Identification of long noncoding RNAs downregulated specifically in ovarian high-grade serous carcinoma.

Purpose: To investigate whether long noncoding RNAs (lncRNAs) are involved in the development or malignant behavior of ovarian high-grade serous carcinoma (HGSC), we attempted to identify lncRNAs specific to HGSC. Methods: Total RNAs were isolated from HGSC, normal ovarian, and fallopian tube tissue samples and were subjected to a PCR array that can analyze 84 cancer-associated lncRNAs. The lncRNAs that were upregulated and downregulated in HGSC in comparison to multiple samples of normal ovary and fallopian tube were validated by real-time RT-PCR. To infer the function, ovarian cancer cell lines that overexpress the identified lncRNAs were established, and the activation of cell proliferation, migration, and invasion was analyzed. Results: Eleven lncRNAs (ACTA2-AS1, ADAMTS9-AS2, CBR3-AS1, HAND2-AS1, IPW, LINC00312, LINC00887, MEG3, NBR2, TSIX, and XIST) were downregulated in HGSC samples. We established the cell lines that overexpress ADAMTS9-AS2, CBR3-AS1, or NBR2. In cell lines overexpressing ADAMTS9-AS2, cell proliferation was suppressed, but migration and invasion were promoted. In cell lines overexpressing CBR3-AS1 or NBR2, cell migration tended to be promoted, although cell proliferation and invasion were unchanged. Conclusion: We identified eleven lncRNAs that were specifically downregulated in HGSC. Of these, CBR3-AS1, NBR2, and ADAMTS9-AS2 had unique functions in the malignant behaviors of HGSC.

Efficacy and safety of olaparib, olaparib plus bevacizumab and niraparib maintenance treatment in Japanese patients with platinum-sensitive advanced ovarian cancer.

OBJECTIVE: To investigate whether maintenance treatment could be safely and effectively performed with olaparib, olaparib plus bevacizumab and niraparib in platinum-sensitive advanced ovarian cancer at multiple institutions in Japan. METHODS: We investigated progression-free survival and adverse events in 117 patients with platinum-sensitive advanced ovarian cancer treated with maintenance therapy. RESULTS: The median progression-free survival of 117 patients was 20.1 months. Patients with germline BRCA pathogenic variants had a significantly better prognosis than the other groups (P < 0.001). Furthermore, in the multivariate analysis, stage IV (P = 0.016) and germline BRCA wild-type (P ≤ 0.001) were significantly associated with worse progression-free survival in patients with advanced ovarian cancer. Regarding adverse events, all three types of maintenance treatment were significantly worse than chemotherapy given before maintenance treatment with respect to renal function (olaparib, P = 0.037; olaparib plus bevacizumab, P < 0.001; and niraparib, P = 0.016). CONCLUSION: Maintenance treatment was performed effectively and safely. Renal function deterioration is likely to occur during maintenance treatment, and careful administration is important in platinum-sensitive advanced ovarian cancer.

Relationship Between Hematological Toxicities During Maintenance Treatment and During Chemotherapy Before Maintenance Treatment in Patients With Platinum-sensitive Relapsed Ovarian Cancer.

BACKGROUND/AIM: To determine if maintenance treatment can be performed effectively and safely in patients with platinum-sensitive relapsed ovarian cancer. PATIENTS AND METHODS: We carried out a multi-center study to investigate progression-free survival (PFS) and adverse events (AEs) in 229 patients receiving maintenance treatment for platinum-sensitive relapsed ovarian cancer. RESULTS: The median PFS in the 229 patients with maintenance treatment was 14.0 months (95% confidence interval=10.3-17.6 months). The hematological toxicities included ≥grade 3 anemia in 33.2% of cases. Anemia during maintenance treatment was significantly more common than anemia during chemotherapy given before maintenance treatment (p<0.001). Anemia during chemotherapy prior to maintenance treatment significantly increased the risk of anemia during maintenance treatment, compared with other clinical features (p<0.001). CONCLUSION: Maintenance treatment can be performed safely and effectively in patients with platinum-sensitive relapsed ovarian cancer. Anemia during chemotherapy given before maintenance treatment significantly increased the risk of developing anemia during maintenance treatment in patients with platinum-sensitive relapsed ovarian cancer.

Different DNA methylome, transcriptome and histological features in uterine fibroids with and without MED12 mutations.

