Actin structural proteins in cell motility

Summary The machinery for cell locomotion is based in a network of polymerized actin filaments supporting the peripheral cytoplasm. This network or gel consists of actin filaments in a variety of configurations, including cables, loose bundles, and branching arrays; all formed by the interaction of actin-associated proteins with actin filaments. For cell locomotion to occur, this network must be reversibly disassembled or solated to allow protrusion, then re-assembled to stabilize the resulting extension. Thus, proteins to promote both solation and gelation of actin are important for efficient cell locomotion. Because of their distribution, control, and in vitro effects on actin filaments, two such proteins, gelsolin and actin-binding protein (ABP) should play especially important roles in cell motility. Support for this premise is found in in vivo studies of mouse kidney fibroblasts which demonstrated increased translocational locomotion after cytoplasmic gelsolin expression was increased genetically and in melanoma cells missing actin-binding protein which behave as expected for a cell unable to achieve efficient actin gelation. Since malignant transformation is known to affect the expression and distribution of several of these actin structural proteins, including gelsolin, further investigations of the role these proteins play in cell motility will be important to the determination of tumor cell motility and hence metastatic propensity.     

Inhibition of cell motility by troglitazone in human ovarian carcinoma cell line

Background  

Troglitazone (TGZ) is a potential anticancer agent. Little is known about the effect of this agent on cancer cell migration.     

Involvement of c-Src in carcinoma cell motility and metastasis.

Carcinoma cells exhibit dysfunction / dysregulation of cell adhesion systems that correlates with their abilities to migrate, invade, and metastasize. Here we show that the tyrosine kinase c-Src is required for motility and metastasis of two carcinoma cell lines. Adherent KYN-2 cells having a high level of c-Src kinase activity become scattered, extend lamellipodia, and exhibit high motility. Expression of a dominant-negative mutant form of c-Src caused formation of stress fibers and focal adhesions, and markedly reduced motility. HCT15 cells extended lamellipodia and became scattered in response to lysophosphatidic acid stimulation in parallel with transient activation of c-Src, which was inhibited by expression of a dominant-negative mutant form of c-Src or treatment with a specific Src kinase inhibitor. Furthermore, implantation of dominant-negative c-Src transfectants into the peritoneal cavity of SCID mice resulted in reduced peritoneal dissemination compared with control transfectants. These findings indicate that c-Src activation is critically involved in carcinoma cell migration and metastasis.     

胃癌组织中p53蛋白及Rasp21蛋白的表达及意义

目的:联合检测p53蛋白及Ras p21蛋白在正常胃组织及胃癌组织中的表达,探讨二者与胃癌分化程度及5年生存率间的关系。方法:应用流式细胞术(FCM)对13例正常胃组织及27例胃癌组织细胞进行p53蛋白及Ras p21蛋白含量的定量检测。结果:p53蛋白在正常胃组织的表达均为阴性,85.2%(23/27)的胃癌组织p53表达阳性,p53蛋白表达量(FI值)和p53蛋白阳性表达率与胃癌组织分化程度及5年生存率有明显相关性(P<0.01)。Ras p21蛋白在正常胃组织中表达均为阴性,71.45%的胃癌组织Ras p21蛋白表达阳性;胃癌组织Ras p21蛋白表达量(FI)和Ras p21蛋白阳性表达率与胃癌组织分化程度及5年生存率有明显相关性(P<0.05)。p53蛋白表达与Ras p21蛋白表达呈正相关(r=0.755)。结论:p53蛋白及Ras p21蛋白过表达与胃癌组织的分化程度及预后有明显相关性,p53蛋白及Ras p21蛋白的高表达与胃癌的发生、发展有密切关系,联合检测p53及Ras p21蛋白可作为判断胃癌恶性程度及预后的有效指标。     

Mutational analysis of K-ras and Ras protein expression in larynx squamous cell carcinoma

