LSD1 inhibitors from the roots of Pueraria lobata.

One new 6a,11a-dehydropterocarpan derivative, 6-O-methyl-anhydrotuberosin (1), one new 6a-hydroxypterocarpan, (6aR,11aR,11bR)-hydroxytuberosone (7), and seven known compounds including two 6a,11a-dehydropterocarpans (2 and 4), two coumestans (3 and 5), one isoflavonoid (6) and two other phenolic compounds (8 and 9) were isolated from the roots of Pueraria lobata. The structures of the isolated compounds were elucidated with spectroscopic and spectrometric methods (1 D and 2DNMR, HRESIMS). Compounds 1, 2, 4-5 showed potent LSD1 inhibitory activities with IC50 values ranging from 1.73 to 4.99 µM. Furthermore, compound 2 showed potent cytotoxicity against gastric cancer cell lines MGC-803 and BGC-823, and lung cancer cell lines H1299 and H460.

[Mechanism of Puerariae Lobatae Radix against lung cancer by inhibiting histone demethylase LSD1].

Histone lysine-specific demethylase 1(LSD1) has become a promising molecular target for lung cancer therapy. Upon the screening platform for LSD1 activity, some Chinese herbal extracts were screened for LSD1 activity inhibition, and the underlying mechanism was preliminarily investigated at both molecular and cellular levels. The results of LSD1 inhibition showed that Puerariae Lobatae Radix extract can effectively reduce LSD1 expression to elevate the expression of H3 K4 me2 and H3 K9 me2 substrates in H1975 and H1299 cells. Furthermore, Puerariae Lobatae Radix was evaluated for its anti-lung cancer activity. It had a potent inhibitory ability against the proliferation and colony formation of both H1975 and H1299 cells. Flow cytometry and DAPI staining assays indicated that Puerariae Lobatae Radix can induce the apoptosis of lung cancer cells. In addition, it can significantly suppress the migration and reverse the epithelial-mesenchymal transition(EMT) process of lung cancer cells by activating E-cadherin and suppressing the expression of N-cadherin, slug and vimentin. To sum up, Puerariae Lobatae Radix displayed a robust inhibitory activity against lung cancer, and the mechanism may be related to the down-regulation of LSD1 expression to induce the cell apoptosis and suppress the cell migration and EMT process. These findings will provide new insights into the action of Puerariae Lobatae Radix as an anti-lung cancer agent and offer new ideas for the study on the anti-cancer action of Chinese medicine based on the epigenetic modification.

Lysine-specific histone demethylase 1A (LSD1) in cervical cancer.

PURPOSE: Demethylation of DNA through enzymes like LSD1 showed a crucial impact on different kind of cancers. Epigenetic modifications in cervical cancer are still not fully investigated nevertheless of high interest for a therapeutic use. METHODS: Tumor samples of 250 cervical cancer patients were immunochemically stained and evaluated based on Immunoreactive Score. Results were statistically analyzed for clinical and pathological parameters. RESULTS: Our patient collective showed a disadvantage for 10-year survival for patients with a strong expression of LSD1 in the cytoplasm of cervical cancer cells. The results of the correlational analysis further revealed a negative correlation of LSD1 to G-protein coupled estrogen receptor (GPER). CONCLUSIONS: Epigenetic changes through enzymes like LSD1 may also be of interest for patients with cervical cancer. A combined therapy with other proteins relayed to cervical cancer like GPER might be of interest for future investigations.

Rapid proteomic analysis for solid tumors reveals LSD1 as a drug target in an end-stage cancer patient.

Recent advances in mass spectrometry (MS)-based technologies are now set to transform translational cancer proteomics from an idea to a practice. Here, we present a robust proteomic workflow for the analysis of clinically relevant human cancer tissues that allows quantitation of thousands of tumor proteins in several hours of measuring time and a total turnaround of a few days. We applied it to a chemorefractory metastatic case of the extremely rare urachal carcinoma. Quantitative comparison of lung metastases and surrounding tissue revealed several significantly upregulated proteins, among them lysine-specific histone demethylase 1 (LSD1/KDM1A). LSD1 is an epigenetic regulator and the target of active development efforts in oncology. Thus, clinical cancer proteomics can rapidly and efficiently identify actionable therapeutic options. While currently described for a single case study, we envision that it can be applied broadly to other patients in a similar condition.

Silencing of LSD1 gene modulates histone methylation and acetylation and induces the apoptosis of JeKo-1 and MOLT-4 cells.

