A Role for LKB1 Gene in Human Cancer Beyond the Peutz–Jeghers Syndrome

M Sanchez-Cespedes

Molecular Pathology Programme, Spanish National Cancer Centre (CNIO), Melchor Fernandez Almagro, Madrid, Spain

Germline LKB1 mutations are responsible for Peutz– development, LKB1 expression becomes more pro- Jeghers syndrome (PJS). Tumors at several locations nounced in heart, esophagus, pancreas, kidney, colon, frequently arise in these patients, confirming that LKB1 is lung, small intestine and stomach (Luukko et al., 1999; linked to cancer predisposition and is therefore a bona fide Rowan et al., 2000). In adult tissues, levels of LKB1 tumor-suppressor gene. In humans, the LKB1 gene is protein are high in most epithelia, in the follicles and located in the short arm of chromosome 19, which is corpus luteum of the ovary, and the seminiferous tubules frequently deleted in many tumors of sporadic origin. of the testis, in myocytes from skeletal muscle and in glia However, LKB1 alterations in tumors other than those of cells (Rowan et al., 2000; Conde et al., 2007; Figure 1a). PJSare rarely reported. Notably, this is not the case for non-small-cell lung cancer, where nearly half of the tumors harbor somatic and homozygous inactivating mutations in LKB1. The present review considers the LKB1 and Peutz–Jeghers syndrome frequency and pattern of LKB1 gene mutations in LKB1 sporadic cancers of various origins, and the role of the Germline mutations of cause the autosomal encoded protein in cancer development. dominant Peutz–Jeghers syndrome (PJS) (OMIM et al et al Oncogene (2007) 26, 7825–7832; doi:10.1038/sj.onc.1210594; 175200) (Hemminki ., 1998; Jenne ., 1998). published online 18 June 2007 The type and pattern of these mutations have been extensively reviewed elsewhere (Alessi et al., 2006; Keywords: LKB1; lung cancer; Peutz-Jeghers syndrome; Launonen, 2005). Individuals with PJS typically exhibit tumor suppressor gene mucocutaneous melanin pigmentation and suffer from hamartomatous polyps in the gastrointestinal tract (Jeghers et al., 1949; Giardiello et al., 2000). However, among the most important associated health-related concerns is the increased risk of cancer development Characteristics of the LKB1 gene and encoded protein: (Giardiello et al., 2000). Gastrointestinal tumors are the patterns of LKB1 expression in human tissues most commonly diagnosed malignancies in PJS patients, but the risk of developing cancer from other origins is The LKB1 gene, also known as STK11, maps to the also significantly higher (Figure 1b) (Giardiello et al., chromosomal region 19p13.3, which is frequently lost in 2000; Hearle et al., 2006a). To some extent, the pattern several types of cancer. The gene spans 23 kb and is made of tumor susceptibility in PJS patients recapitulates the up of nine coding exons and a final noncoding exon. expression of LKB1 in normal adult and embryonic B LKB1 encodes for an mRNA of 2.4kb transcribed in a tissues. For example, gonadal cell types that derive from telomere-to-centromere direction and for a protein of the coelomic epithelium (sex cords) or mesenchymal 433 amino acids and approximately 48 kDa (Hemminki cells of the embryonic gonads include granulosa cells et al., 1998). The protein, which has serine–threonine and theca cells, which have very high levels of LKB1 kinase activity, possesses a nuclear localization signal in protein (Conde et al., 2007). Interestingly, a type of the N-terminal noncatalytic region (residues 38–43) and ovarian cancer arising from these cell types, the ovarian a kinase domain (residues 49–309) (Alessi et al., 2006). sex cord tumors with annular tubules, which occurs Although LKB1 protein expression is mainly cytoplas- rarely in the general population, is present at higher mic, it can also be localized in the nucleus. frequencies in women with PJS. Likewise, the level of LKB1 is widely expressed in embryonic and adult LKB1 immunostaining is moderately high in cells from tissues although at different levels. Early mouse the breast, epithelia from the small intestine, colon and embryos (E7–11) have highly ubiquitous expression in pancreatic islets from human adult normal tissue, and all embryonic and extra-embryonic tissues. During very high in the small intestine and stomach of human embryos. Tumors from these origins are the commonest Correspondence: Dr M Sanchez-Cespedes, Molecular Pathology types in PJS (Figure 1b). Programme, Spanish National Cancer Centre (CNIO), Melchor Most studies of PJS patients have reported that only Fernandez Almagro 3, Madrid 28029, Spain. E-mail: [email protected] about half of the individuals carry LKB1 germline Received 17 March 2007; revised 14May 2007; accepted 14May 2007; mutations, which suggested that other tumor-suppressor published online 18 June 2007 genes must be associated with the syndrome in the Role of LKB1 in sporadic tumors M Sanchez-Cespedes 7826

