Guoqiang Hua
email: g.hua@unistra.fr

Aurélie Bergon

Pierre Cauchy

Brigitte Kahn-Perlès

François Bertucci

Daniel Birnbaum

Nadia Benkirane-Jessel

Jean Imbert
email: jean.imbert@inserm.fr

ERBB2b mRNA isoform encodes a nuclear variant of the ERBB2 oncogene in breast cancer

Keywords: ERBB2, isoform, nuclear translocation, breast cancer, resistance

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INTRODUCTION

The ERBB2/HER2 oncogene encodes a 185 kDa receptor tyrosine kinase (RTK) [START_REF] Akiyama | The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity[END_REF][START_REF] Yarden | Untangling the ErbB signalling network[END_REF]] that belongs to the epithelial growth factor receptor (EGFR) family, including epidermal growth factor receptor (EGFR)/ERBB1 [START_REF] Ullrich | Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells[END_REF], ERBB3 [START_REF] Kraus | Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors[END_REF], and ERBB4 [START_REF] Plowman | Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family[END_REF]. These receptors are composed of an extracellular domain, a transmembrane domain, and an intracellular domain. ERBB2 has no known ligand binding to its extracellular domain [START_REF] Klapper | The ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stromaderived growth factors[END_REF]; however, it becomes the preferred heterodimeric partner of the other three ERBB members [START_REF] Graus-Porta | ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling[END_REF]. Ligand binding to these high-affinity complexes stimulates the kinase activity, phosphorylates tyrosine residues and activates diverse downstream signaling cascades, such as mitogen-activated protein kinase (MAPK), signal transducers and activators of transcription (STAT), and the phosphatidylinositol 3-OH kinase (PI3K) pathway [START_REF] Citri | EGF-ERBB signalling: towards the systems level[END_REF][START_REF] Egan | The pathway to signal achievement[END_REF].

Amplification and overexpression of the ERBB2/HER2 receptor are observed in ~20% of breast cancers, and in other types of cancer with variable proportions, and are associated with poor prognosis and higher metastatic potential [START_REF] Hurst | Update on HER-2 as a target for cancer therapy: the ERBB2 promoter and its exploitation for cancer treatment[END_REF][START_REF] Kim | Palbociclib: A Review in HR-Positive, HER2-Negative, Advanced or Metastatic Breast Cancer[END_REF][START_REF] Vernimmen | Different mechanisms are implicated in ERBB2 gene overexpression in breast and in other cancers[END_REF][START_REF] Yarden | Untangling the ErbB signalling network[END_REF]. Major clinical treatments of ERBB2-positive breast cancers include humanized monoclonal antibodies (trastuzumab and pertuzumab), which bind to the extracellular domain of ERBB2, disrupting the dimerization of the receptor and the downstream signaling [START_REF] Franklin | Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex[END_REF][START_REF] Hudis | Trastuzumab--mechanism of action and use in clinical practice[END_REF], and tyrosine kinase inhibitors (TKI, lapatinib and neratinib), which antagonize the kinase activity of the homo-or heterodimerized receptors, inhibiting phosphorylation of their substrates and downstream signaling [START_REF] Barok | Trastuzumab emtansine: mechanisms of action and drug resistance[END_REF][START_REF] Burris | Dual kinase inhibition in the treatment of breast cancer: initial experience with the EGFR/ErbB-2 inhibitor lapatinib[END_REF][START_REF] Burstein | Neratinib, an irreversible ErbB receptor tyrosine kinase inhibitor, in patients with advanced ErbB2-positive breast cancer[END_REF][START_REF] Diéras | Trastuzumab emtansine versus capecitabine plus lapatinib in patients with previously treated HER2-positive advanced breast cancer (EMILIA): a descriptive analysis of final overall survival results from a randomised, open-label, phase 3 trial[END_REF][START_REF] Xia | Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells[END_REF]. These therapies have greatly improved the overall survival of ERBB2-positive breast cancer patients [START_REF] Slamon | Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2[END_REF]. However, resistances to monoclonal antibody and TKI treatments are observed in the majority of patients [START_REF] Blackwell | Single-agent lapatinib for HER2-overexpressing advanced or metastatic breast cancer that progressed on first-or second-line trastuzumab-containing regimens[END_REF][START_REF] Esteva | Phase II study of weekly docetaxel and trastuzumab for patients with HER-2-overexpressing metastatic breast cancer[END_REF][START_REF] Nahta | Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer[END_REF].

Although different mechanisms have been proposed [START_REF] Esteva | Molecular predictors of response to trastuzumab and lapatinib in breast cancer[END_REF][START_REF] Spector | Understanding the mechanisms behind trastuzumab therapy for human epidermal growth factor receptor 2-positive breast cancer[END_REF], the resistance to anti-ERBB2 treatment remains a major clinical issue.

The presence of nuclear forms of RTKs, including the four members of the EGFR family, has been revealed since the last two decades [START_REF] Carpenter | Nuclear localization and possible functions of receptor tyrosine kinases[END_REF][START_REF] Lin | Nuclear localization of EGF receptor and its potential new role as a transcription factor[END_REF][START_REF] Ni | gamma -Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase[END_REF][START_REF] Offterdinger | c-erbB-3: a nuclear protein in mammary epithelial cells[END_REF][START_REF] Wang | Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2[END_REF] and several mechanisms for the nuclear translocation and the function of those nuclear RTKs have been reported [START_REF] Giri | Endosomal transport of ErbB-2: mechanism for nuclear entry of the cell surface receptor[END_REF][START_REF] Lin | Nuclear localization of EGF receptor and its potential new role as a transcription factor[END_REF][START_REF] Lo | Nuclear-cytoplasmic transport of EGFR involves receptor endocytosis, importin beta1 and CRM1[END_REF][START_REF] Ni | gamma -Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase[END_REF][START_REF] Offterdinger | c-erbB-3: a nuclear protein in mammary epithelial cells[END_REF][START_REF] Rio | Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4/HER4[END_REF][START_REF] Wang | Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2[END_REF][START_REF] Williams | The ERBB4/HER4 receptor tyrosine kinase regulates gene expression by functioning as a STAT5A nuclear chaperone[END_REF]. Interestingly, recent studies shed new light on the role played by the nuclear form of ERBB2 in the resistance to anti-ERBB2 treatments [START_REF] Russo | Targeting ErbB-2 nuclear localization and function inhibits breast cancer growth and overcomes trastuzumab resistance[END_REF], 2019;[START_REF] Li | Nuclear ErbB2 enhances translation and cell growth by activating transcription of ribosomal RNA genes[END_REF][START_REF] Schillaci | Clinical relevance of ErbB-2/HER2 nuclear expression in breast cancer[END_REF][START_REF] Venturutti | Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis[END_REF].

