REV7 is involved in outcomes of platinum-based chemotherapy in pancreatic cancer by controlling the DNA damage response.

REV7 is a multifunctional protein implicated in various biological processes, including DNA damage response. REV7 expression in human cancer cells affects their sensitivity to DNA-damaging agents. In the present study, we investigated the significance of REV7 in pancreatic ductal adenocarcinoma (PDAC). REV7 expression was immunohistochemically examined in 92 resected PDAC specimens and 60 endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) specimens of unresectable PDAC treated with platinum-based chemotherapy, and its association with clinicopathologic features was analyzed. Although REV7 expression was not significantly associated with the progression of primary tumors (T-factor and Stage) in either resected or unresectable PDAC, decreased levels of REV7 expression in EUS-FNAB specimens of unresectable PDAC were significantly associated with better outcomes of platinum-based chemotherapy and a favorable prognosis. REV7-deficient PDAC cell lines showed suppressed cell growth and enhanced sensitivity to cisplatin in vitro. Tumor-bearing mice generated using REV7-deficient PDAC cell lines also showed enhanced sensitivity to cisplatin in vivo. RNA sequencing analysis using WT and REV7-deficient PDAC cell lines revealed that REV7 inactivation promoted the downregulation of genes involved in the DNA repair and the upregulation of genes involved in apoptosis. Our results indicate that decreased expression of REV7 is associated with better outcomes of platinum-based chemotherapy in PDAC by suppressing the DNA damage response. It is also suggested that REV7 is a useful biomarker for predicting the outcome of platinum-based chemotherapy and the prognosis of unresectable PDAC and is a potential target for PDAC treatment.

Upregulation of REV7 correlates with progression of malignant melanoma.

REV7 is a multifunctional protein implicated in DNA damage tolerance, cell cycle control, and gene expression, and is involved in the carcinogenesis of various human tumors. It has been reported that REV7 expression is associated with ultraviolet-induced mutagenesis; however, the role of REV7 expression in skin cancers, including malignant melanomas, remains unclear. In the present study, we investigated the clinical and biological significance of REV7 in malignant melanoma. Levels of REV7 expression in human skin cancers were evaluated immunohistochemically. Positive expression of REV7 was frequently observed in malignant melanomas, as well as in squamous cell carcinomas and basal cell carcinomas. Enhanced immunoreactivity to REV7 was closely linked with cell proliferation assessed by Ki-67 labeling indexes in the three skin cancers, and was related with tumor thickness in malignant melanomas. REV7 depletion in malignant melanoma cells MEWO and G361 suppressed cell proliferation, migration, and invasion abilities. REV7 depletion also affected the expression of intracellular signaling molecules AKT and ERK in MEWO cells, resulting in downregulation of ERK signal activation. In addition, REV7 depletion facilitated sensitivity to cisplatin, but not to dacarbazine, in MEWO cells. Our results suggest that REV7 expression correlates with disease progression of malignant melanoma.

["Exclusivity" and quantifier float in bakari"Exclusivity" and quantifier float in bakari]. / 「ばかり」の「限定」と遊離数量詞.

This paper presents a descriptive study that analyzes the semantic meaning of Toritate focus particle bakari. Previous studies reported that, although bakari expresses exclusivity, it is characterized by the fact that it permits non-applicable cases, thereby drawing the conclusion that the meaning of bakari is not exclusivity. This paper argues that bakari does indeed denote "exclusivity" as bakari is supported by the phenomenon that non-applicable cases are unacceptable when bakari co-occurs with floating quantifiers. Considering existing research on this subject, the following was observed. Even though the subjective set, as established by the speaker's past experiences to interpret the meaning of bakari, may not be consistent with the real world, the number of events that form the said set match the number of real-world events when bakari co-occurs with floating quantifiers due to the characteristics of floating quantifiers. In such cases, bakari does not permit non-applicable cases. The interpretation that permits non-applicable cases applies to situations where the set established by the speaker is fixed at a narrower range than the real world, and the non-applicable cases exist outside the set. We thus conclude that bakari denotes "exclusivity" that does not permit non-applicable cases.

Increased expression of REV7 in small cell lung carcinomas and its association with tumor cell survival and proliferation.