a LKB1 PROTEIN EXPRESSION b PEUTZ-JEGHERS-RELATED TUMORS (Luuko et al. 1999; Rowan et al. 2000 (Giardiello et al.et al. 2000; et al. 2002; Conde et al. 2007) Lim et al. 2004; Hearle et al. 2006a)

ADULT TUMOR TYPE (increased cancer risk)

HIGH Gastroesophageal (15) Cerebral cortex (neurons and glia) Breast (6) Ovary (follicles and corpus luteum) Small bowel (15) Salivary glands (serous acini) Skeletal muscle Colorectal (15) Testis (developing, mature spermatozoa) Pancreas (22-130) Tonsil (epithelia) Gynecological (8.5) Breast (6) Lung(4-9) MODERATE Breast Colon (surface epithelium) Endometrium (glandular epithelium) Heart Kidney (tubules) Pancreas (islets) Respiratory epithelia (except goblet cells) Small Intestine (villous epithelium) Thyroid (follicular cells)

EMBRYO

HIGH Small intestine Stomach

MODERATE Colon Heart Kidney Lung Pancreas Esophagus

Figure 1 LKB1 protein-expression proﬁle in normal tissues and pattern of tumor susceptibility in PJS patients. (a) Levels of LKB1 protein immunostaining in adult and embryonic human normal tissues. (b) Tumor spectrum in patients with PJS. The increased risk of each type of tumor is cited in parentheses. PJS, Peutz–Jeghers syndrome.

remaining patients (Wang et al., 1999a; Alhopuro et al., sporadic origin screened to date. When first identified as 2005). Several chromosomal regions were thought to the gene responsible for PJS, LKB1 was tested for contain a second PJS locus, such as 19q, but genes inactivating mutations in a variety of sporadic tumors. within these regions do not contain LKB1 mutations Although tumor-specific LKB1 alterations have been (Buchet-Poyau et al., 2002). More recently, the use of identified in many of these tumor types, their frequency specific technical approaches, such as multiplex ligation- is extremely low. Table 1 summarizes most of the LKB1 dependent probe amplification, which allows the identi- mutations found in sporadic cancer to date, including fication of large-scale gene deletions, has improved the cancer cell lines and primary tumors of various origins. detection of LKB1 alterations in PJS patients by up to The mutational screening method is also indicated. 80% (Hearle et al., 2006b; Volikos et al., 2006). Thus, LKB1 mutations are present in about 4% of pancreatic these results indicate that LKB1 is the main causal gene cancers (cell lines and primary tumors from xenografts), in PJS, lessening the probability of there being a second but no mutations have been reported in breast, color- PJS locus. ectal or gastric cancer. The exception is non-small-cell lung cancer (NSCLC), in which LKB1 inactivation is a common event (Sanchez-Cespedes et al., 2002; Carretero Is LKB1 involved in the carcinogenesis of sporadic et al., 2004; Matsumoto et al., 2007). These differences tumors? The example of lung cancer are puzzling because lung cancer risk is higher but is not among the commonest tumors that occur in PJS. Before As described above, PJS patients have an increased risk ruling out the LKB1 tumor-suppressor role in sporadic of several types of cancer. Unexpectedly, LKB1 is tumors other than in lung, the obstacles associated with infrequently mutated in most of the tumor types of the detection of mutations in tumors need to be

