Goutham Narla

A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk

Prostate cancer is a leading and increasingly prevalent cause of cancer death in men. Whereas family history of disease is one of the strongest prostate cancer risk factors and suggests a hereditary component, the predisposing genetic factors remain unknown. We first showed that KLF6 is a tumor suppressor somatically inactivated in prostate cancer and since then, its functional loss has been further established in prostate cancer cell lines and other human cancers. Wild-type KLF6, but not patient-derived mutants, suppresses cell growth through p53-independent transactivation of p21. Here we show that a germline KLF6 single nucleotide polymorphism, confirmed in a tri-institutional study of 3,411 men, is significantly associated with an increased relative risk of prostate cancer in men, regardless of family history of disease. This prostate cancer-associated allele generates a novel functional SRp40 DNA binding site and increases transcription of three alternatively spliced KLF6 isoforms. The KLF6 variant proteins KLF6-SV1 and KLF6-SV2 are mislocalized to the cytoplasm, antagonize wtKLF6 function, leading to decreased p21 expression and increased cell growth, and are up-regulated in tumor versus normal prostatic tissue. Thus, these results are the first to identify a novel mechanism of self-encoded tumor suppressor gene inactivation and link a relatively common single nucleotide polymorphism to both regulation of alternative splicing and an increased risk in a major human cancer.

The Kruppel-Like Factor 6 Genotype Is Associated With Fibrosis in Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Although nonalcoholic fatty liver disease (NAFLD) is increasingly common, only a minority of affected individuals develop fibrotic liver disease. Based on its role in liver growth and repair, we explored whether Kruppel-like factor 6 (KLF6) plays a role in NAFLD progression. METHODS KLF6 expression in 31 fibrosis scored NAFLD liver biopsy specimens was assessed by real-time polymerase chain reaction. Transfected minigene constructs were used to study the effect of a polymorphism, KLF6-IVS1-27G>A, that promotes KLF6 alternative splicing in vitro. We genotyped KLF6-IVS1-27G>A in 3 groups of patients (UK group 1, n = 306; Italian group 2, n = 109; trio group 3, n = 61 children and parents). RESULTS KLF6 expression was increased in association with increased steatosis, inflammation, and fibrosis in NAFLD livers. KLF6-IVS1-27G>A promoted alternative splicing of KLF6 and abrogated the up-regulation of both alpha-smooth muscle actin and collagen 1 in LX-2 cells. Group 1 genotyping identified KLF6-IVS1-27G>A in 44 of 306 (14.4%) patients. Notably, KLF6-IVS1-27G>A was associated significantly with milder NAFLD, with only 25% having more advanced fibrosis compared with 45% of wild-type (wt) individuals. This trend was confirmed in group 2. A linear regression analysis including all 415 patients, adjusted for age, sex, body mass index, and blood glucose level, confirmed that presence of the wt KLF6 allele was an independent predictor of fibrotic NAFLD. Furthermore, we have shown preferential transmission of the wt allele to children with fibrotic NAFLD. CONCLUSIONS We report a functional polymorphism in the KLF6 gene associated with advanced NAFLD and believe further study of KLF6 may enhance our understanding of this disease process.

Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread.

Prostate cancer is a leading cause of cancer death in men. Risk prognostication, treatment stratification, and the development of rational therapeutic strategies lag because the molecular mechanisms underlying the initiation and progression from primary to metastatic disease are unknown. Multiple lines of evidence now suggest that KLF6 is a key prostate cancer tumor suppressor gene including loss and/or mutation in prostate cancer tumors and cell lines and decreased KLF6 expression levels in recurrent prostate cancer samples. Most recently, we identified a common KLF6 germ line single nucleotide polymorphism that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Here we show that although wild-type KLF6 (wtKLF6) acts as a classic tumor suppressor, the single nucleotide polymorphism-increased splice isoform, KLF6 SV1, displays a markedly opposite effect on cell proliferation, colony formation, and invasion. In addition, whereas wtKLF6 knockdown increases tumor growth in nude mice >2-fold, short interfering RNA-mediated KLF6 SV1 inhibition reduces growth by approximately 50% and decreases the expression of a number of growth- and angiogenesis-related proteins. Together, these findings begin to highlight a dynamic and functional antagonism between wtKLF6 and its splice variant KLF6 SV1 in tumor growth and dissemination.

microRNA-181a has a critical role in ovarian cancer progression through the regulation of the epithelial-mesenchymal transition.