The ERBB2 gene can be transcribed as at least 17 different transcripts generated by alternative splicing and alternative promoter usage [START_REF] Nezu | Identification of a novel promoter and exons of the c-ERBB-2 gene[END_REF]. All transcript variants are apparently under the control of two major promoters separated by 12 Kb (Fig. 1). The proximal promoter controls the transcription of ERBB2 transcript variant 1 (NM_004448) which encodes the canonical form of the receptor ERBB2a, generally named ERBB2, while the 5'-end distal promoter controls the transcription of ERBB2 transcript variant 2 (NM_001005862.2). ERBB2 transcript variant 1 is composed of 27 exons.

Its first exon codes for 30 amino acids, where the first 22 amino acids constitute a signal peptide which is cleaved during the migration of ERBB2 mature form to the cell membrane. ERBB2 transcript variant 2 is composed of 30 exons where the first 4 exons are non-coding. Consequently, the translation of the ERBB2b isoform starts at an alternative ATG within its fifth exon which is identical to the second exon of variant 1 (Fig. 1). Therefore, sequences coding for the signal peptide in ERBB2a exon 1 are missing in the ERBB2b isoform. Because this signal peptide is cleaved once it transports the protein to the cell membrane, the sequence difference of mature proteins between ERBB2a and ERBB2b isoforms is only eight N-terminal amino acids. Beside its canonical location at cell membrane, alternative forms of the ERBB2/HER2 receptor have been observed into cell nuclei where they can escape current therapeutic approaches. These nuclear forms have been described as the result of at least two different mechanisms: proteolytic shedding of the extracellular domain of the full-length receptor and translation of the mRNA encoding ERBB2 from internal initiation codons [START_REF] Anido | Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation[END_REF][START_REF] Christianson | NH2terminally truncated HER-2/neu protein: relationship with shedding of the extracellular domain and with prognostic factors in breast cancer[END_REF]].

Here, we report a new mechanism for ERBB2 nuclear expression, where the ERBB2b isoform can directly translocate into the nucleus without requirement of an intermediate membrane location. siRNA analyses showed that ERBB2b isoform can repress the ERBB2a isoform, whereas ERBB2a isoform can activate the ERBB2b isoform. In addition, nuclear ERBB2 binding to its own promoter was revealed by chromatin immunoprecipitation (ChIP) assay. Altogether, our results provide new insights into the function of ERBB2 and provide an additional explanation for the resistance to therapies targeting the membrane-anchored ERBB2 in breast cancer.

MATERIALS AND METHODS

Cell lines and antibodies

The non-oncogenic human primary mammary epithelium cells HME-1 (Clontech, Mountain View, CA, USA) and the breast cancer cell lines BT474, BT-483, HCC-202, HCC-1806, HCC-1954, HCC- 2. Twenty cases were ERBB2+, whereas 8 were ERBB2-. Each patient gave written informed consent and the study was approved by our institutional review board. The following antibodies were purchased from a commercial source: ERBB2

(Santa Cruz, sc-284), Sp1 (Upstate, 07-645), anti--tubulin (Abcam, ab11307), IgG Rabbit (ab171870).

ChIP-seq data processing

Reads were aligned to the hg19 genome using bowtie 1.1.1 [START_REF] Langmead | Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[END_REF], with the parameters --all --best --strata -v 2 -m 1 -S. Peak detection was performed using MACS 1.4.2 [START_REF] Zhang | HCC1954 ERBB2 BT474 HCC1954 ERBB2 Sp1 ß-Tubulin Cytosolic, membrane, and nuclear fractions were separated for BT474 and HCC1954 cells, Western blot analyses were performed with ERBB2, SP1 and ß-Tubulin antibodies[END_REF] using -g hs --keep-dup=auto -w -S. Annotation of peaks was performed using Homer annotatePeaks (version 4.7.2) [START_REF] Heinz | Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities[END_REF]. Public BT474 rabbit IgG and RNAPII HCC-1806 data were recovered from the Gene Expression Omnibus (GEO) from accessions GSM1145935 [START_REF] Heinonen | Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer[END_REF] and GSM3317507 [START_REF] Perreault | Epigenetic and transcriptional profiling of triple negative breast cancer[END_REF], respectively. Intersection of peaks was carried out using bedtools intersect 2.19.0 [START_REF] Quinlan | BEDTools: a flexible suite of utilities for comparing genomic features[END_REF]. Annotation to the closest gene was performed using bedtools closest 2.19.0 using -t first as a parameter. Read depth coverage were normalized to BT474 Pol II ChIP-seq data.

siRNA Assay

Three 25-mer duplex small interfering RNAs (siRNAs) to specifically target either ERBB2a or b mRNA variants were designed and ordered (Select Stealth TM RNAi, Invitrogen,

ERBB2a-/ ERBB2b-GFP construction

The full-length cDNAs of the two isoforms ERBB2a and b were obtained by reverse transcription (Super Script ™ First-Strand, Invitrogen) of mRNA extracted from BT474 cells (RNeasy® Micro Kit, Qiagen) and amplified by PCR (Platinum® TaqDNA Polymerase High Fidelity, Invitrogen) using Gateway Specific Primers: ERBB2a-GW-F 5'-AttB2-GGGGACAAGTTTGTACAAAAAAGCAGGCTAGCCGCAGTGAGCACCAT-3'; ERBB2b-GW-F 5'-AttB2-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGTGCACCGGCACAGACAT-3';

ERBB2-GW-R 5'-AttB1-GGGGACCACTTTGTACAAGAAAGCTGGGTGACTTGGCCTTCTGGTTCA-3'. They were subsequently cloned into the expression vectors pEGFP, according to the Gateway protocol (Invitrogen).

