Analysis of the levels of lysine-specific demethylase 1 (LSD1) mRNA in human ovarian tumors and the effects of chemical LSD1 inhibitors in ovarian cancer cell lines
© Konovalov and Garcia-Bassets; licensee BioMed Central Ltd. 2013
Received: 17 July 2013
Accepted: 18 October 2013
Published: 29 October 2013
Lysine-specific demethylase 1 (LSD1, also known as KDM1A and AOF2) is a chromatin-modifying activity that catalyzes the removal of methyl groups from lysine residues in histone and non-histone proteins, regulating gene transcription. LSD1 is overexpressed in several cancer types, and chemical inhibition of the LSD1 activity has been proposed as a candidate cancer therapy. Here, we examine the levels of LSD1 mRNA in human ovarian tumors and the cytotoxicity of several chemical LSD1 inhibitors in a panel of ovarian cancer cell lines.
We measured LSD1 mRNA levels in a cohort of n = 177 normal and heterogeneous tumor specimens by quantitative real time-PCR (qRT-PCR). Tumors were classified by FIGO stage, FIGO grade, and histological subtypes. We tested the robustness of our analyses in an independent cohort of n = 573 serous tumor specimens (source: TCGA, based on microarray). Statistical analyses were based on Kruskal-Wallis/Dunn’s and Mann Whitney tests. Changes in LSD1 mRNA levels were also correlated with transcriptomic alterations at genome-wide scale. Effects on cell viability (MTS/PMS assay) of six LSD1 inhibitors (pargyline, TCP, RN-1, S2101, CAS 927019-63-4, and CBB1007) were also evaluated in a panel of ovarian cancer cell lines (SKOV3, OVCAR3, A2780 and cisplatin-resistant A2780cis).
We found moderate but consistent LSD1 mRNA overexpression in stage IIIC and high-grade ovarian tumors. LSD1 mRNA overexpression correlated with a transcriptomic signature of up-regulated genes involved in cell cycle and down-regulated genes involved in the immune/inflammatory response, a signature previously observed in aggressive tumors. In fact, some ovarian tumors showing high levels of LSD1 mRNA are associated with poor patient survival. Chemical LSD1 inhibition induced cytotoxicity in ovarian cancer lines, which roughly correlated with their reported LSD1 inhibitory potential (RN-1,S2101 >> pargyline,TCP).
Our findings may suggest a role of LSD1 in the biology of some ovarian tumors. It is of special interest to find a correlation of LSD1 mRNA overexpression with a transcriptomic signature relevant to cancer. Our findings, therefore, prompt further investigation of the role of LSD1 in ovarian cancer, as well as the study of its enzymatic inhibition in animal models for potential therapeutic purposes in the context of this disease.
Lysine-specific demethylase 1 (LSD1, also known as KDM1A, AOF2, BHC110, and KIA0601) is a nuclear enzymatic activity that catalyzes the removal of methyl groups from histones and non-histone lysine residues [1–5]. LSD1 demethylates mono- and di-methylated histone H3 at lysines 4 and 9 (H3K4me1/2 and H3K9me1/2, respectively) mediating the transcriptional actions of hormone-liganded nuclear receptors [6–9], oncogene c-myc , and Snail1 [11–13], among others. LSD1 also demethylates non-histone substrates, such as tumor suppressor p53 and cell cycle and apoptosis regulator E2F1 [14, 15]. LSD1 has been found overexpressed in liver cancer , gastric cancer , breast cancer [18, 19], bladder, lung and colorectal cancers , Ewing's sarcoma , and neuroblastoma ; and overexpression of LSD1 is a predictor of poor prognosis in prostate and liver cancer [16, 23]. Together, these and other studies suggest that LSD1 is linked to cancer and could be a target for drug discovery [24, 25].
LSD1 is a flavin adenine dinucleotides-dependent (FAD) amine oxidase . Thus, FDA-approved inhibitors of FAD amine oxidases such as mitochondrial-associated monoamine oxidase (MAO) A and B and polyamine oxidase (PAO) are non-selective inhibitors of the LSD1 activity . Recently, more potent and selective LSD1 inhibitors have been developed, which can be grouped into four different classes based on their chemical structure. One class is based on analogues of (bis)guanidine and (bis)biguanide polyamines and oligoamines . It includes compound CAS 927019-63-4, which is a polyamine analog that selectively inhibits LSD1 in vitro and that induces re-expression of aberrantly silenced genes in human colon carcinoma cells [27, 28]. Polyamine analogs have also been tested in breast cancer cells . A second class of LSD1 inhibitors is based on tranylcypromine (TCP, also known as 2-phenylcyclopropylamine or 2-PCPA). TCP is a non-selective and irreversible MAO inhibitor that forms a covalent adduct with FAD. FAD-approved TCP has been used in the clinical treatment of mood and anxiety disorders [30–32]. Two of the most recently developed TCP-analogues with LSD1 inhibitory activity are S2101  and RN-1 . A third class of LSD1 inhibitors is based on FDA-approved antidepressants pargyline and phenelzine . The LSD1 inhibitory activity of pargyline has been tested in prostate cancer cells . Finally, a fourth class of LSD1 inhibitors mimics the peptide structure of LSD1 substrates, such as histone H3 tails [35, 36]. For example, CBB1007 is an amidino-guanidinium compound that was developed based on the crystal structure of LSD1 associated with a peptide inhibitor derived from the N-terminal tail of H3 . CBB1007 is a potent and reversible substrate competitive inhibitor of LSD1 that in vitro arrests pluripotent cancer cells with minimal effect on non-pluripotent cancer or normal somatic cells .
