Effect of KAI1/CD82 on the β1 integrin maturation in highly migratory carcinoma cells

doi:10.1016/j.bbrc.2007.05.159

Biochemical and Biophysical Research Communications

Volume 359, Issue 3, 3 August 2007, Pages 703-708

https://doi.org/10.1016/j.bbrc.2007.05.159 Get rights and content

Abstract

The KAI1/CD82 protein has been documented as the tumor metastasis suppressor in many types of human cancers. KAI1/CD82 regulates cell motility and invasiveness; however, the mechanism by which this occurs remains to be fully established. Several studies have shown that KAI1/CD82 modulates integrin-dependent signaling. It was suggested that KAI1/CD82 might function to attenuate the β1 integrin function of inducing cellular migration. A wound-healing and modified Boyden chamber assays were performed to investigate the mechanism of the KAI1/CD82-mediated inhibition of cell migration. It was found that the migratory ability of H1299/CD82 was inhibited. The immunoblotting and biotinylation assays revealed that H1299/CD82 showed significantly decreased expression of the mature form of β1, which was functional at the cell surface. It was confirmed that KAI1/CD82 regulates the maturation of the β1 integrin using CD82-specific si-RNA. These results support a model in which KAI1/CD82 attenuates the maturation of the β1 integrin precursor and thereby suppresses cell migration.

Section snippets

Materials and methods

Cell migration assay. The cell migration assay was performed using two methods: the wound-healing and the modified Boyden chamber assays. Each of the control and stable KAI1/CD82-transfectant cells was seeded in one well of a 24-well culture plate for the wound-healing assay. A wound was made in the confluent monolayer with Wide Bore Pipet Tips (Axygen, Union City, CA). The migration of the cells at the wound front was photographed after 3 days using an inverted microscope. The cell motility

KAI1/CD82 inhibits the migration of H1299 lung carcinoma cells

The KAI1/CD82 transfectants were generated as described by Jee et al. [26]. Two of the stable KAI1/CD82 transfectant clones (S1 and S2) and one of the control transfectants (C1) were selected for additional functional assays. We performed using a wound with a plastic micropipette tip and an in vitro cell migration assay using modified Boyden chambers in order to investigate the effect of the KAI1/CD82-mediated inhibition of cellular motility. The wound repairs were significantly inhibited in

Discussion

KAI1/CD82, which is one of the tetraspanins, is a known tumor suppressor gene according to many reports. Various ideas have been put forth to explain the mechanism of the KAI1/CD82-mediated suppression of cell migration [3]. However, none of the explanations fully suffice to explain the details of this mechanism. To examine the attenuation of cell migration by KAI1/CD82, we first constructed a KAI1/CD82-overexpressed H1299 lung carcinoma cell line (p53 null).

In the present report, it was found

Acknowledgments

This work was supported by Grant (01-PJ3-PG6-01GN07-0004), Good Health R&D Project, Ministry of Health Welfare, Republic of Korea and by a grant of the Korea Health 21 R&D Project, Ministry of Health Welfare, Republic of Korea, (00-PJ3-PG6-GN02-0002).

