Analisa DiFeo

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.

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.

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.

Myosin IIA associates with NK cell lytic granules to enable their interaction with F-actin and function at the immunological synapse.

NK cell cytotoxicity requires the formation of an actin-rich immunological synapse (IS) with a target cell and the polarization of perforin-containing lytic granules toward the IS. Following the polarization of lytic granules, they traverse through the actin-rich IS to join the NK cell membrane in order for directed secretion of their contents to occur. We examined the role of myosin IIA as a candidate for facilitating this prefinal step in lytic NK cell IS function. Lytic granules in and derived from a human NK cell line, or ex vivo human NK cells, were constitutively associated with myosin IIA. When isolated using density gradients, myosin IIA-associated NK cell lytic granules directly bound to F-actin and the interaction was sensitive to the presence of ATP under conditions of flow. In NK cells from patients with a truncation mutation in myosin IIA, NK cell cytotoxicity, lytic granule penetration into F-actin at the IS, and interaction of isolated granules with F-actin were all decreased. Similarly, inhibition of myosin function also diminished the penetration of lytic granules into F-actin at the IS, as well as the final approach of lytic granules to and their dynamics at the IS. Thus, NK cell lytic granule-associated myosin IIA enables their interaction with actin and final transit through the actin-rich IS to the synaptic membrane, and can be defective in the context of naturally occurring human myosin IIA mutation.

The role of KLF6 and its splice variants in cancer therapy

The Krüppel-like zinc finger transcription factor (KLF6) gene encodes a family of proteins generated through alternative splicing involved in the regulation of cancer development and progression. Alternative splicing of the KLF6 gene results in the production of at least four alternatively spliced isoforms, two of which are extensively discussed in this review. The full length form of the KLF6 gene is a tumor suppressor gene that is frequently inactivated by loss of heterozygozity (LOH), somatic mutation, and/or decreased expression in human cancer. While the exact mechanisms underlying KLF6s tumor suppressor roles are not completely known, a number of highly relevant, overlapping pathways have been described: transactivation of p21 in a p53-independent manner, reduction of cyclin D1/cdk4 complexes via interaction with cyclin D1, inhibition of c-Jun proto-oncoprotein activities, decreased VEGF expression, and induction of apoptosis. Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Increased KLF6-SV1 expression is associated with poor prognosis in prostate, lung, and ovarian cancer. Furthermore, KLF6-SV1 has been shown to be biologically active, antagonizing the tumor suppressor function of KLF6 and promoting tumor growth and dissemination in both ovarian and prostate cancer models. In addition, a common germline polymorphism in the KLF6 gene associated with increased prostate cancer risk in a large multi-institutional study of 3411 men results in increased expression of KLF6-SV1. Furthermore, recent studies have demonstrated that targeted reduction of KLF6-SV1 results in the induction of spontaneous apoptosis in cell culture, synergizes with chemotherapeutic agents like cisplatin, and results in significant tumor regression in vivo. Combined, these data make the KLF6 gene family a compelling therapeutic target for both the treatment of localized as well as metastatic cancer.