BACKGROUND: The phosphatidylinositol 3 0 -kinase (PI3K) -AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation. METHODS: We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing. RESULTS: We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. INTERPRETATION: Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K -AKTactivating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.

Drosophila tumor suppressor Scribble has been identified as an apical-basolateral polarity determinant in epithelia. A human homolog of Drosophila Scribble, human Scribble (hScrib), has been identified as a protein targeted by human papillomavirus E6 for the ubiquitin-mediated degradation dependent on E6AP, a cellular ubiquitin-protein ligase. Human Scribble is classified as a LAP protein, having leucine-rich repeats (LRRs) and PDZ domains. We investigated whether hScrib, which is thought to have a role in polarity determination based on the data of its Drosophila homolog, is involved in cell-cycle regulation and proliferation control of epithelia. Transfection of hScrib inhibits cell-cycle progression from G1 to S phase, and it up-and down-regulates expression of adenomatous polyposis coli and cyclins A and D1, respectively. Knockdown of hScrib expression by siRNA leads to cell-cycle progression from G1 to S phase. We explored functional domain mapping to reveal which domains of hScrib are critical for its cellular proliferation control and localization at the basolateral membrane. We found that LRRs and PDZ domain 1 are indispensable for hScrib to inhibit cell growth by blocking cell-cycle progression and to keep its proper localization. These data indicate that basolateral membrane localization of hScrib is closely related to its proliferation control. Our findings suggest the possibility that hScrib is involved in signal transduction to negatively regulate cell proliferation by localizing at the basolateral membrane of epithelial cells through LRRs and PDZ domains. Loss of cell polarity due to aberrant expression and localization of junctional proteins causes overgrowth of epithelia.(2) Mutation of a tumor suppressor gene whose encoded protein regulates cell-cycle progression is also characteristic of cancer tissues. Maintenance of tissue polarity and regulation of cell cycle are tightly linked to keep normal growth control and differentiation in epithelia.(3) Loss of tissue architecture and disruption of the cell-cycle control system leads to uncontrolled proliferation of epithelial cells and subsequent tumor formation. Drosophila tumor suppressor Scribble has recently been identified as an apical-basal polarity determinant in epithelia.(5) Drosophila Scribble localizes at the septate junction, which is functionally identical to the vertebrate tight junction, and loss of scribble mutation causes mislocalization of adherens junction to the basolateral membrane.(5) Follicle cells homozygous for scribble mutation are rounded, rather than columnar, and are often multilayered, rather than sheet-like. (6) In Drosophila scribble mutant, disorganized epithelial tissue overproliferates and causes massive tumors in imaginal discs, brain, and follicles. (6) Recently, scribble has been identified as a novel regulator of S-phase entry in Drosophila by a genetic screen for dominant hypomorphic cyclin E mutation. The human homolog of Drosophila tumor suppressor Scribble, hScrib, has recently been identified as a target of...

Endometrial cancer is a ubiquitous gynecological disease with increasing global incidence. Therefore, despite the lack of an established screening technique to date, early diagnosis of endometrial cancer assumes critical importance. This paper presents an artificial-intelligence-based system to detect the regions affected by endometrial cancer automatically from hysteroscopic images. In this study, 177 patients (60 with normal endometrium, 21 with uterine myoma, 60 with endometrial polyp, 15 with atypical endometrial hyperplasia, and 21 with endometrial cancer) with a history of hysteroscopy were recruited. Machine-learning techniques based on three popular deep neural network models were employed, and a continuity-analysis method was developed to enhance the accuracy of cancer diagnosis. Finally, we investigated if the accuracy could be improved by combining all the trained models. The results reveal that the diagnosis accuracy was approximately 80% (78.91–80.93%) when using the standard method, and it increased to 89% (83.94–89.13%) and exceeded 90% (i.e., 90.29%) when employing the proposed continuity analysis and combining the three neural networks, respectively. The corresponding sensitivity and specificity equaled 91.66% and 89.36%, respectively. These findings demonstrate the proposed method to be sufficient to facilitate timely diagnosis of endometrial cancer in the near future.

