👤 Tetsuya Mizutani

Squamous cell carcinoma is the second most prevalent type of non-small cell lung cancer. Analyzing the molecular mechanisms underlying lung carcinoma requires useful tools, such as squamous lung cancer cell lines. A novel new lung squamous cell carcinoma cell line, OMUL-1, was developed from the primary lung cancer of a 74-year-old man. We assessed the characteristics and behavior of OMUL-1 cells were examined, including their growth kinetics, tumorigenicity in mice, histological properties, gene expression profiles using reverse transcription polymerase chain reaction (RT-PCR), and RNA sequencing and invasion assays. OMUL-1-an adherent cell line-resulted in 100% tumor formation when subcutaneously injected into mice. Histological analysis of the subcutaneous tumor using hematoxylin and eosin staining revealed squamous cell carcinoma with characteristics similar to those of the primary tumor (p40 and p63 were positive, and TTF-1 was negative). An invasion assay demonstrated that OMUL-1 had a lower invasion ability compared to that of other developed cell lines. RT-PCR analysis and RNA sequencing indicated that OMUL-1 cells expressed FGFR1, FGFR2, FGFR3, FGFR4, EGFR, HER2, ErbB3, ErbB4, VEGFR3, IGF1R, c-MET, PDGFRa, and PDGFRb. Additionally, picropodophyllin (an IGF1R inhibitor) significantly inhibited the growth of OMUL-1 cells. Immunohistochemistry revealed that IGF1R and PD-L1 were expressed in both the primary and subcutaneous tumors. We developed a novel new squamous cell lung carcinoma cell line, OMUL-1, that expresses IGF1R and PD-L1. Show less

Carcinogenesis results from the sequential acquisition of oncogenic mutations that convert normal cells into invasive, metastasizing cancer cells. Colorectal cancer exemplifies this process through its well-described adenoma-carcinoma sequence, modeled previously using clustered regularly interspaced short palindromic repeats (CRISPR) to induce four consecutive mutations in wild-type human gut organoids. Here, we demonstrate that long-term culture of mismatch-repair-deficient organoids allows the selection of spontaneous oncogenic mutations through the sequential withdrawal of Wnt agonists, epidermal growth factor (EGF) agonists and the bone morphogenetic protein (BMP) antagonist Noggin, while TP53 mutations were selected through the addition of Nutlin-3. Thus, organoids sequentially acquired mutations in AXIN1 and AXIN2 (Wnt pathway), TP53, ACVR2A and BMPR2 (BMP pathway) and NRAS (EGF pathway), gaining complete independence from stem cell niche factors. Quadruple-pathway (Wnt, EGF receptor, p53 and BMP) mutant organoids formed solid tumors upon xenotransplantation. This demonstrates that carcinogenesis can be recapitulated in a DNA repair-mutant background through in vitro selection that targets four consecutive cancer pathways. Show less

Heparan sulfate (HS) is a glycocalyx component present in the extracellular matrix and cell-surface HS proteoglycans (HSPGs). Although HSPGs are known to play functional roles in multiple aspects of tumor development and progression, the effect of HS expression in the tumor stroma on tumor growth in vivo remains unclear. We conditionally deleted Ext1, which encodes a glycosyltransferase essential for the biosynthesis of HS chains, using S100a4-Cre (S100a4-Cre; Ext1f/f) to investigate the role of HS in cancer-associated fibroblasts, which is the main component of the tumor microenvironment. Subcutaneous transplantation experiments with murine MC38 colon cancer and Pan02 pancreatic cancer cells demonstrated substantially larger subcutaneous tumors in S100a4-Cre; Ext1f/f mice. Additionally, the number of myofibroblasts observed in MC38 and Pan02 subcutaneous tumors of S100a4-Cre; Ext1f/f mice decreased. Furthermore, the number of intratumoral macrophages decreased in MC38 subcutaneous tumors in S100a4-Cre; Ext1f/f mice. Finally, the expression of matrix metalloproteinase-7 (MMP-7) markedly increased in Pan02 subcutaneous tumors in S100a4-Cre; Ext1f/f mice, suggesting that it may contribute to rapid growth. Therefore, our study demonstrates that the tumor microenvironment with HS-reduced fibroblasts provides a favorable environment for tumor growth by affecting the function and properties of cancer-associated fibroblasts, macrophages, and cancer cells. Show less

The detoxification of ammonia to urea requires a functional hepatic urea cycle, which consists of six enzymes and two mitochondrial membrane transporters. The initial step of the urea cycle is catalyzed by carbamyl phosphate synthetase 1 (CPS1). CPS1 deficiency (CPS1D) is a rare autosomal recessive disorder. Show less

