Retraction: Oncogenic activity of Cdc6 through repression of the INK4/ARF locus.

This corrects the article DOI: 10.1038/nature04585.

Oncogenic activity of Cdc6 through repression of the INK4/ARF locus.

The INK4/ARF locus encodes three tumour suppressors (p15(INK4b), ARF and p16(INK4a)) and is among the most frequently inactivated loci in human cancer. However, little is known about the mechanisms that govern the expression of this locus. Here we have identified a putative DNA replication origin at the INK4/ARF locus that assembles a multiprotein complex containing Cdc6, Orc2 and MCMs, and that coincides with a conserved noncoding DNA element (regulatory domain RD(INK4/ARF)). Targeted and localized RNA-interference-induced heterochromatinization of RD(INK4/ARF) results in transcriptional repression of the locus, revealing that RD(INK4/ARF) is a relevant transcriptional regulatory element. Cdc6 is overexpressed in human cancers, where it might have roles in addition to DNA replication. We have found that high levels of Cdc6 result in RD(INK4/ARF)-dependent transcriptional repression, recruitment of histone deacetylases and heterochromatinization of the INK4/ARF locus, and a concomitant decrease in the expression of the three tumour suppressors encoded by this locus. This mechanism is reminiscent of the silencing of the mating-type HM loci in yeast by replication factors. Consistent with its ability to repress the INK4/ARF locus, Cdc6 has cellular immortalization activity and neoplastic transformation capacity in cooperation with oncogenic Ras. Furthermore, human lung carcinomas with high levels of Cdc6 are associated with low levels of p16(INK4a). We conclude that aberrant expression of Cdc6 is oncogenic by directly repressing the INK4/ARF locus through the RD(INK4/ARF) element.

A microRNA DNA methylation signature for human cancer metastasis.

MicroRNAs (miRNAs) are small, noncoding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also linked different sets of miRNAs to metastasis through either the promotion or suppression of this malignant process. Interestingly, epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is also emerging as a common hallmark of human tumors. Thus, we wondered whether there was a miRNA hypermethylation profile characteristic of human metastasis. We used a pharmacological and genomic approach to reveal this aberrant epigenetic silencing program by treating lymph node metastatic cancer cells with a DNA demethylating agent followed by hybridization to an expression microarray. Among the miRNAs that were reactivated upon drug treatment, miR-148a, miR-34b/c, and miR-9 were found to undergo specific hypermethylation-associated silencing in cancer cells compared with normal tissues. The reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumor growth, and inhibited metastasis formation in xenograft models, with an associated down-regulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6, and TGIF2. Most important, the involvement of miR-148a, miR-34b/c, and miR-9 hypermethylation in metastasis formation was also suggested in human primary malignancies (n = 207) because it was significantly associated with the appearance of lymph node metastasis. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of human cancer metastasis.

Comprehensive Analysis of SWI/SNF Inactivation in Lung Adenocarcinoma Cell Models.

Mammalian SWI/SNF (SWitch/Sucrose Non-Fermentable) complexes are ATP-dependent chromatin remodelers whose subunits have emerged among the most frequently mutated genes in cancer. Studying SWI/SNF function in cancer cell line models has unveiled vulnerabilities in SWI/SNF-mutant tumors that can lead to the discovery of new therapeutic drugs. However, choosing an appropriate cancer cell line model for SWI/SNF functional studies can be challenging because SWI/SNF subunits are frequently altered in cancer by various mechanisms, including genetic alterations and post-transcriptional mechanisms. In this work, we combined genomic, transcriptomic, and proteomic approaches to study the mutational status and the expression levels of the SWI/SNF subunits in a panel of 38 lung adenocarcinoma (LUAD) cell lines. We found that the SWI/SNF complex was mutated in more than 76% of our LUAD cell lines and there was a high variability in the expression of the different SWI/SNF subunits. These results underline the importance of the SWI/SNF complex as a tumor suppressor in LUAD and the difficulties in defining altered and unaltered cell models for the SWI/SNF complex. These findings will assist researchers in choosing the most suitable cellular models for their studies of SWI/SNF to bring all of its potential to the development of novel therapeutic applications.