Abstract. Cyclin D1 is an important regulator of cell cycle progression. Phosphorylation of cyclin D1 at Thr286 by GSK3β triggers its nuclear export and cytoplasmic proteolysis via the 26S proteasome. Cyclin D1 overexpression is a common event in various types of human cancers; however, reports of mutations are extremely rare. We analyzed mutations of the cyclin D1 gene, CCND1, in 88 endometrial cancer tissue specimens and detected mutations in 2 cases (2.3%). Both were unreported mutations with substitution of threonine to isoleucine at codon 286 (T286I). These two tumors harbored coexisting mutations in K-ras, PIK3CA and/or PTEN and showed accumulation of cyclin D1 in the nucleus by immunohistochemistry. Furthermore, we analyzed the functions of mutant cyclin D1 (T286I) by luciferase assays, immunofluorescence, western blotting and clonogenic cell survival assays in HEK-293T cells. We found that exogenous mutant cyclin D1 (T286I) accumulated in the nuclei in HEK-293T cells, and that it inhibited the expression of pRb. Additionally, the number of colonies was increased by introduction of mutant cyclin D1 (T286I) compared to that of wild-type cyclin D1. In conclusion, we identified an unreported CCND1 mutation (T286I) in two endometrial cancers and revealed that the mutation was functional for inducing cell proliferation in human cells.

A diagnostic biopsy for endometrial cancer is performed via dilation and curettage (D&C). However, D&C may miss endometrial cancer lesions due to of its ‘blind’ approach. Hysteroscopy is a useful method that can be used to detect endometrial cancer lesions. In addition, office hysteroscopy is easy to be scheduled and does not require anesthesia. The patient was a 40-year-old woman with suspected endometrial cancer; however, it could not be diagnosed by D&C and biopsy using hysteroscopy during hospitalization. Office hysteroscopy during the proliferative phase indicated that the suspicious endometrial cancerous lesion was minimal at the isthmus of the uterus with atypical vessels and a white spot, for which biopsy was performed. Pathological diagnosis was endometrioid carcinoma with squamous differentiation, G1. Therefore, total laparoscopic hysterectomy, bilateral salpingo-oophorectomy and pelvic lymphadenectomy were performed. In this case, it was difficult to detect minimal lesion in the secretory phase because the endometrial thickness hid the endometrial cancer. It is easy to perform office hysteroscopy in the proliferative phase. This case indicated that office hysteroscopy is a useful method to diagnose and perform biopsy for minimal lesions.

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235-a dual PI3K/mTOR inhibitor-and RAD001-an mTOR inhibitor-in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n = 9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n = 4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas.

Cancer Research UK Sir,We appreciate the attention given by Drs Dutt, Salvesen, Greulich, Sellers, Beroukhim and Meyerson to our recent publication 'The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas ' (Shoji et al, 2009). In this article, we report a 2% mutational frequency of AKT1 (E17K) among 101 endometrial carcinomas. We also described that these two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. Our report has proposed two issues to be clarified: (1) Are there any 'oncogenic' mutations in other AKT family members in endometrial carcinomas? (2) Are all the AKT family mutations mutually exclusive with other PI3 kinase-AKT-activating mutations?Including the data in their earlier report, Dutt et al (2008) revealed mutations in AKT2 and AKT3, as well as in AKT1. As for AKT1 (E17K) mutations, their data and our data are compatible, showing that AKT1 mutations were detected in 2% of the endometrioid subtype in endometrial cancer. Compared with the accumulative data in AKT1 (E17K) mutations, mutations in AKT2 and AKT3 are not well characterised. Carpten et al (2007) and Kim et al (2008) reported no E17K mutations in AKT2 and AKT3 in breast, colorectal, gastric, hepatocellular, lung carcinomas and acute leukaemias. Davies et al (2008) first reported AKT3 (E17K) mutations in melanoma at 1.5% frequency. Parsons et al (2005) found two mutations of AKT2 (S302G and R371H) in 204 colorectal cancer samples, and Soung et al (2006) reported one missense mutation (A377V) and two possible splice-site mutations in intron 11 of AKT2 in gastric and lung adenocarcinoma. However, the physiological role of AKT2 mutations, including those (D399N, R368C and D32H) reported by Dutt et al (2008), has not been validated yet. In addition, the AKT3 (E438D) mutation has not been reported in any type of tumours. It is important to clarify whether these rare AKT2/3 mutations cause an oncogenic effect in cancer.We previously reported that PIK3CA mutations frequently coexist with mutations in PTEN and/or KRAS, and suggested that the PIK3CA mutation might require another upstream input to fully activate the PI3K kinase -AKT pathway (Oda et al, 2005(Oda et al, , 2008. Their data of coexistent mutations in AKT1 (E17K) and KRAS (G13D) suggest that the KRAS mutation alone is insufficient for a full activation of the PI3 kinase -AKT pathway. Their data are not inconsistent with the hypothesis that the AKT1 (E17K) mutation and the PIK3CA mutation are mutually exclusive. The coexistent mutations of AKT3 and PIK3CA in one sample suggest that some types of AKT mutations (non-E17K) might coexist with PIK3CA mutations to enhance the activation of the PI3 kinase -AKT pathway. Further analyses are necessary to clarify the frequency of coexistent mutations in AKT1 (E17K), AKT2/3 and other mutations.Their current data and our data suggest that the PI3 kinase -AKT pathway is prevalently activated in endometrial cancer through various genetic alterations and their combinations. Further analyses...

