Patent Application
20130295571
The present invention relates to cancer diagnosis and more particularly, to method and kit useful for identifying patients suffering from breast cancer with poor prognosis.
Despite improvements in the treatment of breast cancer, resistance to therapy is a major clinical problem. The molecular taxonomy of breast cancer based on transcriptional profiling has led to a better classification of tumors and pointed out new potential therapeutic targets.
Breast cancers are currently classified into five major molecular subtypes: luminal A, luminal B, basal, ERBB2-like, and normal-like (SORLIE et al., Proc. Natl. Acad. Sci. U.S.A., vol. 100, p: 8418-8423, 2003; SORLIE et al., Proc. Natl. Acad. Sci. U.S.A., vol. 98, p: 10869-10874, 2001). Molecular subtypes are associated with various clinical outcomes, and treatment strategies are being tailored to corresponding groups of patients (e.g. anti-ERBB2 therapies for patients with ERBB2-like tumors or hormone therapy for patients with luminal tumors).
Although luminal B tumors express hormone receptors (estrogen and progesterone), the risk of relapse is dramatically increased in women treated by hormone therapy for a luminal B tumor compared to women with a luminal A tumor. Little is known about specific signaling pathways deregulated in luminal B tumors. This molecular subtype is characterized by the expression of hormone receptors combined with high proliferative index. The specific gene expression signature associated with luminal B tumors is enriched in genes that drive the proliferation of cancer cells, such as CCNE, MKI67, or MYBL2 (SORLIE et al., abovementioned, 2001) and mitotic kinases (FINETTI et al., Cancer Res., 2008). Moreover, the functional loss of the retinoblastoma tumor suppressor gene (RB1) seems to be associated with the luminal B phenotype (HERSCHKOWITZ et al., Breast Cancer Res., vol. 10, p:R75, 2008; JIANG et al., J. Clin. Invest., 2010).
Finally, the identification of key mechanisms that regulate luminal B biology will thus lead to a better diagnostic and treatment of luminal tumors.
The inventors have shown here that luminal B tumors display specific amplifications, including amplification of the 8 p11-12 region in four subamplicons, A1 to A4.
The inventors have also shown that ZNF703, which is included in A1, is significantly amplified in luminal B tumors and that ZNF703 mRNA and protein over-expression is associated with luminal B tumors and defines patients with poor clinical outcome.
Their results suggest that ZNF703 plays a role in the regulation of luminal B cancer stem cell population by controlling key cell processes and has three important features that may explain its involvement in mammary oncogenesis.
First, ZNF703 transcription is induced by estrogen stimulation. In agreement, ZNF703 has been identified as an ER-regulated gene (LIN et al., PLoS Genet., vol. 3, p:e87, 2007; LIN et al., Genome Biol., vol. 5, p:R66, 2004). In contrast, the other 8p12 potential oncogenes have not been shown to be regulated by ER. This could explain the association of A1 (ZNF703) amplification with the luminal B subtype. The 8 p11-12 region is often co-amplified with the 11q13 region, which comprises the Cyclin D-encoding locus CCND1. CCND1 is also under the control of ER. ZNF703 and CCND 1 may thus function in the same pathway and potentiate each other. KWEK et al. have shown that CCND1 overexpression increases ZNF703 expression (KWEK et al., Oncogene, vol. 28, p:1892-1903, 2009).
Second, the ZNF703 protein may be a cofactor in a nuclear corepressor complex composed of NCOR2, PHB2 and DCAF7. Corepressors control the action of many transcription factors including ER. Interestingly, ZNF703 overexpression represses the expression of luminal markers and ER-associated proteins such as GATA3 and FOXA1. Moreover, ZNF703-overexpressing cells display RB1 phosphorylation, P27kip1 downregulation, E2F1 activation and Cyclin E activation. ZNF703 interaction with DCAF7 may explain its role on E2F1 signaling. DCAF7 is a CUL4/DDB1 interacting protein that activates SKP2 to induce P27Kip1 proteolysis. Interestingly, the DCAF7 gene is located in chromosomal region 17q23, which is often gained or amplified in luminal B breast cancers. Our GSEA showed that the same E2F1 activation/ER repression balance is prominent in luminal B tumors.