Transient transfection

Cells were transfected using FuGENE 6 reagent (Roche Diagnostics). 3x10 5 cells were seeded in 6-well plates (Falcon 3046, BD Biosciences, Lincoln Park, NJ) and then transfected the next day using the FuGENE reagent (FuGENE:DNA ratio of 3 µl: 2 µg) for 48 h in complete medium.

RNA extraction and Reverse-Transcription PCR

Total RNA was extracted from cells using RNeasy  Micro Kit (QIAGEN) according to the manufacturer's protocol. Total RNA was quantified using a Nanodrop 1000 Spectrophotometer device. RT-PCR was performed using SuperScript TM II Reverse Transcriptase (Invitrogen) and random hexamer primers.

Subcellular fractionation

The cytosolic, membrane, and nuclear fractions were prepared from fractions 1, 2 and 3 using ProteoExtract ® Subcellular Proteome Extraction (Calbiochem, Merck). The fractionation efficiency was assessed by antibodies against Sp1 and -tubulin.

Immunofluorescence

Cultured cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Tween-20 for 10 min, and then immunostained with primary antibodies against ERBB2 (1:200 dilution in PBS with 1% BSA) for 1 hr at room temperature. After three washes with PBS, the Alexa 488-conjugated secondary antibody was then applied for 45 min at room temperature. The nucleus was stained with DAPI before mounting. For immunofluorescence microscopy, the images were captured with a Zeiss 510 Meta UV confocal microscope. 10 5 cultured cells were lysed in 10 µl of lysis buffer (25 mM Tris pH 7.9, 1% w/v SDS, 1 mM dithiothreitol)

Western blot

and fractionated on a 7.5% SDS-polyacrylamide gel, electrotransferred to a PVDF membrane. Specific polypeptides were revealed with the rabbit polyclonal anti-ERBB2 (C18, sc-284), rabbit polyclonal anti-Sp1 (Upstate, 07-645), and mouse monoclonal anti--tubulin (Abcam, ab11307) at a proper dilution in TBS-Tween containing 5% non-fat milk or BSA according to the recommendations of the supplier, and then reacted with secondary antibodies at a dilution of 1:10 4 in the same buffer.

Chromatin Immunoprecipitation Assay

BT474 cells were fixed with 1% formaldehyde for 15 min, and the cross-linking reaction was stopped by the addition of 125 mM glycine incubating 5 min. Cells were washed and lysed in three Lysis Buffers containing 1x Complete Protease Inhibitors Cocktail (Roche) respectively. 1% Triton X-100 was added to the lysate and all lysates were sonicated at 4°C to generate 100-300 base pair chromatin fragments.

Pretreated mixture of 3 µg of antibody (rabbit polyclonal anti-ERBB2, C18 sc-284 or rabbit polyclonal anti-PolII, N20 sc-899), and protein G-conjugated magnetic Dynabeads (Dynal Biotech, Invitrogen) was added to the supernatant and incubated on a rotating wheel at 4°C for overnight. The immunocomplexes were then washed with RIPA wash buffer (S1) for 6 times and once with TE containing 50 mM NaCl. The Ab-protein-DNA complexes were eluted twice in 100 µl elution buffer at 65°C for 15 min, briefly vortexed every 2 min. The combined eluates as well as the input sample (1% of the amount used in the IP procedure) were treated to reverse the crosslink by incubating overnight at 65°C. After Rnase and proteinase K digestion, the DNA fragments were extracted using phenolchloroform and purified using a Qiagen PCR purification kit.

Quantitative PCR and normalization

DNA was quantified using LC FastStart DNA Master SYBR Green I and read with a Light Cycler ® 2 instrument (Roche Diagnostics) according to the manufacturer. For cDNA analysis, threshold cycles (Ct)

were determined for quantification of the input target number. For comparison between different biological samples, the target gene expression level in each breast cancer cell line and breast carcinoma sample was normalized using a calibrator RNA sample extracted from the HME-1 cell line. The normalized gene expression (NGE) of the target gene was expressed as the N ratio , where each normalized gene expression gene value was divided by a calibrator normalized gene value (TBP): N target = (Ct target,HME-1 -Ct TBP,HME-1 ) -(Ct target,sample -Ct TBP,sample ). Primers used in this study are listed in Table 4.

For RNA interference assay, the relative gene expression (RGE) was calculated as a percentage of the measured mRNA level in cells transfected with target siRNA compared to cells transfected with matched control siRNA. Data were normalized against the TBP signal and the 2 -ΔΔCt method was used to calculate the relative expression level of a given gene [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

F o r P e e r R e v i e w
For chromatin immunoprecipitation (ChIP) assay, the amount of target and endogenous reference DNA was determined from a standard curve. The standard curve was constructed with fourfold serial dilutions of input DNA. The results from specific antibodies were reported to that observed in the corresponding IgG samples. A region between the GAPDH and CNAP1 genes was used as the negative control to standardize the results from COX2 and ERBB2 HAS regions (ChIP-It protocol, Active

Motif). Primers used in ChIP assays are: ERBB2 F 5'-TCCTTTCGATGTGACTGTCTCC -3', ERBB2 R 5'-CTAAATGCAGAGGCTGGTGACT -3'; COX2 F 5'-AACATGGCTTCTAACCCAAAC -3', COX2 R 5'-AGGAAGCCTTTCTCCTCCTC -3'; Neg F 5′-ATGGTTGCCACTGGGGATCT -3′, Neg R 5′-TGCCAAAGCCTAGGGGAAGA-3′.

