Whole-genome sequencing revealed novel prognostic biomarkers and promising targets for therapy of ovarian clear cell carcinoma.

BACKGROUND: Ovarian clear cell carcinoma (OCCC) is mostly resistant to standard chemotherapy that results in poor patient survival. To understand the genetic background of these tumours, we performed whole-genome sequencing of OCCC tumours. METHODS: Tumour tissue samples and matched blood samples were obtained from 55 Japanese women diagnosed with OCCC. Whole-genome sequencing was performed using the Illumina HiSeq platform according to standard protocols. RESULTS: Alterations to the switch/sucrose non-fermentable (SWI/SNF) subunit, the phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway, and the receptor tyrosine kinase (RTK)/Ras signalling pathway were found in 51%, 42%, and 29% of OCCC tumours, respectively. The 3-year overall survival (OS) rate for patients with an activated PI3K/Akt signalling pathway was significantly higher than that for those with inactive pathway (91 vs 40%, hazard ratio 0.24 (95% confidence interval (CI) 0.10-0.56), P=0.0010). Similarly, the OS was significantly higher in patients with the activated RTK/Ras signalling pathway than in those with the inactive pathway (91 vs 53%, hazard ratio 0.35 (95% CI 0.13-0.94), P=0.0373). Multivariable analysis revealed that activation of the PI3K/Akt and RTK/Ras signalling pathways was an independent prognostic factor for patients with OCCC. CONCLUSIONS: The PI3K/Akt and RTK/Ras signalling pathways may be potential prognostic biomarkers for OCCC patients. Furthermore, our whole-genome sequencing data highlight important pathways for molecular and biological characterisations and potential therapeutic targeting in OCCC.

Endoplasmic reticulum-associated degradation of Niemann-Pick C1: evidence for the role of heat shock proteins and identification of lysine residues that accept ubiquitin.

Most cases with Niemann-Pick disease type C carry mutations in NPC1. Some of the mutations, including the most frequent I1061T, give rise to unstable proteins selected for endoplasmic reticulum-associated degradation. The purpose of the current study was to shed mechanistic insights into the degradation process. A proteasome inhibitor MG132 prolonged the life span of the wild-type NPC1 expressed in COS cells. The expressed protein associated with multiple chaperones including heat shock protein 90 (Hsp90), Hsp70, heat shock cognate protein 70 (Hsc70), and calnexin. Accordingly, expression of an E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) enhanced MG132-induced accumulation of ubiquitylated NPC1. Co-expression and RNAi knockdown experiments in HEK cells indicated that Hsp70/Hsp90 stabilized NPC1, whereas Hsc70 destabilized it. In human fibroblasts carrying the I1061T mutation, adenovirus-mediated expression of Hsp70 or treatment with an HSP-inducer geranylgeranylacetone (GGA) increased the level of the mutant protein. In GGA-treated cells, the rescued protein was localized in the late endosome and ameliorated cholesterol accumulation. MALDI-TOF mass spectrometry revealed three lysine residues at amino acids 318, 792, and 1180 as potential ubiquitin-conjugation sites. Substitutions of the three residues with alanine yielded a mutant protein with a steady-state level more than three times higher than that of the wild-type. Introduction of the same substitutions to the I1061T mutant resulted in an increase in its protein level and functional restoration. These findings indicated the role of HSPs in quality control of NPC1 and revealed the role of three lysine residues as ubiquitin-conjugation sites.

Fibroblast growth factor receptor 2 is associated with poor overall survival in clear cell carcinoma of the ovary and may be a novel therapeutic approach.

OBJECTIVE: We previously found that gene and protein expression of fibroblast growth factor receptor (FGFR) 2 were increased in ovarian clear cell carcinoma (CCC); here, we examined FGFR2 expression in CCC tumor tissues and its correlation with clinical parameters. We also analyzed the effect of an FGFR inhibitor on the growth of CCC cells to investigate whether FGFR2 could be a therapeutic target for this disease. METHODS: We analyze the protein expression of FGFR2 by immunohistochemical staining in CCC from 112 patients and evaluated the association of these molecular parameters with clinical outcome. We treated the 11 CCC cell lines with an FGFR inhibitor, and then assessed cell viability, the expression of protein in FGFR2 signaling pathway, and cell cycle distribution. RESULTS: The expressions of FGFR2 were found in 96% of CCC. The 5-year survival rate for patients with a moderate or strong expression of FGFR2 was significantly lower than that for those with an absent or poor expression of FGFR2 (54% vs 79%). Multivariable analysis revealed that FGFR2 expression and disease stage were independent prognostic factors. The FGFR inhibitor effectively suppressed the growth of CCC cells with induction of G1 cell cycle arrest and down-regulated the expression of phosphorylated Akt and phosphorylated ERK. CONCLUSIONS: FGFR2 is an important biomarker predictive of patient outcome and is a potential target for CCC. Further study is warranted for FGFR inhibitor to treat CCC.

