Tumour progression in experimental oral carcinogenesis is associated with changes in EGF and TGF-beta receptor expression and altered responses to these growth factors

This study examined the characteristics of premalignant oral epithelial cell lines derived from non-invasive palatal and lingual mucosa of rats painted with the carcinogen 4-nitroquinoline N-oxide (4NQO) in vivo. In contrast to normal keratinocytes, premalignant epithelial cells had an extended life span, were independent of 3T3 fibroblast support, and expressed variable anchorage independence in gel culture and tumorigenicity in athymic mice. The expression of these functional phenotypes did not correlate with the duration of 4NQO treatment. Keratinocytes from 4NQO-treated tissues predominantly had fewer epidermal growth factor (EGF) receptors than normal controls. The expression of high-affinity EGF receptors paralleled the emergence of the anchorage-independent phenotype and was markedly elevated in tumorigenic cell lines. Cell lines with an extended life span expressed fewer transforming growth factor beta 1 (TGF-beta) receptors than their normal counterparts though the loss of these receptors appeared to be unrelated to either anchorage independence or tumorigenicity. Normal keratinocytes were stimulated and inhibited, in a dose-dependent manner, by EGF and TGF-beta respectively. By contrast, a cell line that was immortal, anchorage dependent and non-tumorigenic showed reduced sensitivity to stimulation by EGF and was inhibited only by high concentrations of TGF-beta. Cells that were immortal, anchorage independent and tumorigenic, however, were refractory to EGF and were inhibited only by high concentrations of TGF-beta. There was no correlation between the expression of EGF or TGF-beta cell surface receptors and the response to ligand binding. The results show that tumour progression in rat oral epithelial cells is associated with a progressive independence of growth factor control. The number and distribution of EGF and TGF-beta receptors may be useful markers in more closely defining the stages of epithelial tumour progression.     

Expression of a decorin-like molecule in human melanoma

Decorin, a member of the family of small leucin-rich proteoglycans, has originally been described as a secreted proteoglycan component of the connective tissues, and has been implicated in the negative regulation of cell proliferation directly or via interactions with TGF-beta. It was reported to be generally absent from tumor cells. Here we show that human melanoma cell lines express a decorin-like molecule. We detected decorin mRNA by RT-PCR in 7 out 7 human melanoma lines characterized by various metastatic potential. Using polyclonal antiserum against the core protein of decorin, the typical 80-120 kD glycanated form as well as a high molecular weight aberrant version (200-210 kD) of decorin were demonstrated by Western blot technique in the culture supernatants as well as in lysates of human melanoma cells. Finally, decorin epitope was also demonstrated immunohistochemically in human melanoma xenografts, as well as in tumor cells of surgically resected melanomas but not in melanocytes of nevi.The expression of this aberrant decorin did not inhibit the in vitroor in vivogrowth of human melanoma cells, and it was independent of their metastatic potential. Human melanoma cell lines expressing aberrant decorin retained sensitivity to the antiproliferative and gelatinase-stimulatory effects of exogenous TGF-beta.     

Expression and nuclear localization of ErbB3 in prostate cancer.

