Technical field

The present invention relates to the use of a peptide derived from ERa (estrogen receptor alpha, ESRl) for restoring sensitivity to antiestrogenic compounds in antiestrogenic-resistant breast tumour cells. State of the art

Breast cancer is the most common cancer in women and the leading cause of death in women 35-51 years of age. The incidence is increasing and today the risk of developing this kind of pathology is estimated 8-10%. Approximately 70% of all breast cancers are dependent for their growth on estrogens and on a functional estrogen receptor α (ERa) .

Estrogen receptor a (ERa) is the primary isoform expressed in breast cancer, even if ERβ is also frequently expressed. Estrogen receptors are members of the Nuclear Receptor superfamily and show a dynamic nuclear-cytoplasmic-membrane localization. Upon 17β- estradiol (E2) binding to ERa, the receptor is released from the complex formed by Hsp90 (Heat shock protein 90) and other chaperones and binds to specific nucleotide sequences called ERE (ERE, estrogen-responsive element) on determined target genes, where it regulates transcription. This regulation depends on the association of the receptor with regulatory proteins, called co-activators.

Commonly, ER-positive breast cancers are treated with therapies involving hormone reduction or antiestrogen administration (AIi and Coombes, 2002, Ghayad et al . , 2008) .

The most convenient pharmacological treatment for estrogenic-responsive breast cancer, at both the adjuvant and metastatic settings, is represented by antiestrogenic endocrine therapy, which combines low toxicity and high efficacy. Among antiestrogenic drugs, the most widely used are Selective Estrogen Receptor

Modulators called SERMs, in particular, tamoxifen (TAM) .

It has been demonstrated that compounds with antagonistic or agonistic activity bind to the ligand binding pocket in the C-terminal domain of ERa (LBD) , inducing binding of the receptor to target genes, and modulate interaction of this domain with co-regulatory proteins . The main estrogen hormone, 17β-estradiol, and other agonistic ligands in general induce a conformational change of LBD that creates a composed patch specifically docking a LXXLL motif, characteristic of known co- activator proteins, such as SRCl and AIBl (Heery et al . , 1997) . Co-activators bring close to the target genes histone-modifying and chromatin remodeler enzymes, necessary to gene transcriptional activation. On the contrary, antagonistic ligands, such as antiestrogen tamoxifen, induce a different conformation of the LBD, which has low affinity for co-activators but shows, instead, high affinity for the CoRNR (co- repressor-nuclear-receptor) box motif that is present in co-repressor proteins NCoR and SMRT (Perissi and Rosenfeld, 2005, Lazar, 1999) . The repression complex recruits histone deacetylases that compact nucleosomes and consequently actively repress transcription, rather than simply blocking activation (Perissi and Rosenfeld, 2005) . In this way, genes that are normally activated by estrogens are repressed, leading to cancer cell growth arrest and apoptosis.

In the clinical use of these kind of compounds (tamoxifen, toremifene) , it is known that secondary resistance almost invariably occurs.

As a matter of fact, even some of the tumours which are initially sensitive to antiestrogenic therapy eventually develop a resistance after a prolonged treatment.

The main molecular alterations which are potentially responsible for the resistance to endocrine therapies are an excessive activation of growth factor or other kinase pathways, the altered activity of co- activators and co-repressors, as well as, in some cases, the receptor loss or the selection of cell clones which do not express the receptor. - A -

The fact that a correct balance between co- repressor and co-activator levels defines the threshold of transcriptional repression or activation is the molecular basis of most forms of resistance to nuclear receptor antagonists (Perissi and Rosenfeld, 2005) . In many tumour-derived or in vitro-selected cells that are resistant to, or even stimulated by, antagonist treatment, a condition that either downregulates NCoR/SMRT co-repressors available in the nucleus, or increases the levels and activity of one or more co- activators, has been described.

There are several mechanisms that may come into play to reduce the available amount and activity of NCoR/SMRT co-repressors in cell nuclei. These proteins are purified as a macromolecular complex, containing fixed and variable subunits, many of which are actually integrators of different signalling pathways and may cause dismissal of the co-repressor complex from target genes. One such mechanism is linked to ubiquitin- dependent degradation, that apparently can be guided by different WD-repeat, F-box proteins, the most important being the TBL1/TBLR1 subunits (Perissi et al., 2004) . TBLl and TBLRl interact directly with NCoR and SMRT within the N-terminus repressor domain (Privalsky, 2004) and act as E3-enzymes of ubiquitination system by recruiting components of the ubiquitin conjugating/19S proteasome to the promoters of target genes, with TBLRl selectively serving to mediate a required exchange of the nuclear receptor co-repressors (Perissi et al . , 2004) . NCoR and SMRT are thus sent to the degradation pathway upon activation of the receptor. Modulation of this degradation pathway may therefore contribute to development of antagonist resistance.

