Up-regulation of E-cadherin and β-catenin in human hepatocellular carcinoma cell lines by sodium butyrate and interferon-α

Summary Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-α. E-cadherin expression was only up-regulated by butyrate and interferon-α (IFN-α) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, β-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-α. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that α-and γ-catenin expression was not changed, while only the expression of β-catenin increased. β-Catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type β-catenin are potentially modulated by butyrate and IFN-α, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.     

Ⅰ型子宫内膜癌组织中HIF-1α、Snail与E-cadherin的表达及临床意义

目的探讨I型子宫内膜癌组织中HIF-1α的表达,以及调控Snail和E-cadherin对肿瘤侵袭性生物学行为的影响。方法采用免疫组化方法,结合组织芯片技术,检测124例I型子宫内膜癌、28例内膜不典型增生、35例正常内膜组织中HIF-1α、Snail和E-cadherin的表达水平,分析三种蛋白表达之间的相关性及与临床病理因素的关系。结果 I型子宫内膜癌组织中HIF-1α、Snail和E-cadherin的表达率分别为61.3%、46.8%、36.3%,与正常内膜和不典型增生内膜组织相比,有显著统计学差异(P<0.01)。HIF-1α表达与病理分级、肿瘤肌层浸润和淋巴结转移明显相关(P<0.05)。Snail表达与FIGO分期、淋巴结转移明显相关(P<0.05)。E-cadherin的缺失与肿瘤肌层浸润和淋巴结转移显著相关(P<0.01)。I型子宫内膜癌组织中HIF-1α和Snail的表达呈明显正相关(r=0.214,P=0.017),而Snail和E-cadherin的表达存在负相关关系(r=–0.203,P=0.024)。结论 HIF-1α可能通过上调Snail的表达和抑制E-cadherin的表达在I型子宫内膜癌发生、侵袭和转移中发挥重要作用。     

Expression of the transmembrane protein tyrosine phosphatase RPTPα in human oral squamous cell carcinoma

 Little is known about the role of protein-tyrosine phosphatases (PTPs), the cellular counterparts of protein-tyrosine kinases, both for normal growth regulation and for its dysregulation in cancer. The receptor-like PTPα (RPTPα) may play a positive role in growth regulation and has been shown to be overexpressed in colon carcinoma. An RNA/RNA in situ hybridisation protocol for RPTPα as well as RPTPα immunohistochemistry was developed to evaluate RPTPα expression in oral squamous cell carcinomas (OSCCs) of different histological grade and to reveal the synthetically active cells and their tissue distribution. In well-differentiated OSCC (G1), RPTPα mRNA could be detected by in situ hybridisation exclusively in stroma cells (fibro/myofibroblasts and inflammatory cells). A higher histological grade (G2/G3) was associated with an increased number of RPTPα-synthesising carcinoma cells haphazardly distributed within invading tumour areas. Consistent results were obtained by immunocytochemistry. Thus, both carcinoma dedifferentiation and stroma recruitment and activation seem to be associated with an upregulation of RPTPα expression in OSCC. The results speak in favour of the important role of activation of stroma fibro/myofibroblasts influencing the biological behaviour of epithelial tumours and also suggest that elevated RPTPα expression may be a more general marker for proliferating or dedifferentiated cells. Accepted: 2 February 1999     

Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines

The epithelial–mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.     

Expression of the E-cadherin repressors Snail, Slug and Zeb1 in urothelial carcinoma of the urinary bladder: relation to stromal fibroblast activation and invasive behaviour of carcinoma cells

Epithelial–mesenchymal transition (EMT) is regulated by interaction of carcinoma and stromal cells and crucial for progression of urinary bladder carcinoma (UBC). Therefore, the influence of activated fibroblasts on the expression of E-cadherin repressors as well as EMT and invasion in UBC was investigated. A correlative analysis of the immunohistochemical expression of fibroblast (ASMA, S100A4, FAP, SDF1, PDGFRβ) and EMT (Snail, Slug, Zeb1, E-cadherin) markers was performed on 49 UBC cases of different stages. The impact of distinguishable growth factor stimulated fibroblasts on invasion, EMT, and E-cadherin repressor expression was investigated in an invasion model. In situ, invasiveness was significantly correlated to the loss of membranous E-cadherin (E-cad_m) and increased Snail, Slug, Zeb1 in tumour cells, as well as to increased ASMA, S100A4, and PDGFRβ in stromal cells. A significant correlation to nodal metastasis could be evidenced for the loss of E-Cad_m, and for an increase in S100A4 and PDGFRβ. Comparison of stromal and EMT markers revealed significant correlations of ASMA to Snail and Slug; of S100A4 to the loss of E-cad_m and Zeb1; and of PDGFRβ to the loss of E-Cad_m, Slug and Zeb1. In vitro, TGFβ1 induced myofibroblasts were the strongest attractants, while aFGF or TGFβ1/aFGF stimulated fibroblasts were the most potent EMT inductors. As shown here for the first time, distinct sub-populations of fibroblasts are to various extents associated with EMT and tumour progression in UBC. These relevant findings might be the basis for the identification of new diagnostic markers and therapeutic targets selectively affecting tumour supporting CAF effects.     