Third, ZNF703 overexpression increases the formation of primary and secondary tumorspheres suggesting a role in the control of cancer stem cell self-renewal. Moreover, the gene expression program activated by ZNF703 overexpression is enriched in genes related to stem cell biology. It has been proposed that luminal tumors might arise through transformation of an ER-positive luminal progenitor. The oncogenic capacity of ZNF703 could thus be exerted on ER-positive mammary progenitors and contribute to re-activation of a self-renewal program.
All these specific features may explain the association of ZNF703 amplification with the luminal B subtype and its frequent association with CCND1.
Thus, a first object of the invention is directed to an in vitro method for diagnosing a breast cancer from the luminal-B subtype in a female, which comprises the steps of analyzing a biological sample from said female by:
i) determining the copies number of the Zinc Finger Protein 703 gene (ZNF703 gene), and/or
ii) determining the expression of the ZNF703 gene,
wherein an increased copies number and/or an over-expression of the ZNF703 gene is indicative of a luminal B tumor.
A second object of the invention is directed to a kit for diagnosing a breast cancer from the luminal-B subtype in a female comprising at least one nucleic acid probe or oligonucleotide or at least one antibody, which can be used in a method as defined previously, for determining the copies number of the ZNF703 gene, and/or determining the expression of the ZNF703 gene.
Finally, a third object of the invention is directed to the use, for diagnosing a breast cancer from the luminal-B subtype in a female, of the abovementioned kit.
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In a first aspect of the invention, there is provided an in vitro method for diagnosing a breast cancer from the luminal-B subtype in a female, which comprises the steps of analyzing a biological sample from said female by:
i) determining the copies number of the Zinc Finger Protein 703 gene (ZNF703 gene), and/or
ii) determining the expression of the ZNF703 gene,
wherein an increased copies number and/or an over-expression of the ZNF703 gene is indicative of a luminal B tumor.
Since luminal B subtype is associated with poor prognosis, the female diagnosed with breast cancer from the luminal-B subtype are preferably not treated with anti-estrogens, but with more aggressive treatments (chemotherapy).
Preferably, the method of the invention further comprises the step of determining the expression of the estrogen receptor, wherein the expression of the estrogen receptor is indicative of a luminal B tumor.
As used herein, the term “female” refers to a mammal, preferably a woman.
Said female may be a healthy, but the method of the invention is particularly useful for testing a female thought to develop or developing a breast cancer. In that case, the method of the invention enables to diagnose a breast cancer from the luminal-B subtype.
As used herein, the expression “biological sample” refers to a breast tissue sample, preferably a breast tumor sample.
Said biological sample can be obtained by various techniques, preferably by biopsy, more preferably by a minimally invasive technique or selected from core biopsy, excisional biopsy, ductal lavage simple, a fine needle aspiration sample or from cells micro dissected from the sample.
Little is known about the ZNF703 protein for “Zinc finger protein 703”, also known as ZNF503L and FLJ14299, which is encoded by a gene located on the 8p12 chromosomal region, more specifically on the 8p11.23 chromosomal region.
The Zinc Finger Protein 703 gene (ZNF703 gene), also known as FLJ 14299, ZEPPO1, ZNF503L or ZPO1, is referenced with the accession number ID 80139, and its cDNA (Accession number NM—025069, SEQ ID no 1) is coding for a protein of 590 amino acids (Accession number NP—079345, SEQ ID no 2).
In a preferred embodiment of the invention, the method comprises the step of determining the copies number of the ZNF703 gene.
This step of determining the copies number of the ZNF703 gene can be done by well known methods such as Southern blot, quantitative PCR, DNA sequencing or fluorescence in situ hybridization (FISH). Primers or probes can be simply designed in view of the sequence of the known ZNF703 gene.
As used herein, the expression “an increased copies number of the ZNF703 gene” refers to a copies number of the ZNF703 gene by genome, which is superior to the two alleles of the ZNF703 gene.
In another preferred embodiment of the invention, the method comprises the step of determining the expression of the ZNF703 gene.
Methods for analyzing the expression of a gene are well known for the man skilled in the art.
In a particular embodiment of the invention, the expression of the ZNF703 gene is assessed by analyzing the expression of mRNA transcript or mRNA precursors, such as nascent RNA, of said gene.
Such analysis can be assessed by preparing mRNA/cDNA from cells in a biological sample from a subject, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA can be used in hybridization or amplification assays that include, but are not limited to, Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TAQMAN), and probes arrays.