More recently, attention was drawn to the role of the shuttle protein TAB2 (MAP3K7IP2) (Takaesu et al . , 2000), that is a facultative component of NCoR (but not SMRT) co-repressor complex. TAB2 displays a double interaction: from one side with NCoR and from the other side with the L/HX7LL motif in the N-terminus sequence of ERa (but not ERβ) , AR, PgR (Zhu et al . , 2006) . A pivotal role in the activity of TAB2 is played by its phosphorylation by the MAP kinase kinase MEKKl, that unmasks a sequence for nuclear export signal (NES) in

TAB2, leading to the export of TAB2/NCoR complex to the cytoplasm (Baek et al . , 2002; Zhu et al . , 2006) . In prostate cancer cells, in response to interleukin-lβ

(IL-lβ) treatment or co-culture with macrophages, MEKKl is recruited near to the NCoR co-repressor complex where it phosphorylates TAB2. In the context of genes where the androgen receptor (AR) is bound in response to antagonists such as bicalutamide, in association with the NCoR co-repressor complex, TAB2 now dissociates from the AR and tethers NCoR away to the cytoplasm, thus converting androgen antagonists to agonists, i.e. a condition of complete antagonist resistance (Zhu et al . , 2006; Baek et al . , 2002) . A similar behaviour has been reported by Zhu et al . , 2006 in connection to the gene response to tamoxifen in cells containing the ERa receptor. Comparing ERa and AR, a conserved peptide has been identified, which has a L/HX7LL structure, which is responsible for the binding of TAB2. A L/HX7LL peptide, derived from AR, restores the response to antiandrogen compounds even in the presence of IL-lβ, when it is microinjected into the nuclei of RWPEl prostate cancer cells. In view of these results, the applicants conclude that the mechanism of delocalisation of the TAB2/NCoR complex, following inflammation, which is very common in prostate cancer, is responsible for the resistance to antiandrogenic compounds. In the case of breast cancer, however, the presence of inflammation has not been associated to a clinical behaviour due to endocrine resistance. The resistance in this case is probably due to other molecular causes. The common observation of the cytoplasmic delocalisation of the co-repressors in endocrine-resistant mammary cancer cells suggests that this mechanism may operate even beyond inflammation.

Therefore, compounds are being sought allowing to restore sensitivity to antiestrogenic therapies in mammary tumour cells in case of resistance, so as to improve the clinical results and the quality of the patient' s life . Disclosure of Invention

It is an object of the present invention to therefore provide a treatment allowing to restore sensitivity to antiestrogenic drugs. This object is obtained by the use of peptides according to claims 1 and 2.

According to the present invention a conjugate of the peptides according to claims 1 and 2 with a peptide carrier is also provided. Definitions

Unless otherwise explicitly stated, the following terms have the meaning indicated in the following.

In the present text, "peptide carrier" means a peptide that, when conjugated to the peptides according to the invention, allows the internalisation into the cell and the localisation in the nuclear compartment .

In the present text, "identity percentage" and "% identity" between two sequences of amino acids (peptides) or nucleic acids (nucleotides) means the percentage of identical amino acid or nucleotide residues in corresponding positions in the two sequences when optimally aligned.

To determine the "identity percentage" of two amino acid or nucleotide sequences, the sequences are aligned with one another; to achieve an optimum comparison, gaps (i.e. deletions or insertions - which may possibly even be arranged at the ends of the sequences) may be introduced in the sequences. The amino acid and nucleotide residues at corresponding positions are therefore compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue that occupies the corresponding position in the second sequence, the molecules are identical in this position. The identity percentage between two sequences is a function of the number of identical positions shared by the sequences [i.e. % identity = (number of identical positions / total number of positions) X 100] .

According to an advantageous embodiment, the sequences have the same length.

Advantageously, the compared sequences do not have any gaps (or insertions) .

The identity percentage may be obtained by using mathematical algorithms. A non-limitative example of a mathematical algorithm used for the comparison of two sequences is the Karlin and Altschul algorithm [Proc. Natl. Acad. Sci. USA 87 (1990) 2264-2268] modified by

Karlin and Altschul [Proc. Natl. Acad. Sci. USA 90

(1993) 5873-5877] . Such an algorithm is incorporated in the BLASTn and BLASTp software by Altschul

[Altschul, et al, J. MoI. Biol. 215 (1990) 403-410] . In order to obtain alignments also in the presence of one or more gaps (or insertions) methods may be used which give a relatively high penalty to each gap (or insertion) and a lower penalty for each additional amino acid or nucleotide residue in the gap

(such an additional amino acid or nucleotide residue is defined as an extension of the gap) . High penalties will obviously determine optimum alignments with a lower number of gaps.

An example of a software adapted to carry out this kind of alignment is the BLAST software as disclosed in Altschul, et al . , Nucleic Acids Res. 25 (1997) 3389-3402. For this purpose the BLASTn and BLASTp software may be used with default parameters. A BLOSUM62 matrix is usually employed with BLAST softwares .