Oncogene Role of LKB1 in sporadic tumors M Sanchez-Cespedes 7827 Table 1 Frequency of LKB1 mutations in primary tumors and cancer cell lines of sporadic origin Tumor type No of tumors % Mutationsa Methodb Reference

Cancer cell lines Breast 17 0% Sequencing Bignell et al. (1998) 5 0% Sequencing Ikediobi et al. (2006) Colorectal 7 0% Sequencing Ikediobi et al. (2006) Gliomas 6 0% Sequencing Ikediobi et al. (2006) HNSCC 8 12.5% Sequencing Qiu et al. (2006) Lung Adenocarcinomas/LCC 11 54% Sequencing Carretero et al. (2004) 7 43% Sequencing Ikediobi et al. (2006) 38 42% Sequencing Matsumoto et al. (2007) Squamous cell carcinomas 11 27% Sequencing Matsumoto et al. (2007) Small-cell lung cancer 11 0% Sequencing Carretero et al. (2004) 19 5% Sequencing Matsumoto et al. (2007) Melanoma 6 0% SSCP Avizienyte et al. (1999) 35 6% DGGE Guldberg et al. (1999) 16 6% SSCP Rowan et al. (1999) 9 0% Sequencing Ikediobi et al. (2006) Myeloma 8 0% SSCP Avizienyte et al. (1999) Pancreas 11 0% Sequencing Su et al. (1999) Prostate 2 50% Sequencing Ikediobi et al. (2006) Ovary 7 0% Sequencing Ikediobi et al. (2006) Renal 8 0% Sequencing Ikediobi et al. (2006) Primary tumors Breast 62 0% Sequencing Bignell et al. (1998) Cervix Squamous cell carcinoma 18 0% SSCP Avizienyte et al. (1999) 15 0% SSCP Kuragaki et al. (2003) Adenocarcinoma 8 12.5% SSCP Avizienyte et al. (1999) 244%SSCP Kuragaki et al. (2003) MDA 11 55% SSCP Kuragaki et al. (2003) 8 0% Sequencing Connolly et al. (2000) Colorectal 33 0% SSCP Avizienyte et al. (1998) Gastric 8 0% SSCP Avizienyte et al. (1999) HNSCC 7 0% Sequencing Qiu et al. (2006) Liver 80 1% SSCP Kim et al. (2004) Lung Adenocarcinomas 12 8% SSCP Avizienyte et al. (1999) 20 30% Sequencing Sanchez-Cespedes et al. (2002) 91 8% Sequencing Matsumoto et al. (2007) Squamous cell carcinomas 12 0% SSCP Avizienyte et al. (1999) 12 0% Sequencing Sanchez-Cespedes et al. (2002) Melanoma 15 7% SSCP Rowan et al. (1999) Ovary Adenocarcinomas 45 0% SSCP Wang et al. (1999b) Granulosa 12 0% SSCP Avizienyte et al. (1999) 12 8% SSCP Wang et al. (1999b) Sex cord tumors 5 0% Sequencing Connolly et al. (2000) Pancreas 100 4% Sequencing Su et al. (1999) 12 8.3% SSCP Avizienyte et al. (1999) Renal 19 0% SSCP Avizienyte et al. (1999) Soft tissue sarcoma 240% SSCP Avizienyte et al. (1999) Testis 28 3% SSCP Avizienyte et al. (1998)