Transgenic mice expressing constitutively active Akt in oral epithelium validate KLFA as a potential biomarker of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a common human neoplasia, of poor prognosis and survival, which frequently displays Akt overactivation. Previously, we reported that mice expressing high levels of constitutively Akt activity (myrAkt) in oral epithelia develop lesions and tumors in the oral cavity. MATERIALS AND METHODS: Functional genomics of primary keratinocytes from different transgenic mouse lines and immunostaining of mouse and human samples were performed in order to identify and validate putative biomarkers of oral cancer progression. RESULTS: The expression of KLF4 was found to be increased only in tumor prone samples from mice bearing overactivation of Akt. Such increased expression was confirmed in oral dysplasias and tumors arising in those mice. Tissue microarray analysis of human samples confirmed the association between active Akt and increased KLF4 expression. CONCLUSION: These data support the notion that KLF4 is potentially a reliable marker of HNSCC, and that myrAkt transgenic mice are valuable tools for preclinical research of HNSCC.

[Prognostic value study of lung cancer molecular markers]. / Valor pronóstico de los marcadores moleculares en el carcinoma broncogénico.

BACKGROUND AND OBJECTIVE: The aim of this study was to determine the prognostic value of molecular markers (proteins) of different paths of lung cancer development in patients with non small cell lung carcinoma (NSCLC) in initial stages. MATERIAL AND METHOD: Observational, cohort study in patients with NSCLC that was initially treated surgically in our hospital between October 1993 and September 1997. Thirty-two proteins were selected. The study consisted of the elaboration of tissue arrays with samples from resected tumour, using a semiquantitative immunohistochemical study. A prognosis analysis was done with the expression of each protein and calculation of the overall 5-year survival rate. The Wilcoxon-Gehan and Log-Rank tests were used for statistical comparisons, with p<.05 being considered to indicate a significant result. RESULTS: One hundred and forty six patients were studied. The overall 5-year survival rate was 37.7%. From 32 proteins studied, three were statistically associated with overall 5-year survival rate. RB protein expression in resected NSCLC was a positive prognostic factor (P=.01). P27 (P=.03) and Ki67 (P=.04) expression in resected NSCLC were negative prognostic factors. There was no protein with prognostic value in epidermoid tumours. CONCLUSIONS: We found three proteins with long-term prognostic value in the long-term in the general population and five adenocarcinoma prognostic proteins in our study of resected non-small cell lung cancer (NSCLC). In the future, genetic-molecular factors should be included along with anatomical (TNM staging) and clinical factors in a multidimensional lung cancer staging.

Genetic unmasking of an epigenetically silenced microRNA in human cancer cells.

The mechanisms underlying microRNA (miRNA) disruption in human disease are poorly understood. In cancer cells, the transcriptional silencing of tumor suppressor genes by CpG island promoter hypermethylation has emerged as a common hallmark. We wondered if the same epigenetic disruption can "hit" miRNAs in transformed cells. To address this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in combination with a miRNA expression profiling. We have observed that DNA hypomethylation induces a release of miRNA silencing in cancer cells. One of the main targets is miRNA-124a, which undergoes transcriptional inactivation by CpG island hypermethylation in human tumors from different cell types. Interestingly, we functionally link the epigenetic loss of miRNA-124a with the activation of cyclin D kinase 6, a bona fide oncogenic factor, and the phosphorylation of the retinoblastoma, a tumor suppressor gene.

VRK1 signaling pathway in the context of the proliferation phenotype in head and neck squamous cell carcinoma.

The vaccinia-related kinase (VRK) proteins are a new family with three members in the human kinome. The VRK1 protein phosphorylates several transcription factors and has been postulated to be involved in regulation of cell proliferation. In normal squamous epithelium, VRK1 is expressed in the proliferation area. Because VRK1 can stabilize p53, the expression of the VRK1 protein was analyzed in the context of the p53 pathway and the proliferation phenotype in a series of 73 head and neck squamous cell carcinomas. VRK1 protein level positively correlated with p53 response proteins, particularly hdm2 and p21. The VRK1 protein also correlated positively with several proteins associated with proliferation, such as cyclin-dependent kinase 2 (CDK2), CDK6, cdc2, cyclins B1 and A, topoisomerase II, survivin, and Ki67. The level of VRK1 protein behaves like a proliferation marker in this series of head and neck squamous cell carcinomas. To identify a possible regulatory role for VRK1 and because it regulates gene transcription, the promoters of two genes were studied, CDK2 and SURVIVIN, whose proteins correlated positively with VRK1. VRK1 increases the activity of both the CDK2 and SURVIVIN gene promoters. The expression of VRK1 was analyzed in the context of regulators of the G1-S transition. VRK1 protein levels increase in response to E2F1 and are reduced by retinoblastoma and p16. These data suggest that VRK1 might play a role in cell cycle regulation and is likely to represent the beginning of a new control mechanism of cell cycle, particularly late in the G1-S phase.

