Cancer stem cells (CSCs), characterized by self-renewal and unlimited proliferation, lead to therapeutic resistance in lung cancer. In this study, we aimed to investigate the expressions of stem cell-related genes in lung adenocarcinoma (LUAD). The stemness index based on mRNA expression (mRNAsi) was utilized to analyze LUAD cases in the Cancer Genome Atlas (TCGA). First, mRNAsi was analyzed with differential expressions, survival analysis, clinical stages, and gender in LUADs. Then, the weighted gene co-expression network analysis was performed to discover modules of stemness and key genes. The interplay among the key genes was explored at the transcription and protein levels. The enrichment analysis was performed to annotate the function and pathways of the key genes. The expression levels of key genes were validated in a pan-cancer scale. The pathological stage associated gene expression level and survival probability were also validated. The Gene Expression Omnibus (GEO) database was additionally used for validation. The mRNAsi was significantly upregulated in cancer cases. In general, the mRNAsi score increases according to clinical stages and differs in gender significantly. Lower mRNAsi groups had a better overall survival in major LUADs, within five years. The distinguished modules and key genes were selected according to the correlations to the mRNAsi. Thirteen key genes (CCNB1, BUB1, BUB1B, CDC20, PLK1, TTK, CDC45, ESPL1, CCNA2, MCM6, ORC1, MCM2, and CHEK1) were enriched from the cell cycle Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, relating to cell proliferation Gene Ontology (GO) terms, as well. Eight of the thirteen genes have been reported to be associated with the CSC characteristics. However, all of them have been previously ignored in LUADs. Their expression increased according to the pathological stages of LUAD, and these genes were clearly upregulated in pan-cancers. In the GEO database, only the tumor necrosis factor receptor associated factor-interacting protein (TRAIP) from the blue module was matched with the stemness microarray data. These key genes were found to have strong correlations as a whole, and could be used as therapeutic targets in the treatment of LUAD, by inhibiting the stemness features.

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S-palmitoylation, catalyzed by a family of 23 zinc finger Asp-His-His-Cys (DHHC) domain-containing (ZDHHC) protein acyltransferases localized on the cell membrane. However, stemness genes modulated by ZDHHCs in lung adenocarcinoma (LUAD) remain to be defined. Previously, we have constructed a network of cancer stem cell genes, including INCENP, based on mRNA stemness indices (mRNAsi) of LUAD. INCENP has the function of a chromosomal passenger complex locating to centromeres, which is performed by the conserved region of its N-terminal domain. INCENP protein with a deletion of the first non-conserved 26 amino acid sequence failed to target centromeres. However, the exact function of the deleted sequence has not been elucidated. To identify novel cancer stem cell-relevant palmitoylated proteins and responsible ZDHHC enzymes in LUAD, we analyzed multi-omics data obtained from the database of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and the Human Protein Atlas (HPA). ZDHHC5 is distinguished from the ZDHHC family for being up-regulated in mRNA and protein levels and associated with malignant prognosis. ZDHHC5 was positively associated with INCENP, and the correlation score increased with LUAD stages. CSS-Palm results showed Cys15 was the S-palmitoylation site of INCENP. Interestingly, Cys15 locates in the 1–26 aa sequence of INCENP, and is a conserved site across species. As INCENP is a nuclear protein, we predicted that the nuclear localization signal of ZDHHC5 was specific to the importin αβ pathway, and the result of immunofluorescence proves that ZDHHC5 is located in the nucleoplasm, in addition to the plasma membrane. Therefore, our study indicates the S-palmitoylation of INCENP mediated by ZDHHC5 as a potential mechanism of S-palmitoylation to modulate CSCs in LUAD.

Background Obesity-related cardiovascular risk, end points, and mortality are strongly related to arterial stiffening. Current therapeutic approaches for arterial stiffening are not focused on direct targeting within the vessel. Perivascular adipose tissue (PVAT) surrounding the artery has been shown to modulate vascular function and inflammation. Peroxisome proliferator-activated receptor γ (PPARγ) activation significantly decreases arterial stiffness and inflammation in diabetic patients with coronary artery disease. Thus, we hypothesized that PPARγ activation alters the PVAT microenvironment, thereby creating a favorable environment for the attenuation of arterial stiffening in obesity. Methods Obese ob/ob mice were used to investigate the effect of PPARγ activation on the attenuation of arterial stiffening. Various cell types, including macrophages, fibroblasts, adipocytes, and vascular smooth muscle cells, were used to test the inhibitory effect of pioglitazone, a PPARγ agonist, on the expression of elastolytic enzymes. Results PPARγ activation by pioglitazone effectively attenuated arterial stiffening in ob/ob mice. This beneficial effect was not associated with the repartitioning of fat from or changes in the browning of the PVAT depot but was strongly related to improvement of the PVAT microenvironment, as evidenced by reduction in the expression of pro-inflammatory and pro-oxidative factors. Pioglitazone treatment attenuated obesity-induced elastin fiber fragmentation and elastolytic activity and ameliorated the obesity-induced upregulation of cathepsin S and metalloproteinase 12, predominantly in the PVAT. In vitro, pioglitazone downregulated Ctss and Mmp12 in macrophages, fibroblasts, and adipocytes—cell types residing within the adventitia and PVAT. Ultimately, several PPARγ binding sites were found in Ctss and Mmp12 in Raw 264.7 and 3T3-L1 cells, suggesting a direct regulatory mechanism by which PPARγ activation repressed the expression of Ctss and Mmp-12 in macrophages and fibroblasts. Conclusions PPARγ activation attenuated obesity-induced arterial stiffening and reduced the inflammatory and oxidative status of PVAT. The improvement of the PVAT microenvironment further contributed to the amelioration of elastin fiber fragmentation, elastolytic activity, and upregulated expression of Ctss and Mmp12. Our data highlight the PVAT microenvironment as an important target against arterial stiffening in obesity and provide a novel strategy for the potential clinical use of PPARγ agonists as a therapeutic against arterial stiffness through modulation of PVAT function.

Although new analogues of immunomodulatory drugs (IMiDs) are being developed for MM, the molecular mechanism of these drugs remains unclear. In the current study, we used MM cell lines as a model to investigate the molecular mechanism of thalidomide and to compare its potency with IMiDs such as pomalidomide. We determined that thalidomide did not inhibit cell proliferation of RPMI8226 and U266 MM cells, whereas pomalidomide showed a significant inhibitory effect on these two MM cell lines. Interestingly, we further demonstrated that although thalidomide down-regulated bFGF translation through the inhibition of IRES even at 0.1 μg/ml, pomalidomide did not have a similar affect bFGF levels. A colony formation assay demonstrated that thalidomide and the bFGF knock-down clones caused a significant reduction in the clonogenic ability of MM cells, and treatment with exogenous bFGF can recover the clonogenic ability of thalidomide-treated cells and knock-down clones, but not that of pomalidomide-treated cells. This implies that thalidomide, but not pomalidomide, targets the IRES of FGF-2.In conclusion, our results highlight a non-cytotoxic anticancer drug target for thalidomide, the IRES of bFGF, and provide the mechanistic rationale for developing IMiDs as anti-cancer therapeutics in MM patients, with improved potency and fewer side effects.