Mutations in the human LKB1/STK11 gene

The human LKB gene (official HUGO symbol, STK11) encodes a serine/threonine protein kinase that is defective in patients with Peutz-Jeghers syndrome (PJS). PJS is an autosomal dominantly inherited syndrome characterized by hamartomatous polyposis of the gastrointestinal tract and mucocutaneous pigmentation. To date, 145 different germline LKB1 mutations have been reported. The majority of the mutations lead to a truncated protein product. One mutational hotspot has been observed. A 1-bp deletion and a 1-bp insertion at the mononucleotide repeat (C6 repeat, c.837-c.842) between the codons 279-281 have been found in six and seven unrelated PJS families, respectively. However, these mutations account only for approximately 7% of all mutations identified in the PJS families (13/193). A review of the literature provides a total of 40 different somatic LKB1 mutations in 41 sporadic tumors and seven cancer cell lines. Mutations occur particularly in lung and colorectal cancer. Most of the somatic LKB1 mutations result in truncation of the protein. A mutational hotspot seems to be a C6 repeat accounting for 12.5% of all somatic mutations (6/48). These results are concordant with the germline mutation spectrum. However, the proportion of the missense mutations seems to be higher among the somatic mutations (45%) than among the germline mutations (21%), and only seven of the mutations are exactly the same in both of the mutation types.     

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

Germline mutations of the LKB1 gene are responsible for the cancer-prone Peutz-Jeghers syndrome (PJS). LKB1 encodes a serine-threonine kinase that acts as a regulator of cell cycle, metabolism and cell polarity. The majority of PJS missense mutations abolish LKB1 enzymatic activity and thereby impair all functions assigned to LKB1. Here, we have investigated the functional consequences of recurrent missense mutations identified in PJS and in sporadic tumors which map in the LKB1 C-terminal non-catalytic region. We report that these C-terminal mutations neither disrupt LKB1 kinase activity nor interfere with LKB1-induced growth arrest. However, these naturally occuring mutations lessened LKB1-mediated activation of the AMP-activated protein kinase (AMPK) and impaired downstream signaling. Furthermore, C-terminal mutations compromise LKB1 ability to establish and maintain polarity of both intestinal epithelial cells and migrating astrocytes. Consistent with these findings, mutational analysis reveals that the LKB1 tail exerts an essential function in the control of cell polarity. Overall, our results ascribe a crucial regulatory role to the LKB1 C-terminal region. Our findings further indicate that LKB1 tumor suppressor activity is likely to depend on the regulation of AMPK signaling and cell polarization.     

Genetic defects underlying Peutz-Jeghers syndrome (PJS) and exclusion of the polarity-associated MARK/Par1 gene family as potential PJS candidates

LKB1/STK11 germline inactivations are identified in the majority (66-94%) of Peutz-Jeghers syndrome (PJS) patients. Therefore, defects in other genes or so far unidentified ways of LKB1 inactivation may cause PJS. The genes encoding the MARK proteins, homologues of the Par1 polarity protein that associates with Par4/Lkb1, were analyzed in this study because of their link to LKB1 and cell polarity. The genetic defect underlying PJS was determined through analysis of both LKB1 and all four MARK genes. LKB1 point mutations and small deletions were identified in 18 of 23 PJS families using direct sequencing and multiplex ligation-dependent probe amplification analysis identified exon deletions in 3 of 23 families. In total, 91% of the studied families showed LKB1 inactivation. Furthermore, a MARK1, MARK2, MARK3 and MARK4 mutation analysis and an MARK4 quantitative multiplex polymerase chain reaction analysis to identify exon deletions on another eight PJS families without identified LKB1 germline mutation did not identify mutations in the MARK genes. LKB1 defects are the major cause of PJS and genes of the MARK family do not represent alternative PJS genes. Other mechanisms of inactivation of LKB1 may cause PJS in the remaining families.     