Despite the abundant literature on LSD1 and the many studies testing LSD1 inhibition in cancer cells, the levels of LSD1 expression in human ovarian tumors and the effects of LSD1 inhibitors in ovarian cancer cells have not yet been investigated. In this study, we examine the levels of LSD1 mRNA expression in two independent cohorts of human ovarian tumors. One analysis is based on quantitative real-time PCR (qRT-PCR) and n = 177 specimens, and the other analysis is based on microarray and n = 573 specimens (source: The Cancer Genome Atlas or TCGA). We also examine transcriptomic profiles associated with changes in levels of LSD1 mRNA in TCGA tumors. Finally, we examine the effects on cell viability of six chemical LSD1 inhibitors (CAS 927019-63-4, TCP, RN-1, S2101, pargyline, and CBB1007) in a panel of ovarian cancer cell lines. Our study is the first systematic analysis of LSD1 in the context of ovarian cancer.
Human cohort of ovarian normal and tumor samples
Ovarian Cancer cDNA Tissue Scan™ was purchased from OriGene Technologies (Rockville, MD, USA). It contains n = 192 ovarian normal and tumor cDNA samples (plate HORT101, Lot#1210; plate HORT102, Lot#0712; plate HORT103, #Lot 0210; and plate HORT104, Lot#0210). For our studies, we reduced the number of specimens in the cohort to n = 177 (see section below, ‘qRT-PCR-based measurement of LSD1 mRNA expression in our study cohort’ for details on how we filtered these data). The clinicopathologic parameters and histological and clinical information of the final cohort can be found at the OriGene website. A summary of this information can also be found in Additional file 1: Table S1.
Human ovarian cancer cell lines SKOV3 and OVCAR3 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human ovarian cancer cell lines A2780 and its cisplatin-resistant clone (A2780-cis) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human breast cancer MCF7 and prostate cancer LNCaP cells were generously provided by Dr. Michael G. Rosenfeld (University of California, San Diego, CA, USA). SKOV3 cells were cultured in McCoy’s 5A Modified Medium (16600–108; Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum, or FBS (Omega Scientific, Tarzana, CA, USA). OVCAR3 cells were cultured in RPMI-1640 Modified Medium (30–2001; ATCC) containing 20% FBS supplemented with 10 μg/mL bovine insulin (Sigma-Aldrich). A2780 and A2780-cis cells were cultured in RMPI-1640 + GlutaMAX-I medium (61870–127; Life Technologies) containing 10% FBS and supplemented with 25 mM HEPES buffer. MCF7 cells were cultured in DMEM(1x) + GlutaMAX-I medium (10566–024; Life Technologies) containing 10% FBS. LNCaP cells were cultured in Advanced DMEM/F12(1x) medium (12634–028; Life Technologies) containing 10% FBS. Trypsin/EDTA (Life Technologies) was used for detachment of SKOV3, MCF7, and LNCaP cells. Accutase (Innovative Cell Technologies, San Diego, CA, USA) was used for the detachment of OVCAR3, A2780, and A2780cis cells. Cells did not exceed 20 passages in any of the experiments shown in this study. Cells were maintained in cell incubators at 37°C and 5% CO2.
LSD1 inhibitors pargyline hydrochloride (also known as pargyline) and trans-2-phenylcyclopropyl-amine hydrochloride (also known as TCP or 2-PCPA) were purchased from Sigma-Aldrich. RN-1 hydrochloride (also known as RN-1 or LSD1 Inhibitor IV, cat. # 489479), S2101 (also known as LSD1 Inhibitor II, cat. # 489477), CBB1007 (also known as LSD1 inhibitor III, cat. # 489478), and CAS 927019-63-4 (also known as LSD1 inhibitor, cat. # 489476) were purchased from Calbiochem (EMD-Millipore, Billerica, MA, USA). Chemicals were dissolved in water at a final concentration of 100 mM for pargyline and TCP, 6 mM for RN-1, and 10 mM for CAS 927019-63-4; or were dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 100 mM for S2010 and 10 mM for CBB1007. Cisplatin was purchased from Sigma-Aldrich and dissolved in dimethylformamide at a final concentration of 40 mM. Dilutions were prepared in the same solvents. Solutions were made fresh in every experiment for pargyline, TCP, and cisplatin. For the rest of chemicals, stock solutions were prepared and stored at -20°C, being thawed/frozen no more than 3 times.
qRT-PCR-based measurement of LSD1 mRNA expression in our study cohort
Dry cDNA pellets from ovarian normal and tumor tissues (see section above, ‘Human cohort of ovarian normal and tumor samples’) were dissolved in 31 μL water at 50°C for 15 minutes, vortexing every 5 minutes. After short spinning, two aliquots of 13 μL and two aliquots of 2 μL were taken for analysis of human LSD1 and ACTB mRNA expression, respectively. The four aliquots were brought to a final volume of 30 μL with 2xFastStart Universal SYBR Green Master mix (Roche Applied Science, Indianapolis, IN, USA) and the solution with primers. Primer sequences were: LSD1-sense 5’-GCTCGGGGCTCTTATTCCTA-3’ and LSD1-antisense 5’-CCCAAAAACTGGTCTGCAAT-3’ and ACTB-sense 5’-GGACTTCGAGCAAGAGATGG-3’ and ACTB-antisense 5’-AGCACTGTGTTGGCGTACAG-3’. The expected sizes of the LSD1 and ACTB PCR amplicons were confirmed by agarose gel. The validity of the LSD1 primers was also confirmed by the observation of a significant downregulation in the levels of a PCR amplicon obtained in cDNA samples from cancer cell lines treated with LSD1 siRNA when compared to control siRNA treatment (data not shown). Standard quantitative real-time PCR (qRT-PCR) reactions were conducted in a 3000 MxPro Instrument (Agilent Technologies, Santa Clara, CA, USA), in 96-well format with adhesive film. PCR settings were the following: 2 min 50°C, 10 min at 95°C followed by 40 cycles of 95°C for 15 sec, 58°C for 15 sec, and 25 sec for 72°C. Cycle threshold (Ct) values were extracted with MxPro qPCR Software (Agilent Technologies) and calculated the difference (ΔCt) between replicates. Those samples in which ΔCt was higher than 0.5, either for LSD1 or control ACTB, were excluded from our analysis, which ensured the inclusions of only robust expression measurements in further analyses. A total of n = 177 out of 192 samples (92.2%) reached this quality requirement.