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To evaluate the effect of imatinib mesylate on KAI1 gene expression and apoptosis properties in human gastric carcinoma AGS cell line.
Cell viability was assessed by MTT assay and quantitative real time PCR method was applied for investigation of Bax, Bcl-2, and KAI1 gene expression in AGS cells. The quantity of KAI1, Bax, and Bcl-2 compared to GAPDH gene expressions were examined using the formula 2^-ΔΔCt. Furthermore, cell apoptosis/necrosis was carried out by annexin V/PI staining and quantified with flow cytometry after treatment with imatinib.
Imatinib mesylate was showed to have a dose-dependent toxicity effect against AGS cells. KAI1/GAPDH gene expression ratios were 1.07 ± 0.02 (P > 0.05), 1.68 ± 0.19 (P > 0.05), 3.60 ± 0.55 (P < 0.05), 6.54 ± 0.27 (P < 0.001) for 20, 50, 80 and 100 μmol/L of imatinib concentrations. The mRNA levels of Bax detected by real-time PCR after treatment with imatinib mesylate were significantly increased. Also, the number of apoptotic cells was increased from 3.72% (statistically significant; P < 0.05) in untreated AGS cells to 21.72%, 83.04% and 85.80%, respectively, following treatment with 20, 40, and 60 μmol/L imatinib mesylate.
The results suggest that imatinib mesylate can induce apoptosis pathway in a dose-dependent mode and might modulate metastasis by up regulating KAI1 gene expression in human gastric carcinoma AGS cell line.
Evaluation of imatinib mesylate (Gleevec) on KAI1/CD82 gene expression in breast cancer MCF-7 cells using quantitative real-time PCR
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Citation Excerpt :
Currently, metastasis suppressor genes are found to play an important role in regulation of cell invasion and metastasis signaling. KAI1 (CD82), a tumor metastasis suppressor gene, has wide-spectrum roles in targeting tumor metastasis [11,12]. Many studies have confirmed that the KAI1/CD82 gene inhibits metastasis in various types of cancers such as endometrial, pancreatic, bladder, breast, ovarian, cervical, lung, hepatic, colorectal and gastric cancer [13–15].
To evaluate the effect of imatinib mesylate on cell viability, anti cancer effect through modulation of KAI1/CD82 gene expression in breast cancer MCF-7 cell line.
The effects of imatinib mesylate on cell viability in MCF-7 cell line were assessed using MTT assay and IC₅₀ value was determined. GAPDH and KAI1/CD82 were selected as reference and target genes, respectively. Quantitative real time PCR technique was applied for investigation of KAI1/CD82 gene expression in human breast cancer MCF-7 cells. Subsequently, the quantity of KAI1 compared to GAPDH gene expressions were analyzed using the formula; 2^−ΔΔCt.
Imatinib was showed to have a dose-dependent inhibitory effect on the viability of MCF-7 cells. CD82/GAPDH gene expression ratios were 1.322 ± 0.030 (P > 0.05), 2.052 ± 0.200 (P < 0.05), 2.151 ± 0.270 (P < 0.05) for 10, 20 and 40 μmol/L of imatinib concentrations.
Based on the present data, imatinib mesylate might modulate metastasis by up-regulating KAI1/CD82 gene expression in human breast MCF-7 cancer cell line.
EGFR over-expression in non-small cell lung cancers harboring EGFR mutations is associated with marked down-regulation of CD82
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Moreover, CD82 expression is correlated with a good prognosis in non-small cell lung cancer (NSCLC) patients [19]. CD82's anti-metastatic ability is attributed to its interaction with integrin molecules [20–23]. CD82 interacts with Duffy antigen receptor for chemokines (DARC) on endothelial cells and causes the growth inhibition of the CD82-expressing tumor cells [24].
Epidermal growth factor receptor (EGFR) gene mutations are strongly associated with lung adenocarcinoma and favorable response to EGFR tyrosine kinase inhibitor. The mutated EGFR proteins (EGFRs) are hyper-phosphorylated and refractory to receptor down-regulation. To address the discrepancy between hyper-phosphorylation and lack of down-regulation of mutant EGFRs, we have examined the expression of EGFR negative regulators in non-small cell lung cancer (NSCLC) cell lines. We found that NSCLC cell lines expressing mutant EGFRs often had low expression of various negative regulators for EGFR. Among them, tumor suppressor CD82 was up-regulated by wild type (WT) EGFR but down-regulated by mutant EGFRs. Reconstitution of CD82 exerted stronger suppressive effects on mutant EGFRs than on WT EGFR. Active exportation of CD82 through the exosome was one of the mechanisms involved in achieving the overall CD82 down-regulation in mutant EGFR-expressing lung cancer cell lines. Over-expression of mutant EGFR protein frequently occurred in the lung cancer tissues of mutant EGFR-transgenic mice and also associated with CD82 down-regulation. Immunoblot analyses on the tumor tissues from 23 lung adenocarcinoma patients (12 with WT EGFR, and 11 with mutant EGFRs) also identified significantly stronger down-regulation of CD82 in tumors with mutant EGFRs than WT. Our data indicate that CD82 down-regulation could be a critical step involved in the EGFR over-expression and the stronger tumorigenic activity triggered by EGFR mutations. Up-regulation of the CD82 level may become a promising new treatment strategy for lung adenocarcinoma.