Loss of ARID1A expression is associated with poor prognosis in patients with stage I/II clear cell carcinoma of the ovary.

BACKGROUND: Recent studies have shown that somatic mutations in the AT-rich interactive domain 1A (SWI-like) gene (ARID1A) are the most common genetic changes in clear cell carcinoma of the ovary (CCC). A gene mutation of ARID1A was found in approximately half of CCC cases, and led to absence of the encoded protein and inactivation of the putative tumor suppressor. Here, we investigated whether ARID1A could be a prognostic biomarker for this disease. METHODS: We analyzed the protein expression of ARID1A in CCC from 112 patients by immunohistochemical staining, and evaluated the association of these molecular parameters with clinical outcome. RESULTS: The loss of ARID1A expression was found in 39 % (44/112) of CCC, and was not associated with patient age, FIGO stage, and status of residual tumor. The 5-year survival rate for FIGO stage I or II patients with negative tumor expression of ARID1A was lower than those with positive tumor expression of ARID1A (74 % vs 91 %), but this difference was not observed in FIGO stage III or IV patients. Multivariable analysis revealed that ARID1A expression was an independent prognostic factor in FIGO stage I or II CCC patients. CONCLUSION: ARID1A may be a biomarker that is predictive of the outcome of FIGO stage I and II CCC.

Checkpoint kinase inhibitor AZD7762 overcomes cisplatin resistance in clear cell carcinoma of the ovary.

OBJECTIVE: Checkpoint kinase (Chk) inhibitors are thought to increase the cytotoxic effects of DNA-damaging agents and are undergoing clinical trials. The present study was aimed to assess the potential to use the Chk1 and Chk2 inhibitor, AZD7762, with other anticancer agents in chemotherapy to treat ovarian clear cell carcinoma. METHODS: Four ovarian clear cell carcinoma cell lines were used in this study. We treated the cells with AZD7762 and anticancer agents, then assessed cell viability, cell cycle distribution, apoptosis, and the expression of protein in apoptotic pathways and molecules downstream of the Chk signaling pathways. We also investigated the effects of these drug combinations on tumor growth in a nude mouse xenograft model. RESULTS: Synergistic effects from the combination of AZD7762 and cisplatin were observed in all 4 cell lines. However, we observed additive effects when AZD7762 was combined with paclitaxel on all cell lines tested. AZD7762 effectively suppressed the Chk signaling pathways activated by cisplatin, dramatically enhanced expression of phosphorylated H2A.X, cleaved caspase 9 and PARP, decreased the proportion of cells in the gap 0/ gap 1 phase and the synthesis-phase fraction, and increased apoptotic cells. Combinations of small interfering RNA against Chk 1 and small interfering RNA against Chk2 enhanced the cytotoxic effect of cisplatin in both RMG-I and KK cells. Finally, treating mice-bearing RMG-I with AZD7762 and cisplatin significantly suppressed growth of tumors in a xenograft model. CONCLUSIONS: The present study indicates that chemotherapy with AZD7762 and cisplatin should be explored as a treatment modality for women with ovarian clear cell carcinoma.

Establishment and characterization of a novel uterine carcinosarcoma cell line, TU-ECS-1, with mutations of TP53 and KRAS.