Analysis of activated EGFR signalling pathways and their relation to laminin-5 γ2 chain expression in oral squamous cell carcinoma (OSCC)

Overexpression of epidermal growth factor receptor (EGFR) was shown for the majority of squamous cell carcinomas. The EGFR expression correlates to tumour size, stage and cytoplasmic accumulation of the laminin-5 γ2 chain (Ln-5/γ2), which is known as a marker of invading tumour cells. There is only limited knowledge if and how EGFR signalling pathways are important for invasion-associated processes and for the regulation of Ln-5/γ2. Therefore the distribution of phosphorylated Erk1/2, p38 MAPK and Akt was immunohistochemically defined in oral squamous cell carcinoma (OSCC) of different histological grade and compared to histological criteria of invasion and cytoplasmic Ln-5/γ2 deposition. With raising histological grade, there is a slight increase in nuclear pErk1/2-stained tumour cells (P=0.398) and a loss of nuclear (P=0.593) and increased cytoplasmic staining (P=0.144) of pAkt mainly in invading OSCC cells. Nuclear pp38 MAPK could only be sporadically detected in few cases. In case of pErk1/2 and pAkt, only a partial co-localisation could be revealed in cases with abundant kinases and Ln-5/γ2. Among the investigated kinases, only pAkt shows a relation to histological grade and invasion in OSCC. pErk1/2, pp38 MAPK and pAkt do not represent a direct link between EGFR and Ln-5 synthesis. Therefore, enhanced Ln-5/γ2 may be a secondary phenomenon of EGFR-induced tumour cell proliferation and dissemination.     

Notch mediated epithelial to mesenchymal transformation is associated with increased expression of the Snail transcription factor

Notch signalling pathway has been implicated as an important contributor to epithelial to myofibroblast transformation (EMT) in tumourigenesis. However, its role in kidney tubular cells undergoing EMT is not defined. This study assessed Notch signalling and the downstream effects on Snail in cultured proximal tubular epithelial cells. EMT was induced by exposure to transforming growth factor beta-1 (TGFβ₁) and angiotensin II (AngII). The expressions of Notch1, Snail, E-cadherin and α-smooth muscle actin (α-SMA) were determined by Western blot. Matrix Metalloproteinase (MMP)-2 and -9 production were determined by zymography. The specific roles of Notch1-ICD and Snail were determined by gene expression or siRNA technique respectively. TGFβ₁ and AngII resulted in EMT as characterized by the expected decrease in E-cadherin expression, an increase in α-SMA, MMP-2 and MMP-9 expression and associated increase of Notch1 and Snail. Over-expression of Notch1-ICD similarly resulted in increased Snail expression, loss of E-cadherin and increasedα-SMA. Inhibiting Snail degradation by pre-treatment with lithium chloride (LiCl) led to a further decrease in E-cadherin expression in cells concurrently exposed to TGFβ₁ + AngII, confirming that Snail is a repressor of E-cadherin. Silencing of Snail blocked TGFβ₁ + AngII induced EMT. Inhibition of Notch activation, by concurrent exposure to DAPT during the induction of EMT attenuated the decrease in E-cadherin expression, limited the increase in α-SMA and MMP-2 and -9 expression and decreased Snail expression. These results suggest a direct role for Notch signalling via the Snail pathway in the development of EMT and renal fibrosis.     

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (P < 0.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G₁ arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC. Xiumei Wang, Wenjing Sun, and Jing Bai contributed equally to this paper.     