Germline and Somatic Mutations of the STK11/LKB1 Peutz-Jeghers Gene in Pancreatic and Biliary Cancers

Peutz-Jeghers syndrome (PJS) is an autosomal-dominant disorder characterized by hamartomatous polyps in the gastrointestinal tract and by pigmented macules of the lips, buccal mucosa, and digits. Less appreciated is the fact that PJS also predisposes patients to an increased risk of gastrointestinal cancer, and pancreatic cancer has been reported in many PJS patients. It was recently shown that germline mutations of the STK11/LKB1 gene are responsible for PJS. We investigated the role of STK11/LKB1 in the development of pancreatic and biliary cancer in patients with and without the PJS. In a PJS patient having a germline splice site mutation in the STK11/LKB1 gene, sequencing analysis of an intestinal polyp and pancreatic cancer from this patient revealed loss of the wild-type allele of the STK11/LKB1 gene in the cancer. Inactivation of STK11/LKB1, by homozygous deletions or somatic sequence mutations coupled with loss of heterozygosity, was also demonstrated in 4-6% of 127 sporadic pancreatic and biliary adenocarcinomas. Our results demonstrate that germline and somatic genetic alterations of the STK11/LKB1 gene may play a causal role in carcinogenesis and that the same gene contributes to the development of both sporadic and familial forms of cancer.     

STRAD in Peutz-Jeghers syndrome and sporadic cancers

BACKGROUND/AIMS: LKB1 is a tumour suppressor gene that is associated with Peutz-Jeghers syndrome (PJS), a rare autosomal dominant cancer predisposition syndrome. However, germline mutations in the LKB1 gene are found in only about 60% of patients with PJS, suggesting the existence of a second PJS gene. The STRAD gene, encoding an LKB1 interacting protein that activates LKB1, which subsequently leads to polarisation of cells, is an interesting candidate for a second PJS gene and a potential tumour suppressor gene in sporadic carcinomas. METHODS: The involvement of STRAD in 42 PJS associated tumours (sporadic lung, colon, gastric, and ovarian adenocarcinomas) was studied using loss of heterozygosity (LOH) analysis of eight microsatellite markers on chromosome 17, including TP53, BRCA1, and STRAD markers. RESULTS: Loss of the marker near the STRAD locus was seen in 13 of 29 informative cases, including all gastric adenocarcinomas. Specific LOH of the STRAD marker was found in four of 29 informative cases. For these patients all exons and exon-intron boundaries of the STRAD gene were sequenced, but no somatic mutations were identified. Furthermore, no germline STRAD mutations were found in 10 patients with PJS and family members without LKB1 germline mutation. CONCLUSIONS: Despite the frequent occurrence of LOH in the STRAD region, these results indicate that inactivation of the STRAD gene is not essential in the sporadic adenocarcinomas studied, although it is possible that STRAD may be inactivated in different ways. In addition, no evidence was found for the hypothesis that STRAD is a second PJS susceptibility gene.     

Conditional deletion of the Lkb1 gene in the mouse mammary gland induces tumour formation

Heterozygous germline mutations in the LKB1 (STK11) gene cause Peutz‐Jeghers syndrome (PJS), an autosomal dominant disorder characterized by hamartomatous polyposis of the gastrointestinal tract and an increased risk of colorectal, breast, and other cancers. To model the role of LKB1 mutation in mammary tumourigenesis, we have used a conditional gene targeting strategy to generate a mouse in which exons encoding the kinase domain of Lkb1 were deleted specifically in the mammary gland. Mammary gland tumours developed in these mice with a latency of 46–85 weeks and occurred in the thoracic or inguinal glands. These tumours were grade 2 invasive ductal carcinomas or solid papillary carcinomas with histological features similar to those described in breast cancers arising in patients with PJS. This mouse model of Lkb1 deficiency provides a potentially useful tool to investigate the role of Lkb1 in tumourigenesis and to guide the development of therapeutic approaches. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Germline mutation screening of the STK11/LKB1 gene in familial breast cancer with LOH on 19p

The recently cloned STK11/LKB1 on chromosome 19p has been shown to be a new tumor suppressor gene. Mutations in the LKB1/STK11 gene on chromosome 19p account for most cases of Peutz-Jeghers syndrome (PJS), in which intestinal hamartomas are associated with elevated risks of several cancer types, including breast cancer. A previous study revealed that familial breast cancer is associated with loss of heterozygosity (LOH) on 19p. To establish whether germline mutations of STK11/LKB1 account for familial breast cancer, 22 patients from 14 breast cancer families with LOH on 19p and one PJS family were selected for screening for germline mutations of LKB1/STK11. A combination of polymerase chain reaction (PCR)-heteroduplex, single-strand conformational polymorphism (SSCP) analyses, Southern blot analysis and direct sequencing were used for mutation detection. No mutations were identified. Germline mutations of LKB1/STK11 did not contribute to breast cancer in these families.     

