Functional role of ALK-related signal cascades on modulation of epithelial-mesenchymal transition and apoptosis in uterine carcinosarcoma.

BACKGROUND: Anaplastic lymphoma kinase (ALK), which is a receptor tyrosine kinase, is essentially and transiently expressed in the developing nervous system. Recently, the deregulated expression of full-length ALK has been observed in some primary solid tumors, but little is known about its involvement in the tumorigenesis of uterine carcinosarcomas (UCSs). Here we examined the functional role of the ALK gene in UCSs. METHODS: Regulation and function of the ALK gene were assessed using two endometrial carcinoma cell lines. Expression of ALK and its related molecules were also investigated using clinical samples of UCSs. RESULTS: In cell lines, ALK promoter activity was significantly increased by transfection of Sox11 and N-myc, which are known to contribute to neuronal properties. Cells stably overexpressing full-length ALK showed an enhancement of EMT properties mediated by TGF-ß1 and HGF, along with an increase in phosphorylated (p) Akt and nuclear p65. Overexpression of p65 also led to transactivation of Twist1 gene, known as an EMT inducer. Finally, treatment of the stable ALK-overexpressing cells with doxorubicin resulted in inhibition of apoptosis with progressive increase in the expression ratio of both pAkt and bcl2 relative to total Akt and bax, respectively. In clinical samples, strong cytoplasmic ALK immunoreactivity and mRNA signals without rearrangement or amplification of the ALK locus were frequently observed in UCSs, particularly in the sarcomatous components. Further, ALK IHC score was found to be positively correlated with Sox11, N-myc, Twist1, and bcl2 scores. CONCLUSION: ALK-related signal cascades containing Akt, NF-κB, Twist1, and bcl2 may participate in initial signaling for divergent sarcomatous differentiation driven from carcinomatous components in UCSs through induction of the EMT process and inhibition of apoptotic features.

Transcription factor Egr1 acts as an upstream regulator of beta-catenin signalling through up-regulation of TCF4 and p300 expression during trans-differentiation of endometrial carcinoma cells.

The beta-catenin/TCF4/p300 pathway is involved in early signalling for trans-differentiation towards the morular phenotype of endometrial carcinoma cells, but little is known about the upstream regulators. Here we show that transcription factor early growth response 1 (Egr1) acts as an initial mediator through up-regulating the expression of TCF4 and p300. In an endometrial carcinoma cell line with abundant oestrogen receptor alpha, Egr1 expression at both mRNA and protein levels was significantly increased by serum and 17beta-oestradiol stimuli. Serum-stimulated cells also showed increased expression of TCF4 and p300, while inhibition of Egr1 by specific siRNAs resulted in decreased expression. Transfection of Egr1 led to transactivation of TCF4 as well as p300 genes, through specific binding to a promoter region, and thus in turn resulted in nuclear accumulation of beta-catenin mediated by the up-regulating TCF4. The overexpression also caused inhibition of beta-catenin/TCF4/p300-mediated transcription, probably through sequestration of p300. Egr1 promoter activity was increased by serum but not 17beta-oestradiol, in contrast to the marked repression associated with TCF4, p300, and Egr1 itself, indicating that the regulation involves several feedback loops. In clinical samples, cells immunopositive for nuclear Egr1, as well as beta-catenin and TCF4, were found to be sporadically distributed in glandular components of endometrial carcinoma with morules. A significant positive correlation between nuclear beta-catenin and TCF4 was observed, but no such link was evident for Egr1, probably due to the existence of negative feedback regulation. Together, these data indicate that Egr1 may participate in modulation of the beta-catenin/TCF4/p300 signalling pathway as an initial event during trans-differentiation of endometrial carcinoma cells, through its impact on several signalling networks.

Crosstalk between NF-kappaB/p65 and beta-catenin/TCF4/p300 signalling pathways through alterations in GSK-3beta expression during trans-differentiation of endometrial carcinoma cells.