An advantageous and non-limitative example of a software to carry out an optimum alignment is GCG Winsconsin Bestfit package (Winsconsin University, USA; Devereux et al., 1984, Nucleic Acids Research 12:387) . Default parameters which provide a penalty of -12 for a gap and a penalty of -4 for each extension for an amino acid sequence are also used in this case. In the present disclosure "homology percentage" and "% homology" between two amino acid or nucleic acid sequences means the percentage of homologous amino acid or nucleotide residues at corresponding positions in the two sequences when ideally aligned. The homology percentage between two sequences is determined in substantially the same manner as described above for the determination of the identity percentage except that homologous positions and not only identical positions are considered in the computation .

As far as nucleotide sequences are concerned, two homologous positions have two nucleotides which are different but lead to the same amino acid.

As far as amino acid sequences are concerned, two homologous positions have two homologous amino acids, i.e. amino acids having similar chemical-physical properties, for instance amino acids belonging to the same groups such as: aromatic (Phe, Trp, Tyr) , acid (GIu, Asp), polar (GIn, Asn) , basic (Lys, Arg, His), aliphatic (Ala, Leu, lie, VaI), with a hydroxy-group (Ser, Thr) , with a short side chain (GIy, Ala, Ser, Thr, Met) . Substitutions among these homologous amino acids are not expected to change the phenotype of the proteins (conservative amino acid substitutions) . Specific examples of conservative substitutions are known in this technical field and are disclosed in literature (for example, Bowie et al . , Science, 247:1306-1310 (1990) ) .

Further examples of software and/or items related to the determination of alignments and of homology and/or identity percentages are indicated for example in US2008003202, US2007093443, WO06048777. In the present text, "corresponding position" means a position in an amino acid or nucleotide sequence corresponding (facing), upon alignment, to a determined position of a reference sequence. Brief description of the figures

For a better understanding of the present invention, the invention will now be described with reference to the accompanying figures, in which: - Figure IA shows morphology MCF7 WT cells; Figure IB shows morphology of MCF7 TAMR-PB9701 cells;

- Figure 2 shows NCoR mRNA expression levels in MCF7 WT, MCF7 WT FBS 1%, MCF7 TAMR-894, -895, -PB9701, -808 cells obtained with qRT-PCR analysis; - Figure 3 shows TAB2 mRNA expression levels in MCF7 WT, MCF7 WT FBS 1%, MCF7 TAMR-894, -895, -PB9701, -808 cells obtained with qRT-PCR analysis;

- Figure 4 shows SMRT mRNA expression levels in MCF7 WT, MCF7 WT FBS 1%, MCF7 TAMR-894, -895, -PB9701, -808 cells obtained with qRT-PCR analysis;

Figure 5 shows TAB2, NCoR, SMRT and ERa expression levels in MCF7 WT, MCF7 WT FBS 1%, MCF7 TAMR- 894, -895, -PB9701, -808 cells obtained with immunoblot analysis ; - Figure 6 shows the levels of expression of NCoR in the two cell compartments: nuclear and cytoplasmic fraction, in the MCF7 WT cell line treated with Et-OH, tamoxifen, Interleukin-lβ and tamoxifen+Interleukin-lβ obtained by means of immunoblot analysis;

- Figure 7 shows the immunocytochemical analysis on MCF7 WT cells treated with Et-OH, TAM and IL for 2 hours, where the cells have been marked with specific antibodies against the C-terminus and the N-terminus of TAB2 and against NCoR. DAPI marking has been used for nuclear marking;

- Figure 8 shows the immunocytochemical analysis on MCF7 TAMR cells treated with Et-OH, TAM and IL for 2 hours, where the cells have been marked with specific antibodies against the C-terminus and the N- terminus of TAB2 and against NCoR. DAPI marking has been used for nuclear marking; - Figures 9 and 10 show mRNA expression levels of TAB2 in MCF7 WT and MCF7 TAMR cell lines transfected with TAB2 siRNA and Control siRNA and treated with 17β- estradiol (E2), tamoxifen (TAM) or Et-OH obtained by qRT-PCR analysis; - Figures 11 and 12 show levels of TAB2 protein in

MCF7 WT and MCF7 TAMR cell lines transfected with TAB2 siRNA and Control siRNA and treated with 17β-estradiol