Analysis of TCGA data
Gene expression data on n = 573 ovarian serous cystadenocarcinoma (OV) samples were downloaded from the TCGA website (datasets UNC_AgilentG4502A_07_2 and UNC_AgilentG4502A_07_3). Matching clinical data were downloaded from the UCSC Cancer Browser  (datasets AgilentG4502A_07_2 and AgilentG4502A_07_3, version: 2013-06-03). Gene expression data for normal ovarian tissue (n = 47) were downloaded from the TCGA website. Of the normal samples, n = 8 belong to “Solid tissue normals” from the “Ovarian Cancer” dataset, and n = 39 belong to “Control” with gene expression data over a total of 546 samples available from the same dataset. The downloaded data were mined with custom Linux scripts to extract data and create tables of gene expression and clinical features for the ovarian tissue (normal and cancerous) samples. Two groups of samples were extracted according to the expression of LSD1 (AOF2): the first group (“LSD1-underexpressed”) included subjects having LSD1 expression under 0.2 percentile, and the second group (“LSD1-overexpressed”) included subjects having LSD1 expression over 0.8 percentile of all samples. Both groups included equal number of samples, n = 115. Gene expression data for the two groups were then imported into R statistical software, and SAM (Significance Analysis for Microarray)-analysis was performed on the data to determine the genes significantly differentially expressed between the groups. The significant genes fell into two groups: genes whose pattern of expression positively correlated with that of LSD1 (i.e. they were overexpressed in the LSD1-overexpressed group and underexpressed in the LSD1-underexpressed group), and genes whose pattern of expression negatively correlated with that of LSD1 (i.e. they were underexpressed in the LSD1-overexpressed group and overexpressed in the LSD1-underexpressed group). SAM analysis (delta = 3, FDR = 3e-05) revealed n = 821 (plus LSD1 itself) genes whose expression correlated with that of LSD1 with the fold change >1.5 (<1/1.5 for negatively correlated genes), including n = 430 positively correlated, and n = 391 negatively correlated genes. This gene set was further filtered to only include the genes that showed certain consistency in their correlation with LSD1 expression; namely, the genes that correlated with the direction of changes in LSD1 expression in more than 50% of high LSD1-expressing samples and in more than 50% of low LSD1-expressing samples. The filtered gene set contained n = 458 genes (plus LSD1): n = 243 positively correlated and n = 215 negatively correlated with LSD1 expression. For each gene, the percent of consistency (percent of samples that exhibited the consistent expression of this gene with LSD1) within LSD1-underexpressed and LSD1-overexpressed groups was calculated (Additional file 2: Table S2). The expression data for the filtered gene set (n = 458) was normalized to (−1:1), hierarchically clustered by centered correlation method, and then visualized as a heatmap using Cluster 3.0/Treeview software . The gene expression data was then manipulated and sorted using Kingsoft Office and Apache OpenOffice software, according to clinical stages, histological subtype, gene expression, and survival data. The statistical analysis of the groups was performed in GraphPad Prism 6 for Windows (for procedures, see Statistical analysis below). Kaplan-Meier analysis was performed on three groups of samples: the LSD1-underexpressed (n = 115), LSD1-overexpressed (n = 115), and the rest of the samples in the dataset (n = 343). The statistical difference between the survival curves was calculated by Mantel-Cox survival test.
Protein extraction and Western blot analysis
For LSD1, protein was isolated directly from culturing 10-cm plates after washing with PBS buffer and adding 400 μL modified IPH buffer (50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 5 mM EDTA, 0.5% (by volume) NP-40, and inhibitor cocktail). Protein extracts were then collected after 30 min lysis at 4°C, and centrifuged for 10 min at 14.000 rpm (4°C). For PARP1 and histones, protein was isolated from 6-well plates in 250 μL modified IPH buffer (as above but 420 mM NaCl). Protein concentration was measured by the standard Bradford Protein assay. Twenty μg (for LSD1) and 7 μg (for PARP1 and H3K4me2) protein were loaded and run on 4-12% Bis-Tris gels with MES running buffer (Life Technologies). After transfer onto 0.2 μm-pore PVDF (BioRad) or nitrocellulose (Whatman) membranes, membranes were blocked with Odyssey blocking solution (LI-COR bioscience) or 5% milk/TBST for 30 min and probed with antibodies (diluted in 5% BSA/TBST) against LSD1 (17721, Abcam), tubulin (DM1A, Sigma), or H3K4me2 (39141, Active Motif), PARP1 (9542, Cell Signaling), actin (MAB1501, Chemicon), respectively, overnight at 4°C. Immunodetection was achieved after incubation with infrared (IR)-dye-conjugated (LI-COR Bioscience, Lincoln, NE, USA) or HRP-conjugated (Invitrogen, Carlsbad, CA, USA), respectively, goat anti-mouse or goat anti-rabbit diluted 1:5,000 in blocking solution. IR-dye immunoreactive bands were scanned using Odyssey Imaging System (LI-COR Bioscience) following manufacturer’s instructions. HRP signal was detected by ECL (Amersham-GE, Pittsburgh, PA, USA) and autoradiography film.
Bright-field images were taken in a Nikon Diaphot microscope at a 10x magnification. Image acquisition was performed with a Canon Digital Rebel XTi camera, using ZoomBrowser EX5.7 and EOS Utility software (Canon) for remote picture shooting.
