METHODS OF TREATMENT OF CANCER DISEASE BY TARGETING AN EPIGENETIC FACTOR
20230070181 · 2023-03-09
Inventors
Cpc classification
A61K31/713
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
A61K31/7105
HUMAN NECESSITIES
C12N15/1135
CHEMISTRY; METALLURGY
International classification
A61K31/7105
HUMAN NECESSITIES
A61K31/713
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention relates to a method for preventing or treating a cancer disease by targeting the epigenetic factor Chromodomain on Y-like 2 (CDYL2). The inventors found that CDYL2 is commonly over-expressed in cancer and high CDYL2 levels correlate with poor prognosis in a number of cancer types even in drug resistant cancer. CDYL2 upregulation in a breast cancer cell line induced migration, invasion, stem-like phenotypes, as well as an epithelial-to-mesenchymal transition (EMT). Due to the importance of EMT and stemness in therapeutic resistance and relapse in cancer, the inventors propose that CDYL2 inhibition will also be beneficial to the treatment of such cancers. Furthermore RNAi inhibition of CDYL2 diminished these same EMT-associated processes in the mesenchymal-like breast cancer cell line. Finally ablating the expression of CDYL2 with RNAi 1) stimulates the expression of genes associated with an anti-tumor immune response (such as gene involved in interferon response) and 2) inhibits lung tumorigenesis in a preclinical model (mouse injected with the triple negative MDA-MB-231 cell line). These results show that CDYL2 as a strong candidate proto-oncogene and therapeutic target in cancer and also contributes to the anti-tumoral immune response escape.
Claims
1. A method of preventing or treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a CDYL2 antagonist.
2. The method according to claim 1 wherein said cancer is a drug resistant cancer.
3. The method according to of claim 1, wherein said CDYL2 antagonist directly binds to CDYL2 protein or a nucleic sequence encoding the CRYL2 protein and promotes the expression of genes that regulate an anti-tumor immune response.
4. The method according to of claim 1 wherein said CDYL2 antagonist is selected from the group consisting of a small organic molecule; an inhibitor of CDYL2 gene expression; an anti-CDYL2 neutralizing antibody; and an anti-CDYL2 aptamer.
5. The method according to claim 4 wherein the inhibitor of CDYL2 gene expression is selected from the group consisting of an antisense oligonucleotide, a nuclease, siRNA, shRNA and a ribozyme nucleic acid sequence.
6. The method according to claim 1 wherein said cancer is a solid tumor or lymphoma/leukemia.
7. The method according to claim 6 wherein the solid tumor is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, oesophagus cancer and renal cancer.
8. A method to activate the anti-tumoral immune response of a patient affected with a cancer, comprising administering to the patient a therapeutically effective amount of a CDYL2 antagonist.
9. The method according to claim 8 wherein said cancer is a drug resistant cancer.
10. The method according to claim 8, wherein said CDYL2 antagonist binds directly to CDYL2 protein or a nucleic acid sequence encoding the CDYL2 protein and promotes the expression of genes that regulate an anti-tumor immune response.
11. The method according to claim 8, wherein said CDYL2 antagonist is selected from the group consisting of a small organic molecule; an inhibitor of CDYL2 gene expression; an anti-CDYL2 neutralizing antibody; and an anti-CDYL2 aptamer.
12. The method according to claim 11 wherein the inhibitor of CDYL2 gene expression is selected from the group consisting of an antisense oligonucleotide, a nuclease, siRNA, shRNA and a ribozyme nucleic acid sequence.
13. The method according to claim 8 wherein said cancer is a solid tumor or lymphoma/leukaemia.
14. The method according to claim 13, wherein the solid tumor is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, oesophagus cancers and renal cancer.
15. The method of claim 3, wherein the nucleic acid sequence is DNA or mRNA.
16. The method of claim 10, wherein the nucleic acid sequence is DNA or mRNA.
Description
FIGURES
[0140]
[0141]
[0142] (A) Western blot analysis of CDYL2 and beta-Actin expression in MCF7-CDYL2 and MCF7-Vector cells. (B) Volcano plot showing genes UP- or DOWN-regulated at least 2.5-fold at an adjusted p-value less than 0.05. (C) Selected molecular signatures over-represented in either the UP- or DOWN-regulated gene sets from (B). (D) qRT-PCR validation of selected differentially expressed genes from (C). Mean of three independent experiments±S.D. Significant at p<0.05 (T-test).
[0143]
[0144] (A) qRT-PCR analysis of EMT markers, normalization to GAPDH. Mean±S.D of three experiments. Significant at p<0.05 (T-test). (B) Western blot analysis of an EMT markers, ER-alpha, CDYL2 and Beta-actin. (C) Diagram of the xCELLigence quantitative, real-time migration and invasion assay. (D) Relative migration efficiency of MCF7-Vector and MCF7-CDYL2 cells. Both (D) and (E) show technical quadruplicates+/−S.D. Repeated at least three times with similar results. (E) Invasion assays were performed as in (D), except that the porous membrane separating the upper and lower chambers of the transwell was first overlaid with Matrigel. (F) Zebrafish embryo cell invasion and migration assay. Shown Percentage of embryos exhibiting tail metastases illustrating the metastasis of fluorescently labelled MCF7-CDYL2 or MCF7-Vector cells from the site of injection to the tail. Repeated three times with similar results. (G) Mammosphere formation in MCF7-CDYL2 cells compared to MCF7-Vector controls. The indicated number of ells were plated in 96-well plates. Mammospheres with size >50 m were counted after 8 days. Assay was repeated three times with similar results. T-test: **** p<0.0001. (H) Mammospheres diameter analysis. Shown is mean+S.D. of eight wells in which 1000 cells were seeded. T-test: *p<0.05. (I-J) FACS analysis of CD44 and CD24 expression. Shown are representative scatter plots (I) and the mean of three independent experiments +/−S.D. (J). T-test: *p<0.05.
[0145]
[0146] (A-B) CDYL2 knock-down validated by RT-qPCR (A) and western blotting (B). (C) Volcano plot showing genes Up- or Down-regulated at least 1.25-fold. (D) Selected GSEA analysis of Up- or Down-regulated genes from (C). (E) qRT-PCR validation of selected genes from (D), normalized to GAPDH. Shown if mean±S.D. of three independent experiments. All significant p<0.05 (T-test). (F) Western blot analysis of a panel of EMT markers, CDYL2 and Beta-actin. (G) Relative migration efficiency of MDA-MB-231 cells treated with esiLuc or esiCDYL2 (esiCD2). Both (G) and (H) show technical quadruplicates+/−S.D. Repeated three times with similar results. (H) Invasion assays were performed as in (G), except measuring migration across a microporous membrane overlaid with Matrigel. (I) Zebrafish embryo cell invasion and migration assay. Shown are micrographs. Percentage of embryos exhibiting tail metastases illustrating the metastasis of fluorescently labelled MDA-MB-231 cells treated with esiLuc or esiCDYL2 from the site of injection to the tail. Repeated three times with similar results. (J) Mammosphere formation in MDA-MB-231 cells treated with esiLuc or esiCDYL2. The indicated number of ells were plated in 96-well plates. Mammospheres with size >50 m were counted after 8 days. Assay was repeated three times with similar results. T-test: **** p<0.0001. (K) Mammospheres diameter analysis. Shown is mean+S.D. of eight wells in which 1000 cells were seeded. T-test: *p<0.05. (K-M) FACS analysis of CD44 and CD24 expression. Shown are representative scatter plots (L) and the mean of three independent experiments+/−S.D. (M). T-test: *p<0.05.
[0147]
[0148] (A) Selected GSEA signatures enriched in the indicated RNA-seq datasets. (B) Western blot of Ser536-phosphorylated p65 and Tyr705-phosphorylated STAT3 in MCF7-Vector versus MCF7-CDYL2. The levels of total p65, STAT3 and 3-actin were also probed. (C) As for (B), except comparing MDA-MB-231 cells treated with esiLuc or esiCDYL2. (D,E) Western blot validation of RNAi knockdown of p65 (D) or STAT3 (E) in MCF7-Vector and MCF7-CDYL2 cells. β-actin, loading control. (F) qRT-PCR analysis of the effect of RNAi knockdown of p65 on the expression of a panel of NF-κB target genes that were up-regulated in MCF7-CDYL2 compared to MCF7-Vector cells. Shown is mean of three experiments ±S.D. T-test, p<0.05. (G) As in (F), except the effect of RNAi knockdown of STAT3 was evaluated. (H,I) Invasion assays of MCF7-CDYL2 in MCF7 cells treated with either control RNAi or siRNA targeting p65 (H) or STAT3 (I). Assay was repeated three times with similar results. Shown is mean+/−S.D. of quadruplicate readings. (J,K) Mammospheres assay of MCF7-CDYL2 in MCF7 cells treated with either control RNAi or siRNA targeting p65 (J) or STAT3 (K). Mammospheres from 1000 seeded cells with size >50 m were counted after 8 days. Shown is the median+/−S.D. of three experiments. T-test: ** n<0.001: *** n<0.0001.