Lysine-specific demethylase 1 (LSD1) has been identified and biochemically characterized in epigenetics; however, the pathological roles of its dysfunction in mantle cell lymphoma (MCL) and T-cell acute lymphoblastic leukemia remain to be elucidated. In this study, we evaluated LSD1, and histone H3 lysine 4 (H3K4)me1 and H3K4me2 expression in patients with MCL and silenced LSD1 in JeKo­1 and MOLT­4 cells, in order to define its role in JeKo­1 and MOLT­4 cell proliferation and apoptosis. We retrospectively analyzed the protein expression of LSD1, and mono- and dimethylated H3K4 (H3K4me1 and H3K4me2), and cyclin D1 and Ki67 in 30 cases of MCL by immunohistochemistry. The correlation of LSD1, H3K4me1 and H3K4me2 with Ki67 was determined by statistical analysis. LSD1 was silenced by small interfering RNA (siRNA). Cell apoptosis and cell proliferation were detected by flow cytometry and 3-(4,5-dimethylthiazol­2-yl)­2,5-diphenyltetrazolium bromide (MTT) assay. The protein expression levels of LSD1, histone methylated H3K4, histone acetylated H3, cyclin D1, apoptotic proteins, p15 and DNA methyltransferase 1 (DNMT1) were examined by western blot analysis. We demonstrated that LSD1 was upregulated, and that H3K4me1 and H3K4me2 were downregulated in the cases with MCL, compared to those with proliferative lymphadenitis (p<0.05). LSD1 positively correlated with Ki67 in MCL [Cohen's kappa (κ)=0.667, p<0.01]. There was no significant correlation between H3K4me1 and H3K4me2, and Ki67 (κ=-0.182, p>0.05, κ=-0.200, p>0.05). The silencing of LSD1 decreased the levels of the apoptosis-related proteins, Bcl-2, pro-caspase-3 and C-myc, and decreased those of DNMT1 and increased p15, and resulted in the loss of cell viability and the induction apoptosis. The silencing of LSD1 increased the expression of H3K4me1 and H3K4me2, and histone acetylated H3 in the JeKo­1 and MOLT­4 cells. LSD1 siRNA also decreased cyclin D1 expression in the JeKo­1 cells. On the whole, our findings demonstrate that the overexpression of LSD1 may be associated with the pathogenesis in MCL. We demonstrated that the silencing of LSD1 is capable of removing the mono- and dimethyl groups from H3K4, and upregulating the histone acetylation of H3 in JeKo­1 and MOLT­4 cells. The silencing of LSD1 inhibited cell growth and induced cell apoptosis. Of note, in JeKo­1 cells, the silencing of LSD1 decreased cyclin D1 expression, which is one of the genes that contribute to the pathogenesis of MCL. LSD1 may thus be a possible therapeutic target in MCL and acute lymphoblastic leukemia MOLT­4 cells.

SETDB1 plays an essential role in maintenance of gonocyte survival in pigs.

Histone methyltransferase SETDB1 suppresses gene expression and modulates heterochromatin formation through H3K9me2/3. Previous studies have revealed that SETDB1 catalyzes lysine 9 of histone H3 tri-methylation and plays essential roles in maintaining the survival of embryonic stem cells and spermatogonial stem cells in mice. However, the function of SETDB1 in porcine male germ cells remains unclear. The aim of the present study was to reveal the expression profile and function of SETDB1 in porcine germ cells. SETDB1 expression gradually increased during testis development. SETDB1 was strongly localized in gonocytes. Knockdown of SETDB1 gene expression led to gonocyte apoptosis and a decrease in H3K27me3, but no significant change in H3K9me3. These observations suggested that SETDB1 is a novel epigenetic regulator of porcine male germ cells, and contributes to the maintenance of gonocyte survival in pigs, probably due to the regulation of H3K27me3 rather than H3K9me3. These findings will provide a theoretical basis for the future study of epigenetic regulation of spermatogenesis.

Role of Histone Demethylases in Cardiomyocytes Induced to Hypertrophy.

Epigenetic changes induced by histone demethylases play an important role in differentiation and pathological changes in cardiac cells. However, the role of the jumonji family of demethylases in the development of cardiac hypertrophy remains elusive. In this study, the presence of different histone demethylases in cardiac cells was evaluated after hypertrophy was induced with neurohormones. A cell line from rat cardiomyocytes was used as a biological model. The phenotypic profiles of the cells, as well as the expression of histone demethylases, were studied through immunofluorescence, transient transfection, western blot, and qRT-PCR analysis after inducing hypertrophy by angiotensin II and endothelin-1. An increase in fetal gene expression (ANP, BNP, and ß-MHC) was observed in cardiomyocytes after treatment with angiotensin II and endothelin-1. A significant increase in JMJD2A expression, but not in UTX or JMJD2C expression, was observed. When JMJD2A was overexpressed in cardiomyocytes through transient transfection, the effect of neurohormones on fetal cardiac gene expression was increased. We conclude that JMJD2A plays a principal role in the regulation of fetal cardiac genes, which increase in expression during the pathological hypertrophic process.