Alternative splicing of KAI1 abrogates its tumor-suppressive effects on integrin αvβ3-mediated ovarian cancer biology
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Decrease or loss of KAI1 has also been observed in many metastatic tumor cell lines. Its reintroduction into tumor cells inhibited cancer cell migration and invasion in vitro as well as suppression of cancer metastasis in animal models [10,22,25–32]. Moreover, a KAI1 splice variant (KAI1-SP) lacking exon 7 has been detected in metastatic tissues of gastric cancer patients correlating with poor prognosis.
Loss or downregulation of the tumor-suppressor KAI1 correlates with poor cancer patient prognosis. KAI1 functions by interacting with other proteins, including integrin cell adhesion and signaling receptors. We previously showed that KAI1 physically and functionally crosstalks with the tumor-biologically relevant integrin αvβ3, thereby suppressing ovarian cancer cell migration and proliferation. Interestingly, in metastases, a KAI1 splice variant had been identified, indicating poor patient prognosis. Thus, we here characterized differential effects of the two KAI1 proteins upon their cellular restoration. Opposite to KAI1, KAI1-splice reduced αvβ3-mediated cell adhesion, thereby inducing cell migration. This was accompanied by elevated αvβ3 levels and drastically elevated focal adhesion kinase activation, however, without any obvious colocalization with αvβ3, as observed for KAI1. Moreover, codistribution of KAI1 with the cell/cell-adhesion molecule E-cadherin was abrogated in KAI1-splice. Whereas KAI1 diminished cell proliferative activity, KAI1-splice prominently enhanced cell proliferation concomitant with elevated transcription and cell-surface expression of the epidermal growth factor receptor. Thus KAI1-splice does not only counteract the tumor-suppressive actions of KAI1, but – beyond that – promotes αvβ3-mediated biological functions in favor of tumor progression and metastasis.
Dissecting the diverse functions of the metastasis suppressor CD82/KAI1
2011, FEBS Letters
Citation Excerpt :
CD82 does not alter the distribution of E-cadherin in these cells; instead, CD82 stabilizes E-cadherin/β-catenin complex formation, which can inhibit cancer cell dissemination from the primary tumor. In vitro studies show that overexpression of CD82 inhibits cell motility and invasion [11,14,15,21–27]. The association of CD82 with α6 integrin results in decreased laminin adhesion and migration [28].
The recent identification of metastasis suppressor genes, the products of which inhibit metastasis but not primary tumor growth, distinguishes oncogenic transformation and tumor suppression from a hallmark of malignancy, the ability of cancer cells to invade sites distant from the primary tumor. The metastasis suppressor CD82/KAI1 is a member of the tetraspanin superfamily of glycoproteins. CD82 suppresses metastasis by multiple mechanisms including inhibition of cell motility and invasion, promotion of cell polarity as well as induction of senescence and apoptosis in response to extracellular stimuli. A common feature of these diverse effects is CD82 regulation of membrane organization as well as protein trafficking and interactions, which affects cellular signaling and intercellular communication.
Tumor suppressor KAI1 affects integrin αvβ3-mediated ovarian cancer cell adhesion, motility, and proliferation
2009, Experimental Cell Research
The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin αvβ3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin αvβ3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with β1-integrins, also colocalizes with integrin αvβ3. Functionally, elevated KAI1 levels drastically increased integrin αvβ3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin αvβ3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin αvβ3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.

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View full text

Effect of KAI1/CD82 on the β1 integrin maturation in highly migratory carcinoma cells

Abstract

Section snippets

Materials and methods

KAI1/CD82 inhibits the migration of H1299 lung carcinoma cells

Discussion

Acknowledgments

Genomics

Cancer Lett.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Cell

J. Biol. Chem.

A new superfamily of lymphoid and melanoma cell proteins with extensive homology to Schistosoma mansoni antigen Sm23

Eur. J. Immunol.

KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2

Science

Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer

Cancer Res.

Down-regulation of the KAI1 metastasis suppressor gene during the progression of human prostatic cancer infrequently involves gene mutation or allelic loss

Cancer Res.

KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases

Cancer Res.