Autocrine galectin-1 promotes collective cell migration of squamous cell carcinoma cells through up-regulation of distinct integrins

We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and β5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.     

VILIP-1 Expression In Vivo Results in Decreased Mouse Skin Keratinocyte Proliferation and Tumor Development

VILIP-1, a member of the neuronal Ca²⁺ sensor protein family, is able to act as a tumor suppressor in carcinoma cells by inhibiting cell proliferation and migration. In order to study the role of VILIP-1 in skin carcinogenesis we generated transgenic mice overexpressing VILIP-1 in epidermis under the control of the bovine keratin K5 promoter (K5-VILIP-1). We studied the susceptibility of FVB wild type and VILIP-1 transgenic mice to chemically mediated carcinogenesis. After 30 weeks of treatment with a two-stage carcinogenesis protocol, all animals showed numerous skin tumors. Nevertheless, K5-VILIP-1 mice showed decreased squamous cell carcinoma (SCC) multiplicity of ∼49% (p<0.02) with respect to the corresponding SCC multiplicity observed in wild type (WT) mice. In addition, the relative percentage of low-grade cutaneous SCCs grade I (defined by the differentiation pattern according to the Broders grading scale) increased approximately 50% in the K5-VILIP1 mice when compared with SCCs in WT mice. Similar tendency was observed using a complete carcinogenesis protocol for skin carcinogenesis using benzo(a)pyrene (B(a)P). Further studies of tumors and primary epidermal keratinocyte cultures showed that matrix metalloproteinase 9 (MMP-9) levels and cell proliferation decreased in K5-VILIP-1 mice when compared with their wild counterparts. In addition tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was higher in K5-VILIP-1 keratinocytes. These results show that VILIP-1 overexpression decreases the susceptibility to skin carcinogenesis in experimental mouse cancer models, thus supporting its role as a tumor suppressor gene.     

Expression of Snail is associated with myofibroblast phenotype development in oral squamous cell carcinoma

Snail is a regulator of epithelial–mesenchymal transition (EMT) and considered crucial to carcinoma metastasis, myofibroblast transdifferentiation, and fibroblast activation. To investigate the role of Snail in oral squamous cell carcinoma (OSCC), its immunohistochemical expression was analysed in 129 OSCC samples and correlated to nodal metastasis, histological grade, E-cadherin, and alpha smooth-muscle-actin (αSMA). The results were compared to findings in 23 basal cell carcinomas (BCC). Additionally, the influence of TGFβ1 and EGF on Snail, E-cadherin, vimentin, and αSMA expression was analysed in two OSCC cell lines. As a result, Snail-positive cells were mainly found in the stroma of the OSCC invasive front without statistically significant correlation to histological grade or nodal metastasis. Snail was co-localised to αSMA but not to E-cadherin or cytokeratin and showed a significant correlation to the loss of membranous E-cadherin. All BCCs were Snail negative. In OSCC culture, the growth-factor-mediated EMT-like phenomenon was accompanied by αSMA down-regulation. In summary, Snail expression in OSCC is a stromal phenomenon associated with the myofibroblast phenotype and not related to growth-factor-mediated transdifferentiation of the carcinoma cells themselves. Consequently, Snail immunohistochemistry cannot contribute to the prediction of the metastatic potential. Furthermore, stromal Snail expression is suggested to be the result of mutual paracrine interaction of fibro-/myofibroblasts and dedifferentiated carcinoma cells leading to the generation of a special type of carcinoma-associated fibroblasts. M. Franz and K. Spiegel have contributed equally to the study.     

Functional characterization of cultured cells derived from an intraepidermal carcinoma of the skin (IEC-1)

We have successfully isolated a cell line (IEC-1) from an intraepidermal carcinoma of the skin of a patient and compared its behavior, in vitro, to normal human epidermal keratinocytes (HEK) and squamous cell carcinoma cell lines (SCCs). HEK differentiation comprises an initial growth arrest followed by an induction of squamous differentiation-specific genes such as transglutaminase type 1 (TG-1). Using thymidine uptake and TG-1 induction as markers of proliferation and differentiation, respectively, we were able to show that HEKs and the IEC-1 cells undergo growth arrest and induce TG-1 mRNA expression in response to various differentiation-inducing stimuli, while neoplastic SCC cell lines did not. However, differentiation in HEKs was an irreversible process whereas differentiation of the IEC-1 cells was reversible. Furthermore, growth of IEC-1 cells in organotypic raft cultures revealed differences in their ability to complete a squamous differentiation program compared with that of normal HEKs. The IEC-1 cells also exhibited a transitional phenotype with respect to replicative lifespan; HEKs had a lifespan of 4-6 passages, IEC-1 cells of 15-17 passages, and SCC cells were immortal. These alterations in IEC-1 cell behavior were not associated with functional inactivation or mutations of the p53 gene. These data indicate that the IEC-1 cells, derived from a preneoplastic skin tumor, exhibit differences in their ability to undergo terminal differentiation and have an extended replicative lifespan.     