Somatic mutations in Mediator complex subunit 12 (MED12m) have been reported as a biomarker of uterine fibroids (UFs). However, the role of MED12m is still unclear in the pathogenesis of UFs. Therefore, we investigated the differences in DNA methylome, transcriptome, and histological features between MED12m-positive and -negative UFs. DNA methylomes and transcriptomes were obtained from MED12m-positive and -negative UFs and myometrium, and hierarchically clustered. Differentially expressed genes in comparison with the myometrium and co-expressed genes detected by weighted gene co-expression network analysis were subjected to gene ontology enrichment analyses. The amounts of collagen fibers and the number of blood vessels and smooth muscle cells were histologically evaluated. Hierarchical clustering based on DNA methylation clearly separated the myometrium, MED12m-positive, and MED12m-negative UFs. MED12m-positive UFs had the increased activities of extracellular matrix formation, whereas MED12m-negative UFs had the increased angiogenic activities and smooth muscle cell proliferation. The MED12m-positive and -negative UFs had different DNA methylation, gene expression, and histological features. The MED12m-positive UFs form the tumor with a rich extracellular matrix and poor blood vessels and smooth muscle cells compared to the MED12m-negative UFs, suggesting MED12 mutations affect the tissue composition of UFs.

Docetaxel and carboplatin chemotherapy for treating patients with stage IVB or recurrent non-squamous cell carcinoma of the uterine cervix: a phase II study.

BACKGROUND: This phase II study evaluated the efficacy and safety of docetaxel/carboplatin chemotherapy for treating patients with stage IVB or recurrent non-squamous cell carcinoma of the uterine cervix. METHODS: A total of 50 patients with International Federation of Gynecology and Obstetrics stage IVB or recurrent non-squamous cell carcinoma of the uterine cervix were enrolled and administered docetaxel at a dose of 60 mg/m2, followed by carboplatin at a dose based on the area under the receiver operating characteristic curve of 6. The treatments were repeated every 21 days until disease progression or unacceptable adverse events. Except for two patients, 48 were eligible for evaluation. Another patient withdrew consent before treatment; adverse events were evaluated in 47. RESULTS: The response rate was 47.9% with 5 patients achieving complete response, 18 partial response, 14 stable disease, and 6 progressive disease. The disease control rate was 77.1%. With a median follow-up duration of 368 days, the median progression-free survival and overall survival were 6.1 months (95% CI 5.5-8.6) and 15.8 months (95% CI 18.2-28.3), respectively. The most frequent grade 3 and grade 4 hematological toxicity was neutropenia, with 38 patients (81%) having grade 4 and 4 (9%) having grade 3 neutropenia. The non-hematological toxicities were mainly grade 1 or 2 in severity. CONCLUSION: Docetaxel/carboplatin chemotherapy was effective, with a higher disease control rate and well-tolerated chemotherapeutic regimen for patients with stage IVB or recurrent non-squamous cell carcinoma of the uterine cervix.

Identification of aberrantly expressed long non-coding RNAs in ovarian high-grade serous carcinoma cells.

PURPOSE: To identify the aberrantly expressed long non-coding RNAs (lncRNAs) in ovarian high-grade serous carcinoma (HGSC). METHODS: Total RNA was isolated in HGSC cell lines, ovarian surface epithelial cells, and normal ovaries. Aberrantly expressed lncRNAs in HGSC were identified by PCR array, which analyzes 84 kinds of lncRNAs. To infer their functions, HGSC cell lines with different levels of expression of the identified lncRNAs were established, and then, activities of proliferation, migration, and apoptosis were examined. Expression levels of the identified lncRNAs were also examined in multiple ovarian HGSC tissues. RESULTS: Ten aberrantly expressed lncRNAs, six upregulated and four downregulated, were identified in the HGSC cell lines. The authors established four HGSC cell lines: in two of the cell lines, one of the upregulated lncRNAs was knocked down, and in two other cell lines, one of the downregulated lncRNAs (MEG3 and POU5F1P5) was overexpressed. Migration activities were inhibited in the HGSC cell lines overexpressing MEG3 or POU5F1P5 while there were no differences in proliferation and apoptosis between the established and control cell lines. The four lncRNAs downregulated in the HGSC cell lines were also observed to be downregulated in ovarian HGSC tissues. CONCLUSION: The authors identified four downregulated lncRNAs in ovarian HGSC.