The p53 family of transcription factors includes p63, which is a master regulator of gene expression in epithelial cells. Determining whether p63 is tumor-suppressive or tumorigenic is complicated by isoform-specific and cellular context-dependent protein associations, as well as antagonism from mutant p53. ΔNp63 is an amino-terminal-truncated isoform, that is, the predominant isoform expressed in cancer cells of epithelial origin. In HaCaT keratinocytes, which have mutant p53 and ΔNp63, we found that mutant p53 antagonized ΔNp63 transcriptional activity but that activation of Ras or transforming growth factor-β (TGF-β) signaling pathways reduced the abundance of mutant p53 and strengthened target gene binding and activity of ΔNp63. Among the products of ΔNp63-induced genes was dual-specificity phosphatase 6 (DUSP6), which promoted the degradation of mutant p53, likely by dephosphorylating p53. Knocking down all forms of p63 or DUSP6 and DUSP7 (DUSP6/7) inhibited the basal or TGF-β-induced or epidermal growth factor (which activates Ras)-induced migration and invasion in cultures of p53-mutant breast cancer and squamous skin cancer cells. Alternatively, overexpressing ΔNp63 in the breast cancer cells increased their capacity to colonize various tissues upon intracardiac injection in mice, and this was inhibited by knocking down DUSP6/7 in these ΔNp63-overexpressing cells. High abundance of ΔNp63 in various tumors correlated with poor prognosis in patients, and this correlation was stronger in patients whose tumors also had a mutation in the gene encoding p53. Thus, oncogenic Ras and TGF-β signaling stimulate cancer progression through activation of the ΔNp63 transcriptional program. Show less

Heterochromatin protein 1 (HP1) is associated with heterochromatin formation and the regulation of gene expression. In this study, we demonstrated that decreased HP1beta, but not HPla, mRNA and protein expression, correlates with invasive potential in five human melanoma cell lines, and we used immunohistochemistry to confirm that HP1beta expression is suppressed during melanoma progression. HPIP levels are decreased in (V600E)B-RAF-transformed mouse melanocytes, suggesting that HP1beta-mediated suppressive mechanisms correlate with melanoma oncogenesis. Expression of microphthalmia associated-transcription factor (MITF), an important melanocyte differentiation factor, is reduced in melanoma, which is correlated with poor prognosis. In CRL1579, SK-MEL-28 and HMV-II human melanoma cells in which HP1beta expression is reduced by RNAi, MITF RNA levels and invasiveness activities are differentially altered and are not correlated with each other. Our findings indicate that the (V600E)B-RAF mutation induces HPIbeta down-regulation, which causes epigenetic gene regulation associated with melanoma progression. Show less

In the yeast, Saccharomyces cerevisiae, cell size is affected by the kind of carbon source in the medium. Here, we present evidence that the Gpr1 receptor and Gpa2 Galpha subunit are required for both maintenance and modulation of cell size in response to glucose. In the presence of glucose, mutants lacking GPR1 or GPA2 gene showed smaller cells than the wild-type strain. Physiological studies revealed that protein synthesis rate was reduced in the mutant strains indicating that reduced growth rate, while the level of mRNAs for CLN1, 2 and 3 was not affected in all strains. Gene chip analysis also revealed a down-regulation in the expression of genes related to biosynthesis of not only protein but also other cellular component in the mutant strains. We also show that GPR1 and GPA2 are required for a rapid increase in cell size in response to glucose. Wild-type cells grown in ethanol quickly increased in size by addition of glucose, while little change was observed in the mutant strains, in which glucose-dependent cell cycle arrest caused by CLN1 repression was somewhat alleviated. Our study indicates that the yeast G-protein coupled receptor system consisting of Gpr1 and Gpa2 regulates cell size by affecting both growth rate and cell division. Show less

Transcription of the rat fatty acid synthase (FAS) gene in the rat liver can be regulated by feeding a high carbohydrate diet. A carbohydrate response element (ChoRE) located on the rat FAS gene promoter has been identified. Using multiple copies of the ChoRE as the bait in a yeast one-hybrid system, a rat liver cDNA library was screened, and the cDNA of ChoRE-binding proteins was cloned. A positive clone that encodes a basic helix-loop-helix protein, enhancer of split- and hairy-related protein-2 (SHARP-2), was obtained. Northern blot analysis revealed that the levels of SHARP-2 mRNA increase when a high carbohydrate diet is fed to normal rats or when insulin is administered to diabetic rats. In primary cultured rat hepatocytes, insulin rapidly induced an accumulation of SHARP-2 mRNA even in the absence of glucose. A time course for the increase in SHARP-2 mRNA levels indicated that it followed by those of FAS and L-type pyruvate kinase mRNAs and that the initial time course of SHARP-2 mRNA was similar to changes in the levels of glucokinase mRNA and phosphoenolpyruvate carboxykinase mRNA. Although wortmannin, LY294002, and actinomycin D blocked the increase in SHARP-2 mRNA levels by insulin, rapamycin, staurosporine, PD98059, okadaic acid, and 8-bromocyclic AMP had no effect. In addition, nuclear run-on assay revealed that transcription of the rat SHARP-2 gene was induced by insulin. Thus, we conclude that insulin induces the transcription of the rat SHARP-2 gene via a phosphoinositide 3-kinase pathway. Show less