Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors

Rac1 is a member of the Ras superfamily of small GTPases involved in signal transduction pathways that induce the formation of lamellipodia, stimulate cell proliferation and activate the JNK/SAPK protein kinase cascade. Here we describe that amplification by RT-PCR of the entire Rac1 coding sequence from a series of human adult and fetal tissues revealed beside the expected Rac1 cDNA, a variant product which contained additional 57 nucleotides between codons 75 and 76. This variant resulted in an in-frame insertion of 19 new amino acids immediately behind the switch II region, including two potential threonine phosphorylation sites for casein kinase II and protein kinase C. Primers designed within and downstream of the inserted nucleotide sequence allowed isolation of a genomic clone with intronic consensus sequences demonstrating that the insertion corresponds to a novel, yet undescribed exon 3b. This Rac1 splice variant, designated Rac1b, was predominantly identified in skin and epithelial tissues from the intestinal tract. Most notably, the expression of rac1b versus rac1 was found to be elevated in colorectal tumors at various stages of neoplastic progression, as compared to their respective adjacent tissues. We suggest that the 19 amino acid-insertion following the switch II region may create a novel effector binding site in rac1b, and thus participate in signaling pathways related to the normal or neoplastic growth of the intestinal mucosa.     

Sequence variation in the I-like domain of the beta1 integrin subunit in human oral squamous cell carcinomas

We recently identified a heterozygous mutation in the beta1 integrin subunit of a squamous cell carcinoma (SCC) that maps to the I-like domain and activates ligand binding. To investigate the frequency of such mutations we screened 124 human oral SCCs. We identified six single nucleotide changes, all of which were also present in normal tissue, suggestive of polymorphisms. Two were in non-coding intronic sequences. Three were silent changes in exons. One caused a change in amino acid (A239V) that is unlikely to disturb integrin structure. We conclude that mutations in the beta1 I-like domain are uncommon in SCCs. However, population based studies of the polymorphisms we found may reveal an association with SCC development or prognosis.     

Detection of a hypersialylated beta1 integrin endogenously expressed in the human astrocytoma cell line A172

Gliomas are the most common deadly brain tumors. Human cerebral tumors express high level of alpha5beta1 integrins. As a potential new target, alpha5beta1 was investigated here in two human astrocytoma cell lines, A172 and U87MG. We found that a hypersialylated beta1 integrin was endogenously expressed in A172 cells. It forms heterodimers with alpha5 subunits, localizes at the cell membrane and allows adhesion to fibronectin. This form of beta1 integrin was only recognized by the 9EG7 anti-beta1 antibody and appeared devoid of other specific antibody epitopes (12G10, TS2/16 and mAb13 shown here to be N-glycosylation sensitive). Overexpression of the beta1 integrin subunit in A172 cells not only increased the hypersialylated form but also led to the appearance of a non-hypersialylated beta1 form also addressed to the cell surface. Compared to wild-type A172 cells, beta1-A172 cells showed increased adhesion to fibronectin and decreased sensitivity to SJ749, a non-peptidic alpha5beta1 antagonist. In addition, beta1-A172 cells exhibited increased matrix dependence for normal cell cycling. Collectively, the data add new evidence for the role of beta1 glycosylation/sialylation in the regulation of integrin functions.     

Transforming growth factor-beta1 increases cell migration and beta1 integrin up-regulation in human lung cancer cells

Transforming growth factor-beta1 (TGF-beta1) plays a crucial role in adhesion and migration of human cancer cells. Besides, integrins are the major adhesive molecules in mammalian cells. Here we found that TGF-beta1 increased the migration and cell surface expression of beta1 integrin in human lung cancer cells (A549 cells). TGF-beta1 stimulation increased phosphorylation of p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) and Ser(473) of Akt was determined. Besides, we performed that PI3K inhibitor (Ly294002) or Akt inhibitor suppressed the TGF-beta1-induced migration activities of A549 cells. Treatment of A549 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also repressed TGF-beta1-induced cells migration and beta1 integrins expression. In addition, treatment of A549 cells with TGF-beta1 induced IkappaB kinase alpha/beta (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the TGF-beta1-mediated increases in IKKalpha/beta, IkappaBalpha phosphorylation and p65 Ser(536) phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85alpha and Akt mutants also reduced the TGF-beta1-induced kappaB-luciferase activity. Taken together, our results suggest that TGF-beta1 acts through PI3K/Akt, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of beta1 integrins and contributing the migration of human lung cancer cells.     