Cell viability assay
Cells were seeded at a 1/12 dilution in 96-well plates (200 μL/well) from 10 cm confluent plates. After 24 hrs, 100 μL medium was removed and replaced with 100 μL fresh medium containing the indicated small compound concentrations in figure legends. After additional 48 hrs, 150 μL medium was removed and replaced with 30 μL medium plus 20 μL of MTP/PMS (20:1, v:v) solution We purchased MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] from Promega (Madison, WI, USA), and PMS (phenazine methosulfate) from Sigma-Aldrich. Plates were maintained in cell incubators at 37°C (5% CO2) for 2–2.30 hours and, afterwards, absorbance was measured at 490 nm in a microQuant plate reader (Biotek Instruments, Winooski, VT, USA). Values were obtained with the KCjunior Software (Biotek). Wells in the periphery of each 96-well plate were not used for measurements (A1-A12, H1-12, B-G1, and B-G12). Each experiment was performed in triplicate with six replicates each (total data point, n = 18). We show a representative example of one of them. For estimation of IC50 values and visualization we used Excel (Microsoft) and Prism 6 (GraphPad) software. Cells under vehicle conditions were used to determine 100% viability (C1). Wells without cells (in the case of pargyline and TCP treatments) or at conditions in which no cells remained alive after treatment (visually determined, in the case of RN-1 and S2101) were used to determine 0% viability (C2). Viability percentage (C3) was calculated as: ((C3-C2)/(C1-C2))*100 in Excel. Cell viability graph show the average (mean) value of viable cells under each condition, ± s.e.m. After exporting these data to Prism, half maximum inhibitory concentration (IC50, or best fit) values and 95% confidence intervals (95% CI) were then estimated by calculating the nonlinear regression (curve fit, dose response-inhibition variable slope) with Prism 6.
Statistical analysis in our sample cohort was conducted with the GraphPad Prism 6 version for Mac (GraphPad). For two-groups-only comparisons (n = 2), we conducted two-tailed Mann Whitney tests. For analysis of multiple groups (n > 2), we conducted one-way non-parametric ANOVA (Kruskal-Wallis test) followed by the Dunn’s post hoc test (unless indicated otherwise, p-values were calculated comparing the mean of each group to the mean of control or normal tissue). For the Kaplan-Meier analysis, we used Mantel-Cox survival test. Differences were considered significant at p≤0.05 (*). Other values were indicated as: p≤0.01 (**), p≤0.001 (***), and p≤0.0001 (****).
Results and discussion
qRT-PCR-based profiling of LSD1 mRNA in human ovarian specimens
Moderate LSD1 mRNA overexpression in stage IIIC and grades G2/G3 ovarian tumors
The issue with this analysis is whether normal ovarian tissue is the best (or even a permissible) reference to establish abnormalities with regard to gene expression levels in ovarian tumors. Ovarian normal tissue is largely constituted of stromal cells, whereas ovarian tumors mainly derive from epithelial cells . Although the distinction between stromal and epithelial cells is not always obvious at molecular level (for example, well-established tumors sometimes display a robust stromal gene signature even in the absence of visually apparent stroma ), it is obvious that a difference in cell type composition per se may cause differences in gene expression that might not be attributable to disease. On a different note, multiple studies suggest that ovarian tumors may derive from the fimbria of the fallopian tube, thus ovarian normal epithelium might not be a good reference control [40, 42]. One known workaround to the use of normal (either ovarian or fallopian) tissue is to use cultured primary or immortalized normal epithelial cells. However, it is known that normal cells may quickly acquire aberrant features once in culture (reported in different systems, e.g. [43–45]), those expression differences not being associated with disease. Therefore, any reference control seems to be problematic in one or another way , as it might also be in our case (Figure 2A). Nevertheless, we suspect, that these aspects might be less relevant in the particular case of LSD1, because this gene is highly expressed in many cell and tissue types apparently without a strong cell/tissue-specific pattern of expression (i.e. being a housekeeping-like gene). Perhaps in agreement with this comment is the observation that LSD1 mRNA levels are very similar in a comparison of borderline tumors (which are largely epithelial), different tumors at different stages (epithelial), mucinous tumors, and normal tissue (stromal). We show these comparisons later in this study.
In any case, to potentially avoid issues regarding the different origin of normal and tumor specimens, we subdivided the TCGA and our study cohort by FIGO stage, which allows us direct comparisons between tumor groups. In the TCGA cohort, we observed higher average levels of LSD1 mRNA in stage II, III, and IV than in stage I tumors (Additional file 4: Figure S2A), but these differences did not reach significance likely because of the small number of stage I specimens in the TCGA cohort (n = 16). The differences (specially for stage III and IV) reached significance when compared to the larger group of normal tissue (n = 47), which shows similar average levels of LSD1 mRNA to stage I tumors (Additional file 4: Figure S2A). In our cohort (in which, from now on, the few tumors of the mucinous subtype were removed from analysis), we could see statistically significant higher levels of LSD1 mRNA in stage III tumors than in control and stage I tumors, although only after the application of a less stringent pair-comparison test (p = 0.0447, Mann–Whitney), but not after the application of a more stringent multi-comparison test (Kruskal-Wallis followed post hoc by Dunn’s, Figure 2B). Therefore, we suspect that the small size and high dispersion of tumor groups such as stage I diminish the power of our statistical analyses. Together, these analyses suggest moderately higher levels of LSD1 mRNA in at least stage III ovarian tumors. The differences were robust compared to normal tissue, but not as robust in the direct comparison to stage I tumors.
We further subdivided stage III tumors into (sub)stages IIIA, IIIB, and IIIC. In the TCGA cohort, stage IIIC specimens showed the higher average levels of LSD1 mRNA of the three (sub)stages, and the difference compared to control reached statistical significance (p < 0.0001 with restrictive Kruskal-Wallis/Dunn’s test; Additional file 4: Figure S2B). In our cohort, we observed a similar result (p = 0.0003, Kruskal-Wallis/Dunn’s test; Figure 2C). We also observed a statistically significant difference between stage IIIC tumors and tumors classified as stage IIIA or IIIB, although only after these two groups were combined (IIIA/IIIB; p = 0.0400, Mann-Whitney test; Figure 2C), thus reinforcing the argument that small group sizes limit the power of our statistical analyses. These results suggest that LSD1 mRNA levels are high in stage IIIC ovarian tumors.