(E2), tamoxifen (TAM) or Et-OH obtained by immunoblot analysis ; - Figure 13 shows the immunocytochemical analysis on MCF7 TAMR cells after transfection with TAB2 siRNA, treated with TAM for 2 hours, where the cells have been marked with antibodies specific against TAB2 and NCoR. DAPI marking has been used for nuclear marking; - Figure 14 shows results of a proliferation assay in MCF7 WT cell line in response to 17β- estradiol (E2), tamoxifen (Tarn) or control after transfection of TAB2 siRNA or Control siRNA; Figure 15 shows results of a proliferation assay in MCF7 TAMR cell line in response to 17β- estradiol (E2), tamoxifen (Tarn) or control after transfection of TAB2 siRNA or Control siRNA; - Figures 16, 17, 18, 19, and 24 illustrate results of proliferation assays in MCF7 TAMR-PB9701, MCF7 TAMR-AL894, MCF7 TAMR-AL895, and -BT474 cultured in the presence of tamoxifen, after "treatment" with peptides having SEQ ID No: 3 and SEQ ID No: 5; - Figures 20 and 21 show the results of gene expression analysis on MCF7 TAMR cells (TAMR-PB9701 and TAMR -894 cells) in response to E2 or tamoxifen after transfection of TAB2 siRNA or Control siRNA. TAB2, pS2, ERBB2 and CXCR4 mRNA expression levels were analyzed by Real Time PCR;

Figure 22 shows results of a proliferation assay in a BT474 cell line in the same conditions as in Figures 14 and 15, i.e. in response to 17β- estradiol (E2), tamoxifen (Tarn) or control after transfection of TAB2 siRNA or Control siRNA;

- Figure 23 shows results of an in vitro binding assay ("pull-down") between recombinant TAB2 and ERa proteins in the presence of peptides (SEQ ID NO: 10) or (SEQ ID NO: 11) . Detailed description of the invention

To study the role of TAB2/NCoR in breast tumour cells, tamoxifen resistant breast cancer cell strains, derived from the MCF7 cell line by continuous exposure to tolerable doses of the drug were selected (Lykkesfeldt and Briand, 1986; Madsen et al . , 1997) . Four different resistant cell lines (TAMR) called MCF7 TAMR-894, -895, -PB9701, -808, were used together with two different sub-cultures of wild-type, estrogen- dependent and tamoxifen-sensitive MCF7 cells: MCF7 WT FBS1% (continuously maintained with a low serum concentration) , and normal MCF7 WT cell line, cultured in DMEM medium supplemented with 10% FBS. The MCF7 WT FBS 1% cell line and the resistant cell lines were propagated in control growth medium DMEM/F12 supplemented with 1% FBS. Moreover, the resistant cell lines were propagated in control growth medium supplemented with 10 ^~6 M 4-OH-tamoxifen . None of these cell lines showed significant evidence of constitutive activation of one or more components of the inflammatory signal transduction pathways, as was shown by the analysis of the gene expression with a DNA microarray containing 45,000 probes representative of the whole human transcriptome .

Though maintaining the characteristic epithelial aspect of MCF7 (Figure IA) , the MCF7 TAMR cells were slightly smaller and more irregular and grew as tightly packed colonies with limited cell spreading (in Figure IB MCF7 TAMR-PB9701 are shown, as an example of the characteristic aspect of MCF7 TAMR cells) . Morphological alterations observed in the TAM-resistant cell lines are likely due to an adaptive process in response to the primary drug action.

The expression levels of TAB2, NCoR, SMRT and ERa mRNA and proteins in these different cell lines were studied by qRT-PCR and immunoblot analysis respectively (Figures 2-5) .

TAB2, NCoR and SMRT mRNA levels in the four MCF7 TAMR cell lines were lower than in MCF7 WT, in particular when compared to MCF7 cells grown in medium supplemented with 10% FBS (Figures 2-4) .

Immunoblot analysis showed a constant expression level of the ERa and NCoR proteins in all cell lines analyzed, whereas the expression of the TAB2 co- repressor in MCF7 TAMR cells was less abundant than in MCF7 WT cells and SMRT markedly reduced (Figure 5) .

This demonstrates that tamoxifen resistance would not result from either the absence of the estrogen receptor or downregulation of main components of NCoR or TAB2 co-repression complexes. However, it is noteworthy that in TAMR cell lines, SMRT expression was significantly reduced, thus enhancing the role of NCoR in resistance.

