity.

1. CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme superfamily, we recently identified 22 CYP2D6 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of venlafaxine in vitro.2. The wild-type and 24 CYP2D6 variants were expressed in insect cells, and each variant was characterized using venlafaxine as the substrate. Reactions were performed at 37 degrees C with 5-500 mu M substrate (three variants was adjusted to 1000 mu M) for 50 min. By using high-performance liquid chromatography to detect the products, the kinetic parameters K-m, V-max, and intrinsic clearance (V-max/K-m) of O-desmethylvenlafaxine were determined.3. Among the 22 CYP2D6 variants, the intrinsic clearance (V-max/K-m) values of all variants were significantly decreased (from 0.2% to 84.5%) compared with wild-type CYP2D6*1. In addition, the kinetic parameters of two CYP2D6 variants could not be detected because they have no detectable enzyme activity.4. The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards venlafaxine in vivo.

AIMS:Cytochrome P450 (CYP450) 2D6 is an important member of the P450 enzyme superfamily and responsible for clearing 25% of clinically important drugs. The aim of this study was to assess the catalytic characteristics of 24 CYP2D6 allelic isoforms found in the Chinese population and their effects on the metabolism of risperidone in vitro.METHODS:Insect microsomes expressing wild-type CYP2D6 and 24 CYP2D6 allelic variants were incubated with 20-1,000 μmol/l risperidone for 40 min at 37°C. After termination, risperidone and 9-OH risperidone, the metabolite of risperidone, were precipitated and used for signal collection by ultra-performance liquid-chromatography tandem mass spectrometry.RESULTS:Among 24 CYP2D6 variants tested, 2 variants (CYP2D6*92 and CYP2D6*96) were found to be with no detectable activity. Two variants (E215K and R440C) exhibited higher intrinsic clearance values than the wild-type protein, while the remaining 20 CYP2D6 allelic variants exhibited significantly decreased clearance values (2.01-87.56%) compared to CYP2D6*1.CONCLUSION:These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2D6 alleles when administering risperidone in the clinic. This is the first report of all these novel alleles for risperidone metabolism, providing fundamental data for further clinical studies on CYP2D6 alleles.

www.karger.com/pha J. Asai, Kyoto, Japan Gen-ichi Atsumi, Teikyo, Japan Jie Bai, Kunming, China B. Bermas, Boston, Mass., USA Pangala V. Bhat, Montreal, Que., Canada De-Liang Cao, Springfield, Ill., USA D. Cheng, Princeton, N.J., USA Young Sik Cho, Daegu, Korea Hartmut Derendorf, Gainesville, Fla., USA Salvatore Di Somma, Rome, Italy Dong Hailong, Xi’an, China Erol Kevser, Eskisehir, Turkey S. Espuelas, Pamplona, Spain Jun Fang, Kumamoto, Japan Patrice Forget, Brussels, Belgium Masanori Fujii, Kyoto, Japan T. Fujii, Kyotanabe, Japan Li Gan, Madison, Wisc., USA Luis Granero, Valencia, Spain Ki Baik Hahm, Seongnam, Korea Abid Hamid, Jammu, India Philippe Haouzi, Hershey, Pa., USA R. Hardeland, Göttingen, Germany Chaoyong He, Oklahoma City, Okla., USA Christopher Henstridge, Budapest, Hungary Stan Heptinstall, Nottingham, UK Thomas Hohlfeld, Düsseldorf, Germany Masatoshi Hori, Tokyo, Japan T.M. Hu, Taipei, Taiwan Longshuang Huang, Chicago, Ill., USA Christian Humpel, Innsbruck, Austria Iacobazzi Dominga, Bristol, UK K. Iga, Kyotanabe, Japan S. Iino, Fukui, Japan N. Inagaki, Gifu, Japan N. Inatomi, Fijisawa, Japan Keiichi Ishihara, Kyoto, Japan Y. Isohama, Chiba, Japan Yukako Ito, Kyoto, Japan B. Jugdutt, Edmonton, Alta., Canada H. Katsuki, Kumamoto, Japan Atsufumi Kawabata, Higashiosaka, Japan Naoyuki Kawao, Osaka, Japan So Hee Kim, Suwon, Korea Yoshihisa Kitamura, Kyoto, Japan Heidemarie Kletzl, Basel, Switzerland Ipek Komsuoglu Celikyurt, Kocaeli, Turkey Veerapol Kukongviriyapan, Khon Kaen, Thailand S.W.S. Leung, Hong Kong, Hong Kong Karen Liby, Hanover, N.H., USA Zhen-Guo Liu, Columbus, Ohio, USA Kexin Liu, Dalian, China Xiaowen Liu, Houston, N.Y., USA Z. Lu, New York, N.Y., USA Christopher J. Lynch, Hershey, Pa., USA Hisashi Matsuda, Kyoto, Japan K. Matsumoto, Kyoto, Japan S. Matsumoto-Okano, Fujisawa, Japan Shogo Matsuyama, Himeji, Japan K. Miyata, Tsukuba, Japan D. Miyazawa, Nagoya, Japan Nobuaki Mizutani, Kobe, Japan E. Morii, Suita, Japan H. Motohashi, Kyoto, Japan V. Murthy, Rochester, N.Y., USA Rais Mustafa, Kuala Lumpur, Malaysia Takeshi Nabe, Kyoto, Japan T. Nabe, Hirakata, Japan Y. Naito, Kyoto, Japan Tsutomu Nakahara, Tokyo, Japan Tetsuo Nakata, Kyoto, Japan Yuji Nakayama, Kyoto, Japan H.J. Nam, Scottsdale, Ariz., USA Pierluigi P. Navarra, Rome, Italy Hiroyuki Nishikawa, Osaka, Japan Tomoyuki Nishizaki, Nishinomiya, Japan R. Noland, Baton Rouge, La., USA S. Ohya, Kyoto, Japan A. Overby, Trondheim, Norway Y. Oyama, Tokushima, Japan Vera Petricevich, Cuernavaca, Mexico Nikolaos Pitsikas, Larissa, Greece Y. Qi, Temple, Pa., USA D.L. Quinn, Tucson, Ariz., USA S. Reisman, Irving, Tex., USA M. Ruggero, New Haven, Conn., USA Samir Saha, Stockholm, Sweden Y. Sawada, Fukuoka, Japan

Warfarin is the most frequently prescribed anticoagulant for the long-term treatment in the clinic. Recent studies have shown that polymorphic alleles within the CYP2C9, VKORC1, and CYP4F2 genes are related to the warfarin dosage requirement. In this study, a novel non-synonymous mutation (1009C>A) in CYP2C9 was detected in a warfarin-hypersensitive patient, while the other two candidate genes were both found to be homozygous for the wild-type alleles. The newly identified point mutation results in an amino acid substitution at position 337 of the CYP2C9 protein (P337T) and has been designated as the novel allele CYP2C9*58. When expressed in insect cell microsomes, the relative intrinsic clearance values of the CYP2C9.58 variant for tolbutamide and losartan were quite similar to those of the typical defective variant CYP2C9.3, whereas the clearance value of CYP2C9.58 for diclofenac was slightly higher than that of another typical defective variant CYP2C9.2. These data suggested that when compared with wild-type CYP2C9.1, the enzymatic activity of the novel allelic variant has been greatly reduced by the 1009C>A mutation. If patients carrying this allele take drugs metabolized by CYP2C9, their metabolic rate might be slower than that of wild-type allele carriers and thus much more attention should be paid to their clinical care.