Cancer-predisposition gene KLLN maintains pericentric H3K9 trimethylation protecting genomic stability.

Maintenance of proper chromatin states and genomic stability is vital for normal development and health across a range of organisms. Here, we report on the role of KLLN in maintenance of pericentric H3K9 trimethylation (H3K9me3) and genomic stability. Germline hypermethylation of KLLN, a gene uncovered well after the human genome project, has been linked to Cowden cancer-predisposition syndrome (CS) in PTEN wild-type cases. KLLN first identified as a p53-dependent tumor suppressor gene, was believed to bind randomly to DNA and cause S-phase arrest. Using chromatin immunoprecipitation-based sequencing (ChIP-seq), we demonstrated that KLLN binds to DNA regions enriched with H3K9me3. KLLN overexpression correlated with increased H3K9 methyltransferase activity and increased global H3K9me3, while knockdown of KLLN had an opposite effect. We also found KLLN to localize to pericentric regions, with loss of KLLN resulting in dysregulation of pericentric heterochromatin, with consequent chromosomal instability manifested by increased micronuclei formation and numerical chromosomal aberrations. Interestingly, we show that KLLN interacts with DBC1, with consequent abrogation of DBC1 inhibition of SUV39H1, a H3K9 methyltransferase, suggesting the mode of KLLN regulating H3K9me3. These results suggest a critical role for KLLN as a potential regulator of pericentric heterochromatin formation, genomic stability and gene expression.

Germline and somatic KLLN alterations in breast cancer dysregulate G2 arrest.

PTEN is a well-described predisposition gene for Cowden syndrome (CS), a familial cancer syndrome characterized by a high risk of breast and other cancers. KLLN, which shares a bidirectional promoter with PTEN, causes cell cycle arrest and apoptosis. We previously identified germline hypermethylation of the KLLN promoter in 37% of PTEN mutation-negative CS/CS-like (CSL) patients. Patients with germline KLLN hypermethylation have an increased prevalence of breast and renal cancers when compared with PTEN mutation carriers. We have consequently sought to identify and characterize germline KLLN variants/mutations in CS/CSL and in apparently sporadic breast cancer patients. KLLN variants in CS/CSL patients are rare (1 of 136, 0.007%). Interestingly, among 438 breast cancer patients, 13 (3%) have germline KLLN variants when compared with none in 128 controls (P = 0.049). Patients with KLLN variants have a family history of breast cancer when compared with those without (P = 0.02). We demonstrate that germline KLLN variants dysregulate the cell cycle at G2. Of 24 breast carcinomas analyzed, 3 (13%) have somatic KLLN hemizygous deletions, with somatic loss of the wild-type allele in a patient with germline KLLN p.Leu119Leu. Of 452 breast carcinomas in The Cancer Genome Atlas project, 93 (21%) have KLLN hemizygous or homozygous deletions. This is the first study to associate germline KLLN variants with sporadic breast cancer and to recognize somatic KLLN deletions in breast carcinomas. Our observations suggest that KLLN may be a low penetrance susceptibility factor for apparently sporadic breast cancer.

Germline PTEN, SDHB-D, and KLLN alterations in endometrial cancer patients with Cowden and Cowden-like syndromes: an international, multicenter, prospective study.

BACKGROUND: Endometrial cancer has been recognized only recently as a major component of Cowden syndrome (CS). Germline alterations in phosphatase and tensin homolog (PTEN; PTEN_mut+), succinate dehydrogenase B/C/D (SDHB-D; SDHx_var+), and killin (KLLN_Me+) cause CS and Cowden syndrome-like (CSL) phenotypes. This study was aimed at identifying the prevalence and clinicopathologic predictors of germline PTEN_mut+, SDHx_var+, and KLLN_Me+ in CS/CSL patients presenting with endometrial cancer. METHODS: PTEN and SDHB-D mutation and KLLN promoter methylation analyses were performed for 371 prospectively enrolled patients (2005-2011). PTEN protein was analyzed from patient-derived lymphoblast lines. The PTEN Cleveland Clinic (CC) score is a weighted, regression-based risk calculator giving the a priori risk for PTEN_mut+. Demographic and clinicopathologic features were correlated with the specific gene. RESULTS: Germline PTEN_mut+, SDHx_var+, and KLLN_Me+ were found in 7%, 9.8%, and 10.5% of informative samples, respectively. Predictors of PTEN_mut+ included an age ≤ 50 years (odds ratio [OR] for an age < 30 years, 6.1 [P = .015]; OR for an age of 30-50 years, 4.4 [P = .001]), macrocephaly (OR, 14.4; P < .001), a higher CC score (OR for a 1-U increment, 1.35; P < .001), a PTEN protein level within the lowest quartile (OR, 5.1; P = .039), and coexisting renal cancer (OR, 5.7; P = .002). KLLN_Me+ patients were on average 8 years younger than KLLN_Me- patients (44 vs 52 years, P = .018). Predictors of KLLN_Me+ were a younger age and a higher CC score. On the other hand, no clinical predictors of SDH_var+ were found. CONCLUSIONS: Clinical predictors of PTEN and KLLN alterations, but not SDHx_var+, were identified. These predictors should alert the treating physician to potential heritable risk and the need for referral to genetic professionals. High-risk cancer surveillance and prophylactic surgery of the uterus may be considered for KLLN_Me+ patients similarly to PTEN_mut+ patients.

KLLN epigenotype-phenotype associations in Cowden syndrome.

Germline KLLN promoter hypermethylation was recently identified as a potential genetic etiology of the cancer predisposition syndrome, Cowden syndrome (CS), when no causal PTEN gene mutation was found. We screened for KLLN promoter methylation in a large prospective series of CS patients and determined the risk of benign and malignant CS features in patients with increased methylation both with and without a PTEN mutation/variant of unknown significance. In all, 1012 CS patients meeting relaxed International Cowden Consortium criteria including 261 PTEN mutation-positive CS patients, 187 PTEN variant-positive CS patients and 564 PTEN mutation-negative CS patients, as well as 111 population controls were assessed for germline KLLN promoter methylation by MassARRAY EpiTYPER analysis. KLLN promoter methylation was analyzed both as a continuous and a dichotomous variable in the calculation of phenotypic risks by stepwise logistic regression and Kaplan-Meier/standardized incidence ratio methods, respectively. Significantly increased KLLN promoter methylation was seen in CS individuals with and without a PTEN mutation/VUS compared with controls (P<0.001). Patients with high KLLN promoter methylation have increased risks of all CS-associated malignancies compared with the general population. Interestingly, KLLN-associated risk of thyroid cancer appears to be gender and PTEN status dependent. KLLN promoter methylation associated with different benign phenotypes dependent on PTEN status. Furthermore, increasing KLLN promoter methylation is associated with a greater phenotype burden in mutation-negative CS patients. Germline promoter hypermethylation of KLLN is associated with particular malignant and benign CS features, which is dependent on the PTEN mutation status.