Genetic heterogeneity in Peutz-Jeghers syndrome

LKB1, the human gene encoding a serine threonine kinase, was recently identified as a susceptibility gene for Peutz-Jeghers syndrome (PJS), a disease characterized by the constellation of intestinal hamartomata, oral mucocutaneous hyperpigmentation, and an increased risk for gastrointestinal as well as extraintestinal malignancies. To date, the majority of individuals with PJS have been found to have genetic alterations in LKB1, most of which result in protein truncation. Additionally, linkage analyses have suggested a modicum of genetic heterogeneity, with the majority of PJS families showing linkage to the LKB1 locus. In this study, we evaluated five kindreds with greater than two affected family members, five PJS probands with only one other affected family member, as well as 23 individuals with sporadic PJS for mutations within the LKB1 gene. Conformation sensitive gel electrophoresis was utilized for the initial screen, followed by direct sequence analysis for characterization. Long-range PCR was used for the detection of larger genetic insertions or deletions. Mutation analysis revealed genetic alterations in LKB1 in two probands who had a family history of PJS. LKB1 mutations were detected in only four of the remaining 23 cases of sporadic PJS. These data suggest the presence of significant genetic heterogeneity for PJS and the involvement of other loci in this syndrome.     

Mutation analysis of the STK11/LKB1 gene and clinical characteristics of an Australian series of Peutz-Jeghers syndrome patients

Peutz-Jeghers syndrome (PJS) is a rare cancer predisposition, which is characterized by the presence of hamartomatous polyposis and mucocutaneous pigmentation. A significant proportion of both familial and sporadic forms of this disorder are associated with mutations in the STK11 (serine/threonine kinase 11)/LKB1 gene. In this report we present a series of Australian PJS cases, which suggest that mutations in the STK11 gene do not account for many families or patients without a family history. The most likely explanation is either the presence of another susceptibility gene or genetic mosaicism in the non-familial patients.     

Nasal polyposis in Peutz-Jeghers syndrome: a distinct histopathological and molecular genetic entity

BACKGROUND: Peutz-Jeghers syndrome (PJS) is an autosomal dominant hamartomatous polyposis syndrome of the gastrointestinal tract, caused by a germline STK11/LKB1 mutation. Nasal polyposis was described in the original report by Peutz. Recently, a molecular-genetic association between nasal polyposis and PJS has been reported. OBJECTIVE: To further explore the occurrence and pathogenesis of PJS-related nasal polyposis. METHODS: 51 patients with PJS, 84 unaffected family members and 36 spouses from 18 families with PJS were questioned for the presence of nasal polyposis. 12 PJS-related nasal polyps, 1 carcinoma of the nasal cavity and 28 sporadic nasal polyps were analysed for loss of (wild type) STK11/LKB1, eosinophilia, squamous metaplasia, dysplasia and expression of cyclo-oxygenase 2 and p53. RESULTS: Nasal polyps occurred in 8 of 51 patients with PJS, and were not reported by non-affected family members (p<0.001). Germline STK11/LKB1 mutations were identified in all patients with PJS and nasal polyposis. Loss of heterozygosity was found in four of eight PJS-related nasal polyps, but not in sporadic nasal polyps (p = 0.002). PJS-related nasal polyps showed less eosinophilia than sporadic nasal polyps (p<0.001). Expression of cyclo-oxygenase 2 was found in 11 of 12 PJS-related nasal polyps and 19 of 28 sporadic nasal polyps (p>0.05). Overexpression of p53 was not found. CONCLUSIONS: Nasal polyposis occurs in a significant number of Dutch patients with PJS, one of whom developed a carcinoma in the nasal cavity. The loss of heterozygosity, and the absence of eosinophilia suggest a distinct pathogenesis compared with sporadic nasal polyposis.     

