Identification of effective CCR2 inhibitors for cancer therapy using humanized mice.

C-C chemokine receptor type 2 (CCR2) is the receptor for C-C motif chemokine 2 (CCL2) and is associated with various inflammatory diseases and cancer metastasis. Although many inhibitors for CCR2 have been developed, it remains unresolved which inhibitors are the most effective in the clinical setting. In the present study, we compared 10 existing human CCR2 antagonists in a calcium influx assay using human monocytic leukemia cells. Among them, MK0812 was found to be the most potent inhibitor of human CCR2. Furthermore, we generated a human CCR2B knock-in mouse model to test the efficacy of MK0812 against a lung metastasis model of breast cancer. Oral administration of MK0812 to humanized mice did indeed reduce the number of monocytic myeloid-derived suppressor cells and the rate of lung metastasis. These results suggest that MK0812 is the most promising candidate among the commercially available CCR2 inhibitors. We propose that combining these two screening methods may provide an excellent experimental method for identifying effective drugs that inhibit human CCR2.

Modulation of host glutamine anabolism enhances the sensitivity of small cell lung cancer to chemotherapy.

Small cell lung cancer (SCLC) is one of the deadliest human cancers, with a 5-year survival rate of ∼7%. Here, we performed a targeted proteomics analysis of human SCLC samples and thereby identified hypoxanthine phosphoribosyltransferase 1 (HPRT1) in the salvage purine synthesis pathway as a factor that contributes to SCLC malignancy by promoting cell survival in a glutamine-starved environment. Inhibition of HPRT1 by 6-mercaptopurine (6-MP) in combination with methotrexate (MTX), which blocks the de novo purine synthesis pathway, attenuated the growth of SCLC in mouse xenograft models. Moreover, modulation of host glutamine anabolism with the glutamine synthetase inhibitor methionine sulfoximine (MSO) in combination with 6-MP and MTX treatment resulted in marked tumor suppression and prolongation of host survival. Our results thus suggest that modulation of host glutamine anabolism combined with simultaneous inhibition of the de novo and salvage purine synthesis pathways may be of therapeutic benefit for SCLC.

FOXK1 promotes nonalcoholic fatty liver disease by mediating mTORC1-dependent inhibition of hepatic fatty acid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disorder caused by overnutrition and can lead to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). The transcription factor Forkhead box K1 (FOXK1) is implicated in regulation of lipid metabolism downstream of mechanistic target of rapamycin complex 1 (mTORC1), but its role in NAFLD-NASH pathogenesis is understudied. Here, we show that FOXK1 mediates nutrient-dependent suppression of lipid catabolism in the liver. Hepatocyte-specific deletion of Foxk1 in mice fed a NASH-inducing diet ameliorates not only hepatic steatosis but also associated inflammation, fibrosis, and tumorigenesis, resulting in improved survival. Genome-wide transcriptomic and chromatin immunoprecipitation analyses identify several lipid metabolism-related genes, including Ppara, as direct targets of FOXK1 in the liver. Our results suggest that FOXK1 plays a key role in the regulation of hepatic lipid metabolism and that its inhibition is a promising therapeutic strategy for NAFLD-NASH, as well as for HCC.

Molecular evolution of Keap1 was essential for adaptation of vertebrates to terrestrial life.

Reactive oxygen species (ROS) posed a risk for the transition of vertebrates from aquatic to terrestrial life. How ancestral organisms adapted to such ROS exposure has remained a mystery. Here, we show that attenuation of the activity of the ubiquitin ligase CRL3Keap1 for the transcription factor Nrf2 during evolution was key to development of an efficient response to ROS exposure. The Keap1 gene was duplicated in fish to give rise to Keap1A and the only remaining mammalian paralog Keap1B, the latter of which shows a lower affinity for Cul3 and contributes to robust Nrf2 induction in response to ROS exposure. Mutation of mammalian Keap1 to resemble zebrafish Keap1A resulted in an attenuated Nrf2 response, and most knock-in mice expressing such a Keap1 mutant died on exposure as neonates to sunlight-level ultraviolet radiation. Our results suggest that molecular evolution of Keap1 was essential for adaptation to terrestrial life.

Potentials of C-C motif chemokine 2-C-C chemokine receptor type 2 blockers including propagermanium as anticancer agents.

Inflammation plays an essential role in the development and progression of most cancers. Chemokine C-C motif chemokine 2 (CCL2) and its receptor C-C chemokine receptor type 2 (CCR2) constitute a key signaling axis in inflammation that has recently attracted much interest on the basis of evidence showing its association with cancer progression. Propagermanium (3-oxygermylpropionic acid polymer) is an organogermanium compound that is given for the treatment of hepatitis B in Japan and which inhibits the CCL2-CCR2 signaling pathway. Herein, we review the importance of the CCL2-CCR2 axis as a target in cancer treatment as shown by studies in mice and humans with pharmacological agents including propagermanium.

SCFFbxw7 ubiquitylates KLF7 for degradation in a manner dependent on GSK-3-mediated phosphorylation.

The biological relation between ubiquitin ligases and their substrates has been largely unclear. We previously developed a method-differential proteomics-based identification of ubiquitylation substrates (DiPIUS)-for the comprehensive identification of substrates for a given ubiquitin ligase. We have now applied DiPIUS to the F-box protein Fbxw7 in three cell lines (mHepa, Neuro2A and C2C12) and thereby identified Krüppel-like factor 7 (KLF7) as a candidate substrate of the SCFFbxw7 ubiquitin ligase complex. KLF7 was shown to interact with Fbxw7 and to undergo Fbxw7-mediated polyubiquitylation. The stability of KLF7 was increased by depletion of Fbxw7, mutation of a putative Cdc4 phosphodegron (CPD) of KLF7 or exposure to inhibitors of glycogen synthase kinase-3 (GSK-3). Over-expression of Fbxw7 in Neuro2A cells down-regulated expression of the p21Cip1 gene, which is a transcriptional target of KLF7 in neuronal differentiation and maintenance. Despite the presence of an almost identical CPD sequence in KLF6, the closest paralog of KLF7, mutation of this sequence affected neither the interaction of KLF6 with Fbxw7 nor its half-life. Our results suggest that KLF7, but not KLF6, is a bona fide substrate of SCFFbxw7 , and that control of KLF7 abundance by SCFFbxw7 might contribute to the regulation of neuronal differentiation and maintenance.