J E Bluff

M Amarzguioui

J Slattery

N J Brown

Mwr Reed

Dr Carolyn A Staton
email: c.a.staton@sheffield.ac.uk

Submitted to Breast Cancer Research and Treatment

Keywords: Angiogenesis, breast cancer, haemostasis, Tissue Factor, VEGF CCS -Cumulative Chalkley Score, GFgrowth factor, HuDMEC -Human dermal microvascular endothelial cells, MVDmicrovessel density, PECAMplatelet endothelial cell adhesion molecule, TF -Tissue Factor, VEGFvascular endothelial growth factor

Purpose: In breast cancer there is a correlation between tissue factor (TF) expression, angiogenesis and disease progression. TF stimulates tumour angiogenesis, in part, through up-regulation of vascular endothelial growth factor (VEGF). Therefore, this study aim ed to establish whether TF stimulates angiogenesis and tumour progression directly and independent of VEGF up-regulation.

Methods: Initially the effects of TF and VEGF were assessed on endothelial cell migration (Boyden chamber) and differentiation (tubule formation on Matrigel). Susbequently MDA-MB-436 breast cancer cells, which produce high levels of both TF and VEGF (western blot analysis), were established in vivo, following which tumours were treated three times per week for three weeks with intra-tumoural injections of either anti-VEGF siRNA, anti-TF shRNA, the two treatments combined or relevant controls.

Results: Both VEGF and TF significantly stimulated endothelial cell migration and tubule formation (P<0.02). Breast cancer xenografts (MDA-MB-436) treated with TF or VEGFspecific agents demonstrated significant inhibition in tumour growth (VEGFsiRNA 61%; final volume: 236.2+/-23.2 mm 3 vs TFshRNA 89%; 161.9+/-17.4 mm 3 vs combination 93%; 136.3+/-9.2 mm 3 vs control 400.4+/-32.7 mm 3 : p<0.005). Microvessel density, a measure of angiogenesis, was also significantly inhibited in all groups (MVD in control=29+/-2.9; TFshRNA=18+/-1.1; VEGFsiRNA=16.7+/-1.5; both=12+/-2.1; p<0.004), whereas the proliferative index of the tumours was only reduced in the TFshRNA treated groups (control=0.51+/-0.011; TFshRNA=0.41+/-0.014; VEGFsiRNA=0.49+/-0.013; both=0.41+/-0.004; p<0.008).

Conclusions: These data suggest that TF has a direct effect on primary breast cancer growth and angiogenesis, and that specific inhibition of the TF signalling pathway has potential for the treatment of primary breast cancer.

Introduction

Angiogenesis, the outgrowth of new capillaries from an existing vascular bed, and haemostasis, the coagulation cascade leading to clot formation, are amongst the most consistent host responses associated with cancer. These interrelated processes have important implications for cancer therapy as hypercoagulability is exhibited by most cancer patients and contributes to the pathogenesis of tumour growth and metastasis by promoting angiogenesis [START_REF] Bluff | Tissue Factor, angiogenesis and tumour progression (review)[END_REF]. Tissue factor (TF) is a 47kDa transmembrane glycoprotein that safeguards the vascular integrity of tissues by initiating the coagulation cascade following vessel injury [START_REF] Bluff | Tissue Factor, angiogenesis and tumour progression (review)[END_REF].

In normal physiological situations, TF expression is restricted to the tunica adventitia and subendothelial cells, however during tumourigenesis, expression within vascular endothelial cells is also observed. Indeed, up-regulation of TF protein by cancer cells and associated stromal cells has been well documented in breast cancer and other malignant tumours [START_REF] Bluff | Angiogenesis is associated with hyperplasia in human ductal breast disease[END_REF][START_REF] Staton | The angiogenic switch occurs at the adenoma stage of the adenoma carcinoma sequence in colorectal cancer[END_REF][START_REF] Ueno | Tissue factor expression in breast cancer tissues: its correlation with prognosis and plasma concentration[END_REF].

TF levels have been correlated with advanced stages of malignancy and metastatic potential in different cancers, including breast [START_REF] Bluff | Angiogenesis is associated with hyperplasia in human ductal breast disease[END_REF][START_REF] Ueno | Tissue factor expression in breast cancer tissues: its correlation with prognosis and plasma concentration[END_REF], suggesting that TF may play a role in tumour progression, which may be related to the putative role of TF in metastatic disease as TF inhibition reduces both melanoma and breast cancer cell metastasis in vivo [START_REF] Ngo | CNTO 859, a humanized anti-tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models[END_REF][START_REF] Amarzguioui | Ex vivo and in vivo delivery of anti-Tissue Factor siRNA inhibits mouse pulmonary metastasis of B16 melanoma cells[END_REF].

TF indirectly contributes to tumour angiogenesis via generation of thrombin, which releases pro-angiogenic factors from activated platelets and converts circulating fibrinogen to an insoluble fibrin clot, providing a provisional matrix to facilitate blood vessel infiltration (reviewed by [START_REF] Rak | Tissue factor in cancer and angiogenesis: the molecular link between genetic tumor progression, tumour neovascularization, and cancer coagulopathy[END_REF]). TF also up-regulates the pro-angiogenic protein, vascular endothelial growth factor (VEGF) [START_REF] Abe | Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor[END_REF], the most potent known stimulator of angiogenesis, and correlates with both microvessel density (MVD; a surrogate marker of angiogenesis) and VEGF expression in breast and colon cancer [START_REF] Bluff | Angiogenesis is associated with hyperplasia in human ductal breast disease[END_REF][START_REF] Staton | The angiogenic switch occurs at the adenoma stage of the adenoma carcinoma sequence in colorectal cancer[END_REF].

Despite increasing evidence that TF participates in many tumour-related processes, the precise role is incompletely understood, although evidence suggests that therapeutic targeting of TF may inhibit tumour angiogenesis and hence disease progression. This study demonstrates that TF stimulates angiogenesis independent of VEGF up-regulation and provides evidence for the first time that TF-specific knockdown using shRNA technology significantly reduces primary breast tumour growth in vivo.

Methods

Cells

Adult human dermal microvascular endothelial cells (HuDMEC; Promocell, Heidelberg), were cultured in complete microvascular endothelial cell growth medium (EGM; Promocell), and used for a maximum of 5 passages. MDA-MB-436, MCF-7 and T47D human breast cancer cell lines (ATCC) were routinely tested for key characteristics (including ER & PR status). MDA-MB-436/MCF-7 and T47D cells were cultured in high glucose RPMI 1640 (GIBCO , Paisley, UK) and DMEM (Invitrogen) medium respectively, containing 1% Lglutamine, 10% FCS and 1% penicillin-streptomycin. All cells were grown at 37 C in a 100% humidified incubator with a gas phase of 5% CO 2 and routinely screened for Mycoplasma.

Tubule formation assay

The HuDMEC tubule formation assay was performed as previously described [START_REF] Krajewska | A novel fragment derived from the beta chain of human fibrinogen, 43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo[END_REF] using EGM and 1% heat inactivated FCS containing VEGF, TF or both (2-50 ng/ml). Tubule formation was measured as the number of cord-like structures and the number of branch points seen within an entire well. Each experiment contained 3 replicate wells for each treatment and was repeated 3 times.

Migration assay

A 48 well microchemotaxis chamber (Neuro Probe, Cabin John, MD) was used as previously described [START_REF] Krajewska | A novel fragment derived from the beta chain of human fibrinogen, 43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo[END_REF] with VEGF or TF alone or in combination (2-50 ng/ml) placed in the lower chamber, and serum starved HuDMEC in the top chamber. Migrated cells were then fixed, stained and counted at x40 magnification in 3 random fields per well in 3 replicate wells and repeated 3 times.

Cloning of pFRT-H1 shRNA expression vectors.

Expression plasmids pFRT-H1T-hTFsh (referred to hereafter as hTFshRNA) and pFRT-H1T-mTFsh (referred to hereafter as non-targeting vector control) were generated by directional oligonucleotide cassette mediated cloning into the BglII/KpnI linearised expression vector pFRT-H1tetO, as previously described [START_REF] Aagaard | A facile lentiviral vector system for expression of doxycycline-inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha[END_REF]. The shRNAs were designed to target previously identified optimal human-specific [START_REF] Wang | Down-regulation of Tissue Factor by RNA interference in human melanoma LOX-L cells reduce pulmonary metastasis in nude mice[END_REF] and murine-specific [START_REF] Amarzguioui | Ex vivo and in vivo delivery of anti-Tissue Factor siRNA inhibits mouse pulmonary metastasis of B16 melanoma cells[END_REF] TF siRNA target sites.

The following oligonucleotides were used for cloning of the shRNA cassettes: hTFsh-A: 5'-gatcgcgcttcaggcactacaaatactgtcctgacccaacagtatttgtagtgcctgaagcgctttttgtac-3'; hTFsh-B: 5'-aaaaagcgcttcaggcactacaaatactgttgggtcaggacagtatttgtagtgcctgaagcgc-3'; mTFsh-A: 5'-gatcgcattccagagaaagcgtttaatttcctgacccaaaattaaacgctttctctggaatgctttttgtac-3'; mTFsh-B: 5'-aaaaagcattccagagaaagcgtttaattttgggtcaggaaattaaacgctttctctggaatgc-3'

The oligos were annealed pairwise, phosphorylated, and ligated into complementary BglII/KpnI-linearised vector, as previously described [START_REF] Aagaard | A facile lentiviral vector system for expression of doxycycline-inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha[END_REF]. Correct sequence of restrictionpositive transformants was confirmed by sequencing in both directions, using the primers 5'caaggtcgggcaggaagag-3' and 5'-aggggcaaacaacagatggc-3'.

Quantitative Real Time PCR (qRT-PCR)

The mRNA was extracted using the tri-reagent system (Sigma, Dorset, UK), from directly lysed cells, and the concentration and purity of total RNA determined by UV light absorption.

cDNA was synthesised from 1 g total RNA using the Taqman reverse transcriptase reagents (Applied Biosystems, Foster City, CA, USA) and SuperScript® II Reverse Transcriptase (Invitrogen). qRT-PCR was performed using PCR Master Mix (Eurogentec Ltd, Hampshire, UK), with predesigned sequence specific Taqman gene probes (Applied Biosystems). qRT-PCR cycling conditions were 2 minutes at 50 C and then 95 C for 10 minutes, followed by 40 cycles of 15 seconds at 95 C, followed by 1 minute at 60 C. qRT-PCR was detected by ABI-Prism 7900HT Sequence Detection System and data processed using GeneAmp (Applied Biosystems). Target gene expression (VEGF and TF mRNA) was normalised to levels of GAPDH endogenous control.

Protein analysis

Protein extracts were prepared by lysing breast cancer cells with a triple cell lysis buffer [START_REF] Krajewska | A novel fragment derived from the beta chain of human fibrinogen, 43-63, is a potent inhibitor of activated endothelial cells in vitro and in vivo[END_REF].

