Manabu Hashimoto

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.

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.

Distinct telomere length regulation in premalignant cervical and endometrial lesions : Implications for the roles of telomeres in uterine carcinogenesis

Mouse models show that progressive shortening of telomeres with ageing causes chromosomal instability, which can lead to the initiation of cancer. However, it is unclear what roles telomere shortening plays in human carcinogenesis. The present study has investigated the involvement of telomere dynamics in uterine carcinogenesis. Using telomere‐FISH (telo‐FISH) assays, telomere lengths in premalignant and malignant cervical and endometrial lesions were measured and compared with chromosomal arm loss or gain. Telo‐FISH signals were visualized with Cy3‐labelled telomere‐specific probes and presented as telomere intensity (TI). Early‐stage cervical intraepithelial neoplasias (CINs), especially CIN2, had significantly shorter telomeres than corresponding normal squamous epithelia (p = 0.019), together with increased rates of chromosomal arm loss/gain (p < 0.001). Cervical cancers had relatively short telomeres, but they also showed greater heterogeneity than other sampled tissues, including those with long telomeres. In contrast, there was no significant difference between the telomere length of normal endometrium and of endometrial hyperplasia and endometrial cancer. There was no significant difference in the rate of chromosomal arm loss/gain between normal endometrium and endometrial hyperplasia. These findings suggest that progressive shortening of telomeres occurs in CIN, in association with chromosomal instability, which may play critical roles in cervical carcinogenesis. In contrast, endometrial hyperplasias have relatively stable telomeres without widespread chromosome alteration, implying that endometrial carcinogenesis involves mechanisms distinct from those of cervical carcinogenesis, possibly including microsatellite instability. Copyright

Expression of HER-2 affects patient survival and paclitaxel sensitivity in endometrial cancer

Background:Disabled phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase signalling is involved in endometrial carcinogenesis, and there is evidence that expression of epidermal growth factor receptor (EGFR) family members has a role in such intracellular signalling pathways. This study analysed the prognostic impact of EGFR family expression in endometrial cancer in relation to PI3K–AKT and MAPK–ERK signalling, as well as drug sensitivity.Methods and results:Immunohistochemical analysis using 63 surgical specimens of endometrioid-type endometrial cancers revealed that EGFR, human epidermal growth factor receptor (HER)-2 and HER-4 were expressed in 25 (39.7%) of 63, 26 (41.3%) of 63 and 31 (49.2%) of 63 tumours, respectively. Gene amplification of HER-2 was observed in 2 of 26 patients with high HER-2 expression. Kaplan–Meier analysis revealed that high HER-2 expression was a factor that negatively influenced the progression-free and overall survival rate (P<0.05), and multivariate analysis showed high HER-2 expression to be an independent prognostic factor. Subsequently, we performed in vitro knockdown analysis to investigate the linkage between HER-2 expression and PI3K–AKT pathways. Short interfering RNA (siRNA)-based knockdown of HER-2 in endometrial cancer cells led to a significant reduction in phosphorylated AKT (p-AKT) expression, indicating the existence of a HER-2/PI3K-AKT axis. As the PI3K–AKT pathway is known to have crucial roles in anticancer drug sensitivity, we examined the involvement of HER-2 in sensitivity to paclitaxel. Short interfering RNA-based knockdown of HER-2 conferred increased sensitivity to paclitaxel in endometrial cancer cells, attenuating the induction of p-AKT on paclitaxel stimulation, which was cancelled by inactivating AKT by the introduction of a dominant-negative form.Conclusion:HER-2 is a significant prognostic factor of endometrioid-type endometrial cancer, as well as a key molecule that affects paclitaxel sensitivity by HER-2 interaction with the PI3K–AKT pathway.

Analysis of telomeric single-strand overhang length in human endometrial cancers.

The 3′ single‐strand telomeric overhang (3′‐OH) is a key component of telomere structure. Although telomere length has been well analyzed in a variety of human cancers, no information is available on the 3′‐OH length in cancers. In the present study, we examined the 3′‐OH length in normal and malignant endometria using telomere‐oligonucleotide ligation assay. Although 3′‐OH lengths varied among patients, 3′‐OH length observed in endometrial cancers was significantly shorter than that found in samples derived from normal endometria (P < 0.001: Students t‐test), suggesting that erosion of 3′‐OH length induces impaired telomeric integrity and genomic instability, leading to carcinogenesis. Interestingly, we found that the most aggressive subtypes of endometrial cancers harbored significantly longer 3′‐OH length than those with non‐aggressive subtypes (P < 0.001: Sheffes test), suggesting that cancer cells with long 3′‐OH length have growth advantage due to their stabilized telomere ends. In contrast, we failed to observe an association between overall telomere length and any clinicopathological characteristics of endometrial cancers. These findings suggest that erosion of 3′‐OH length, rather than overall telomere length, play roles in endometrial carcinogenesis. Furthermore, long 3′‐OH may serve as a molecular marker for aggressive phenotype of tumors.

Ultrasound‐guided culdotomy for vaginal ovarian cystectomy using a renal balloon dilator catheter

To evaluate the feasibility and utility of performing ultrasound‐guided culdotomy using a renal balloon dilator catheter for transvaginal ovarian cystectomies.