PURPOSE: The ErbB1 and ErbB2 receptors have been implicated in prostate cancer progression, but less is known about the role and biology of other ErbB receptor family members in prostate cancer. The aim of this study was to analyze the expression and localization of ErbB3 in prostate tissues and prostate cancer cell lines. EXPERIMENTAL DESIGN: Immunohistochemistry of ErbB3 was done on prostate cancer tissue sections from 143 patients and on a tissue microarray containing 390 cores of radical prostatectomy-derived specimens representing normal, prostatic intraepithelial neoplasia, and malignant tissues from 81 patients. ErbB3 subcellular localization was studied by Western blot analysis in LNCaP, 22Rv1, PC-3, and DU145 prostate cancer cell lines. RESULTS: Immunohistochemistry analysis of prostate cancer tissues revealed that >90% of prostate cancer tissues displayed cytoplasmic ErbB3 staining. Minimal ErbB3 nuclear staining was observed in normal prostate tissues and benign prostatic hyperplasia tissues; in contrast, ErbB3 was frequently localized in the nucleus of cancerous tissues. This nuclear localization was more frequent (P < 0.001) in hormone-refractory tissues (17 of 17, 100%) compared with hormone-sensitive samples (37 of 92, 40.2%). Additionally, in the tissue microarray, increased nuclear ErbB3 was associated with increasing Gleason grade. Interestingly, Western blot analysis of cytoplasmic and nuclear subcellular fractions showed that ErbB3 nuclear localization was more prevalent in hormone-sensitive prostate cancer cell lines (LNCaP and 22Rv1) compared with hormone-insensitive cell lines (PC-3 and DU145). CONCLUSIONS: ErbB3 nuclear localization discriminates normal from malignant prostate tissues and between tumors from hormone-sensitive versus hormone-refractory prostate cancer. ErbB3 nuclear staining seems to be associated with risk of disease progression. The high frequency of ErbB3 nuclear localization in hormone-refractory tissues indicates that ErbB3 warrants further study to understand its association with prostate cancer disease progression.     

The aberrant methylation of TSP1 suppresses TGF-beta1 activation in colorectal cancer

Colorectal cancer arises from the progressive accumulation of mutations and epigenetic alterations in colon epithelial cells. Such alterations often deregulate signaling pathways that affect the formation of colon cancer, such as the Wnt, RAS-MAPK and TGF-beta pathways. The tumor promoting effects of mutations in genes, such as APC, have been demonstrated in cancer cell lines and in mouse models of intestinal cancer; however, the biological effects of most epigenetic events identified in colorectal cancer remain unknown. Consequently, we assessed whether the aberrant methylation of TSP1, the gene for thrombospondin 1, a regulator of TGF-beta ligand activation, is an epigenetic mechanism for inhibiting the TGF-beta signaling pathway. We found methylated TSP1 occurs in colon cancer cell lines (33%), colon adenomas (14%) and colon adenocarcinomas (21%). In primary colorectal cancers, loss of TSP1 expression correlated with impaired TGF-beta signaling as indicated by decreased Smad2 phosphorylation and nuclear localization. Furthermore, methylation-induced silencing of TSP1 expression reduced the concentration of secreted active TGF-beta1 and attenuated TGF-beta signaling. Reversal of TSP1 methylation resulted in increased TSP1 mediated activation of the latent LAP:TGF-beta complex and subsequent TGF-beta receptor activation. Our results demonstrate that the aberrant methylation of TSP1 has biological consequences and provide evidence that the aberrant methylation of TSP1 is a novel epigenetic mechanism for suppressing TGF-beta signaling in colorectal cancer.     

p21-Activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer

Overexpression of p21-activated kinase 1 (PAK1) occurs during the progression of human breast cancer. We have investigated the role of PAK1 in the premalignant progression of the MCF10 series of human breast epithelial cell lines. Levels of PAK1 expression and activation increased with premalignant progression, and expression of dominant-negative (DN) PAK1 reduced both cell proliferation and migration/invasion. In three-dimensional (3D) overlay cultures in reconstituted basement membrane, the MCF10 series produced an in vitro model for premalignant progression. MCF10AneoT cells formed a hyperplastic morphology in which some spheroids developed abnormal lumens. The MCF10.AT1 line exhibited an atypical hyperplastic morphology of abnormal spheroid clusters that did not form lumens. The MCF10.DCIS cells exhibited dysplastic growth. Expression of DN-PAK1 promoted lumen formation in 3D-cultured MCF10A, NeoT, and AT1 structures, suggesting partial reversion of the premalignant phenotype, but did not affect the atypical budding of AT1 structures or the dysplastic growth of ductal carcinoma in situ structures. Aberrant proteolysis is another important characteristic of breast cancer progression and invasion. DN-PAK1 or knock-down of PAK1 reduced pericellular proteolysis of DQ-collagen IV in the 3D cultures. Treatment of cells with an inhibitor of Rac1 also reduced pericellular proteolysis, and this reduction was reversed by the expression of activated PAK1. Our conclusion is that overexpressed and activated PAK1 may be a key coordinator of aberrant cell survival and proteolysis in breast cancer progression.     