To examine the peculiar feature of the tamoxifen resistance in MCF7 TAMR cells, the sub-cellular localisation of the repressor complex proteins TAB2 and NCoR in MCF7 WT and MCF7 TAMR cells was studied and secondly MCF7 TAMR cells were evaluated for the effect of TAB2 silencing. The stimulation of the cells with IL-lβ was used as a positive control, as, in prostate cancer cells (Zhu et al., 2006) and in MCF7 cells (Rosenfeld MG, personal communication) , it induces the nuclear delocalisation of the repressor complex, upon activation of a cascade of MAPKs that culminates with the phosphorylation of TAB2 by MEKKl (Baek et al . , 2002; Zhu et al . , 2006) . At first, the sub-cellular localisation of NCoR in MCF7 WT cells was investigated by biochemical analysis with cellular fractionation. MCF7 WT cells were exposed to TAM (10 ^~6 M), IL-lβ (10 ng/ml), TAM (10 ^~6 M) + IL-lβ (10 ng/ml) , or solvent alone as a control, for 2 hours and the cytoplasmic and nuclear fractions from each sample were prepared. Equal amounts of the obtained nuclear and cytosolic fractions were analyzed by immunoblotting with anti-NCoR, anti-Lamin Bl, anti-β-tubulin antibodies. As shown in Figure 6, when MCF7 WT cells were stimulated by IL-lβ or TAM + IL-lβ, it was possible to observe an increase of NCoR localisation in the cytoplasmic fractions, even if a residual nuclear localisation of the protein was observed. A very similar result was obtained also by indirect immunofluorescence. It was observed that in MCF7 WT cells cultured in the presence of TAM, TAB2 and NCoR were mainly localised in the nucleus (Figures 7C-D-J) , while, when cells were stimulated by IL-lβ or TAM + IL-lβ, TAB2 and NCoR showed a cytoplasmic localisation (Figures 7E-F-G-H-K) . These data confirm that, in MCF7 WT cell lines, TAB2 and NCoR are prevalently in the nucleus and that the activation of the IL-lβ-dependent signalling pathway delocalised TAB2 and NCoR to the cytoplasm. Indirect immunofluorescence experiments were also carried out in MCF7 TAMR cells. Contrary to the situation observed for MCF7 WT, TAB2 was found significantly located in the cytoplasm (Figure 8 A-B-C- D) . Treatment of MCF7 TAMR cells with IL-I β alone or TAM + IL-lβ enhanced the cytoplasmic localisation of TAB2 (Figures 8E-F-G-H) .

This demonstrates that, in clear contrast with respect to tamoxifen-sensitive MCF7 WT cells, in breast tumour cells spontaneously resistant to tamoxifen, there is a delocalisation of the TAB2-NCoR complex, which is independent of inflammatory processes.

Altogether, these data demonstrate that in MCF7 cells and in their MCF7 TAMR derivatives, the dismissal mechanism of NCoR is functional.

The actual dependency of the suggested resistance mechanism on TAB2 was then evaluated, by analysing the localisation of NCoR following the silencing of TAB2 by RNA interference. The efficiency of the silencing of TAB2, which may be obtained by transfecting small specific double-strand RNAs (siRNA), was measured in MCF7 WT and MCF7 TAMR cells, both in terms of mRNA, by means of qRT-PCR analysis and in terms of protein by means of immunoblotting. In order to see whether the silencing efficiency was influenced by treatments with growth- modulating agents, the effect of TAB2 silencing was studied following stimulation with estradiol and tamoxifen .

The cells were transfected with TAB2 siRNA and Control siRNA and, 48 hours after transfection, were treated with 17βestradiol (10 ^~8 M), TAM (10 ^~6 M), or EtOH (vehicle), as a control, for 6 hours. RNA and proteins were extracted and analyzed by qRT-PCR and immunoblotting, respectively. In both cell lines, TAB2 siRNA transfection demonstrated a silencing by at least 50% at the TAB2 mRNA levels (Figures 9-10) and at the respective protein level (Figures 11-12) in all experimental conditions.

To obtain the evidence of TAB2 functional role, MCF7 TAMR-PB9701 cells were transfected with TAB2 siRNA and, 48 hours after transfection, were treated with TAM for 2 hours and subsequently used for indirect immunofluorescence analyses. In MCF7 TAMR cells, TAB2 siRNA transfection induced complete relocalisation of NCoR into the nuclear compartment (Figures 13E-F vs G- H) . Comparable results were obtained with other MCF7 TAMR cell lines.

These results demonstrate that TAB2 activity is responsible of incorrect localisation of the repression complex in cell lines rendered resistant to tamoxifen, and so targeting this mechanism can restore tamoxifen response .

Gene expression modulation by TAB2 silencing in MCF7 TAMR cells Tamoxifen acts as antagonist to estrogen action on a large number of genes (Frasor et al . , 2004; Scafoglio et al . , 2006) . In tamoxifen-resistant cells, tamoxifen is expected to be inactive on the expression of estrogen target genes, or to act in agonistic fashion. Therefore, the inventors investigated whether by suppressing TAB2 expression by siRNA in MCF7 TAMR cells, tamoxifen regulation of a set of estrogen-dependent genes is restored. Three known estrogen target genes, where tamoxifen is known to act antagonistically, were chosen, i.e. the pS2 gene (TFFl), CXCR4 and ERBB2. Using MCF7 TAMR-PB9701 and MCF7 TAMR-AL894, cells were transfected with TAB2 siRNA and Control siRNA and 48 hours after transfection, cells were treated for 6 hours with 4-OH- TAM (10 ^~6 M) or vehicle. As shown in Figures 20 and 21, both cell lines showed a 70-90% downregulation of TAB2 mRNA by siRNA treatment. pS2 mRNA, the most well described target of estrogen, is completely insensitive to tamoxifen in cells transfected with control siRNA, but recovered 60-70% inhibition in cells transfected with TAB2 siRNA. Analogously, downregulation of CXCR4 and ERBB2 gene mRNAs by tamoxifen was increased in cells with TAB2 silencing as compared to control cells. These results confirm that in TAMR cells, TAB2 renders at least in part, tamoxifen ineffective in regulating estrogen-target genes, whereas TAB2 downregulation restores response.