The ras gene family (H, K and N-ras) encodes the Ras protein, a GTPase-activating protein that regulates several signal transduction pathways including cellular proliferation and differentiation. Mutations in codons 12, 13 and 61 of the ras genes constitute one of the most frequent alterations in human cancer. In the Western Hemisphere, a low frequency of mutations in these genes has been observed in head and neck carcinomas; a higher frequency has been found in countries such as India and Taiwan. Increased protein expression is a relatively frequent event in larynx carcinomas. This study was aimed to evaluate the participation of the k-ras gene and Ras expression in 20 Mexican patients with larynx squamous carcinoma, 2 with dysplasia and 4 with normal mucosa. Samples (of 26 patients) were embedded in paraffin and immunohistochemical analysis was performed for the Ras protein, as well as amplification of the k-ras gene exon 1 (108 bp) by laser capture microdissection. Then, DNA extraction, PCR and sequencing were performed looking for possible mutation in codons 12 and 13. All patients with larynx carcinoma were men, median age 62 years. Eighty-five percent of the patients had risk factors such as smoking and/or alcohol consumption, 25% were in clinical stages I and II, and 75% in stages III and IV; 45% of the patients presented tumor recurrence or persistence. In this study, no mutations were found in codons 12 or 13 of the k-ras gene; however, protein expression was observed in 95% of the samples and a higher expression of the protein was associated with tumor recurrence or persistence, although this was not statistically significant. Unexpectedly, well-differentiated carcinomas and dysplasias presented an increase in protein expression. These results suggest that ras may be involved in early stages of larynx carcinogenesis and may be activated by other mechanisms different from mutations, such as epigenetic events.     

Retinoblastoma protein in human renal cell carcinoma in relation to alterations in G1/S regulatory proteins

The retinoblastoma gene product (pRb) is the main substrate for cyclin-dependent kinases (CDKs) during the G1/S transition. Aberrations in cell cycle regulatory proteins, which have been observed in many malignancies, can theoretically cause increased phosphorylation of pRb due to unbalanced CDK activities. The expression and phosphorylation of pRb and potential associations to cell cycle aberrations in renal cell carcinomas (RCC) has only partly been clarified. We therefore evaluated the presence of pRb and the level of pRb-phosphorylation in 216 RCCs arranged in tissue microarrays by using different pRb-antibodies, including pRb-phosphospecific antibodies. Most RCCs (95%) expressed pRb, while cases with the low pRb levels, potentially indicative for pRb-inactivation, were few. In order to detect secondary alterations to a potential pRb-inactivation, the p16 expression was also monitored. None of the tumors exhibited increased p16 levels, confirming that pRb-inactivation is rare in RCC. Phosphorylated pRb was detected in approximately 50% of the RCCs, using Western blotting or immunohistochemistry. The immunohistochemical ppRb(ser807/811) levels were associated with high proliferation, cyclin D1, cyclin E and p27 protein content. Surprisingly, there was no association between pRb-phosphorylation and clinicopathological data. In summary, pRb seemed to be functional and aberrations in G1/S-regulatory proteins were associated with increased phosphorylation of pRb and proliferation. The data supports that pRb might be one of the main cell cycle regulators in RCC.     

Coordinate regulation of cell growth and differentiation by TGF-beta superfamily and Runx proteins

Runx proteins regulate various biological processes, including growth and differentiation of hematopoietic cells, lymphocytes, osteoblasts, and gastric epithelial cells. Some of the biological activities of Runx proteins are reminiscent of those of transforming growth factor (TGF)-beta superfamily cytokines. Consistent with this notion, receptor-regulated Smads (R-Smads), signal mediators of the TGF-beta superfamily cytokines, and Runx proteins have been shown to physically interact with each other. R-Smads activated by TGF-beta and Runx proteins cooperatively induce synthesis of IgA in B lymphocytes, and those activated by bone morphogenetic proteins and Runx2 induce osteoblastic differentiation. Moreover, the R-Smad-Runx signaling pathways are regulated by an E3 ubiquitin ligase Smurf1, as well as a signal transducer of interferons, STAT1. Since Runxl and Runx3 are involved in the development of some cancers including acute leukemia and gastric cancer, it will be of interest to examine in detail whether TGF-beta-specific R-Smads and Runx proteins coordinately regulate growth and differentiation of hematopoietic cells and gastric epithelial cells.     

Bag-1 proteins in oral squamous cell carcinoma

Bag-1 is an anti-apoptotic protein that exhibits altered expression in many malignancies, including oral squamous cell carcinoma. The bag-1 gene gives rise to different protein products with different subcellular localisations through alternative translational initiation sites. In oral squamous cell carcinoma, cytoplasmic expression has been associated with metastasis to regional lymph nodes and poor prognosis. In contrast, the longest Bag-1 isoform is nuclear and may regulate differentiation in oral epithelium. In this review, the functions of the three isoforms of Bag-1 expressed in oral epithelial cells are discussed in relation to their contribution to oral carcinogenesis.     