Advantageously, the analysis of the expression level of mRNA transcribed from the ZNF703 gene involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in U.S. Pat. No. 4,683,202), ligase chain reaction (BARANY, Proc. Natl. Acad. Sci. USA, vol. 88, p: 189-193, 1991), self sustained sequence replication (GUATELLI et al., Proc. Natl. Acad. Sci. USA, vol. 87, p: 1874-1878, 1990), transcriptional amplification system (KWOH et al., 1989, Proc. Natl. Acad. Sci. USA, vol. 86, p: 1173-1177, 1989), Q-Beta Replicase (LIZARDI et al., Biol. Technology, vol. 6, p: 1197, 1988), rolling circle replication (U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
In another particular embodiment, the expression of the ZNF703 gene is assessed by analyzing the expression of the ZNF703 protein translated from said gene.
Such analysis can be assessed using an antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the ZNF703 gene. Said analysis can be assessed by a variety of techniques well known by one of skill in the art including, but not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, immunohistochemistry and enzyme linked immunoabsorbant assay (ELISA).
Polyclonal antibodies can be prepared by immunizing a suitable animal, such as mouse, rabbit or goat, with the ZNF703 protein (SEQ ID no 2) or a fragment thereof (e.g., at least 10 or 15 amino acids). The antibody titer in the immunized animal can be monitored over time by standard techniques, such as with an ELISA using immobilized polypeptide. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody producing cells can be obtained from the animal and used to prepare monoclonal antibodies (mAb) by standard techniques, such as the hybridoma technique originally described by KOHLER and MILSTEIN (Nature, vol. 256, p:495-497, 1975), the human B cell hybridoma technique (KOZBOR et al., Immunol., vol. 4, p: 72, 1983), the EBV-hybridoma technique (COLE et al., In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., p: 77-96, 1985) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, COLIGAN et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cells producing the desired monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA.
Commercial ZNF703 antibodies are available, as an example, one can cite the ones available from ABCAM, ABNOVA CORPORATION, ATLAS ANTIBODIES, AVIVA SYSTEMS BIOLOGY, EVEREST BIOTECH, GENETEX, GENWAY BIOTECH, INC., IMGENEX, LIFESPAN BIOSCIENCES, NOVUS BIOLOGICALS, PROSCI, INC, SANTA CRUZ BIOTECHNOLOGY, INC., or SIGMA-ALDRICH.
Alternatively, the step of determining the expression of the ZNF703 gene can done indirectly by determining the expression of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185 or 189 of the 189 genes listed in table 1, wherein the induction and/or downregulation of said genes are indicative of the expression of the ZNF703 gene.
In fact, the examples established that a gene signature can be associated with ZNF703 gene expression.
As used herein, the expression “over-expression of the ZNF703 gene” occurs when the transcription and/or the translation of the gene leads to an expression level in a biological sample that is at least 20% superior to the normal level of expression of said gene, preferably at least 50% superior to the normal level of expression of said gene, and most preferably at least 100% superior to the normal level of expression of said gene.
Therefore, the method of the invention may comprise comparing the level of expression of the ZNF703 gene in a biological sample from a female with its expression level in a control (i.e., normal expression level). A significantly greater level of expression of said gene in the biological sample of a female as compared to the normal expression level is an indication that the female suffers from a breast cancer of luminal-B subtype.
As used herein, a “control” corresponds preferably to a control sample comprising non-tumoral cells. Preferably, said control corresponds to normal breast tissues.
Thus, the “normal” level of expression of the ZNF703 gene is the level of expression of said gene in a biological sample of non-tumoral cell. Preferably, said normal level of expression is assessed in a control sample and preferably, the average expression level of said gene in several control samples.
Determining the normal expression of the ZNF703 gene may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid or translated protein as previously described.
In a second aspect, the present invention refers to a kit for diagnosing a breast cancer with the luminal-B subtype in a female comprising at least one nucleic acid probe or oligonucleotide or at least one antibody, which can be used in a method as defined previously, for determining the copies number of the ZNF703 gene, and/or determining the expression of the ZNF703 gene or protein.
Preferably, the oligonucleotide is at least one PCR primer, preferably a set of PCR primers is provided, which allows amplifying the ZNF703 gene or a fragment thereof. The skilled person readily provides such an oligonucleotide or set of PCR primers which allows to amplify a region of the ZNF703 gene, provided that the nucleic acid sequence of ZNF703 is well known (Current Protocols in Molecular Biology; edited by Fred M. Ausubel et al., supra).