Ovarian cancer is a leading cause of cancer deaths among women. Effective targets to treat advanced epithelial ovarian cancer (EOC) and biomarkers to predict treatment response are still lacking because of the complexity of pathways involved in ovarian cancer progression. Here we show that miR-181a promotes TGF-β-mediated epithelial-to-mesenchymal transition via repression of its functional target, Smad7. miR-181a and phosphorylated Smad2 are enriched in recurrent compared with matched-primary ovarian tumours and their expression is associated with shorter time to recurrence and poor outcome in patients with EOC. Furthermore, ectopic expression of miR-181a results in increased cellular survival, migration, invasion, drug resistance and in vivo tumour burden and dissemination. In contrast, miR-181a inhibition via decoy vector suppression and Smad7 re-expression results in significant reversion of these phenotypes. Combined, our findings highlight an unappreciated role for miR-181a, Smad7, and the TGF-β signalling pathway in high-grade serous ovarian cancer.

Mutations in Capillary Morphogenesis Gene-2 Result in the Allelic Disorders Juvenile Hyaline Fibromatosis and Infantile Systemic Hyalinosis

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are autosomal recessive syndromes of unknown etiology characterized by multiple, recurring subcutaneous tumors, gingival hypertrophy, joint contractures, osteolysis, and osteoporosis. Both are believed to be allelic disorders; ISH is distinguished from JHF by its more severe phenotype, which includes hyaline deposits in multiple organs, recurrent infections, and death within the first 2 years of life. Using the previously reported chromosome 4q21 JHF disease locus as a guide for candidate-gene identification, we identified and characterized JHF and ISH disease-causing mutations in the capillary morphogenesis factor-2 gene (CMG2). Although CMG2 encodes a protein upregulated in endothelial cells during capillary formation and was recently shown to function as an anthrax-toxin receptor, its physiologic role is unclear. Two ISH family-specific truncating mutations, E220X and the 1-bp insertion P357insC that results in translation of an out-of-frame stop codon, were generated by site-directed mutagenesis and were shown to delete the CMG-2 transmembrane and/or cytosolic domains, respectively. An ISH compound mutation, I189T, is predicted to create a novel and destabilizing internal cavity within the protein. The JHF family-specific homoallelic missense mutation G105D destabilizes a von Willebrand factor A extracellular domain alpha-helix, whereas the other mutation, L329R, occurs within the transmembrane domain of the protein. Finally, and possibly providing insight into the pathophysiology of these diseases, analysis of fibroblasts derived from patients with JHF or ISH suggests that CMG2 mutations abrogate normal cell interactions with the extracellular matrix.

Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1

Kruppel-like factor 6 (KLF6) is a tumor suppressor gene inactivated in prostate and colon cancers, as well as in astrocytic gliomas. Here, we establish that KLF6 mediates growth inhibition through an interaction with cyclin D1, leading to reduced phosphorylation of the retinoblastoma protein (Rb) at Ser795. Furthermore, introduction of KLF6 disrupts cyclin D1-cyclin-dependent kinase (cdk) 4 complexes and forces the redistribution of p21Cip/Kip onto cdk2, which promotes G1 cell cycle arrest. Our data suggest that KLF6 converges with the Rb pathway to inhibit cyclin D1/cdk4 activity, resulting in growth suppression.

Frequent inactivation of the tumor suppressor Kruppel-like factor 6 (KLF6) in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, reflecting incomplete characterization of underlying mechanisms and lack of early detection. Krüppel‐like factor 6 (KLF6) is a ubiquitously expressed zinc finger transcription factor that is deregulated in multiple cancers through loss of heterozygosity (LOH) and/or inactivating somatic mutation. We analyzed the potential role of the KLF6 tumor suppressor gene in 41 patients who had HCC associated with hepatitis C virus (16 patients), hepatitis B virus (12 patients, one of whom was coinfected with hepatitis C virus), and other etiologies (14 patients) by determining the presence of LOH and mutations. Overall, LOH and/or mutations were present in 20 (49%) of 41 tumors. LOH of the KLF6 gene locus was present in 39% of primary HCCs, and the mutational frequency was 15%. LOH and/or mutations were distributed across all etiologies of HCC evaluated, including patients who did not have cirrhosis. Functionally, wild‐type KLF6 decreased cellular proliferation of HepG2 cells, while patient‐derived mutants did not. In conclusion, we propose that KLF6 is deregulated by loss and/or mutation in HCC, and its inactivation may contribute to pathogenesis in a significant number of these tumors. (HEPATOLOGY 2004;40:1047–1052.)

KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis

Metastatic prostate cancer (PCa) is one of the leading causes of death from cancer in men. The molecular mechanisms underlying the transition from localized tumor to hormone-refractory metastatic PCa remain largely unknown, and their identification is key for predicting prognosis and targeted therapy. Here we demonstrated that increased expression of a splice variant of the Kruppel-like factor 6 (KLF6) tumor suppressor gene, known as KLF6-SV1, in tumors from men after prostatectomy predicted markedly poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that KLF6-SV1 levels were specifically upregulated in hormone-refractory metastatic PCa. In 2 complementary mouse models of metastatic PCa, KLF6-SV1-overexpressing PCa cells were shown by in vivo and ex vivo bioluminescent imaging to metastasize more rapidly and to disseminate to lymph nodes, bone, and brain more often. Interestingly, while KLF6-SV1 overexpression increased metastasis, it did not affect localized tumor growth. KLF6-SV1 inhibition using RNAi induced spontaneous apoptosis in cultured PCa cell lines and suppressed tumor growth in mice. Together, these findings demonstrate that KLF6-SV1 expression levels in PCa tumors at the time of diagnosis can predict the metastatic behavior of the tumor; thus, KLF-SV1 may represent a novel therapeutic target.

Roles of KLF6 and KLF6-SV1 in Ovarian Cancer Progression and Intraperitoneal Dissemination

Purpose: We investigated the role of the KLF6 tumor suppressor gene and its alternatively spliced isoform KLF6-SV1 in epithelial ovarian cancer (EOC). Experimental Design: We first analyzed tumors from 68 females with EOC for KLF6 gene inactivation using fluorescent loss of heterozygosity (LOH) analysis and direct DNA sequencing and then defined changes in KLF6 and KLF6-SV1 expression levels by quantitative real-time PCR. We then directly tested the effect of KLF6 and KLF6-SV1 inhibition in SKOV-3 stable cell lines on cellular invasion and proliferation in culture and tumor growth, i.p. dissemination, ascites production, and angiogenesis in vivo using BALB/c nu/nu mice. All statistical tests were two sided. Results: LOH was present in 59% of samples in a cell type–specific manner, highest in serous (72%) and endometrioid (75%) subtypes, but absent in clear cell tumors. LOH was significantly correlated with tumor stage and grade. In addition, KLF6 expression was decreased in tumors when compared with ovarian surface epithelial cells. In contrast, KLF6-SV1 expression was increased ∼5-fold and was associated with increased tumor grade regardless of LOH status. Consistent with these findings, KLF6 silencing increased cellular and tumor growth, angiogenesis, and vascular endothelial growth factor expression, i.p. dissemination, and ascites production. Conversely, KLF6-SV1 down-regulation decreased cell proliferation and invasion and completely suppressed in vivo tumor formation. Conclusion: Our results show that KLF6 and KLF6-SV1 are associated with key clinical features of EOC and suggest that their therapeutic targeting may alter ovarian cancer growth, progression, and dissemination.

Ribosome-inactivating proteins isolated from dietary bitter melon induce apoptosis and inhibit histone deacetylase-1 selectively in premalignant and malignant prostate cancer cells

Epidemiologic evidence suggests that a diet rich in fruits and vegetables is associated with a reduced risk of prostate cancer (PCa) development. Although several dietary compounds have been tested in preclinical PCa prevention models, no agents have been identified that either prevent the progression of premalignant lesions or treat advanced disease. Momordica charantia, known as bitter melon in English, is a plant that grows in tropical areas worldwide and is both eaten as a vegetable and used for medicinal purposes. We have isolated a protein, designated as MCP30, from bitter melon seeds. The purified fraction was verified by SDS‐PAGE and mass spectrometry to contain only 2 highly related single chain Type I ribosome‐inactivating proteins (RIPs), α‐momorcharin and β‐momorcharin. MCP30 induces apoptosis in PIN and PCa cell lines in vitro and suppresses PC‐3 growth in vivo with no effect on normal prostate cells. Mechanistically, MCP30 inhibits histone deacetylase‐1 (HDAC‐1) activity and promotes histone‐3 and ‐4 protein acetylation. Treatment with MCP30 induces PTEN expression in a prostatic intraepithelial neoplasia (PIN) and PCa cell lines resulting in inhibition of Akt phosphorylation. In addition, MCP30 inhibits Wnt signaling activity through reduction of nuclear accumulation of β‐catenin and decreased levels of c‐Myc and Cyclin‐D1. Our data indicate that MCP30 selectively induces PIN and PCa apoptosis and inhibits HDAC‐1 activity. These results suggest that Type I RIPs derived from plants are HDAC inhibitors that can be utilized in the prevention and treatment of prostate cancer.