Expression of autocrine motility factor receptor in human esophageal squamous cell carcinoma

Autocrine motility factor and its receptor (gp78) have been shown to play an important role in tumor cell migration, invasion and metastasis. We have detected gp78 expression in buffered-formalin-fixed, paraffin-embedded sections of esophageal squamous cell carcinomas using an anti-gp78 monoclonal antibody (MAb), 3F3A, and examined the relationship between gp78 expression and clinicopathological and prognostic factors. In 55 of 101 (54%) patients, gp78 was detected in the tumor cells. The frequency of gp78-positive expression was significantly associated with tumor size, infiltrative growth, depth of invasion and lymph node metastasis. The cumulative survival rate of patients with gp78 was significantly lower than that of patients without gp78. Our results suggest that autocrine motility factor receptor (gp78) expression could be a useful biomarker for malignancy grading and prognosis in patients with esophageal squamous cell carcinoma. © 1995 Wiley-Liss, Inc.     

FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequent in superficial urothelial cell carcinoma (UCC). Ras gene mutations are also found in UCC. As oncogenic activation of both FGFR3 and Ras is predicted to result in stimulation of the mitogen-activated protein kinase (MAPK) pathway, we hypothesized that these might be mutually exclusive events. HRAS mutation has been widely studied in UCC, but all three Ras gene family members have not been screened for mutation in the same sample series. We screened 98 bladder tumours and 31 bladder cell lines for mutations in FGFR3, HRAS, NRAS and KRAS2. FGFR3 mutations were present in 54 tumours (55%) and three cell lines (10%), and Ras gene mutations in 13 tumours (13%) and four cell lines (13%). These included mutations in all three Ras genes; ten in HRAS, four in KRAS2 and four in NRAS and these were not associated with either tumour grade or stage. In no cases were Ras and FGFR3 mutation found together. This mutual exclusion suggests that FGFR3 and Ras gene mutation may represent alternative means to confer the same phenotype on UCC cells. If these events have biological equivalence, Ras mutant invasive UCC may represent a novel subgroup.     

EBV-expressing AGS gastric carcinoma cell sublines present increased motility and invasiveness

Tumor invasion marks a critical point in cancer progression; it is a harbinger of morbidity and mortality. Thus, the cellular events that enable the invasive phenotype are under intense investigation. Epstein-Barr virus (EBV) is associated with a number of cancers, including Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) and is suspected to contribute to their tumorigenesis. On average, 8% of gastric carcinomas have been shown to carry this virus. To explore whether the presence of EBV in gastric carcinoma contributes to tumor progression in this predominantly invasive carcinoma, we examined a panel of 2 in vitro EBV-infected human gastric cancer cell line sublines and their mock-infected AGS parental control line. We found EBV infection caused a marked increase in transmigration of a Matrigel barrier (415% and 303%, p < 0.05, for the 2 infected lines). This correlated with increased motility of these sublines (233% and 140%, p < 0.05). As this pattern of increased motility leading to a more pronounced enhancement of invasion has been noted in other tumor cells, we explored the roles of autocrine signaling pathways previously implicated in carcinoma motility and invasion. Inhibitors to the epidermal growth factor receptor (EGFR) (PD153035), phospholipase C (PLC) (U73122), extracellular-signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) (PD089035) and PI-3 kinase (Wortmannin) were not informative. These data suggest that EBV increases migration of AGS cells by a mechanism independent of these autocrine growth factor-induced pathways. Instead, we found that the EBV-infected cells presented increased focal adhesion kinase (FAK) phosphorylation. These findings suggest a role for integrin-mediated signaling in promoting EBV-associated invasiveness.     

