Yasunari Mizumoto

Activation of ERK1/2 occurs independently of KRAS or BRAF status in endometrial cancer and is associated with favorable prognosis

The extracellular‐regulated kinase (ERK) signaling pathway plays important roles in regulating the malignant potential of cancer cells in vitro. However, the effect of ERK signaling on the prognosis of human tumors is not clearly understood. The present study examined the expression of phosphorylated ERK1/2 (p‐ERK1/2) as a hallmark of ERK activation, in relation to KRAS and BRAF mutations, in 63 endometrial cancer specimens with endometrioid‐subtype, in order to clarify the prognostic value of p‐ERK1/2 expression. Immmunohistochemical analysis revealed that 40 tumors (63%) expressed p‐ERK1/2, with varying levels of expression. Total ERK1/2 expression was also evaluated in a subset of tumors; most cases expressed ERK1/2 constitutively but no correlation was observed with p‐ERK expression, indicating that p‐ERK1/2 staining was not due to ERK overexpression but to hyperactivation of ERK1/2. There was no statistically significant correlation between p‐ERK1/2 expression and clinicopathological features, including patient age, International Federation of Gynecology and Obstetrics stage, pathological grade, myometrial invasion and lymph node metastasis. Sequencing analysis indicated that 23% of patients had a mutation in exon 1 of KRAS, whereas none of the patients had a mutation in exons 11 or 15 of BRAF, which are reportedly hot spots for mutation in many tumor types. There was no significant correlation between KRAS or BRAF status and p‐ERK1/2 expression. Unexpectedly, patients with low p‐ERK1/2 expression had significantly lower relapse‐free survival (P = 0.041) and overall survival (P = 0.020). Multivariate Cox regression analysis indicated that p‐ERK1/2 expression was an independent prognostic indicator for overall survival (P = 0.047). These findings suggest that ERK activation occurs in a KRAS‐ and BRAF‐independent manner in endometrial cancer, and is associated with favorable prognosis. (Cancer Sci 2007; 98: 652–658)

Prognostic impact of CD133 expression as a tumor-initiating cell marker in endometrial cancer

Tumor-initiating cells are known to be the major source of tumor propagation and might be an attractive therapeutic target. The present study dissected the roles of CD133 as a tumor-initiating cell marker in endometrial cancer and investigated the prognostic impact of this marker expression. Flow cytometry using 6 endometrial cancer cell lines revealed that the frequency of CD133(+) cells varied widely among the cell types and that Ishikawa and MFE280 cells contained significantly higher ratio (10%-20%) of such cells; therefore, these were subjected to the subsequent analyses. Sorted CD133(+) cells showed more aggressive proliferative potential in vitro and more increased tumorigenicity in nude or NOD/SCID mice than CD133(-) cells and generated both CD133(+) and CD133(-) cells. Furthermore, they showed apparent resistance to cisplatin- or paclitaxel-induced cytotoxicity compared with CD133(-) cells. CD133(+) cells had a greater S-phase fraction than CD133(-) cells, and the serum starvation that induced G0/G1 accumulation decreased the population of CD133(+) cells. Finally, we immunohistochemically analyzed the CD133 expression in endometrial cancer specimens from 62 patients. CD133 expression was not significantly associated with any of the clinicopathologic characteristic of tumors. However, the Kaplan-Meier analysis revealed that tumors with high CD133 expression showed worse overall survival (P = .023, log-rank test) than those with low CD133 expression; and the Cox regression hazard model found that high CD133 expression was an independent prognostic factor (P = .045). Thus, the present study demonstrates that CD133 is not only a tumor-initiating cell marker but also a critical prognostic marker in endometrial cancer.

Intraperitoneal administration of telomerase-specific oncolytic adenovirus sensitizes ovarian cancer cells to cisplatin and affects survival in a xenograft model with peritoneal dissemination.

