Shunsuke Nakagawa

Human Scribble (Vartul) Is Targeted for Ubiquitin-Mediated Degradation by the High-Risk Papillomavirus E6 Proteins and the E6AP Ubiquitin-Protein Ligase

ABSTRACT The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of theDrosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 proteins.

Analysis of the expression and localisation of a LAP protein, human scribble, in the normal and neoplastic epithelium of uterine cervix

Recently, a LAP protein, scribble, was identified in Drosophila epithelia as a basolateral protein that controls the apical-basolateral polarity. Loss of scribble causes disorganisation and overgrowth of the epithelia. Scribble has a human homologue, human scribble (hScrib), which is a substrate of ubiquitin-mediated degradation by human papillomavirus E6 and the E6AP ubiquitin-protein ligase. In the present study, we revealed that hScrib localised to the basolateral regions of the epithelial cell line MDCK and human uterine cervical epithelial tissues by immunofluorescence. Human scribble colocalised rather with the adherens junction protein E-cadherin, but not with the tight junction protein ZO-1. Histochemical analysis showed a dramatic decrease in the expression of hScrib with the progression of disease from normal uterine cervical tissues to invasive cervical cancers through the precursor lesions. In contrast, the expression of hScrib was retained in the throughout epithelial layer of the HPV-negative cervical high-grade squamous intraepithelial lesions (H-SIL). Although quantitative RT–PCR revealed no significant downregulation of hScrib mRNA expression in the H-SIL, it revealed a clear downregulation in the invasive cancers. These results suggest the possibility that degradation by HPV E6 is one of the causal roles for the progressive decrease of hScrib expression during the disease progression from low-grade squamous intraepithelial lesions to H-SIL, and a cooperative role of downregulation of hScrib mRNA expression and ubiquitin-mediated degradation of hScrib by E6 and E6AP led to the complete decrease of hScrib expression during the process of carcinogenesis from H-SIL to invasive cancer. These data underscore the importance of hScrib in the construction of tissue architecture and prevention of cancer development.

Methylation-associated silencing of the Wnt antagonist SFRP1 gene in human ovarian cancers

The SFRP1 gene on chromosome 8p11.2 encodes a Wnt signaling antagonist, and was recently demonstrated to be a new tumor suppressor that is inactivated by promoter methylation in human colon cancers. Here, we analyzed promoter methylation of the SFRP1 gene in human ovarian cancers, in which loss of heterozygosity in 8p is frequently observed and involvement of the Wnt signaling pathway has been suggested. Methylation‐specific PCR (MSP) analysis showed that four of 13 ovarian cancer cell lines and two of 17 primary ovarian cancers had methylated SFRP1, while an immortalized ovarian epithelial cell line, HOSE, and seven ovarian endometrial cyst samples did not. In the four ovarian cancer cell lines with the methylation, SFRP1 was not expressed at all as determined by quantitative RT‐PCR analysis. A cell line with SFRP1 methylation, MCAS, was treated with a demethylating agent, 5‐aza‐2′‐deoxycytidine, and demethylation of the promoter and re‐expression of SFRP1 were observed. These results show that SFRP1 is inactivated by promoter methylation in human ovarian cancers, as well as colon cancers.

Human scribble, a novel tumor suppressor identified as a target of high‐risk HPV E6 for ubiquitin‐mediated degradation, interacts with adenomatous polyposis coli

Recently, we have identified human scribble (hScrib), human homolog of the Drosophila tumor suppressor Scribble, as a substrate of human papillomavirus E6 oncoproteins for ubiquitin‐mediated degradation dependent on ubiquitin‐protein ligase E6AP. Human Scribble, classified as a LAP protein containing leucine‐rich repeats and PDZ domains, interacts with E6 through its PDZ domains and C‐terminal PDZ domain‐binding motif of E6 protein. Interaction between human Discs Large (hDlg), which is a substrate of E6 for the ubiquitin‐mediated degradation, and adenomatous polyposis coli (APC) has been shown. Here, we investigated whether hScrib and APC interact with each other in vitro and in vivo. Interaction between hScrib and APC is mediated by the PDZ domains 1 and 4 of hScrib and C‐terminal PDZ domain‐binding motif of APC. Human Scribble co‐localized with APC at the synaptic sites of hippocampal neuron and at the tip of membrane protrusion in the epithelial cell line. Interference of the interaction between hScrib and APC caused disruption of adherens junction. Knockdown of hScrib expression by RNAi disrupts localization of APC at the adherens junction. These data suggest that hScrib may participate in the hDlg‐APC complex through its PDZ domains and regulate cell cycle and neural function by associating with APC.