[0149]
[0150] (A) CDYL2 ChIP-seq peaks upstream of the MIR124 genes in MCF7-Vector cells (top graph) and MCF7-CDYL2 (lower graph). (B) ChIP-qPCR validation of the MIR124-proximal CDYL2 peaks represented in (A). A non-specific antibody (IgG) was used as negative control. All ChIP-qPCRs (B,C) show the mean enrichment as a percentage of Input of three experiments, ±S.D. T-test: * p<0.05; ** p<0.01. (C) As in (B), except CDYL2 or IgG ChIP was performed using chromatin prepared from MDA-MB-231 cells treated with esiLuc or esiCDYL2. (D) Selected GSEA signatures enriched in the indicated RNA-seq datasets. (E) qRT-PCR analysis of pre-mir-124 and miR-124-3p levels in MCF7-CDYL2 and MCF7-Vector cells. Expression was normalized to an unrelated miRNA. Data represent the mean of three independent experiments±S.D. T-test: * p<0.05; ** p<0.01. (F) As in (E), except qRT-PCR analysis was performed using miRNA prepared from MDA-MB-231 cells treated with esiLuc or esiCDYL2. (G) qRT-PCR analysis of the expression of miR-124-3p target genes in MCF7-CDYL2 and MCF7-Vector cells. Shown is mean±S.D. of three experiments. T-test: * p<0.05. (H) As in (G), except qRT-PCR analysis was performed using RNA prepared from MDA-MB-231 cells treated with esiLuc or esiCDYL2. (I) Western blot of phosphorylated p65 (Ser 536), Total p65, phosphorylated STAT3 (Tyr 705), and total STAT3 in MCF7-CDYL2 cells treated with a miR124-3p mimic or miR control. Repeated three times with similar results. (J) Western blot of phosphorylated p65 (Ser 536), Total p65, phosphorylated STAT3 (Tyr 705), and total STAT3 in MDA-MB-231 cells co-treated with esiCDYL2 and either an anti-miR-124-3p oligonucleotide, or a control anti-miR. Repeated three times with similar results.
[0151]
[0152] (A) Immunoblots of immunoprecipitates of non-specific IgG, CDYL2, EZH2 and G9a from MCF7 cell lysates. Repeated three times with similar results. (* specific band; ** IgG heavy chain). (B) ChIP-qPCR analysis of the relative occupancy of CDYL2, EZH2 and G9a upstream of MIR124 genes in MCF7-CDYL2 and MCF7-Vector cells. IgG, negative control ChIP. qPCR analysis was also performed at an unrelated negative control sequence. Shown is the mean enrichment as a percentage of Input of three independent experiments, ±S.D. T-test (* p<0.05; ** p<0.01; *** p<0.001). (C) As in (B), except using antibodies specific to H3K9me2, H3K27me3, H3 and IgG. These ChIP analyses were conducted using the same lysates as in (B), so are paired analyses. (D,E) Experiments were conducted as described in (B) and (C), except using chromatin lysates prepared from MDA-MB-231 cells treated with esiCDYL2 or esiLuc. (F) Schematic model of the proposed contribution of CDYL2 to epigenetic regulation of MIR124, cell signaling, and malignancy-associated cellular processes.
[0153]
[0154] (A) Oncomine analysis cancer cohorts revealed upregulation of CDYL2 mRNA in breast, colorectal, esophagus cancers and leukemia, but a downregulation in lymphoma. (B,C) Kaplan-Meier overall survival (OS) analysis performed from TCGA colorectal cancer (B), or rectal adenocarcinoma (C). High or low CDYL2 mRNA was based best cutoff. (D,E) Kaplan-Meier overall survival (OS) analysis performed from TCGA lung squamous cell carcinoma (D) or lung adenocarcinoma (E). High or low CDYL2 mRNA was based respectively on best cutoff of and highest versus lowest quartiles. Significance using LogRank p-value and Hazard Ratio (CI) are indicated.
[0155]
[0156] (A) Immunoblots (IB) of whole cell extract from MCF-7 show that CDYL2 antiserum reacts with one band of the expected molecular weight. (B) RNAi of CDYL2 in MCF-7 and HEK293T cells diminished the anti-CDYL2 immunoblot band intensity. (C) Transfection of MCF-7 with an HA-CDYL2 expression plasmid, but not empty vector, increased the anti-CDYL2 IB band intensity. Anti-HA blotting confirmed that the transfected and endogenous proteins co-migrate on SDS-PAGE gels. (D) Transfection of HEK293T cells with HA-CDYL (CDYL1) expression plasmids confirmed the CDYL2 antiserum does not cross-react with CDYL, which migrated at a higher molecular weight. HA blotting indicates the position of the CDYL band.
[0157]
EXAMPLE 1: IDENTIFICATION OF CDYL2 AS THERAPEUTIC TARGET IN CANCER
[0158] Material & Methods
[0159] Cell Culture
[0160] MCF7 cells (ATCC, HTB-22) and their derivatives were grown in DMEM Low Glucose (Gibco, 31885-023) supplemented with 10% of FBS (Gibco, 10270-106), 40 μg/mL of gentamicin (Gibco, 15710-049) and 0.6 μg/mL of insulin (NovoRapid, 3525909). MDA-MB-231 cells (ATCC, HTB-26) were grown in DMEM GlutaMAX (Gibco, 10566016) supplemented with 10% of FBS (Gibco, 10270-106), 1% penicillin/streptomycin (Gibco, 15140122). Cells were grown at 37° C. and 5% C02 in a humidified incubator and passaged every 2-4 days by trypsinization. Sustained expression of ER-alpha in MCF7 was validated regularly by western blotting and immunofluorescence. Cells were regularly tested for mycoplasma using a commercial kit (ATCC, 30-1012K), and cultures renewed from low passage stocks every two months or less.
Stable Expression of CDYL2 in MCF7 Cells
[0161] CDYL2 cDNA was cloned by PCR from an MCF7 cDNA library using primers and Phusion polymerase (NEB, M0530), and inserted into the Gateway pENTR-D-TOPO vector (Invitrogen, K240020). Sequencing on both strands confirmed that the cDNA corresponded to a published CDYL2 sequence (Genbank, NM_152342.2). The cloned cDNA was then transferred into MSCV plasmid (Addgene #41033) using LR Clonase (Invitrogen, 11791100), and the resulting expression construct validated by sequencing. MCF7 were then stably transduced with MSCV (Vector) or MSCV-CDYL2 retroviruses and selected for 14 days using 2 μg/mL puromycin (Sigma, P8833). Expression was confirmed by western blotting and immunofluorescence using CDYL2 antibody.
RNA Interference and microRNA Treatments
[0162] For transient RNAi, MDA-MB-231 cells were transfected with CDYL2 esiRNA (Sigma, EHU042511), esiLuciferase (Sigma, EHUFLUC), on-target plus p65 siRNA (Dharmacon, L-003533-00), or on-target plus STAT3 siRNA (Dharmacon, L-003544-00-0005) or on-target plus control siRNA (Dharmacon, D-001810-01-05) using Interferin reagent (Polyplus, 409-10) according to the manufacturer's instructions. Cellular assays and analysis were performed between 48 and 72 h post-transfection, according to the experiment. For stable RNAi, MCF7 cells were transduced with non-targeting control shRNA pLKO.1 lentiviruses (Sigma, SHC016) or shRNA-pLKO targeting CDYL2 (Sigma, shCDYL2 #1, TRCN0000359078; shCDYL2 #2, TRCN0000130741; shCDYL2 #3, TRCN0000129278), selected for 14 days using 2 μg/mL puromycin (Sigma, P8833). Cellular assays and analysis were then performed between weeks 2 and 4 post-transduction. The Hsa-miR-124-3p MISSION microRNA Mimic (Sigma, HMI0086) and its corresponding miRNA mimic Negative Control (Sigma, HMC0002) were transfected into MCF7-Vector or MCF7-CDYL2 cells using interferin. Samples were harvested for analysis 48-72 h post-transfection. The hsa-miR-124-3p Inhibitor (Qiagen, YT04102198-ADA), and its corresponding negative control (Qiagen, YT00199006-ADA) were co-transfected into MDA-MB-231 cells along with either esiCDYL2 or esiLuciferase siRNA, using Interferin reagent. Samples were harvested 72 h later for analysis.