Despite tremendous development in chemotherapy for ovarian cancer over the past few decades, the prognosis of advanced cases with massive peritoneal dissemination is still unsatisfactory, and novel treatment modalities that can combine with chemotherapy are urgently needed. We recently developed virotherapy for solid tumors using telomerase-specific replication-selective adenoviruses (Telomelysin: OBP-301), in which the human telomerase reverse transcriptase (hTERT) gene promoter has been inserted to direct tumor-specific E1 gene expression. In this study, we investigated the anti-tumor effects of OBP-301, combined with cisplatin (CDDP), on ovarian cancer cells. In vitro treatment of SKOV3 cells with OBP-301 at a multiplicity of infection (MOI) of 0.01–100 induced significant cell death in a dose-dependent manner, with moderate cytotoxicity at an MOI of 1–10 and maximal cytotoxicity at an MOI of 100. In contrast, OBP-301 treatment of normal human cells showed no significant cell death at an MOI of 1–10 and exhibited modest cytotoxicity at an MOI of 100. The effects of low-dose CDDP at 0.5–1 μM, which induced only 20% cell death, were significantly augmented by combination with OBP-301 at an MOI of 1–10, finally achieving 40% cell death. Such enhancement of CDDP sensitivity was also observed in CDDP-resistant ovarian cancer cells. The combinatorial effects were further tested using a xenograft mouse model of SKOV3 with peritoneal dissemination. After intraperitoneal administration of OBP-301, we confirmed that injected OBP-301 fused with the green fluorescent protein (GFP) gene (OBP-401) was preferentially localized to peritoneal disseminations, as determined by fluorescence imaging. Treatment of mice with CDDP at low dose (0.5 mg kg–1) had modest effects, showing a 10% decrease in disseminations, whereas combination with intraperitoneal administration of OBP-301 at an MOI of 10 led to enhanced effects, achieving an approximately 80% decrease in disseminations. Kaplan–Meier analysis showed improved overall survival of mice treated with CDDP plus OBP-301 compared with CDDP alone. These findings support the therapeutic potential of intraperitoneal administration of OBP-301 to sensitize ovarian cancer cells to CDDP.

Creation of immortalised epithelial cells from ovarian endometrioma

Background:Epithelial cells of endometriotic tissues are difficult to propagate in vitro as experimental material is scarce owing to their limited life span. However, there is an increasing concern regarding their malignant transformation in ovaries. The present study sought to generate their stable culture system.Methods and results:Purified epithelial cells isolated from ovarian endometriomas using microscopic manipulation were successfully immortalised by combinatorial transfection of human cyclinD1, cdk4 and human telomerase reverse transcriptase (hTERT) genes, whereas the introduction of hTERT alone, or together with cdk4, was insufficient for immortalisation, leading to cellular senescence. We confirmed stable cytokeratin expression in the immortalised cells, proving their epithelial origin. These cells expressed progesterone receptor B and showed significant growth inhibition by various progestins. Oestrogen receptor (ER) expression was detected in these cells, albeit at low levels. Additional overexpression of ERα generated stable cells with oestrogen-dependent growth activation. Soft-agar colony formation assay and nude mice xenograft experiments demonstrated that these cells, even those with additional inactivation of p53, did not have transformed phenotypes.Conclusion:We for the first time generated immortalised epithelial cells from ovarian endometrioma that retained sex steroid responsiveness. These cells are invaluable tools not only for the consistent in vitro work but also for the study of molecular pathogenesis or carcinogenesis of endometriosis.

Forkhead transcription factor FOXO1 is a direct target of progestin to inhibit endometrial epithelial cell growth

Purpose and experimental design: Despite the therapeutic utility of progestin in invasive and preinvasive endometrial neoplasias, the molecular mechanisms through which it exerts inhibitory effects on endometrial epithelial growth are largely unknown. The aim of the study was to clarify the molecular mechanisms of progestin action to endometrial epithelial cells using originally established in vitro and in vivo treatment models for immortalized and transformed endometrial epithelial cell lines that express progesterone receptor. Results: In this model, progestin effectively inhibited the cell growth, inducing G0/G1 arrest rather than apoptosis without p21/WAF-1 induction. Using DNA microarray analysis, we identified 24 genes whose expression increased more than 10-fold on progestin treatment. Of these genes, we paid special attention to forkhead box transcription factor FOXO1, known as a key gene for endometrial decidualization. Progestin markedly induced FOXO1 gene expression mainly in the nuclei in vitro and in vivo. This induction was not due to the canonical activation of FOXO1 via protein dephosphorylation but due to FOXO1 promoter activation and mRNA induction. siRNA inhibition of FOXO1 significantly attenuated the effects of progestin to inhibit endometrial epithelial cell growth. Disrupting Akt activity by the introduction of the dominant negative form of Akt increased nuclear FOXO1 accumulation and enhanced the effect of progestin. Conclusion: These findings suggest that FOXO1 is a direct target of progestin, implicating novel molecular mechanisms of progestin to eradicate endometrial neoplasia. Clin Cancer Res; 17(3); 525–37. ©2010 AACR.