Protein concentrations were determined using the bicinchoninic acid (BCA) assay. Equal amounts of protein extracts (20 g) were separated via SDS polyacrylamide gel electrophoresis (PAGE), electrotransferred to nitrocellulose membranes and exposed to anti-TF (4509; American Diagnostica Inc. Connecticut, USA; 2 g/ml) or anti-VEGF (A-20;

Santa Cruz Biotechnology, Wiltshire, UK; 5 g/ml). Immunoreactive bands were detected by enhanced chemiluminescence (Amersham), with -actin as a loading control (Sigma).

Alternatively, conditioned media was collected from relevant samples and centrifuged before TF and VEGF were measured in duplicate using commercial two-antibody sandwich assays kits (TF: Immubind, American Diagnostica; VEGF: Quantikine, R&D Systems, Abingdon, Oxfordshire, UK). The detection limits of the TF and VEGF assays were 10 pg/ml and 30 pg/ml, respectively.

Transfections and flow cytometry

MDA-MB-436 cells were seeded in six-well plates and grown to 40% confluence before co- doses were based on previous studies with siRNA [START_REF] Filleur | SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth[END_REF] or shRNA [START_REF] Lin | Overexpresion of PKC is required to impart estradiol inhibition and tamoxifen-resistance in a T47D human breast cancer tumour model[END_REF]. Another group of mice received no treatment (WT), while a non-targeting shRNA vector and a scrambled (non-targeting) siRNA were used as negative controls. Tumour growth was assessed using calipers every 2/3 days. At the end of the study tumours and control tissues were excised and bisected for immunohistochemical analysis.

Immunohistochemical staining

Tissues were fixed in either 10% neutral buffered formalin or zinc-based fixative for 48 hours, then processed into paraffin wax. Immunohistochemistry was performed using a panel of antibodies to murine platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31; 1:100 using zinc-fixed tissues) (Pharmingen, San Diego, CA, USA), human Ki67 (1:100; Dakocytomation Ltd, Cambridgeshire, UK), VEGF (A-20; 1:150; Santa-Cruz Biotechnology Inc. Santa Cruz, CA, USA), TF (1:150; R&D Systems) using a standard horse radish peroxidase staining procedure [START_REF] Staton | Identification of key residues involved in mediating the in vivo anti-tumour/antiendothelial activity of Alphastatin[END_REF]. Negative controls were achieved by omission of the primary antibody and replacement with normal mouse IgG (DAKO), normal goat IgG (DAKO)

or normal rabbit IgG (DAKO) at a dilution of 1:100.

Histological analysis

Formalin-fixed tissue sections were stained using Haematoxylin & Eosin and tumour necrosis was assessed using a Chalkley grid method [START_REF] Tozer | Phosphorus-31 magnetic resonance spectroscopy and blood perfusion of the RIF-1 tumor following X-irradiation[END_REF]. Microvascular density (MVD), a surrogate marker of angiogenesis, was assessed by obtaining a Chalkley grid score for 5 hotspots per section and the sum of the Chalkley scores for each section was termed the cumulative Chalkley score (CSS) [START_REF] Staton | Alphastatin, a 24 amino-acid fragment of human fibrinogen, is a potent new inhibitor of activated endothelial cells in vitro and in vivo[END_REF]. Ki67 staining was assessed as the percentage of tumour cells showing positive staining for Ki67 antigen by counting a minimum of 1000 cells per tumour section in a total of four areas of viable tumour per section [START_REF] Morrone | In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model[END_REF].

Statistical analysis

Statistical analysis was conducted using SPSS v16 software (Chicago, IL, USA). All data presented are means +/-SEM, and representative data from one of 3 replicate experiments are shown. Statistical analysis was performed using a one-way analysis of variance (ANOVA) and either the Dunnet's or Mann-Whitney U post hoc test for nonparametric data.

Data were considered statistically significant at p<0.05.

Results

Effects of TF on HuDMECs in vitro

Tubule formation: HuDMECs elongated and formed tubules on GF reduced Matrigel in the absence of exogenous stimuli (due to the residual level of growth factors remaining in this matrix), but this was significantly enhanced with the addition of VEGF (p<0.01) or to a lesser extent by TF (p<0.02) as assessed by the number of tubules and branch points/well (Table 1, Figure 1A). The effects of TF on tubule formation were not attributable to stimulation of VEGF production by HuDMECs as an ELISA showed no change in secreted VEGF levels in response to 2-50 ng/ml TF concentrations (untreated=31+/-3.2 pg/ml, TF treated=28+/-4.5 pg/ml). When added in combination there was a non-significant increase in tubule number and no difference in the number of branch points compared to either factor alone (Figure 1).

Migration: HuDMECs migrated across the fibronectin coated filters in the absence of exogenous stimuli, however this was significantly increased in the presence of VEGF (p<0.007) or TF (p<0.001) (Table 2). Moreover, VEGF stimulated TF to a significantly greater extent than TF (p<0.01) and the combination of both factors enhanced migration more than either factor alone, although this failed to reach significance (Figure 2).

Effect of p-FRT-H1-TFShRNA on TF/VEGF expression

In order to identify a breast cancer cell line that expressed both TF and VEGF for subsequent study, qRT-PCR and western blot analysis was used to determine basal TF and VEGF gene and protein expression in MDA-MB-436, MCF-7 and T47D breast cancer cell lines. VEGF mRNA was detected in all cell lines examined (Figure 3A). In contrast, only MDA-MB-436 cells expressed TF mRNA (higher than VEGF mRNA levels), with negligible TF mRNA detected in either MCF-7 or T47D cells. Western blot analysis was consistent with the gene expression data, showing VEGF expressed by all cell lines, but only detectable levels of TF expressed in MDA-MB-436 cells (Figure 3B).

The effects of TF knockdown on TF/VEGF gene and protein expression in MDA-MB-Transfection with hTFshRNA significantly decreased both TF mRNA (77+/-3.3%; p<0.001) and protein release (80.3+/-3.7%; p<0.003) (Figure 3C & D), whereas VEGF gene expression was unaffected by TF knockdown. Furthermore, there was no difference in VEGF/TF mRNA and protein expression between cells in all control conditions. TF knockdown in MDA-MB-436 cells had no significant effect on proliferation or cell viability assessed using a Vi-cell counter over 4 days (data not shown). Essentially similar results

were obtained using an alternative TF expressing breast cancer cell line, MDA-MB-231, showing ~75% knockdown in expression.

Intra-tumoural injections of anti-TFshRNA and anti-VEGFsiRNA inhibits tumor growth in vivo

Direct intra-tumoural injections of anti-TFshRNA or anti-VEGFsiRNA alone and in combination, were performed on established MDA-MB-436 tumours to determine whether TF expression plays a key role in primary breast tumour growth and angiogenesis that is functionally distinct from the role in VEGF production (Figure 4). Importantly, the nontargeting vector and scrambled siRNA control treatments had no significant effect on final tumour volume compared to the untreated group (367.2+/-35.9, 352.1+/-23.1 and 400.4+/-32.7 mm 3 respectively at day 21). In contrast, the TF or VEGF-specific treatments all significantly (p<0.005) inhibited tumour growth; treatment with anti-VEGFsiRNA by 61%

(final volume: 236.2+/-23.2 mm 3 ) or TFshRNA by 89% (161.9+/-17.4 mm 3 ) alone, while the combination resulted in 93% growth inhibition (136.3+/-9.2 mm 3 ). Interestingly the tumour growth inhibition induced by TFshRNA alone or in combination with VEGFsiRNA was significantly greater than treatment with VEGFsiRNA alone (p=0.018 and p=0.016 respectively; Figure 4).

Histological Analysis of tumours

Histological analysis of control tumour tissue revealed the presence of a central area of necrosis surrounded by a viable rim of tumour cells, which was unchanged any of the treatment regimes (data not shown). To determine whether the reduced volume of both TFshRNA and VEGFsiRNA treated tumours was due to an effect on the tumour vasculature, endothelial cells were identified using CD31 staining (Figure 5A). Microvessel density in the viable rim of tumour cells was significantly reduced compared to relevant controls in VEGFsiRNA (p=0.002), TFshRNA (p=0.002) and combined (p=0.004) treatment groups (Figure 5A). In order to determine whether the reduced tumour volume was also due to an effect on tumour cell proliferation, the proliferation index was calculated on Ki67 stained sections [START_REF] Morrone | In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model[END_REF]. In contrast to MVD, only TFshRNA treatment had a significant effect on proliferation index (control: 0.51+/-0.01 vs TFshRNA: 0.41+/-0.02; p=0.008, Figure 5B).

VEGF staining revealed a relatively homogenous pattern throughout the cytoplasm of tumour cells, with the intensity of staining reduced, but not completely absent in the VEGFsiRNA and combined treated tumours only (Online Resource 1A). In contrast, TF staining was seen in the membrane of tumour cells and was heterogeneous, with more intense staining adjacent to the necrotic regions of the tumours. TFshRNA treated tumours showed less staining than any other tumours and contained some areas where TF staining was absent (Online Resource 1B).

Discussion

Elevated TF expression in breast cancer has been associated with disease progression, poor survival, increased VEGF expression and angiogenesis [START_REF] Bluff | Angiogenesis is associated with hyperplasia in human ductal breast disease[END_REF][START_REF] Ueno | Tissue factor expression in breast cancer tissues: its correlation with prognosis and plasma concentration[END_REF]. VEGF is known to up-regulate the expression of TF in cancer cells and associated endothelial cells, potentially creating a feedback loop whereby TF in turn up-regulates VEGF expression [START_REF] Bluff | Tissue Factor, angiogenesis and tumour progression (review)[END_REF]. It has therefore been proposed that inhibition of TF in breast cancer may result in reduced angiogenesis due to down-regulation of VEGF expression. The data presented here however, provide evidence that TF has the ability to modulate angiogenic mechanisms in vitro in the absence of VEGF up-regulation. Moreover, we demonstrate for the first time that direct intra-tumoural injections of TFshRNA prevents the growth of established primary breast cancer xenografts in vivo by inhibiting both tumour cell proliferation and angiogenesis.

