Hiroyasu Kashima

Overexpression of Hedgehog Signaling Molecules and Its Involvement in the Proliferation of Endometrial Carcinoma Cells

Purpose: Research has revealed abnormal activation of the hedgehog pathway in human malignancies. The present study was undertaken to examine the expression and functional involvement of the hedgehog pathway in endometrial tissues. Experimental Design: The expression of sonic hedgehog (Shh), patched (Ptch), Smoothened (Smo), and Gli1 was examined in various endometrial tissues and endometrial carcinoma cell lines. The effect of hedgehog signaling on the proliferation of endometrial carcinoma cell lines was also examined. Results: The expression of Shh, Ptch, Smo, and Gli1 was very weak in normal endometrium, but was increased in endometrial hyperplasia and carcinoma stepwisely with significant differences. There was no marked difference in the expression of these molecules in carcinomas according to stages and histologic grades. Treatment with cyclopamine, a specific inhibitor of the hedgehog pathway, for endometrial carcinoma Ishikawa and HHUA cells suppressed growth by 56% and 67%, respectively, compared with the control. The addition of recombinant Shh peptide to HHUA cells enhanced their proliferation by 41%. The silencing of Gli1 using small interfering RNA (siGli1) resulted in the growth suppression and down-regulation of Ptch expression. In addition, the cyclopamine/siGli1-induced growth suppression was associated with the down-regulation of cyclins D1 and A and N-myc. No somatic mutations for ptch and smo genes were detected in the endometrial carcinoma cases examined. Conclusions: The abnormal activation of this pathway is involved in the proliferation of endometrial carcinoma cells possibly in an auto-/paracrine fashion, suggesting the possibility of the hedgehog pathway being a novel candidate for molecular targeting.

BRAF Mutation in Endometrial Carcinoma and Hyperplasia: Correlation with KRAS and p53 Mutations and Mismatch Repair Protein Expression

Purpose: Although several gene abnormalities have been reported in endometrial carcinoma, the genetic alterations have not fully been elucidated. Recent studies have revealed frequent activating mutations of the gene for BRAF, an effector of Ras protein in the mitogen-activated protein kinase pathway, in several malignancies. However, the prevalence and significance of BRAF mutations in endometrial carcinoma remain unclear. Experimental Design: We examined BRAF mutations in exons 11 and 15 in 97 cases of endometrial carcinoma (endometrioid type, 78; nonendometrioid type, 19), 9 cases of atypical endometrial hyperplasia, and 20 cases of normal endometrium by direct sequencing. In addition, mutations of KRAS and p53 and the immunohistochemical expression of hMLH1 and hMSH2 were also examined. Results: Of the 97 carcinomas and 9 hyperplasias, 20 (21%) and 1 (11%) had BRAF mutations, most of them at previously unreported sites. Twenty samples of normal endometrium and 21 samples of normal endometrium obtained from sites adjacent to neoplastic lesions had no BRAF mutations. There was no apparent difference in the prevalence of BRAF mutation among stages, histologic subtypes, or grades. Mutations of KRAS and p53 were found in 18 (19%) and 22 (23%) cases, and 65 (67%) and 92 (95%) cases showed positive immunostaining for hMLH1 and hMSH2, respectively. BRAF mutation was more frequently found in hMLH1-negative cases (12 of 32, 41%) than in hMLH1-positive cases (7 of 65, 11%; P = 0.008), suggesting that it is associated with an abnormal mismatch repair function. Conclusions: These findings suggest that mutations of the BRAF gene are partly involved in the malignant transformation of the endometrium.

Estrogen-induced proliferation of normal endometrial glandular cells is initiated by transcriptional activation of cyclin D1 via binding of c-Jun to an AP-1 sequence.

To explore the mechanism of estrogen-induced growth of normal endometrium, the transactivation system of the cyclin D1 gene was analysed using cultured normal endometrial glandular cells. Estradiol (E2) treatment of cultured normal endometrial glandular cells induced upregulation of c-Jun, and then cyclin D1 proteins, followed by serial expressions of cyclins E, A and B1 proteins. Increase in the mRNA expression of cyclin D1 preceded the protein expression of cyclin D1 under E2 treatment. A luciferase assay using deletion constructs of the cyclin D1 promoter indicated that E2-induced increase in transcriptional activity was observed in reporters containing AP-1-binding site sequence, and that in the absence of E2, cotransfection of c-Jun also showed increase of transcriptional activity in the same reporters with AP-1 sequence. A gel shift assay using nuclear extract from E2-treated endometrial glandular cells and AP-1 sequences of the cyclin D1 promoter indicated specific binding between c-Jun protein and the promoter. Transfection of c-jun antisense oligonucleotides to the glandular cells resulted in the suppression of the E2-induced upregulation of cyclin D1 mRNA and protein. These findings suggest that E2-induced proliferation of normal endometrial glandular cells is initiated by transcriptional activation of cyclin D1 via binding of c-Jun to the AP-1 sequences.