Motif discovery and average profiles

A custom position weight matrix was created for HAS motifs [START_REF] Wang | Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2[END_REF]. Mapping of motifs to specific and shared peaks was performed via Homer annotatePeaks using -hist 10 -m <motif> -size 2000 as parameters. Average profiles were computed for each peak population. Motif discovery was performed using Homer findMotifsGenome using hg19 as a parameter.

Gene Set Enrichment Analyses

Publicly available gene expression data for BT474 and HCC-1806 cells were retrieved from GEO accessions GSM1230347 and GSM1230072 [START_REF] Dezső | Gene expression profiling reveals epithelial mesenchymal transition (EMT) genes can selectively differentiate eribulin sensitive breast cancer cells[END_REF][START_REF] Milosevic | Clinical instability of breast cancer markers is reflected in long-term in vitro estrogen deprivation studies[END_REF] and were processed in R using the affy package. Gene Set Enrichment Analyses (GSEAs) were computed using GSEA 2.2.4 [START_REF] Subramanian | Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles[END_REF], using the top RNAPII 500 peaks in summit height for BT474 and HCC-1806 as the genesets, the GENE_SYMBOL.chip chip platform, gene_set permutation for p-value computation, log 2 ratio of classes as the ranking metric, and weighted statistic.

Statistical Analysis

Statistical analyses were done using the SPSS 16.0 program for Windows. The Spearman's correlation coefficient (ρ) was used to compare the correlation between two target gene expression levels in breast cancer cell lines and tumor samples. A p-value < 0.05 was set as the criteria for statistical significance, while a p-value < 0.01 was considered highly significant. For p-values pertaining to motif densities, paired, two-tailed student t-tests were performed.

RESULTS

Both proximal and distal ERBB2 gene promoters are actively used in breast cancer cells

To confirm that both proximal and distal ERBB2 gene promoters are actively used in breast cancer cells, we performed a PolII ChIP-seq assay using BT474 cells and extracted data pertinent to the ERBB2 gene locus. As shown in Figure 1A, PolII was significantly recruited at both promoters with predominance for the well characterized proximal promoter that controls the expression of the canonical transmembrane form of this RTK. Pol II signal was indeed enriched at both promoters as compared to a publicly available IgG ChIP-Seq control dataset performed in BT474 cells [START_REF] Heinonen | Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer[END_REF]. The recruitment of PolII at the distal promoter clearly indicates that both promoters might contribute to ERBB2 overexpression in breast cancer cells.

Prevalent presence of ERBB2b isoform in breast cancer

To determine the relative expression of the ERBB2a and ERBB2b mRNA isoforms in human breast cancer, mRNAs were extracted from 16 breast cancer cell lines and 28 clinical tumour samples (see Table 1 andTable 2). The human normal epithelial breast cell line (HME-1) was used as a reference to measure the relative gene expression (RGE). Quantitative-RT-PCRs using primers specific for either ERBB2a or ERBB2b isoform showed that both ERBB2a and ERBB2b isoforms were expressed in 15 breast cancer cell lines and all carcinoma samples (Fig. 2A-B), whereas the ERBB2b isoform was not expressed in the HCC-1569 cell line. Importantly, ERBB2b

expression was highly correlated to that of ERBB2a with a Spearman rank correlation coefficient equal to 0.912 and 0.882 for cell lines (without HCC-1569) and carcinoma samples, respectively (Fig. 2C-D).

Because the amplification of the chromosome 17q12 region is often observed in various solid tumours [START_REF] Kao | RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes[END_REF][START_REF] Maqani | Molecular dissection of 17q12 amplicon in upper gastrointestinal adenocarcinomas[END_REF], three genes adjacent to the ERBB2 locus within this chromosome region, PERLD1 (PGAP, NM_033419.5), C17orf37 (MIEN1, NM_032339.5), and GRB7 (NM_005310.5) were also analysed for their RGEs in this series of breast cancer samples. Highly positive correlations were observed among all five transcripts. The highest correlation was observed around the ERBB2 locus (Fig. 2E). These results indicated the prevalent presence of ERBB2b isoform in breast cancer.

ERBB2b isoform is a nuclear form of ERBB2

To study the protein expression of the two ERBB2 isoforms in different cell compartments, four breast cancer cell lines were selected based on the RGE of the two ERBB2 isoforms: BT474 and HCC-1954 overexpress both isoforms, whereas HCC-1569 overexpresses only ERBB2a, and Br-Ca-Mz-01 weakly expresses ERBB2b but not ERBB2a (Fig. 2A). Cytosolic, membrane, and nuclear fractions were separated for each of these four cell lines. ERBB2 protein expression was observed mainly in the membrane fraction, but also in the cytosolic and nuclear fractions in BT474 and HCC-1954 cells, which is expected for a transmembrane receptor (Fig. 3A). Interestingly, no ERBB2 nuclear expression was revealed in HCC-1569 cells (Fig. 3A). However, no detectable expression of ERBB2 by Western-blot was observed for Br-Ca-Mz-01 cell lines probably due to its very weak expression level (Fig. 3A).

Immunofluorescence assays were used to precisely determine the expression location of ERBB2a and ERBB2b in single breast cancer cells. Confocal microscopy analyses revealed that clear membrane ERBB2 expression was observed in BT474, HCC-1954, and HCC-1569 cells, but not in Br-Ca-Mz-01 cells, which is in agreement with the lack of ERBB2a isoform expression (Fig. 3B). However, nuclear ERBB2 expression was observed in all the four tested cell lines, including HCC-1569 in which

ERBB2b mRNA was not detected by transcription analysis (Fig. 3B). Altogether these results demonstrated that ERBB2b isoform does not contribute to a detectable membrane expression of ERBB2.