CIP2A expression and localization in oral carcinoma and dysplasia

Aims

Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy of the oral cavity resulting in severe morbidity and mortality. To date only few proteins have been suggested as potential biomarkers or targets for this type of cancer. Cancerous inhibitor of PP 2A (CIP2A) is a protein expressed in epithelial tissues that stabilizes the oncogene c-Myc and causes cell transformation. This study was designed to investigate the expression of CIP2A in OSCC cell lines and tissues representing human normal, dysplasia and OSCC.

Results

CIP2A was significantly increased in the human carcinoma cell lines compared to the primary gingival cell line. CIP2A was overexpressed in the human oral dysplasia and OSCC tissues compared to normal oral tissues. CIP2A was also preferentially localized in the dysplastic and OSCC epithelial areas compared to EGFR that was expressed mainly in areas of relatively normal epithelium and in dysplastic tissues above the basal layers.

Methods

Using quantitative real time PCR, mRNA quantification for CIP2A was performed in a primary gingival cell line and OSCCs CAL 27 and SCC-25. Paraffin embedded human specimen classified as normal, dysplastic or OSCC were immunohistochemically stained for CIP2A expression. EGFR and CIP2A were also stained by immunofluorescence for colocalization. Samples of human normal oral tissue and OSCC were studied by PCR for mRNA expression of CIP2A.

Conclusions

CIP2A may play a significant role in oral malignant transformation and therefore, it may be a potential target for chemotherapy of OSCC.Key words: oral cancer, EGFR, CIP2A, dysplasia, C MYC     

Cyclin G2 dysregulation in human oral cancer

Using expression microarray, we have previously shown that human cyclin G2 (hCG2) is significantly down-regulated in laser capture microdissected oral cancer epithelia. Western analysis showed detectable hCG2 protein in normal (2 of 2) but not in malignant (4 of 4) oral keratinocyte cell lines. Immunohistochemistry analysis done on oral cancers showed that normal oral mucosa (100%, 12 of 12) and 69.1% (47 of 68) of dysplastic oral epithelia expressed readily detectable hCG2 in the nuclei. However, only 11.1% of oral cancer epithelia (14 of 126) showed mild hCG2 nuclear staining. Interestingly, of the oral cancers devoid of nuclear hCG2 (112 cases), 58 cases (52%) showed cytoplasmic hCG2 immunostaining, whereas the other 54 cases (48%) exhibited neither nuclear nor cytoplasmic hCG2 staining. In vitro functional study by ectopic restoration of hCG2 expression in the human malignant squamous cell carcinoma (SCC) line SCC15 resulted in a significant inhibition of cellular proliferation (P < 0.001) and colony formation (P < 2 x 10(-5)) with increased population of G(1) phase and decreased in S phase (P < 0.01). Furthermore, stable down-regulation of hCG2 by short interference RNA-based gene silencing in immortalized normal oral keratinocytes resulted in enhanced cell growth with increase in S and prominently in G(2) phase. Because hCG2 has been implicated as a negative regulator in cell cycle progression, our results support that hCG2 dysregulation may play an important role in epithelial transformation and the early stages of human oral cancer development.     

Expression and mutation analysis of heterogeneous nuclear ribonucleoprotein G in human oral cancer

We previously reported that wild type (wt) hnRNP G exhibited tumor suppressive activity in human oral squamous cell carcinoma (HOSCC) cell lines lacking hnRNP G. Wt hnRNP G markedly inhibited the proliferation capacity, anchorage independency and in vivo tumorigenicity of HOSCC cells and notably enhanced the DNA repair capabilities of these cells. In the present study, we studied the genetic and expression states of hnRNP G in normal, premalignant and malignant human oral tissues to further understand the relationship between the hnRNP G alterations and the development of human oral cancer. To correlate the cancer development and the level of hnRNP G expression, we performed an immunohistochemistry staining of hnRNP G in normal, premalignant and malignant human oral tissues. Moreover, we examined the entire coding regions of hnRNP G from selected samples to understand the cause of the alterations of the gene expression.The expression of hnRNP G was notably decreased or completely abolished in 80% of premalignant-dysplastic and malignant oral epithelial tissues, whereas 100% of normal and 90% of hyperplastic non-dysplastic epithelium showed high level of hnRNP G in the nucleus of the basal cell layers. Approximately 80% of HOSCC lacking the expression of hnRNP G showed genetic alteration in hnRNP G, i.e., point mutation and exonic deletion. This study suggest that genetic alterations and aberrant expression of hnRNP G occurring during oral carcinogenesis might be useful markers for the early detection of human oral cancer.     