Antibodies and Reagents
[0163] The following antibodies were used: CDYL2 (Sigma, HPA041016), ERa (Santa Cruz, sc-8002), β-Actin-HRP (Sigma, A3854), Vimentin (Dako, M0725), E-cadherin (BD 610682), Snail/Slug (Abeam, ab85936), Twist (Abeam, ab50887), Phospho-NF-κB p65 (Ser536) (Cell Signaling Technologies, #3031), total p65 (Cell Signaling Technologies, #3034), phosphor-STAT3 (Tyr705) (Cell Signaling Technologies, #9145), total STAT3 (Cell Signaling Technologies, #9139), CD44-FITC (Miltenyl Biotec, 130-113-341), CD24-PE (Miltenyl Biotec, 130-095-953), EZH2 (Cell Signaling Technologies #5246S), ChIP-grade EZH2 (Diagenode, C15410039), SUZ12 (Cell Signaling Technologies #3737S), H3K9me2 (Abeam, Ab1220), H3K27me3 (Diagenode, C15410069), rabbit IgG (Bethyl, P120-101), H3 (Abeam, Ab1791). The G9a and GLP antibodies were gifts from Y. Nakatani lab (PMID: 12004135).
Immunoblot
[0164] Cells were washed with PBS, lysed in ice-cold RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF, all from Sigma) containing protease inhibitor cocktail (Roche, 04693132001) phosphatase inhibitors cocktail (Roche, 4906845001), and sonicated briefly. Cleared lysates were resolved on Bis-Tris NuPage Gels (Invitrogen), transferred to Nitrocellulose (GE Healthcare), and probed according to the primary antibody manufacturer's protocols. Images were collected using the ChemiDoc system (BioRad).
Co-Immunoprecipitation
[0165] Cells were grown to sub-confluence in 15 cm tissue culture treated plates (Corning). Monolayers were washed three times with ice-cold PBS, scraped in cold PBS containing a protease inhibitor cocktail (Roche) and pelleted. The resulting cell pellets were lysed in lysis buffer (50 mM Tris pH8; 150 mM NaCl; 1% NP40; 2 mM EDTA) containing protease inhibitor cocktail (Roche, 04693132001) phosphatase inhibitors cocktail (Roche, 4906845001). The lysates mechanically homogenized using 25G syringes (BD, #300600), then incubated for 30 min at 4° C. with rotation. Lysates were centrifuged at 12000 rpm 15 min 4° C. to clear debris, treated with DNase I (Qiagen, #79254) and RNase A (Sigma, R4875), then precleared with protein A agaroses beads (for 1 hour 4° C. with rotation. Immunoprecipitation was performed by incubating indicated antibodies with the lysates overnight at 4° C. with rotation, the prewashed protein A agaroses beads were incubated with the lysates for 2 hours 4° C. with rotation. The beads were washed 5 times with wash buffer (10 mM Tris pH8; 1 mM EDTA; 1 mM EGTA; 150 mM NaCl; 1% Triton) containing protease inhibitor cocktail (Roche, 04693132001) phosphatase inhibitors cocktail (Roche, 4906845001). The immune-precipitated beads were boiled in Laemmli buffer and then subjected to immunoblotting.
Immunofluorescence
[0166] Cells were seeded on sterilized coverslips. Forty-eight hours after seeding, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed cells were permeabilized by NP-40 0.5% at room temperature for 15 min and blocked with 5% FBS in PBS+0.1% NP-40 at room temperature for 1 h. Cells were then probed with the indicated primary antibodies 1 h at room temperature and then Alexa dye tagged secondary antibody as mentioned for 1 h at room temperature. Coverslips were mounted using Dapi mounting medium (Vectashield; VECTOR Laboratories) and observed under the upright microscope (ZEISS AXIOIMAGER, SIP 60549), images were analyzed using Zen software.
Flow Cytometry
[0167] For CSC analysis, the cells were labeled with anti-CD44-PerCP-Cy 5.5 and anti-CD24-PE antibodies according to the manufacturer's instructions. All analyses were performed using a BD FACSCalibur flow cytometer and BD CellQuest software (BD Biosciences).
Gene Expression
[0168] RNA was extracted using TRI-reagent (Sigma, T9424), and cDNA synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368814). miRNA was extracted using the miRNeasy Mini Kit (Qiagen, #217004) following the manufacturer's instructions, and cDNA synthesized using miScript II RT Kit (Qiagen, #218161). The RT-qPCR was performed using the Fast SYBR Green 2× Master Mix (Applied Biosystems, #4385610) and an LC480 PCR machine (Roche).
RNA-Seq and Enrichment Analysis
[0169] RNA libraries were prepared with the TruSeq Stranded Total-RNA kit and sequenced on a Illumina NextSeq sequencing machine. After careful quality controls, raw data were aligned on the human genome (hg38) with STAR v2.7.0f (Dobin et al., 2013) and default parameters. Read counts on each genes of the Gencode annotation v29 were produced by STAR.
[0170] Unless otherwise specified, the analyses were performed using R (v 3.4.4) and illustrations produced with the ggplot2 (Wickham, H., (2016) Elegant Graphics for Data Analysis) and ggpubr packages. Starting from raw counts, we used the R package DESeq2 (Love et al, 2014) (v1.14) to perform the differential expression analyses. Each time the design was set as ˜REP +TYPE, where REP refers to the replicate number (paired analysis) and TYPE to the treatment group. Differential expression was tested using the Wald test, and p-values were corrected with the Benjamini-Hochberg method. To test the pathway enrichment of a list of genes, we used the R packages clusterProfiler (Yu et al, 2012) (v 3.8.1), msgidbr (v 6.2.1), org.Hs.eg.db (v 3.5.0) and reactomePA (v 1.24) (Carlson, M, 2013). We tested the list of genes against pathways from msigdb hallmarks, GO molecular functions, KEGG and Reactome. Over-representation p-values were corrected with the Benjamini-Hochberg method.
Chromatin Immunoprecipitation
[0171] For MDA-MB-231 ChIP-qPCR analysis, cells grown in 6-well plates were treated as described, then cross-linked by the addition of 1% methanol-free formaldehyde to the cell culture medium for 10 minutes, followed by the addition of glycine to a final concentration of 125 mM for 5 minutes. Monolayers were washed three times with ice-cold PBS, scraped in cold PBS containing a protease inhibitor cocktail (Roche) and pelleted. Chromatin lysates were prepared from the cross-linked pellets, and ChIP assays performed using a commercial kit (Cell Signaling Technologies, #9003), with the indicated antibodies. Quantitative PCR was performed. Input chromatin was purified in parallel in each ChIP assay and used to determine the percentage of input recovered in each ChIP assay.
[0172] For MCF7 ChIP-seq and ChIP-qPCR analysis, cells were grown to sub-confluence in 15 cm tissue culture treated plates (Corning), cross-linked, washed and pelleted as described for MDA-MB-231 cells, above. The resulting cell pellets were pre-treated by incubating in lysis buffer A (Lysis Buffer 1 (50 mM HEPES pH 7.5; 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1× protease inhibitors) for 10 minutes at 4° C., then lysis buffer B (10 mM Tris-HCl pH 8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1× protease Inhibitors) for 10 minutes at room temperature, as previously described (Lee et al., 2006). They were then lysed in buffer C (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1%; Na-Deoxycholate, 0.5% SDS, 1× protease inhibitors), incubated on ice for 30 minutes with occasional vortexing, then sonicated on ice to an average fragment size of 150 bp using a Branson sonicator. The sonicated lysate was centrifuged at 12,000 r.p.m. in a benchtop centrifuge at 4° C., the supernatant diluted five times in buffer C, and 1 mL aliquots of this added to 50 μL of magnetic protein A beads (Invitrogen) pre-coated with 5 μg of anti-CDYL2 IgG. These were incubated overnight at 4° C. with rotation, then pelleted and washed with two sequential additions each of wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8, 150 mM NaCl), wash buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8, 500 mM NaCl), wash buffer 3 (0.25M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8), and TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0, 50 mM NaCl). Chromatin was eluted by incubating for 30 minutes at 65° C. in elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA pH 8, 1% SDS) with frequent vortexing. Crosslinks were reversed by overnight incubation at 65° C., and eluates treated with RNAse A (Sigma, (Yu and He, 2016)) for 2 h, followed by Proteinase K for 2 h, then extracted using a classical Phenol-Chloroform/Ethanol precipitation protocol.
ChIP-seq
[0173] Pair end DNA sample libraries were sequenced using Illumina. Raw sequences were aligned to human genome hgl9, using Bowtie 2.0 (Langmead and Salzberg, 2012) with paired-end parameters. Normalized and subtraction bigwig files were obtaining using deepTools (Ramirez et al., 2016). Analysis of ChIP-Seq data was in the galaxeast.fr instance. Significant peaks were called using MACS2 (Zhang et al., 2008). Called peaks were annotated using Homer_AnnotatePeaks.
Colony Formation Assay
[0174] To form adherent colonies, 3000 cells were seeded in 12 well tissue-culture treated plates. After 14 days of growth, all colonies were stained with crystal violet solution (crystal violet 0.05%, formaldehyde 1%, methanol 1%) for 20 min, washed extensively with water. Colonies were counted and their size was measured using Fiji software.
