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Molecular Biology and Genetics |
Molecular Pathology Program [A. I. J., P. F., M. S-C.], Genomics Unit [O. D.], and Microarray Analysis Unit [A. D.], Spanish National Cancer Center, 28029 Madrid, Spain
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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15 times more likely than the general population). Malignant neoplasms may occur in a variety of tissues, including colon, small intestine, breast, cervix, ovary, pancreas, and lung (2)
. PJS is caused by germ-line mutations in the LKB1 gene, also known as STK11 (3
, 4)
, which encodes a conservative and ubiquitously expressed serine/threonine kinase the substrates of which have yet to be characterized. Human LKB1 shows strong homology with the cytoplasmic serine/threonine kinase of several organisms (5)
, including mouse (6)
. LKB1 is a 436-amino-acid protein with a kinase domain (residues 50–337) and a putative COOH-terminal regulatory domain. Moreover, the protein contains two potential nuclear localization signals located between amino acids 38–43 and 81–84. Additionally, cytoplasmic and mitochondrial localization signals have been reported (7
, 8) . Several authors have observed that LKB1 overexpression in different tumor cell lines induced cell growth suppression, in some cases by blocking the cell cycle in G1 (8
, 9)
. However, the intrinsic mechanism by which LKB1 activity is regulated in cells and how it leads to the suppression of cell growth is still unknown. It has been proposed that growth suppression by LKB1 is mediated through p21 in a p53-dependent mechanism (7)
. In addition, it has been observed that LKB1 binds to brahma-related gene 1 protein (BRG1) and this interaction is required for BRG1-induced growth arrest (10)
. Similar to what happens in the PJS, Lkb1 heterozygous knockout mice show gastrointestinal hamartomatous polyposis and frequent hepatocellular carcinomas (11
, 12)
. Interestingly, the hamartomas, but not the malignant tumors, arising in Lkb1+/- mice had no inactivation of the remaining Lkb1 wild-type allele, suggesting a haploinsufficient mechanism in the hamartoma formation. The Lkb1 gene knockout mouse is lethal in embryos and causes multiple developmental defects, including aberrant vessel formation in the yolk sac and in the placenta, coupled with a marked dysregulation of vascular endothelial growth factor expression (13)
. Occasional LKB1 gene mutations or promoter hypermethylation have been reported in some sporadic tumors from the pancreas, breast, and cervix, among others (14, 15, 16) . In LACs, losses of heterozygosity in chromosome 19p are very common (17) , and we have recently shown that LKB1 is the gene target because at least one-third of LACs harbor inactivating LKB1 mutations (18) . Despite its relevance and, partly because LKB1 was not cloned until recently (3 , 4) , its biological function has not been completely elucidated nor have its upstream or downstream components been identified. This information is critical because it will allow us to understand whether LKB1 belongs to any of the already known cancer pathways or, alternatively, to identify new cancer cascades, relevant to the design of specific cancer therapeutic targets.
To investigate further the biological role of LKB1 and its relevance in lung cancer development, we expressed wild-type and mutant LKB1 ectopically in A549 lung cancer cells, which are LKB1-deficient (18) , and analyzed changes in cell growth and gene expression patterns by the use of cDNA microarrays.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Western Blotting.
Cells were scraped from the dishes into lysis buffer at various times after transfection. Eight µg of total protein was separated by SDS-PAGE, as described previously (19)
, and blotted with goat anti-LKB1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-actin (1:500; Santa Cruz Biotechnology), mouse anti-hemagglutinin (1:1000; Babco, Berkeley, CA), rabbit anti-PTEN (1:300; Zymed, San Francisco, CA) or mouse anti-cyclin A (1:300; Novocastra, Newcastle, United Kingdom). The secondary antibody antigoat-IgG:HRP, antirabbit-IgG:HRP or anti-mouse-IgG:HRP (Santa Cruz Biotechnology) was added to give a final dilution of 1:5000.
RT-PCR.