To confirm that ERBB2b is a bona fide nuclear form of ERBB2, we constructed two expression vectors that contain either ERBB2a or ERBB2b cDNA fused to green fluorescent protein (GFP) using the Gateway cloning system (Invitrogen Tm). These two vectors were then transiently transfected into BT474 (high ERBB2 expression) and HCC-1806 (low ERBB2 expression) cell lines. ERBB2-GFP expression was observed in whole cells (Fig. 3C left panels) with the transfection of ERBB2a, whereas ERBB2-GFP expression was specifically located in cell nucleus upon transfection of the ERBB2b isoform (Fig. 3C right panels). These results clearly demonstrated that ERBB2 mRNA variant 2 (ERBB2b) encodes a bona fide nuclear isoform of ERBB2.

ERBB2a isoform upregulates ERBB2b whereas ERBB2b isoform inhibits ERBB2a

To study putative functional relations between ERBB2a and ERBB2b isoforms, RNA interference assays were carried out in BT474 and HCC-1954 cell lines, in which both isoforms are overexpressed. Three specific custom Stealth™ siRNAs (Invitrogen) were designed against each isoform and transiently transfected into two cell lines (Fig. 4A). Control siRNAs were simultaneously transfected into cells and resulting RGEs were defined as 100%. qRT-PCR showed that ERBB2a siRNAs inhibited 30-40% of ERBB2a isoform expression, and 50-70% of ERBB2b isoform expression in both cell lines (Fig. 4B).

However, in marked contrast, ERBB2b siRNAs induced a significant increase of ERBB2a expression, while efficiently inhibited ERBB2b expression (Fig. 4B). These results suggested that the ERBB2a isoform upregulates the expression of the ERBB2b isoform, whereas ERBB2b inhibits the expression of ERBB2a.

Immunofluorescence assay were further carried out in HCC-1954 and Br-Ca-Mz-01 cell lines treated with siRNAs against either ERBB2a or ERBB2b isoforms. In HCC-1954 cells, the diffusion of ERBB2 protein expression was observed upon transfection of ERBB2a siRNAs: less ERBB2 membrane protein was observed and nuclear proteins were present (Fig. 4C mid panels). In marked contrast, ERBB2 protein expression was strongly increased in membranes, and reduced in nuclei when cells were transfected with ERBB2b siRNAs (Fig. 4C lower panels). In Br-Ca-Mz-01 cells, the already weak cytoplasmic and nuclear ERBB2 expressions were further reduced by both ERBB2a and ERBB2b siRNAs (Fig. 4D). However, no convincing ERBB2 membrane accumulation was observed when ERBB2b expression was inhibited, due to the low expression of ERBB2a (Fig. 4D). These observations, together with the siRNA assays, strongly suggest a 2-stage feedback interaction between ERBB2a and ERBB2b

where the ERBB2b isoform upregulated by ERBB2a can subsequently downregulates ERBB2a expression.

Binding of nuclear ERBB2 to a specific HAS motif of the ERBB2 gene proximal promoter

Nuclear ERBB2 was reported to bind to the COX2 gene promoter via an HER2/ERBB2-associated sequence (HAS), a specific AT-rich DNA motif [START_REF] Wang | Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2[END_REF]. By searching a similar HAS regulatory element in the vicinity of the ERBB2 gene promoter, a potential ERBB2-binding sequence was found 500 bps upstream of the transcriptional start site of ERBB2a isoform (NM_004448) (Fig. 5A). Chromatin immunoprecipitation assays in BT474 cells confirmed the binding of nuclear ERBB2 to the COX2 gene promoter via HAS (Fig. 5B). Most importantly, our ChIP assay also revealed the binding of nuclear ERBB2 to ERBB2 proximal promoter region (Fig. 5B).

Regulatory regions are enriched in HAS motifs and correlate with increased gene expression in BT474 cells

We next sought to determine whether HAS motifs were enriched in other regulatory regions in BT474 cells. To this end, we used the RNAPII ChIP-seq that we performed above (Fig. 1A) and performed peak detection. This analysis identified 13,897 peaks, which mostly corresponded to distal regions, with ~1/5 of peaks annotated to promoter and 5' UTR regions (Fig. 6A). We hypothesized that if ERBB2b binds to certain regulatory regions in BT474 cells, those regions would not be present in ERBB2b lowexpressing cells. We thus compared our genome-wide mapping of RNAPII with that performed in HCC-1806 [START_REF] Perreault | Epigenetic and transcriptional profiling of triple negative breast cancer[END_REF], a triple-negative, low-ERBB2a and ERBB2b expressing breast cancer cell line (Fig. 2A and Fig. 6B). By mapping HAS motifs to BT474-, HCC-1806-specific and shared peaks, we observed that BT474-specific peaks were significantly more enriched in HAS motifs than shared or HCC-1806-specific peaks (Fig. 6C). Further, unsupervised motif discovery revealed a compatible motif enriched in BT474-specific RNAPII peaks (Fig. 6D), consistent with putative ERBB2b binding in those regions. We next aimed to determine whether the elements identified via genome-wide mapping of RNAPII targets corresponded to putative enhancers regions. Importantly, gene set enrichment analysis (GSEAs) [START_REF] Subramanian | Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles[END_REF] done using previously published BT474 and HCC-1806 gene expression data [START_REF] Dezső | Gene expression profiling reveals epithelial mesenchymal transition (EMT) genes can selectively differentiate eribulin sensitive breast cancer cells[END_REF][START_REF] Milosevic | Clinical instability of breast cancer markers is reflected in long-term in vitro estrogen deprivation studies[END_REF] showed positive, significant correlations between RNAPII peaks and gene expression fold change in BT474 as well as in HCC-1806 cells, consistent with a trans-activating roles for those regions (Fig. 6E).