[0175] Migration and invasion assays Real-time cell migration and invasion were measured using the xCELLigence RTCA DP apparatus (Acea Biosciences, Inc.) according to the manufacturer's protocol. Briefly, 40,000 cells were prepared in serum-deprived medium and then added to the upper chambers of CIM-16 plates (Aceabio, #5665817001). Complete medium containing 10% FBS was added to the lower chamber. For invasion assays, Matrigel diluted in serum-free medium at 500 μg/mL (BD Biosciences, #354234) was added to the upper chamber and allowed to set before adding cells. Migration across the membrane separating the two chambers was expressed as the Cell Index (CI).
Mammosphere Formation Assay
[0176] Cells were seeded at several densities (10, 50, 100, 1000 cells per well) in 96-well ultra-low attachment plates (Corning, #3474) with MEBM Basal Medium (Lonza, CC-3151) containing 2% B-27 (Invitrogen, 17504-044), 20 ng/mL EGF (Sigma, E9644), 4 μg/mL insulin (Novo Nordisk, #3525909), and 2 μg/mL hydrocortisone (Sigma, H0135). Mammospheres were cultured for 1-2 weeks, with image collection approximately every three days starting at day 8. Whole-well images were taken with the IncuCyte ZOOM System (Essen Bioscience) using a 4× phase contrast objective. Mammosphere diameter and the number of mammospheres >50 μm were determined by image analysis using Fiji software (Fiji).
Zebrafish Embryo Metastasis Assay
[0177] Zebrafish embryos were raised under standard experimental conditions. Cells trypsinated, resuspended in serum-free media, and stained with lipophilic dyes DiO or DiD from the Vybrant Multicolor Cell-Labeling Kit (Invitrogen, V22889) for 20 minutes at 37° C., then washed resuspended in PBS 1×. 48 hours post-fecundation, the embryos were dechorionated and anesthetized with tricaine (Sigma-Aldrich, E10521). The anesthetized embryos were subjected to microinjection. 20 nl of cell suspension, which represent approximately 300 labeled human cells, were injected into perivitelline space of each embryo. The injected zebrafish embryos were immediately placed at 30° C. for 24 hours in presence of N-phenylthiourea (Sigma-Aldrich, P7629) to inhibit melanocyte formation. For metastasis assessment, the anesthetized embryos were evaluated using the fluorescent microscope Axio Observer Zeiss microscope (Zeiss).
Statistical Analysis
[0178] CDYL2 expression levels from TCGA were stratified based on molecular markers such as ERa and HER2 expression by IHC. Correlation analysis between CDYL2 RNA and protein levels were performed using GraphPad Prism7. For overall survival analysis, patients were divided into two groups (low and high CDYL2) using the expression level of CDYL2 and best cutoff. Kaplan-Meier survival plots, log-rank p-values, hazard ratios were calculated using GraphPad Prism7.
Results
[0179] High CDYL2 Expression Level in Breast Cancer is Associated with Poor Prognosis
[0180] Datamining revealed that CDYL2 mRNA is upregulated in four breast cancer cohorts within The Cancer Genome Atlas (TCGA) 34 (
CDYL2 Over-Expression in the Non-Invasive Breast Cancer Cell Line MCF7 Induces Transcriptional Changes Associated with Malignant Progression
[0181] To ask if CDYL2 upregulation could induce oncogenic transcriptional and cellular changes, we stably expressed a CDYL2 cDNA in the non-invasive breast cancer cell line MCF7 (MCF7-CDYL2), or empty vector (MCF7-Vector) (
CDYL2 Over-Expression in MCF7 Cells Induces EMT-Like Changes, Migration, Invasiveness and Mammosphere Formation
[0182] Further probing if CDYL2 might induce EMT-like changes in MCF7 cells, we assessed the expression of a panel of established EMT markers. qRT-PCR analysis revealed CDYL2 upregulation of mesenchymal markers TWIST1, SNAI1, FN1, VIM, CTNNB1 and SNAI2 (
[0183] Among the primary contributions of EMT to malignant progression is increased cancer cell migration and invasion. In vitro assays revealed that the MCF7-CDYL2 cells migrated more proficiently across a microporous membrane compared to controls (
[0184] We then probed the effect of CDYL2 on MCF7 cell invasion and metastasis in vivo. Both MCF7-CDYL2 and control cells were fluorescently labeled and injected into the perivitelline space of zebrafish embryos. The presence of tail metastases was monitored by fluorescence microscopy 24 h later. Whereas MCF7-Vector cells rarely produced metastases (3.57% of fish), MCF7-CDYL2 cells did so in 21.57% of cases (
[0185] To test if CDYL2 overexpression additionally induced stem-like characteristics in MCF7 cells, we first performed a mammosphere assay. This is a functional assay to assess the enrichment of stem-like cells in a population. MCF7-CDYL2 cells yielded both more and larger mammospheres compared to controls (
RNAi Knockdown of CDYL2 in the Invasive Breast Cancer Cell Line MDA-MB-231 Diminishes the Expression of EMT Markers and Inhibits Migration, Invasion, and Mammosphere Formation
[0186] We next analyzed the effect of CDYL2 loss of function in the highly invasive, cancer stem cell enriched, mesenchymal-like breast cancer line MDA-MB-231. CDYL2 expression was inhibited by RNAi (
[0187] We first confirmed by qRT-PCR that CDYL2 RNAi reduced the levels of a number of transcripts associated with EMT, namely JUN, MYC, SNAI2, FOSL1 and TWIST1 (
Regulation of p65/NF-κB and STAT3 Signaling by CDYL2
[0188] GSEA analysis of the effects of CDYL2 over-expression in MCF7 cells or knockdown in MDA-MB-231 revealed a potential role in regulating NF-κB/TNF-alpha and STAT3/Interleukin-6 signaling (
[0189] We then asked if CDYL2 induction of genes associated with EMT, invasion, and sternness in MCF7 cells might be dependent on signaling via p65/NF-κB and STAT3. qRT-PCR analysis revealed that p65 RNAi downregulated several genes associated with NF-κB signaling, namely CTGF, EGR1, FOS, IL6, CXCL8, INHBA, JUN, MYC, SNAI1, KLF4, SOX2 and TWIST1 (
[0190] CDYL2 binds upstream of MIR124-2 gene and regulates miR-124 expression Consistent with the possibility that CDYL2 might be an epigenetic regulator of transcription, we found that it was enriched in the nucleus of both MCF7 and MDA-MB-231 cells, with a significant fraction present in the chromatin fraction (Data not shown). To identify where on chromatin CDYL2 is bound, we performed CDYL2 Chromatin Immunoprecipitation in both MCF7-Vector and MCF7-CDYL2 cells followed by Illumina sequencing (ChIP-seq). This revealed several genomic loci that were more enriched in CDYL2 in the MCF7-CDYL2 cells compared to vector controls, including upstream of all three members of the MIR124 gene family (
CDYL2 Interacts with G9a, GLP, and PRC2 Complex Components EZH2 and SUZ12
[0191] Because CDYL2 is enriched at MIR124 genes and negatively regulates miR-124 expression, we asked if it might promote an epigenetically repressive chromatin environment at these loci. However, the epigenetic mechanism of CDYL2 is not known. By analogy with CDYL1, we speculated that it may form a complex with the H3K9 di-methyltransferases G9a, GLP or SETDB1.sup.25 and the Polycomb Repressive Complex 2 (PRC2) core components EZH2 and SUZ12.sup.27. Using immunoprecipitation (IP) assays we found that Anti-CDYL2, but not a control IgG, efficiently recovered endogenous CDYL2 from MCF7 lysates and co-immunoprecipitated (CoIP) G9a and GLP (
CDYL2 Regulates the Enrichment of G9a and EZH2 Upstream of MIR124 Genes, as Well as that of their Cognate Methylation Marks H3K9Me2 and H3K27Me3
[0192] We next asked if CDYL2 might control the levels of G9a and EZH2 at a promoter-proximal region upstream of MIR124 genes. ChIP-qPCR assays indicated that CDYL2, G9a and EZH2 were enriched upstream of these genes in both MCF7 and MDA-MB-231 cells (
Discussion
[0193] Despite the emergence of epigenetic factors as important regulators of cancer cell plasticity and malignant progression, the underlying molecular mechanisms remain poorly understood. This is due in part to insufficient characterization of several putative epigenetic factors, including CDYL2. Our study shows that CDYL2 is frequently misexpressed in breast cancer, and provides a proof-of-principle that this could promote cellular phenotypes associated with malignant progression. We present the first insights into the genes and cellular pathways CDYL2 controls, and the epigenetic mechanisms it engages. Based on our findings, we propose that CDYL2 upregulation contributes to poor prognosis in breast cancer by inducing epigenetic deregulation of genes and pathways important in tumorigenesis (MIR124, NF-κB, STAT3), resulting in cellular changes central to malignant progression (EMT, migration, invasion, stemness).