细胞周期调节蛋白在食管鳞癌组织中的表达

背景与目的:研究表明肿瘤细胞周期分析显示细胞具高增殖率的肿瘤,其临床病情发展快;细胞周期调节蛋白Ki-67、细胞周期蛋白A及p27介入到细胞的增殖,但这些因子与食管癌之间的关系的研究在我国尚未见报道。本研究观察细胞周期调节蛋白Ki-67、细胞周期蛋白A及p27在中国食管癌患者中的表达特征,以探讨这些分子标志物与临床病理因素之间的关系。方法:60例(48例男性、12例女性)食管鳞癌患者行外科手术切除肿瘤,其标本行免疫组织化学染色。Ki-67及细胞周期蛋白A表达程度以染色指数表示,p27以标记指数表示。结果:Ki-67、细胞周期蛋白A及p27免疫组化染色在瘤组织与非瘤组织中均固定于细胞核。Ki-67及细胞周期蛋白A的染色指数在低分化鳞癌(27.2±4.9;15.4±5.3)明显高于高分化鳞癌(20.6±6.3;11.3±6.4,P<0.05);p27免疫组化染色的阳性率在高分化鳞癌(36%)高于其它病理类型(29%及18%),但相互之间的差异无统计学意义(P>0.05)。结论:Ki-67、细胞周期蛋白A及p27的表达程度可反映食管鳞癌细胞的增殖状况。细胞周期调节蛋白Ki-67及细胞周期蛋白A的过度表达提示食管鳞癌细胞分化差。     

Pituitary tumor-transforming 1 increases cell motility and promotes lymph node metastasis in esophageal squamous cell carcinoma

Human pituitary tumor-transforming 1 (PTTG1)/securin is a putative oncoprotein that is overexpressed in various tumor types. However, the involvement of PTTG1 in gastrointestinal cancer development and progression remains unclear. In this study, we investigated the clinical significance and biological effects of PTTG1 in esophageal squamous cell carcinoma (ESCC). Immunohistochemical studies performed on 113 primary ESCC specimens revealed a high prevalence of PTTG1 overexpression (60.2%), which was significantly associated with lymph node metastasis (regional, P = 0.042; distant, P = 0.005), advanced tumor stage (P = 0.028), and poorer overall survival (P = 0.017, log-rank test; P = 0.044, Cox proportional hazard model). Eleven ESCC cell lines expressed PTTG1 protein at levels 2.4 to 6.6 times higher than those in normal esophageal epithelial cells (HEEpiC). PTTG1 protein expression was confined to the nucleus in HEEpiC cells but present in both the cytoplasm and nucleus in ESCC cells. Two small interfering RNAs (siRNA) inhibited PTTG1 mRNA and protein expression in three ESCC cell lines by 77% to 97%. In addition, PTTG1 down-regulation by these siRNAs significantly reduced cell motility in all three ESCC cell lines (P < 0.01) in vitro, as well as popliteal lymph node metastases of ESCC cells in nude mice (P = 0.020). Global gene expression profiling suggested that several members of the Ras and Rho gene families, including RRAS, RHOG, ARHGAP1, and ARHGADIA, represented potential downstream genes in the PTTG1 pathway. Taken together, these findings suggest that PTTG1 overexpression promotes cell motility and lymph node metastasis in ESCC patients, leading to poorer survival. Thus, PTTG1 constitutes a potential biomarker and therapeutic target in ESCCs with lymph node metastases.     

Glycosylation of a novel member of the immunoglobulin gene superfamily expressed in rat carcinoma cell lines

MAb E4 recognizes a 66-kDa glycoprotein, pE4, which is a member of the immunoglobulin gene superfamily. This protein is expressed at the cell surface in rat colon and mammary carcinomas, but only in trace amounts in normal adult rat tissues. Since expression of aberrant carbohydrate structures is often associated with malignant transformation, glycosylation of pE4 was analyzed. Reactivity of lectins with pE4 suggested the absence of N-acetylneuraminic acid, terminal galactose and O-linked glycan, and the presence of N-linked glycans. Tunica-mycin treatment reduced the binding of MAb E4 to cancer cells suggesting that the E4 epitope is at least partially glycosylated. Digestions with neuraminidases, O-glycosidase and peptide-N-glycosidase F confirmed these results. Pronase treatment abolished the binding of MAb E4, indicating that E4 epitope involves not only a carbohydrate determinant but also a peptide moiety. Mild periodate oxidation abolished the binding of MAb E4, indicating that non-reducing terminus carbohydrates are part of the E4 epitope. Neutral sugar analysis revealed the absence of galactose and the presence of fucose. Since fucose is sensitive to periodate oxidation, this sugar could be the carbohydrate part of the determinant recognized by MAb E4. Reactivity of lectins specific for fucose indicated the presence of ø(1-6)-fucose on pE4. © 1995 Wiley-Liss, Inc.     

Tumor cell motility

Tumor cell motility is required for invasion and metastasis. The locomotory machinery of the cell includes cell projections called pseudopodia which are regulated by a complicated linkage between cell surface receptors or sensors and the internal cytoskeleton. Recently a new class of motility stimulating cytokines have been identified. These cytokines can function as autocrine motility factors and require a pertussis toxin sensitive G protein pathway to transduce a random motile response.     