Germline mutations of the LKB1 (STK11) gene in Peutz-Jeghers patients

Germline mutations of the LKB1 (STK11) serine/threonine kinase gene (chromosome 19p13.3) cause Peutz-Jeghers syndrome, which is characterised by hamartomas of the gastrointestinal tract and typical pigmentation. Peutz-Jeghers syndrome carries an overall risk of cancer that may be up to 20 times that of the general population. Here, we report the results of a screen for germline LKB1 mutations by DNA sequencing in 12 Peutz-Jeghers patients (three sporadic and nine familial cases). Mutations were found in seven (58%) cases, in exons 1, 2, 4, 6, and 9. Five of these mutations, two of which are identical, are predicted to lead to a truncated protein (three frameshifts, two nonsense changes). A further mutation is an in frame deletion of 6 bp, resulting in a deletion of lysine and asparagine; the second of these amino acids is conserved between species. The seventh mutation is a missense change in exon 2, converting lysine to arginine, affecting non-conserved amino acids and of uncertain functional significance. Despite the fact that Peutz-Jeghers syndrome is usually an early onset disease with characteristic clinical features, predictive and diagnostic testing for LKB1 mutations will be useful for selected patients in both familial and non-familial contexts.     

STK11/LKB1 Peutz-Jeghers Gene Inactivation in Intraductal Papillary-Mucinous Neoplasms of the Pancreas

Despite the growing awareness of intraductal papillary-mucinous neoplasms (IPMNs) of the pancreas among clinicians, the molecular features of IPMNs have not been well characterized. Previous reports suggest that inactivation of the STK11/LKB1, a tumor-suppressor gene responsible for Peutz-Jeghers syndrome (PJS), plays a role in the pathogenesis of gastrointestinal hamartomas as well as several cancers, including pancreatic adenocarcinoma. Using polymerase chain reaction amplification of five microsatellite markers from the 19p13.3 region harboring the STK11/LKB1 gene, we analyzed DNA from 22 IPMNs for loss of heterozygosity (LOH). LOH at 19p13.3 was identified in 2 of 2 (100%) IPMNs from patients with PJS and 5 of 20 (25%) from patients lacking features of PJS (7 of 22, 32% overall). Sequencing analysis of the STK11/LKB1 gene in these IPMNs with LOH revealed a germline mutation in one IPMN that arose in a patient with PJS and a somatic mutation in 1 of the 20 sporadic IPMNs. None of the 22 IPMNs showed hypermethylation of the STK11/LKB1 gene. These results suggest that the STK11/LKB1 gene is involved in the pathogenesis of some IPMNs.     

Genetic predisposition to phaeochromocytoma: analysis of candidate genes GDNF, RET and VHL