Overexpression of carbonyl reductase 1 inhibits malignant behaviors and epithelial mesenchymal transition by suppressing TGF-ß signaling in uterine leiomyosarcoma cells.

Carbonyl reductase 1 (CBR1) has been reported to be involved in cancer progression. Recently, we found that CBR1 overexpression inhibited malignant behaviors and the epithelial mesenchymal transition (EMT) in uterine cervical cancer. It remained unclear whether this was also the case in uterine leiomyosarcoma (uLMS), which is derived from mesenchymal cells and is a much more malignant gynecological tumor. A number of previous studies suggested that malignant behaviors are associated with EMT, even in mesenchymal malignant tumors. In the present study, we investigated whether CBR1 inhibits malignant behaviors and EMT in uLMS. We established clones of uLMS cells (SKN cells) and uterine sarcoma cells (MES-SA cells) that overexpressed CBR1. Cell proliferative, migratory and invasive activities were suppressed by CBR1 overexpression, accompanied by increases in the expressions of epithelial markers (E-cadherin and cytokeratin) and decreases in the expressions of mesenchymal markers (N-cadherin and fibronectin), suggesting that CBR1 overexpression inhibits malignant behaviors and EMT in uLMS cells. In addition, transforming growth factor-ß (TGF-ß) production and the subsequent signaling and phosphorylation of Smad were suppressed in the clones. To investigate the association between TGF-ß and EMT, SKN cells were treated with TGF-ß or a TGF-ß receptor blocker (SB431542). EMT was promoted by TGF-ß and inhibited by SB431542. In conclusion, this is the first study, to the best of the authors' knowledge, showing that CBR1 overexpression inhibits malignant behaviors and EMT in uLMS cells. The present study provided novel insight demonstrating that the suppressive effect of CBR1 is mediated through TGF-ß signaling.

Decreased carbonyl reductase 1 expression promotes tumor growth via epithelial mesenchymal transition in uterine cervical squamous cell carcinomas.

PURPOSE: Carbonyl reductase 1 (CBR1) is involved in cancer progression. Recently, the authors reported that the loss of CBR1 expression is associated with a poor prognosis in uterine cervical cancer. Here, we investigated whether the decreased CBR1 expression promotes cancer progression by inducing the epithelial mesenchymal transition (EMT). METHODS: Antisense constructs of CBR1 complementary DNA (antisense clones) and the empty vectors (control clones) were transfected into human uterine cervical squamous cell carcinoma cell lines (SKG II and SiHa) and the proliferation and EMT marker expression of these clones were analyzed in vitro. In an in vivo study, 107 cells of the antisense and control clones were subcutaneously injected into nude mice and the tumorigenesis was observed for 8 weeks. RESULTS: With the decreased CBR1 expression, the proliferation of the antisense clones increased, accompanied by a decrease in epithelial markers (E-cadherin and cytokeratin) and an increase in mesenchymal markers (fibronectin, alpha-smooth muscle actin, and N-cadherin), which suggests EMT induction. In the in vivo study, the tumor volume in the antisense group was significantly larger than that in the control group. CONCLUSION: Decreased CBR1 expression promotes tumor growth by inducing EMT in uterine cervical squamous cell carcinomas.

Functional significance of transgelin-2 in uterine cervical squamous cell carcinoma.

AIM: Transgelin-2 (TAGLN2) has previously been found to be highly expressed in uterine cervical squamous cell carcinoma (SCC) tissues by proteomic analyses. The present study investigated the role of TAGLN2 in the malignant behaviors of cervical SCC cells in vitro and in vivo, and the clinical significance of TAGLN2 using immunohistochemistry for human cervical SCC tissues. METHODS: Antisense (AS) constructs of TAGLN2 cDNA (AS clones) and the empty vector (control clone) were transfected into a human uterine SCC cell line (SKG IIIa), and malignant behaviors were analyzed in vitro. In an in vivo experiment, 10(7) cells of the AS and control clones were subcutaneously inoculated into female BALB/c nude mice. In immunohistochemistry with anti-TAGLN2 antibodies for human cervical SCC, FIGO stage IA and IB (n = 75), the expression patterns of TAGLN2 were divided into two groups: weak and strong. The relation between expression pattern and prognosis was analyzed. RESULTS: Suppression of TAGLN2 inhibited cancer cell migration and secretion of matrix metalloproteinases. Tumors in the control clone group continued to grow, whereas those in the AS clone group clearly stopped growing. Six weeks after injection, the tumor size was significantly smaller in the AS clone group than in the control clone group. Immunohistochemistry revealed that the strong pattern was associated with poor overall survival compared with the weak pattern by the Kaplan-Meier method. CONCLUSION: TAGLN2 plays functional roles in the progression of cervical SCC. Suppression of TAGLN2 may be a new strategy for the treatment of cervical SCC.