[0194] Although we predicted CDYL2 to be an epigenetic repressor of transcription due to its homology to CDYL1.sup.22,23, this was not previously demonstrated. Our data support a mechanism whereby CDYL2 regulates the levels of G9a and EZH2 and their cognate histone methyl-lysine marks upstream of MIR124 genes, creating a local epigenetic environment repressive to transcription (schematic diagram,
[0195] MIR124 genes are emerging tumor suppressors commonly silenced in various cancers including breast.sup.15,20,48,55. MiR-124-3p directly targets STAT3 mRNA and antagonizes p65/NF-κB by inhibiting multiple components of its signaling pathway. It also regulates EMT, migration, invasion, and stemness.sup.13-15 Importantly, we show that CDYL2 positively regulated the active forms of both STAT3 and p65 in a manner reliant on miR-124 levels. We conclude that CDYL2 regulation of miR-124 expression substantially accounts for CDYL2 regulation of p65/NF-κB and STAT3 signaling, though we cannot exclude the possibility that other factors also contribute.
[0196] Both STAT3 and p65/NF-κB signaling are known drivers of cancer cell plasticity and malignant progression 3-5,7,15,16,18 In addition to positively regulating these pathways, CDYL2 also induced several cellular phenotypes associated with plasticity and aggressiveness in breast cancer, namely increased migration, invasiveness and stem-like behavior. Significantly, the ability of CDYL2 to induce MCF7 cell invasion and mammosphere formation was suppressed by inhibition of either p65/NF-κB or STAT3, indicating a crucial role for these pathways in its putative oncogenic mechanism.
[0197] It has been proposed that in certain malignancies, including breast cancer, molecular and cellular changes that promote the emergence of mesenchymal-like cells constitute a key enabling step in the process of malignant progression.sup.56 59. The EMT paradigm now encompasses a diversity of molecular and cellular expressions, several of which were positively regulated by CDYL2. These include changes in established EMT markers, as well as in cell morphology, migration, invasion and stemness. Notably, as was the case for invasion and stemness, CDYL2 induction of an EMT-like gene expression program in MCF7 cells was partially reversed by inhibition of STAT3 or p65, indicating it is downstream of these pathways. Overall, our studies are consistent with an oncogenic effect of CDYL2 over-expression in breast cancer. This might contribute to the poor prognosis of the ER+/HER2− and TN breast cancer patients whose cancers express high levels of CDYL2. Although not studied in depth here, we also observed a correlation between high CDYL2 expression and poor prognosis in lung and colorectal carcinomas, hinting at a wider role in cancer. Given the emergence of epigenetic factors as viable therapeutic targets in cancer, our study supports the further evaluation of CDYL2 as a candidate drug target in breast cancer, and potentially other malignancies. Notably, molecular and cellular changes associated with EMT and stemness in cancer cells were proposed to underlie resistance to a range of cancer therapies, as well as increased propensity to form invasive and metastatic tumors (42-44; 57, 59). Therefore, CDYL2 inhibition may also be effective in treating therapy resistant or malignant cancers.
EXAMPLE 2: CDYL2 INHIBITION AS A STRATEGY TO INCREASE TUMOR CELL IMMUNOGENICITY AND REDUCE CANCER IMMUNE EVASION
Materials and Methods:
Cell Culture
[0198] MCF7 cells (ATCC, HTB-22) and their derivatives were grown in DMEM Low Glucose (Gibco, 31885-023) supplemented with 10% of FBS (Gibco, 10270-106), 40 μg/mL of gentamicin (Gibco, 15710-049) and 0.6 μg/mL of insulin (NovoRapid, 3525909). MDA-MB-231 cells (ATCC, HTB-26) were grown in DMEM GlutaMAX (Gibco, 10566016) supplemented with 10% of FBS (Gibco, 10270-106), 1% penicillin/streptomycin (Gibco, 15140122). Cells were grown at 37° C. and 5% C02 in a humidified incubator and passaged every 2-4 days by trypsinization. Sustained expression of ER-alpha in MCF7 was validated regularly by western blotting and immunofluorescence. Cells were regularly tested for mycoplasma using a commercial kit (ATCC, 30-1012K), and cultures renewed from low passage stocks every two months or less.
Stable Expression of CDYL2 in MCF7 Cells
[0199] CDYL2 cDNA was cloned by PCR from an MCF7 cDNA library using the primers in Table S3 and Phusion polymerase (NEB, M0530), and inserted into the Gateway pENTR-D-TOPO vector (Invitrogen, K240020). Sequencing on both strands confirmed that the cDNA corresponded to a published CDYL2 sequence (Genbank, NM_152342.2). The cloned cDNA was then transferred into MSCV plasmid (Addgene #41033) using LR Clonase (Invitrogen, 11791100), and the resulting expression construct validated by sequencing. MCF7 were then stably transduced with MSCV (Vector) or MSCV-CDYL2 retroviruses and selected for 14 days using 2 μg/mL puromycin (Sigma, P8833). Expression was confirmed by western blotting and immunofluorescence using CDYL2 antibody.
RNA Interference
[0200] MCF-7 or MDA-MB-231 cells were transfected with CDYL2 esiRNA (Sigma, EHU042511) or esiLuciferase (Sigma, EHUFLUC) using Interferin reagent (Polyplus, 409-10) according to the manufacturer's instructions. Cellular assays and analysis were performed between 48 and 72 h post-transfection, according to the experiment.
RNA-Seq and Enrichment Analysis
[0201] RNA was extracted using TRI-reagent (Sigma, T9424) and libraries were prepared with the TruSeq Stranded Total-RNA kit and sequenced on a Illumina NextSeq sequencing machine. After careful quality controls, raw data were aligned on the human genome (hg38) with STAR v2.7.0f (Dobin et al., (2013) Bioinformatics) and default parameters. Read counts on each genes of the Gencode annotation v29 were produced by STAR. Unless otherwise specified, the analyses were performed using R (v 3.4.4) and illustrations produced with the ggplot2 (Wickham, H., (2016) Elegant Graphics for Data Analysis) and ggpubr packages. Starting from raw counts, we used the R package DESeq2 (v1.14) (Love et al, Genome Biology, 2014) to perform the differential expression analyses. Each time the design was set as ˜REP+TYPE, where REP refers to the replicate number (paired analysis) and TYPE to the treatment group. Differential expression was tested using the Wald test, and p-values were corrected with the Benjamini-Hochberg method. GSEA analysis was performed as described (Subramanian et al., (2005) Proc. Natl. Acad. Sci. U.S.A.), using the MSigDB collection of gene signatures (Liberzon et al., (2011) Bioinformatics).
Anti-CDYL2 Production:
[0202] CDYL2 cDNA was cloned from MCF-7 cells into pENTR(D)-Topo plasmid (Invitrogen), then transferred using LR Clonase (Invitrogen) into an N-terminal 6-Histidine tagging bacterial expression plasmid (pET-28a(+), EMD Biosciences) that was previously adapted for use as an LR Clonase Destination plasmid using the gateway system (Invitrogen). Next we expressed this plasmid in DE3-pLysS E. coli (Promega), and purified the resulting soluble, non-denatured His6-CDYL2 using NiNTA agarose beads (Qiagen) essentially as described in the NiNTA bead manual. The resulting purified Hisr-CDYL2 was used to immunise two rabbits and generate polyclonal anti-CDYL2 sera (immunization conducted by Covalab, Lyon, France). The antisera thus produced were tested at various points along the immunisation protocol.
Results and Discussion:
[0203] Both MCF-7 and MDA-MB-231 cell lines were treated with either siRNA targeting CDYL2 (esiCDYL2) or control siRNA targeting the firefly luciferase gene (esiLuc), and RNA extracted after 48 h in the case of MCF-7 and after 72 h in the case of MDA-MB-231 cells. The time of RNA harvesting was determined by doing a time course experiment to identify the point of maximum CDYL2 knock-down efficiency. In both cases, the experiment was repeated three times. The resulting triplicate samples were analyzed by paired-end (PE) total RNA sequencing (RNA-seq), and differences in gene expression between the esiCDYL2 and esiLuc groups determined. From the resulting data we derived a list of genes ranked from the most up-regulated in the esiCDYL2 dataset relative to esiLuc, to the most down-regulated. Comparison of the ranked gene lists to previously published gene expression signatures in the Molecular Signatures Database (MSigDB) using the Broad Institute Gene Set Enrichment Analysis (GSEA) software revealed similarities with many other gene expression signatures. In the case of MCF-7 cells, the most striking similarities were with IFN response gene signatures. These include signatures from the MSigDB Hallmark collection corresponding to Interferon alpha response, Interferon gamma response, and inflammatory response (Table 1). They also include numerous signatures associated with IFN responses from the MSigDB C2 curated gene set collection (Table 2). GSEA comparison of esiCDYL2-regulated genes and the MSigDB C2 collection further revealed strong similarities with gene expression signatures associated with the KEGG ‘antigen processing and presentation’ signature, suggesting CDYL2 inhibition might promote this central aspect of cancer cell immunogenicity (Table 2). In addition, CDYL2 RNAi induced gene expression signatures associated with the response to dsRNA (Table 2, ‘Geiss Response to dsRNA UP’ and ‘KEGG RIG-1-like receptor signaling pathway’). This suggests that, as was the case for loss or inhibition of DNMT1, HDACs, LSD1 and SETDB1 (Chiappinelli et al., (2015) Cell; Cuellar et al., (2017) Cell Biol; Sheng et al., (2018) Cell; Topper et al., (2019) Nat Rev Clin Oncol; Woods et al., (2015) Cancer Immunol Res), increased dsRNA levels might contribute to the observed IFN responses resulting from RNAi inhibition of CDYL2.