As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.
The present kits can also include one or more reagents, buffers, hybridization media, nucleic acids, primers, nucleotides, probes, molecular weight markers, enzymes, solid supports, databases, computer programs for calculating dispensation orders and/or disposable lab equipment, such as multi-well plates, in order to readily facilitate implementation of the present methods. Enzymes that can be included in the present kits include nucleotide polymerases and the like. Solid supports can include beads and the like whereas molecular weight markers can include conjugatable markers, for example biotin and streptavidin or the like.
In one embodiment, the kit is made up of instructions for carrying out the method described herein for diagnosing a breast cancer from the luminal-B subtype in a female. The instructions can be provided in any intelligible form through a tangible medium, such as printed on paper, computer readable media, or the like.
Still a further aspect of the present invention refers to the use, for diagnosing a breast cancer from the luminal-B subtype in a female, of the abovementioned kit comprising at least one nucleic acid probe or oligonucleotide or at least one antibody, which can be used in a method as defined previously, for determining the copies number of the ZNF703 gene, and/or determining the expression of the ZNF703 gene.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
In the following, the invention is described in more detail with reference to amino acid sequences, nucleic acid sequences and the examples. Yet, no limitation of the invention is intended by the details of the examples. Rather, the invention pertains to any embodiment which comprises details which are not explicitly mentioned in the examples herein, but which the skilled person finds without undue effort.
To identify molecular mechanisms and signaling pathways specifically deregulated in luminal B tumors, we compared the genomic profiles from of luminal B and luminal A cases by using high density aCGH.
More especially, the tumor tissues were initially collected from 266 patients with invasive adenocarcinoma who underwent initial surgery at the Institute Paoli-Calmettes between 1987 and 2007, and from 22 samples from the Jules Bordet Institute. These samples were macrodissected and frozen in liquid nitrogen within 30 minutes of removal. DNA and RNA were extracted from frozen tumor samples by using guanidium isothiocynanate and cesium chloride gradient and their integrity was controlled on AGILENT BIOANALYZER (AGILENT TECHNOLOGIES) and agarose gel electrophoresis, respectively.
The genome profiling was then established on a subset of 100 luminal breast tumors (59 luminal A and 41 luminal B) for which RNA and DNA samples were both available. Because luminal B tumors have been associated with the amplifier genomic profile, we focused on recurrent amplifications. Genomic imbalances of 100 breast tumors were analyzed by aCGH using 244K CGH Microarrays (Hu-244A, AGILENT TECHNOLOGIES) according to the manufacturer's instruction. The final data set contained 225,388 unique probes covering 22,509 genes and intergenic part following the hg17 human genome mapping.
The
The results show that five genomic regions of recurrent high-level amplification (8p12, 8q22, 11q13, 17q24, 20q13) are associated with luminal B tumors (
We found a strong association of 8p12 amplification with luminal B tumors with 32% of tumors amplified; only 8% of luminal A tumors showed the 8p12 amplification, which was below the threshold retained by the aCGH statistical analysis (See
We searched to identify the 8 p11-12 genes targeted by amplification by comparing for each gene, the association between copy number and mRNA expression level for the 38 genes comprised in the amplicon defined by the aCGH analysis. These genes are localized in a 4.6 Mb region spanning between ZNF703 and ANK1.
For this analysis, the expression profiles of tumor samples wee obtained using AFFYMETRIX U133 Plus 2.0 human oligonucleotide microarrays and scanning was done with AFFYMETRIX GENEARRAY scanner. The data were analyzed by ‘Robust Multichip Average’ (RMA) with the non-parametric quantile algorithm as normalization parameter in R/Bioconductor and associated packages.
The
The results identify a hot spot of amplification frequency for the genomic segment that contains only the ZNF703 gene (Fisher's exact test, p<0.01) (data not shown). The results established that nineteen genes showed amplification correlated with upregulation (Fisher's exact test, p<0.01) (See
In 11 breast cancer cell lines (BCL), we did a similar integrated analysis of genomic amplification (using this time quantitative PCR) and gene overexpression by both DNA microarrays and reverse-transcribed PCR for each of the 38 genes. These BCLs are derived from normal or malignant epithelium and include 184B5, CAMA-1, HCC1500, HME-1, MCF7, MCF10A, MDA-MB-134, T47D, ZR-75-1, SUM52 and S68. BCLs were grown using the recommended culture conditions. The DNA and RNA form BCLs were isolated utilizing RNEASY MINI KIT (QIAGEN) and used for QPCR and RTQPCR in a LightCycler FastStart DNA Masterplus SYBR Green I (ROCHE) according to manufacturers' instruction.