Abbreviations: DGGE, denaturing gradient gel electrophoresis; HNSCC, head and neck squamous cell carcinomas; LCC, large cell carcinoma; MDA, Minimal Deviation Adenocarcinoma of the uterine cervix; SSCP, single-strand conformation polymorphism. aFor primary tumors, only mutations reported to be somatic are included. bSequencing, direct sequencing of PCR products. considered. These may have adversely affected the intragenic deletions, most of which cannot be detected detection of LKB1 mutations. In PJS patients, a by common mutational screening of primary tumors. substantial percentage of LKB1-inactivating events are Second, in primary tumors the admixture of non- large deletions (Hearle et al., 2006b; Volikos et al., malignant cells also hinders the detection of point 2006), and the same may be the case in sporadic cancer. mutations, and small insertions and deletions (indels). If so, such deletions would have gone undetected in a Consistent with this, in lung cancer, we and others have simple mutational screening, masked by normal DNA in observed higher rates of LKB1 mutation in cell lines primary tumors or mistaken as PCR failures in cancer than in primary tumors (Sanchez-Cespedes et al., 2002; cell lines. As shown in Table 2 and Figure 2, about half Carretero et al., 2004; Matsumoto et al., 2007). This of the LKB1 alterations in lung cancer cell lines are large would not be the case, however, for pancreatic cancer,

Oncogene Role of LKB1 in sporadic tumors M Sanchez-Cespedes 7828 Table 2 LKB1 mutations found to date in sporadic lung tumors Tumor identiﬁcation Histology Exon Mutation type Nucleotide changea Predicted effect Reference

Point mutations+indels PTb AC E1 Nonsense 109C>T Q37X Sanchez-Cespedes et al. (2002) A549b AC E1 Nonsense 109C>T Q37X Sanchez-Cespedes et al. (2002) H460b AC E1 Nonsense 109C>T Q37X Carretero et al. (2004) PTb AC E1 Nonsense 109C>T Q37X Matsumoto et al. (2007) PTb AC E1 Nonsense 109C>T Q37X Greenman et al. (2007) PTb LCC E1 Nonsense 109C>T Q37X Greenman et al. (2007) PT AC E1 Nonsense 130A>T K44X Sanchez-Cespedes et al. (2002) PT AC E1 Frameshift 150insT Stop at 162 Matsumoto et al. (2007) PT AC E1 Frameshift 153insG Stop at 162 Matsumoto et al. (2007) HCC44 AC E1 Frameshift 153_154insC Stop at 162 Matsumoto et al. (2007) PTb AC E1 Frameshift 157_158insG Stop at 162 Fernandez et al. (2004) PTb AC E1 Frameshift 157_158delG Stop at 64J Sanchez-Cespedes (unpublished.) H1395 AC E1 Frameshift 167_168delG Stop at 64Carretero et al. (2004) PTb AC E1 Nonsense 180C>A Y60X Fernandez et al. (2004) PT AC E1 Nonsense 193G>T E65X Fernandez et al. (2004) PT AC IVS1 Splicing IVS1+1G>T Truncated Matsumoto et al. (2007) PT AC E2 Nonsense 358G>T E120X Fernandez et al. (2004) VMRC-LCD AC IVS3 Splicing IVS3-1G>T Truncated Matsumoto et al. (2007) PT AC E4Nonsense 475C>TQ159X Matsumoto et al. (2007) PTb AC E4Missense 581A>T Sanchez-CespedesD194VJ (unpublished) PT AC E4Missense 581A>T D194VAvizienyte et al. (1999) PTb AC IVS4Splicing IVS4-2A>GTruncated J Sanchez-Cespedes (unpublished) PT AC E5 Nonsense 630C>A C210X Sanchez-Cespedes et al. (2002) PT LCC E5 Nonsense 640C>T Q214X J Sanchez-Cespedes (unpublished) PT AC E5 Nonsense 667G>T E223X Sanchez-Cespedes et al. (2002) PC13 LCC E5 Missense 668A>T E223V Matsumoto et al. (2007) HCC515 AC IVS5 Splicing IVS5+1G>T Truncated Matsumoto et al. (2007) PC7 AC E6 Missense 747A>C+749G>T T250P Matsumoto et al. (2007) PT AC E6 Nonsense 766G>T E256X Matsumoto et al. (2007) Ma25 LCC E6 Nonsense 782C>G Y261X Matsumoto et al. (2007) PT AC E6 Frameshift 837_842delC Stop at 286 Matsumoto et al. (2007) PTb AC E6 Frameshift 837_842delC Stop at 286 Sanchez-Cespedes et al. (2002) PTb AC E6 Frameshift 837_842delC Stop at 286 Matsumoto et al. (2007) H2122 AC E6 Frameshift 837_842delC Stop at 286 Matsumoto et al. (2007) Ma29 AC E6 Missense 847C>G L283V Matsumoto et al. (2007) H23 AC E8 Nonsense 996G>A W332X Sanchez-Cespedes et al. (2002) PT LCC E8 Frameshift 1034insTGCA Stop at360 J Sanchez-Cespedes (unpublished)