Total RNA was collected using the Quiagen RNase. RT was performed (Reverse Transcription System, Promega) with 1 µg of Rnasy following the manufacturer’s protocol. The following primers were used to amplify two different regions of the LKB1-cDNA: Primer set 1, reverse 5'-CACATCCACCAGCTGGATG-3' and forward 5'-TCCACCAGGTCATCTACC-3' (the bold base indicates complementarily with the wild-type sequence, not for the mutation present in A549); Primer set 2, reverse 5'-CTCTGAGCCGTTCATACACA-3' and forward 5'-GTTCATCCACCGCATCGAC-3'. cDNA was subjected to PCR, and products were analyzed by electrophoresis on a 2% agarose gel.
Semiquantitative RT-PCR.
A549 cells were transiently transfected with full-length cDNA of LKB1. Total RNA was isolated and subjected to RT as described above. Equal amounts of cDNA were PCR-amplified for 20–35 cycles with specific primers at the following cycling conditions: denaturing at 94°, annealing at 58°C, and extension at 72°C. As an internal control, we amplified ß-actin. The sequence of the primers used for RT-PCR were as follows: for PIK3R4, forward 5'-GATGATGGAAAATGCTGAATG-3' and reverse 5'-ACCGTCCTCCTTCTGATCTAG-3'; for STK17B, forward 5'-CAGCCTGTGTTTACCTGAGT-3' and reverse 5'-TCCACATATCTGTTGCTGTGG-3'; for Vav3, forward 5'-CTGGTGAACAAGGGACACTC-3' and reverse 5-'TGCTTGCAATCTTTCCATTG-3'; and for GADD45A, forward 5'-GACCGAAAGGATGGATAAGG-3' and reverse, 5'-CCATTGATCCATGTAGCG-3'. Products were analyzed by electrophoresis on 2% agarose gels.
Immunocytochemical Identification of LKB1/STK11 Protein.
For subcellular localization of LKB1 protein, we performed an immunocytochemistry assay. The cells were fixed with acetone/methanol (1:1) for 5 min and kept frozen at -20°C until processing. Immunofluorescence was performed as described previously (20)
, following the manufacturer’s protocol. Cells were transfected with pCI-LKB1 and stained with DAPI and anti-HA. Labeling was revealed with anti-IgG-Alexa594 (red) for HA. Fluorescence was analyzed by confocal microscopy, and the colocalization of both markers was electronically evaluated. All of the preparations were mounted with Prolong antifade medium. Coverslides were visualized using an MRC-1024 confocal microscope (Bio-Rad, Hercules, CA).
Preparations of Labeled cDNA and Hybridization of Microarray.
A549 cells were plated on a 75-cm2 flask at 70% confluence and transfected with pCINeo alone, LKB1, or LKB1-SL8 sequence. At different times (from 0 to 72 h) after transfection, the cells were washed with PBS, and total RNA was obtained and treated with 1 unit of DNase I (Quiagen). Thirty-five µg of total RNA were used for cDNA microarray analysis. Fluorescent-labeled cDNA was synthesized and hybridized to the CNIO OncoChip as described previously (21)
. The CNIO OncoChip is a cDNA microarray that has been specially designed for looking at genes involved in cancer and includes a core of 2489 cancer-relevant genes in addition to genes involved in drug-response, tissue-specific genes, and control genes. There are a total of 6386 genes represented by 7237 clones. It was prepared as described previously (21)
. Slides were scanned for Cy3 and Cy5 fluorescence using Scanarray 5000 XL (GSI Lumonics Kanata, Ontario, Canada) and quantified using the Quantarray (GSI Lumonics) and/or GenePix Pro 4.0 programs (Axon instruments Inc., Union City, CA). The A549 wild-type (Cy3) versus transfected A549 (Cy5) hybridizations were performed in duplicate using different target preparations.
Data Analysis.