Specific pattern of LKB1 and phospho-acetyl-CoA carboxylase protein immunostaining in human normal tissues and lung carcinomas.

The LKB1 tumor suppressor gene codes for a serine/threonine protein kinase, and among its substrates is the adenosine monophosphate-dependent protein kinase, a sensor of intracellular energy levels. LKB1 is genetically inactivated in several types of tumors, especially lung adenocarcinomas. Here we used immunohistochemistry to evaluate the levels of LKB1 and the phosphorylated form of the acetyl-CoA carboxylase (ACC) protein in a variety of human adult normal tissues and in 159 lung carcinomas. The enzyme ACC becomes inactive upon phosphorylation by adenosine monophosphate-dependent protein kinase. Our analysis in normal tissues revealed strong LKB1 immunostaining in most epithelia, in the seminiferous tubules of the testis, in myocytes from skeletal muscle, and in glia cells. In contrast to the cytosolic location of LKB1 found in most tissues, glia cells carried mainly nuclear LKB1. Some epithelial cells showed apical accumulation of LKB1, supporting its role in cell polarity. Regarding phospho-ACC (p-ACC), strong immunostaining was observed in myocytes from the skeletal muscle and heart, and in Leydig cells of the testis. In lung tumors, LKB1 immunostaining was absent, moderate, and high in 20%, 61%, and 19% of the tumors, respectively, whereas p-ACC immunostaining was found to be absent/low, moderate, and high in 35%, 34%, and 31% of the tumors, respectively. High levels of LKB1 and p-ACC immunostaining predominated in lung adenocarcinomas compared with squamous cell carcinomas. Finally, high p-ACC was an independent marker for prediction of better survival in lung adenocarcinoma patients. Median overall survival was longer in patients with p-ACC-positive than those with p-ACC-negative tumors (96 versus 44 months, P = .04). In conclusion, our observations provide complete information about the pattern and levels of LKB1 and p-ACC immunostaining in normal tissues and in lung tumors, and highlight the special relevance of abnormalities of the LKB1 pathway in lung adenocarcinoma.

Alteration of the VRK1-p53 autoregulatory loop in human lung carcinomas.

Human VRK1 (vaccinia-related kinase 1) is a novel serine-threonine kinase that regulates several transcription factors, including p53, ATF2 and c-Jun; and its loss results in defects of cell proliferation. VRK1 stabilizes p53 and the accumulated p53 downregulates VRK1 forming an autoregulatory loop. Wild-type p53, but not mutant p53, was able to downregulate VRK1 in the A549 lung carcinoma cell line. VRK1 expression has been studied in human lung carcinomas. VRK1 protein level was significantly higher in squamous cell lung carcinomas than in adenocarcinomas, and inversely correlated with p16. Tumours with p53 mutations have a positive trend with those having very high levels of VRK1 protein, particularly in squamous cell lung carcinomas. These data indicate that the VRK1-p53 autoregulatory loop was not functional in a group of lung carcinomas. The accumulation of VRK1 in tumours with mutant p53 could result in stimulation of other signalling pathways that can contribute to tumour growth and progression in addition to those resulting from loss of p53 function.

Prognostic value of KIT expression in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is a very aggressive disease, with poor survival rates despite standard treatment with combination chemotherapy with or without radiotherapy. Further insights into the molecular biology of this malignant tumour are needed to improve the therapeutic approaches and outcome. KIT protein is expressed in SCLC, and its kinase activity has been implicated in the pathophysiology of many tumours, including SCLC. The purpose of this study was to evaluate the prevalence of KIT expression in patients with SCLC and its prognostic value. METHODS: We performed an inmunohistochemical analysis of 204 SCLC samples to determine KIT protein expression. The relationship between KIT expression and clinicopathological parameters was evaluated. Univariate and multivariate analyses were performed to define its prognostic significance. RESULTS: KIT expression was observed in 149 of 204 tumour tissues (73%). KIT expression was associated with advanced disease and with decreased incidence of bone metastases. No significant differences were observed for time to disease progression (TTP) (9.1% versus 6.2% at 3 years, p=0.6) or overall survival (OS) (10.7% versus 6.9% at 3 years, p=0.37) among patients with KIT positive versus negative tumours, respectively. Multivariate analysis showed that sex, tumour stage, albumin levels and response to therapy were the only independent predictors for survival. CONCLUSION: KIT protein is expressed in a high percentage of SCLC tumours. In our study population, however, the expression of KIT had no significant impact on survival.