Large deletions 11–18 AC BÀ25 to BÀ0.5 kb Deletion No protein Matsumoto et al. (2007) HCC15 SCC BÀ15 kb-E1 Deletion No protein Matsumoto et al. (2007) H1184SCLC BÀ15 kb-E1 Deletion No protein Matsumoto et al. (2007) HCC366 AC-SCC BÀ2.5 kb-E1 Deletion No protein Matsumoto et al. (2007) Sq5 SCC BÀ9 kb-E3 Deletion No protein Matsumoto et al. (2007) Ma29 AC E1 Deletion No protein Matsumoto et al. (2007) A427 AC E1–5 Deletion No protein Carretero et al. (2004) H157 SCC E2 Deletion Truncated Matsumoto et al. (2007) Ma24AC E2–7 Deletion Truncated Matsumoto et al. (2007) Lu65 LCC E4–5 Deletion Truncated Matsumoto et al. (2007) H2126 AC E4–6 Deletion Truncated Carretero et al. (2004) PT AC E4–7 Deletion Truncated J Sanchez-Cespedes (unpublished) PT AC E8 Deletion Truncated Fernandez et al. (2004)

Abbreviations: AC, adenocarcinoma; LCC, large-cell carcinoma; PT, primary tumor; SCC, squamous cell carcinoma; SCLC, small-cell lung cancer. aNumbering is according to the coding DNA starting at the A of the start codon. bMutations or residues reported also to be altered in tumors from PJS patients.

where all the primary tumors examined were xenograft It is crucial to ﬁll these gaps in our knowledge of samples and the mutation frequency of LKB1 is far tumor biology. In addition, in the era of targeted lower than that of the lung (Su et al., 1999). Although therapies, these obstacles may have dire consequences the risk for pancreatic cancer is strongly increased in PJS for the selection of patients for future individualized patients, the low frequency of LKB1 mutations in the treatments based on the presence of speciﬁc gene sporadic cases may indicate that other components of mutations. Thus, in the coming years, the development the LKB1 pathway are preferentially altered in these of novel technologies that allow accurate molecular tumors. diagnosis of heterogeneous cancer specimens will be a