Fluorescence intensity measurements from each array element were compared with local background and background subtraction was performed. To normalize the data, the Cy5:Cy3 ratio was adjusted to a normalized factor equal to the median ratio value of all spots in the array. In addition, spots with background-subtracted signal intensities lower than 500 fluorescence units (sum of the two channels) were excluded from the analysis, and bad spots or areas of the array with obvious defects were manually flagged. The Cy5:Cy3 ratios of the duplicated spots of the array were averaged. For the analysis, all of the expressed sequence tags and genes of unknown function were excluded. Biological functions were assigned using the GENECARDS database.4
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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To confirm that A549 transfected cells expressed exogenous LKB1, we performed RT-PCR and Western blotting experiments (Fig. 1, A and B)
. Exogenous wild-type and mutant LKB1 protein were detected in A549 cells transfected with LKB1 and LKB1/SL8, respectively, whereas no LKB1 protein expression was detectable in the parental cells (Fig. 1B)
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. These observations are in agreement with previous data concerning overexpression of LKB1 in other cancer cell lines (8
, 10)
and with the reported potential nuclear localization signals supporting a role for LKB1 protein in the nuclear compartment.
To investigate the effect of ectopic LKB1 expression on cell growth we selected transfected cells using the Neomicin/G418 resistance gene contained in the pCI/Neo expression vector. Sixteen days after selection with G418, there was a clear reduction in the number of colonies expressing wild-type LKB1 compared with those expressing LKB1/SL8 or with those carrying the empty vector (Fig. 1, D and E)
. No reduction in the number of colonies was observed after overexpression of LKB1 in the H522 lung cancer cells (data not shown), which carries endogenous wild-type LKB1 (18)
. Thus, reintroduction of wild-type, but not mutant LKB1, in LKB1-deficient lung cancer cells clearly triggered significant cell-growth suppression and further implies that LKB1 is a critical tumor suppressor gene in LAC development. This is consistent with previous observations after reintroduction of LKB1 into the G361 melanoma and into the HeLa S3 cell lines (8)
, both deficient in wild-type-LKB1 protein.
cDNA Microarray Analysis of A549 Cells Expressing Ectopic LKB1 Reveals an Expression Profile Consistent with Growth Suppression and Apoptosis.
To investigate the differences in gene expression after reintroduction of wild-type LKB1 in A549 cells, we performed a profiling analysis using cDNA microarrays. Total RNA was isolated from parental A549 cells and from cells transfected with wild-type LKB1 at different times (0, 6, 16, 24, 48, and 72 h). RNA was obtained also from cells transfected with the mutant LKB1/SL8 and with the empty vector (24 h after transfection). cDNA from parental A549 cells and from transfected cells was labeled with Cy3 (green) and Cy5 (red), respectively, and hybridized to the OncoChip v.2 microarray. Differences in gene expression patterns between parental and transfected cells were then compared. Genes were defined as up-regulated or down-regulated if they matched the following criteria: (a) their expression varied only in cells transfected with wild-type LKB1 (we discarded those genes whose expression varied also in cells transfected with empty vector or with LKB1/SL8); (b) their expression had a ratio of signal intensity green:red >3 or <0.3 for induced and repressed genes, respectively; and (c) their expression appeared two or more different times. By using these criteria we detected 100 genes showing differential patterns of gene expression in LKB1-transfected cells compared with parental cells. A total of 69 genes were up-regulated, whereas 31 showed severe down-regulation. To determine the reliability of our results, we repeated the microarray hybridization using RNA from an independent LKB1-transfection (at 24 h). We observed a correlation coefficient of 0.76 in patterns of gene expression. Moreover, and as anticipated, expression profiles of cells at 0 and 72 h after LKB1 transfection were very similar to those of the parental cells because they either did not express exogenous LKB1 (0 h) or the percentage of transient LKB1 expression was already very low (3% of positive ß-galactosidase cells at 72 h).
As expected, LKB1 expression increased during the course of the experiments after transfection (Table 1
), whereas LKB1 cDNA levels remained unchanged in cells carrying the empty vector (Cy5:Cy3 = 0.94). Genes showing changes in gene expression were classified into different functional categories (Table 1)
. As can be observed, many of these genes show an obvious connection. The global patterns of gene expression observed after restoring LKB1 wild-type expression in A549 cells showed a marked deregulation of transcripts that control cell growth, an up-regulation of transcripts that promote apoptosis, and a decrease of molecules involved in cell motility and adhesion. Because we selected those genes whose expression vary only in cells transfected with wild-type LKB1 (not those transfected with empty vector or with mutant LKB1), the effects observed are unlikely to be a mere consequence of cellular stresses. However, it should be noted that the use of other microarray systems containing larger number of genes coupled with a lower stringent criteria for gene selection may increase the list of deregulated genes in A549 cells after ectopic expression of LKB1. We corroborated the expression changes either by semiquantitative RT-PCR or Western blotting, in all seven targets selected for validation (Fig. 2)
, indicating a very high confidence for our cDNA microarrays analysis.