Next, we subclassified tumors by FIGO grade (borderline malignant, highly differentiated or grade G1, moderately differentiated or grade G2, and low differentiated/undifferentiated or grade G3). In the TCGA cohort (which lacks borderline tumors), we observed higher levels of LSD1 mRNA in G2 and G3 than in G1 tumors by multi-comparison analysis (p = 0.0085 and p = 0.0071, Kruskal-Wallis/Dunn’s test; Additional file 4: Figure S2C). In our cohort, we observed very similar average levels and dispersion of LSD1 expression in normal ovarian tissue and borderline tumors, which are mainly epithelial in origin (Figure 2D). As in the TCGA cohort, we also observed the highest levels of LSD1 mRNA in G2, and the difference in this case with respect to G3 tumors reached significance (p = 0.0467 by Mann–Whitney, Figure 2D). These results suggest that LSD1 mRNA levels are abnormally high in at least grade G2 tumors, a trend that was similarly observed in both independent cohorts.
Overall, our analyses of LSD1 mRNA levels in ovarian tumors suggest moderate LSD1 mRNA overexpression in certain FIGO stages and grades, often observed consistently in two independent cohorts. In particular, we observed LSD1 mRNA overexpression in stage IIIC tumors, as well as associated with high-grade tumors (G2 and, to a certain point, G3). In the TCGA cohort, we also observed LSD1 mRNA upregulation in stage II and IV tumors, which may have been missed in our cohort due to the limited size of these two particular groups (n = 14 and n = 10 in our cohort, respectively, versus n = 27 and n = 85 in the TCGA cohort). Inconsistencies between cohorts might be attributed to disparities in tumor collections (sample size, donors, and composition of tumor subtypes), and to the use of different profiling techniques (qRT-PCR and microarray). In any case, we admit that the strength of our conclusions will strongly depend on the suitability of using normal tissue as reference control in the study of LSD1 mRNA levels.
Transcriptomic features of high LSD1 mRNA-expressing ovarian tumors
To functionally characterize the filtered gene set, we next performed gene ontology (GO) analysis finding that the group of genes positively correlated with LSD1 (i.e. those overexpressed in high LSD1-expressing tumors and underexpressed in low LSD1-expressing tumors) is enriched in GO terms of cell cycle and mitosis (Figure 4B, top). Interestingly, LSD1 is known to control cell proliferation in cancer cells . On the other hand, GO analysis of the group of genes that showed a pattern of expression that negatively correlated with that of LSD1 (i.e. those underexpressed in high LSD1-expressing tumors and overexpressed in low LSD1-expressing tumors) showed enrichment in GO terms related with the defense/immune/inflammatory response (Figure 4B, bottom). This last observation is of special interest due to the importance of the immune response in the pathology of ovarian cancer, particularly in contributing to the pattern of T-cell infiltration in ovarian tumors [49, 50]. In fact, a set of high-grade serous ovarian tumors (known as subtype C5) has been recently defined by the simultaneous overexpression of cell proliferation genes and underexpression of an immune signature (among other features), and it is clinically associated with poor overall survival . Based on this report, we also examined whether high LSD1-expressing tumors are associated with poor overall patient survival, but failed to find such association with our set of n = 115 high LSD1-expressing tumors (Additional file: 6 Figure S4D). In contrast, we found, using the cBioPortal for Cancer Genomic , poor patient survival and reduced disease free survival associated with high LSD1-expressing tumors (fold-change > 1.20, n = 30) in the cohort of n = 580 TCGA specimens (log-rank test p = 0.002644 and p = 0.012440, respectively; Additional file 7: Figure S5). Together, we have identified a rich molecular signature of differentially expressed cell cycle and immune/inflammatory genes associated with LSD1 mRNA overexpression in ovarian tumors. Whether this molecular (transcriptomic) signature is functionally (biologically) relevant and, if so, whether it results directly from alterations in LSD1 expression or instead is induced by the same mechanisms that alter LSD1 expression is yet to be determined.
Chemical inhibitors of LSD1 activity are cytotoxic for ovarian cancer cells
LSD1 is a lysine demethylase whose activity is involved in cancer biology, but a link to ovarian cancer has not yet been directly explored. Histone lysine methylation and demethylation have gained significant attention since their discovery [1, 56]. These two antagonistic activities are emerging as important in cancer biology via regulating histone and non-histone substrates . The list of methylated proteins is continuously expanding , and the field seems to be flourishing with intriguing new data. Here, we have studied LSD1 in the context of ovarian tumors and cancer cells. Our studies suggest the moderate overexpression of LSD1 mRNA in stage IIIC and high-grade ovarian tumors. The lack of a strong association between the levels of LSD1 mRNA and the disease could be due to the fact that we limited our analyses to the measurement of the transcript. This association could be stronger (or weaker or even absent) at the level of the functional entities: the LSD1 protein and its activity. Future studies, therefore, will be required to tests these associations. That said, LSD1 might be important in ovarian cancer independently whether its levels/activity change. In breast and prostate cancer cells, for example, LSD1 is required for the stimulation of the hormonal signaling response and cell proliferation [7, 8]. In fact, our observation that the four ovarian cancer lines tested in this study show robust levels of LSD1 (protein) expression may suggest a functional role of LSD1 also in these cells.
At molecular level, our study suggests that tumors showing high levels of LSD1 mRNA also exhibit a signature of relative overexpression of genes involved in cell cycle and underexpression of genes involved in the immune response, which in the past has been associated to aggressive tumors in the context of other cohorts . Perhaps in agreement, we found a set of high LSD1 mRNA-expressing tumors associated with poor patient survival (Additional file 7: Figure S5), although we failed to observe the same association with the full set of tumors that we analyzed to derive the mentioned signature (Additional file 6: Figure S4D). We also report the cytotoxic effects of a panel of chemical LSD1 inhibitors in a panel of ovarian cancer cell lines. Additional studies will be necessary to determine the robustness of these inhibitory effects in vivo, as well as their secondary effects, before we can consider these compounds as potential therapies for the disease. Together, our study prompts further exploration of LSD1 and the LSD1 demethylase activity in ovarian cancer and their targeting for potential therapeutic purposes.