Assembly of methylated KDM1A and CHD1 drives androgen receptor-dependent transcription and translocation.

Prostate cancer evolution is driven by a combination of epigenetic and genetic alterations such as coordinated chromosomal rearrangements, termed chromoplexy. TMPRSS2-ERG gene fusions found in human prostate tumors are a hallmark of chromoplexy. TMPRSS2-ERG fusions have been linked to androgen signaling and depend on androgen receptor (AR)-coupled gene transcription. Here, we show that dimethylation of KDM1A at K114 (to form K114me2) by the histone methyltransferase EHMT2 is a key event controlling androgen-dependent gene transcription and TMPRSS2-ERG fusion. We identified CHD1 as a KDM1A K114me2 reader and characterized the KDM1A K114me2-CHD1 recognition mode by solving the cocrystal structure. Genome-wide analyses revealed chromatin colocalization of KDM1A K114me2, CHD1 and AR in prostate tumor cells. Together, our data link the assembly of methylated KDM1A and CHD1 with AR-dependent transcription and genomic translocations, thereby providing mechanistic insight into the formation of TMPRSS2-ERG gene fusions during prostate-tumor evolution.

Integrating Tumor and Stromal Gene Expression Signatures With Clinical Indices for Survival Stratification of Early-Stage Non-Small Cell Lung Cancer.

BACKGROUND: Accurate survival stratification in early-stage non-small cell lung cancer (NSCLC) could inform the use of adjuvant therapy. We developed a clinically implementable mortality risk score incorporating distinct tumor microenvironmental gene expression signatures and clinical variables. METHODS: Gene expression profiles from 1106 nonsquamous NSCLCs were used for generation and internal validation of a nine-gene molecular prognostic index (MPI). A quantitative polymerase chain reaction (qPCR) assay was developed and validated on an independent cohort of formalin-fixed paraffin-embedded (FFPE) tissues (n = 98). A prognostic score using clinical variables was generated using Surveillance, Epidemiology, and End Results data and combined with the MPI. All statistical tests for survival were two-sided. RESULTS: The MPI stratified stage I patients into prognostic categories in three microarray and one FFPE qPCR validation cohorts (HR = 2.99, 95% CI = 1.55 to 5.76, P < .001 in stage IA patients of the largest microarray validation cohort; HR = 3.95, 95% CI = 1.24 to 12.64, P = .01 in stage IA of the qPCR cohort). Prognostic genes were expressed in distinct tumor cell subpopulations, and genes implicated in proliferation and stem cells portended poor outcomes, while genes involved in normal lung differentiation and immune infiltration were associated with superior survival. Integrating the MPI with clinical variables conferred greatest prognostic power (HR = 3.43, 95% CI = 2.18 to 5.39, P < .001 in stage I patients of the largest microarray cohort; HR = 3.99, 95% CI = 1.67 to 9.56, P < .001 in stage I patients of the qPCR cohort). Finally, the MPI was prognostic irrespective of somatic alterations in EGFR, KRAS, TP53, and ALK. CONCLUSION: The MPI incorporates genes expressed in the tumor and its microenvironment and can be implemented clinically using qPCR assays on FFPE tissues. A composite model integrating the MPI with clinical variables provides the most accurate risk stratification.

Expression of Lysine-specific demethylase 1 in human epithelial ovarian cancer.