Clinical implications of human leukocyte antigen class I expression in endometrial cancer.

Decreased expression of human leukocyte antigen (HLA) class I molecules, which is found in several types of cancer, is associated with worse clinical prognosis in cancer patients. The present study was undertaken to investigate the association of immunohistochemical HLA class I expression patterns with clinicopathological factors and prognosis in 96 endometrial cancer patients. HLA class I is composed of a heavy chain (HC-10) and a ß2-microglobulin (ß2-m) light chain. The HLA class I expression patterns were classified as positive when both HC-10 and ß2-m were strongly stained and negative in all other cases. The negative staining pattern was associated with advanced International Federation of Gynecology and Obstetrics stage (P<0.001), lymphovascular space involvement (LVSI) (P=0.003) and lymph node metastasis (P=0.005). Moreover, these cases exhibited worse progression-free survival (PFS) and overall survival (OS) rates compared with positive cases (P=0.005 and P=0.014, respectively). However, the multivariate analysis did not identify HLA class I expression as an independent predictive factor for PFS and OS. In conclusion, HLA class I expression may be useful for predicting postoperative outcome in endometrial cancer, as well as well-known predictive prognostic factors, such as lymph node metastasis and LVSI.

Prognosis for endometrial cancer patients treated with systematic pelvic and para-aortic lymphadenectomy followed by platinum-based chemotherapy.

OBJECTIVE: The purpose of this study was to analyze the prognosis for endometrial cancer patients treated with systematic pelvic and para-aortic lymphadenectomy (PLA and PALA) followed by platinum-based chemotherapy. MATERIALS AND METHODS: From 1994 to 2004, in the Cancer Institute Hospital, 502 patients who were surgically treated with systematic PLA and PALA were enrolled in this study. Their prognosis and clinicopathological features were retrospectively reviewed. RESULTS: One hundred ninety-one (38.0%) patients received adjuvant platinum-based chemotherapy. Lymph node (LN) metastasis was observed in 80 (15.9%) patients, pelvic-only LN metastasis in 27 (5.4%), para-aortic-only LN metastasis in 15 (3.0%), and both pelvic and para-aortic LN metastasis in 38 (7.6%). The median number of metastatic LNs was 2 (range, 1-57), 1 (range, 1-4), and 6 (range, 2-50) in patients with pelvic-only, para-aortic-only, and both pelvic and para-aortic LN metastasis, respectively (P < 0.001). Only 2.6% (2/76) of patients with no myometrial invasion had LN metastasis, and no less than 8.9% (22/247) of patients with myometrial invasion (limited to the inner half) had LN metastasis. Five-year overall survival (OS) for LN metastasis-negative and -positive patients was 96.7% and 76% (P < 0.001), respectively. Five-year OS for patients with metastasis in 1 or 2 LNs was 84.8% and was significantly higher than that for patients with metastasis in 3 or more LNs (57.8%; P = 0.011). In patients with LN metastasis, 5-year OS of endometrioid adenocarcinoma and non-endometrioid adenocarcinoma cell types was 90.2% and 56.7% (P = 0.0016), respectively. CONCLUSIONS: Under the settings of thorough PLA and PALA followed by intensive platinum-based chemotherapy for endometrial cancer, metastasis in 1 or 2 LNs seems to have little effect on survival, although para-aortic LNs are involved. This therapeutic strategy could not improve the prognosis of patients with metastasis in 3 or more LNs or patients with non-endometrioid adenocarcinoma cell types along with LN involvement.

Heat shock protein 70 is involved in malignant behaviors and chemosensitivities to cisplatin in cervical squamous cell carcinoma cells.