A new human uterine carcinosarcoma (UCS) cell line, TU-ECS-1, was established and characterized. The morphological appearance of the cultured cells was an insular of epithelial-like cells arranged in the form of a jigsaw puzzle and mesenchymal-like cells with a spindle-shaped or fibroblast-like morphology. A relatively high proliferation rate was observed with a doubling time of 18.2 h. The chromosome number ranged from 44 to 49 and had an extra chromosome 12 (trisomy 12). The respective half-maximal inhibitory concentrations of cisplatin, paclitaxel, and doxorubicin were 2.9 µM, 154 nM, and 219 ng/mL, respectively. Mutational analysis revealed that TU-ECS-1 cells have mutations of TP53 in exons 4, 6, and 8 and of KRAS at codon 12 (G12D) in exon 2, which is a mutation hot spot on this gene. Western blot analysis showed that p53 protein was overexpressed in TU-ECS-1 cells. Immunostaining of the cultured cells and in vivo tumors showed that the TU-ECS-1 cells and xenografts were positive for epithelial marker cytokeratin AE1/3 and mesenchymal marker vimentin. These results suggested that TU-ECS-1 cells might have both epithelial and mesenchymal characteristics. This cell line may be useful to study the carcinogenesis of UCS and contribute to the development of novel treatment strategies.

Establishment and characterization of a novel ovarian clear cell carcinoma cell line, TU-OC-2, with loss of ARID1A expression.

A new cell line of human ovarian clear cell carcinoma (CCC), TU-OC-2, was established and characterized. The cells were polygonal in shape, grew in monolayers without contact inhibition and were arranged in islands like pieces of a jigsaw puzzle. The chromosome numbers ranged from 41 to 96. A low rate of proliferation was observed and the doubling time was 37.5 h. The IC50 values of cisplatin, 7-ethyl-10-hydroxycamptothecin (SN38), which is an active metabolite of camptothecin, and paclitaxel were 7.7 µM, 17.7 nM and 301 nM, respectively. The drug sensitivity assay indicated that TU-OC-2 was sensitive to SN38, but resistant to cisplatin and paclitaxel. Mutational analysis revealed that TU-OC-2 cells have no mutations of PIK3CA in exons 9 and 20 and of TP53 in exons 4-9. We observed the loss of ARID1A protein expression in TU-OC-2 cells by western blot analysis and in the original tumor tissue by immunohistochemistry. This cell line may be useful for studying the chemoresistant mechanisms of CCC and exploring novel therapeutic targets such as the ARID1A-related signaling pathway.

Establishment and mutation analysis of a novel malignant peritoneal mesothelioma cell line, TU-MM-1, using whole genome sequencing.

A new cell line of human malignant peritoneal mesothelioma (MPM), TU-MM-1, was established and characterized. The cells showed polygonal morphology, grew in monolayers without contact inhibition and were arranged like a jigsaw puzzle. The chromosome numbers ranged from 41 to 44. A low rate of proliferation was observed and the doubling time was 67.9 h. Genomic DNA sequencing revealed that TU-MM-1 cells harbored missense mutations in APC, LATS2, BRCA1/2, and TP53, and mutation of a splice donor site in BAP1 and loss of CDKN2A gene. We observed the absence of BAP1 and p16(INK4a) proteins, underexpression of LATS2 protein, and overexpression of p53 protein in TU-MM-1 cells in western blot analysis. Heterotransplantation to nude mice produced tumors that had the characteristics of the original tumor. This cell line may be useful for studying biological properties and contribute to novel treatment strategies.

The PI3K/mTOR dual inhibitor NVP-BEZ235 reduces the growth of ovarian clear cell carcinoma.

Patients with clear cell carcinoma of the ovary (OCCC) have poor survival due to resistance to standard chemotherapy. OCCC has frequent activating mutations of the PIK3CA gene. The present study was conducted to clarify the efficacy of the inhibition of the PI3K-AKT-mTOR pathway in OCCC. We used 8 OCCC cell lines and 5 ovarian serous adenocarcinoma (OSAC) cell lines. The mutation status of the PIK3CA and KRAS genes was examined by direct sequencing. The IC50 values of NVP-BEZ235 (BEZ235) and temsirolimus were determined by WST-8 assay. Protein expression levels of PI3K-AKT-mTOR pathway molecules were examined by western blotting. Cell cycle distribution was analyzed by flow cytometry. Annexin V staining was used for detecting apoptosis. We also investigated the effects of BEZ235 on OCCC tumor growth in a nude mouse xenograft model. Four of the 8 OCCC cell lines showed a PIK3CA mutation while none of the 5 OSAC cell lines showed a mutation. The IC50 values of BEZ235 for the OCCC cell lines were lower than these values for the OSAC cell lines. The IC50 value of temsirolimus was higher than BEZ235 in the OCCC cell lines. The PIK3CA mutation was more frequently noted in OCCC than OSAC cells, but the sensitivity of these cell lines to BEZ235 or temsirolimus was not related to the mutation status. pHER3 and pAkt proteins were expressed more frequently in OCCC compared with OSAC. However, protein expression levels were distributed widely, and were not related to the sensitivity. Treatment with BEZ235 suppressed expression of pAkt, although treatment with temsirolimus did not. OCCC cells exhibited G1 phase arrest after treatment with BEZ235 and apoptosis with a higher concentration of the agent. BEZ235 significantly inhibited tumor growth in mice bearing OVISE and TU-OC-1 cell tumors. The present study indicated that the PI3K-AKT-mTOR pathway is a potential target for OCCC, and that BEZ235 warrants investigation as a therapeutic agent.