It has been well documented that VEGF stimulates angiogenic activities in endothelial cells (reviewed in [START_REF] Ferrara | The biology of VEGF and its receptors[END_REF]) and our data is consistent with other studies showing a dose-dependent increase in HuDMEC migration and tubule formation in vitro. TF also stimulates HuDMEC migration and tubule formation/differentiation, although to a lesser extent, and appears not to be dose dependent or resulting from VEGF up-regulation. This is in agreement with studies demonstrating that soluble TF promotes EC migration and tubule formation [START_REF] He | Soluble tissue factor has unique angiogenic activities that selectively promote migration and differentiation but not proliferation of endothelial cells[END_REF] and that an alternatively spliced form of TF (asTF) induces tubule formation independently of VEGF or PAR signaling [START_REF] Hobbs | Alternatively spliced human tissue factor promotes tumor growth and angiogenesis in a pancreatic cancer tumor model[END_REF]. In vitro assays from the current study were carried out in the absence of activated factor VII (FVIIa), by using low levels of heat inactivated serum, and data indicate a direct role for TF signaling in EC migration and tubule formation, although the precise mechanisms mediating this response are as yet undetermined. Interestingly, there was only a small additional increase in tubule formation or migration in the presence of both TF and VEGF, compared to VEGF alone, suggesting that VEGF, the most potent angiogenic factor identified to date [START_REF] Abe | Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor[END_REF], is likely to be saturating the endothelial cell activation and functional responses in these assays.

In breast cancer, TF is associated with metastatic potential and poor prognosis [START_REF] Ueno | Tissue factor expression in breast cancer tissues: its correlation with prognosis and plasma concentration[END_REF] in addition to playing a role in angiogenesis [START_REF] Bluff | Angiogenesis is associated with hyperplasia in human ductal breast disease[END_REF]. The current study demonstrates that TF is highly expressed in metastatic breast carcinoma cells (MDA-MB-436), in contrast to nonmetastatic breast carcinoma cells (MCF-7, T47D), although MCF-7 expresses TF under hypoxic conditions (data not shown; [START_REF] Amirkhosravi | Pentoxifylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor[END_REF]). These observations are in agreement with previous studies, reporting elevated TF expression in metastatic cell lines compared to nonmetastatic cell lines of the same tissue origin [START_REF] Sawada | Expression of tissue factor in non-smallcell lung cancers and its relationship to metastasis[END_REF][START_REF] Kataoka | Analysis of tissue factor and tissue factor pathway inhibitor expression in human colorectal carcinoma cell lines and metastatic sublines to the liver[END_REF][START_REF] Mueller | Expression of tissue factor by melanoma cells promotes efficient hematogenous metastasis[END_REF]. Following TF expression knockdown of ~80% in the MDA-MB-436 cell line there was no effect on VEGF expression, suggesting that TF is not involved in regulating VEGF expression in this cell line. Furthermore, TF knockdown had no effect on cell proliferation or viability in vitro over a time course of 4 days, in agreement with other studies using a similar mouse specific construct in B16 melanoma cells [START_REF] Amarzguioui | Ex vivo and in vivo delivery of anti-Tissue Factor siRNA inhibits mouse pulmonary metastasis of B16 melanoma cells[END_REF].

As previous studies had investigated the effects of murine-specific TF shRNA on metastasis [START_REF] Amarzguioui | Ex vivo and in vivo delivery of anti-Tissue Factor siRNA inhibits mouse pulmonary metastasis of B16 melanoma cells[END_REF], we used a model of established primary MDA-MB-436 subcutaneous tumours treated with intra-tumoural injections of TFshRNA or VEGFsiRNA alone and in combination, to achieve direct homology-dependent post-transcriptional gene silencing, with subsequent knockdown of protein translation. Our data show that VEGF inhibition significantly reduces primary breast tumour growth and angiogenesis (measured by MVD), in agreement with results from relevant mouse xenograft studies [START_REF] Matsumoto | Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma[END_REF][START_REF] Tao | Inhibiting the growth of malignant melanoma by blocking the expression of vascular endothelial growth factor using an RNA interference approach[END_REF][START_REF] Filleur | SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth[END_REF]. Interestingly, treatments with TFshRNA (either alone or in combination with VEGFsiRNA) were more potent in reducing primary tumour growth than VEGFsiRNA treatment alone. However, shRNA requires cellular transcription in the nucleus, further processing, then transport to the cytoplasm before being incorporated into the RNA-interfering silencing complex, therefore data generated from administration of siRNA and shRNA are not directly comparable. What is interesting is that addition of TFshRNA to VEGFsiRNA significantly increases tumour inhibition compared to VEGFsiRNA alone. Moreover, these data suggest that TF has a direct role in promoting primary tumour growth that is functionally distinct from the role in VEGF production. This is in agreement with previous in vivo studies using stable TF knockdown in colorectal cancer cells grown in Matrigel plugs which showed a reduction in angiogenesis, but only minimal effects on VEGF expression [START_REF] Yu | Oncogenic events regulate tissue factor expression in colorectal cancer cells: implications for tumor progression and angiogenesis[END_REF]. Although other groups have previously shown that inhibition of TF signaling suppresses hypoxia-induced retinal angiogenesis [START_REF] Uusitalo-Jarvinen | Role of protease activated receptor 1 and 2 signaling in hypoxia-induced angiogenesis[END_REF] and breast tumour growth in vivo [START_REF] Ngo | CNTO 859, a humanized anti-tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models[END_REF][START_REF] Versteeg | Inhibition of tissue factor signaling suppresses tumor growth[END_REF], this is the first study to evaluate TF inhibition alone and in combination with VEGF inhibition, in established primary breast tumours.

Our in vivo data show that both treatments (TF and VEGF knockdown) inhibit angiogenesis, but only tumours treated with TFshRNA showed a significantly reduced tumour proliferative index, suggesting that TF knockdown in breast tumour cells reduces proliferation in vivo, in addition to inhibiting angiogenesis. These data therefore indicate that down-regulation of VEGF expression can inhibit co-option or invasion of murine ECs, whilst the anti-tumour response to TF knockdown may also, in part, be attributed to direct effects on breast tumour cell proliferation. This data is consistent with observations from previous studies on gene expression in a human melanoma cell line demonstrating down-regulation of genes involved in cell growth, invasion and metastasis and an up-regulation of genes involved in apoptosis, and reduced cell proliferation following TF knockdown using TFshRNA [START_REF] Wang | Down-regulation of Tissue Factor by RNA interference in human melanoma LOX-L cells reduce pulmonary metastasis in nude mice[END_REF]. Differences between in vitro and in vivo proliferation responses are likely to be attributable to other tumour microenvironmental factors in vivo. Although breast cancer cell lines express PAR-2, which becomes activated in the presence of TF-FVIIa stimulating signaling pathways potentially resulting in proliferation [START_REF] Morris | Protease-activated receptor-2 is essential for factor VIIa and Xa-induced signaling, migration, and invasion of breast cancer cells[END_REF], data relating to the direct TF effects on proliferation are contradictory [START_REF] Yu | Oncogenic events regulate tissue factor expression in colorectal cancer cells: implications for tumor progression and angiogenesis[END_REF][START_REF] Zhang | Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice[END_REF][START_REF] Ge Ler | Inhibition of tissue factor/protease-activated receptor-2 signaling limits proliferation, migration and invasion of malignant glioma cells[END_REF].

Whilst the current study using TFshRNA, and others using inactivating peptides and antibodies [START_REF] Ngo | CNTO 859, a humanized anti-tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models[END_REF][START_REF] Versteeg | Inhibition of tissue factor signaling suppresses tumor growth[END_REF], show a definite effect of TF inhibition on primary tumour growth in vivo, clinical applications of systemic anti-TF therapy are likely to be associated with significant risks, due to the essential role of the haemostatic pathway. Indeed, a phase I clinical trial using a monoclonal antibody against TF showed an increase in the frequency of haemorrhagic events such as oral mucosal bleeding, cutaneous bleeding and spontaneous tongue haematomas in a dose dependent manner [START_REF] Morrow | Potent inhibition of thrombin with a monoclonal antibody against tissue factor (Sunol-cH36): results of the PROXIMATE-TIMI 27 trial[END_REF]. These side effects may be circumvented by the development of specific inhibitors that suppress TF-FVIIa signaling in pathological angiogenesis, but not in TF-FVIIa coagulant function [START_REF] Versteeg | Inhibition of tissue factor signaling suppresses tumor growth[END_REF], although such an approach requires full evaluation. Alternative approaches such as using atellocollagen or nanoparticles to specifically target shRNA/siRNAs to tumours following systemic administration, are also beginning to show some efficacy in vivo [START_REF] Kim | Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer[END_REF][START_REF] Mu | Systemic delivery of siRNA specific to tumor mediated by atelocollagen: Combined therapy using siRNA targeting Bcl-xL and cisplatin against prostate cancer[END_REF][START_REF] Yagi | A nanoparticle system specifically designed to deliver short interfering RNA inhibits tumor growth in vivo[END_REF]. These methods require further validation, but may prove effective for tumour-specific targeting of TF with shRNA or siRNA via systemic delivery, thereby circumventing the haemostatic side-effects associated with systemic anti-TF treatment.

In conclusion, these data suggest for the first time that modulation of TF has a direct effect on established primary breast cancer growth and angiogenesis that is distinct from the role in VEGF production. Therefore, similar to a potential use suggested for gliomas [START_REF] Ge Ler | Inhibition of tissue factor/protease-activated receptor-2 signaling limits proliferation, migration and invasion of malignant glioma cells[END_REF],

specific inhibition of the TF signaling pathway with shRNA or siRNA represents a powerful tool for inhibiting cancer cell proliferation and angiogenesis in vivo with potential therapeutic utility for the effective treatment of primary breast cancers using targeted therapy. Further work is now required to investigate the effects of TF knockdown in models relevant to breast cancer metastasis. There is no evidence of VEGFsiRNA reducing TF expression in these tumours.

Concentration (ng/ml) VEGF TF

Number of tubules

Number of branch points

Number of tubules

VEGFsiRNAA

  VEGF Stealth TM Select 3 RNAi pre-validated siRNA set (including three 25bp duplexes targeting non-overlapping sequences specific to VEGF) and Stealth RNAi TM siRNA negative control kit were purchased from Invitrogen Ltd (Renfrew, UK). The latter had no known homology to the target sequence. The target sequences of the VEGF siRNAs were:

1 (

 1 transfection with Block-IT fluorescent (fluorescein[FITC]) oligo (Invitrogen) and hTFshRNA vector targeting human TF (1.2 g/well) under serum-free conditions in Opti-MEM-Invitrogen). Untransfected, mock transfected (MT) (Block-IT fluorescent oligo) and cells transfected with a non-targeting vector (pFRT-H1T-mTFsh) served as controls. All experiments were performed in triplicate and repeated at least 3 times. Flow cytometry (FACSAria TM [BD Biosciences, San Jose, CA, USA]) was used to select transfected cells based on FITC expression. Transfected cells were plated and cell proliferation and viability measured using a Vi-cell counter at 2 and 4 days post transfection. RNA was also extracted at 2 and 4 days post transfection for qRT-PCR analysis and conditioned media was collected at 4 days post transfection for protein analysis via ELISA.In vivo tumourigenicity assayAnimal experiments were conducted in accordance with the Home Office License Authority of the United Kingdom Animals (Scientific procedures) Act 1986 (PPL 40/2972; holder NJB) and with local research ethics committee approval. MDA-MB-436 cells (1x10 7 ) were grown subcutaneously in athymic nude mice until the tumour reached approximately 100mm3 (range =104-119 mm 3 , mean +/-SE = 108.9+/-3.2). Mice were randomly assigned to treatment groups (6/group) and received intra-tumoural injections, 3 times per week for 3 weeks, of either anti-VEGFsiRNA (6 g in 20 l lipofectamine 2000; Invitrogen), or anti-TFshRNA (10 µg in 20 l lipofectamine LTX; Invitrogen), or the two treatments combined;

Figure 1 :

 1 Figure 1: Tubule formation in response to VEGF and TF. (A) the number of tubes and

Figure 2 :

 2 Figure 2: HuDMEC migration in response to VEGF and TF. (A) Graph showing effect of VEGF or TF alone or in combination on cell migration at 10 or 20ng/ml. *p<0.001 with respect to relevant control group. (B) Representative photographs of cell migration in

Figure 3 :

 3 Figure 3: Endogenous VEGF and TF expression in breast cancer cell lines and TF knockdown studies. (A) qRT-PCR analysis of VEGF and TF mRNA expression (n=3). (B)

Figure 4 .