REV7 is involved in multiple biological processes including DNA damage tolerance, cell cycle regulation and gene expression, and is an accessory subunit of the mutation-prone DNA polymerase ζ. It has been reported that REV7 expression is associated with poor prognosis in several human cancers. The aim of this study is to investigate the significance of REV7 in lung carcinogenesis. Immunohistochemical analyses of surgically resected lung cancer specimens revealed that REV7 shows an increased expression in small cell lung carcinomas (SCLCs) when compared with other histological types of lung carcinoma. Association between REV7 expression levels and clinicopathological factors was investigated using SCLC cases with or without surgical resection. Our analyses revealed that high REV7 expression significantly correlated with tumor cell proliferation, assessed by Ki-67 labeling indices, and was negatively associated with distant metastasis and extensive-stage disease. No significant association was detected between REV7 expression and other factors, including prognosis or response to chemoradiotherapy in SCLC. Increase in REV7 expression in SCLC was confirmed using SCLC cell lines. In addition, siRNA-mediated depletion of REV7 activated the apoptotic pathway and suppressed cell growth in SCLC cells. These results suggest that REV7 plays an important role in tumor cell survival and proliferation in SCLC.

Inactivation of REV7 enhances chemosensitivity and overcomes acquired chemoresistance in testicular germ cell tumors.

REV7 is a multitasking protein involved in replication past DNA lesions, cell cycle regulation, and gene expression. REV7 is highly expressed in the adult testis and plays an essential role in primordial germ cell maintenance in mice. In this study, we analyzed whether REV7 can be a molecular target for the treatment of testicular germ cell tumors (TGCTs), in which acquired chemoresistance is a major cause of treatment failure. Strong expression of REV7 was detected in human TGCT tissues by immunohistochemistry. REV7 depletion in the TGCT cell lines suppressed cell proliferation and increased sensitivity to cisplatin and doxorubicin. cDNA microarray analysis revealed that REV7 depletion downregulated genes in the DNA repair gene set and upregulated genes in the apoptosis gene set. REV7 depletion-provoked chemosensitivity was associated with DNA double-strand break accumulation and apoptosis activation. In addition, inactivation of REV7 in cisplatin-resistant TGCT cells recovered chemosensitivity at almost equal levels as parental cells in vitro and in vivo. Our results indicate that inactivation of REV7 enhances chemosensitivity and overcomes chemoresistance in TGCT cells, suggesting REV7 as a potential therapeutic target in chemoresistant TGCTs.

CD109, a negative regulator of TGF-ß signaling, is a putative risk marker in diffuse large B-cell lymphoma.

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein that negatively regulates TGF-ß signaling. CD109 was originally identified in hematopoietic tumors; however, the significance of CD109 in hematopoietic malignancies remains unclear. Here, we study the association of CD109 with diffuse large B-cell lymphoma (DLBCL) prognosis. Eighty-four DLBCL specimens were immunohistochemically analyzed for CD109 expression, and 31 and 53 cases were classified into low- and high-CD109 expression groups, respectively. CD109 expression was not associated with overall survival using the Kaplan-Meier analysis and log-rank tests (P = 0.17); however, a significant association was observed between high-CD109 expression and low-1-year survival (P = 0.01). Moreover, in combination with the revised International Prognostic Index (R-IPI), R-IPI-poor/CD109-high was associated with poorer prognosis compared with R-IPI-poor alone. We assessed TGF-ß signaling in CD109-depleted Nalm6 cells (a human B-lymphoblastic leukemia/lymphoma cell line), and found prolonged Smad2 phosphorylation compared with control cells after TGF-ß1 stimulation, suggesting that CD109 attenuates TGF-ß1 signaling in human B-cell tumors. These results suggest that CD109 is a putative biomarker for identifying a high-risk group among DLBCL patients.

High expression of REV7 is an independent prognostic indicator in patients with diffuse large B-cell lymphoma treated with rituximab.