Expression of steroid receptor coactivators and corepressors in human endometrial hyperplasia and carcinoma with relevance to steroid receptors and Ki‐67 expression

To examine the steroid hormone dependent growth mechanism of human endometrial hyperplasia and carcinoma, expression levels of steroid receptor cofactors, such as coactivators (steroid receptor coactivator 1 [SRC‐1] and p300/cyclic AMP‐response element‐binding protein (p300/CBP]) and corepressors (nuclear receptor corepressor [NCoR] and silencing mediator for retinoid and thyroid‐hormone receptors [SMRT]), were investigated.

Autocrine stimulation of IGF1 in estrogen-induced growth of endometrial carcinoma cells: involvement of the mitogen-activated protein kinase pathway followed by up-regulation of cyclin D1 and cyclin E.

To examine estrogen-induced growth mechanisms of endometrial carcinoma, we investigated the estrogen-induced activation of the mitogen-activated protein kinase (MAPK) pathway and cell cycle regulators. Estradiol (E(2)) treatment at concentrations of 10(-8) M and 10(-6) M to estrogen receptor (ER)-positive endometrial carcinoma Ishikawa cells for 24 h resulted in increased cell proliferation by 20% and 28% respectively. The E(2)-induced proliferation was associated with the activation of extracellular signal-regulated kinase (MAPK)3/1 and up-regulation of cyclin D1 and E, which were suppressed by the addition of an MAP2K inhibitor (U0126) or an ER antagonist (ICI 182 780). Then, our screening for estrogen-inducible growth factors identified that IGF1 was up-regulated remarkably by E(2). Immunoprecipitation using conditioned medium of Ishikawa cells after E(2) treatment confirmed the E(2)-induced secretion of IGF1 protein. Treatment with recombinant IGF1 stimulated cell proliferation in a dose-dependent fashion, in association with MAPK3/1 phosphorylation and up-regulation of cyclin D1 and E. These IGF1-induced responses were suppressed by treatment with MAP2K inhibitor or anti-IGF1 receptor antibody. Immunohistochemical staining confirmed the expression of activated MAPK3/1 in normal proliferative phase endometria and endometrial carcinomas, indicating the involvement of this pathway in actively proliferating endometrial tissues in vivo. These findings suggest that E(2)-induced proliferation of endometrial carcinoma cells is mediated by the MAPK3/1 pathway via autocrine stimulation of IGF1.

Prognostic significance of Notch signalling molecules and their involvement in the invasiveness of endometrial carcinoma cells

Mitsuhashi Y, Horiuchi A, Miyamoto T, Kashima H, Suzuki A & Shiozawa T 
(2012) Histopathology 60, 826–837

Expression of cyclins, p53, and Ki-67 in cervical squamous cell carcinomas: overexpression of cyclin A is a poor prognostic factor in stage Ib and II disease

We previously reported the overexpression of cyclins in uterine cervical carcinoma; however, their clinicopathological significance remained undetermined. In the present study, we examined the immunohistochemical expression of cyclins (D1, E, A, B1), p53 and Ki-67 in squamous cell carcinoma (stage Ib+II; 80 cases, stage III+IV; 23 cases). Correlations between the expression of cyclins and clinicopathological parameters and patient survival were statistically evaluated. The results indicated that in the normal squamous epithelium, the expression of cyclins and Ki-67 was sporadically observed in the parabasal layer. Of the 103 cervical carcinomas, overexpression of cyclins D1, E, A, B1 and p53 was observed in 13 (13%), 23 (22%), 25 (24%), 18 (18%) and 23 (22%) cases, respectively, with a slight predominance in advanced stage tumors. The expression of cyclin D1, E, A and p53 significantly correlated with that of Ki-67 (Spearman’s rank correlation). Univariate and multivariate analyses revealed that lymph node metastasis and cyclin A overexpression were independent prognostic factors for unfavorable outcomes in stage Ib+II patients. These findings suggest that the overexpression of various cyclins is involved in the acquisition of the vigorous growth potential of cervical carcinoma cells, and that cyclin A is an independent prognosticator of cervical carcinoma in early stages.