AIM: Heat shock protein 70 (HSP70) was previously found to be highly expressed in cervical squamous cell carcinoma (SCC) tissues compared with normal tissues by proteomic analyses. The present study investigated the roles of HSP70 in malignant behaviors and chemosensitivity to cisplatin in cervical SCC cells in vitro. METHODS: Effects of HSP70 knockdown on activities of cell migration, cell invasion and cell proliferation, cell cycle status, and apoptosis were examined using siRNA in two human cervical cancer cells (SiHa and SKG II). Furthermore, the effect of HSP70 knockdown on the apoptotic effect by cisplatin was examined. RESULTS: HSP70 knockdown significantly suppressed activities of cell migration, cell invasion and cell proliferation. The cell cycle was arrested at the S- and G2/M-phases with the increase in apoptosis by HSP70 knockdown. Although cisplatin had no apoptotic effect in the control cell, it showed a dose-dependent apoptotic effect in the HSP70 knockdown cells in SiHa. In SKG II, 5 µg/mL of cisplatin induced apoptosis, and the apoptotic effect by cisplatin was enhanced by HSP70 knockdown. CONCLUSION: It was suggested that HSP70 played functional roles in the progression of uterine cervical SCC, and HSP70 knockdown enhanced chemosensitivities to cisplatin in cervical SCC cells.

Decreased carbonyl reductase 1 expression promotes malignant behaviours by induction of epithelial mesenchymal transition and its clinical significance.

Human carbonyl reductase 1 (CBR1) is an enzyme that catalyse the reduction of many compounds by using NADPH-dependent oxydoreductase activity. Although CBR1 is known to regulate the tumour progression, the molecular mechanisms of CBR1 in cancer progression and the clinical significance of CBR1 status remain unclear. Here, we investigated the molecular mechanisms by which CBR1 affects cancer cell behaviour in vitro and the clinical significance of CBR1 using immunohistochemical analyses in endometrial cancer. Here, the role of CBR1 in cancer cell invasion and metastasis, and its molecular mechanisms were investigated by transfection of sense and antisense CBR1 cDNAs into a human endometrial adenocarcinoma cell line. The relationship between CBR1 expression analysed by immunohistochemistry and prognosis such as progression free survival (PFS) and overall survival (OS) was examined in endometrial cancer tissues from FIGO stage I-IV (n=109). Suppression of CBR1 by antisense CBR1 cDNA increased cancer cell invasion, and suppressed E-cadherin expression and capacity for cellular aggregation. In contrast, over-expression of CBR1 by sense CBR1 cDNA increased E-cadherin expression and capacity for cellular aggregation, and suppressed cancer cell invasion. The expression of transcriptional suppressors of E-cadherin, Snail and ZEB1, were increased by CBR1 suppression, but suppressed by CBR1 over-expression. Immunohistochemical analyses showed that decreased CBR1 expression is significantly related with poor PFS and OS compared with strong CBR1 expression. In multivariate analyses, decreased CBR1 expression was an independent prognostic factor for PFS and OS. CBR1 regulates cancer cell invasion in endometrial adenocarcinomas by regulating the epithelial mesenchymal transition. A decreased CBR1 expression can be a useful marker of an unfavourable clinical outcome in patients with endometrial cancer.

Suppression of carbonyl reductase expression enhances malignant behaviour in uterine cervical squamous cell carcinoma: carbonyl reductase predicts prognosis and lymph node metastasis.

Carbonyl reductase (CR) is an NADPH-dependent, mostly monomeric, cytosolic enzyme with broad substrate specificity for carbonyl compounds. CR appears to be involved in the regulation of tumour progression. However, molecular mechanisms of CR in tumour progression and clinical significance of CR status remain unclear in human uterine squamous cell carcinoma (SCC). Here, we investigated the clinical significance of CR using immunohistochemical analyses of human uterine cervical SCC tissues and how CR affects cancer cell behaviour in vitro. Paraffin sections from uterine cervical SCC tissues, FIGO stage Ib1-IIb (n = 67) were immunostained with anti-CR antibodies. Overall survival (OS) and progression-free survival (PFS) were analyzed by the Kaplan-Meier method. Sense and antisense CR cDNAs were transfected into a human uterine SCC cell line (SiHa) to investigate the role of CR in cancer cell invasion and metastasis. Immunohistochemical analyses showed that reduced CR expression patterns in primary cancer lesions were closely associated with a high incidence of pelvic lymph node metastasis, poor OS, and poor PFS. In an in vitro experiment, suppression of CR increased cancer cell invasion, secretion of MMP-2, -9 and cyclooxygenase-2 (COX-2) expression and decreased E-cadherin expression. On the other hand, over-expression of CR increased E-cadherin expression and decreased MMP-2, -9 secretion and COX-2 expression. The reduced CR expression pattern, as measured by immunohistochemistry, can be a useful predictor of lymph node metastasis and poor prognosis in patients with uterine SCC. This clinical result is supported by the in vitro data which show that suppression of CR expression promotes cancer cell invasion with decreased E-cadherin expression and increased MMP-2, -9 secretion.