DISCUSSION

Nuclear translocation of receptor tyrosine kinases (RTKs) has been described for twenty years. Here, we report a new mechanism for a constant and ubiquitous presence of a nuclear form of ERBB2. The ERBB2b isoform, lacking a signal peptide as compared to the canonical ERBB2a isoform (see Fig. 1), most probably cannot be expressed at the cell membrane. This was confirmed by immunofluorescence analyses of Br-Ca-Mz-01 cells, which do not express the ERBB2a isoform (Fig. 3B). Our results also indicate that membrane ERBB2 is mostly if not exclusively expressed by the ERBB2a isoform.

Importantly, transient transfection of an ERBB2b expression vector coupled with a GFP reporter gene clearly demonstrated that ERBB2b is a nuclear form of ERBB2 (Fig. 3C). It was reported that membrane ERBB2 could contribute to the nuclear translocation as a consequence of endocytic internalization [START_REF] Giri | Endosomal transport of ErbB-2: mechanism for nuclear entry of the cell surface receptor[END_REF], which could explain the nuclear expression of ERBB2 observed in HCC-1569 cells which express only ERBB2a isoform, but not ERBB2b isoform (Fig. 3B). Nuclear ERBB2 has been reported to regulate target gene expression through binding to HASs or tethered binding to other transcription factors [START_REF] Béguelin | Progesterone Receptor Induces ErbB-2 Nuclear Translocation To Promote Breast Cancer Growth via a Novel Transcriptional Effect: ErbB-2 Function as a Coactivator of Stat3[END_REF][START_REF] Diaz Flaqué | Progestin drives breast cancer growth by inducing p21(CIP1) expression through the assembly of a transcriptional complex among Stat3, progesterone receptor and ErbB-2[END_REF][START_REF] Li | Nuclear ErbB2 enhances translation and cell growth by activating transcription of ribosomal RNA genes[END_REF][START_REF] Proietti | Activation of Stat3 by heregulin/ErbB-2 through the co-option of progesterone receptor signaling drives breast cancer growth[END_REF][START_REF] Venturutti | Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis[END_REF][START_REF] Wang | Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2[END_REF]. Accordingly, we observed that nuclear ERBB2 binds to a HAS located in the promoter region of the ERBB2 gene (Fig. 5). However, we could not exclude that nuclear ERBB2 binds to this sequence through tethered binding to other transcription factors, since no DNA-binding domain has been yet identified in the ERBB2 protein.

Transcriptional analyses from both breast cancer cell lines and clinical samples revealed a positive correlation between the expression of ERBB2a and ERBB2b mRNAs (Fig. 2A-D). Using siRNAs targeting specifically and independently both mRNA species, we have established a functional link between these two isoforms, where ERBB2a could upregulate the ERBB2b, in agreement with the positive correlation observed between these two isoforms (Fig. 4). However, our siRNA assays provided an unexpected result when overexpressed ERBB2b inhibited ERBB2a mRNA expression in breast cancer cell lines. This apparent discrepancy could be explained when considering that the positive correlation between these two isoforms were observed in breast cancer cell lines and carcinomas samples under near physiological condition, whereas siRNA assays knock-out target gene expression and alter its downstream gene expression which subsequently break the physiological balance between these two ERBB2 isoforms. In addition and perhaps more importantly, the well-

F o r P e e r R e v i e w
known amplification of the ERBB2 locus in chromosome region 17q12 in breast cancers may also contribute to this positive correlation between the two isoforms by overcoming ERBB2b repressive activity as previously hypothesized for several other transcriptional repressors of the ERBB2 gene such as FOXP3, GATA4, MYB, PAX3 [START_REF] Birnbaum | A reason why the ERBB2 gene is amplified and not mutated in breast cancer[END_REF]. Further in vivo experiments using transgenic mouse models in which the expression of either isoform would be knocked-out or knocked-in should help to further decipher the respective functional role played by each ERBB2 isoform.

Nuclear ERBB2 has been reported to be involved in tumour growth and metastasis as well as resistance to anti-ERBB2 treatment in breast cancers [START_REF] Béguelin | Progesterone Receptor Induces ErbB-2 Nuclear Translocation To Promote Breast Cancer Growth via a Novel Transcriptional Effect: ErbB-2 Function as a Coactivator of Stat3[END_REF][START_REF] Russo | Nuclear ErbB-2: a Novel Therapeutic Target in ErbB-2-Positive Breast Cancer?[END_REF][START_REF] Diaz Flaqué | Progestin drives breast cancer growth by inducing p21(CIP1) expression through the assembly of a transcriptional complex among Stat3, progesterone receptor and ErbB-2[END_REF][START_REF] Flaqué | Progesterone receptor assembly of a transcriptional complex along with activator protein 1, signal transducer and activator of transcription 3 and ErbB-2 governs breast cancer growth and predicts response to endocrine therapy[END_REF][START_REF] Venturutti | Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis[END_REF][START_REF] Wang | Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2[END_REF]. C-terminal fragments of ERBB2 generated by alternative initiation from the internal AUG codon have also been

shown to have tumorigenic property [START_REF] Anido | Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation[END_REF][START_REF] Arribas | p95HER2 and breast cancer[END_REF][START_REF] Vernieri | Resistance mechanisms to anti-HER2 therapies in HER2-positive breast cancer: Current knowledge, new research directions and therapeutic perspectives[END_REF]. In this study, we provide evidence that nuclear ERBB2, in addition to the two known origins (proteolytic shedding of the extracellular domain of the full-length receptor, translation of the mRNA encoding ERBB2/HER2 from internal initiation codons) also result from the translation of the ERBB2 mRNA variant 2 lacking the exons encoding the first 30 amino acids that are present in the ERBB2a full-length isoform. Our results together with published observations strongly indicate that the ERBB2a and ERBB2b isoforms may have different functions both in normal and breast cancer cells. Thus, a specific antibody against the small peptide encoded by amino acids 23-30 of the ERBB2a isoform might help to further study the role of nuclear ERBB2. With such discriminative tool, the composition of ERBB2 nuclear fraction would be further characterize, and specifically study each ERBB2 isoform both in normal and breast cancer cells, which could contribute to the screen and identification of new potential treatments in HER2-positive breast cancer.