Immunohistochemical detection of steroid receptor cofactors in ovarian endometriosis: involvement of down-regulated SRC-1 expression in the limited growth activity of the endometriotic epithelium

To study the steroid hormone-induced growth mechanisms of endometriosis, the immunohistochemical expression of steroid hormone receptor cofactors was investigated in 37 cases of endometriotic epithelia and was compared with that of eutopic endometria of identical patients. The expression of steroid receptor coactivators (p300/CBP and SRC-1) and corepressors (NCoR and SMRT) was examined in relation to the estrogen receptor (ER), the progesterone receptor (PR), and Ki-67. Results of immunostaining were indicated as a “positivity index” (PI, full score; 100). The expression of ER and PR in endometriotic epithelia largely resembled that in eutopic endometria, however, the expression of Ki-67 in the proliferative phase (PI 13.8 ± 2.4, mean ± SD) was significantly lower than that in eutopic endometria (32.6 ± 10.6). The expression of SRC-1 in eutopic endometria was increased in the proliferative phase (56.5 ± 16.8) and decreased in the secretory phase (14.8 ± 6.9). In endometriosis, however, the PI for SRC-1 did not show apparent cyclic changes during the menstrual cycle. Moreover, the expression of SRC-1 in endometriotic epithelia in the proliferative phase was significantly lower than that in eutopic endometria. These findings suggested the reduced proliferative activity in endometriotic epithelia to be related to the reduced expression of SRC-1.

Preoperative differential diagnosis of minimal deviation adenocarcinoma and lobular endocervical glandular hyperplasia of the uterine cervix: a multicenter study of clinicopathology and magnetic resonance imaging findings.

Objective: To clarify the preoperative differential diagnosis and management of minimal deviation adenocarcinoma (MDA) and lobular endocervical glandular hyperplasia (LEGH), a multicenter study was performed. Methods: A total of 112 patients who underwent conization or a hysterectomy for suspected MDA were collected from 24 hospitals. The pathological diagnosis in each case was determined by a central pathological review board. The diagnostic significance of clinicopathologic findings including results of magnetic resonance imaging (MRI), Papanicolaou (Pap) smears, and testing for gastric mucin was analyzed. Results: The central pathological review identified 37 cases of Nabothian cyst or tunnel cluster, 54 cases of LEGH, 6 cases of MDA, 11 cases of adenocarcinoma, and 4 cases of benign disease. Lobular endocervical glandular hyperplasia was often associated with adenocarcinoma in situ, MDA, and mucinous adenocarcinoma. Three MDA patients had a recurrence, whereas none of LEGH patients had a recurrence irrespective of the type of surgery. On MRI, LEGH appeared as a characteristic multicystic lesion with an inner solid component, whereas MDA showed a predominantly solid pattern. A Pap smear or gastric mucin alone had limited diagnostic power. However, a combination of these findings is useful; that is, a cystic structure with inner solid components on MRI associated with mild glandular atypia and gastric mucin strongly suggested LEGH (24/26, 92%). A solid structure with atypical glandular cells was indicative of MDA or adenocarcinoma (5/5, 100%). Conclusions: The combination of MRI, Pap smears, and gastric mucin will improve the accuracy of the preoperative diagnosis of MDA and LEGH. Patients suspected of having LEGH may need to be treated with less aggressive methods. Abbreviations: MDA - Minimal deviation adenocarcinoma, LEGH - Lobular endocervical glandular hyperplasia, NC - Nabothian cyst, TC - Tunnel cluster, NILM - Negative for intraepithelial lesion, AGCs - Atypical glandular cells, AIS - Adenocarcinoma in situ, CPR - Central pathological review, Pap - Papanicolaou, MRI - Magnetic resonance imaging

Laser-captured microdissection-microarray analysis of the genes involved in endometrial carcinogenesis: stepwise up-regulation of lipocalin2 expression in normal and neoplastic endometria and its functional relevance

Endometrial carcinoma often arises from normal endometrial glandular cells via a precursor, atypical endometrial hyperplasia. However, the genetic changes involved in this carcinogenetic process are not fully understood. Differentially expressed genes were selected from glandular cells of normal proliferative-phase endometria, atypical endometrial hyperplasia, and endometrial carcinoma using laser-captured microdissection and microarray. The microarray analysis revealed a total of 51 genes to be up-regulated and 23 genes to be down-regulated in neoplastic endometrial epithelia. We focused on lipocalin2 (LCN2), which showed the largest magnitude of up-regulation. Immunostaining for lipocalin2 confirmed a stepwise increase in its expression in endometrial hyperplasia and carcinoma. In addition, elevated expression of lipocalin2 was correlated with the poor outcome of endometrial carcinoma patients. The subcellular distribution of lipocalin2 was both cytoplasmic and nuclear, despite reports that lipocalin2 is a secretory protein. Treatment of endometrial carcinoma cells with 5-azacytidine increased the expression of lipocalin2, suggesting the expression to be controlled by methylation of the promoter. The forced expression of lipocalin2 resulted in the enhanced cell proliferation and invasion in vitro. The expression of lipocalin2 increased with the endometrial carcinogenesis, and accumulation of the protein conferred biological aggressiveness to endometrial carcinoma cells. These results suggest lipocalin2 to be a novel target in the treatment of endometrial carcinoma.