The results published here are in part based upon data generated by The Cancer Genome Atlas (TCGA) established by the NCI and NHGRI. Information about TCGA and the investigators and institutions that constitute The Cancer Genome Atlas Research Network can be found at http://cancergenome.nih.gov/. Some results are also based on the cBioPortal for Cancer Genomics developed by the Computational Center at Memorial Sloan-Kettering Cancer Center and the i-Vis Research Group of the Computer Engineering Department at Bilkent University. We would like to thank these institutions/programs and their investigators for developing these resources and computational tools. We would like to also thank Dr. Stephen Howell (UCSD), Dr. Angels Almenar-Queralt (UCSD), Dr. Julie McDowell and her team at Origene, Dr. Carlos Mackintosh (UCSD), Dr. Angeles Secord (Duke), Dr. Rex Bentley (Duke), and reviewers for their valuable input during preparation of this manuscript. We would like to thank the Ovarian Cancer Research Foundation (OCRF) and its donors for their support. This work was funded by OCRF and the Estate of Agatha Fort. I.G.B. is a Liz Tilberis Scholar (2012).
- Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA, Shi Y: Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 2004, 119: 941–953.PubMedView ArticleGoogle Scholar
- Culhane JC, Cole PA: LSD1 and the chemistry of histone demethylation. Curr Opin Chem Biol 2007, 11: 561–568.PubMed CentralPubMedView ArticleGoogle Scholar
- Nicholson TB, Chen T: LSD1 demethylates histone and non-histone proteins. Epigenetics 2009, 4: 129–132.PubMedView ArticleGoogle Scholar
- Forneris F, Battaglioli E, Mattevi A, Binda C: New roles of flavoproteins in molecular cell biology: histone demethylase LSD1 and chromatin. FEBS J 2009, 276: 4304–4312.PubMedView ArticleGoogle Scholar
- Shi Y-J, Matson C, Lan F, Iwase S, Baba T, Shi Y: Regulation of LSD1 histone demethylase activity by its associated factors. Mol Cell 2005, 19: 857–864.PubMedView ArticleGoogle Scholar
- Metzger E, Imhof A, Patel D, Kahl P, Hoffmeyer K, Friedrichs N, Müller JM, Greschik H, Kirfel J, Ji S, Kunowska N, Beisenherz-Huss C, Günther T, Buettner R, Schüle R: Phosphorylation of histone H3T6 by PKCbeta(I) controls demethylation at histone H3K4. Nature 2010, 464: 792–796.PubMedView ArticleGoogle Scholar
- Metzger E, Wissmann M, Yin N, Müller JM, Schneider R, Peters AHFM, Günther T, Buettner R, Schüle R: LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 2005, 437: 436–439.PubMedGoogle Scholar
- Garcia-Bassets I, Kwon Y-S, Telese F, Prefontaine GG, Hutt KR, Cheng CS, Ju B-G, Ohgi KA, Wang J, Escoubet-Lozach L, Rose DW, Glass CK, Fu X-D, Rosenfeld MG: Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors. Cell 2007, 128: 505–518.PubMed CentralPubMedView ArticleGoogle Scholar
- Nair SS, Nair BC, Cortez V, Chakravarty D, Metzger E, Schüle R, Brann DW, Tekmal RR, Vadlamudi RK: PELP1 is a reader of histone H3 methylation that facilitates oestrogen receptor-alpha target gene activation by regulating lysine demethylase 1 specificity. EMBO Rep 2010, 11: 438–444.PubMed CentralPubMedView ArticleGoogle Scholar
- Amente S, Bertoni A, Morano A, Lania L, Avvedimento EV, Majello B: LSD1-mediated demethylation of histone H3 lysine 4 triggers Myc-induced transcription. Oncogene 2010, 29: 3691–3702.PubMedView ArticleGoogle Scholar
- Lin Y, Wu Y, Li J, Dong C, Ye X, Chi Y-I, Evers BM, Zhou BP: The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1. EMBO J 2010, 29: 1803–1816.PubMed CentralPubMedView ArticleGoogle Scholar
- Lin T, Ponn A, Hu X, Law BK, Lu J: Requirement of the histone demethylase LSD1 in Snai1-mediated transcriptional repression during epithelial-mesenchymal transition. Oncogene 2010, 29: 4896–4904.PubMed CentralPubMedView ArticleGoogle Scholar
- McDonald OG, Wu H, Timp W, Doi A, Feinberg AP: Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition. Nat Struct Mol Biol 2011, 18: 867–874.PubMed CentralPubMedView ArticleGoogle Scholar
- Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, Richter M, Opravil S, Shiekhattar R, Bedford MT, Jenuwein T, Berger SL: p53 is regulated by the lysine demethylase LSD1. Nature 2007, 449: 105–108.PubMedView ArticleGoogle Scholar
- Kontaki H, Talianidis I: Lysine methylation regulates E2F1-induced cell death. Mol Cell 2010, 39: 152–160.PubMedView ArticleGoogle Scholar
- Zhao Z-K, Yu H-F, Wang D-R, Dong P, Chen L, Wu W-G, Ding W-J, Liu Y-B: Overexpression of lysine specific demethylase 1 predicts worse prognosis in primary hepatocellular carcinoma patients. World J Gastroenterol 2012, 18: 6651–6656.PubMed CentralPubMedView ArticleGoogle Scholar
- Magerl C, Ellinger J, Braunschweig T, Kremmer E, Koch LK, Höller T, Büttner R, Lüscher B, Gütgemann I: H3K4 dimethylation in hepatocellular carcinoma is rare compared with other hepatobiliary and gastrointestinal carcinomas and correlates with expression of the methylase Ash2 and the demethylase LSD1. Hum Pathol 2010, 41: 181–189.PubMedView ArticleGoogle Scholar