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provides a schematic representation of some relevant genes showing altered expression in A549 cells after reintroduction of wild-type LKB1 and their putative functional consequences. Among the characteristic changes in gene expression, we observed an up-regulation of GADD45A/B and down-regulation of TOP2A, which strongly implies that cell cycle arrest is induced at G2-M, probably because ectopic LKB1 triggers a p53-mediated response. This is possible because p53 gene is wild type in A549 cells (22)
. Tianen et al. (9)
observed a G1 arrest after restoring LKB1 activity in G361 melanoma cells (because of the increased expression of p21) that was also mediated by p53. We looked specifically for the p21 transcript in our databases and found an increase in p21 expression, 6 h after LKB1 reintroduction, that clearly diminished with time (median of ratios Cy5:Cy3 at different times after transfection: 0.95 at 0 h; 2.8 at 6 h; 1.7 at 16 h; 1.4 at 24 h; 1.3 at 48 h; and 0.85 at 72 h; and, in A549 cells transfected with empty vector, 0.9). Other genes related with G1-S-phase cell cycle arrest may remain undetectable under our highly stringent selection criteria. Thus, it is likely that G1-S-phase cell cycle arrest also takes place in A549 cells after restoring LKB1 wild-type activity. Karuman et al. (7)
demonstrated that LKB1 physically associates with p53 and regulates specific p53-dependent apoptosis pathways. In agreement with this, we detected an up-regulation of SMAC/DIABLO, NOD1 (the same family as Apaf-1; contains a caspase recruitment domain, CARD), STK17B/DRAK, and TNF-family members, indicating that wild-type LKB1 triggers an apoptotic response in A549 cells. The cell cycle arrest and apoptosis responses observed after LKB1 overexpression occurred in cells with wild-type p53 gene (e.g., A549 and G361 cells), and it is reasonable to conclude that wild-type p53 is needed for LKB1 biological function. However, previous observations have shown that p53 and LKB1 gene mutations often coexist in lung primary tumors (18)
, indicating that the LKB1 and p53 biochemical functions are not functionally equivalent. It is also possible that the timing of the mutations are also critical and when LKB1 is inactivated first, subsequent p53 mutations confer additional advantage to the loss of LKB1 activity.
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Interestingly, reintroduction of wild-type LKB1 into A549 cells triggered a clear up-regulation of the PTEN expression, which was also confirmed at the protein level (Fig. 2B)
, indicating a blockage of the PI3K signaling. PTEN increased expression was accompanied by down-regulation of PIK3R4, an adaptor protein for the human PI3K protein (24)
, and vav3 whose down-regulation attenuates phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation (25)
. Fig. 3
depicts a schematic representation of the most representative changes in gene expression and their biological roles. These results are intriguing because some researchers have speculated about PTEN and LKB1 proteins acting in the same biochemical pathway (5)
. Several observations support this hypothesis: (a) germ-line mutations at the PTEN gene in humans lead to Cowden's disease, a type of multiple hamartomatous polyposis syndrome with high predisposition to cancer, similar to the PJS; (b) Pten-deficient mice are embryonic lethal and have some common features with Lkb1-/- mice; and (c) the expression patterns of the Lkb1 and Pten genes overlap significantly (26)
. Among the genes differentially expressed in our cDNA microarray system after LKB1 reintroduction, there was a remarkable down-regulation of transcripts involved in cell adhesion and motility, including ITGB3BP, an integrin family member. Several indications link PTEN with cell migration, and it has been shown that PTEN protein can reduce phosphorylation of focal adhesion kinase (FAK), which is involved in integrin-induced migration (27)
. While we were preparing this article, Bardeesy et al. (28)
reported the transcriptome of mouse embryonic fibroblast from Lkb1-/-mice showing a remarkable increase in factors linked to extracellular matrix remodeling and cell adhesion, which strongly supports our observations.