In BCLs, the results have shown that ZNF703 expression levels measured by DNA microarrays and by quantitative RT-PCR were correlated (Pearson test, r=0.96), and were correlated with an increase in protein expression level (data not shown). Thus the integrated analysis on cell lines confirmed the results obtained on luminal B primary tumors, with ZNF703 gene presenting the most significant correlation between DNA copy number and mRNA expression (Pearson test, r=0.9) (data not shown).
We next examined ZNF703 expression measured by DNA microarrays according to the distribution of molecular subtypes distribution in 1172 breast cancers from 11 different published datasets.
The
The results confirmed that ZNF703 overexpression was associated with luminal B tumors when compared with luminal A tumors (T test, p=4.10−6) or with other subtypes (ANOVA, p=1.2−53) (See
To determine whether ZNF703 overexpression had clinical impact, we looked for association with clinical and pathologic features in 561 luminal tumors (A and B).
The
The results have shown no correlation between ZNF703 overexpression and any histoclinical factor. However, a high level of ZNF703 mRNA was associated with a decrease in disease-free survival (DFS) with a 5-years DFS of 68% versus 78% for a low level of ZNF703 (p=0.0136) (see
Taken together our results identified ZNF703 as a plausible oncogene candidate.
Some studies have described ZNF703 as a target gene of ER transcriptional activity, which is the master transcriptional regulator of luminal tumor phenotype, and have identified estrogen response elements (ERE) in its promoter region.
To firmly establish that ZNF703 expression is under the control of ER transcriptional activity, we cultured MDA-MB-134 and HCC1500 breast cancer cell lines in medium depleted or supplemented in 17β-estradiol (E2).
The results have shown that, in these cells, ZNF703 transcription and protein production was induced by addition of E2 in a time-dependent manner (data not shown).
Consequently, ZNF703 is a potential oncogene under the control of ER activity in luminal B tumors.
To identify signaling pathways regulated by ZNF703, ZNF703 was overexpressed in MCF7, a luminal breast cancer cell line that does not present 8p12 amplification, utilizing a GFP-ZNF703 fusion protein expression. Human ZNF703 cDNA full length (IMAGE: 5527569) was obtained by. cDNA sequence was PCR-amplified using attB1-TCACCATGAGCGATTCGCCCGCTGG-3′ (SEQ ID no 3) and attB2-CCTGGTATCCCAGCGCCGAGG-5′ (SEQ ID no 4) oligonucleotides. The resulting PCR fragment was inserted into the vector pDONR™/Zeo using BP-reaction gateway technology (INVITROGEN). pDONR™/Zeo-ZNF703 was then subcloned in pEGFP-C1-DEST using LR-reaction (INVITROGEN). As control a pEGFP-C1 vector that produces only a GFP protein was used. MCF7 cells were transfected with pEGFP-ZNF703 or pEGFP-C1 by electroporation as described by the manufacturer (AMAXA). Individual clones stably expressing GFP-ZNF703 fusion protein or GFP were obtained by constant geneticin selection at 0.5 mg/ml, the resulting clones were confirmed by Western blotting.
For immunofluorescence, the MCF7 GFP and GFP-ZNF703 cells were grown on LABTEK chambers. After 48 hours, cells were fixed 20 minutes in 3.7% paraformaldehyde, PBS; permeabilized 5 minutes with PBS, 0.1% TRITON X-100®; blocked with protein block serum-free (DAKO); and incubated 1 hour with primary antibodies (anti-GFP (ROCHE, 1:250)), and with corresponding secondary antibody Alexa Fluor 594 (INVITROGEN) conjugated for 20 minutes incubation. Images of the nuclei were counterstained with DAPI/antifade (INVITROGEN) and coverslipped. Sections were examined with a fluorescent microscope (LEICA).
The
The Immunofluorescence results reveal nuclear dot-like structures in MCF7 GFP-ZNF703 cells whereas GFP was detected in all subcellular structures in MCF7 GFP cells. To avoid potential artefactual subcellular location of ZNF703 due to the fusion protein product with GFP, we confirmed that endogenous ZNF703 is present in the same structure (data not shown). The size and the morphology of these nuclear structures are reminiscent of promyelocytic leukemia (PML) oncogenic domains (PODs) and RNA splicing bodies (SBs), which contain different sets of proteins.
