Expression profiling of integrins in lung cancer cells

doi:10.1016/j.prp.2009.07.005

Pathology - Research and Practice

Volume 205, Issue 12, 15 December 2009, Pages 847-853

https://doi.org/10.1016/j.prp.2009.07.005 Get rights and content

Abstract

Integrins are heterodimeric transmembrane receptors consisting of 18 α and 8 β subunits. Heterodimer composition of α and β subunits has a potential for determining tumor subtypes of human lung cancer. The purpose of this study was to investigate the expression profile of integrins in lung cancer cells. Expression profiling of integrins in a panel of lung cancer cell line, including A549 (adenocarcinoma, ADC), Calu-1 (squamous carcinoma, SCC), H1650 (bronchioloalveolar carcinoma, BAC) and DMS-53 (small cell lung cancer, SCLC), was analyzed by cDNA microarrays, restriction analysis of gene expression (RAGE) and flow cytometry. Seventy-nine lung cancer specimens were used to further validate the data from cell lines using immunohistochemistry. Integrins are obviously expressed in a cell type-specific manner, such as α3 in A549, Calu-1 and H1650 except in DMS53, α4 in H1650, α5 and β1 in all cell lines. The integrins detected with cDNA microarrays were all unequivocally detected with RAGE and by flow cytometry at the protein level. In all lung cancer specimens, α3 integrin was strongly expressed in ADC, SCC and BAC, but was infrequent in SCLC. α4 integrin was solely expressed in BAC. α5 and β1 integrins were expressed in all four histological types of lung cancer specimens. Integrin α3 and α4 may be useful as diagnostic markers for adenocarcinoma, squamous cell carcinoma and bronchioloalveolar carcinoma. RAGE is a promising technique for studying the expression profiles of genes, such as integrins in cancer cells.

Introduction

Integrins are well-known mediators in the attachment of cells to each other and to their surrounding extracellular matrix (ECM). Owing to their cell membrane localization and their dual function, integrins not only preserve tissue integrity by connecting the intracellular actin cytoskeleton with the ECM but also mediate signals for the control of diverse cell functions, including survival, proliferation, differentiation, adhesion and migration [1], [2], [3]. So far, 18 α and 8 β subunits of integrins have been identified, which covalently bind to form 24 different transmembrane heterodimers [4]. Each heterodimer is composed of a single α and a single β subunit, and both subunits participate in ligand recognition. Specificity of an integrin in interacting with extracellular ligands is determined by heterodimer composition of α and β subunits. For example, α3β1 integrin predominantly recognizes a receptor for laminin 5, whereas αVβ3 is reported mainly as a ligand of fibronectin and vitronectin and α2β1 as a collagen receptor [4], [5], [6], [7].

Lung cancer is one of the most common malignant tumors with more aggressive and high metastatic potential. Evidence now suggests that these features may be due to important links between the cancer cells and the ECM in their local environment [8], [9]. Low activation state of integrins on cancer cells is thought to account for the highly metastatic and motile behavior. Investigations have shown that down-regulation of α3 integrin subunit may contribute to the enhanced tumorigenicity of c-myc-overexpressing small cell lung carcinoma, while the loss of αv expression is correlated with the recurrence in node-negative lung carcinoma [8], [9]. Increased expression of α1β1 and α2β1 integrins has been shown to be positively correlated with the increased metastatic ability in squamous cell carcinoma [10]. Reduced α3β1 integrin expression is probably related to the increased aggressiveness and a poor factor of prognosis in patients either with small cell lung cancer (SCLC) or non-small cell lung cancer [10], [11]. For β1 integrin, it has also been reported that its mediated adhesion of SCLC cells to ECM proteins promotes tyrosine phosphorylation. This blocks chemotherapy-induced activation of the caspase pathway and, hence, apoptosis [13]. Thus, it appears that integrins are potential targets for specific diagnosis and therapy of lung cancer. It will then be necessary to delineate the expression profile of integrins expressed on a particular type of cancer in order to identify a unique target.

In this study, we investigated for the first time the profiling expression of integrins in a panel of lung cancer cell lines using cDNA microarray analysis. Furthermore, we developed a novel method for restriction analysis of gene expression (RAGE) to confirm the results from cDNA microarray analysis.

Section snippets

Cell cultures

Cell lines of human lung cancers, including A549 (adenocarcinoma, ADC), Calu-1 (squamous carcinoma, SCC), H1650 (bronchioloalveolar carcinoma, BAC) and DMS-53 (SCLC), were obtained from American Tissue Culture Collection (ATCC). Cells were grown in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO).

RNA extraction

RNAs were extracted from the cell lines using standard techniques. The total RNA was isolated by TRIzol (Invitrogen) according to

Integrin profiling analysis with cDNA microarrays

We analyzed integrin expression in a panel of lung cancer cell lines including A549, Calu-1, H1650 and DMS-53 using commercially available cDNA microarrays for adhesion and extracellular matrix molecules. A typical array of expression of 112 genes encoding for these and house-keeping molecules is shown in Fig. 1. As expected, the house-keeping genes were expressed at relatively high levels. The expression profile of integrin by microarray analysis is summarized in Table 1. Integrins are

Discussion

Integrins represent very attractive molecular targets for the diagnosis and therapy of lung cancer because of their contribution to cancer progression by mediating cellular functions such as tumor cell proliferation, invasion, metastatsis and the development of resistance to chemotherapy [8], [9], [14], [15], [16], [17], [18]. Therefore, identification of the integrin expression profiles of lung cancer cells is the first step to identify the appropriate candidate molecules for such purposes.

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ORIGINAL ARTICLEExpression profiling of integrins in lung cancer cells

Abstract

Introduction

Section snippets

Cell cultures

RNA extraction

Integrin profiling analysis with cDNA microarrays

Discussion

Methods

J. Biol. Chem.

Lung Cancer

Surg. Oncol. Clin. N. Am.

Lancet Oncol.

Semin. Cancer Biol.

Hum. Pathol.

The integrin–actin connection, an eternal love affair

Eur. Mol. Biol. Org. J.

Positional control of cell fate through joint integrin/receptor protein kinase signaling

Annu. Rev. Cell Dev. Biol.

Integrin-mediated signal transduction pathways

Histol. Histopathol.

Integrins: bidirectional, allosteric signaling machines

Cell

ORIGINAL ARTICLE
Expression profiling of integrins in lung cancer cells