Cyclooxygenase-2 (COX-2) expression in high-risk premalignant oral lesions

Emerging data indicate a link between genetic instability and up-regulation of cyclooxygenase-2 (COX-2). To see if individuals at high risk of oral cancer are candidates for treatment with selective COX-2 inhibitors (coxibs), levels of COX-2 expression in healthy, premalignant and cancerous oral mucosa were compared with the occurrence of DNA ploidy status as a genetic risk marker of oral cancer. COX-2 gene product was evaluated immunohistochemically in 30 healthy persons, in 22 patients with dysplastic lesions without previous or concomitant carcinomas, and in 29 patients with oral carcinomas. The immunohistochemical findings were verified by western blotting. COX-2 expression was correlated to DNA content as a genetic risk marker of oral cancer. COX-2 was up-regulated from healthy to premalignant to cancerous oral mucosa. Thus, COX-2 expression was found in 1 case of healthy oral mucosa (3%). All specimens from healthy mucosa had a normal DNA content. In patients with premalignancies. In 29 patients with oral carcinomas, cyclooxygenase-2 expression was observed in 26 (88%), and aneuploidy was observed in 25 cases (94%, P=0.04). Notably, of 22 patients with dysplastic lesions, COX-2 was exclusively expressed in a subgroup of nine patients (41%) identified to be at high risk of cancer by the aberrant DNA content of their lesions. Seven of these patients were followed for 5 years or more. An oral carcinoma developed in six of them (85%; P=0.02). These findings emphasize the need to determine whether coxibs can reduce the risk of oral cancer in patients with high-risk precancerous lesions.     

Decorin-mediated inhibition of colorectal cancer growth and migration is associated with E-cadherin in vitro and in mice

Previous studies have shown that decorin expression is significantly reduced in colorectal cancer tissues and cancer cells, and genetic deletion of the decorin gene is sufficient to cause intestinal tumor formation in mice, resulting from a downregulation of p21, p27(kip1) and E-cadherin and an upregulation of β-catenin signaling [Bi,X. et al. (2008) Genetic deficiency of decorin causes intestinal tumor formation through disruption of intestinal cell maturation. Carcinogenesis, 29, 1435-1440]. However, the regulation of E-cadherin by decorin and its implication in cancer formation and metastasis is largely unknown. Using a decorin knockout mouse model (Dcn(-/-) mice) and manipulated expression of decorin in human colorectal cancer cells, we found that E-cadherin, a protein that regulates cell-cell adhesion, epithelial-mesenchymal transition and metastasis, was almost completely lost in Dcn(-/-) mouse intestine, and loss of decorin and E-cadherin accelerated colon cancer cell growth and invasion in Dcn(-/-) mice. However, increasing decorin expression in colorectal cancer cells attenuated cancer cell malignancy, including inhibition of cancer cell proliferation, promotion of apoptosis and importantly, attenuation of cancer cell migration. All these changes were linked to the regulation of E-cadherin by decorin. Moreover, overexpression of decorin upregulated E-cadherin through increasing of E-cadherin protein stability as E-cadherin messenger RNA and promoter activity were not affected. Co-immunoprecipitation assay showed a physical binding between decorin and E-cadherin proteins. Taken together, our results provide direct evidence that decorin-mediated inhibition of colorectal cancer growth and migration are through the interaction with and stabilization of E-cadherin.     