Beta-catenin/TCF4/p300 signalling loops play an important role in trans-differentiation towards the morular phenotype of endometrial carcinomas. Crosstalk between NF-kappaB and beta-catenin pathways has been proposed and we focused here on associations between these two pathways during trans-differentiation. In normal endometrium, nuclear phosphorylated p65 (pp65), the active form NF-kappaB subunit, was found to be significantly increased in the secretory phase, correlating positively with vimentin and E-cadherin and inversely with Snail mRNA expression. On transfection of p65, vimentin, E-cadherin, and Snail were transcriptionally altered, indicating possible roles in establishment and maintenance of the secretory phenotype. In endometrial carcinomas with morules, levels of nuclear pp65, Snail mRNA, vimentin, and cytoplasmic TNF-alpha were reduced during trans-differentiation, correlating inversely with nuclear beta-catenin. Nuclear accumulation of GSK-3beta, along with beta-catenin, was observed in morules. In cell lines, overexpression of p65 inhibited beta-catenin/TCF4-mediated transcription, while transfection of GSK-3beta resulted in repression of TNF-alpha-induced NF-kappaB activity. Moreover, nuclear GSK-3beta was increased by overexpression of beta-catenin, as well as induction of G1-cell cycle arrest. These findings provide evidence that a shift from NF-kappaB to beta-catenin signalling pathways through alterations in GSK-3beta expression may be essential for the induction of trans-differentiation of endometrial carcinoma cells, leading to a shut-down of mesenchymal markers.

beta- Catenin mutations and aberrant nuclear expression during endometrial tumorigenesis.

To clarify the possible role of aberrant beta-catenin expression during endometrial tumorigenesis, a total of 199 cases of endometrial carcinomas (endometrioid type), as well as 37 cases of simple/complex and 32 of atypical hyperplasias, was consecutively investigated for immunohistochemistry, along with 141 normal endometrial samples distant from carcinomas. Of 199 carcinoma cases, 73 tumours as well as 44 normal samples were also analysed using a combination of RT-PCR and Southern blot hybridization, Western blot, and mutation gene assays. Cell membrane beta-catenin immunoreactivity showed a stepwise decrease from normal, through atypical hyperplasia, to grade 3 carcinomas. In contrast, the nuclear accumulation in atypical hyperplasias and grade 1 or 2 tumours was higher than in simple/complex hyperplasias. Mutations in exon 3 of the beta-catenin gene involving codons 33, 34, 37, 41, and 45 were observed in 16 (22.9%) of 70 endometrial carcinomas, as well as 3 (12.5%) of 24 atypical hyperplasias, the results being significantly related to low membrane and high nuclear immunoreactivity but not relative mRNA expression levels, suggesting that the gene mutations may be closely associated with changes in subcellular distribution. In addition to significant association between beta-catenin mutation and low grade histological malignancy (P = 0.048), the mutations were detected in none of 15 and 13 (26%) of 50 tumours with or without lymph node metastasis, the difference being significant (P = 0.027). These findings suggest that beta-catenin abnormalities may play an important role in a relatively early event during the endometrial hyperplasia-carcinoma sequence.

Up-regulation of pS2 expression during the development of adenocarcinomas but not squamous cell carcinomas of the uterine cervix, independently of expression of c-jun or oestrogen and progesterone receptors.

The pS2 gene product was firstly identified as an oestrogen-induced molecule in a breast cancer cell line, while recent studies demonstrate a close association with mucus-secreting epithelia. To assess pS2 expression in uterine cervical adenocarcinomas (C-ACas) and invasive squamous cell carcinomas (C-ISCCs), a series of 94 and 86 cases, respectively, as well as 77 samples of normal cervix, were immunohistochemically investigated and the results compared with data for expression of oestrogen and progesterone receptors (ER and PR) and c-jun. RT-PCR and western blot assays were also applied to 21 cervical carcinomas and 24 normal tissues. With cervical glandular lesions, significant up-regulation of pS2 expression at both the mRNA and the protein levels was observed for adenocarcinomas in situ (AISs) and overt carcinomas, closely linked with mucinous differentiation and tumour grades. pS2 scores were inversely related to ERalpha status for all cervical glandular categories, while there was no association with ERbeta and PR values. In squamous lesions, pS2 values did not differ between normal and malignant lesions, in contrast to the significant down-regulation of ERalpha expression with tumour development. Although c-jun expression significantly correlated with ERalpha values for all squamous categories, it did not relate to pS2 status in either C-ACas or ISCCs. These results indicate that alterations in pS2 expression may occur relatively early in the development of cervical glandular, but not squamous lesions, independently of factors known to promote transcription of the pS2 gene.

