Background and purpose: Targeted therapy and immunotherapy have led to dramatic change in the treatment of lung cancer, however, the overall 5-year survival rate of lung cancer patients is still suboptimal. It is important to exploit new potential of molecularly targeted therapies. High-frequency somatic mutations in KEAP1/NRF2 (27.9%) have been identified in lung squamous cell carcinoma. In this research, we explored the role of KEAP1 somatic mutations in the development of LSCC and whether a nuclear factor erythroid 2-related factor 2(NRF2) inhibitor be potential to target lung cancer carrying KEAP1/NRF2 mutations. Methods: Lung cancer cell lines A549 and H460 with loss-of-function mutations in KEAP1 stably transfected with wild-type (WT) KEAP1 or somatic mutations in KEAP1 were used to investigate the functions of somatic mutations in KEAP1. Flow cytometry, plate clone formation experiments, and scratch tests were used to examine reactive oxygen species, proliferation, and migration of these cell lines. Results: The expression of NRF2 and its target genes increased, and tumor cell proliferation, migration, and tumor growth were accelerated in A549 and H460 cells stably transfected with KEAP1 mutants compared to control cells with a loss-of-function KEAP1 mutation and stably transfected with WT KEAP1 in both in vitro and in vivo studies. The proliferation of A549 cell line trasfected with the R320Q KEAP1 mutant was inhibited more apparent than that of the A549 cell line trasfected with WT KEAP1 after treatment with NRF2 inhibitor ML385. Conclusion: Somatic mutations of KEAP1 identified from patients with LSCC likely promote tumorigenesis mediated by activation of the KEAP1/NRF2 antioxidant stress response pathway. NRF2 inhibition with ML385 could inhibit the proliferation of tumor cells with KEAP1 mutation.

Mimecan, also known as an osteoinductive factor or osteoglycin, is a 12 kDa secreted protein corresponding to the 105 C-terminal amino acids (residues 176–280) of the preproprotein encoded by the osteoinductive factor gene (Oif), first identified in the organic matrix of bovine bone by chromatography ( Bentz et al., 1989 x Bentz et al., 1989 Bentz, H., Nathan, R.M., Rosen, D.M., Armstrong, R.M., Thompson, A.Y., Segarini, P.R., Mathews, M.C., Dasch, J.R., Piez, K.A., and Seyedin, S.M. Purification and characterization of a unique osteoinductive factor from bovine bone. J. Biol. Chem. 1989; 264: 20805–20810 PubMed | Google Scholar See all References Bentz et al., 1989 ). Furthermore, a 25 kDa keratin sulfate proteoglycan in bovine cornea was shown to be encoded by the cDNA of this gene, representing the 223 C-terminal amino acids of the encoded protein (residues 58–280) ( Funderburgh et al., 1997 x Funderburgh et al., 1997 Funderburgh, J.L., Corpuz, L.M., Roth, M.R., Funderburgh, M.L., Tasheva, E.S., and Conrad, G.W. Mimecan, the 25-kDa corneal keratan sulfate proteoglycan, is a product of the gene producing osteoglycin. J. Biol. Chem. 1997; 272: 28089–28095 Crossref | PubMed | Scopus (150) | Google Scholar See all References Funderburgh et al., 1997 ). The full-length mimecan cDNA was identified from the human pituitary, and was submitted to GenBank (accession no. AF100758) in our previous study ( Hu et al., 2000 x Hu et al., 2000 Hu, R.M., Han, Z.G., Song, H.D., Peng, Y.D., Huang, Q.H., Ren, S.X., Gu, Y.J., Huang, C.H., Li, Y.B., Jiang, C.L., Fu, G., Zhang, Q.H., Gu, B.W., Dai, M., Mao, Y.F., Gao, G.F., Rong, R., Ye, M., Zhou, J., Xu, S.H., Gu, J., Shi, J.X., Jin, W.R., Zhang, C.K., Wu, T.M., Huang, G.Y., Chen, Z., Chen, M.D., and Chen, J.L. Gene expression profiling in the human hypothalamus–pituitary–adrenal axis and full-length cDNA cloning. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9543–9548 Crossref | PubMed | Scopus (91) | Google Scholar See all References Hu et al., 2000 ). The genomic structure of mimecan is highly conserved among different species. The human mimecan precursor (298 amino acids) exhibits 92% homology with bovine mimecan (299 amino acids), suggesting a functional importance ( Madisen et al., 1990 x Madisen et al., 1990 Madisen, L., Neubauer, M., Plowman, G., Rosen, D., Segarini, P., Dasch, J., Thompson, A., Ziman, J., Bentz, H., and Purchio, A.F. Molecular cloning of a novel bone-forming compound: osteoinductive factor. DNA Cell Biol. 1990; 9: 303–309 Crossref | PubMed | Scopus (70) | Google Scholar See all References Madisen et al., 1990 ). However, the physiological functions of mimecan remain elusive. Several studies suggest mimecan may be an essential component of normal vascular extracellular matrix, and it is involved in atherosclerosis ( Kampmann et al., 2009 x Kampmann et al., 2009 Kampmann, A., Fernandez, B., Deindl, E., Kubin, T., Pipp, F., Eitenmuller, I., Hoefer, I.E., Schaper, W., and Zimmermann, R. The proteoglycan osteoglycin/mimecan is correlated with arteriogenesis. Mol. Cell. Biochem. 2009; 322: 15–23 Crossref | PubMed | Scopus (30) | Google Scholar See all References Kampmann et al., 2009 ). Mimecan is considered a major putative regulator of the left ventricular mass ( Petretto et al., 2008 x Petretto et al., 2008 Petretto, E., Sarwar, R., Grieve, I., Lu, H., Kumaran, M.K., Muckett, P.J., Mangion, J., Schroen, B., Benson, M., Punjabi, P.P., Prasad, S.K., Pennell, D.J., Kiesewetter, C., Tasheva, E.S., Corpuz, L.M., Webb, M.D., Conrad, G.W., Kurtz, T.W., Kren, V., Fischer, J., Hubner, N., Pinto, Y.M., Pravenec, M., Aitman, T.J., and Cook, S.A. Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass. Nat. Genet. 2008; 40: 546–552 Crossref | PubMed | Scopus (105) | Google Scholar See all References Petretto et al., 2008 ), and the absence of mimecan has been observed in the development of colorectal cancer ( Wang et al., 2007 x Wang et al., 2007 Wang, Y., Ma, Y., Lu, B., Xu, E., Huang, Q., and Lai, M. Differential expression of mimecan and thioredoxin domain-containing protein 5 in colorectal adenoma and cancer: a proteomic study. Exp. Biol. Med. (Maywood). 2007; 232: 1152–1159 Crossref | PubMed | Scopus (61) | Google Scholar See all References Wang et al., 2007 ). In our previous study, mimecan was coexpressed with adrenocorticotropic hormone (ACTH) in corticotroph cells of the pituitary, and was up-regulated by glucocorticoids ( Ma et al., 2010 x Ma et al., 2010 Ma, Q.Y., Zuo, C.L., Ma, J.H., Zhang, X.N., Ru, Y., Li, P., Pan, C.M., Liu, Z., Cao, H.M., Chen, M.D., and Song, H.D. Glucocorticoid up-regulates mimecan expression in corticotroph cells. Mol. Cell. Endocrinol. 2010; 321: 239–244 Crossref | PubMed | Scopus (12) | Google Scholar See all References Ma et al., 2010 ). Moreover, mimecan regulates ACTH secretion in corticotroph cells, and may play roles in the coordination of the hypothalamus–pituitary–adrenal axis ( Ma et al., 2011 x Ma et al., 2011 Ma, Q.Y., Zhang, X.N., Jiang, H., Wang, Z.Q., Zhang, H.J., Xue, L.Q., Chen, M.D., and Song, H.D. Mimecan in pituitary corticotroph cells may regulate ACTH secretion and the HPAA. Mol. Cell. Endocrinol. 2011; 341: 71–77 Crossref | PubMed | Scopus (6) | Google Scholar See all References Ma et al., 2011 ). These previous results suggest that mimecan is an important factor involved in the physiological and biological functions. In the current study, we report that mimecan is highly expressed in adipose tissue, and it acts as a satiety factor inhibiting food intake by inducing interleukin (IL)-1β and IL-6 expression in the hypothalamus.