IMP-3 expression in keratoacanthomas and squamous cell carcinomas of the skin: an immunohistochemical study

The protein insulin-like growth factor II mRNA binding protein 3 (IMP-3) is an important factor for cell migration and adhesion in malignancies. Recent studies have shown a remarkable overexpression of IMP-3 in different human malignant neoplasms and also revealed it as an important prognostic marker in some tumor entities. The purpose of this study is to compare IMP-3 immunostaining in cutaneous squamous cell tumors and determine whether IMP-3 can aid in the differential diagnosis of these lesions. To our knowledge, IMP-3 expression has not been investigated in skin squamous cell proliferations thus far. Immunohi-stochemical staining for IMP-3 was performed on slides organized by samples from 67 patients, 34 with keratoacanthoma (KA) and 33 with primary cutaneous squamous cell carcinoma (SCC) (16 invasive and 17 in situ). Seventyfour percent of KAs (25/34) were negative for IMP-3 staining, while 57% of SCCs (19/33) were positive for IMP-3 staining. The percentage of IMP-3 positive cells increased significantly in the invasive SCC group (P=0.0111), and particularly in the SCC in situ group (P=0.0021) with respect to the KA group. IMP-3 intensity staining was significantly higher in invasive SCCs (P=0.0213), and particularly in SCCs in situ (P=0.008) with respect to KA. Our data show that IMP-3 expression is different in keratoacanthoma with respect to squamous cell carcinoma. IMP-3 assessment and staining pattern, together with a careful histological study, can be useful in the differential diagnosis between KA e SCC.Key words: IGF-II mRNA-binding protein-3 (IMP-3), keratoacanthoma, squamous cell carcinoma.     

Progression of Oral Squamous Cell Carcinoma Accompanied with Reduced E-Cadherin Expression but Not Cadherin Switch

The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (n = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (P<0.01) and enhanced invasion (P<0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins in vitro reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.     

鼻咽癌ALDH1~+细胞具有上皮细胞-间质转化的性质

目的:探讨鼻咽癌ALDH1+细胞是否具有上皮细胞-间质转化的性质。方法:利用ALDEFLUOR试剂分选鼻咽癌ALDH1+细胞和ALDH1-细胞,Transwell及Boyden小室实验分析ALDH1+细胞和ALDH1-细胞的侵袭、迁移能力;实时定量PCR及Western blot检测E-cadherin、Vimentin、Snail及Twist基因在ALDH1+细胞和ALDH1-细胞mRNA和蛋白表达水平。结果:与ALDH1-细胞相比,ALDH1+细胞侵袭及迁移能力明显高于ALDH1-细胞;而且ALDH1+细胞中E-cadherin的mRNA和蛋白水平明显低于ALDH1-细胞,相反,ALDH1+细胞中Vimentin、Snail及Twist的mRNA和蛋白水平明显高于ALDH1-细胞。结论:鼻咽癌ALDH1+细胞可能具有上皮-间质转化的性质,这将为阐明肿瘤细胞在侵袭、转移中的机制提供理论基础。     

Celastrol inhibits TGF-β1-induced epithelial–mesenchymal transition by inhibiting Snail and regulating E-cadherin expression

The epithelial–mesenchymal transition (EMT) is a pivotal event in the invasive and metastatic potentials of cancer progression. Celastrol inhibits the proliferation of a variety of tumor cells including leukemia, glioma, prostate, and breast cancer; however, the possible role of celastrol in the EMT is unclear. We investigated the effect of celastrol on the EMT. Transforming growth factor-beta 1 (TGF-β1) induced EMT-like morphologic changes and upregulation of Snail expression. The downregulation of E-cadherin expression and upregulation of Snail in Madin–Darby Canine Kidney (MDCK) and A549 cell lines show that TGF-β1-mediated the EMT in epithelial cells; however, celastrol markedly inhibited TGF-β1-induced morphologic changes, Snail upregulation, and E-cadherin expression. Migration and invasion assays revealed that celastrol completely inhibited TGF-β1-mediated cellular migration in both cell lines. These findings indicate that celastrol downregulates Snail expression, thereby inhibiting TGF-β1-induced EMT in MDCK and A549 cells. Thus, our findings provide new evidence that celastrol suppresses lung cancer invasion and migration by inhibiting TGF-β1-induced EMT.     

Vascular Endothelial Growth Factor Receptor-1 Activation Promotes Migration and Invasion of Breast Cancer Cells through Epithelial-Mesenchymal Transition

Vascular endothelial growth factor receptor-1 (VEGFR-1 or Flt-1), a tyrosine kinase receptor, is highly expressed in breast cancer tissues, but near absent in normal breast tissue. While VEGFR-1 expression is associated with poor prognosis of women with breast cancer, it is not clear whether it is involved in the aggressiveness of breast cancer. Thus, the present study examined whether VEGFR-1 activation is associated with the invasiveness of breast cancer. We reported that VEGFR-1 was detected in 60.6% of invasive breast carcinoma tissue sections. In addition, VEGFR-1 expression positively correlated with lymph node-positive tumor status, low expression level of membranous E-cadherin, and high expression levels of N-cadherin and Snail. We found that PlGF-mediated VEGFR-1 activation promoted migration and invasion in MCF-7 (luminal) cells and led to morphologic and molecular changes of epithelial-mesenchymal transition (EMT). This was blocked by the down-regulation of VEGFR-1. Conversely, down-regulation of VEGFR-1 in MDA-MB-231 (post-EMT) cells resulted in morphologic and molecular changes similar to mesenchymal-epithelial transition (MET), and exogenous PlGF could not reverse these changes. Moreover, VEGFR-1 activation led to an increase in nuclear translocation of Snail. Finally, MDA-MB-231 cells expressing shRNA against VEGFR-1 significantly decreased the tumor growth and metastasis capacity in a xenograft model. Histological examination of VEGFR-1/shRNA-expressing tumor xenografts showed up-regulation of E-cadherin and down-regulation of N-cadherin and Snail. These findings suggest that VEGFR-1 may promote breast cancer progression and metastasis, and therapies that target VEGFR-1 may be beneficial in the treatment of breast cancer patients.