CD44 expression in benign, premalignant, and malignant ovarian neoplasms: relation to tumour development and progression.

To clarify the possible role of CD44 expression in ovarian tumour development and progression, an immunohistochemical investigation was undertaken of a series of 115 carcinomas, 32 tumours with low malignant potential (LMP), and 53 cystadenomas. A combination of the reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization (SBH) assays was also performed for 17 malignant, four LMP, six cystadenoma, and seven normal ovarian samples. Immunoreactivity scores for CD44s, CD44v3, and CD44v6 were significantly higher in LMP and malignant tumours than in the benign or normal cases, in line with the results of gross mRNA-based assays. Exon-specific RT-PCR/SBH assays revealed that the expression of large CD44 transcripts containing v6 to v8 exons and small isoforms containing v2 and v3 was common among normal and neoplastic tissues, while a simultaneous increase of large isoforms containing v2 to v5 was also revealed in LMP and malignant tumours. In ovarian carcinomas, the scores for CD44s, CD44v3, and CD44v6 were inversely related to the FIGO stage, but there was no association with lymph node status or expression of hormone receptors. Multivariate analysis revealed loss of CD44v3 expression to be an independent factor for poor survival. The findings indicate that CD44 is up-regulated during the development of ovarian carcinomas but is subsequently down-regulated during their progression, resulting in aggressive behaviour and an unfavourable prognosis.

Down-regulation of p27Kip1 expression is correlated with increased cell proliferation but not expression of p21waf1 and p53, and human papillomavirus infection in benign and malignant tumours of sinonasal regions.

AIMS: Although p27Kip1(p27) is a cyclin-dependent kinase inhibitor and a contribution to tumorigenesis has been hypothesized, the possible role in tumours arising in the nasal and paranasal sinus regions is still unknown. METHODS AND RESULTS: Seventy-six sinonasal tumours, including 28 inverted papillomas (IPs) and 48 squamous cell carcinomas (SCCs), were immunohistochemically investigated, along with 46 exophytic papillomas (EPs) of upper respiratory tract and 34 samples of normal paranasal sinus epithelium. The results were also compared with expression of p21WAF1 (p21) and p53, cell proliferation assessed in terms of Ki67 labelling indices (LIs), and human papillomavirus (HPV) infection. The average p27 scores decreased from normal through to malignant lesions, while Ki67 LI scores showed a stepwise increase, the inverse correlation between scores for all categories being significant (r = - 0.639, P < 0. 0001). In the SCCs, p27 expression was significantly higher in keratinizing than nonkeratinizing type tumours (P < 0.05), while there was no association with p21 and p53 expression. Although HPV DNAs for type 16 and 18 were detected in two (7.4%) of 27 EPs, six (35.8%) of 28 IPs, and nine (28.1%) of 32 SCCs, no relation with p27 scores was evident. CONCLUSION: Loss of p27 expression correlates with increased cell proliferation in sinonasal tumours. Moreover, the expression appears to be associated with keratinization in SCCs of the paranasal sinus. These findings indicate that p27 expression may be a useful marker for the dysregulation of cell kinetics in these tumours.

Down-regulation of CD44 standard and variant isoforms during the development and progression of uterine cervical tumours.

To clarify the possible role of CD44 in the development or progression of uterine cervical tumours, an immunohistochemical investigation was carried out on 125 cases of cervical intraepithelial neoplasia (CIN), 78 invasive squamous cell carcinomas (ISCC), 61 cervical adenocarcinomas (AC), nine adenosquamous carcinomas (ASq), and 15 carcinomas with co-existent SCC and AC components, as well as 87 samples of normal cervix. A combination of reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization (SBH) was also applied to 16 cervical carcinomas and 24 normal cervical specimens. Immunoreactivity for CD44s, CD44v3, and CD44v6 did not alter during the progression of CIN, while significantly decreased expression was observed in ISCC, associated with invasive features in some tumours. Reduced levels of CD44 expression in AC were also found, compared with normal cervical glandular epithelia. The average immunoreactivity scores for CD44s, CD44v3, and CD44v6 were significantly higher in ISCC than in AC, in line with the RT-PCR/SBH assay results. However, CD44 scores did not correlate with any clinicopathological factors or with survival in ISCC or AC. The ASq and AC CD44 scores were similar, while staining patterns in mixed tumours were dependent on the morphological phenotype, suggesting a close association between CD44 expression and the cell types. The results suggest that whereas CD44 is down-regulated during cervical tumourigenesis, positivity may not be useful as a consistent prognostic indicator.