 4 Figure 4. Response of tumours to anti-VEGFsiRNA and/or anti-TFshRNA treatment.Established MDA-MB-436 subcutaneous tumours (~100mm 3 ) were treated by intra-tumoural

Figure 5 .

 5 Figure 5. Histological analysis of tumours treated with anti-VEGFsiRNA and/or anti-

1 .

 1 photographs of tumours stained for Ki67 (dark grey-black nuclei) to assess proliferation index.

Table 1 . Effect of concentration curves of VEGF or TF on HuDMEC tubule formation as measured by number of tubules or number of branch points

 1 . *P<0.02 with respect to relevant no factor control.

	Number of
	branch points

Table 2 . Effect of concentration curves of VEGF or TF on HuDMEC migration.

 2

*P<0.007 with respect to relevant no factor control.

Acknowledgements:

We gratefully acknowledge the technical support of Jenny Globe, Carmel Nichols and Yvonne Stephenson. This work was supported by Yorkshire Cancer Research (grant number S287).


Christine Desmedt

Stefan Michiels

Benjamin Haibe-Kains

Sherene Loi

M.D Christos Sotiriou
email: christos.sotiriou@bordet.be

Time to move forward from "first-generation" prognostic gene signatures in early breast cancer

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

For clinicians, it has been clear for many years that breast cancer patients with the same clinical and pathological characteristics can have diverse clinical outcomes. The advances in the annotations of the genome and the emergence of high through-put gene expression technologies during the last decade have offered researchers powerful means to investigate breast cancer biology and to determine the mechanisms of this clinical heterogeneity [START_REF] Sotiriou | Taking gene-expression profiling to the clinic: When will molecular signatures become relevant to patient care?[END_REF][START_REF] Sotiriou | Gene-expression signatures in breast cancer[END_REF]. For this purpose, several research groups have developed "firstgeneration" gene expression signatures that could help to better identify which breast cancers are at high or low risk of cancer recurrence, such as MammaPrint [START_REF] Van 't Veer | Gene expression profiling predicts clinical outcome of breast cancer[END_REF], the Oncotype Dx [START_REF] Paik | A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer[END_REF] and the Gene Expression Grade Index [START_REF] Sotiriou | Gene expression profiling in breast cancer: Understanding the molecular basis of histologic grade to improve prognosis[END_REF]. The prognostic value of these 3 scores have all stood the test in independent validation series [START_REF] Buyse | Validation and clinical utility of a 70gene prognostic signature for women with node-negative breast cancer[END_REF][START_REF] Dowsett | Prediction of risk of distant recurrence using the 21-gene recurrence score in node-negative and node-positive postmenopausal patients with breast cancer treated with anastrozole or tamoxifen: A transatac study[END_REF][START_REF] Haibe-Kains | Comparison of prognostic gene expression signatures for breast cancer[END_REF] and are reported to provide further prognostic information to standard clinical and pathological factors, although their precise added value needs to be studied [START_REF]Recommendations from the egapp working group: Can tumor gene expression profiling improve outcomes in patients with breast cancer?[END_REF]. For two of these prognostic scores, prospective worldwide clinical trials are ongoing to estimate their impact on clinical practice for the prescription of adjuvant chemotherapy [START_REF] Cardoso | Clinical application of the 70-gene profile: The mindact trial[END_REF][START_REF] Sparano | Development of the 21-gene assay and its application in clinical practice and clinical trials[END_REF].

In this article, Naoi et al. developed a 95-gene score to predict recurrences in nodenegative (N0) patients with ER-positive tumors. They developed their score using publicly available gene expression data from 549 node negative (N0) breast cancer patients with ERpositive disease and validated their predictor in a Japanese cohort of 105 N0 with ER-positive disease. They reported that their 95-gene score was the most significant factor in a multivariate analysis which included the classical clinical and pathological characteristics, including other proliferation markers such as Ki-67, as well as the previously identified gene expression grade signature (GGI), another gene expression prognostic score [START_REF] Sotiriou | Gene expression profiling in breast cancer: Understanding the molecular basis of histologic grade to improve prognosis[END_REF]. The authors deserve credit for their study which investigated if currently available prognostic gene expression signatures derived from Caucasian breast cancer patients are also be applicable to the Japanese population.

However, there are some limitations to their study that should be highlighted. First, there exists heterogeneity in the treatment of the patients selected for the training and validation set. In other words, some patients did and did not receive adjuvant hormonal treatment. This could have considerably affected the clinical outcome of some of these patients. Additionally, although the type of endocrine therapy seems to be quite homogeneous in the training set, a variety of different hormonal agents were administered to patients in the validation set. This issue could have been addressed by adjustment in the statistical models or by using other publicly available datasets of systemically untreated N0 ER+ patients [START_REF] Haibe-Kains | A fuzzy gene expression-based computational approach improves breast cancer prognostication[END_REF].

Secondly, the validation set of Japanese patients used comprised of only 24 relapse events. Even if we assume that the validation set only was used in the multivariate analyses (as including the training set would provide an overoptimistic advantage to the new 95 -gene score), such a small number of events is really too small to draw definitive conclusions. More importantly, in order to show that the new 95-gene score could add prognostic information to established clinical or pathological factors (and GGI), further statistical methods are required.

It is not sufficient to just perform a multivariate regression analysis comparing the effects of the established prognostic factors and of the gene score under study, and to show that the new gene score is 'more significant' than the others factors in this model [START_REF] Kattan | Judging new markers by their ability to improve predictive accuracy[END_REF]. A new gene score is of interest only if it provides additional prognostic value, over and above that of easy-to measure clinico-pathological characteristics (nodal involvement, tumour size, age, histological grade and immunochemistry markers) or gene signatures. The gain in prognostic ability should therefore be quantified by comparing the predictive accuracy of two multivariate models -one with and one without the newly developed gene score-, using specific statistical measures such as the concordance index [START_REF] Dunkler | Gene expression profiling: Does it add predictive accuracy to clinical characteristics in cancer prognosis?[END_REF][START_REF] Haibe-Kains | A comparative study of survival models for breast cancer prognostication based on microarray data: Does a single gene beat them all?[END_REF].

From a clinical point of view, this signature will most likely not add important information to the existing gene scores. Previously, a pooled analysis of prognostic gene expression signatures using publicly available gene expression data from over 1000 breast cancer patients reported that proliferation and cell-cycle genes appear to be the common denominator underlying the prognostic abilities of the vast majority of the first generation prognostic signatures [START_REF] Wirapati | Metaanalysis of gene expression profiles in breast cancer: Toward a unified understanding of breast cancer subtyping and prognosis signatures[END_REF]. Notable also was the fact that they were only clinically relevant for the ER-positive/ HER2-negative breast cancers [START_REF] Wirapati | Metaanalysis of gene expression profiles in breast cancer: Toward a unified understanding of breast cancer subtyping and prognosis signatures[END_REF][START_REF] Desmedt | Biological processes associated with breast cancer clinical outcome depend on the molecular subtypes[END_REF]. Since Noai et al. report that a) the new 95-gene score is significantly correlated with both the histological grade and Ki67, b) the hazard ratios of histological grade, Ki67 and GGI dramatically change in the multivariate analysis as compared to the univariate one, and c) the 95-gene score is significantly enriched in cell cycle proliferation (22 genes), it is highly likely that their gene signature is tracking the same biological mechanism as the above mentioned prognostic gene scores.

It also should be highlighted that whilst proliferation-based prognostic signatures have considerable potential to refine the risk of recurrence for breast cancer patients, they have several important limitations. Even though these signatures display high performances in identifying early relapses (up to 5 years after the initial diagnosis), their performance decreases with increasing follow-up years [START_REF] Desmedt | Strong time dependence of the 76 -gene prognostic signature for node-negative breast cancer patients in the transbig multicenter independent validation series[END_REF]. Despite seeming to add prognostic value to established clinical prediction systems (such as AdjuvantOnline!), there is still a lot of variation in the occurrence of distant metastases that remains unexplained (such as those that occur in the HER2 overexpressing or triple negative breast cancer populations). It is therefore hoped that the "second generation" gene signatures that will be reported in the near future can provide better fine-tuning. Advancements in the understanding of signaling, tumor microenrivronment and host-tumor immune interplay are also likely to aid prognostic and therapy prescription for breast cancer patients [START_REF] Reis-Filho | Molecular profiling: Moving away from tumor philately[END_REF].

Additionally, it has now become a standard practice for microarray data to become publicly available after publication and to provide full details of the statistical methods used.

Whole genome gene expression data provides a valuable and powerful resource for breast cancer researchers worldwide and its global accessibility also acknowledges the altruism of the women that consent for their tumors to be used for the advancement of breast cancer research. Given that there are few datasets of non-Caucasian breast cancer samples available, we encourage these authors and others to publicly release their data.

Conflict of interest

Christos Sotiriou is a named inventor on the patent of the Gene Expression Grade Index (GGI).