Receptor-type tyrosine kinases (RTK) are major regulators of cellular processes that are often mutated resulting in their constitutive activation in human cancers. In contrast to many oncogenic RTKs oncogenes, some others such as ERBB2 are amplified without mutation or rearrangement. We have previously hypothesized that ERBB2 gene amplification is used to overcome repression of its expression by several sequence-specific transcription factors [START_REF] Birnbaum | A reason why the ERBB2 gene is amplified and not mutated in breast cancer[END_REF]. The present characterization of a new negative feedback autoregulatory loop where ERBB2b can repress ERBB2a fits with this model, adding one more member in the list of the transcription factors known to repress ERBB2 gene expression [START_REF] Hua | A Negative Feedback Regulatory Loop Associates the Tyrosine Kinase Receptor ERBB2 and the Transcription Factor GATA4 in Breast Cancer Cells[END_REF].

F

FIGURE 2 FIGURE 3

 23 FIGURE 2The expression of ERBB2a and ERBB2b isoforms are positively correlated in breast cancer. Quantitative-RT-PCR was performed from breast cancer cell lines and tumor samples.Relative gene expressions (RGE) of ERBB2a and ERBB2b were presented: as 2 ΔΔCt in breast cancer cell lines (A) and carcinomas samples (B); or as ΔΔCt in breast cancer cell lines (C) and tumor samples (D). E, The correlation coefficient and significant rate were calculated using ΔΔCt of relative gene expressions of three genes co-amplified at the 17q12 locus PERLD1, C17orf37 and GRB7 in breast cancer carcinomas samples.

FIGURE 4 FIGURE 5 FIGURE 6

 456 FIGURE 4 ERBB2a activates ERBB2b whereas ERBB2b inhibits ERBB2a. A, Location of specific siRNAs targeting either ERBB2a or 2b isoform. B, Transcription analyses upon transient transfection of siRNA in breast cancer cell line BT474 or HCC-1954. C, Immunofluorescence analysis was performed upon transient transfection of siRNA in HCC-1954 cells. D, Immunofluorescence analysis was performed upon transient transfection of siRNA in Br-Ca-Mz-01 cells.

F

  Twenty-eight breast carcinoma samples were obtained from patients treated at the Institut Paoli-Calmettes (Marseille, France), and corresponded to surgical specimens in case of early-stage disease and/or diagnostic biopsies in case of advanced stage. Their characteristics are summarized in Table1and detailed in Table

							1569, MDA-
	MB-175,	MDA-MB-361,	MDA-MB-453,	SK-BR-3,	UACC-812,	ZR-75-30,	Br-Ca-Mz-01
	(http://www.atcc.org)	and	SUM-185,	SUM-190,	SUM-206
	(http://www.cancer.med.umich.edu/breast_cell/production) were grown according to the
	recommendations of the supplier.

Table 3

 3

	). A mixed of each three

  with the ChiP-seq assays. This work was supported by Association pour la recherche sur le cancer (ARC), Fondation Groupe EDF, Institut national du cancer (INCA), Ligue Nationale Contre le Cancer (EL2016 Birnbaum and EL2019 Bertucci), the European Molecular Biology Organization (EMBO Short Term Fellowship ASTF 25.00-2009 HUA) and Boehringer Ingelheim Fonds (Travel Grant 2008 HUA). ERBB2 gene proximal and distal promoter usage and schematic representation of its two major mRNA isoforms. A, UCSC genome browser screenshot of RNAPII and IgG read depth (2 upper black profiles) at the ERBB2 gene locus in BT474 cells. Vertical arrows indicate the genomic location of both distal and proximal ERBB2 promoters. Known isoforms are annotated underneath, with mammal conservation at the bottom (blue). B, Domain structure of ERBB2a isoform: signal peptide in grey; transmembrane domain in black which separates the extracellular domain from intracellular domain; tyrosine kinase domain in red. C, Exon map for ERBB2a isoform, the Exon 1 containing sequences coding the signal peptide in red. D, Domain structure of ERBB2b isoform with transmembrane domain in black and tyrosine kinase domain in red. E, Exon map for ERBB2b isoform, the first four non-coding exons in brown.

	Figure legends
	FIGURE 1
	F F F F F
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	r r r r r r
	P e P e P e P e P e P e
	e r e r e r e r e r e r
	R R R R R R
	e v e v e v e v e v e v
	i e i e i e i e i e i e
	w w w w w w

Table 1 :

 1 The expression of ERBB2a and ERBB2b isoforms are positively correlated in breast cancer. Clinicopathological characteristics of 28 breast cancer samples

	o
	r
	P
	e e
	r
	R
	e
	v i
	e w
	ERBB2 gene proximal and distal promoter usage and schematic representation of its two major mRNA
	isoforms.
	190x254mm (307 x 307 DPI)

RNAPII peaks in BT474 cells are enriched in HAS motifs and correlate with increased gene expression.

Table 3 .

 3 Targeted sequences used for siRNA Assay.

	RNA	Target Name	Sequence
		NM_004448_stealth_14 sense strand	GAGGAGGGCUGCUUGAGGAAGUAUA
		NM_004448_stealth_14 antisense strand	UAUACUUCCUCAAGCAGCCCUCCUC
	ERBB2 isoform a	NM_004448_stealth_15 sense strand	AGGAGGGCUGCUUGAGGAAGUAUAA
	(NM_004448)	NM_004448_stealth_15 antisense strand	UUAUACUUCCUCAAGCAGCCCUCCU
		NM_004448_stealth_17 sense strand	GAGGGCUGCUUGAGGAAGUAUAAGA
		NM_004448_stealth_17 antisense strand	UCUUAUACUUCCUCAAGCAGCCCUC
		NM_001005862_stealth_90 sense strand	CCUGAUGGGUUAAUGAGCAAACUGA
		NM_001005862_stealth_90 antisense strand	UCAGUUUGCUCAUUAACCCAUCAGG
	ERBB2 isoform b	NM_001005862_stealth_97 sense strand	GGUUAAUGAGCAAACUGAAGUGUUU
	(NM_001005862)	NM_001005862_stealth_97 antisense strand	AAACACUUCAGUUUGCUCAUUAACC
		NM_001005862_stealth_187 sense strand	GGUAGAACCUUUGCUGUCCUGUUCA
		NM_001005862_stealth_187 antisense strand UGAACAGGACAGCAAAGGUUCUACC

Table 4 .