In contrast with our observations, it has recently been demonstrated that PTEN or phospho-Akt expression in polyps from Lkb1+/- mice is no different from that in the normal stomach of unaffected mice (29)
. Instead, we observed up-regulation of 
-Cox-2 protein, mediated by the Ras/Raf-1/MEK/ERK signal transduction pathway. The discrepancy with our observations may reflect the intrinsic genetic characteristic of the A549 cell line, which has a constantly activated RAS/RAF-1/MEK/ERK pathway as consequence of KRAS gene mutation (23)
.
Finally, we cannot rule out the possibility that PTEN up-regulation after LKB1 overexpression is due caused by p53 activation. A bidirectional connection between p53 and PTEN has previously been observed: p53 protein is able to induce PTEN gene expression (30) , whereas PTEN protein inhibits the PI3K/Akt signaling that promotes translocation of MDM2 to the nucleus, thus protecting p53 from degradation (31) .
In summary, we have demonstrated the growth suppression ability of ectopic LKB1 in lung cancer cell lines that are LKB1 deficient, further highlighting the relevance of LKB1 in lung cancer development. In addition, we have identified genes the products of which may be involved in the biological function of LKB1.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a grant from the Spanish Ministerio de Ciencia y Tecnología (SAF2002-01595). A. I. J. is supported by a fellowship from the Spanish National Cancer Center (CNIO). M. S-C. is supported by the Ramon y Cajal Program, Ministerio de Ciencia y Tecnología, Spain. 
2 To whom requests for reprints should be addressed, at Molecular Pathology Program, Spanish National Cancer Center. C/Melchor Fernández Almagro, 3, 28029 Madrid, Spain. E-mail: msanchez{at}cnio.es
3 The abbreviations used are: PJS, Peutz-Jeghers syndrome; LAC, lung adenocarcinoma; HRP, horseradish peroxidase; RT, reverse transcription; DAPI, 4',6-diamidino-2-phenylindole; CNIO, Spanish National Cancer Center; PI3K, phosphatidylinositol 3'-kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein (MAP)/ERK kinase.
4 Internet address: http://bioinformatics.weizmann.ac.il/cards.
Received 10/ 2/02. Accepted 1/15/03.
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in cutaneous T-cell lymphoma. Am. J. Pathol., 161: 1825-1837, 2002.This article has been cited by other articles:
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  P. Song, Z. Xie, Y. Wu, J. Xu, Y. Dong, and M.-H. Zou Protein Kinase C{zeta}-dependent LKB1 Serine 428 Phosphorylation Increases LKB1 Nucleus Export and Apoptosis in Endothelial Cells J. Biol. Chem., May 2, 2008; 283(18): 12446 - 12455. [Abstract] [Full Text] [PDF]
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A. Tzatsos and P. N. Tsichlis Energy Depletion Inhibits Phosphatidylinositol 3-Kinase/Akt Signaling and Induces Apoptosis via AMP-activated Protein Kinase-dependent Phosphorylation of IRS-1 at Ser-794 J. Biol. Chem., June 22, 2007; 282(25): 18069 - 18082. [Abstract] [Full Text] [PDF]
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D. Wang and M. You Five Loci, SLT1 to SLT5, Controlling the Susceptibility to Spontaneously Occurring Lung Cancer in Mice Cancer Res., September 15, 2005; 65(18): 8158 - 8165. [Abstract] [Full Text] [PDF]
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C. Forcet, S. Etienne-Manneville, H. Gaude, L. Fournier, S. Debilly, M. Salmi, A. Baas, S. Olschwang, H. Clevers, and M. Billaud Functional analysis of Peutz-Jeghers mutations reveals that the LKB1 C-terminal region exerts a crucial role in regulating both the AMPK pathway and the cell polarity Hum. Mol. Genet., May 15, 2005; 14(10): 1283 - 1292. [Abstract] [Full Text] [PDF]
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