Caroline Brünner-Kubath

Waheed Shabbir

Victoria Saferding

Renate Wagner

Christian F Singer

Peter Valent

Walter Berger

Brigitte Marian

Christoph C Zielinski

Michael Grusch

Dr Thomas W Grunt
email: thomas.grunt@meduniwien.ac.at

The PI3 Kinase/mTOR Blocker NVP-BEZ235 Overrides Resistance against Irreversible ErbB Inhibitors in Breast Cancer Cells

Keywords: Breast cancer, drug resistance, irreversible EGFR/ErbB/HER inhibitor, ovarian cancer, NVP-BEZ235, PI3K/AKT/mTOR

Resistance against first and second generation (irreversible) ErbB inhibitors is an unsolved problem in clinical oncology. The purpose of this study was to examine the effects of the irreversible ErbB inhibitors pelitinib and canertinib on growth of breast and ovarian cancer cells. Although in vitro growth-inhibitory effects of both drugs exceeded by far the effects of all reversible ErbB blockers tested (lapatinib, erlotinib, gefitinib), complete growth-inhibition was usually not reached. To define the mechanism of resistance, we examined downstream signaling pathways in drugexposed cells by Western blot analysis. Although ErbB phosphorylation was reduced by pelitinib and canertinib, activation of the AKT/mTOR pathway remained essentially unaltered in drug-resistant cells. Correspondingly, transfection of tumor cells with constitutively activated AKT was found to promote resistance against all ErbB inhibitors tested, whereas dominant negative AKT reinstalled s ensitivity in drugresistant cells. In a next step, we applied PI3K/AKT/mTOR blockers including the dual PI3K/mTOR kinase inhibitor NVP-BEZ235. These agents were found to cooperate with pelitinib and canertinib in producing in vitro growth inhibition in cancer cells resistant against ErbB-targeting drugs. In conclusion, our data show that ErbB drugrefractory activation of the PI3K/AKT/mTOR pathway plays a crucial role in resistance against classical and second-generation irreversible ErbB inhibitors, and NVP-BEZ235 can override this form of resistance against pelitinib and canertinib.

Introduction

ErbB receptors have been implicated as key triggers of oncogenesis in various solid tumors [START_REF] Yarden | Molecular approach to breast cancer treatment[END_REF]. Therefore, these receptors represent potential molecular targets for therapeutic intervention [START_REF] Citri | EGF-ERBB signalling: towards the systems level[END_REF]. Indeed, several ErbB-targeting antibodies and small molecule-type inhibitors of ErbB receptors are currently tested in clinical trials and some of these agents are already used in clinical practice [START_REF] Yarden | Molecular approach to breast cancer treatment[END_REF][START_REF] Citri | EGF-ERBB signalling: towards the systems level[END_REF]. One highlighting example is the ErbB2-targeting antibody trastuzumab that has improved treatment outcome in patients with breast cancer expressing ErbB2 [START_REF] Geyer | Lapatinib plus capecitabine for HER2-positive advanced breast cancer[END_REF]. Unfortunately, however, about 70% of these patients progress despite treatment, suggesting that resistance is an important issue. Some of the trastuzumab-resistant patients (20-30%) may still respond to the ErbB1/2 inhibitor lapatinib [START_REF] Geyer | Lapatinib plus capecitabine for HER2-positive advanced breast cancer[END_REF]. However, responses are usually shortlived and most patients relapse after a variable latency period. Therefore, research is focusing on the development of more potent ErbB receptor blockers. One novel type of targeted drugs are the irreversible ErbB receptor inhibitors. These agents may exert higher anticancer activity compared to first-generation (reversible) ErbB-targeting drugs [START_REF] Ocaña | Irreversible pan-ErbB tyrosine kinase inhibitors and breast cancer: current status and future directions[END_REF][START_REF] Rabindran | Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase[END_REF], and there was some hope that resistance against these inhibitors would occur less frequently [START_REF] Ocaña | Novel tyrosine kinase inhibitors in the treatment of cancer[END_REF]. Unfortunately, however, ErbB-expressing tumor cells may become resistant even against irreversible ErbB-targeting drugs. Although the exact mechanisms of resistance against these novel (irreversible) ErbB blockers remain unknown, it has been hypothesized that additional pro-oncogenic signaling pathways may play an important role [START_REF] Ocaña | Novel tyrosine kinase inhibitors in the treatment of cancer[END_REF]. Therefore, a better understanding of mechanisms underlying resistance against irreversible ErbB inhibitors is crucial for improvement in therapy of ErbB expressing neoplasms.

All ErbB receptors except ErbB3 activate various downstream signaling molecules including the phosphatidylinositol 3-kinase (PI3K) and its downstream mediators AKT and mTOR, and the mitogen-activated protein kinase (MAPK) cascade proteins C-RAF, MEK, and ERK [START_REF] Testa | AKT plays a central role in tumourigenesis[END_REF][START_REF] Maurer | 3-Phosphoinositide-dependent kinase 1 potentiates upstream lesions on the phosphatidylinositol 3-kinase pathway in breast carcinoma[END_REF]. Both, the MAPK-and the PI3K signaling pathway, have been involved in resistance of tumor cells against ErbB-targeting drugs [START_REF] Moasser | The tyrosine kinase inhibitor ZD1839 ("Iressa") inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells[END_REF][START_REF] Moulder | A phase I study of EKB-569 in combination with capecitabine in patients with advanced colorectal cancer[END_REF][11].

However, the relative contribution of these two pathways to the development of resistance is still a matter of debate.

We examined the effects of two potent irreversible ErbB inhibitors, pelitinib (EKB-569) and canertinib (CI-1033), on MAPK and PI3K/AKT downstream cascades in ErbB drug-sensitive and drug-resistant breast and ovarian cancer cells. Pelitinib blocks ErbB1 by covalent interaction with the ATP binding pocket (IC 50 8 -83nM) and inhibits ErbB2 to lesser extent (IC 50 378 -1255 nM) [START_REF] Ocaña | Irreversible pan-ErbB tyrosine kinase inhibitors and breast cancer: current status and future directions[END_REF][12][13][14]. Canertinib inactivates ErbB1, -2 and -4 at IC 50 -concentrations of 0.8 -30nM (ErbB1), 19 -127nM (ErbB2), and 7 -388nM (ErbB4), respectively [START_REF] Ocaña | Irreversible pan-ErbB tyrosine kinase inhibitors and breast cancer: current status and future directions[END_REF]15,[16][17][18].

We here show that wiping out AKT phosphorylation is essential and sufficient, whereas abrogation of ERK phosphorylation is not required for the anticancer effects of these ErbB-targeting drugs. Thus, pAKT may be a biomarker for resistance against irreversible ErbB inhibitors in breast and ovarian cancer cells. Most importantly, resistance against ErbB blockers can be overcome by co-application of PI3K/AKT/mTOR inhibitors such as NVP-BEZ235. These data suggest that further studies exploring the effects of targeting of both ErbB receptors and PI3K/AKT/mTOR in breast and ovarian cancer are warranted. All kinase blockers were dissolved in DMSO and diluted 1:1,000 or 1:2,000 in medium before use. Fetal calf serum (FCS), DMEM, α-MEM, and RPMI 1640 were purchased from Gibco (Karlsruhe, Germany).

Materials and methods

Reagents

Cell lines

Ten mammary cancer cell lines (BT-20, BT-474, MCF-7, MDA-MB-231, MDA-MB-361, MDA-MB-453, SK-BR-3, SW527, T47D, ZR-75-30) and 11 ovarian cancer cell lines (A2774, A2780, A2780ADR, CAOV3, OVCAR-3, PA-1, SKOV3, H134, HEY, HOC-7, TR-170), as well as CEM leukemia cells were used in this study. A specification of cell lines is shown in Supplementary Table S1. Cells were maintained in DMEM, α-MEM, or RPMI 1640 medium (Supplementary Table S1) with 10% FCS, antibiotics, and 2 mM glutamine (Gibco). Cells were cultured for up to 20 passages. They were tested for absence of Mycoplasma (Venor GeM, Minerva Biolabs, Berlin, Germany). Cell morphology and expression of marker antigens (ErbBs, estrogen receptor, retinoic acid receptors, fatty acid synthase) were checked to exclude cross -contamination of cell lines (Supplementary Table S1).

Cell growth assays

For growth stimulation, cells were plated at 1.5x10³/well (SK-BR-3) or 3x10³/well (T47D) in 96-well plates, were allowed to attach, and were then exposed to 100 ng/ml EGF for up to 6 days in culture medium containing 5% FCS. Cell numbers were examined using a formazan dye assay (Biomedica, Vienna, Austria). For assessing growth inhibition, cells were plated at 1.5x10³/well in 96-well culture plates. After adhesion, kinase inhibitors were added at various concentrations in 5% FCS in the absence of EGF. After 72 hours, cell numbers were determined. In a separate set of experiments, cell growth and inhibitory drug effects were confirmed by determining cellular DNA content by CyQUANT assay (Invitrogen, Paisley, UK). The cell numbers, given in percent of untreated control, were plotted against the logarithm of the concentration of each drug. Sigmoidal curves were calculated with the Boltzmann equation using GraphPad Prism 4.03 software and the drug concentrations causing 50% reduction of the cell numbers (IC 50 ) were obtained from these curves. The type of interaction (synergistic, additive or antagonistic) between ErbB drugs combined with PI3K/AKT/mTOR antagonists in inhibition of cell growth was determined at the IC 50levels using the geometric isobologram method. To this end, the IC 50 -values of the single drugs were plotted on the x or y axis, respectively, and a line connecting these two points was drawn. Synergism between both drugs is encountered if the isoeffective point of the drug combination falls below that line, whereas antagonism occurs if the point lies above it [START_REF] Grunt | Effects of retinoic acid and fenretinide on the c-erbB-2 expression, growth and cisplatin sensitivity of breast cancer cells[END_REF].

Western blotting

Cells plated in media containing 5% FCS at 5x10 5 in 60 mm dishes were allowed to adhere and were either immediately exposed to various concentrations of kinase inhibitors for various time periods, or were depleted from FCS for 24 hours before adding drugs. In select experiments, drug-treated cells were challenged with 100 ng/ml EGF alone or together with 1 nM HRG-β1 for up to 20 minutes before lysis.

Ligand-dependent phosphorylation of the ErbB system and of downstream effectors was examined in untreated, serum-depleted cells that had been challenged with EGF and/or HRG-β1 as described above. Cells were then lysed, and protein fractions (30 µg/lane) subjected to SDS-PAGE, blotted onto PVDF membranes, and immunostained as described [START_REF] Grunt | An EGF receptor inhibitor induces RAR-beta expression in breast and ovarian cancer cells[END_REF] using antibodies against total or phosphorylated (p) ErbB1, ErbB2, ErbB3, ErbB4, MEK1/2, ERK1/2, PTEN, AKT, GSK3ß, mTOR, S6, / tubulin, and actin. Membranes were then incubated with peroxidase-tagged secondary antibodies.

Antibody-binding was made visible by enhanced chemiluminescence. Specifications of antibodies are shown in Supplementary Table S2.