 4 Sequences of the oligonucleotide primers used for qRT-PCR.

	Page 29 of 33
	Gene	Oligonucleotide	Sequence	PCR product size (bp)
	ERBB2a mRNA	Forward Primer Reverse Primer	5'-CTCCTCCTCGCCCTCTTG -3' 5'-GACCTGCCTCACTTGGTTGT -3'	253
	ERBB2b mRNA	Forward Primer Reverse Primer	5'-ACGCCTGATGGGTTAATGAG -3' 5'-CCAAATTCTGTGCTGGAGGT -3'	154
	TBP mRNA	Forward Primer Reverse Primer	5'-CTTGTGCTCACCCACCAAC -3' 5'-GGAGGCAAGGGTACATGAGA -3'	228
	PERLD1 mRNA	Forward Primer Reverse Primer	5'-AGTTTTCCACACCAGGGACA -3' 5'-CATAGTCGAAGCGGATGAGG -3'	203
	C17orf37 mRNA	Forward Primer Reverse Primer	5'-TTCGAGGCGACCTACCTG -3' 5'-ACGGCTGTTGGTGATCTTTT -3'	225
	GRB7 mRNA	Forward Primer Reverse Primer	5'-CGTGTGTGAAATGCTGGTG -3' 5'-TCTGGGAACAGGGAGTGTG -3'	225

Table 2

 2 Detailed clinicopathological characteristics of 28 breast cancer samples , IHC status with 10% positivity cut-off; **, status defined using IHC, then array-CGH

	ID	Age at diagnosis	Menopausal status	AJCC stage	Pathological tumor type	Pathological tumor grade	Pathological tumor size	Pathological axillary lymph node status	ER status* PR status*	ERBB2 status**	Molecular subtype
	12781	<=50	no	1	other	NA	pT1	neg			pos	ERBB2+
	9934	>50	yes	3	ductal	3			neg	neg	pos	ERBB2+
	9948	>50	yes	3	ductal	3	pT1		pos	pos	pos	ERBB2+
	2933	>50	yes	2	ductal	3	pT1	pos	neg	neg	pos	ERBB2+
	11041	>50	yes	2	ductal	3	pT2	pos	neg	neg	pos	ERBB2+
	13008	<=50	no	2	ductal	3	pT3	pos	pos	pos	pos	ERBB2+
	13591	>50	yes	4	ductal	3	pT3	pos	neg	neg	pos	ERBB2+
	7462	<=50	no	2	ductal	3	pT3	pos	neg	neg	pos	ERBB2+
	9983	<=50	no	2	ductal	2	pT1	neg	neg	neg	pos	ERBB2+
	10797	>50	yes	1	ductal	3	pT2	pos	neg	neg	pos	ERBB2+
	8035	<=50	no	2	ductal	2	pT3	pos	neg	neg	pos	ERBB2+
	12652	<=50	no	3	ductal	3		pos	pos	pos	pos	ERBB2+
	8584	>50	yes	2	other	2	pT2	neg	pos	pos	pos	ERBB2+
	12710	<=50	no	3	ductal	3		neg	neg	neg	pos	ERBB2+
	9991	>50	yes	1	ductal	2	pT2	neg	neg	neg	pos	ERBB2+
	9725	<=50	no	1	ductal	3	pT3	pos	neg	neg	pos	ERBB2+
	9317	>50	yes	2	ductal	3	pT2	pos	pos	neg	pos	ERBB2+
	10982	>50	yes	2	ductal	3	pT3	pos	neg	neg	pos	ERBB2+
	7780	<=50	no	3	ductal	3	pT2	pos	neg	neg	pos	ERBB2+
	5520	>50	yes	1	ductal	3	pT1	neg	neg	neg	pos	ERBB2+
	8719	<=50	no	1	other	2	pT1	neg	pos	pos	neg	HR+/ERBB2-
	6604S	>50	yes	2	ductal	1	pT1	pos	pos	pos	neg	HR+/ERBB2-
	9091	>50	yes	1	ductal	1	pT1	neg	pos	neg	neg	HR+/ERBB2-
	9840	>50	yes	1	ductal	1	pT3	neg	neg	neg	neg	TN
	9670	<=50	no	1	ductal	1	pT2	neg	pos	pos	neg	HR+/ERBB2-
	7876T	>50	yes	2	lobular	1	pT1	neg	neg	neg	neg	TN
	4700	>50	yes	2	other	3	pT2	pos	neg	neg	neg	TN
	9745	>50	yes	1	lobular	2	pT1	pos	neg	neg	neg	TN

*HR: hormone receptor; TN: triple-negative

John Wiley & Sons, Inc.Journal of Cellular Biochemistry

ACKNOWLEDGEMENTS

We thank Karim Ben Salah, Hélène Holota (TAGC, Marseille) and José Adélaïde (CRCM, Marseille) for excellent technique support. We thank Keiji Zhao (NHLBI/NIH, Bethesda) for his invaluable assistance

CONFLICT OF INTEREST

The authors here declare that no conflicts of interest exist during the completion of the paper. Br-Ca-MZ-01 HCC1569

AUTHOR CONTRIBUTIONS

G

Cytosolic, membrane, and nuclear fractions were separated for Br-Ca-Mz-01 and HCC1569 cells, Western blot analyses were performed with ERBB2, SP1 and ß-Tubulin antibodies.