[0204] RNAi knock-down of CDYL2 followed by RNA-seq and GSEA analysis also revealed up-regulation of genes associated with IFN responses in MDA-MB-231 cells (Table 3, Table 5 and Table 4). A gene expression signature associated with the dsRNA response was also observed in this experiment (Table 5). Although the effect of CDYL2 RNAi on IFN response and dsRNA response gene expression signatures was less striking than in the case of MCF-7 cells, it was nonetheless statistically significant at the level of Nominal p-value. The reasons why CDYL2 RNAi more potently induced IFN and dsRNA response gene signatures in MCF-7 cells remain to be determined but could be related to the efficiency of RNAi knock-down, time of harvesting of RNA after transfection of cells with esiRNA, or simply differences in the intrinsic ability of each cell line to activate an IFN response.
[0205] Further evidence supporting a role for CDYL2 in regulating tumor cell immunogenicity came from RNA-seq analysis of the effects of stable over-expression of CDYL2 on MCF-7 cells. GSEA analysis of the resulting data-sets revealed that genes associated with IFN responses and antigen presentation were down-regulated in MCF-7 cells over-expressing CDYL2 compared to controls stably transfected with an empty vector (Table 7; note, the Enrichment Score (ES) and Normalized ES (NES) have negative values). Hence, while CDYL2 inhibition with RNAi induces IFN responses and related processes in MCF-7 cells, CDYL2 over-expression had the opposite effect. This further supports our hypothesis that CDYL2 levels negatively regulate the expression of IFN response and antigen processing and presentation genes, and that CDYL2 inhibition could be exploited therapeutically to induce expression of such genes in breast cancer.
[0206] Based on these gene expression changes, we propose that therapeutic inhibition of CDYL2 in breast cancer will induce an IFN response resulting in increased presentation of tumor antigens on MHC class 1 receptors and increased tumor cell attraction of T cells into the tumor microenvironment. While this could potentially be sufficient to increase T cell-directed elimination of cancer cells. This is expected to be especially the case in breast cancers expressing high levels of CDYL2 relative to normal breast tissue.
[0207] From a therapeutic point of view, we could envision the use of anti-CDYL2 antibodies—or modified versions thereof—to inhibit CDYL2 and induce an anti-tumour immune response in cancer patients. Towards this goal, we generated and tested an anti-CDYL2 antiserum. First, we cloned CDYL2 cDNA from MCF-7 cells into an N-terminal 6-Histidine tagging bacterial expression plasmid. Next we expressed this plasmid in DE3-pLysS E. coli, and purified CDYL2 using NiNTA agarose beads. The resulting purified protein was used to immunise two rabbits and generate polyclonal anti-CDYL2 sera (immunization conduced by Covalab, Lyon, France). The antisera thus produced were tested at various points along the immunisation protocol. One of the immunized rabbits produced an anti-CDYL2 antiserum that specifically reacted with a band of the expected molecular weight (
EXAMPLE 3: CDYL2 RNAI INHIBITS MDA-MB-231 LUNG TUMORIGENESIS IN VIVO
Materials and Methods
Preparation of Cells for the Assay
[0208] MDA-MB-231 cell lines stably transduced with either a negative control RNAi lentiviral vector (MISSION® pLKO.1-puro Non-Mammalian shRNA Control Plasmid DNA, SHC002, Sigma Aldrich) or a vector containing one of two distinct shRNA targeting CDYL2 (Sigma, shCDYL2 #1, TRCN0000359078; shCDYL2 #2, TRCN0000130741). The resulting transduced cells were then selected for 14 days using 2 μg/mL puromycin (Sigma, P8833) to eliminate cells that did not stably express the lentiviral vector. Knockdown of CDYL2 was validated by western blot using the antibody anti-CDYL2 (MyBiosource, MBS821304) according to standard protocols. CDYL2 knockdown was also validated by RT-PCR analysis using the primers (forward) ACCAACGGGGGATTGAACCTGC (table 8, SEQ ID NO:3) and (reverse) GGTGTCAGGGCATTGTTATCCGAGG (table 8, SEQ ID NO:4) in a Fast SYBR Green Master Mix (Applied Biosystems, #4385610) and an LC480 PCR machine (Roche). On the day of injection of cells into the mice, sub-confluent cell cultures were harvested by trypsinization and cells counted using a haemocytometer. Trypan blue staining of the cells at the time of counting confirmed the viability of the cells, as the vast majority excluded trypan blue dye. Cells were washed in ice-cold Phosphate Buffered saline (PBS), counted, and solutions of cells were prepared in cold PBS and kept on ice until the time of injection.
Murine Tumorigenesis Assays
[0209] These assays were carried out in accordance with the protocols approved under the ethics committee application CECCAPP CLB-2020-002. CECCAPP is an ethics committee in animal experimentation in the Rhône-Alpes region, based in Lyon and registered with the Ministry of Higher Education and Research of France under number C2EA15.
[0210] The experiment was performed as follows: three groups of MDA-MB-231 cells described above (shControl, shCDYL2 #1, shCDYL2 #2) injected intravenously into the lateral tail vein of 8 weeks old nude (NMRI nu) mice and their ability to form tumors in the lungs was determined. Each mouse was injected with 2×10E+5 cells in 0.1 ml of PBS, and each group consisted of 7 mice. These analyses were carried out broadly according to previously published methods (Minn A J, et al. Nature. 2005; 436 (7050): 518-524; Kuperwasser et al., Cancer Research, 2005, Volume 65, Issue 14), except that we used CT imaging to detect lung tumor formation in living mice (as detailed below).
Imaging Step
[0211] On the day of imaging, the anesthetized mouse (isoflurane) was slipped into the bed through which the gas anesthesia arrives. Thus the mouse remains motionless throughout the acquisition. Moreover, the receptacle also keeps the temperature of the mouse at 37° C. The testing period for the pulmonary observation is 2 minutes which induces an exposure of 746mGy (this dose is well less than the maximum tolerated dose of 2.6 Gy per exposure). The mouse was then returned to its cage. Acquisitions were made no more than once every two weeks.
Results and Discussion:
[0212] These assays show that the shCDYL2-expressing MDA-MB-231 cells formed fewer and/or smaller lung tumours compared to the control cells. This was revealed visually in the CT scans (
[0213] In other words, CDYL2 inhibition by RNAi inhibited the ability of these cells to form tumours in the lungs after injection into the tail vein of mice. By day 56, two of the mice injected with shControl cells had to be euthanised for ethical reasons as they had too many tumours in their lungs. This is why this group has fewer mice than the two shCDYL2 treated groups.
[0214] The results of this assay are consistent with the idea that CDYL2 inhibition can reduce the tumorigenicity of breast cancer cells, in this case a triple-negative breast cancer cell line. They further support the attractiveness of CDYL2 as a therapeutic target in breast cancer.