Recovering of the effect of tamoxifen on cell proliferation in MCF7 TAMR cells by TAB2 silencing

To assess the effect on cell proliferation of TAB2 downregulation in response to tamoxifen, MCF7 WT and MCF7 TAMR cells were transfected with TAB2 siRNA or Control siRNA and 48 hours after transfection, cells were treated for 24 hours with 17βestradiol (10 ^~8 M), 17-βestradiol (10 ^~8 M) + 4-OH-TAM (10 ^~6 M) or Et-OH, as a control. The cells were then used in the proliferation assay (Figures 14-15) .

Estrogen is mitogenic in MCF7 cells and stimulates cell proliferation through activation of ERa. The antiestrogen tamoxifen, on the other hand, blocks ER- mediated transactivation because it induces ERa conformation having high co-repressor affinity, instead of co-activator affinity, resulting in growth inhibition of breast cancer cells.

In both cases, in fact, treatment with estrogen increased basal cell growth, while tamoxifen inhibited proliferation (Figure 14) . By contrast, in MCF7 TAMR cells (Figure 15) tamoxifen did not inhibit the proliferation rates when cells were transfected with Control siRNA. On the other hand, upon downregulation of TAB2, tamoxifen was able to inhibit cell growth, again. The same analysis was repeated with other MCF7 TAMR cell lines and the results were reliably confirmed.

Efficacy of TAB2 silencing in restoring tamoxifen response in one additional model of breast cancer cells, showing primary (non-induced) resistance

In order to generalize the above observations, a screening was carried out to identify available human breast cancer cell lines possessing tamoxifen resistance while maintaining expression of functional ERa. The ER+ BT474 human breast cancer cell line (ATCC Number HTB-20) that contains an amplified ERBB2 gene, fulfilled the inventors expectations. BT474 cells were transfected with TAB2 siRNA or Control siRNA and 48 hours after transfection, cells were treated for 6 hours with 10 ^~8 M E2, 10 ^"6 M TAM or Et-OH, as a control, and then used in the proliferation assay (Figures 15 and 22) .

Also in BT474 cells, as in MCF7 TAMR cells, estrogen is mitogenic and stimulates cell proliferation through activation of ERa, while tamoxifen did not inhibit the proliferation rates when cells were transfected with Control siRNA. On the other hand, upon downregulation of TAB2, tamoxifen was again able to inhibit cell growth (Figures 15 and 22) .

These results provide a definitive proof of concept on the efficacy of this molecular target to revert tamoxifen resistance in breast cancer. Peptides blocking ERα/TAB2 interaction restore and stabilize the antiestrogenic action of tamoxifen in MCF7 TAMR cells

In the IL-lβ model, nuclear microinjection experiments into prostatic cancer cells showed that the peptide of sequence L/HXXAXXXXLL (referred to as

L/HX7LL) derived from AR blocked IL-lβ-induced dismissal of NCoR (Zhu et al . , 2006) .

Surprisingly, the L/HX7LL peptides were shown to be effective even for restoring sensitivity to antiestrogenic compounds in antiestrogenic resistant breast tumour in the absence of interleukin-1 and in the absence of inflammatory activation.

In particular, according to the present invention, there is provided the use of a peptide having a sequence with at least 90% identity with SEQ ID NO: 1 for the preparation of a medicament for restoring sensitivity to antiestrogenic compounds in antiestrogenic-resistant breast tumour cells. Alternatively, there is provided the use of a peptide having SEQ ID NO: 1 for the preparation of a medicament for recovering sensitivity to antiestrogenic compounds in antiestrogenic-resistant breast tumour cells . The applicants of the present patent application have shown that such peptides may be used in case of breast tumours in which the resistance has developed in the absence of interleukin-lβ or other inflammatory stimuli .

The antiestrogenic compounds to which the breast tumour cells have developed a resistance are preferably tamoxifen, toremifene and other SERMs (selective

Estrogen Receptor Modulators) with a similar pharmacological mechanism, preferably tamoxifen.

It was demonstrated that a cellular membrane permeable compound, expressly designed to interfere with ERα/TAB2 interaction could revert tamoxifen resistance in breast cancer cells spontaneously resistant to tamoxifen, with no indication of inflammatory pathway activation .