DNA constructs and transfections

Hyperactive (HA) MEK1 lacking nuclear export signal domain (amino acids [START_REF] Jones | Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells[END_REF][START_REF] Serra | NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations[END_REF][START_REF] Sarbassov | Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB[END_REF][START_REF] Huang | A complex interplay between Akt, TSC2 and the two mTOR complexes[END_REF][START_REF] Nelson | Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition[END_REF][START_REF] Crespo | Redesigning kinase inhibitors to enhance specificity[END_REF][START_REF] Slichenmyer | CI-1033, a pan-erbB tyrosine kinase inhibitor[END_REF][START_REF] Kumar | Structure and clinical relevance of the epidermal growth factor receptor in human cancer[END_REF][START_REF] Grunt | Interaction between fatty acid synthase-and ErbB-systems in ovarian cancer cells[END_REF][START_REF] Hollestelle | Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines[END_REF][START_REF] García | Promoter methylation of the PTEN gene is a common molecular change in breast cancer[END_REF][START_REF] Wang | NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN[END_REF][START_REF] She | Resistance to gefitinib in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3'kinase/Akt pathway signaling[END_REF][START_REF] Sergina | Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3[END_REF][46][47][48][49][50][51] and harboring activating amino acid substitutions at two phosphorylation sites (S218E, S222D) was kindly provided by H. Kiyama (Osaka City Univ., Japan) [START_REF] Namikawa | Akt/protein kinase B prevents injuryinduced motoneuron death and accelerates axonal regeneration[END_REF] and murine HA AKT1 carrying the myristoylated signal of Lck, which targets it to the membrane was kindly provided by D. Efremov (International Center for Genetic Engineering and Biotechnology, Rome, Italy) [START_REF] Longo | The Akt signaling pathway determines the different proliferative capacity of chronic lymphocytic leukemia Bcells from patients with progressive and stable disease[END_REF]. Dominant negative, kinase-inactive AKT that harbors a K179M mutation was obtained from M. Sibilia (Medical Univ. Vienna, Austria) [START_REF] Bellacosa | Akt activation by growth factors is a multiple-step process: the role of the PH domain[END_REF].

Vectors with or without green fluorescent protein (GFP) were used as negative control plasmids. For transient transfection, 8x10 5 SK-BR-3, BT-474 or T47D cells were plated in 6-well plates and allowed to attach. Then, plasmid DNA diluted in Opti-MEM I (Invitrogen) containing Lipofectamine LTX (Invitrogen) was incubated for 30 minutes at room temperature to allow formation of Opti-MEM-DNA-LTX transfection complexes, added to cells and left in cultures for 24 hours. Cells were then allowed to grow for another 24 hours in the absence of exogenous plasmid DNA. These transfected, polyclonal cell populations were trypsinized, and subjected to cell growth assays or to Western blot analysis as described above without further clonal selection or enrichment.

Statistical analyses

The levels of significance in differences between IC 50 -values produced by pelitinib in individual SK-BR-3 transfectants were determined by one-factorial analysis of variance (ANOVA) followed by Scheffe test. In all other experiments, the Student's t-test for unpaired samples was applied to examine the significance in differences of growth in drug-treated cells. p-values 0.05 were considered significant.

Results

Irreversible ErbB blockers are stronger inhibitors of growth of breast and ovarian cancer cells compared to reversible ErbB-targeting drugs

The growth-inhibitory effects of irreversible and reversible ErbB-targeting drugs were examined and compared in various breast and ovarian cancer cell lines. Results obtained by formazan dye assay were found to match well with the CyQUANT assay in all cases tested. As shown in Table 1, the first generation ErbB1 inhibitors gefitinib and erlotinib, and the ErbB1/2 inhibitor lapatinib were marginally or not at all effective in inducing growth arrest (IC 50 -values: >5 µM). However, the irreversible ErbB inhibitors pelitinib and canertinib were found to block growth of most breast and ovarian cancer cells. The IC 50 -values produced by canertinib, a drug targeting all three ErbB kinases (ErbB1, -2, and -4) [START_REF] Ocaña | Irreversible pan-ErbB tyrosine kinase inhibitors and breast cancer: current status and future directions[END_REF]15,[16][17][18], were comparable to those produced by pelitinib, which targets ErbB1 and -2 [START_REF] Ocaña | Irreversible pan-ErbB tyrosine kinase inhibitors and breast cancer: current status and future directions[END_REF][12][13][14]. Despite their more potent effects, IC 50 -values produced with pelitinib and canertinib varied from cell line to cell line (IC 50 :

1.20-15.40 µM). Whereas the breast cancer cell lines MDA-MB-453, SK-BR-3, ZR-75-30 and MDA-MB-361, and the ovarian carcinoma lines H134, A2780 and HOC-7 were inhibited at concentrations (IC 50 : 1.2-4.4 µM) that are within a pharmacological range [START_REF] Burris Ha 3rd | Phase I safety, pharmacokinetics, and clinical activity study of lapatinib (GW572016), a reversible dual inhibitor of epidermal growth factor receptor tyrosine kinases, in heavily pretreated patients with metastatic carcinomas[END_REF][START_REF] Wang | Targeting endoplasmic reticulum stress and Akt with OSU-03012 and gefitinib or erlotinib to overcome resistance to epidermal growth factor receptor inhibitors[END_REF], the breast carcinoma cell lines BT20, MCF-7, and T47D, and the ovarian carcinoma line CAOV3 showed no response at reasonable drug concentrations (IC 50 : >5µM) (Table 1, Supplementary Fig. S1). None of the examined cell lines were found to harbor mutations in the EGFR/ErbB1 gene (W. Shabbir and T.W. Grunt unpublished observation). In control experiments, proliferation of ErbB-negative CEM leukemia cells [START_REF] Grunt | Upregulation of retinoic acid receptorbeta by the epidermal growth factor-receptor inhibitor PD153035 is not mediated by blockade of ErbB pathways[END_REF] was not affected by pelitinib or canertinib (Supplementary Fig. S1) suggesting that drug-dependent growth inhibition was indeed mediated by blockade of the ErbB system and not by interference of the drugs with other kinases. The sensitivity of breast cancer cells against the growth inhibitory action of irreversible ErbB drugs correlated with ErbB2 (pelitinib p<0.05, canertinib p<0.01), but not with ErbB1, -3, or -4 protein levels (Table 2). Unfortunately, in the clinical setting, only about 30% of ErbB2 overexpressing breast cancers actually do respond to ErbBtargeted therapy [START_REF] Vogel | Efficacy of trastuzumab[END_REF][START_REF] Neve | A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes[END_REF]. This indicates that ErbB2 overexpression is not the only molecular determinant for sensitivity against ErbB-specific drugs.

Irreversible ErbB blockers inhibit ErbB receptor phosphorylation in drugsensitive SK-BR-3 and in drug-resistant T47D cells

The efficacy of pelitinib and canertinib in abolishing ErbB receptor phosphorylation was examined in SK-BR-3 and T47D cells. Serum-depleted, quiescent cell cultures were exposed to the drugs and then challenged with EGF, which binds ErbB1 and primarily activates ErbB1 and -2, and with heregulin-1 (HRG-1) that binds ErbB3 and -4 and activates ErbB2, -3, and -4, and to lesser extent ErbB1 [START_REF] Tzahar | A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor[END_REF][START_REF] Troyer | Regulation of mouse mammary gland development and tumorigenesis by the ERBB signaling network[END_REF]. As shown in Fig. 1a, the drugs completely abrogated both ligand-activated and constitutive phosphorylation of ErbB1 and ErbB3 as well as ErbB4 in the two cell lines, and they blocked ligand-activated phosphorylation of ErbB2 in T47Dcells. In contrast, neither pelitinib nor canertinib could abolish constitutive phosphorylation of ErbB2 in SK-BR-3 and T47D cells. Instead, the drugs were found to downregulate the steady-state level of ErbB2 in a dose-dependent manner in both cell lines. Next, we examined ErbB family receptors and their downstream mediators in ErbB drug-sensitive SK-BR-3 cells and in drug-resistant T47D cells. Notably, despite marked differences in ErbB receptor expression, SK-BR-3 (much ErbB1 and -2, less ErbB3, no ErbB4) and T47D (little ErbB1 and -2, much ErbB3 and -4) exhibited similar levels of spontaneous AKT and ERK1/2 expression/phosphorylation, and expressed equal amounts of PI3K antagonist PTEN in the presence of serum (Fig. 1b). In serum-free conditions, however, cells were unable to maintain high levels of pAKT and pERK1/2 despite appreciable baseline phosphorylation of ErbB2 and -3, and required EGF to fully activate ErbB1, -2, and -3 (but not ErbB4) phosphorylation and to induce AKT and ERK1/2 phosphorylation (Fig. 1c). This suggests that a certain threshold level of composite phosphorylation of ErbB1, -2, and -3 is required for full phosphorylation of AKT and ERK1/2. Surprisingly, EGF-induced stimulation of serum-supported cell growth, which is characterized by appreciable levels of AKT and ERK1/2 phosphorylation (Fig. 1b), was stronger in T47D than in SK-BR-3 (Fig. 1d). Thus, although T47D cells are resistant to the growth-inhibitory action of ErbB drugs, they are responsive to EGF stimulation. In both cell lines, the ErbB system is functional and responsive to ligandand inhibitor-mediated modulation, irrespective of the relative sensitivity/resistance of the cells against the growth inhibitory action of the irreversible ErbB-targeting drugs.

Resistance of breast and ovarian cancer cells against irreversible ErbBtargeting drugs correlates with drug-refractory PI3K/AKT signaling

Pelitinib and canertinib rapidly and completely erased phosphorylation of AKT in sensitive (MDA-MB-453, SK-BR-3 and BT-474), but not in resistant breast cancer cells (MDA-MB-231, MCF-7 and T47D). In fact, drug-refractory cells up-rather than downregulated pAKT levels upon drug exposure and maintained these levels during the whole treatment period (Fig. 2a-c). We asked whether the residual AKT activity can be caused by autocrine insulin-like growth factor-I (IGF-I) activity, which might cause stimulation of PI3K/AKT and thus may cause resistance against ErbB-targeting drugs [START_REF] Lu | Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin)[END_REF][START_REF] Jones | Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells[END_REF]. However, as visible in Supplementary Fig. S2, the IGF-IR inhibitor picropodophyllin (PPP) was unable to impair pelitinib-resistant phosphorylation of AKT in T47D cells, although it moderately reduced the growth of these cells. Thus, IGF-IR is not contributing to constitutive pAKT levels in these cells. PI3K/AKT signaling also appears to determine ErbB drug resistance in ovarian carcinoma cells. In fact, Figure 2d demonstrates that the ability of pelitinib to block pAKT was more pronounced in sensitive HOC-7 than in resistant CAOV-3 cells (see also Table 1 and Supplementary Fig. S1E,F). By contrast, drug-sensitivity of breast and ovarian cancer cells did not correlate with drug-mediated inhibition of p-ERK1,2 levels (Fig. 2, Table 2). These data suggest that silencing phosphorylation of AKT, but not of ERK, may be crucial for the growth inhibitory effects of ErbB-blocking drugs in breast and ovarian cancer cells, and that AKT-related signaling cascades may contribute to resistance against these drugs.