Loss of expression of the gene deleted in colon carcinoma (DCC) is closely related to histologic differentiation and lymph node metastasis in endometrial carcinoma.

BACKGROUND: Although frequent loss of the tumor suppressor gene deleted in colon carcinoma (DCC) has been demonstrated in endometrial carcinoma, an alteration of the expression during normal menstrual cycle and tumorigenesis from hyperplastic lesions is still unclear. METHODS: A total of 151 endometrial carcinomas (endometrioid type), along with 90 hyperplasias (23 simple, 30 complex, and 37 atypical) and 143 normal endometria (28 atrophic, 44 proliferative, and 71 secretory), were immunohistochemically investigated for expression of DCC as well as for estrogen and progesterone receptors (ER and PR). Analysis for DCC mRNA levels was also performed on 37 endometrial carcinomas and 14 normal endometria. RESULTS: DCC expression was observed in endometrial glandular cells in both proliferative and secretory stages; the immunoreactivity scores were not related to values for either ER or PR. The values for DCC were significantly higher in hyperplasia than in normal endometria, and then decreased in the sequence leading to Grade 3 carcinoma. In endometrial carcinoma, reduction or loss of DCC expression was significantly related to the histologic evidence of malignancy and lymph node metastasis, and this was in keeping with the results of mRNA analysis. The transcripts derived from alternative splicing in the extracellular domain were not observed in any tumor samples. CONCLUSIONS: The findings of this study indicate that DCC expression may be linked to the maintenance of differentiated glandular cells during the normal menstrual cycle without any relation to immunoreactivity for ovarian hormone receptors. Moreover, loss or reduction of expression may be a significant event in the progression of endometrial carcinoma through metastatic features.

CD44 expression in normal, hyperplastic, and malignant endometrium.

A total of 140 endometrial carcinomas (endometrioid type), as well as 72 hyperplasias (41 of simple or complex and 31 of atypical type) and 141 normal endometria (35 in the proliferative and 106 in the secretory phase), were immunohistochemically investigated for expression of the standard and variant-3 and -6 isoforms of CD44, and the results compared with several known prognostic factors. A combination of the reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization (SBH) for CD44 mRNA levels was also carried out on 27 endometrial carcinomas. In normal endometrium, the expression of standard and variant CD44 forms was remarkably higher in the secretory than in the proliferative phase, with immunoreactivity scores being inversely correlated with numbers of oestrogen and progesterone receptors. Significantly elevated levels of CD44 expression in endometrial carcinomas compared with the proliferative phase and hyperplasia were also revealed by both the immunohistochemical and the RT-PCR/SBH assays, while no association was noted with any prognostic factors. The results indicate that CD44 expression in the normal menstrual cycle is closely related to the secretory differentiation of the glandular epithelium. Moreover, detection of aberrant expression may be useful for the early diagnosis of endometrial carcinoma, but not as an indicator of tumour progression.

Detection of the Epstein-Barr virus genome in cervical neoplasia is closely related to the degree of infiltrating lymphoid cells: a polymerase chain reaction and in situ hybridization approach.

To cast light on the significance of Epstein-Barr virus (EBV) infection in cervical tumorigenesis, 44 cervical intraepithelial neoplasia (CIN) types I/II, and 70 CIN III lesions, 60 invasive squamous cell carcinomas (ISCC), and 20 normal cervical samples were investigated by polymerase chain reaction (PCR) and RNA in situ hybridization (RISH) assays. The EBV genome was detected by PCR using primers targeting the IR region in three (6.8%) of the CIN I/II, 15 (21.4%) of the CIN III and 13 (21.7%) of the ISCC lesions, while using an EBER oligonucleotide probe RISH revealed positive signals in infiltrating lymphocytes located in the cervical stroma, but not in dysplastic or tumor cells. There was a significant correlation between the presence of EBV DNA and the degree of lymphoid cell infiltration (P = 0.0223). In contrast, none of the normal cervical samples that were without inflammation demonstrated any EBV infection. Thus, the results indicated that a positive result for EBV on PCR may be simply dependent on the amount of lymphocytes in cervical stroma, suggesting that this virus does not play a major role in the etiology of cervical neoplasia.