BACKGROUND: Lysine-specific demethylase 1(LSD1) is implicated in the tumorigenesis and progression in various cancers. However, the expression of LSD1 in epithelial ovarian cancer and its clinical significance has not been examined in detail. METHODS: Immunohistochemical was used to detect the expression of LSD1 in normal ovarian epithelial tissues, cystadenoma, borderline cystadenoma, and cystadenocarcinoma. Next, the correlations between expression of LSD1 and clinicopathological features was assessed in 96 species of serous cystadenocarcinoma and 36 species of mucinous cystadenocarcinoma. RESULTS: Immunohistochemical results showed that the expression of LSD1 was gradually increased from benign cystadenoma and borderline cystadenoma to cystadenocarcinoma. The positive ratio of LSD1 expression was 50% in normal ovarian epithelial tissues, 72% in serous cystadenoma, 73% in mucinous cystadenoma, 82% in borderline serous cystadenoma, 83% in borderline mucinous cystadenoma, 94% in serous cystadenocarcinoma and 92% in mucinous cystadenocarcinoma, respectively. LSD1 expression levels were associated with International Federation of Gynecology and Obstetrics stage and lymphatic metastasis in both serous and mucinous cystadenocarcinoma samples. Kaplan-Meier curves suggested that overall survival time of patients with high LSD1 expression was significantly shorter than that of patients with low LSD1 expression. Multivariate Cox proportional hazard regression indicated that higher LSD1 expression was a significant independent predictor of poor survival of EOC patients (P = 0.016). CONCLUSIONS: These results suggest that LSD1 may be involved in carcinogenesis and progression with promising therapeutic potential for epithelial ovarian cancer.

Relationship between LSD1 expression and E-cadherin expression in prostate cancer.

PURPOSE: To investigate the relationship between the expression of LSD1 and E-cadherin in prostate cancer and their prognostic significance. METHODS: The expression of LSD1 and E-cadherin in prostate cancer was detected using immunohistochemistry, and the relationship between the expressions of these two molecules was analyzed by correlation analysis. Furthermore, LNCap cell line was treated with Pargyline (an inhibitor of LSD1), and Western blot was used to analyze LSD1 and E-cadherin expression. RESULTS: LSD1 expression increased significantly in prostate cancer specimens compared with benign prostatic hyperplasia (P < 0.05). Further analysis testified that LSD1 expression was positively correlated with higher Gleason Score, distant metastases, and poor prognosis (P < 0.05). Nevertheless, E-cadherin expression decreased significantly in prostate cancer specimens compared with benign prostatic hyperplasia (P < 0.05) and was negatively correlated with higher Gleason Score, distant metastases (P < 0.05). Correlation analysis revealed that LSD1 expression was negatively correlated with E-cadherin expression in prostate cancer (rs = -0.486, P = 0.001). Positive LSD1 expression and negative E-cadherin expression were significantly correlated with high 2-year progression (occurrence of castration-resistant prostate cancer) rate and low 5-year survival rate (P < 0.05). Moreover, Pargyline inhibited activity of LSD1 and up-regulated E-cadherin expression. CONCLUSION: High LSD1 expression combined with low E-cadherin expression might be predictors of prostate cancer progression and metastasis. Inhibition of LSD1 may be a potential therapeutic target for prevention of prostate cancer.

High expression of the histone demethylase LSD1 associates with cancer cell proliferation and unfavorable prognosis in tongue cancer.

BACKGROUND: The histone lysine-specific demethylase (LSD1) is a key chromatin modifier mediating the demethylation of both H3K4me1/me2 and H3K9 me1/me2. Recently, its deregulation has been implicated in the initiation and progression of various cancers. The aim of this study was to investigate the expression pattern of LSD1 in tongue squamous cell carcinoma (SCC) and determine its prognostic significance in predicting patients' prognosis. METHODS: LSD1 expression was examined by RT-PCR and western blotting in three tongue cancer cell lines and by immunohistochemistry in 63 primary tongue SCC specimens with detailed clinical, pathological, and follow-up data. Its associations with various clinicopathological parameters, Ki-67 expression, and patients' survival were further assessed. RESULTS: Upregulated LSD1 expression was observed in tongue cancer cells and a major fraction of tongue SCC samples. Overexpression of LSD1 significantly associated with tumor size (P = 0.0357), pathological grade (P = 0.0323), Ki-67 abundance (P = 0.0148), and reduced overall and disease-free survival (Kaplan-Meier analysis, P = 0.0351, 0.0479, respectively). The Cox regression survival analyses identified LSD1 as an important independent predictor for patients' overall survival. CONCLUSION: Our data indicate that aberrant LSD1 overexpression associates with key clinicopathological features and unfavorable prognosis in patients with tongue cancer. LSD1 might play critical roles during tongue tumorigenesis and represent a novel biomarker and potential therapeutic target for this malignancy.

Genomic and proteomic dissection and characterization of the human sperm chromatin.