Inherited predisposition to phaeochromocytoma (MIM No 171300) occurs in multiple endocrine neoplasia type 2 (MEN 2) (MIM No 171400), von Hippel- Lindau (VHL) disease (MIM No 199300), and neurofibromatosis type 1 (NF1) (MIM No 162200). In addition, familial phaeochromocytoma alone has also been reported and we and others have identified germline VHL mutations in five of six kindreds analysed previously. Germline mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, and in the VHL tumour suppressor gene cause MEN 2 and VHL disease, respectively. To further investigate the genetics of phaeochromocytoma predisposition, we analysed three groups of patients with no evidence of VHL disease, MEN 2 or NF1: Group A, eight kindreds with familial phaeochromocytoma; Group B, two patients with isolated bilateral phaeochromocytoma; and Group C, six cases of multiple extra- adrenal phaeochromocytoma or adrenal phaeochromocytoma with a family history of neuroectodermal tumours. Germline missense VHL mutations were identified in three of eight kindreds with familial phaeochromocytoma. A germline VHL mutation was also characterised in one of the two patients with bilateral phaeochromocytoma. No VHL or RET mutations were detected in the final group of patients with multiple extra-adrenal phaeochromocytoma or adrenal phaeochromocytoma with a family history of neuroectodermal tumours. The absence of germline VHL and RET gene mutations in many of these families suggested that other phaeochromoeytoma susceptibility loci may exist. Glial cell line- derived neurotrophic factor (GDNF) has been recently identified as a natural ligand for RET. Thus, it seems plausible that GDNF is a good candidate gene to play a role in phaeochromocytoma susceptibility. We searched for germline mutations in GDNF in 16 cases of familial phaeochromocytoma (groups A, B and C) and looked for evidence of somatic change in GDNF in 28 sporadic phaeochromocytomas, 12 MEN 2 phaeochromocytomas and five VHL phaeochromocytomas. No GDNF mutations were identified in patients with familial phaeochromocytoma disease, but a c277C-->T (R93W) sequence variant was identified in one of 28 sporadic tumours. This candidate mutation was identified in the germline and tumour tissue but was not present in 104 control GDNF alleles. GDNF sequence variants including R93W have been suggested previously to represent low penetrance susceptibility mutations for Hirschsprung disease and the R93W was not identified in 376 control alleles studied by others. These findings suggest that although GDNF mutations do not appear to have a major role in the pathogenesis of familial or sporadic phaeochromocytomas, allelic variation at the GDNF locus may modify phaeochromocytoma susceptibility.      

LKB1 interacts with and phosphorylates PTEN: a functional link between two proteins involved in cancer predisposing syndromes

Germline mutations of the LKB1 (STK11) tumor suppressor genelead to Peutz-Jeghers syndrome (PJS) and predisposition to cancer.LKB1 encodes a serine/threonine kinase generally inactivatedin PJS patients. We identified the dual phosphatase and tumorsuppressor protein PTEN as an LKB1-interacting protein. SeveralLKB1 point mutations associated with PJS disrupt the interactionwith PTEN suggesting that the loss of this interaction mightcontribute to PJS. Although PTEN and LKB1 are predominantlycytoplasmic and nuclear, respectively, their interaction leadsto a cytoplasmic relocalization of LKB1. In addition, we showthat PTEN is a substrate of the kinase LKB1 in vitro. As PTENis a dual phosphatase mutated in autosomal inherited disorderswith phenotypes similar to those of PJS (Bannayan–Riley–Ruvalcabasyndrome and Cowden disease), our study suggests a functionallink between the proteins involved in different hamartomatouspolyposis syndromes and emphasizes the central role played byLKB1 as a tumor suppressor in the small intestine.     

Novel mutations in the LKB1/STK11 gene in Dutch Peutz‐Jeghers families

The Peutz‐Jeghers syndrome (PJS) is a rare hereditary disorder in which gastrointestinal hamartomatous polyposis, mucocutaneous pigmentation, and a predisposition for developing cancer are transmitted in an autosomal dominant fashion. The recently identified LKB1/STK11 gene located at chromosome 19p13.3 is mutated in a number of PJS pedigrees. We performed mutation analysis in 19, predominantly Dutch, PJS families. In 12 of these families, we identified LKB1/STK11 mutations, none of which has been described before. These 12 novel LKB1/STK11 mutations consist of one nonsense mutation, three frameshift deletions, three frameshift insertions, two acceptor splice site mutations, and three missense mutations. In addition, we detected four polymorphisms in LKB1/STK11. In the remaining seven PJS families, we found no apparent abnormalities of the LKB1/STK11 gene, which could reflect the existence of locus heterogeneity in PJS. None of the mutations occurred in more than one family, and a number were demonstrated to have arisen de novo. The diverse array of mutations found, the apparent high mutation rate, as well as the existence of a possible second PJS locus, renders diagnostic or predictive genetic testing in individual patients difficult, although future identification of additional mutations or even gene(s) will help in increasing the yield of direct mutation analysis. Hum Mutat 13:476–481, 1999. © 1999 Wiley‐Liss, Inc.     