Absence of human papillomavirus genomic sequences detected by the polymerase chain reaction in oesophageal and gastric carcinomas in Japan.

AIMS: To estimate the occurrence of human papillomavirus (HPV) infection in oesophageal or gastric carcinomas in patients in Japan. METHODS: 103 oesophageal and 99 gastric carcinomas were investigated by polymerase chain reaction (PCR) assays using two consensus (targeting either the L1 or the E6-E7 regions) and two type specific (type 16 and 18) primer sets. A beta globin gene specific primer set was also applied to examine the quality of the extracted DNA. RESULTS: Amplification of beta globin gene was clearly visible in 92 (89.3%) of the 103 oesophageal, and 89 (89.8%) of the 99 gastric carcinoma specimens. However, the entire series of tumour DNA was negative for HPV sequences by PCR assay using all four primer sets. CONCLUSION: HPV is not likely to be involved in oesophageal or gastric tumorigenesis in Japanese patients.

The possible role of bcl-2 expression in the progression of tumors of the uterine cervix.

BACKGROUND: A close link between human papillomavirus (HPV) and the development of uterine cervical neoplasias has been proposed. However, other cofactors also are required for malignant transformation. METHODS: Forty-six cervical intraepithelial neoplasias (CIN) I/II, 75 CIN III, and 60 invasive squamous cell carcinomas (ISCC) were investigated by immunohistochemical staining for bcl-2 protein (bcl-2), bax protein (Bax), estrogen receptor (ER), and progesterone receptor (PR). The presence of HPV-DNA was examined using the polymerase chain reaction assay. RESULTS: bcl-2 immunoreactivity was found in 17 of 46 (37%) CIN I/II, 48 of 75 (64%) CIN III, and 12 of 60 (20%) ISCC, the positivity in CIN III being significantly higher than in CIN I/II or ISCC (P < 0.004, P = 0.0001). The bcl-2 immunostaining pattern could be subdivided into two groups, basal type and diffuse type, with the incidence of the latter being clearly increased, in line with tumor progression. In the Bax or HPV-DNA positive groups, bcl-2 positive cases in CIN I/II were in the minority, whereas in CIN III they constituted the majority. In ISCC, bcl-2 positive was significantly lower than negative cases, not being associated with Bax and HPV-DNA. Estrogen receptor and PR immunoreactivity were rare. CONCLUSIONS: These results indicate that bcl-2 may play an important role in a relatively early stage of cervical tumorigenesis, in association with Bax expression and HPV infection.

HPV type 16 in conjunctival and junctional papilloma, dysplasia, and squamous cell carcinoma.

AIMS: To clarify the role of human papillomavirus (HPV) infection in the development of papilloma, dysplasia, squamous cell carcinoma, and basal cell epithelioma arising from the eyelids, including the tunica conjunctiva palpebrum (conjunctiva), its junction to epidemis of eyelid skin (junction), and eyelid skin. METHODS: Sixteen cases of papilloma, four of dysplasia, four of squamous cell carcinoma, and 12 of basal cell epithelioma were examined using formalin fixed and paraffin embedded samples. Detection of HPV-DNA was performed by PCR-RFLP and in situ hybridisation (ISH) methods. RESULTS: HPV-16 was detected in 12/16 papillomas (75%), 2/4 dysplasias (50%), and 1/4 squamous cell carcinomas (25%) but in none of the basal cell epitheliomas. No other HPV subtypes were found. ISH assay showed positive signals in only two cases of dysplasia and squamous cell carcinoma. The mean age of HPV-16 positive dysplasia and squamous cell carcinoma cases (81.7 years) was significantly higher than that of HPV-16 positive papilloma cases (p < 0.01). CONCLUSIONS: Based on the presence of HPV-16 in both benign and malignant lesions and the age distribution, it seems likely that HPV-16 alone may be incapable of causing development of conjunctival and junctional dysplasia and squamous cell carcinoma, and that any correlation between the papilloma-squamous cell carcinoma sequence and HPV infection may be due to rare events.