To examine the potential overlap of ZNF703 localization with these nuclear structures, MCF7 GFP-ZNF703 cells were subjected to immunohistochemistry with antibody against PML (SANTA-CRUZ, 1:20) as described previously and to DNase 1 (100 μg/ml) or RNase A (80 μg/ml) treatment for 30 min and 15 min respectively. The nuclei were counterstained with DAPI/antifade (INVITROGEN) and coverslipped. Sections were examined with a fluorescent microscope (LEICA).
The
The results established that the staining pattern of PML did not overlap with that of ZNF703 (data not shown). The RNase A treatment did not affect ZNF703 nuclear structures (
We next sought to identify proteins that associate with ZNF703 in the nucleus. This identification was done by immunoprecipitated with anti-GFP antibody (IP GFP) or beads only (No Ab), separated by SDS-PAGE, and stained with colloidal Coomassie blue. The copreciptated proteins were then identified by mass spectrometry as HSP60 (accession number, NP 955472); DCAF7 (NP 005819) and PHB2 (NP 001138303), respectively.
The
Co-immunoprecipitation experiments confirmed the interaction of ZNF703 with HSP60 and DCAF7 in HCC1500 and MDA-MB-134 (endogenous conditions) and in MCF7 GFP-ZNF703 (
The
The results show that DCAF7 and PHB2 colocalized with ZNF703 in the dot-like nuclear structures (
Interestingly, when we questioned the multi-experiment matrix (MEM) web interface for ZNF703 gene expression similarities across 432 published gene expression datasets analyzed with AFFYMETRIX GENECHIP® from cell lines and primary tumors studies, we identified NCOR2 to be the most co-expressed gene with ZNF703 (data not shown).
Altogether, our results suggest that nuclear ZNF703 is a cofactor of a nuclear corepressor complex and may contribute to transcriptional regulation.
To further characterize ZNF703 overexpression effect, modification of gene expression program in MCF7 GFP-ZNF703 was analyzed compared to MCF7 GFP using DNA microarrays.
The
A total of 189 genes were differentially expressed between the two cell lines with 160 genes induced and 29 genes downregulated by ZNF703 overexpression (
Among the 160 genes induced by ZNF703, several genes were related to WNT (such as LEF1, TCF12, WNT4) or NOTCH (such as ASCL1, HEY2, TLE4) signaling pathways. Activation of these pathways is known to regulate self-renewal program in breast cancer stem cells (CSC). To better characterize the association between genes induced by ZNF703 and stem cell-related genes, we measured the enrichment of 26 published stem cell-related gene sets in genes differentially expressed in MCF7 GFP-ZNF703. Ten out of 26 gene sets were enriched in the ZNF703-related genes (data not shown).
These results suggest that ZNF703 overexpression may be implicated in the regulation of breast CSC. To directly test this hypothesis, we used an in vitro tumorsphere assay, a powerful surrogate method to evaluate cancer stem/progenitor cells. MCF7 GFP-ZNF707 and MCF7 GFP cell lines were plated as single cells in 1% agarose coated plates at a density of 1000 cells/ml and grown for 5 days. The number of primary spheres was counted under microscope. Subsequent cultures after dissociation of primary spheres were plated in 1% coated plates at a density of 1000 cells/ml. As for primary spheres, the number of secondary spheres was determined under microscope. Experiments were done in triplicate. Tumorsphere cultures were grown in a serum-free mammary epithelium basal medium as previously described (GINESTIER et al., Cell Cycle, vol. 8, p:3297-3302, 2009).
The
The results show that when stimulated by E2, MCF7 GFP-ZNF703 cells presented a significant increase in primary tumorsphere formation. Similar results were observed for secondary tumorsphere formation (
The above observations suggested that ZNF703 could regulate breast CSCs self-renewal activity by interacting with nuclear corepressor complex PHB2/NCOR2. Nuclear corepressors modulate the activity of master transcriptional regulators, such as ER or E2F1, and thus regulate key cell processes. Both ER and E2F1 have been implicated in the control of differentiation and self-renewal programs in stem cells. Therefore, we speculated that ZNF703 overexpression may regulate breast CSC biology through regulation of ER and E2F1 transcriptional activities. To test this hypothesis we first studied ER protein expression by immunostaining in MCF7 GFP-ZNF703.