RNA干扰抑制Snail表达对于胃癌细胞上皮间充质转化以及体外侵袭的影响

目的用RNA干扰(RNAinterference,RNAi)技术抑制转录因子Snail表达,观察其对人胃腺癌SGC-7901细胞上皮-间充质转化表型和体外侵袭能力的影响。方法构建能表达针对Snail的小干扰RNA(Small interferingRNA,si RNA)的RNA干扰载体(Snail si RNAvector)和表达不针对任何已知mRNA的si RNA的阴性对照RNA干扰载体(control si RNAvector),分别转染SGC-7901细胞,筛选得到Snail表达受抑制的SGC-7901-siSnail细胞和Snail表达未受影响的SGC-7901-siControl细胞。分别采用RT-PCR和Western blot技术检测非转染组、SGC-7901-siSnail、SGC-7901-siControl三组细胞Snail、α-平滑肌肌动蛋白(α-SMA)和E-cadherin表达,用Boyden chamber模型检测细胞侵袭能力。结果 SGC-7901-siSnail组与SGC-7901-nontransfection组相比,Snail和α-SMA表达显著减弱(P0.01),E-cadherin表达显著增强(P0.01),Boyden chamber穿膜细胞数显著减少(P0.01);SGC-7901-siControl组中Snail、α-SMA、E-cadherin表达、Boyden chamber穿膜细胞数分别和SGC-7901-nontransfection组比较无显著差异(P0.05)。结论通过RNA干扰阻滞Snail表达能有效地抑制SGC-7901细胞上皮-间充质转化及体外侵袭能力。Snail可能在胃腺癌上皮-间充质转化及侵袭过程中扮演重要角色,抑制Snail表达可能成胃腺癌治疗的可行策略。     

TGF-β1 influence on TNF-α production and sTNF-Rs shedding in a coculture of colon carcinoma cell spheroids with normal cells

In this study, the levels of TNF-α and its soluble receptors sTNF-Rp55 and sTNF-Rp75 were analyzed in cocultures of human colon carcinoma cell spheroids prepared from different grades of tumors with normal human colon epithelium, myofibroblast, and endothelial cell monolayers. Additionally, the influence of exogenously added rhTGF-β1 (2 ng/ml) on the TNF-α and sTNF-Rs levels was tested. Direct interactions of colon carcinoma spheroids with normal cells caused decreases in TNF-α levels and normal cell-dependent changes in sTNF-Rs amounts as compared to normal cells cultured alone. The addition of rhTGF-β1 to the cocultures caused a significant increase in TNF-α levels with a simultaneous decrease in the amounts of both sTNF-Rs. During direct interactions of colon carcinoma cells with normal tissue, paracrine effects are very important. We showed that TGF-β1 acts synergistically with TNF-α and significantly limits sTNF-Rs shedding. Therefore, TNF-Rs bound to cellular membranes, but not their soluble forms, play an important role in tumor/normal cell interactions. TGF-β1 and sTNF-Rs, in turn, may be valuable factors in colon cancer development and metastasis.     

SRSF1和Survivin在口腔鳞状细胞癌中的表达及临床意义

目的:研究人丝氨酸/精氨酸富有剪接因子1(SRSF1)和凋亡抑制因子(Survivin)在口腔鳞状细胞癌(OSCC)组织和正常口腔黏膜(NOM)中的表达,探究两因子在OSCC中的相关性及其临床意义。方法:采用免疫组织化学SP法,检测两因子在60例OSCC组织、20例NOM组织中的表达,并结合组织学分级、淋巴结转移及临床分期等相关的信息进行相关性分析。结果:在NOM中SRSF1和Survivin低表达,在OSCC组织中均高表达,比例分别为68.3%和60%。SRSF1和Survivin与淋巴结转移、组织学分级以及临床分期之间存在统计学差异(P0.05);并且在OSCC组织中二者之间表达存在着显著正相关性,(r=0.541,P0.05)。结论:SRSF1和Survivin在OSCC中均高表达,与口腔鳞癌的发生、发展密切相关并具有协同作用。     

Variable Stromal Periductular Expression of CD34 and Smooth Muscle Actin (SMA) in Intraductal Carcinoma of the Breast

In breast carcinoma, the stromal loss of CD34 expression and acquisition of SMA myofibroblastic features may constitute a prerequisite for tumor invasiveness. However, this hypothesis remains controversial, with some authors describing the loss of CD34 fibrocytes in the absence of SMA myofibroblastic-like cells in the stroma of invasive carcinoma. Others have also described the disappearance of CD34 fibrocytes from in situ carcinoma. To clarify this issue, we compared the distribution of CD34 fibrocytes and SMA reactive myofibroblasts between stromal areas of tumor-free mammary tissue, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). In addition to 28 IDC, 300 normal duct–lobular units and 600 ducts with DCIS (158 low-grade, 266 intermediate, and 176 high-grade) were scored. The relationships between staining patterns and different histological features (grade of DCIS and presence or absence of necrosis) were compared. Loss of CD34 expression and acquisition of SMA expression were more frequent in high-grade in situ lesions than in intermediate and low-grade lesions (p<0.001). When necrosis was found in association with grade 2 or 3 DCIS, the decrease in CD34 expression was higher than in lesions without necrosis and that independently of the grade of DCIS (p<0.05). Necrosis did not appear to play a significant role in the expression of SMA (p = 0.35). In all cases, the stroma of invasive carcinomas showed a complete loss of CD34 fibrocytes. Future research on both CD34 fibrocytes and mechanisms stromal changes are essential in the future and may potentially lead to new treatment approaches.     