REV7 is a multifunctional protein involved in DNA damage tolerance, cell-cycle regulation, gene expression, and carcinogenesis. Although its expression is reportedly associated with poor prognosis in human solid tissue cancers, the significance of REV7 expression in hematopoietic malignancies is unclear. This study evaluated the prognostic significance of REV7 expression in patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab-combined chemotherapy. Using immunohistochemistry, we analyzed 83 specimens of de novo DLBCL [38 germinal center B-cell-like (GCB) and 45 non-GCB DLBCLs] treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone for REV7 expression. Aberrant REV7 expression was detected in DLBCL cell nuclei. High REV7 expression was associated with significantly shorter overall survival (OS) and progression-free survival (PFS) using Kaplan-Meier analysis and log-rank tests (P < 0.01 and P < 0.01, respectively). Multivariate analysis revealed that REV7 expression is an independent prognostic factor for both OS and PFS. Additionally, when patients were divided into four groups using a combination of REV7 expression and international prognostic index (IPI) or Bcl-2 expression, REV7(High)/IPI(Poor) and REV7(High)/Bcl-2(High) patients showed the poorest outcome. These results indicate that REV7 may be a useful biomarker to predict the prognosis of patients with DLBCL treated with rituximab.

CD109 attenuates TGF-ß1 signaling and enhances EGF signaling in SK-MG-1 human glioblastoma cells.

CD109 is a glycosylphosphatidylinositol-anchored cell surface protein that is frequently detected in squamous cell carcinomas. CD109 is a negative regulator of TGF-ß1 signaling in human keratinocytes, and the N-terminal fragment of CD109 secreted from cells after cleavage by the furin protease is important for modulating TGF-ß1 signaling. Previously, we found that CD109 is expressed in human glioblastoma cells; however, the role of CD109 in glioblastoma cells is not established. Here, we describe the effects of CD109 in human glioblastoma cell lines. Three glioblastoma cell lines, SK-MG-1, U251MG and MG178, were tested and CD109 overexpression attenuated TGF-ß1 signaling and enhanced EGF signaling in SK-MG-1, but not in U251MG or MG178. The N-terminal CD109 fragment in SK-MG-1 was hyperglycosylated compared with that in MG178 or U251MG. The conditioned medium of CD109-overexpressing SK-MG-1, containing the secreted N-terminal CD109, had a negative effect on TGF-ß1 signaling in wild-type SK-MG-1 and MG178, whereas it did not show any effect on EGF signaling. In addition, cell surface CD109 interacts with EGF receptor in SK-MG-1 overexpressing CD109, and exhibited enhanced cell migration and invasion. These findings suggest that CD109 attenuates TGF-ß1 signaling and enhances EGF signaling in SK-MG-1 cells and that the membrane-anchored CD109 may play major roles in the EGF signaling pathway.

Association of D2-40 and MMP-1 expression with cyst formation in lung metastatic lesions of cutaneous angiosarcoma on the scalp: immunohistochemical analysis of 23 autopsy cases.

Cutaneous angiosarcoma of the scalp can rapidly develop into pulmonary metastasis. The pulmonary metastatic lesions display a unique appearance, so-called thin-walled cysts, which cause a fatal relapsed pneumothorax by rupturing. We analyzed 23 autopsy cases of angiosarcoma with pulmonary metastasis to elucidate the mechanism of the thin-walled cyst development. Of the 23 cases of cutaneous angiosarcoma of the scalp with pulmonary metastasis, radiological examination revealed pulmonary metastatic lesions as thin-walled cysts (39%), nodules (39%), mixed cysts and nodules (13%), and ground-glass opacity (9%). All the cases but one with cystic metastases were complicated by pneumothorax. The cystic lesions were accompanied by podoplanin (D2-40)-positive tumor cells in the luminal surface of the cysts. In both primary cutaneous lesions and pulmonary metastatic lesions, the D2-40 expression was positive for angiosarcoma cells in 100% and 92% of the cases, respectively. While the estrogen-regulated gene (ERG) expression was also positive for most of the primary and metastatic pulmonary angiosarcomas, D2-40 was a more useful marker to differentiate tumor cells from the background than was the ERG expression of the vascular endothelium. Matrix metalloproteinase-1 (MMP-1) expression was also predominant in primary lesions (95%) and pulmonary metastatic lesions (82.6%). Proteinases, like MMP-1, might be associated with a developing thin-walled cyst, although there were no differences in the MMP-1 expression in either the cystic or nodular metastasis. Two extremely aggressive cases showed cystic metastasis with central necrosis that was not observed in other cases. These results suggest a pathogenesis of thin-walled cysts in some progressive cases.