Establishment and characterization of a novel ovarian clear cell adenocarcinoma cell line, TU-OC-1, with a mutation in the PIK3CA gene.

A new cell line of human ovarian clear cell adenocarcinoma (CCC), TU-OC-1, was established and characterized. The cells showed a polygonal-shaped morphology and grew in monolayers without contact inhibition and were arranged like a jigsaw puzzle. The chromosome numbers ranged from 64 to 90. A low rate of proliferation was observed, similar to other CCC cell lines tested (OVTOKO, RMG-I, and OVAS), and the doubling time was 38.4 h. The respective IC50 values of cisplatin and paclitaxel were 12.2 µM and 58.3 nM. Mutational analysis revealed that TU-OC-1 cells harbored a PIK3CA mutation at codon 542 (E542K) in exon 9, which is a mutation hot spot on this gene. We observed that phosphorylated Akt protein was overexpressed in TU-OC-1 cells by western blot analysis. Heterotransplantation to nude mice produced tumors that reflected the original. This cell line may be useful to study the chemoresistant mechanisms of CCC and contribute to novel treatment strategies.

A crucial role of bone morphogenetic protein signaling in the wound healing response in acute liver injury induced by carbon tetrachloride.

Background. Acute liver injury induced by administration of carbon tetrachloride (CCl(4)) has used a model of wound repair in the rat liver. Previously, we reported transient expression of bone morphogenetic protein (Bmp) 2 or Bmp4 at 6-24 h after CCl(4) treatment, suggesting a role of BMP signaling in the wound healing response in the injured liver. In the present study, we investigated the biological meaning of the transient Bmp expression in liver injury. Methods. Using conditional knockout mice carrying a floxed exon in the BMP receptor 1A gene, we determined the hepatic gene expressions and proliferative activity following CCl(4)-treated liver. Results. We observed retardation of the healing response in the knockout mice treated with CCl(4), including aggravated histological feature and reduced expressions of the albumin and Tdo2 genes, and a particular decrease in the proliferative activity shown by Ki-67 immunohistochemistry. Conclusion. Our findings suggest a crucial role of BMP signaling in the amelioration of acute liver injury.

Establishment and characterization of a transgenic mouse model for in vivo imaging of Bmp4 expression in the pancreas.

Type-2 diabetes results from the development of insulin resistance and a concomitant impairment of insulin secretion. Bone morphogenetic protein 4 (Bmp4)-Bmp receptor 1A signaling in ß cells has recently been reported to be required for insulin production and secretion. In addition, Bmp4 blocks the differentiation and promotes the expansion of endocrine progenitor cells. Bmp4 therefore regulates the maintenance of homeostasis in the pancreas. In this study, we constructed a reporter plasmid carrying 7-kb enhancer and promoter region of the Bmp4 gene upstream of the firefly luciferase gene. We used this construct to produce transgenic mice by pro-nuclear microinjection, for subsequent in vivo monitoring of Bmp4 expression. The bioluminescent signal was detected mainly in the pancreas in three independent lines of transgenic mice. Furthermore, the bioluminescent signal was enhanced in association with the autophagy response to 24-h fasting. These results suggest that pancreatic expression of Bmp4 is involved in responding to the physiological environment, including through autophagy. These mouse models represent useful tools for toxicological screening, and for investigating the mechanisms responsible for pancreatic Bmp4 functions in vivo, with relevance to improving our understanding of pancreatic diseases.