Human papillomavirus type 16 E6 variants and HLA class II alleles among Japanese women with cervical cancer

The enhanced oncogenicity of particular human papillomavirus type 16 (HPV16) E6 variants is population‐dependent, implying the involvement of additional genetic cofactors. This study was designed to investigate the association between E6 variants and human leukocyte antigen (HLA) polymorphism within a Japanese population. Fifty‐seven women with HPV16‐positive cervical cancer were analyzed for E6 sequence variation and its relationship to HLA class II alleles. Compared with local controls (n = 138) and published controls (n = 916), DRB1*1501 and DQB1*0602 frequencies were significantly increased among patients with HPV16 E6 prototype (n = 11). Additionally, DRB1*1502 was positively associated with a particular E6 variant designated D25E (n = 25), although we could not find a significant association between HLA class II alleles and L83V variants (n = 16). Our observations suggest that a specific match between E6 variant proteins and HLA types may contribute to HPV16‐related cervical carcinogenesis.

Human homolog of Drosophila tumor suppressor Scribble negatively regulates cell-cycle progression from G1 to S phase by localizing at the basolateral membrane in epithelial cells

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. (Cancer Sci 2006; 97: 1217–1225)

Loss of Hugl-1 expression associates with lymph node metastasis in endometrial cancer

Mutation of neoplastic tumor suppressor genes, scribble, discs large, and lethal giant larvae (lgl), causes disruption of cell polarity and overproliferation of Drosophila epithelial cells and neuroblasts. Reduced expression of human homologue of lgl, Hugl-1, has been reported to be involved in development and progression of human colon cancer and malignant melanoma. To explore the association between Hugl-1 expression and clinical character in endometrial cancer, we examined the expression of Hugl-1 in primary endometrial cancer tissues. The expression of Hugl-1 mRNA in 86 primary endometrial cancer tissues was examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). All samples were categorized into two groups: Hugl-1 positive and Hugl-1 negative. Clinical data of each group were analyzed by Fishers exact probability test and survival rates of each group were compared by Kaplan-Meier method and Log-rank test. Loss of Hugl-1 expression had correlation with the higher incidence of lymph node metastasis, but not to the patients age at onset, distant metastasis, clinical stage, lymph or venous vessel invasion, or histopathological grade of differentiation. The Hugl-1-positive group had poorer prognosis compared with the Hugl-1-negative group. These results indicate that loss of Hugl-1 expression in endometrial cancer may contribute to lymph node metastasis and it can be a factor of poor prognosis.

Preoperative diagnosis of colouterine fistula secondary to diverticulitis by sonohysterography with contrast medium

Colouterine fistulae secondary to sigmoid diverticulitis are unusual. Methods for diagnosis remain to be established. We report a case with a colouterine fistula in which sonohysterography detected the flow of ultrasound contrast medium between the uterine cavity and the sigmoid colon through the posterior uterine wall, thus confirming the diagnosis. The diagnosis was further substantiated by a charcoal challenge test. The patient underwent en bloc resection of the uterus, Fallopian tubes, ovaries and sigmoid colon, the organs involved with diverticulitis. This is the first report to describe a colouterine fistula successfully diagnosed by sonohysterography using ultrasound contrast medium. Copyright

Abnormal Fhit expression is an independent poor prognostic factor for cervical cancer

We analysed the expression of the fragile histidine triad (FHIT) gene in cervical cancer to evaluate its clinical relevance in relation to human papillomavirus (HPV) infection. A total of 73 women with cervical cancer of stage Ib or more advanced (67 squamous cell carcionomas, four adenocarcinomas, two adenosquamous carcinomas) were examined for Fhit expression by immunohistochemistry. They were further analysed for the presence of HPV and its subtype. Abnormal expression of Fhit (absent or reduced Fhit expression) was observed in 52 cases (71.2%). The high-risk HPV DNAs for cervical cancer, including type 16, 18, 31, 33, 51, 52, 58, 68, were identified in 63 cases (86%). The abnormal Fhit expression was not related to the clinicopathological factors including histology, tumour stage, and HPV type. Notably, the 5-year survival of patients showing the abnormal Fhit expression was significantly poorer than those showing normal Fhit expression (64 versus 87%, P=0.035). Interestingly, the mean age of the patients with the abnormal Fhit expression was significantly less than those with the normal Fhit expression (51.6 versus 58.7 years of age, P=0.027, students t-test). These data imply that the aberrant Fhit expression could be a poor prognostic factor independent of HPV. In the light of a high incidence of abnormal Fhit expression in younger patients and HPV as a key player in cervical carcinogenesis, abnormal Fhit expression may accelerate carcinogenesis in concert with HPV.

Ovarian choriocarcinoma arising from partial mole as evidenced by deoxyribonucleic acid microsatellite analysis.

BACKGROUND Recent developments in genetic analysis allow determination of the origin of choriocarcinoma (ie, gestational or nongestational), which helps determine the strategy for clinical treatment of the disease. CASE We present a case of ovarian choriocarcinoma forming a huge ovarian mass 40 days after the patients last menstrual period. Deoxyribonucleic acid microsatellite analysis of the tumor revealed that it contained a single maternal and two paternal alleles at several independent loci, consistent with the tumor resulting from ovarian pregnancy of a partial hydatidiform mole. CONCLUSION This is the first description of an ovarian pregnancy of a partial hydatidiform mole–derived ovarian choriocarcinoma.