Molecular context of the EGFR mutations: evidence for the activation of mTOR/S6K signaling.

PURPOSE: Activating somatic mutations in the epidermal growth factor receptor (EGFR) gene are present in a small subset of lung adenocarcinomas. These mutations cluster in specific regions and confer sensitivity to inhibitors of the tyrosine kinase activity of EGFR. To further determine the genetic and molecular characteristics of tumors carrying EGFR gene mutations, we investigated the EGFR gene status in lung adenocarcinomas and evaluated its association with specific characteristics of the patients and tumors, such as mutations at KRAS and p53, EGFR and ErbB2 gene amplification, levels of EGFR and HER2 proteins, and levels of downstream effectors of EGFR, such as phospho-extracellular signal-regulated kinase and phospho-S6 proteins. EXPERIMENTAL DESIGN: The mutational status of EGFR was determined by direct sequencing in 86 primary lung adenocarcinomas and 12 lung cancer cell lines, and was correlated with a number of variables relating to the tumor and patient. A tissue microarray containing 37 lung tumors was constructed to determine, by fluorescence in situ hybridization analysis, the number of copies of EGFR and ErbB2 genes and, by immunohistochemistry, the levels of EGFR, HER2, phospho-ERK, and phospho-S6 proteins. RESULTS: EGFR gene mutations were identified in 13% of the primary tumors. The type and clustering of the mutations were identical to those previously reported. Amplification of the EGFR occurred in 14% of the tumors and could arise in tumors with EGFR mutations. Interestingly, mTOR activation, as measured indirectly by augmented levels of phospho-S6 protein, was more frequent in tumors with gene alterations in either EGFR or KRAS (P = 0.00005; Fisher's exact test) than in their wild-type counterparts. CONCLUSIONS: Our data agree with the accumulation of EGFR mutations in a subset of patients with lung cancer. Moreover, we report EGFR gene amplification in EGFR-mutant tumors and a positive correlation between EGFR or KRAS alterations and activation of mTOR signaling.

Growth and molecular profile of lung cancer cells expressing ectopic LKB1: down-regulation of the phosphatidylinositol 3'-phosphate kinase/PTEN pathway.

Germ-line mutations in LKB1 gene cause the Peutz-Jeghers syndrome (PJS), a genetic disease with increased risk of malignancies. Recently, LKB1-inactivating mutations have been identified in one-third of sporadic lung adenocarcinomas, indicating that LKB1 gene inactivation is critical in tumors other than those of the PJS syndrome. However, the in vivo substrates of LKB1 and its role in cancer development have not been completely elucidated. Here we show that overexpression of wild-type LKB1 protein in A549 lung adenocarcinomas cells leads to cell-growth suppression. To examine changes in gene expression profiles subsequent to exogenous wild-type LKB1 in A549 cells, we used cDNA microarrays. We detected deregulation of 100 genes involved in cell proliferation, apoptosis, and cell adhesion. Strikingly, modification of the expression of well-known p53-responsive genes such as GADD45, TOP2A, and p21 suggests that growth suppression in A549 cells overexpressing LKB1 may be mediated by p53. In addition, PTEN up-regulation indicates that LKB1 could be involved in the PTEN/phosphatidylinositol-3'-kinase(PI3K)/AKT molecular pathway. Thus, our results give some insights into the understanding of how LKB1 inactivation contributes to lung carcinogenesis.

Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung.

Frequent losses of chromosome 19p have recently been observed in sporadic lung adenocarcinomas, targeting the location of a critical tumor suppressor gene. Here we performed fine mapping of the short arm of chromosome 19 and found that the LKB1/STK11 gene mapped in the minimal-deleted region. Because germ-line mutations at LKB1/STK11 result in the Peutz-Jeghers syndrome and an increased risk of cancer, we performed a detailed genetic screen of the LKB1/STK11 gene in lung tumors. We detected a high frequency of somatic alterations (mainly nonsense mutations) in primary lung adenocarcinomas and in lung cancer cell lines. Thus, our findings demonstrate for the first time that LKB1/STK11 inactivation is a very common event and may be integrally involved in the development of sporadic lung adenocarcinoma.