VEGF-C contributes to head and neck squamous cell carcinoma growth and motility

Previous work from our laboratory has demonstrated overexpression of chemokines in head and neck cancer and the utility of targeting these proteins for tumor therapy in a preclinical model. However, the mechanisms involved are unexplored. Through gene expression analysis, we found that expression of vascular endothelial growth factor (VEGF-C) was elevated in HN12 cells expressing high levels of CXCL5. In the present study, we have investigated the contribution of VEGF-C to tumor cell growth and motility. RNAi-mediated knockdown of VEGF-C expression in HN12 cells, which express high levels of CXCL5, resulted in a decrease in proliferation. Conversely, forced expression of VEGF-C in HN4 tumor cells with low endogenous CXCL5 levels increased cell growth. Suppression of VEGF-C inhibited migration of HN12 cells. Similarly, HN4 cells showed reduced migration towards conditioned media collected from HN12 cells with VEGF-C knockdown compared to controls, while HN4/VEGF-C conditioned media stimulated cell migration. Moreover, tumor growth in vivo was markedly reduced when VEGF-C expression was blocked by shRNA. Finally, determination of VEGF-C expression in squamous carcinoma cell lines revealed universal overexpression compared to normal keratinocytes. These findings support a role for VEGF-C in head and neck squamous cell carcinogenesis.     

Inhibition of carcinoma cell motility by epoxyeicosatrienoic acid (EET) antagonists

Cytochrome P450 (CYP) epoxygenases, CYP2C8, 2C9 and 2J2 mRNA and proteins, were expressed in prostate carcinoma (PC‐3, DU‐145 and LNCaP) cells. 11,12‐Epoxyeicosatrienoic acid (11,12‐EET) was the major arachidonic acid metabolite in these cells. Blocking EET synthesis by a selective CYP epoxygenase inhibitor (N‐methylsulfonyl‐6‐(2‐propargyloxyphenyl)hexanamide [MS‐PPOH]) inhibited tonic (basal) invasion and migration (motility) while exogenously added EET induced cell motility in a concentration‐dependent manner. An epidermal growth factor receptor (EGFR) kinase inhibitor (AG494) or a PI3 kinase inhibitor (LY294002) inhibited cell migration and reduced 11,12‐EET‐induced cell migration. Importantly, synthetic EET antagonists (14,15‐epoxyeicosa‐5(Z)‐enoic acid [14,15‐EEZE], 14,15‐epoxyeicosa‐5(Z)‐enoic acid 2‐[2‐(3‐hydroxy‐propoxy)‐ethoxy]‐ethyl ester [14,15‐EEZE‐PEG] and 14,15‐epoxyeicosa‐5(Z)‐enoic‐methylsulfonylimide [14,15‐EEZE‐mSI]) inhibited EET‐induced cell invasion and migration. 11,12‐EET induced cell stretching and myosin‐actin microfilament formation as well as increased phosphorylation of EGFR and Akt (Ser473), while 14,15‐EEZE inhibited these effects. These results suggest that EET induce and EET antagonists inhibit cell motility, possibly by putative EET receptor‐mediated EGFR and PI3K/Akt pathways, and suggest that EET antagonists are potential therapeutic agents for prostate cancer. (Cancer Sci 2010; 101: 2629–2636)     

Role of bone morphogenetic proteins in transitional cell carcinoma cells

Bone morphogenetic proteins (BMPs) are pleiotropic growth factors that signal through an interaction with the membrane receptors-type--IA, -IB, and -II (BMP-RIA, -RIB, and -RII, respectively). Although the prototypical members of this group of growth factors were isolated as osteoinductive factors, recently accumulated data have suggested that these factors regulate malignant cells. Herein, we review the data concerning BMPs in transitional cell carcinoma cells.     

RAS AND p53 EXPRESSION IN HUMAN THYROID CARCINOMA

Both benign and malignant nodular disorder of the thyroid gland may give rise to similar symptomatology. Even though clinical background and pathologic criteria may predict prognosis, there are still patients without these adverse prognosis indicators who develop subsequent local invasion or distant metastasis after surgical intervention and eventually succumb to the disease.[1] In recent years it has become apparent that malignant transformation is a result of the accumulation of genetic abno…