Oncogene Role of LKB1 in sporadic tumors M Sanchez-Cespedes 7829 a Kinase domain

∗ ∗ ∗ ∗ ^ b ∗ ^^^ ^ ∗ ∗ ^^^∗∗ ++∗ ∗ +∗∗∗ +∗∗^ ∗ ^ 1357924 6 8

c 0255075 100

GC:TA 10 GC:AT 9 TA:AT 4 GC:CG 2 TA:GC 1 TA:CG 1 Indels 11 Large deletions 13

Figure 2 Genetic profile of LKB1 mutations in sporadic lung cancer. (a) Schematic representation of the LKB1 protein structure. (b) Location and type of LKB1 mutations in lung tumors. Above: nucleotide substitutions and indels. The different types of mutations are indicated as follows: nonsense*; frameshift4; splicing þ ; and missense’. Below: large deletions. (c) LKB1 mutation spectrum in lung tumors. Absolute numbers are indicated inside the bars. major challenge. Considerable effort is already being (Mehenni et al., 1998; Ylikorkala et al., 1999) or to made in this regard, as exemplified by microreactor- phosphorylate AMP-activated protein kinase (AMPK), based pyrosequencing, by which low-abundance onco- a direct LKB1 substrate (Hawley et al., 2003). Similar to gene mutations may be detected in complex samples PJS, LKB1 mutations in lung tumors are scattered with low tumor content (Thomas et al., 2006). throughout exons 1–8, and no mutations have been identified in exon 9. The LKB1 mutational profile in lung tumors includes the presence of mutations in exon Genetic profile of LKB1 mutations in sporadic lung cancer 8 that retain the kinase activity but lack the C-terminal regulatory domain. Recurrent mutations such as the The mutational pattern of LKB1 in lung tumors of Q37X, the 837-842delC and frameshift indels at codons sporadic origin is that of a classical tumor-suppressor 51–53, also reported in PJS patients, are mutational hot gene. First of all, mutations are homozygous, as spots. LKB1 deletions involving one or more exons, predicted by Knudson’s two hit hypothesis (Knudson, common in PJS, are also present in lung tumors. These 1971). Second, a large proportion of mutations lead to abnormalities account for about half of the LKB1 the generation of truncated proteins, indicative of an alterations in lung cancer cell lines. The frequency of inactivating event. Third, the mutations in tumors of such deletions is considerably lower in lung primary sporadic origin arise somatically, and so are only present tumors, probably due to the masking effect of mixed in the tumor tissue. normal DNA. Table 2 summarizes the LKB1 mutations found to The mutational signature of LKB1 in lung tumors is date in lung tumors. As indicated, there is a large illustrated in Figure 2c. There is a high prevalence of proportion of nonsense, indel frameshift mutations, GC:TA and GC:AT substitutions among the six types large deletions and intronic mutations in splicing- of nucleotide substitutions. Although a large number of conserved sites (Figure 2). These changes lead to the mutations would be needed to be confident of the generation of truncated and, therefore, inactive LKB1 conclusion, the high percentage of GC:TA substitutions proteins. Remarkably, of the 51 known mutations in may reflect the influence of polycyclic aromatic hydro- lung tumors, only five are amino-acid substitutions, and carbon adducts from tobacco carcinogens, similar to all of these are located in the kinase domain of the what happens with KRAS and TP53 mutations in lung protein (Figures 2a and b). Missense mutations in this tumors (Ahrendt et al., 2001; Hainaut and Pfeifer, region abolish the ability of LKB1 to autophosphorylate 2001). Consistent with this explanation, most lung