The
The results show a decrease of ER expression in cells with ZNF703 dot-like structures (
Then, a specific luciferase reporter assay was used for the detection of estrogen transcriptional activity (p17M-ERE-Luc). MCF7 GFP and GFP-ZNF703 stable cell lines were electroporated (AMAXA) with 250 ng TK-R-Luc (pGL3; PROMEGA) as an internal control and 1 μg of one luciferase reporter vector according to the manufacturer's protocol. For ER-related transcriptional activity, cells were seeded overnight in 60-mm dishes containing phenol red-free RPMI 1640 (INVITROGEN) supplemented with 10% charcoal dextran-treated fetal bovine serum (HYCLONE). 16 hours later, 10 nM of 17-β estradiol (E2) were added to the medium and luciferase activity was determined after 24 hours. For luciferase activity detection, transfected cells lysate were prepared and THE DUAL-LUCIFERASE REPORTER ASSAY System was used (PROMEGA) according to the manufacturer's instructions. Each luciferase activity value was then normalized by protein concentration determined by Bradford assay and TK-renilla luciferase activity value. Each experiment was done in duplicate.
The
The results show a decrease of ER transcriptional activity in MCF7 GFP-ZNF703 compared to MCF7 GFP cells, independently of E2 induction (see
The
The results show then that MCF7 GFP-ZNF703 presented a decrease in expression for the two main ER-associated proteins, FOXA1 and GATA3 (See
The effect of ZNF703 overexpression on the other classical nuclear target of corepressors, E2F1 was next examined.
Two different E2F1 reporter systems (p3XE2F1-Luc and pCCNE-Luc) was then used according to the luciferase assay protocol described previously. Both vectors code for the luciferase protein either under the control of three E2F1 responsive elements or under the control of Cyclin E (CCNE) promoter, a direct E2F1 target.
The
Both reporter assays show an increase of E2F1 transcriptional activity induced by ZNF703 overexpression (See
We further tested the functional activation of E2F1 by western blot analysis of RB1 and P27kip1 protein status. Both proteins are implicated in E2F1 regulation. RB1 binds E2F1 and represses cell cycle progression; phosphorylations inactivate RB1 that releases E2F1 leading to proteolysis of P27kip1 and to transcription of several target genes implicated in cell cycle progression from G1 to S.
The
The results show an increase of RB 1 phosphorylation and a decrease of P27kip1 protein expression in MCF7 GFP-ZNF703 attesting of E2F1 activation induced by ZNF703 overexpression (See
Altogether, these results suggest that ZNF703 may be implicated in the regulation of ER and E2F1 transcriptional regulators and thus may modulate gene expression program implicated in breast CSC biology through these two major pathways.
To extend these results to clinical samples of luminal B primary tumors, the Gene Set Enrichment Analysis (GSEA) {http://www.broadinstitute.org/gsea/} algorithm was used to screen the activated transcription factors from the Broad Institute (MSigDB C3: motif gene sets TFT) for identifying a priori defined sets of genes that were differentially expressed between luminal A and luminal B samples. GSEA was applied on the IPC series with 138 luminal BCs (i.e. the 49 luminal B and 89 luminal A). Prior the GSEA, data were filtered to remove low and poorly measured expression probe sets as defined by an expression value inferior to 6.64 log 2 units across the 138 BCs. GSEA was done using signal-to-noise metric for ranking genes, weighted enrichment statistic to computed enrichment score (ES) of each gene sets tested and 1000 phenotype permutations to evaluate significance. As gene sets database, we chose the transcription factor targets list (C3, TFT) from the Molecular Signatures database (http://www.broadinstitute.org/gsea/msigdb). The C3 TFT list contains gene sets that share a transcription factor-binding site defined in the TRANSFAC database {http://www.gene-regulation.com}. Gene sets were defined as significant at the 5% level with a False Discovery Rate (FDR) under 25%. Relation between each transcription factor and E2F1 or ESR1/ERa was established by mining PubMed abstracts using Chilibot.
The
The results show a total of 119 gene sets that were differentially regulated between luminal B and luminal A tumors (FDR q-val<0.25; Nominal p-value<0.05) (See
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP11/05450 | 10/28/2011 | WO | 00 | 7/10/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61407947 | Oct 2010 | US |