Differential expression of peroxisome proliferator-activated receptor in histologically different human gastric cancer tissues

Gastric cancer cell lines express peroxisome proliferator-activated receptor gamma (PPARgamma), and treatment with PPARgamma ligands suppresses growth of subgroup of these cell lines. However, expression and subcellular distribution of PPARgamma in human gastric cancer tissues is still unknown. Therefore, expression and subcellular localization of PPARgamma were examined among different histological types of gastric cancer tissues. Immunohistochemical staining for PPARgamma was performed using biopsy specimens of human gastric cancer of various histological types, gastric adenomas, and intestinal metaplasia. All samples of intestinal metaplasia and most samples of gastric tumors, except for signet ring cell carcinoma, expressed PPARgamma in the epithelial cells. Most samples of signet ring cell cancer lacked PPARgamma expression. All samples of intestinal metaplasia expressed PPARgamma only in the cytosol. For adenoma, 90% was positive for PPARgamma in cytosol, and 40% was positive in nuclei, for well-differentiated adenocarcinoma, 80% was positive in cytosol, and 20% was positive in nuclei. For moderately differentiated adenocarcinomas, 70% was positive for cytosol, and 80% was positive for nuclei; for poorly differentiated adenocarcinoma, 30% was positive in cytosol, and 70% was positive in nuclei. The frequency of samples with positive cytosolic staining decreased as the differentiation stage turned from intestinal metaplasia to adenoma, well-, moderately-, and poorly-differentiated cancers. Simultaneously, there was a tendency toward an increased frequency of samples with positive nuclear PPARgamma staining as the differentiation stage transformed from intestinal metaplasia to poorly-differentiated cancer. There was a striking difference in subcellular localization according to the differentiation levels of gastric dysplastic cells. The findings also supported an intestinal metaplasia-adenoma-well-differentiated gastric cancer sequence, and signet ring cell cancer was suggested to be of a different lineage from other types of gastric cancers.     

Experimental oral carcinoma of the tongue and buccal mucosa: possible biologic markers linked to cancers at two anatomic sites

The application of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) can initiate and promote the development of oral squamous cell carcinoma of the tongue and buccal mucosa. In this study the level of expression of various markers related to the development of programmed cell death (PCD) in the respective oral carcinomas was analyzed. Sixteen male and female Syrian hamsters (Mesocrietus auratus) were treated with 0.05% DMBA for 16 weeks. Immunohistochemistry was used to observe the expression of p53, proliferating cell nuclear antigen (PCNA), Bcl-2, and nucleosome formation. Single-strand conformational polymorphism (SSCP) for exons 2-9 and sequence analysis of exon 9 of the p53 gene from normal buccal or tongue mucosa as well as the squamous cell carcinomas from the buccal mucosa or the tongue were determined. p53 (wild type) expression was significantly reduced in the tongue dysplastic mucosa or squamous cell carcinoma. The SSCP disclosed banding shifts or new bands in exons 2/3, 4, 8, and 9 for the tongue or buccal oral carcinomas (five of each). In exon 9 the mutation in codon 307 (ala)GCC-GTC(val) was present in the tongue but not in the buccal carcinoma. Other markers included the level of PCNA. PCNA was initially lower in the premalignant tongue lesions but increased in oral squamous cell carcinoma at both sites. In contrast, the amount of nucleosome formation in the tongue carcinomas was less than the level noted for buccal cancers but premalignant dysplasias in the tongue mucosa exhibited higher levels. The inhibitor of PCD, Bcl-2 was lower for dysplasias and carcinomas of the tongue compared to similar lesions of the buccal mucosa. These results indicate that oral carcinomas of different anatomical sites can exhibit differences in growth, oncogene mutation expression, and the development of PCD. The differences in Bcl-2 and nucleosome formation may signify their influence on oncogene expression and growth potential for developing transformed clones and established oral carcinomas.     