Oncogene Role of LKB1 in sporadic tumors M Sanchez-Cespedes 7830 tumors with LKB1 mutations are from smokers confirmed that wild-type LKB1 is also required for (Matsumoto et al., 2007). LKB1 mutant tumors also modulating AMPK activity and therefore that of AMPK- carry concomitant mutations at other cancer genes downstream targets, including mTOR, in energetically such as KRAS, TP53, EGFR and p16, indicating depleted lung cancer cells (Carretero et al., 2007). The that their role in carcinogenesis is not functionally identification of AMPK as a key substrate of LKB1 equivalent (Sanchez-Cespedes et al., 2002; Matsumoto kinase activity provides definitive evidence that the et al., 2007). abrogation of energetic checkpoints, probably with the LKB1 mutations accumulate in NSCLC as compared purpose of maintaining energetically costly processes to small cell lung cancer. The loss of LKB1 protein such as DNA replication and cell division, is an during the development of lung adenocarcinomas has obligatory event for cancer development. LKB1, how- also been evaluated. Immunostaining of LKB1 in ever, functions as a master upstream protein kinase of adenomatous atypical hyperplasia, the proposed pre- many AMPK-related kinases (Lizcano et al., 2004), cursor lesions for the adenocarcinomas of the lung, indicating that LKB1 modulates other important cell shows that loss of protein expression is already observed processes. Some of these AMPK-related kinases are the in severe dysplasia, suggesting that LKB1 inactivation MAP/microtubule affinity-regulating kinases (MARK1, occurs early on in the development of this type of lung MARK2, MARK3 and MARK4) involved in micro- cancer (Ghaffar et al., 2003). tubule stabilization, which may be related with the proposed role of LKB1 in cell polarity. Supporting this, LKB1 is homologous to the Par-4genes of Caenorhab- LKB1 as an energetic checkpoint controller ditis elegans, essential for establishing anterior–posterior (A–P) axis during embryonic development (Guo and Functionally, the tumor-suppressor role of LKB1 is Kemphues, 1995), and to the Drosophila lkb1, also indicated by its ability to suppress the growth of tumor required for the early A–P polarity of the oocyte, and cells (Tiainen et al., 1999, 2002; Marignani et al., 2001; for the repolarization of the oocyte cytoskeleton that Jimenez et al., 2003), or to bring about apoptosis defines the embryonic A–P axis (Martin and St through its physical interaction with p53 (Karuman Johnston, 2003). In mammalians, LKB1 induces com- et al., 2001). LKB1 has also been implicated in plete polarization of intestinal epithelial cells in a cell- angiogenesis (Ylikorkala et al., 2001) and in the autonomous fashion (Baas et al., 2004). For a better regulation of cyclooxygenase 2 levels through the understanding of the roles of LKB1 as an energetic regulation of the levels of the transcription factor checkpoint controller and in cell polarity, we refer the PEA3 (Rossi et al., 2002; Upadhyay et al., 2006). reader to several excellent recent reviews (Spicer and Knock-out LKB1 mice are lethal in embryos, whereas Ashworth, 2004; Alessi et al., 2006; Katajisto et al., the heterozygous counterparts have a PJS-like pheno- 2007). In addition, other LKB1 substrates are known type, highlighting the critical role of LKB1 in cell and others will surely be found in the upcoming years growth and survival (Ylikorkala et al., 2001; Miyoshi that will help to understand other cell processes et al., 2002; Nakau et al., 2002). modulated by LKB1. LKB1 functions in a heterotrimeric complex with the inactive pseudokinase, STE20-related adaptor, and the armadillo repeat scaffolding-like protein, MO25 (Baas Conclusions et al., 2003; Boudeau et al., 2003). The best character- ized LKB1 substrate is the AMPK, a sensor of cellular The frequency and type of LKB1 gene inactivation energy status (Hawley et al., 2003). An increase in the unequivocally attests to its tumor-suppressor role in intracellular AMP/ATP ratio triggers the phosphoryla- lung tumorigenesis and demonstrates its significance tion and activation of AMPK and subsequently in cancer development in other circumstances that modulates the activity of multiple downstream targets merely the cancer-prone PJS. In addition to lung cancer, in normalizing ATP levels (Hardie, 2003). Among these the use of highly sensitive screenings for mutations and targets is the tuberin protein, the product of the large deletions may reveal that LKB1 gene alterations tuberous sclerosis complex 2 gene (TSC2) that leads to occur in a broad range of tumor types. Finally, the the repression of mTOR (Corradetti et al., 2004; Shaw critical involvement of LKB1 in energetic control et al., 2004). Germline mutations of TSC1 or TSC2 checkpoints highlights the importance of these processes genes cause the tuberous sclerosis syndrome, which in carcinogenesis and provides novel potential targets shares similarities with PJS, including the presence of for gene screening in tumors and for therapeutic hamartomas (Consortium TECS, 1993). We recently intervention.

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