Prognostic value of beta1 integrin expression in metastatic melanoma

The expression of integrin-type cell adhesion receptors is frequently changed in malignant transformation. Despite their important role in cancer cell behaviour, the value of integrins as prognostic markers is mostly unknown. We have examined the expression of beta1 integrins in 38 metastatic melanomas obtained from 27 patients treated with combined chemoimmunotherapy. On the basis of beta1 integrin expression, the melanoma samples were divided into two groups: beta1-negative tumours (<10% beta1 integrin immunostained cells) and beta1-positive tumours (with > or = 10% positive cells). Patients with beta1-positive tumours (n = 15) had significantly longer disease-free survival (median 38 versus 7 months, P < 0.0001) and overall survival (median 70 versus 23 months, P = 0.0001) evaluated after the diagnosis of primary disease compared with patients with beta1-negative metastases (n = 11). Moreover, the survival of the patients with beta1-positive tumours after the initiation of chemoimmunotherapy was significantly prolonged (median 18 versus 9 months, P = 0.017). The independent nature of beta1 integrin expression as a significant prognostic factor for survival after therapy was confirmed using Cox's multivariate analysis (P = 0.014). Our results indicate that the expression of beta1 integrins might have some major tumour growth regulatory role and can be used as a predictor for prognosis in patients with metastatic melanoma.     

Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread

Prostate cancer is a leading cause of cancer death in men. Risk prognostication, treatment stratification, and the development of rational therapeutic strategies lag because the molecular mechanisms underlying the initiation and progression from primary to metastatic disease are unknown. Multiple lines of evidence now suggest that KLF6 is a key prostate cancer tumor suppressor gene including loss and/or mutation in prostate cancer tumors and cell lines and decreased KLF6 expression levels in recurrent prostate cancer samples. Most recently, we identified a common KLF6 germ line single nucleotide polymorphism that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Here we show that although wild-type KLF6 (wtKLF6) acts as a classic tumor suppressor, the single nucleotide polymorphism-increased splice isoform, KLF6 SV1, displays a markedly opposite effect on cell proliferation, colony formation, and invasion. In addition, whereas wtKLF6 knockdown increases tumor growth in nude mice >2-fold, short interfering RNA-mediated KLF6 SV1 inhibition reduces growth by approximately 50% and decreases the expression of a number of growth- and angiogenesis-related proteins. Together, these findings begin to highlight a dynamic and functional antagonism between wtKLF6 and its splice variant KLF6 SV1 in tumor growth and dissemination.     

An osteopontin splice variant induces anchorage independence in human breast cancer cells

In malignant tumors, metastasis genes are typically deregulated by aberrant expression or splicing. Osteopontin is expressed at high levels by various cancers and contributes importantly to their invasive potential. In contrast, osteopontin derived from host cells induces cellular immunity and could bolster antitumor protection by cytotoxic T lymphocytes. Here we show that breast cancer cells express multiple splice variants of osteopontin. According to RT-PCR analysis of human breast tissue specimens, the splice variant osteopontin-c is a highly specific marker for transformed cells, which is not expressed in their surrounding normal tissue. The full-length form of osteopontin aggregates in the presence of physiologic amounts of calcium and, in this state, leads to enhanced cell adhesion. Ostensibly, this effect is inhibitory for tumor cell dissemination. The shortest splice variant, osteopontin-c, does not aggregate in the presence of calcium and enhances clone formation in soft agar. According to microarray analysis, osteopontin-c induces the expression of oxidoreductases, consistent with protection from anoikis during anchorage-independent growth. These studies define a third functional domain of osteopontin, beside the C-terminal CD44-binding site and the central integrin-binding site. They also provide evidence for a bifunctional character of osteopontin, with the soluble form supporting invasiveness and the aggregated form promoting adhesion.     

整合素α6β1增强人肝癌细胞侵袭性机制的探讨

目的 研究层粘连蛋白受体α６β１整合素在人肝癌细胞侵行为中的作用。方法 采用ｄｏｍｉｎａｎｔ ｎｅｇａｔｉｖｅ策略,以无功能的α６β４－ＴＲ代替细胞表面α６β１受体,从而消除了人肝癌细胞Ｂｅｌ－７０４２ α６β１受体的功能。用划痕法和Ｂｏｒｄｅｎ小室法分别检测细胞的迁移和侵袭,明胶酶谱分析法检测细胞基质金属蛋白ｍａｔｒｉｘ ｍｅｔａｌｌｏｐｒｏｖｔｅｉｎａｓｅｓ,ＭＭＰａ）的分泌。结果 消除α６β