Concomitant activation of AKT with extracellular-regulated kinase 1/2 occurs independently of PTEN or PIK3CA mutations in endometrial cancer and may be associated with favorable prognosiss

Deregulated signaling via the phosphatidylinositol 3‐kinase (PI3K) pathway is common in many types of cancer, but its clinicopathological significance in endometrial cancer remains unclear. In the present study, we examined the status of the PI3K signaling pathway, especially in relation to PTEN and PIK3CA status, in endometrioid‐type endometrial cancer. The immunohistochemical analysis revealed a high level of phosphorylated (p)‐AKT expression, which is a hallmark of activated PI3K signaling, in approximately 60% of endometrial cancers. There was no correlation between p‐AKT expression and clinicopathological characteristics, such as International Federation of Gynecology and Obstetrics stage, tumor grade, and myometrial invasion. Unexpectedly, a high level of p‐AKT expression occurred independently of the presence of PTEN or PIK3CA mutations. Furthermore, p‐AKT expression did not correlate with the expression of potential downstream targets, including p‐mTOR and p‐FOXO1/3a. In turn, p‐AKT expression was strongly associated with extracellular‐regulated kinase 1/2 expression (P = 0.0031), which is representative of the activated RAS–MAP kinase pathway. Kaplan–Meier analysis suggested that low p‐AKT expression was associated with low rates of relapse‐free survival, although the difference was not statistically significant, indicating that AKT activation does not confer worse prognosis. The present study demonstrates the presence of complex signaling pathways that might mask the conventional tumorigenic PTEN–PI3K–AKT–mTOR pathway, and strongly suggests a close association between the extracellular‐regulated kinase and PI3K pathways in this tumor type. (Cancer Sci 2007; 98: 1881–1888)

Creation of tumorigenic human endometrial epithelial cells with intact chromosomes by introducing defined genetic elements

Several genetic mutations have been identified in human endometrial cancers, but the specific combinations of mutations required to form endometrial cancer cells remain unknown. In the present study, we established an in vitro model of endometrial carcinogenesis, in which defined genetic elements were introduced into endometrial epithelial cells to create transformed endometrial cells at different stages. Introduction of the human papillomavirus type 16 E6/E7 gene and the human telomerase reverse transcriptase (hTERT) gene into human primary endometrial epithelial cells was sufficient to generate immortalized cells. Introduction of hTERT in early passages stabilized telomeres and created immortalized cells with normal karyotype, whereas introduction of hTERT in later passages generated immortalized cells but with widespread chromosome abnormalities. However, neither of those two immortalized cell lines exhibited tumorigenic phenotypes. Tumorigenic endometrial epithelial cells with invasive capacity were created by introducing a mutant K-ras allele into immortalized cells, keeping their chromosomes intact. Inhibiting the PTEN gene and activating Akt pathways did not create tumorigenic phenotypes, although the latter conferred anchorage-independent growth capacity. These findings suggest that neoplastic transformation of human endometrial cells can occur in the absence of widespread chromosomal abnormality, and that the combination of Rb inactivation, telomerase activation and altered K-ras signaling is sufficient for in vitro neoplastic transformation. The present experimental model can help clarify the genetic requirements for endometrial carcinogenesis, and it is useful for testing and developing specific inhibitors of specific oncogenic pathways.