Mimecan mRNA was present in a limited number of mouse and human tissues, however, abundant mimecan mRNA was observed in the lung tissue. Therefore, we hypothesize that mimecan could serve as a biomarker for differentiating various histological types of lung cancers. In humans, the mimecan mRNA was found most abundant in ovary and less abundant in lung by using Northern blot analysis. Moreover, the mimecan was expressed strongly in the epithelial cells of the bronchial wall and weaker in the epithelial cells of the alveolar sacs by in situ hybridization and immunohistochemical analysis. Furthermore, the mimecan immunoreactivity was found in 103 (97.2%) of 106 non-small cell lung cancers (NSCLCs). Nevertheless, a large majority of small cell lung cancers (SCLCs) (50/56, 89.3%) showed negative immunoreactivity to mimecan polyclonal antibody. A significant difference of mimecan immunoreactivity was found between NSCLC and SCLC (P<0.00001). This is the first study showing that mimecan could serve as an excellent pathological biomarker to distinguish NSCLCs from SCLCs.

Mutations of the epidermal growth factor receptor (EGFR) in patients with non-small cell lung cancer (NSCLC) were identified by re-sequencing all exons of this gene to evaluate the frequencies of EGFR gene mutation and identify rare or novel EGFR mutations. A total of 55 NSCLC samples from 55 patients were included in the study. Genomic DNA was extracted and exons 1–28 of the EGFR gene were sequenced to identify mutations. The cDNA of the EGFR gene with P848L and T790M double mutants was constructed by introducing point mutations into the wild-type EGFR vector using a site-directed mutagenesis kit. Among the 55 patients with NSCLC, 8 patients carried mutations of the EGFR gene. Notably, of the mutation-harboring patients with a pathological type of adenocarcinoma, 6 were non-smokers. The in vitro study demonstrated that the P848L mutant had a similar response to that of the wild-type EGFR after gefitinib treatment, and the P848L and T790M double mutant exhibited high resistance to gefitinib. These EGFR mutations preferentially occurred in lung adenocarcinoma patients, most of whom were non-smokers. In the in vitro study, P848L mutant EGFR had a similar response as the wild-type EGFR to gefitinib treatment, suggesting that lung cancer patients with a rare mutation of EGFR, such as the P848L mutation, do not respond to gefitinib treatment.

Background: Streptococcus pneumoniae is the major cause of life‐threatening infections. Toll‐like receptors (TLRs) and NOD‐like receptors (NLRs) could recognise S. pneumoniae and regulate the production of pro‐inflammatory cytokines. UGRP1, highly expressed in lung, is predominantly secreted in airways. However, the function of UGRP1 in pneumonia is mainly unknown.Methods and results: We showed that upon TLR2/TLR4/NOD2 agonists stimulation or S. pneumoniae infection, treatment with UGRP1 could promote phosphorylation of p65 and enhance IL‐6, IL‐1β and TNFα production in macrophages. We further elucidated that after binding with cell‐surface receptor PDPN, UGRP1 could activate RhoA to enhance interaction of IKKγ and IKKβ, which slightly activated NF‐κB to improve expression of TLR2, MyD88, NOD2 and NLRP3. Deletion of UGRP1 or blocking UGRP1 interaction with PDPN protected mice against S. pneumoniae‐induced severe pneumococcal pneumonia, and activating RhoA with agonist in UGRP1‐deficient mice restored the reduced IL‐6 production.Conclusion: We demonstrated that UGRP1–PDPN–RhoA signaling could activate NF‐κB to promote expression of TLR2, MyD88, NOD2 and NLRP3, which enhanced inflammatory cytokines secretion during S. pneumoniae infection. Antibodies, which could interrupt interaction of UGRP1 and PDPN, are potential therapeutics against S. pneumoniae.