Germ-line variants in methyl-group metabolism genes and susceptibility to DNA methylation in normal tissues and human primary tumors.

Aberrant DNA methylation is recognized as being a common feature of human neoplasia.CpG island hypermethylation and global genomic hypomethylation occur simultaneously in the cancer cell. However, very little is known about the interindividual inherited susceptibility to these epigenetic processes. To address this matter, we have genotyped in 233 cancer patients (with colorectal, breast, or lung tumors), four germ-line variants in three key genes involved in the metabolism of the methyl group, methylene-tetrahydrofolate reductase, methionine synthase, and cystathionine beta-synthase, and analyzed their association with DNA methylation parameters. The epigenetic features analyzed were the 5-methylcytosine content in the genome of the tumors and their normal counterparts, and the presence of CpG island hypermethylation of tumor suppressor genes (p16(INK4a), p14(ARF), hMLH1, MGMT, APC, LKB1, DAPK, GSTP1, BRCA1, RAR beta 2, CDH1, and RASSF1). Two positive associations were found. First, carriers of genotypes containing the methylene-tetrahydrofolate reductase 677T allele show constitutive low levels of 5-methylcytosine in their genomes (P = 0.002), and tumors in these patients do not achieve severe degrees of global hypomethylation (P = 0.047). Second, tumors occurring in homozygous carriers of the methionine synthase 2756G allele show a lower number of hypermethylated CpG islands of tumor suppressor genes (P = 0.029). The existence of these associations may provide another example of the interplay between genetic and epigenetic factors in the cancer cell.

Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS-driven lung adenocarcinoma.

Patients with advanced Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutant lung adenocarcinoma are currently treated with standard chemotherapy because of a lack of efficacious targeted therapies. We reasoned that the identification of mediators of Kras signaling in early mouse lung hyperplasias might bypass the difficulties that are imposed by intratumor heterogeneity in advanced tumors, and that it might unveil relevant therapeutic targets. Transcriptional profiling of Kras(G12V)-driven mouse hyperplasias revealed intertumor diversity with a subset that exhibited an aggressive transcriptional profile analogous to that of advanced human adenocarcinomas. The top-scoring gene in this profile encodes the tyrosine kinase receptor DDR1. The genetic and pharmacological inhibition of DDR1 blocked tumor initiation and tumor progression, respectively. The concomitant inhibition of both DDR1 and Notch signaling induced the regression of KRAS;TP53-mutant patient-derived lung xenografts (PDX) with a therapeutic efficacy that was at least comparable to that of standard chemotherapy. Our data indicate that the combined inhibition of DDR1 and Notch signaling could be an effective targeted therapy for patients with KRAS-mutant lung adenocarcinoma.

CpG island promoter hypermethylation of the Ras-effector gene NORE1A occurs in the context of a wild-type K-ras in lung cancer.

Imbalance of the Ras signaling pathway is a major hallmark of human cancer. In this context, activating point mutations of the K-ras oncogene are a common feature of many tumor types. The discovery of methylation-mediated silencing of the Ras-effector homologue RASSF1A has revealed another way by which this cellular pathway may be altered. Inactivation by hypermethylation of a RASSF1A homologue, NORE1A, has recently been observed in human cancers. If both K-ras and NORE1A act in the same pathway, simultaneous molecular lesions in the two genes in the same tumor should be a rare event. To test whether this inverse association exists, we have analysed the K-ras mutational status and NORE1A CpG island hypermethylation of 61 non-small-cell lung carcinomas and the methylation status of the two other Ras effectors, RASSF1A and HRASLS. No association was found between the methylation status of NORE1A, RASSF1A and HRASLS or the status of K-ras with respect to the latter two genes. However, our results demonstrate that the epigenetic alteration of NORE1A is confined to lung tumors with a wild-type K-ras: 88% (15 of 17) of the tumors with NORE1 hypermethylation did not harbor a K-ras mutation (P=0.008, Fisher's exact test). Thus, the mutual exclusivity of the epigenetic and genetic alterations in the two genes of the Ras pathway suggests that they play a critical and cooperative role in human tumorigenesis.

Novel and natural knockout lung cancer cell lines for the LKB1/STK11 tumor suppressor gene.