Microsatellite instability and mutation analysis of hMSH2 and hMLH1 in patients with sporadic, familial and hereditary colorectal cancer

To date, at least four genes involved in DNA mismatch repair, hMSH2, hMLH1, hPMS1 and hPMS2, have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colorectal cancer (HNPCC). Additionally, defective mismatch repair is thought to account for the observation of microsatellite instability (MIN) in tumors from these patients. The genetic defect responsible for the MIN+ phenotype in sporadic colorectal cancer, however, has yet to be clearly delineated. In order to better understand the role of somatic and germline alterations within hMSH2 and hMLH1 in the process of colorectal tumorigenesis, we examined the entire coding regions of both of these genes in seven patients with MIN+ sporadic colorectal cancer, 19 patients with familial colorectal cancer, and 20 patients meeting the strict Amsterdam criteria for HNPCC. Thirteen germline, two somatic, and four neutral alterations were identified. The two somatic mutations occurred in patients having familial cancer, while the germline mutations were distributed among one sporadic (14%), three familial (16%), and nine HNPCC (45%) cases. All patients with identified mutations in the mismatch repair genes, whose tumors were available for analysis, demonstrated MIN. On the other hand, we could not identify mutations in the subset of clinically defined HNPCC patients with MIN negative tumors nor in the majority (6/7) of MIN+ sporadic tumors.      

Desmoglein 2 is expressed abnormally rather than mutated in familial and sporadic gastric cancer

Alterations of the cell adhesion molecule E-cadherin have been demonstrated in sporadic and hereditary gastric carcinomas. A cell adhesion molecule with functional similarity to E-cadherin is desmoglein 2 (Dsg2), a major component of the desmosomes. In this study, we investigated whether alterations of Dsg2 are involved in gastric carcinogenesis and whether germline mutations contribute to a genetic predisposition in familial gastric cancer patients with no germline mutations in the E-cadherin gene. Seventy-five formalin-fixed, paraffin-embedded tissues from 37 familial and 38 sporadic gastric carcinomas were analysed for Dsg2 expression by immunohistochemistry. DNA from 31 familial gastric cancer patients was analysed for germline mutations and five sporadic tumours were analysed for somatic mutations by DHPLC. Of the 75 tumours, 25 (33%) demonstrated abnormal (reduced and/or non-membrane-associated) Dsg2 expression. There was a trend towards more frequent abnormal expression in diffuse type (42%) than in intestinal type tumours (18%) (p = 0.066). One germline missense variant leading to a non-conservative amino acid change (c. 2810 C > A, Thr 937 Asn) was found in a familial gastric cancer patient with a diffuse type tumour. No somatic mutations were identified. The observed abnormal expression of Dsg2 protein suggests that this molecule is involved in the carcinogenesis of a subset of gastric carcinomas, in particular of the diffuse type. Somatic mutations in the gene do not seem to be a very frequent inactivation event and the finding of no clear pathogenic germline mutation rules out Dsg2 as a major gastric cancer predisposition gene.     

Growth arrest by the LKB1 tumor suppressor: induction of p21(WAF1/CIP1)

Germline mutations of the LKB1 tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS), with a predisposition to cancer. LKB1 encodes for a nuclear and cytoplasmic serine/threonine kinase, which is inactivated by mutations observed in PJS patients. Restoring LKB1 activity into cancer cell lines defective for its expression results in a G(1) cell cycle arrest. Here we have investigated molecular mechanisms leading to this arrest. Reintroduced active LKB1 was cytoplasmic and nuclear, whereas most kinase-defective PJS mutants of LKB1 localized predominantly to the nucleus. Moreover, when LKB1 was forced to remain cytoplasmic through disruption of the nuclear localization signal, it retained full growth suppression activity in a kinase-dependent manner. LKB1-mediated G(1) arrest was found to be bypassed by co-expression of the G(1) cyclins cyclin D1 and cyclin E. In addition, the protein levels of the CDK inhibitor p21(WAF1/CIP1) and p21 promoter activity were specifically upregulated in LKB1-transfected cells. Both the growth arrest and the induction of the p21 promoter were found to be p53-dependent. These results suggest that growth suppression by LKB1 is mediated through signaling of cytoplasmic LKB1 to induce p21 through a p53-dependent mechanism.