Comparative proteomic profiling in squamous cell carcinoma of the uterine cervix.

PURPOSE: Proteomic profiling of human uterine cervical squamous cell carcinoma (SCC) tissues was performed to find new biomarkers for the early diagnosis and molecular target therapies. EXPERIMENTAL DESIGN: Proteins extracted from five of normal tissues and five of the SCC tissues were subjected to 2-DE followed by LC-MS/MS. RESULTS: Twenty-seven of the protein spots were increased, and five of them were identified by LC-MS/MS to be cytokeratin-19, HSP70, HSP27, glyceraldehyde-3-phosphate dehydrogenase, and transgellin-2. The upregulation of HSP70, HSP27, and transgelin-2 was examined by Western blot of cultured SCC cell lines, ten additional normal tissues, ten additional SCC tissues, and three paired samples of the SCC tissue and its corresponding noncancerous tissue. The positive rate of HSP70 expression was higher in the SCC tissues than in normal tissues. Transgelin-2 was observed only in the SCC tissues, and also those in the early stage. CONCLUSIONS AND CLINICAL RELEVANCE: The present study successfully identified three upregulated proteins in the uterine cervical SCC by proteomic analyses, and suggested that these proteins could be the candidates for new biomarkers for early diagnosis of SCC and molecular target therapies.

Tumor-related protein, the squamous cell carcinoma antigen binds to the intracellular protein carbonyl reductase.

Squamous cell carcinoma antigen (SCCA) is a useful tumor marker for diagnosis and management of squamous cell carcinoma. Recent reports have shown that SCCA can influence the invasion or metastasis of cancer cells. However, it remained unclear how SCCA acts to mediate these biological functions. To solve this question, at first, SCCA1- and SCCA2-glutathione S-transferase fusion protein were used to purify a protein which binds to SCCA1 or SCCA2, and the combined protein was identified by proteomic analysis. Secondly, immunocytochemical and immunohistochemical analyses were performed to investigate the localization of this protein. Third, Western blotting was performed to analyze the expression levels of this protein in keratinocytes and six kinds of uterine squamous cell carcinoma cell lines. Both SCCA1 and SCCA2 molecules bind to the cytoplasmic protein, which was identified to be carbonyl reductase (CR). The immunostaining analyses revealed that CR is located in the cytoplasm of keratinocytes and the normal squamous epithelial cells of the uterine cervix as well as SCCA1 and SCCA2. The CR expression levels in six kinds of squamous cell carcinoma cell lines were lower compared to those in keratinocytes. In conclusion, CR binds to SCCA1 and SCCA2 and they are co-located in the same layer of the squamous epithelium, suggesting that CR may collaborate with SCCA1 and SCCA2 to mediate cancer behavior such as invasion or metastasis.

Relationship between decreased expression of squamous cell carcinoma antigen 2 and E-cadherin in primary cervical cancer lesions and lymph node metastasis.