Small gastrointestinal stromal tumor in the stomach: identification of precursor for clinical gastrointestinal stromal tumor using c-kit and α-smooth muscle actin expression.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. To find precursors for clinical GISTs of the stomach, small gastric stromal tumors of less than 3 cm were collected and examined immunohistochemically with analysis of the KIT mutation. Sixty-eight of 74 lesions were classified into 4 representative groups according to the expression of c-kit and α-smooth muscle actin (αSMA): group A, c-kit diffusely positive and αSMA negative (18 cases); group B, c-kit diffusely positive and αSMA focally positive (13); group C, c-kit focally positive and αSMA diffusely positive (27); and group D, c-kit negative and αSMA diffusely positive (10). Of the 4 groups, groups A and B of c-kit diffuse expression showed higher cellularity and labeling indices of p27(Kip1) and Ki-67 than did groups C and D of diffuse αSMA expression. Incidence of KIT exon 11 mutation in groups A and B was 86% (25/29), whereas that in groups C and D was 0% (0/20). Small gastric stromal tumors with c-kit diffuse expression were considered precursors for clinical GIST because they were significantly different from c-kit focally positive or negative tumors. The mutation of KIT is considered as an early event in tumorigenesis of GIST.

Papillary thyroid carcinoma with heterotopic ossification is a special subtype with extensive progression.

An immunohistochemical study was conducted of 108 papillary carcinoma cases, including 48 cases with intratumoral heterotopic ossification (IHO). In 48 cases, papillary carcinoma with IHO was accompanied by nodular fibrosis. Cases of papillary carcinoma with IHO or nodular fibrosis showed higher incidences of lymph node metastasis, multifocal lesions, and extrathyroidal invasion than those without IHO and nodular fibrosis. A higher number of stromal myofibroblasts was observed in papillary carcinoma with IHO or nodular fibrosis than in that without fibrosis. Expression of both basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP)-2 was the highest in papillary carcinoma with IHO. Papillary carcinoma with IHO showed higher vascular invasion and higher numbers of capillaries expressing nestin, which is associated with high expression of vascular endothelial growth factor (VEGF). Papillary carcinoma with IHO is a unique subtype with extensive progression including frequent lymph node metastasis, multifocality, and invasive behavior. Papillary carcinoma with IHO was correlated with expression of bFGF, BMP-2, and VEGF in the carcinoma cells, leading to neovascularization.

Invasive behavior of ulcerative colitis-associated carcinoma is related to reduced expression of CD44 extracellular domain: comparison with sporadic colon carcinoma.

BACKGROUND: To elucidate relations of invasion of ulcerative colitis (UC)-associated carcinoma with its prognosis, the characteristics of invasive fronts were analyzed in comparison with sporadic colonic carcinomas. METHODS: Prognoses of 15 cases of UC-associated colonic carcinoma were compared with those of sporadic colon carcinoma cases, after which 75 cases of sporadic invasive adenocarcinoma were collected. Tumor budding was examined histologically at invasive fronts using immunohistochemistry (IHC) of pancytokeratin. Expressions of beta-catenin with mutation analysis, CD44 extracellular domain, Zo-1, occludin, matrix matalloproteinase-7, laminin-5γ2, and sialyl Lewis X (LeX) were immunohistochemically evaluated. RESULTS: UC-associated carcinoma showed worse prognosis than sporadic colon carcinoma in all the cases, and exhibited a tendency to become more poorly differentiated when carcinoma invaded the submucosa or deeper layers than sporadic carcinoma. When the lesions were compared with sporadic carcinomas considering differentiation grade, reduced expression of CD44 extracellular domain in UC-associated carcinoma was apparent. Laminin-5γ2 and sialyl-LeX expression showed a lower tendency in UC-associated carcinomas than in their sporadic counterparts. There were no differences in the numbers of tumor budding foci between the two lesion types, with no apparent relation to nuclear beta-catenin levels in IHC. CONCLUSIONS: UC-associated carcinoma showed poorer differentiation when the carcinoma invaded submucosa or deeper parts, which may influence the poorer prognosis. The invasive behavior of UC-associated carcinoma is more associated with CD44 cleavage than with basement membrane disruption or sialyl-Lewis-antigen alteration.

Epithelial and stromal genetic instability linked to tumor suppressor genes in ulcerative colitis-associated tumorigenesis.