- Lim S, Janzer A, Becker A, Zimmer A, Schüle R, Buettner R, Kirfel J: Lysine-specific demethylase 1 (LSD1) is highly expressed in ER-negative breast cancers and a biomarker predicting aggressive biology. Carcinogenesis 2010, 31: 512–520.PubMedView ArticleGoogle Scholar
- Serce N, Gnatzy A, Steiner S, Lorenzen H, Kirfel J, Buettner R: Elevated expression of LSD1 (Lysine-specific demethylase 1) during tumour progression from pre-invasive to invasive ductal carcinoma of the breast. BMC Clin Pathol 2012, 12: 13.PubMed CentralPubMedView ArticleGoogle Scholar
- Hayami S, Kelly JD, Cho H-S, Yoshimatsu M, Unoki M, Tsunoda T, Field HI, Neal DE, Yamaue H, Ponder BAJ, Nakamura Y, Hamamoto R: Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers. Int J Cancer 2011, 128: 574–586.PubMedView ArticleGoogle Scholar
- Bennani-Baiti IM, Machado I, Llombart-Bosch A, Kovar H: Lysine-specific demethylase 1 (LSD1/KDM1A/AOF2/BHC110) is expressed and is an epigenetic drug target in chondrosarcoma, Ewing’s sarcoma, osteosarcoma, and rhabdomyosarcoma. Hum Pathol 2012, 43: 1300–1307.PubMedView ArticleGoogle Scholar
- Schulte JH, Lim S, Schramm A, Friedrichs N, Koster J, Versteeg R, Ora I, Pajtler K, Klein-Hitpass L, Kuhfittig-Kulle S, Metzger E, Schüle R, Eggert A, Buettner R, Kirfel J: Lysine-specific demethylase 1 is strongly expressed in poorly differentiated neuroblastoma: implications for therapy. Cancer Res 2009, 69: 2065–2071.PubMedView ArticleGoogle Scholar
- Kahl P, Gullotti L, Heukamp LC, Wolf S, Friedrichs N, Vorreuther R, Solleder G, Bastian PJ, Ellinger J, Metzger E, Schüle R, Buettner R: Androgen receptor coactivators lysine-specific histone demethylase 1 and four and a half LIM domain protein 2 predict risk of prostate cancer recurrence. Cancer Res 2006, 66: 11341–11347.PubMedView ArticleGoogle Scholar
- Lohse B, Kristensen JL, Kristensen LH, Agger K, Helin K, Gajhede M, Clausen RP: Inhibitors of histone demethylases. Bioorg Med Chem 2011, 19: 3625–3636.PubMedView ArticleGoogle Scholar
- Suzuki T, Miyata N: Lysine demethylases inhibitors. J Med Chem 2011, 54: 8236–8250.PubMedView ArticleGoogle Scholar
- Huang Y, Marton LJ, Woster PM, Casero RA: Polyamine analogues targeting epigenetic gene regulation. Essays Biochem 2009, 46: 95–110.PubMed CentralPubMedView ArticleGoogle Scholar
- Huang Y, Greene E, Murray Stewart T, Goodwin AC, Baylin SB, Woster PM, Casero RA Jr: Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes. Proc Natl Acad Sci U S A 2007, 104: 8023–8028.PubMed CentralPubMedView ArticleGoogle Scholar
- Huang Y, Stewart TM, Wu Y, Baylin SB, Marton LJ, Perkins B, Jones RJ, Woster PM, Casero RA Jr: Novel oligoamine analogues inhibit lysine-specific demethylase 1 and induce reexpression of epigenetically silenced genes. Clin Cancer Res 2009, 15: 7217–7228.PubMed CentralPubMedView ArticleGoogle Scholar
- Zhu Q, Huang Y, Marton LJ, Woster PM, Davidson NE, Casero RA Jr: Polyamine analogs modulate gene expression by inhibiting lysine-specific demethylase 1 (LSD1) and altering chromatin structure in human breast cancer cells. Amino Acids 2012, 42: 887–898.PubMed CentralPubMedView ArticleGoogle Scholar
- Lee MG, Wynder C, Schmidt DM, McCafferty DG, Shiekhattar R: Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications. Chem Biol 2006, 13: 563–567.PubMedView ArticleGoogle Scholar
- Schmidt DMZ, McCafferty DG: trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1. Biochemistry 2007, 46: 4408–4416.PubMedView ArticleGoogle Scholar
- Yang M, Culhane JC, Szewczuk LM, Jalili P, Ball HL, Machius M, Cole PA, Yu H: Structural basis for the inhibition of the LSD1 histone demethylase by the antidepressant trans-2-phenylcyclopropylamine. Biochemistry 2007, 46: 8058–8065.PubMedView ArticleGoogle Scholar
- Mimasu S, Umezawa N, Sato S, Higuchi T, Umehara T, Yokoyama S: Structurally designed trans-2-phenylcyclopropylamine derivatives potently inhibit histone demethylase LSD1/KDM1. Biochemistry 2010, 49: 6494–6503.PubMedView ArticleGoogle Scholar
- Neelamegam R, Ricq EL, Malvaez M, Patnaik D, Norton S, Carlin SM, Hill IT, Wood MA, Haggarty SJ, Hooker JM: Brain-penetrant LSD1 inhibitors can block memory consolidation. ACS Chem Neurosci 2012, 3: 120–128.PubMed CentralPubMedView ArticleGoogle Scholar
- Tortorici M, Borrello MT, Tardugno M, Chiarelli LR, Pilotto S, Ciossani G, Vellore NA, Bailey SG, Cowan J, O’Connell M, Crabb SJ, Packham G, Mai A, Baron R, Ganesan A, Mattevi A: Protein Recognition by Short Peptide Reversible Inhibitors of the Chromatin-Modifying LSD1/CoREST Lysine Demethylase. ACS Chem Biol 2013, 8: 1677–1682.PubMedView ArticleGoogle Scholar
- Wang J, Lu F, Ren Q, Sun H, Xu Z, Lan R, Liu Y, Ward D, Quan J, Ye T, Zhang H: Novel histone demethylase LSD1 inhibitors selectively target cancer cells with pluripotent stem cell properties. Cancer Res 2011, 71: 7238–7249.PubMed CentralPubMedView ArticleGoogle Scholar
- Goldman M, Craft B, Swatloski T, Ellrott K, Cline M, Diekhans M, Ma S, Wilks C, Stuart J, Haussler D, Zhu J: The UCSC Cancer Genomics Browser: update 2013. Nucleic Acids Res 2013,41(Database issue):D949-D954.PubMed CentralPubMedView ArticleGoogle Scholar