Squamous cell carcinoma antigen (SCCA) has been used for the management of squamous cell carcinoma, especially in order to evaluate therapeutic effects and monitor recurrence. Recent studies have shown that SCCA performs several biological functions and can influence the behavior of cancer cells. It is well known that altered expression of E-cadherin is involved in the process of cancer invasion and metastasis. The present study was therefore undertaken to investigate the relationship between the expression of SCCA, E-cadherin and lymph node metastasis in advanced cervical squamous cell carcinoma patients. We studied 70 patients who had undergone radical hysterectomy and pelvic lymphadenectomy for stage IB, IIA and IIB of the disease, without pretreatments. Immunohistochemistry, using monoclonal antibodies against SCCA2 and E-cadherin, was performed to examine the relationship between SCCA2 and E-cadherin expression patterns in primary cancer lesions and lymph node metastasis. There was a significant positive relationship between the two expression patterns in primary cancer lesions (p<0.01). Both exhibited a heterogeneous expression pattern in the primary tumor which indicated a significant relationship with lymph node metastasis (p<0.01). Our data clearly show that SCCA2 expression is significantly related to E-cadherin expression and that the heterogeneous pattern of SCCA and E-cadherin in primary lesions is strongly associated with the high incidence of lymph node metastasis in cervical squamous cell carcinoma. These findings suggest that SCCA2 may be involved in cancer behavior such as metastasis, and as such can be a useful marker in predicting lymph node metastasis.

Different expression patterns of intact forms of squamous cell carcinoma antigens between normal and malignant cervical squamous epithelial tissues: nondenaturing polyacrylamide gel electrophoretic analysis.

Squamous cell carcinoma antigen (SCCA), a 45-kDa tumor-associated serpin, mainly consists of two highly homologous molecules, SCCA1 and SCCA2, which possess unique proteinase inhibitory properties. Importantly, our previous study demonstrated that an intact structure of SCCAs, and not a cleaved form yielded by interacting with target proteinase, is essential for their function as a serpin. The aim of this study is therefore, to develop a simple method of analyzing expression patterns of intact forms of SCCAs (functional SCCAs) in cervical squamous epithelial tissues and to investigate whether there are any differences in the expression of intact forms of SCCAs between normal and malignant cervical squamous epithelial tissues. We used nondenaturing polyacrylamide gel electrophoresis (PAGE) with immunoblotting. The newly generated antibody, Pab Y2, recognizes only intact form of SCCAs, while the conventional antibody, Mab 27, reacts with the cleaved form of SCCA1 as well as intact forms of SCCAs. Nondenaturing PAGE using Pab Y2 showed that an intact form of SCCAs in the heat-treated tissue extract at 60 degrees C for 2 h was separated into at least five bands, termed as bands A-E from cathode to anode. By comparison with two-dimensional electrophoresis patterns of SCCAs, it was found that the first three bands, i.e. bands A-C, are derived from the intact form of SCCA1, while the other two bands, i.e. band D and E are from the intact form of SCCA2. Specifically, band E, but not band D, of SCCA2 is apparently increased in squamous cell carcinomas compared with normal squamous epithelium. In conclusion, this novel analytical approach will be useful for investigating the different expression patterns of functional SCCAs between normal and malignant cervical squamous epithelial tissues.

Tumor-associated serpin, squamous cell carcinoma antigen stimulates matrix metalloproteinase-9 production in cervical squamous cell carcinoma cell lines.

Squamous cell carcinoma antigen (SCCA) is clinically used as a tumor marker for patients with squamous cell carcinoma of various organs. SCCA1 and its highly homologous molecule, SCCA2, belong to the serine proteinase inhibitors (serpins) family, suggesting that these proteins may be involved in the malignant behavior of squamous cell carcinoma cells. The aim of this study is to functionally characterize these tumor-associated serpins regarding the potential to influence the production of matrix metalloproteinases (MMPs), which play a key role in tumor cell invasion and metastasis. Cervical squamous cell carcinoma cell lines, CaSki cells and SKG-IIIa cells were incubated with SCCA1 or SCCA2 and MMP production was analyzed by gelatin zymography. Both SCCA1 and SCCA2 significantly increased production of proMMP-9, but not proMMP-2. These stimulatory effects were still observed when cells were treated with SCCA mutants lacking the proteinase inhibitory activity of serpins. Furthermore, treatments with various forms of SCCAs, which are generated by interacting with their target proteinases, diminished the stimulatory effect of SCCAs, implying the importance of the conformational structure of SCCAs in the stimulatory effects of SCCAs on proMMP-9 production. In addition, in vitro invasion assay showed that SCCA1 and SCCA2 significantly promoted the activity of cell invasion. It is concluded that SCCAs can alter the invasive phenotype of cervical squamous cell carcinoma cells, probably by stimulating proMMP-9 production, and that intact conformational structure of SCCAs, but not proteinase inhibitory activity of serpins, is required for its stimulatory activity on proMMP-9 production.