The mammalian spermatozoon has a unique chromatin structure where the majority of DNA is packaged by protamines, while a small fraction (∼8%) remains associated with nucleosomes. However, the chromatin affinity and repertoire of the additional proteins constituting the different sperm chromatin fractions have not yet been explored. To address this we have carried out a genomic and proteomic characterization of human sperm samples subjected to chromatin fractionation using either 0.65 M NaCl extraction followed by EcoRI/BamHI DNA restriction enzyme digestion, or micrococcal nuclease digestion. DNA fractions corresponding to the nucleosome-packaged DNA were sequenced, confirming an appropriate dissection of the sperm chromatin. In addition we detected and sequenced a subnucleosomal particle. Although both fractions were highly enriched at gene promoters, some sequences were found to be exclusively associated with one of those. The results of the proteomic analyses demonstrate that there are two distinct sets of sperm proteins which differ in chromatin affinity. Histone variants, transcription factors, chromatin-associated and modifying proteins involved in regulatory roles were identified as weakly attached to the sperm DNA, whereas proteins with structural roles were identified in the condensed fraction. Many factors, such as the histone lysine demethylase PHF8 identified for the first time in the human sperm cell in this study, were identified exclusively in soluble fraction. Our results provide additional support to the possibility that all of these factors may constitute additional layers of sperm epigenetic information or have structural or regulatory roles transmitted by the sperm cell to the oocyte at fertilization.

The lysine specific demethylase-1 (LSD1/KDM1A) regulates VEGF-A expression in prostate cancer.

Recurrent prostate cancer remains a major clinical challenge. The lysine specific demethylase-1 (LSD1/KDM1A), together with the JmjC domain-containing JMJD2A and JMJD2C proteins, have emerged as critical regulators of histone lysine methylation. The LSD1-JMJD2 complex functions as a transcriptional co-regulator of hormone activated androgen and estrogen receptors at specific gene promoters. LSD1 also regulates DNA methylation and p53 function. LSD1 is overexpressed in numerous cancers including prostate cancer through an unknown mechanism. We investigated expression of the LSD1 and JMJD2A in malignant human prostate specimens. We correlated LSD1 and JMJD2A expression with known mediators of prostate cancer progression: VEGF-A and cyclin A1. We show that elevated expression of LSD1, but not JMJD2A, correlates with prostate cancer recurrence and with increased VEGF-A expression. We show that functional depletion of LSD1 expression using siRNA in prostate cancer cells decreases VEGF-A and blocks androgen induced VEGF-A, PSA and Tmprss2 expression. We demonstrate that pharmacological inhibition of LSD1 reduces proliferation of both androgen dependent (LnCaP) and independent cell lines (LnCaP: C42, PC3). We show a direct mechanistic link between LSD1 over-expression and increased activity of pro-angiogenic pathways. New therapies targeting LSD1 activity should be useful in the treatment of hormone dependent and independent prostate cancer.

Application of MassSQUIRM for quantitative measurements of lysine demethylase activity.

Recently, epigenetic regulators have been discovered as key players in many different diseases (1-3). As a result, these enzymes are prime targets for small molecule studies and drug development( 4). Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity. Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate (5). This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format (5).

LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing.

Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on the chromatin structure of the integrated proviruses. We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter. In the present work, we report that the histone demethylase LSD1 represses HIV-1 transcription and viral expression in a synergistic manner with CTIP2. We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks. Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.

In vitro histone demethylase assay.

Post-translational modifications of histones play an important role in regulating chromatin dynamics and function. One of the modifications, methylation, occurs on both lysine and arginine residues, and methylation status defines the epigenetic program of a cell by determining chromatin structure and thereby regulating DNA-dependent processes such as transcription. Until recently, histone methylation was considered to be irreversible. However, the discovery of histone demethylases revealed that histone methylation is more dynamic than previously recognized. This protocol describes two different in vitro histone demethylase enzyme reactions and three different methods for measuring histone demethylase activity. The first reaction (type I) uses the Fe(II)- and α-ketoglutarate-dependent dioxygenase family of histone demethylase (represented by JmjC domain-containing histone demethylase [JHDM]); the second (type II) is for the flavin adenine dinucleotide (FAD)-dependent amine oxidase family (represented by lysine-specific demethylase 1 [LSD1]). Histone demethylase activity can then be detected by measuring the release of radiolabeled formaldehyde from (3)H-labeled methylated histone substrates, by monitoring the change in methylation levels of histone substrates by immunoblotting with site-specific methylhistone antibodies, or by using mass spectrometry to detect reductions in histone peptide masses that correspond to methyl groups. These assays can be applied to a wide range of histone demethylase studies, including the measurement of histone demethylase activity in tissue and cell lysates, identification of novel histone demethylases, and screening for inhibitors of histone demethylases.