Constitutively active AKT1 induces resistance, whereas dominant negative AKT promotes sensitivity of breast cancer cells against pelitinib

To assess the potential role of AKT as a resistance-related signaling molecule in cancer cells, we employed a constitutively active variant of AKT1. Drug-sensitive SK-BR-3 were transfected with this constitutively active AKT1 gene [START_REF] Longo | The Akt signaling pathway determines the different proliferative capacity of chronic lymphocytic leukemia Bcells from patients with progressive and stable disease[END_REF] or with MEK1 [START_REF] Namikawa | Akt/protein kinase B prevents injuryinduced motoneuron death and accelerates axonal regeneration[END_REF]. Overexpression/hyperphosphorylation of transgenes and downstream effector molecules (GSK3 , mTOR, S6, ERK1,2) was confirmed by Western blot analysis of the unselected polyclonal transfectant cell populations. Unlike wildtype, control-vector-, and MEK1-transfected cells, AKT1-transfectants exhibited an excess of pAKT that was refractory to the inhibitory activity of pelitinib. Accordingly, phosphorylation of AKT-downstream targets (GSK3 , mTOR, S6) was not or only slightly affected by pelitinib in these cells. In turn, forced expression of hyperactive MEK1 was found to phosphorylate ERK1/2 and to make pERK1/2 expression unresponsive to pelitinib (Fig. 3a). These transfectants were used to directly examine the relative contribution of MAPK and PI3K signaling to drug resistance against ErbB-targeting drugs. As shown in Fig. 3b and3c, pelitinib was found to inhibit growth of wildtype, sham-, vector-, GFP-, and MEK1-transfected SK-BR-3 cells with similar potency, but did not inhibit the growth of AKT1-transfected cells. Induction of pelitinib-resistance by transient transfection of constitutively active AKT1 was also observed in unselected, polyclonal BT-474 cells (Fig. 3d,e). In contrast, introduction of the dominant negative, kinaseinactive K179M mutant of AKT into T47D cells reduces phosphorylation of downstream S6 ribosomal protein and induces sensitivity against pelitinib in these cells (Fig. 3f-h). Collectively, our data demonstrate 1) that forced expression of dominant active MEK1 and AKT1 effectively stimulates MAPK and PI3K pathways in SK-BR-3 cells, respectively, and renders these pathways resistant to pelitinib, 2) that PI3K/AKT-, but not MAPK-hyperactivity, confers growth-resistance of the cells against pelitinib, and 3) that blockade of AKT downstream signaling can reinstall pelitinibsensitivity in drug-resistant cells.

NVP-BEZ235 and other PI3K/AKT/mTOR antagonists abrogate PI3K signaling in ErbB drug-resistant breast cancer cells

This prompted us to examine drugs that silence the PI3K signaling cascade at distinct levels in SK-BR-3 and T47D cells. NVP-BEZ235 inhibits all four PI3K isoforms and both mTOR complexes (mTORC1, mTORC2) [START_REF] Serra | NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations[END_REF], rapamycin blocks mTORC1 and mTORC2 [START_REF] Sarbassov | Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB[END_REF], whereas Akti-1/2 inhibits AKT1 and AKT2. These agents decreased phosphorylation of AKT and of S6 in a dose-dependent manner, but left pERK1/2 levels unaffected (Supplementary Fig. S3). Interestingly, low concentrations of NVP-BEZ235 inhibited pAKT(Ser473), but not pAKT(Thr308), which was even up-regulated in T47D cells. This may be explained by higher affinity of NVP-BEZ235 to mTOR than to PI3K [START_REF] Serra | NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations[END_REF]. NVP-BEZ235-mediated blockade of mTORC1 abolishes mTORdependent negative feedback-regulation of PI3K and PDK1 and thus stimulates phosphorylation of AKT at Thr308. In contrast, concurrent NVP-BEZ235-mediated blockade of mTORC2 leads to reduced pAKT(Ser473) levels [START_REF] Huang | A complex interplay between Akt, TSC2 and the two mTOR complexes[END_REF]. Accordingly, dephosphorylation of Thr308 was observed only at higher doses of NVP-BEZ235, which inhibit both mTOR and PI3K. In addition, we examined the MEK1/2 antagonists AZD6244 and U0126 and found that both inhibitors efficiently silenced the MAPK cascade in SK-BR-3 and T47D cells (Supplementary Fig. S4).

NVP-BEZ235 and other PI3K/AKT/mTOR antagonists selectively block growth in ErbB drug-refractory breast cancer cells and override resistance against ErbB blockers

ErbB drug-resistant T47D were more responsive to growth arrest by the PI3K pathway blockers NVP-BEZ235, rapamycin and Akti-1/2 than ErbB drug-sensitive SK-BR-3 cells (Fig. 4a-c). Moreover, cotreatment of T47D cells with PI3K-and ErbB drugs significantly reduced the IC 50 -values for the ErbB drugs from 13-15µM in the absence, to a pharmacologically useful dose range of <4.6 µM in the presence of low amounts (0.01 -0.5µM) of PI3K antagonists. The responsiveness of ErbB drug-sensitive SK-BR-3 cells was also significantly enhanced by some of the PI3K-silencing drugs (Fig. 4a,b,d). Notably, isobologram analysis revealed strong synergisms for the combinations of NVP-BEZ235 or rapamycin with ErbB drugs, and weak synergism for Akti-1/2 plus ErbB blockers (Fig. 4e). Moreover, growth of MDA-MB-231 breast cancer cells, which are resistant to ErbB drugs (Table 1, Supplementary Fig. S1) and express wildtype PIK3CA (Table 2), was also synergistically blocked by cotreatment with pelitinib or canertinib and NVP-BEZ235 (Fig. 4f). On the other hand, the MEK1/2 inhibitors AZD6244 and U0126 did not inhibit the growth of T47D or SK-BR-3 cells (IC 50 -values far beyond 60 µM), nor did they improve the responsiveness of these cells against pelitinib and canertinib (Fig. 4a-d); they rather induced slight antagonism (Fig. 4e). Thus, unlike MEK1/2 inhibitors, PI3K/AKT/mTOR antagonists cooperate with ErbB drugs and render breast cancer cells sensitive to pelitinib and canertinib. This demonstrates that PI3K/AKT/mTOR antagonists, but not MEK1/2 inhibitors, can overcome resistance against novel irreversible ErbB blockers.

Discussion

Novel small molecular drugs that irreversibly inhibit ErbB family receptors reveal higher efficacy than first-generation reversible ErbB inhibitors (Table 1) [START_REF] Ocaña | Irreversible pan-ErbB tyrosine kinase inhibitors and breast cancer: current status and future directions[END_REF][START_REF] Rabindran | Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase[END_REF], and there was some hope that ErbB-positive tumor cells would less frequently exhibit resistance against these drugs [START_REF] Ocaña | Novel tyrosine kinase inhibitors in the treatment of cancer[END_REF]. Unfortunately, however, we demonstrate that several breast and ovarian cancer cells are insensitive to the growth inhibitory effects of irreversible ErbB blockers pelitinib and canertinib, which target ErbB1/2 or ErbB1/2/4, respectively [START_REF] Ocaña | Irreversible pan-ErbB tyrosine kinase inhibitors and breast cancer: current status and future directions[END_REF][12][13][14][15][16][START_REF] Nelson | Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition[END_REF]. Very high concentrations of pelitinib may block several other non-ErbB kinases as well, whereas canertinib appears to retain specificity for the ErbB kinases, even at high concentrations [START_REF] Crespo | Redesigning kinase inhibitors to enhance specificity[END_REF][START_REF] Slichenmyer | CI-1033, a pan-erbB tyrosine kinase inhibitor[END_REF]. Interestingly, the growth of ErbB-negative CEM leukemia cells was completely unresponsive to ErbB inhibitors, even at very high doses (Supplementary Fig. S1), suggesting that the biological drug effects seen in the tumor cells were not caused by non-ErbB related inhibitory activities of the drugs. We thought that elucidating the molecular mechanisms of unresponsiveness of ErbB-positive tumors against such irreversible ErbB inhibitors may be helpful for the development of strategies to overcome resistance. In line with previous studies [START_REF] Moasser | The tyrosine kinase inhibitor ZD1839 ("Iressa") inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells[END_REF][START_REF] Moulder | A phase I study of EKB-569 in combination with capecitabine in patients with advanced colorectal cancer[END_REF], the sensitivity of breast cancer cells to ErbB-targeting drugs significantly correlated with ErbB2 expression levels (pelitinib p<0.05, canertinib p<0.01), but did not correlate with expression of ErbB1, -3, or -4 (Table 2). Moreover, the lack of activity of the ErbB1 blockers erlotinib and gefitinib argues against ErbB1 inhibition as a critical determinant of ErbB drug sensitivity in breast and ovarian cancer cells. Pelitinib and canertinib inhibit both ligand-stimulated and constitutive phosphorylation of ErbB1, -3, and -4. However, the drugs cannot abrogate constitutive phosphorylation of ErbB2. This suggests that pelitinib and canertinib bind to the ErbB2 kinase only, if the domain is in the inactive, but not if it is in the active conformation. Interestingly, there is a precedent to this. Kumar et al. [START_REF] Kumar | Structure and clinical relevance of the epidermal growth factor receptor in human cancer[END_REF] recently reported that lapatinib binds to the inactive, but not to the active, conformation of ErbB1. Nevertheless, pelitinib and canertinib attenuated the steady-state level of the ErbB2 protein in both cell lines. Interestingly, a similar pelitinib-and canertinibdependent downregulation of ErbB2 has recently been observed in ovarian cancer [START_REF] Grunt | Interaction between fatty acid synthase-and ErbB-systems in ovarian cancer cells[END_REF]. This lets us speculate that complexing between inhibitor and target may result in a conformational change that would trigger target degradation. This entails reduced overall levels of pErbB2 leading to deficient transphosphorylation/activation of ErbB3 (Fig. 1a). ErbB3, when dimerized with other ErbB members (mainly ErbB2), is known to predominantly stimulate the PI3K/Akt growth and survival pathway [START_REF] Sergina | Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3[END_REF]. Overall, our data are consistent with the notion that interference with the ErbB3-PI3K axis is essential for ErbB drugs to affect cell growth. The ErbB system was functional and responsive to EGF in both ErbB blocker-sensitive SK-BR-3 cells and -resistant T47D cells. Pelitinib and canertinib blocked ErbB phosphorylation in responsive and in drugresistant cancer cell lines (Fig. 1a). Thus, differential ErbB inhibitory efficacy or differential cellular drug uptake did not account for the variable sensitivity of various cancer cells to ErbB-targeting drugs.