Association of mismatch repair deficiency with PTEN frameshift mutations in endometrial cancers and the precursors in a Japanese population

We studied mismatch repair deficiency and PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutations in endometrial cancers and hyperplasias in a Japanese population. Methylation-sensitive restriction enzyme polymerase chain reaction revealed MLH1 hypermethylation in 21 (38%) of 56 endometrial cancers. Sequencing analysis revealed PTEN mutations in 22 patients with cancer (39%) in exons 5 and 8. A PTEN frameshift mutation was associated significantly with MLH1 hypermethylation (P = .01) and a highly positive phenotype with microsatellite instability (P < .001) but not with a PTEN missense mutation. In hyperplasia, MLH1 hypermethylation was similarly observed (11/27 [41%]), but the PTEN mutation was less frequent (5/27 [19%]), observed only in atypical hyperplasias; among the 5 patients with a PTEN mutation, the 2 patients with frameshift mutations had MLH1 hypermethylation, but the 3 patients with missense mutations had unmethylated MLH1. These findings indicate that MLH1 hypermethylation is an early event frequently occurring in hyperplasia without atypia, whereas the PTEN mutation occurs later, mostly in atypical hyperplasia, possibly caused by MLH1 hypermethylation.

Activation of NF-κB Is a Novel Target of KRAS-Induced Endometrial Carcinogenesis

Purpose: Although the KRAS mutation is one of critical genetic alterations in endometrial carcinogenesis, the downstream targets are not known. Experimental Design: In this study, we investigated the molecular targets of KRAS signals, using tumorigenic cells with oncogenic KRAS mutation established from telomerase reverse transcriptase (TERT)-immortalized endometrial epithelial cells. Results: We first confirmed that the RAF-ERK pathway, but not the PI3K-Akt pathway, was activated in KRAS tumorigenic cells. However, the introduction of constitutively active MAP/ERK kinase into immortalized cells to mimic RAF-ERK activation failed to obtain tumorigenic phenotypes, indicating the existence of other carcinogenic pathways triggered by KRAS. Recent evidence suggestive of linkage with KRAS signals prompted us to examine the involvement of NF-κB in endometrial carcinogenesis. We found that the DNA-binding activity of NF-κB was markedly elevated in KRAS tumorigenic cells compared with TERT-immortalized cells. Furthermore, the ability of NF-κB to activate the target gene promoters significantly increased in KRAS tumorigenic cells. Introduction of a mutant IκB that is resistant to degradation and thereby enhances the inhibitory effect on NF-κB largely abrogated the transformed phenotypes of KRAS tumorigenic cells. Thus, oncogenic KRAS signals contributed to the tumorigenic phenotypes of endometrial cells by activating the transcription function of NF-κB. Conclusions: These findings clearly show that NF-κB activation is a novel target of oncogenic KRAS in endometrial carcinogenesis, implying the potential utility of NF-κB inhibitors for endometrial cancer chemoprevention, especially with KRAS mutation. Clin Cancer Res; 17(6); 1341–50. ©2011 AACR.

Circulating tumour cells detected by a novel adenovirus-mediated system may be a potent therapeutic marker in gynaecological cancers

Background:Recently developed detection system for circulating tumour cells (CTCs) using a telomerase-specific replicative adenovirus generated nonspecific green fluorescent protein (GFP) signals because of the co-presence of white blood cells (WBCs) nonspecifically infected by viruses. Here, we established a unique detection system for CTCs that completely excludes nonspecific signals.Methods:Blood obtained from the patients was subjected to haemolytic processes to eliminate red blood cells. The cell pellets were then infected with OBP-401, fixed, incubated with fluorescence-labelled anti-CD45 antibody to mark white blood WBCs, and examined on slides under a microscope.Results:Preparatory experiments with cancer cells artificially added to healthy donor samples confirmed that CD45 labelling could distinguish GFP-positive cancer cells from WBCs. In 53 patients with gynaecological cancers, CTCs were detected in 21 patients (39.6%) when CD45-positive cells were excluded as WBCs among GFP-positive cells. No CTCs were detected in samples from healthy volunteers. There was no significant correlation between CTC counts and known clinicopathological factors. The CTCs rapidly vanished after surgery or chemotherapy in most patients whose treatments were effective. In contrast, the persistence of CTCs even after treatments was tightly associated with poor response to the treatments (P<0.005).Conclusion:The presence of CTCs in our system may potentially be a novel therapeutic marker in gynaecological cancers.