In order to obtain these results, first direct evidence of a competing activity of ERα-derived peptides on TAB2/ERα interaction was obtained "in vitro", using recombinant proteins. Figure 23 shows results of an "in vitro" binding assay (also called "pull-down") in which a bacterially expressed C-terminus fragment of TAB2 fused to a maltose binding protein (MBP-ΔsinoS-TAB2 ) and ERa from lysates of HEK 293T cells transfected with a ERa expression vector, were co-precipitated using maltose-agarose, in the presence of competing peptides, i.e. the N-terminus HX7LL motif of the estrogen receptor alpha (ERa, SEQ ID No: 10) and its mutated form (ERa mut, SEQ ID No: 11) . As shown in Figure 23, the wild type ERa peptide (SEQ ID No: 10) competes for ERa/TAB2 interaction as compared to the mutated peptide (ERa mut, SEQ ID No: 11) . The controls with the MBP protein alone are shown, demonstrating no aspecific interaction. This result confirms that this motif is implicated in TAB2/ERα molecular interaction. Two different types of peptides, one having SEQ ID No: 1, that is containing ERa HX7LL motif or the other having SEQ ID No: 2, that is containing the same sequence with H→A and L→A substitutions (AX ₇ AA) , conjugated with a peptide vehicle, preferably selected in the group consisting of TAT (SEQ ID No: 7), Antennapedia (Antp) (SEQ ID No: 8) e Model Amphipathic Peptide (MAP) (SEQ ID No: 9), were designed.

Two conjugates were synthesized for the experiment: a peptide having SEQ ID NO: 1 was conjugated with a carrier peptide derived from the TAT protein fused at the N-terminus or C-terminus, respectively having SEQ ID NO: 3 (ERα/TAT) and SEQ ID NO: 4 (TAT/ERα) . The conjugates were designed in this manner so as to be permeable to the cell membrane, and so as to be tested as soluble compounds in the culture medium of tamoxifen- resistant cells. These conjugates were compared with two conjugates of a peptide having SEQ ID NO: 2 with a nuclear vehicle derived from the TAT protein fused at the N-terminus or C-terminus, respectively having SEQ ID NO: 5 (ERαMut/TAT) and SEQ ID NO: 6 (TAT/ERαMut) .

MCF7 TAMR-PB9701 cells were treated with 1 μM or 100 μM of the ERα/TAT peptide (SEQ ID NO: 3) in DMEM/F12 1:1 without serum and supplemented with 10 ^~6 M TAM for 24 hours. After the first hour of treatment 1% serum was either added or not. At the end of the treatment, cell proliferation was evaluated.

It has been observed that the ERα/TAT peptide (SEQ ID No: 3) allows the recover of inhibitory effect of tamoxifen on cell proliferation (Figure 16), while the mutated conjugates (SEQ ID No: 5 and SEQ ID No: 6), failed to rescue the inhibition of cell growth. This experiment was repeated three times and biological variations are shown in figure 16.

The same experiment was run in other cell lines, as well, and results are reported in Figures 17-19. In all cases, the ERα/TAT peptide (SEQ ID No: 3) restores tamoxifen response, at variable level, whereas in no case the mutated peptide (SEQ ID No: 5 and SEQ ID No: 6) show any activity (Figure 9) . As an additional proof, the experiment was repeated on BT474 cell line described above, and the results are shown in Figure 24.

A reverse peptide, TAT/ERα (SEQ ID No: 4), carrying the carrier TAT peptide at the N-terminus, together with its cognate mutated version, was also used in these assay. This peptide consistently shows the same activity as the previous, albeit to a lesser extent. These results demonstrate the efficacy of a cell permeable peptide, interfering with the ERα/TAB2 complex, in restoring tamoxifen sensitivity in resistant breast cancer cells.

Discussion The tamoxifen resistant cell lines studied (MCF7 TAMR-894, -895, -PB9701, -808) have shown very similar characteristics both in morphology and in the expression of the proteins involved in the mechanism of resistance. The comparison with parental MCF7 WT cells reveals a slightly smaller dimension, a more irregular appearance and a growth as tightly packed colonies with limited cell spreading. Morphological alterations observed in the TAM-resistant cell lines are likely due to an adaptive process in response to the primary drug action. It has been observed a constant expression level of the proteins in all cell lines analyzed, while the expression of the co-repressor proteins TAB2 and SMRT in MCF7 TAMR cells is lower than in MCF7 WT cells. Thus, the tamoxifen resistance is not to be induced by either the absence of the estrogen receptor or suppression of components of co-repressor complexes. However, it is noteworthy that in MCF7 TAMR cell lines, SMRT expression was particularly reduced. In these cells, thus, transcriptional repression in response to tamoxifen is predominantly NCoR-dependent . The repressor complexes SMRT and NCoR are not completely equivalent, but they can interact with different co-activators and co- repressors, in order to regulate the expression of different genes. Indeed, it was demonstrated that SMRT complex does not contain the co-repressor TAB2 (Perissi and Rosenfeld, 2005) . Thus, in these TAMR cell lines the TAB2 dependence could be enhanced by the low level of SMRT available.

