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.

Identification of the promoter region regulating the transcription of the REV7 gene.

REV7 is involved in various biological processes including DNA repair and mutagenesis, cell cycle regulation, gene transcription, and carcinogenesis. REV7 is highly expressed in adult testicular germ cells as well as several malignant tumors. REV7 expression levels are associated with prognosis in several human cancers, however, the mechanism of REV7 transcriptional regulation has not been elucidated. In this study, we characterized the promoter region of the REV7 gene. A luciferase reporter assay using the human germ cell tumor cell line NEC8 was utilized to examine the upstream genomic region of REV7 for transcriptional activity, and two transcriptional activation regions were identified. We determined a small genomic region important for transcriptional activation using site-directed mutagenesis; this region is shared by several putative binding motifs for transcription factors, including the cAMP-responsive element modulator (CREM), cAMP-response element binding protein (CREB), and B-lymphocyte-induced maturation protein-1 (BLIMP-1). Exogenous CREM and CREB expression had no effect on the transcriptional activity in NEC8 cells or the human embryonic kidney cell line HEK293T. In contrast, exogenous BLIMP-1 expression increased luciferase reporter activity in HEK293T cells but unexpectedly decreased activity in NEC8 cells. Chromatin immunoprecipitation analysis demonstrated that BLIMP-1 binds to the genomic region near the binding motif in the REV7 promoter. Additionally, BLIMP-1 overexpression promoted endogenous REV7 expression in HEK293T cells. These findings suggest that BLIMP-1 may be a putative transcriptional regulator of REV7 in mammalian cells.

REV7 in Cancer Biology and Management.

DNA repair and cell cycle regulation are potential biological fields to develop molecular targeting therapies for cancer. Human REV7 was originally discovered as a homologous molecule to yeast Rev7, which is involved in DNA damage response and mutagenesis, and as the second homolog of yeast Mad2, involved in the spindle assembly checkpoint. Although REV7 principally functions in the fields of DNA repair and cell cycle regulation, many binding partners of REV7 have been identified using comprehensive analyses in the past decade, and the significance of REV7 is expanding in various other biological fields, such as gene transcription, epigenetics, primordial germ cell survival, neurogenesis, intracellular signaling, and microbial infection. In addition, the clinical significance of REV7 has been demonstrated in studies using human cancer tissues, and investigations in cancer cell lines and animal models have revealed the greater impacts of REV7 in cancer biology, which makes it an attractive target molecule for cancer management. This review focuses on the functions of REV7 in human cancer and discusses the utility of REV7 for cancer management with a summary of the recent development of inhibitors targeting REV7.

Clinicopathological Significance of BRCAness in Resectable Pancreatic Ductal Adenocarcinoma and Its Association With Anticancer Drug Sensitivity in Pancreatic Cancer Cells.

OBJECTIVE: The concept of BRCAness has been proposed as a homologous recombination repair dysfunction triggered by a genetic defect in the BRCA pathway including the BRCA1/2 mutations. A certain number of pancreatic ductal adenocarcinoma (PDAC) patients have BRCAness. However, a large-scale analysis of BRCAness in PDAC has not been performed. In addition, no basic studies have examined the significance of BRCAness in PDAC cell lines. METHODS: Ninety-two patients who underwent surgery for PDAC were enrolled. Formalin-fixed and paraffin-embedded specimens of resected PDACs were used to analyze BRCAness by multiplex ligation-dependent probe amplification. We also analyzed BRCAness in pancreatic cancer cell lines and the sensitivity to cisplatin and olaparib using a colony formation assay. RESULTS: Of the 92 patients with PDAC, 6 were detected to have BRCAness-positive PDAC (6.5%). No significant differences in overall survival and progression-free survival were observed between the BRCAness-positive and BRCAness-negative groups. One PDAC cell line, KP-2, was positive for BRCAness and was more sensitive to cisplatin and olaparib than the BRCAness-negative cell lines. CONCLUSIONS: Our results revealed that a considerable number of PDACs are positive for BRCAness, suggesting that BRCAness status could be a useful biomarker for selecting anticancer treatments for advanced or relapsed PDAC.

CD109 expression in tumor cells and stroma correlates with progression and prognosis in pancreatic cancer.