Purpose; PI3K (phosphatidylinositol-3-kinase) and its downstream mTOR (mammalian target of rapamycin) pathway play a critical role in diverse cellular functions, including proliferation, growth and cell survival. PI3K-mTOR pathway is frequently activated in endometrial cancer through various PI3K/AKT activating genetic alterations, such as mutations in PTEN, PIK3CA, and K-Ras. It has not been well analyzed whether mTOR inhibition alone is as effective as dual PI3K/mTOR inhibition to cancer cells with the activating PI3K pathway mutations, especially in vivo. In this study, we compared in vivo anti-tumor activity of a dual PI3K.inhibitor, NVP-BEZ235, with an mTOR inhibitor, RAD001, in endometrial cancer cell lines with the PI3K/AKT activating mutations.Experimental procedure; We screened 13 endometrial cancer cell lines, 11 of which possess one or more PI3K/AKT activating mutations. Among them, we selected two cell lines; AN3CA with a nonsense mutation in PTEN, and Hec-59 with double mutations in PIK3CA and PTEN. The IC50 values for cell proliferation by RAD001 were 14nM in AN3CA and 220nM in Hec-59, whereas the values by NVP-BEZ235 were 20nM in AN3CA and 24nM in Hec-59. We examined the effect of RAD001 (2.5mg/kg/day) and BEZ235 (40mg/kg/day) on tumor growth in vivo, using mice inoculated with endometrial cancer cells. We analyzed the phosphorylation levels of Akt, FOXO, GSK3beta and S6 in the tumors by immunoblotting. Results;Both BEZ and RAD001, compared with placebo, significantly suppressed the tumor growth in xenograft mice in the two cell lines. No significant adverse effects were observed with either compounds in any of the mice. Inconsistent with the in vitro data, the effect was comparable between BEZ235 and RAD001 even in Hec-59 cells, although the IC50 value was much higher with RAD001 than NVP-BEZ235. In Hec-59, NVP-BEZ235 clearly suppressed all the phosphorylation level of Akt, FOXO, GSK3beta and S6 in one hour from the treatment. However, all the phosphorylation levels were completely recovered to the base level within 24 hours. RAD001 clearly suppressed the p-S6 level in 1 hour and the effect partly remained even after 24 hours from the treatment. Conclusion; We demonstrated that both NVP-BEZ235 and RAD001 suppressed tumor growth in vivo in endometrial cancer cell lines with PIK3CA and/or PTEN mutations. The comparable effect of NVP-BEZ235 and RAD001 might be explained by the recovery of the phosphorylation levels of the target proteins, such as p-Akt, suggesting that sufficient suppression of the PI3K/mTOR pathway for full time-course might be required to robustly induce the anti-tumor effect by these inhibitors. In clinical trials, both pharmacokinetic and pharmacodinamic analyses would be important to appropriately assess the effect of these inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4494. doi:10.1158/1538-7445.AM2011-4494