OBJECTIVE: We have previously documented not only epithelial but also stromal genetic instability in ulcerative colitis (UC)-associated lesions, including adenocarcinomas, using microsatellite markers close to the p53 gene on chromosome 17 (Chr.17). However, about half of the UC-associated tumors do not have p53 gene alterations. The purpose of this study was to detect early genetic instability (loss of heterozygosity (LOH) and microsatellite instability (MSI)) of both epithelial and stromal cells in UC-associated tumorigenesis, using different microsatellite markers from the p53 gene. MATERIAL AND METHODS: The laser-captured microdissection-PCR-GeneScan method was applied to investigate genetic instability in both the epithelial and stromal elements of early UC-associated lesions (regenerative mucosa and dysplasia) and carcinomas using multiple microsatellite markers, chiefly close to tumor suppressor genes (TSGs: p16(INK4A), Rb, Smad4 and fragile histidine triad (FHIT)). Furthermore, expression of their gene products was analyzed by immunohistochemistry. RESULTS: In epithelium, although LOH for Chr.17 markers increased along with histological progression, the frequencies of LOH or MSI for TSG markers were found to be almost constantly increased in both stromal and epithelial components of all lesion types. In contrast, genetic instability of National Cancer Institute (NCI)-recommended standard markers was not found to be significantly correlated with UC-associated tumorigenesis. Immunohistochemically, epithelial p16(INK4A) expression tended to be decreased in LOH-positive lesions (p = 0.0780) and Smad4 expression was significantly decreased (p < 0.05). CONCLUSIONS: These results suggest that genetic instability in the stroma, especially regarding TSG markers, may play an important role in early-phase, UC-associated tumorigenesis. In addition, decreased expression of TSG due to genetic alteration might contribute to tumorigenesis.

Low frequency of promoter methylation of O6-methylguanine DNA methyltransferase and hMLH1 in ulcerative colitis-associated tumors: comparison with sporadic colonic tumors.

To cast light on the contribution of methylation to genesis of ulcerative colitis (UC)-associated tumors, promoter methylation and expression of O6-methylguanine DNA methyltransferase (MGMT), hMLH1, p16INK4, and E-cadherin were examined in 14 low-grade dysplasias (LGDs), 15 high-grade dysplasias (HGDs), and 14 adenocarcinomas associated with UC and, for comparison, in 30 sporadic adenomas with LGD, 30 adenomas with HGD, and 60 adenocarcinomas, using methylation-specific polymerase chain reaction and immunohistochemical analysis. The frequency of MGMT and hMLH1 methylation in UC-associated tumors was low, with a significant difference between HGD and sporadic adenomas with HGD of the left hemicolon. The methylation frequency of p16INK4 in UC-associated tumors was also relatively low compared with sporadic colonic tumors. For E-cadherin, methylation was limited in both types of tumor. Decrease of expression of MGMT, hMLH1, and p16INK4 was significantly correlated with methylation. Thus, compared with the sporadic type, contribution of methylation to UC-associated tumorigenesis seems to be low.

Disruption of the p53-p53r2 DNA repair system in ulcerative colitis contributes to colon tumorigenesis.

With ulcerative colitis (UC)-associated tumorigenesis, p53 gene alteration is considered to be a key event. To clarify whether the p53-checkpoint is operating in foci of inflammation and that its disruption is a feature of UC-associated neoplasms, the present immunohistochemical study was conducted. Since accumulation of butyric acid with active UC is associated with apoptosis, effects of in vitro exposure of newly established UC-cancer derived cell lines to organic acids were also assessed. The regulatory subunit of ribonucleotide reductase, p53R2, was found to be localized with p53 in situ, and levels of p53, phospho-p53, p53R2 and inducible nitric oxide synthase were significantly intercorrelated. However, p53R2 expression was clearly reduced with progression through UC-associated dysplasia to carcinoma, demonstrating an inverse relation with p53 overexpression. In vitro treatment with butyrate or propionic acid, but not succinic acid, elicited a positive response in the p53-p53R2 system. Moreover, p53-dependent DNA repair, investigated by radioactive nucleotide incorporation, was induced by butyric acid and inhibited by short-interfering p53 and p53R2 RNAs. Therefore, it was concluded that the p53-p53R2-dependent DNA repair system is constitutively stimulated by butyric acid, which accumulates in UC inflammatory lesions. Since failure of the p53-G(1) checkpoint may cause dysfunction of repair under the influence of butyrate, gene alterations may increase and spread through the genome, leading to tumorigenesis.