N-Cadherin Promotes Adhesion Between Invasive Breast Cancer Cells and the Stroma

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number of tissues including the mammary gland. In breast and other tumor types, loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stromal cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells: in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells, thereby facilitating invasion and metastasis.     

Identification,paracrine generation,and possible function of human breast carcinoma myofibroblasts in culture

Summary Myofibroblasts from human breast carcinomas were identified and experimentally generated in culture, and a possible function was examined. The frequency ofα-smooth muscle actin immunoreactive cells was evaluated as a measure of myofibroblast differentiation in primary culture. Few or noα-smooth muscle actin-positive stromal cells (6.1 ± 8.4%) were identified in primary cultures from normal breast tissue (n=9). In contrast, high frequencies (68.8 ± 15.1%) were observed in primary cultures from carcinomas (n=19). The frequencies of myofibroblasts in primary cultures were almost identical to those obtained in the corresponding cryostat sections (69.1 vs. 68.8%). A possible precursor cell to the myofibroblast was looked for among typical fibroblasts and vascular smooth muscle cells. Purified blood vessels containing both fibroblasts and vascular smooth muscle cells were embedded in collagen gel and incubated with medium conditioned by breast epithelial cells. Fibroblasts rather than smooth muscle cells were recruited from the blood vessels. In medium conditioned by carcinoma cell lines or in co-cultures of carcinoma cell lines and purified fibroblasts,α-smooth muscle actin and the typical myofibroblast phenotype were induced in otherwiseα-smooth muscle actin-negative fibroblasts. The effect of myofibroblasts on cellular movement—essential to neoplastic cells—was analyzed. Spontaneous motility of tumor cells (MCF-7) was entirely suppressed in a collagen gel assay. Under these conditions tumor cell motility was selectively mediated by direct cell-to-cell interaction between tumor cells and myofibroblasts. Under chemically defined conditions, interaction was dependent on the presence of plasminogen. Anti-plasminogen, soybean trypsin inhibitor, and anti-fibronectin partly neutralized the effect of plasminogen. It is concluded that elements of myofibroblast differentiation and function may be studied in culture.     

14-3-3ε Overexpression Contributes to Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma

Background

14-3-3ε is implicated in regulating tumor progression, including hepatocellular carcinoma (HCC). Our earlier study indicated that elevated 14-3-3ε expression is significantly associated with higher risk of metastasis and lower survival rates of HCC patients. However, the molecular mechanisms of how 14-3-3ε regulates HCC tumor metastasis are still unclear.

Methodology and Principal Findings

In this study, we show that increased 14-3-3ε expression induces HCC cell migration and promotes epithelial-mesenchymal transition (EMT), which is determined by the reduction of E-cadherin expression and induction of N-cadherin and vimentin expression. Knockdown with specific siRNA abolished 14-3-3ε-induced cell migration and EMT. Furthermore, 14-3-3ε selectively induced Zeb-1 and Snail expression, and 14-3-3ε-induced cell migration was abrogated by Zeb-1 or Snail siRNA. In addition, the effect of 14-3-3ε-reduced E-cadherin was specifically restored by Zeb-1 siRNA. Positive 14-3-3ε expression was significantly correlated with negative E-cadherin expression, as determined by immunohistochemistry analysis in HCC tumors. Analysis of 14-3-3ε/E-cadherin expression associated with clinicopathological characteristics revealed that the combination of positive 14-3-3ε and negative E-cadherin expression is significantly correlated with higher incidence of HCC metastasis and poor 5-year overall survival. In contrast, patients with positive 14-3-3ε and positive E-cadherin expression had better prognostic outcomes than did those with negative E-cadherin expression.

Significance

Our findings show for the first time that E-cadherin is one of the downstream targets of 14-3-3ε in modulating HCC tumor progression. Thus, 14-3-3ε may act as an important regulator in modulating tumor metastasis by promoting EMT as well as cell migration, and it may serve as a novel prognostic biomarker or therapeutic target for HCC.