In vitro growth inhibition of ovarian cancer cells by decorin: synergism of action between decorin and carboplatin

In vitro studies showed that decorin, a small proteoglycan that is a normal component of the cell matrix involved in tissue scaffolding, effectively inhibited the growth of two ovarian cancer lines, SKOV3 and 2774. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay to measure cell growth, IC50s for decorin ranged from 150 to 400 microg/ml for the two cell lines. In contrast, the growth of tumor cells grown on an artificial cell matrix (Matrigel) was unaffected by decorin treatment, perhaps because of the decorin being irreversibly bound by matrix-associated collagen. Decorin-induced inhibition of ovarian tumor cells appeared to be associated with the increased expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1. Up-regulation of p21 expression was shown by Western blot analysis in decorin-treated ovarian cancer cells. No decorin-induced up-regulation of c-myc was seen, although decorin was reported to activate the epidermal growth factor receptor. Decorin was also shown to synergize with carboplatin to inhibit the growth of ovarian tumor cells. Additional studies are warranted to determine the role of decorin in the treatment of ovarian cancer.     

Up‐regulation of CD109 expression is associated with carcinogenesis of the squamous epithelium of the oral cavity

CD109 is a glycosylphosphatidylinositol (GPI)–anchored glycoprotein whose expression is up‐regulated in squamous cell carcinomas (SCCs) of the lung, esophagus, and uterus. The purpose of this study was to evaluate CD109 expression in oral tumors, including premalignant lesions, and to assess the clinical application of CD109 in oral cancer. CD109 expression in oral normal and tumor tissues from 124 patients was examined by immunohistochemical staining with anti‐CD109 antibody, and significant relations between clinical features and CD109 expression were statistically assessed. We found that high levels of CD109 expression were frequently detected in SCCs and premalignant lesions of the oral cavity, but not in normal squamous epithelia. The CD109 expression level was higher in well‐differentiated SCCs than in poorly differentiated SCCs. Furthermore, premalignant lesions highly expressing CD109 showed higher risk to progress to SCCs. Oral SCC cell lines overexpressing CD109 exhibited accelerated cell growth in vitro compared with control cell lines. In addition, overexpression of CD109 impaired the transforming growth factor (TGF)–β1‐mediated suppression of cell growth. These findings suggest that CD109 plays a role in the development of oral cancers, and is a useful prognostic marker to predict malignant transformation of premalignant lesions. (Cancer Sci 2008; 99: 1916–1923)     

Alterations of Smad signaling in human breast carcinoma are associated with poor outcome: a tissue microarray study.

Based largely on studies of cell lines in vitro and of transgenic mouse models, disruptions of transforming growth factor (TGF) beta signaling are thought to contribute to the development and progression of human breast cancer. However, whether and how TGF-beta signaling becomes disrupted during human breast cancer development in vivo remains largely unknown. To address this question, we have compared the patterns of expression and activation of the postreceptor components of the TGF-beta signaling pathway, the so-called Smads, in human breast cancer cell lines with those in breast carcinoma specimens. None of the breast carcinoma cell lines were growth arrested by TGF-beta in vitro. Each of the tumor cell lines expressed normal levels of Smad2 and -3. Moreover, TGF-beta treatment induced phosphorylation of Smad2 (Smad2P) in each of these lines, except those that lacked TGF-beta type II receptors. Moreover, only one of the cell lines failed to express Smad4. Among 456 cases of human breast carcinoma assembled in tissue microarrays, the majority (92%) expressed Smad2, Smad2P, as well as Smad4, indicating their ability to proliferate within a microenvironment that contains bioactive TGF-beta. Thirty cases (6.6%) failed to express Smad2P, suggesting the loss of TGF-beta receptor signaling. Nine cases (2%) failed to express Smad4, and 3 of these also failed to express Smad2P. Thus, the phenotypes of breast tumors in vivo paralleled that of human breast cancer cell lines in terms of Smad2P and Smad4 expression. Loss of Smad signaling was not associated with any particular histological subtype, histological or nuclear grade, estrogen- or progesterone receptor expression, or HER2/neu expression. Loss of Smad4 was inversely correlated with the presence of axillary lymph node metastases. Most importantly, among patients with stage II breast cancer, lack of Smad2P expression in the tumor was strongly associated with shorter overall survival. Finally, analysis of a small cohort of hereditary breast cancers failed to reveal any association between BRCA1 or BRCA2 genotype and alterations in Smad signaling.