By immunocytochemical and biochemical analyses, the applicants verified the peculiar feature of the tamoxifen resistance in their proposed model. In fact, MCF7 TAMR cells show a clear cytoplasmic localisation of NCoR/TAB2, in contrast with the situation observed in MCF7 WT cells. The nuclear delocalisation of NCoR to the cytoplasm is caused by the IL-lβ-dependent phosphorylation of TAB2 in prostate cancer cells (Zhu et al . , 2006) . Thus, as a positive control, IL-lβ was used to induce the nuclear translocation of NCoR outside the nucleus in MCF7 WT cells. The export was verified with both cellular fractionation experiments and indirect immunofluorescence experiments. When MCF7 WT cells are stimulated by IL-lβ or TAM + IL-lβ, an increase of NCoR localisation in the cytoplasmic fractions is observed. In MCF7 WT cells, cultivated with tamoxifen, TAB2 is mainly localised in the nucleus, while, when cells were stimulated by IL-lβ or TAM + IL-lβ, TAB2 showed a cytoplasmic localisation. In MCF7 TAMR cells in the presence of tamoxifen, TAB2 is significantly located in the cytoplasm and IL-lβ alone or TAM + IL-lβ stimulation increases its cytoplasmic localization. These data confirmed that, in resistant cell lines, the activation of the IL-lβ-dependent signalling pathway delocalises TAB2 in the cytoplasm. Comparing MCF7 WT and MCF7 TAMR cells, it has been observed that NCoR and TAB2 distributions are completely different. Indeed, in MCF7 WT cells, NCoR and TAB2 are mainly localised in the nucleus, while in MCF7 TAMR cells, the co-repressor complex is significantly located in the cytoplasm. Also in this case, the stimulation with IL-I β or TAM + IL-I β induces NCoR localisation in the cytoplasm. Thus, it has been provided evidence indicating that MCF7 TAMR show a constitutive nuclear export of NCoR mediated by TAB2. However, residual abundant nuclear localisation of NCoR was observed, both in response to IL-I β, and in MCF7 TAMR cells. In fact, TAB2 is a facultative component of the NCoR complex and TAB2-less NCoR complexes are equally functional in repressing steroid receptor and target genes, e.g. TR and RAR (Zhu et al . , 2006), as also demonstrated by the fact that downregulation of TAB2 restores antiproliferative response to tamoxifen in these cells and, upon TAB2 silencing, complete relocalisation of NCoR into the nuclear compartment occurs .

As a whole, these results show that in MCF7 cells and their MCF7 TAMR derivatives, the mechanism of NCoR dismissal from the target genes is functional. Furthermore, and more importantly, MCF7 TAMR cells show a constitutive delocalisation of the co-repression complex .

The central role of TAB2 in the mechanism of resistance was also strongly demonstrated by cell proliferation assays. Indeed, it has been observed that in MCF7 TAMR cells tamoxifen does not inhibit the proliferation rates, but, upon downregulation of TAB2, tamoxifen is able again to block ERα-mediated transactivation, resulting in growth inhibition of breast cancer cells.

In contrast to what was known in the art, the inventors have shown the mechanism of resistance in breast tumour cells which have acquired the resistance following a prolonged exposure to the drug, without involving the original model of stimulation with IL-lβ (or other inflammatory stimuli, published by Zhu et al . , 2006) .

Advantageously, while it was known that IL-lβ activates the inflammatory pathway, in turn leading to MEKKl phosphorylation of TAB2, unmasking its NES motif and its NCoR shuttling activity, the inventors demonstrated that, on the contrary, in cells that are not exposed to the cytokine (IL-lβ) mechanisms other than the inflammatory pathway lead to a similar mechanism of resistance.

The experiments carried out show that the suppression of TAB2 in these cells allows the nuclear relocalisation of NCoR, associated to the recovery of the antiproliferative effect of tamoxifen, clearly showing that the action of TAB2 is not only limited to the stimulation paradigm of the cytokines, but is most probably active in cells which have acquired resistance to tamoxifen by other mechanisms.

The inventors further report for the first time that the ERa N-terminus HX7LL peptide, conjugated with a peptide function that allows the penetration in cells and the localisation in the cell nucleus, is effective in restoring the antiproliferative response of tamoxifen in spontaneously resistant breast cancer cells.

This peptide is provided as a pharmaceutical compound to be administered together with SERM drugs to patients with breast cancer, to stabilise and revert resistance; or as an alternative, as a guide compound for the development of similar molecules which can penetrate cells, target TAB2 and interfere with the dismissal of NCoR from the target genes and with the transcriptional derepression.

SEQUENCE LISTING

<110> BIOINDUSTRY PARK CANAVESE SPA

<120> USE OF A PEPTIDE DERIVED FROM ER-ALPHA FOR RESTORING SENSITIVITY TO ANTIESTROGENIC COMPOUNDS IN BREAST TUMOUR CELLS

<130> 934-08

<160> 11 <170> Patentln version 3.3

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