- De Hoon MJL, Imoto S, Nolan J, Miyano S: Open source clustering software. Bioinformatics 2004, 20: 1453–1454.PubMedView ArticleGoogle Scholar
- Cho KR, Shih I-M: Ovarian cancer. Annu Rev Pathol 2009, 4: 287–313.PubMed CentralPubMedView ArticleGoogle Scholar
- Jones PM, Drapkin R: Modeling high-grade serous carcinoma: how converging insights into pathogenesis and genetics are driving better experimental platforms. Front Oncol 2013, 3: 217.PubMed CentralPubMedView ArticleGoogle Scholar
- Tothill RW, Tinker AV, George J, Brown R, Fox SB, Lade S, Johnson DS, Trivett MK, Etemadmoghadam D, Locandro B, Traficante N, Fereday S, Hung JA, Chiew Y-E, Haviv I, Gertig D, DeFazio A, Bowtell DDL: Novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome. Clin Cancer Res 2008, 14: 5198–5208.PubMedView ArticleGoogle Scholar
- Karst AM, Drapkin R: The new face of ovarian cancer modeling: better prospects for detection and treatment. F1000 Med Rep 2011, 3: 22.PubMed CentralPubMedView ArticleGoogle Scholar
- Maines-Bandiera SL, Kruk PA, Auersperg N: Simian virus 40-transformed human ovarian surface epithelial cells escape normal growth controls but retain morphogenetic responses to extracellular matrix. Am J Obstet Gynecol 1992, 167: 729–735.PubMedView ArticleGoogle Scholar
- Berthon P, Waller AS, Villette JM, Loridon L, Cussenot O, Maitland NJ: Androgens are not a direct requirement for the proliferation of human prostatic epithelium in vitro. Int J Cancer 1997, 73: 910–916.PubMedView ArticleGoogle Scholar
- Speirs V, Green AR, Walton DS, Kerin MJ, Fox JN, Carleton PJ, Desai SB, Atkin SL: Short-term primary culture of epithelial cells derived from human breast tumours. Br J Cancer 1998, 78: 1421–1429.PubMed CentralPubMedView ArticleGoogle Scholar
- O’Shannessy DJ, Jackson SM, Twine NC, Hoffman BE, Dezso Z, Agoulnik SI, Somers EB: Gene expression analyses support fallopian tube epithelium as the cell of origin of epithelial ovarian cancer. Int J Mol Sci 2013, 14: 13687–13703.PubMed CentralPubMedView ArticleGoogle Scholar
- Wang J, Scully K, Zhu X, Cai L, Zhang J, Prefontaine GG, Krones A, Ohgi KA, Zhu P, Garcia-Bassets I, Liu F, Taylor H, Lozach J, Jayes FL, Korach KS, Glass CK, Fu X-D, Rosenfeld MG: Opposing LSD1 complexes function in developmental gene activation and repression programmes. Nature 2007, 446: 882–887.PubMedView ArticleGoogle Scholar
- Chen Y, Jie W, Yan W, Zhou K, Xiao Y: Lysine-specific histone demethylase 1 (LSD1): a potential molecular target for tumor therapy. Crit Rev Eukaryot Gene Expr 2012, 22: 53–59.PubMedView ArticleGoogle Scholar
- Facciabene A, Motz GT, Coukos G: T-regulatory cells: key players in tumor immune escape and angiogenesis. Cancer Res 2012, 72: 2162–2171.PubMed CentralPubMedView ArticleGoogle Scholar
- Motz GT, Coukos G: Deciphering and reversing tumor immune suppression. Immunity 2013, 39: 61–73.PubMed CentralPubMedView ArticleGoogle Scholar
- Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Jacobsen A, Byrne CJ, Heuer ML, Larsson E, Antipin Y, Reva B, Goldberg AP, Sander C, Schultz N: The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discovery 2012, 2: 401–404.PubMedView ArticleGoogle Scholar
- Shi L, Cui S, Engel JD, Tanabe O: Lysine-specific demethylase 1 is a therapeutic target for fetal hemoglobin induction. Nat Med 2013, 19: 291–294.PubMedView ArticleGoogle Scholar
- Schenk T, Chen WC, Göllner S, Howell L, Jin L, Hebestreit K, Klein H-U, Popescu AC, Burnett A, Mills K, Casero RA Jr, Marton L, Woster P, Minden MD, Dugas M, Wang JCY, Dick JE, Müller-Tidow C, Petrie K, Zelent A: Inhibition of the LSD1 (KDM1A) demethylase reactivates the all-trans-retinoic acid differentiation pathway in acute myeloid leukemia. Nat Med 2012, 18: 605–611.PubMed CentralPubMedView ArticleGoogle Scholar
- Whyte WA, Bilodeau S, Orlando DA, Hoke HA, Frampton GM, Foster CT, Cowley SM, Young RA: Enhancer decommissioning by LSD1 during embryonic stem cell differentiation. Nature 2012, 482: 221–225.PubMed CentralPubMedGoogle Scholar
- Hino S, Sakamoto A, Nagaoka K, Anan K, Wang Y, Mimasu S, Umehara T, Yokoyama S, Kosai K-I, Nakao M: FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure. Nat Commun 2012, 3: 758.PubMed CentralPubMedView ArticleGoogle Scholar
- Rea S, Eisenhaber F, O’Carroll D, Strahl BD, Sun ZW, Schmid M, Opravil S, Mechtler K, Ponting CP, Allis CD, Jenuwein T: Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 2000, 406: 593–599.PubMedView ArticleGoogle Scholar
- He Y, Korboukh I, Jin J, Huang J: Targeting protein lysine methylation and demethylation in cancers. Acta Biochim Biophys Sin (Shanghai) 2012, 44: 70–79.View ArticleGoogle Scholar
- Moore KE, Carlson SM, Camp ND, Cheung P, James RG, Chua KF, Wolf-Yadlin A, Gozani O: A general molecular affinity strategy for global detection and proteomic analysis of lysine methylation. Mol Cell 2013, 50: 444–456.PubMed CentralPubMedView ArticleGoogle Scholar
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