Germline mutations of the LKB1 gene are responsible for Peutz-Jeghers syndrome (PJS), an autosomal dominant inherited disorder bestowing an increased risk of cancer. We have recently demonstrated that LKB1 inactivating mutations are not confined to PJS, but also appear in lung adenocarcinomas of sporadic origin, including primary tumors and lung cancer cell lines. To accurately determine the frequency of inactivating LKB1 gene mutations in lung tumors we have sequenced the complete coding region of LKB1 in 21 additional lung cancer cell lines. Here we describe the mutational status of LKB1 gene in 30 lung cancer cell lines from different histopathological types, including 11 lung adenocarcinomas (LADs) and 11 small cell lung cancers (SCLCs). LKB1 gene alterations were present in six (54%) of the LAD cell lines tested but in none of the other histological types. Similar to our previous observations in primary tumors, all point mutations were of the nonsense or frameshift type, leading to an abnormal, truncated protein. Moreover, 2 cell lines (A427 and H2126) harbored large gene deletions that spanned several exons. Hence, we have identified additional lung cancer cell lines carrying inactivating mutations of the LKB1 tumor suppressor gene, further attesting to the significance of this gene in the development of LADs and providing new natural LKB1 knockouts for studies of the biological function of the LKB1 protein.

Distinctive gene expression of human lung adenocarcinomas carrying LKB1 mutations.

LKB1, a tumor-suppressor gene that codifies for a serine/threonine kinase, is mutated in the germ-line of patients affected with the Peutz-Jeghers syndrome (PJS), which have an increased incidence of several cancers including gastrointestinal, pancreatic and lung carcinomas. Regarding tumors arising in non-PJS patients, we recently observed that at least one-third of lung adenocarcinomas (LADs) harbor somatic LKB1 gene mutations, supporting a role for LKB1 in the origin of some sporadic tumors. To characterize the pattern of LKB1 mutations in LADs further, we first screened for LKB1 gene alterations (gene mutations, promoter hypermethylation and homozygous deletions) in 19 LADs and, in agreement with our previous data, five of them (26%) were shown to harbor mutations, all of which gave rise to a truncated protein. Recent reports demonstrate that LKB1 is able to suppress cell growth, but little is known about the specific mechanism by which it functions. To further our understanding of LKB1 function, we analysed global expression in lung primary tumors using cDNA microarrays to identify LKB1-specific variations in gene expression. In all, 34 transcripts, 24 of which corresponded to known genes, differed significantly between tumors with and without LKB1 gene alterations. Among the most remarkable findings was deregulation of transcripts involved in signal transduction (e.g. FRAP1/mTOR, ARAF1 and ROCK2), cytoskeleton (e.g. MPP1), transcription factors (e.g. MEIS2, ATF5), metabolism of AMP (AMPD3 and APRT) and ubiquitinization (e.g. USP16 and UBE2L3). Real-time quantitative RT-PCR on 15 tumors confirmed the upregulation of the homeobox MEIS2 and of the AMP-metabolism AMPD3 transcripts in LKB1-mutant tumors. In addition, immunohistochemistry in 10 of the lung tumors showed the absence of phosphorylated FRAP1/mTOR protein in LKB1-mutant tumors, indicating that LKB1 mutations do not lead to FRAP1/mTOR protein kinase activation. In conclusion, our results reveal that several important factors contribute to LKB1-mediated carcinogenesis in LADs, confirming previous observations and identifying new putative pathways that should help to elucidate the biological role of LKB1.

An efficient expression system for the production of functionally active human LKB1.

Human LKB1, also known as STK11, is a tumour-suppression protein that mediates important functions in cellular proliferation and polarization. It might constitute an important target in cancer therapy. In order to produce large amounts of recombinant protein for biochemical and functional studies, a full-length cDNA clone was subcloned and expressed in Escherichia coli and insect cells. Although fusion proteins corresponding to LKB1 with 6xHis, GST and MBP tags could be overexpressed in E. coli, only MBP-LKB1 was recovered in a soluble, but heavily degraded form. Further studies demonstrated that this protein was not functional. Subsequent expression in insect cells of LKB1 with 6xHis and GST tags yielded insoluble products also. However, when chaperones Hsp70 and its cofactors Hsp40 and Hsdj were co-expressed with GST-LKB1, a clear increase in the solubility of the final protein was obtained. Moreover, this soluble, purified recombinant GST-LKB1 demonstrated to be a phosphoprotein, with at least residue Ser325 phosphorylated. The purified protein was functionally active as being able to demonstrate autophosphorylation in the absence of any associated kinase.