Recent data suggest that mutations in PTEN may contribute to resistance of tumor cells against various anti-cancer agents [START_REF] Hollestelle | Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines[END_REF][START_REF] García | Promoter methylation of the PTEN gene is a common molecular change in breast cancer[END_REF][START_REF] Wang | NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN[END_REF]. In the current study, mutation in PTEN, rarely seen in breast cancer patients [START_REF] Hollestelle | Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines[END_REF], was found in one of nine cell lines studied. This line (MDA-MB-453) revealed an E307K exchange in PTEN and was most sensitive to the ErbB-targeting drugs. This suggests that PTEN mutations not always confer resistance against ErbB inhibitors. Moreover, we found that drugsensitive SK-BR-3 cells and drug-resistant T47D cells express similar levels of wildtype PTEN protein (Fig. 1b). Therefore, loss of PTEN e.g. due to promoter hypermethylation [START_REF] García | Promoter methylation of the PTEN gene is a common molecular change in breast cancer[END_REF] or proteasomal degradation [START_REF] Wang | NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN[END_REF] apparently did not account for differential drug sensitivity. Another resistance-related gene is PIK3CA. However, in this study, PIK3CA mutations [START_REF] Hollestelle | Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines[END_REF] were found in both drug-sensitive and drug-resistant breast cancer cells (Table 2) suggesting that PIK3CA mutations are not the sole determinant for development of resistance against ErbB-targeting drugs.

The relative contribution of the MAPK and the PI3K pathways to resistance against ErbB-targeting drugs in breast and ovarian cancer cells is still a matter of debate.

Several reports suggested that constitutive PI3K signaling confers ErbB drug resistance [START_REF] Moasser | The tyrosine kinase inhibitor ZD1839 ("Iressa") inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells[END_REF][START_REF] Jones | Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells[END_REF][START_REF] She | Resistance to gefitinib in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3'kinase/Akt pathway signaling[END_REF][START_REF] Sergina | Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3[END_REF]. Other studies have identified MAPK signaling as a potential driving force that contributes to resistance [46][47][48][49]. Depending on the tumor cell type other mechanisms may also play a role in drug resistance [50,51]. The issue gets further complicated by the occurrence of multiple crosstalk regulations between MAPK and PI3K pathways [52,53]. We observed that resistance of breast and ovarian cancer cells against ErbB blocking agents correlates with drug-refractory phosphorylation of AKT, but not with drug effects on ERK1,2. In some resistant cells, pelitinib and canertinib even caused a dose-dependent increase in expression of pAKT (Fig. 2). This may be explained by drug-mediated silencing of MAPK, which abolishes negative MAPK-PI3K feedback loops [52,53]. Therefore, our data suggest that silencing of AKT signaling is crucial for ErbB drugs to exert anticancer activity in malignant breast and ovarian disease.

Persistent AKT signaling can generally be caused by several mechanisms including activation by other ligand/receptor systems. However, the IGF-I/IGF-IR cascade, which has been linked to resistance against ErbB blockers [START_REF] Lu | Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin)[END_REF][START_REF] Jones | Insulin-like growth factor-I receptor signalling and acquired resistance to gefitinib (ZD1839; Iressa) in human breast and prostate cancer cells[END_REF], did not account for unresponsiveness of T47D against irreversible ErbB drugs, since the IGF-IR inhibitor PPP, though moderately impairing cell proliferation, did not diminish pAKT levels in these cells. Notably, T47D cells harbor the H1047R activating mutation in the PIK3CA catalytic domain of PI3K, which could contribute to constitutive AKT activity. However, our data show that cell lines with mutant (e.g. MDA-MB-453) and wildtype PIK3CA (e.g. SK-BR-3) not only can reveal similar constitutive and drug-modulated pAKT levels, but may also be equally sensitive to pelitinib and canertinib (compare Fig. 1b,c and 2a with Table 2). On the other hand, MDA-MB-231 contain wildtype, whereas T47D express mutant PIK3CA; yet both cell lines are resistant to pelitinib and canertinib. Moreover, the PI3K/mTOR inhibitor NVP-BEZ235 efficiently blocks growth of both cell lines on its own (T47D: IC 50 0.058µM, MDA-MB-231: IC 50 0.067µM) and synergizes with the ErbB targeting drugs pelitinib and canertinib (Fig. 4f), irrespective of the mutation status of PIK3CA. Thus, the activation status of AKT appears to be independent of the presence of PIK3CA mutations in the cell lines studied. In fact, it is quite questionable whether activating PIK3CA mutations stimulate AKT downstream signaling and exert oncogenic activity. It has also been shown that PIK3CA mutations exert relatively mild effects on tumor cell growth [54]. Moreover, recent reports [START_REF] Neve | A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes[END_REF]55,56] failed to demonstrate a correlation between the presence of PIK3CA mutations and expression of pAKT, nor did they provide evidence that such mutations shorten survival or elevate chemoresistance in breast tumor patients [55]. Consistently, recent evidence indicates that when PDK1, which is overexpressed in a large proportion of breast cancers [START_REF] Maurer | 3-Phosphoinositide-dependent kinase 1 potentiates upstream lesions on the phosphatidylinositol 3-kinase pathway in breast carcinoma[END_REF], is activated by mutant PIK3CA, it phosphorylates SGK3 rather than AKT [56]. Therefore, PIK3CA mutations may at best contribute to drug-refractory AKT signaling and growth resistance, but are definitely not the sole causes for it. This is further supported by the fact that the PI3K/mTOR inhibitor NVP-BEZ235 sensitizes resistant breast cancer cells against ErbB drugs irrespective of their PIK3CA mutation status.

In order to directly examine the relative contribution of MAPK and PI3K pathways in ErbB drug resistance we overexpressed constitutively active MEK1 or constitutively active AKT1 in ErbB inhibitor-sensitive SK-BR-3 and BT-474 cells. AKT1 rendered the cells fully resistant to pelitinib, whereas MEK1 had no effect (Fig. 3b-e). Conversely, dominant negative AKT is able to reinstall drug-sensitivity in pelitinib-resistant T47D cells (Fig. 3f-h). Importantly, these experiments directly demonstrate 1) that pAKT down-regulation is required and is sufficient for growth arrest induced by ErbB inhibitors, 2) that ErbB drug sensitivity is independent from MAPK blockade, and 3) that resistance occurs independent of the molecular mechanisms of AKT hyperactivity. Thus, drug-dependent downregulation of pAKT may serve as a biomarker for breast and ovarian cancer cell sensitivity against irreversible ErbB inhibitors. Notably, we have found that ErbB drug-resistant cells are particularly sensitive to the PI3K/AKT/mTOR inhibitors NVP-BEZ235, rapamycin and Akti-1/2. In contrast, the MEK1/2 antagonists AZD6244 or U0126 did not affect growth of these cells (Fig. 4a,c).

Most importantly, NVP-BEZ235, rapamycin and Akti-1/2, when used in combination with ErbB blockers, significantly increased the antiproliferative efficacy and reduced the IC 50 -values of pelitinib and canertinib in T47D cells into a pharmacologically reasonable range (Fig. 4a,d). This effect was obviously dependent on mTOR inhibition, since rapamycin, which targets mTOR, was as efficient as the other compounds, which either block upstream PI3K along with mTOR (NVP-BEZ235) or inhibit AKT, which is also upstream of mTOR (Akti-1,2). In summary, our data show that 1) abrogation of PI3K/AKT/mTOR reinstalls ErbB drug sensitivity and 2) that ErbB/PI3K/mTOR co-targeting using irreversible ErbB inhibitors along with NVP-BEZ235 or mTOR blockers represents a promising strategy to overcome resistance against irreversible ErbB inhibitors. Thus, from our data it may be tempting to speculate that NVP-BEZ235, which is currently in phase I/II clinical trials for advanced breast cancer, may have clinical utility when combined with novel ErbBtargeting drugs.

  Recombinant epidermal growth factor (EGF) and heregulin-β1 (HRG-β1) were purchased from Sigma (St. Louis, MO) or Thermo Fisher Scientific (Fremont, CA). The irreversible ErbB inhibitor pelitinib (EKB-569) was a kind gift from Wyeth (Cambridge, MA) and the irreversible ErbB inhibitor canertinib (CI-1033, PD-183805) was kindly provided by Pfizer (Groton, CT). The reversible ErbB inhibitors gefitinib, erlotinib, and lapatinib, the PI3K/mTOR inhibitor NVP-BEZ235, and MEK1/2 inhibitor AZD6244 were purchased from ChemieTek (Indianapolis, IN), the IGF-IR inhibitor picropodophyllin, AKT1/2 inhibitor Akti-1/2, and mTOR inhibitor rapamycin from Calbiochem (San Diego, CA), and the MEK1/2 inhibitor U0126 from Cell Signaling Technology (Boston, MA).

(

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Fig. 1 3 Fig. 2 Fig. 3 Fig. 4

 13234 Fig. 1 Ligand-and drug-dependent modulation of ErbB-and downstream effector

Table 1

 1 The antiproliferative efficacy (IC 50 ) of novel, irreversible ErbB inhibitors compared to first-generation, reversible ErbB blockers

	IC 50 (µM)

Table 2

 2 Overview on ErbB expression, downstream pathways and responsiveness of breast cancer cells against the irreversible ErbB1/2 inhibitor pelitinib and the irreversible pan-ErbB

	inhibitor canertinib

b own data, obtained from densitometry of Western blot autoradiographs and expressed as optical density relative to BT474; c not determined; d not significant; e Ref. 41; f wildtype; g this report

by irreversible ErbB Inhibitors (IC 50 , µM) g

	Pelitinib	2,95	3,65	4,26	4,38	4,45	5,43	6,43	8,16	15,40
	Canertinib	2,38	2,45	3,54	4,34	5,70	6,85	8,88	7,86	12,64
	Downstream Effect of irreversible ErbB Inhibitors	g
	pAKT	decreased decreased	?	?	decreased increased	?	increased increased
	pERK	not affected decreased	?	?	decreased not affected	?	decreased decreased

Acknowledgements

Pelitinib (EKB-569) and canertinib (CI-1033) were kindly provided by Wyeth and Pfizer, respectively. A2774, A2780, A2780ADR, H134, HEY, HOC-7, TR170 and CEM cells were generous gifts from M. Krainer (Medical Univ. Vienna, Austria), H.J.

Broxterman (Free Univ. Hospital, Amsterdam, The Netherlands), R. N. Buick (Univ.

Toronto, ON, Canada), B.T. Hill (Imperial Cancer Research Fund, London, UK) and U.

Jaeger (Medical Univ. Vienna, Austria), respectively. Human myc-HA-MEK1/pBluescript KS was kindly obtained from H. Kiyama (Osaka City Univ., Osaka, Japan), murine dominant active AKT1 was generously provided by D. Efremov (International Center for Genetic Engineering and Biotechnology, Rome, Italy), and human dominant negative AKT was a kind gift from M. Sibilia (Medical Univ. Vienna, Austria). This work was supported by the 'Medical Scientific Fund of the Mayor of the City of Vienna' (#08037) and by the 'Initiative Krebsforschung' of the Medical University Vienna.