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, whose expression is upregulated in some types of malignant tumors. High levels of CD109 in tumor cells have been reported to correlate with poor prognosis; however, significance of CD109 stromal expression in human malignancy has not been elucidated. In this study, we investigated the tumorigenic properties of CD109 in pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analysis of 92 PDAC surgical specimens revealed that positive CD109 expression in tumor cells was significantly associated with poor prognosis (disease-free survival, p = 0.003; overall survival, p = 0.002), and was an independent prognostic factor (disease-free survival, p = 0.0173; overall survival, p = 0.0104) in PDAC. Furthermore, CD109 expression was detected in the stroma surrounding tumor cells, similar to that of α-smooth muscle actin, a histological marker of cancer-associated fibroblasts. The stromal CD109 expression significantly correlated with tumor progression in PDAC (TNM stage, p = 0.033; N factor, p = 0.024; lymphatic invasion, p = 0.028). In addition, combined assessment of CD109 in tumor cells and stroma could identify the better prognosis group of patients from the entire patient population. In MIA PaCa-2 PDAC cell line, we demonstrated the involvement of CD109 in tumor cell motility, but not in PANC-1. Taken together, CD109 not only in the tumor cells but also in the stroma is involved in the progression and prognosis of PDAC, and may serve as a useful prognostic marker in PDAC.

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.

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.

Expression of CA2 and CA9 carbonic anhydrases in ulcerative colitis and ulcerative colitis-associated colorectal cancer.

Ulcerative colitis (UC) is characterized by chronic inflammation in the colonic mucosa and submucosa with repeating relapse and remission, but the pathogenesis is unknown. Patients with long-standing UC are at high risk of neoplasm development. The aim of the present study was to identify molecules whose expression is associated with UC and UC-associated colorectal cancer (UCCA). Biopsy specimens from UC and normal colonic mucosae were analyzed using a proteomics approach, in which carbonic anhydrase 2 (CA2) was identified as a molecule downregulated in UC mucosae. Immunohistochemically, CA2 expression was detected in normal and diverticulitis mucosal epithelia, and its expression decreased as UC activity increased. CA2 expression was almost undetectable in UCCA. We also analyzed the expression of another carbonic anhydrase, CA9, and its upstream molecule, hypoxia-inducible factor-1α (HIF-1α), both of which are induced under hypoxic conditions. It was revealed that CA9 expression was relatively low in normal, diverticulitis and UC mucosae, and was upregulated in UCCA. HIF-1α expression was consistently low in all tissue types examined. In conclusion, these results suggest that CA2 and CA9 may be possible indicators of UC activity and UCCA development, respectively.

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.

UV-induced mutations in epidermal cells of mice defective in DNA polymerase η and/or ι.

Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to sunlight-induced skin cancer, which is caused by deficiency in DNA polymerase η (Polη). Polη catalyzes accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions. DNA polymerase ι (Polι) is a paralog of Polη that has been suggested to participate in TLS past UV-induced lesions, but its function in vivo remains uncertain. We have previously reported that Polη-deficient and Polη/Polι double-deficient mice showed increased susceptibility to UV-induced carcinogenesis. Here, we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Polη- and/or Polι-deficient mice. While Polη-deficient mice showed significantly higher UV-induced mutation frequencies than wild-type mice, Polι deficiency did not influence the frequencies in the presence of Polη. Interestingly, the frequencies in Polη/Polι double-deficient mice were statistically lower than those in Polη-deficient mice, although they were still higher than those of wild-type mice. Sequence analysis revealed that most of the UV-induced mutations in Polη-deficient and Polη/Polι double-deficient mice were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T pairs associated with Polη deficiency suggests that Polη contributes to accurate TLS past both thymine- and cytosine-containing dimers in vivo. A significant decrease in G:C to A:T transition in Polη/Polι double-deficient mice when compared with Polη-deficient mice suggests that Polι is involved in error-prone TLS past cytosine-containing dimers when Polη is inactivated.

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.

Stalled Polη at its cognate substrate initiates an alternative translesion synthesis pathway via interaction with REV1.

DNA polymerase η (Polη), whose gene mutation is responsible for the inherited disorder xeroderma pigmentosum variant (XP-V), carries out accurate and efficient translesion synthesis (TLS) across cyclobutane pyrimidine dimer (CPD). As Polη interacts with REV1, and REV1 interacts with other TLS polymerases including Polι, Polκ and Polζ, Polη may play a role in recruitment of these TLS polymerases at lesion site. But it is unclear whether UV sensitivity of XP-V patients is caused not only by defect of Polη activity but also by dysfunction of network between Polη and other TLS polymerases. Here, we examined whether the TLS polymerase network via Polη is important for replicative bypass of CPDs and DNA damage tolerance induced by UV in mouse cells. We observed that UV sensitivity of Polη-deficient mouse cells was moderately rescued by the expression of a catalytically inactive Polη. Moreover, this recovery of cellular UV sensitivity was mediated by the interaction between Polη and REV1. However, expression of the inactive mutant Polη was not able to suppress the incidence of UV-induced mutation observed in Polη-deficient cells. We propose the model that REV1 and Polκ are involved in DNA damage tolerance via Polη-REV1 interaction when Polη fails to bypass its cognate substrates.