Table Section
[0215]
TABLE-US-00001 TABLE 1 GSEA MSigDB Hallmarks enriched upon CDYL2 RNAi in MCF-7 cells compared to control RNAi. RNA-seq was used to compare the relative expression of genes in MCF-7 cells treated with CDYL2 RNAi versus those treated with a control RNAi. The resulting gene list was ranked from the most over-expressed in the CDYL2 RNAi dataset to the most down-regulated. This ranked gene list was then compared to the GSEA MSigDB Hallmark gene set collection. Shown are the selected enriched gene expression signatures ranked in order of their Normalised Enrichment Score (NES). This revealed enrichment of gene expression signatures associated with the interferon response, as well as a number of other inflammation-associated gene signatures. NOM FDR FWER RANK LEADING NAME SIZE ES NES p-val q-val p-val AT MAX EDGE HALLMARK_INTERFERON_ALPHA_RESPONSE 93 0.85202414 2.3963137 0 0 0 4551 tags = 86%, list = 11%, signal = 97% HALLMARK_INTERFERON_GAMMA_RESPONSE 194 0.8040638 2.3467538 0 0 0 3906 tags = 58%, list = 10%, signal = 64% HALLMARK_TNFA_SIGNALING_VIA_NFKB 196 0.71396554 2.082239 0 0 0 7748 tags = 59%, list = 19%, signal = 72% HALLMARK_ALLOGRAFT_REJECTION 183 0.6662855 1.9243379 0 0 0 6411 tags = 40%, list = 16%, signal = 47% HALLMARK_INFLAMMATORY_RESPONSE 190 0.6495075 1.8971618 0 0 0 5134 tags = 37%, list = 13%, signal = 43% HALLMARK_IL6_JAK_STAT3_SIGNALING 85 0.68244517 1.893703 0 0 0 5944 tags = 48%, list = 15%, signal = 56% HALLMARK_COMPLEMENT 190 0.6153743 1.7986497 0 8.89E−05 0.001 4897 tags = 29%, list = 12%, signal = 33% HALLMARK_APOPTOSIS 157 0.594564 1.7259465 0 1.52E−04 0.002 5643 tags = 25%, list = 14%, signal = 30% HALLMARK_IL2_STAT5_SIGNALING 190 0.5235451 1.527428 0 0.00647235 0.112 7565 tags = 33%, list = 19%, signal = 41%
TABLE-US-00002 TABLE 2 GSEA MSigDB C2 curated gene sets collection signatures that were enriched upon CDYL2 RNAi in MCF-7 cells compared to control RNAi. RNA-seq was used to compare the relative expression of genes in MCF-7 cells treated with CDYL2 RNAi versus those treated with a control RNAi. The resulting gene list was ranked from the most over-expressed in the CDYL2 RNAi dataset to the most down-regulated. This ranked gene list was then compared to the GSEA MSigDB ‘C2 curated gene sets’ collection. Shown are selected enriched gene expression signatures ranked in order of their Normalised Enrichment Score (NES). This revealed enrichment of gene expression signatures associated with the interferon alpha, beta and gamma responses, as well as an interferon signature in cancer. NOM FDR FWER RANK LEADING NAME SIZE ES NES p-val q-val p-val AT MAX EDGE BROWNE_INTERFERON.sub.— 65 0.92116886 2.5035763 0 0 0 2132 tags = 83%, RESPONSIVE_GENES list = 5%, signal = 88% SANA_RESPONSE.sub.— 74 0.85650414 2.357083 0 0 0 4433 tags = 74%, TO_IFNG_UP list = 11%, signal = 83% SANA_TNF_SIGNALING_UP 76 0.8561752 2.3413148 0 0 0 1353 tags = 53%, list = 3%, signal = 54% MOSERLE_IFNA_RESPONSE 31 0.9504861 2.3155174 0 0 0 1772 tags = 97%, list = 4%, signal = 101% HECKER_IFNB1_TARGETS 87 0.8162935 2.2935858 0 0 0 2451 tags = 54%, list = 6%, signal = 57% DER_IFN_ALPHA.sub.— 73 0.8277265 2.269248 0 0 0 2663 tags = 52%, RESPONSE_UP list = 7%, signal = 56% RADAEVA_RESPONSE.sub.— 52 0.86436087 2.2681239 0 0 0 3702 tags = 62%, TO_IFNA1_UP list = 9%, signal = 68% BOSCO_INTERFERON.sub.— 72 0.82344323 2.2433825 0 0 0 3702 tags = 56%, INDUCED_ANTIVIRAL_MODULE list = 9%, signal = 61% DER_IFN_BETA.sub.— 101 0.790648 2.2273357 0 0 0 3237 tags = 43%, RESPONSE_UP list = 8%, signal = 46% REACTOME_INTERFERON.sub.— 56 0.8338998 2.202736 0 0 0 3060 tags = 55%, ALPHA_BETA_SIGNALING list = 8%, signal = 60% REACTOME_INTERFERON.sub.— 146 0.76679814 2.2004812 0 0 0 3906 tags = 38%, SIGNALING list = 10%, signal = 42% ZHANG_INTERFERON_RESPONSE 23 0.93506724 2.1814709 0 0 0 2343 tags = 91%, list = 6%, signal = 97% GEISS_RESPONSE.sub.— 37 0.79821616 2.0357206 0 0 0 1891 tags = 43%, TO_DSRNA_UP list = 5%, signal = 45% KEGG_ANTIGEN_PROCESSING.sub.— 67 0.73790246 2.0054493 0 0 0 4421 tags = 42%, AND_PRESENTATION list = 11%, signal = 47% WORSCHECH_TUMOR_EVASION.sub.— 29 0.80273026 1.934113 0 1.32E−05 0.001 2308 tags = 31%, AND_TOLEROGENICITY_UP list = 6%, signal = 33% KEGG_RIG_I_LIKE.sub.— 62 0.6545148 1.7591536 0 0.00422434 0.484 5643 tags = 32%, RECEPTOR_SIGNALING_PATHWAY list = 14%, signal = 37%
TABLE-US-00003 TABLE 3 Enrichment of gene expression signatures associated with antigen presentation and processing. Selected enrichment plots visualizing the indicated GSEA MSigDB C2 curated gene sets collection signatures. These data relate to MCF-7 cells treated with CDYL2 RNAi compared to control cells treated with a non-targeting siRNA. Shown are profiles of the Running ES Score & Positions of GeneSet Members on the Rank Ordered List. The associated statistics are shown in the table Dataset preranked_MCF7_KD_alone Phenotype NoPhenotypeAvailable Upregulated in class na_pos GeneSet KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION Enrichment Score (ES) 0.5307957 Normalized Enrichment 3.4366443 Score (NES) Nominal p-value 0.0 FDR q-value 1.530637E−5 FWER p-Value 0.003 Dataset preranked_MCF7_KD_alone Phenotype NoPhenotypeAvailable Upregulated in class na_pos GeneSet REACTOME_ANTIGEN_PROCESSING_CROSS_PRESENTATION Enrichment Score (ES) 0.43565646 Normalized Enrichment 2.7975485 Score (NES) Nominal p-value 0.0 FDR q-value 0.0029249415 FWER p-Value 0.794
TABLE-US-00004 TABLE 4 CDYL2 RNAi induces an interferon response gene signature in MDA-MB-231 cell line. Selected GSEA MSigDB Hallmarks enriched upon CDYL2 RNAi in MDA-MB-231 cells compared to control RNAi. These data relate to MDA-MB-231 cells treated with CDYL2 RNAi compared to control cells treated with a non-targeting siRNA. Shown are profiles of the Running ES Score & Positions of GeneSet Members on the Rank Ordered List. The associated statistics are shown in the table Dataset MDAMB231_esiCD2_versus_MDAMB231_esiLuc Phenotype cdyl2_all_samples_cat.cls#MDAMB231_esiCD2_versus_MDAMB231_esiLuc_repos Upregulated in class MDAMB231_esiCD2 GeneSet HALLMARK_INTERFERON_ALPHA_RESPONSE Enrichment Score (ES) 0.504692 Normalized Enrichment 1.5324306 Score (NES) Nominal p-value 0.004470939 FDR q-value 0.08714676 FWER p-Value 0.103 Dataset MDAMB231_esiCD2_versus_MDAMB231_esiLuc Phenotype cdyl2_all_samples_cat.cls#MDAMB231_esiCD2_versus_MDAMB231_esiLuc_repos Upregulated in class MDAMB231_esiCD2 GeneSet HALLMARK_INTERFERON_GAMMA_RESPONSE Enrichment Score (ES) 0.44447333 Normalized Enrichment 1.472826 Score (NES) Nominal p-value 0.0013888889 FDR q-value 0.09225965 FWER p-Value 0.209
