Junko Sakaguchi

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)

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.

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

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.