TABLE-US-00005 TABLE 5 GSEA MSigDB C2 curated gene sets collection signatures that were enriched upon CDYL2 RNAi in MDA-MB-231 cells compared to control RNAi. RNA-seq was used to compare the relative expression of genes in MDA-MB-231 cells treated with CDYL2 RNAi versus those treated with a control RNAi. The resulting gene list was ranked from the most over-expressed in the CDYL2 RNAi dataset to the most down-regulated. This ranked gene list was then compared to the GSEA MSigDB ‘C2 curated gene sets’ collection. Shown are selected enriched gene expression signatures ranked in order of their Normalised Enrichment Score (NES). This revealed enrichment of gene expression signatures associated with the interferon signalling and tumor evasion and tolerogenicity. NOM FDR FWER p- RANK LEADING NAME SIZE ES NES p-val q-val val AT MAX EDGE ZHANGI_INTERFERON.sub.— 23 0.72339386 1.7277623 0.00168919 0.57765883 0.888 8162 tags = 65%, RESPONSE list = 20%, signal = 82% MOSERLE_IFNA_RESPONSE 31 0.6615628 1.6825045 0 0.5698889 0.988 9337 tags = 74%, list = 23%, signal = 97% BROWNE_INTERFERON.sub.— 65 0.5806052 1.6543516 0 0.7417494 0.997 9103 tags = 57%, RESPONSIVE_GENES list = 23%, signal = 73% CHIANG_LIVER_CANCER.sub.— 24 0.6324977 1.5248917 0.03529412 0.98659104 1 8834 tags = 54%, SUBCLASS_INTERFERON_UP list = 22%, signal = 69% WORSCHECH_TUMOR.sub.— 29 0.6030707 1.5053093 0.03204047 0.9703892 1 6630 tags = 41%, EVASION_AND.sub.— list = 16%, TOLEROGENICITY_UP signal = 50% GEISS_RESPONSE.sub.— 37 0.5679429 1.4921762 0.01597444 0.95655155 1 9506 tags = 54%, TO_DSRNA_UP list = 24%, signal = 71% EINAV_INTERFERON.sub.— 26 0.6105456 1.4813169 0.02684564 1 1 8162 tags = 50%, SIGNATURE_IN_CANCER list = 20%, signal = 63% BOSCO_INTERFERON.sub.— 72 0.50043344 1.4688625 0.01225115 1 1 9080 tags = 47%, INDUCED_ANTIVIRAL_MODULE list = 23%, signal = 61%
TABLE-US-00006 TABLE 6 Stable transgenic over-expression of CDYL2 represses expression of genes involved in the interferon response in MCF-7 cell line. The indicated GSEA MSigDB Hallmarks negatively correlated with CDYL2 over-expression in MCF-7 cells compared to control cells stably transfected with an empty vector. Shown are profiles of the Running ES Score & Positions of GeneSet Members on the Rank Ordered List. The associated statistics are shown in the table Dataset MCF7_CDYL2_versus_MCF7_Vector Phenotype cdyl2_all_samples_cat.cls#MCF7_CDYL2_versus_MCF7_Vector_repos Upregulated in class MCF7_Vector GeneSet HALLMARK_INTERFERON_ALPHA_RESPONSE Enrichment Score (ES) −0.40990403 Normalized Enrichment −1.5868864 Score (NES) Nominal p-value 0.0 FDR q-value 0.01045875 FWER p-Value 0.028 Dataset MCF7_CDYL2_versus_MCF7_Vector Phenotype cdyl2_all_samples_cat.cls#MCF7_CDYL2_versus_MCF7_Vector_repos Upregulated in class MCF7_Vector GeneSet HALLMARK_INTERFERON_GAMMA_RESPONSE Enrichment Score (ES) −0.32045266 Normalized Enrichment −1.369899 Score (NES) Nominal p-value 0.0 FDR q-value 0.042379666 FWER p-Value 0.155
TABLE-US-00007 TABLE 7 GSEA MSigDB C2 curated gene sets collection signatures the expression of which was repressed in MCF-7 cells stably over-expressing CDYL2 compared to control cells stably transfected with the empty vector. RNA-seq was used to compare the relative expression of genes in MCF-7 cells over-expressing CDYL2 versus controls. The resulting gene list was ranked from the most over-expressed in the CDYL2 RNAi dataset to the most down-regulated. This ranked gene list was then compared to the GSEA MSigDB ‘C2 curated gene sets’ collection. This revealed an inverse correlation between CDYL2 over-expression and expression of gene expression signatures associated with the interferon response, as well as antigen processing and cross-presentation. Nominal (NOM) p-value, False Discovery Rate (FDR) q-value and FWER p-values indicated as zero are less than 0.001. NOM FDR FWER RANK LEADING NAME SIZE ES NES p-val q-val p-val AT MAX EDGE MOSERLE_IFNA_RESPONSE 31 −0.7358488 −2.5625768 0 0 0 5560 tags = 81%, list = 20%, signal = 101% EINAV_INTERFERON.sub.— 25 −0.7354067 −2.3354008 0 2.84E−05 0.002 4471 tags = 64%, SIGNATURE_IN_CANCER list = 16%, signal = 76% REACTOME_CROSS.sub.— 45 −0.6152415 −2.3066409 0 5.26E−05 0.004 8487 tags = 80%, PRESENTATION_OF.sub.— list = 31%, SOLUBLE_EXOGENOUS.sub.— signal = 115% ANTIGENS_ENDOSOMES RADAEVA_RESPONSE.sub.— 48 −0.5988618 −2.2925148 0 5.00E−05 0.004 5560 tags = 52%, TO_IFNA1_UP list = 20%, signal = 65% REACTOME_ANTIGEN.sub.— 70 −0.5071787 −2.0585754 0 0.00103037 0.124 9034 tags = 69%, PROCESSING_CROSS.sub.— list = 32%, PRESENTATION signal = 101% BROWNE_INTERFERON.sub.— 64 −0.5185457 −2.03764 0 0.00134007 0.165 4050 tags = 41%, RESPONSIVE_GENES list = 15%, signal = 47% REACTOME_ANTIVIRAL.sub.— 65 −0.473695 −1.9114428 0.00367647 0.0046838 0.568 8410 tags = 48%, MECHANISM_BY_IFN.sub.— list = 30%, STIMULATED_GENES signal = 68% ZHANG_INTERFERON.sub.— 23 −0.5935327 −1.8828089 0 0.00590645 0.672 4292 tags = 65%, RESPONSE list = 15%, signal = 77%
TABLE-US-00008 TABLE 8 Useful nucleotide and amino acid sequences for practicing the invention SEQ ID NO Nucleotide or amino acid sequence 1 (CDYL2 MASGDLYEVERIVDKRKNKKGKWEYLIRWKGYGSTEDTWEPEHH AA LLHCEEFIDEFNGLHMSKDKRIKSGKQSSTSKLLRDSRGPSVEKLSH sequence) RPSDPGKSKGTSHKRKRINPPLAKPKKGYSGKPSSGGDRATKTVSY RTTPSGLQIMPLKKSQNGMENGDAGSEKDERHFGNGSHQPGLDLN DHVGEQDMGECDVNHATLAENGLGSALTNGGLNLHSPVKRKLEA EKDYVFDKRLRYSVRQNESNCRFRDIVVRKEEGFTHILLSSQTSDNN ALTPEIMKEVRRALCNAATDDSKLLLLSAVGSVFCSGLDYSYLIGRL SSDRRKESTRIAEAIRDFVKAFIQFKKPIVVAINGPALGLGASILPLCD IVWASEKAWFQTPYATIRLTPAGCSSYTFPQILGVALANEMLFCGR KLTAQEACSRGLVSQVFWPTTFSQEVMLRVKEMASCSAVVLEESK CLVRSFLKSVLEDVNEKECLMLKQLWSSSKGLDSLFSYLQDKIYEV 2 (CDYL2 atggcttctggggacctttacgaggttgaaaggattgtagacaagaggaagaacaagaaaggaaaatggg nucleic agtatcttatccgatggaaaggctacgggagcaccgaggacacgtgggagccggagcaccacctcttgca acid ctgtgaggagtttattgatgaattcaatgggttgcacatgtccaaggacaagaggatcaagtcagggaagca sequence) gtccagtacctccaagctgctgcgtgacagtcgaggcccgtcggttgagaaactgtcccacagaccttcag atcctggaaagagcaaggggacctcccataaacggaagcgaattaaccctcccctggccaagccaaaaa aagggtattcaggcaagccctcttcaggaggtgacagggccaccaagacggtgtcttacaggactacccc cagtggtttgcaaataatgcccctgaaaaagtctcagaacgggatggaaaatggggacgccggctctgag aaggatgagaggcactttggaaatgggtcccatcagcctggcttggatttgaatgatcatgttggagagcaa gatatgggtgaatgtgacgtgaatcacgctacactggcggagaacgggctcggctctgctctgaccaacg ggggattgaacctgcacagtccagtgaagaggaagctggaagcggagaaggactacgtctttgacaaaa ggctcagatacagtgtccgccagaatgaaagcaactgtcggtttcgagacatcgttgtgcggaaggaagaa gggttcacgcacatcctgctgtccagtcagacctcggataacaatgccctgacacctgagatcatgaaaga agtccggcgagcgctctgcaacgcagccacagacgacagcaaactgctgctcctcagcgcagtgggga gcgtgttctgcagcggcctggattattcctacctaattggccggttgtccagcgaccggcgaaaggagagc actcggattgcagaagccatcagggactttgtgaaggcctttatccagtttaagaagcctatcgtggtggcca tcaatgggccggccctgggcctgggtgcctccatcctgcccctctgtgacatcgtgtgggccagtgagaag gcctggttccagacgccctacgccaccatccgcctcacgcctgctggctgctcctcctacaccttcccccag atcctgggcgtcgcgctggccaatgagatgctgttctgtgggcggaagctcaccgcccaggaggcctgca gcagggggctggtgtcgcaggtcttctggcccaccacgttcagccaggaggtcatgctgcgggtcaagga gatggcatcctgcagtgccgtggtgttagaggagtccaaatgcctcgtgcggagcttcctgaaatcagtgct ggaagacgtgaacgagaaggaatgcctcatgctcaagcagctctggagctcctccaaaggccttgactcc cttttcagctacctgcaggacaaaatttatgaagtctga 3 (CDYL2 accaacgggggattgaacctgc forward PCR primer) 4 (CDYL2 ggtgtcagggcattgttatccgagg PCR reverse primer)
TABLE-US-00009 TABLE 9 Quantification of mean lung volume (mm3) by CT scan 56 days after injection of shControl, shCDYL2 #1 or shCDYL2 #2 MDA-MB-231 cells into the tail vein of nude mice. shRNA plasmid Mean Lung Vol (day 56) Mann-Whitney p-val shControl (n = 5) 429,615 mm3 shCDYL2 #1 (n = 7) 512,380 mm3 <0.05 shCDYL2 #2 (n = 7) 596,88 mm3 <0.05
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