CYR61-Avb3 Integrin Loop’ Regulates Breast Cancer Cell Survival and Chemosensitivity Through Activation of ERK1/ERK2 MAPK Signaling Pathway

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A novel CYR61-triggered ‘CYR61-avb3 integrin loop’ regulates breast cancer cell survival and chemosensitivity through activation of ERK1/ERK2 MAPK signaling pathway

Javier A Menendez1,2, Luciano Vellon1,2, Inderjit Mehmi1, Poh K Teng1, David W Griggs3 and Ruth Lupu*,1,2

1Department of Medicine, Breast Cancer Translational Research Laboratory, Evanston Northwestern Healthcare Research Institute, Evanston, IL, USA; 2Northwestern University Feinberg School of Medicine, Chicago, USA; 3Oncology Pharmacology, Discovery Research, Pharmacia Corporation, St Louis, MO, USA

The angiogenic inducer CYR61 is differentially over- increased their sensitivity to Taxol. Our data provide expressed in breast cancer cells exhibiting high levels of evidence that CYR61 is sufficient to promote breast Heregulin (HRG), a growth factor closely associated with cancer cell proliferation, cell survival, and Taxol resis- a metastatic breast cancer phenotype. Here, we examined tance through a avb3-activated ERK1/ERK2 MAPK whether CYR61, independently of HRG, actively reg- signaling. The identification of a ‘CYR61-avb3 autocrine ulates breast cancer cell survival and chemosensitivity, and loop’ in the epithelial compartment of breast carcinoma the pathways involved. Forced expression of CYR61 in strongly suggests that targeting avb3 may simultaneously HRG-negative MCF-7 cells notably upregulated the prevent breast cancer angiogenesis, growth, and chemo expression of its own integrin receptor avb3 (>200 times). resistance. Small peptidomimetic avb3 integrin antagonists dramat- Oncogene (2005) 24, 761–779. doi:10.1038/sj.onc.1208238 ically decreased cell viability of CYR61-overexpressing Published online 13 December 2004 MCF-7 cells, whereas control MCF-7/V remained insensitive. Mechanistically, functional blockade of avb3 Keywords: CYR61; CCN1; Heregulin; avb3; paclitaxel; specifically abolished CYR6-induced hyperactivation of Taxol; ERK1/ERK2 MAPK; chemotherapy; breast ERK1/ERK2 MAPK, whereas the activation status of cancer AKT did not decrease. Moreover, CYR61 overexpression rendered MCF-7 cells significantly resistant (>10-fold) to Taxol-induced cytotoxicity. Remarkably, avb3 inhibition converted the CYR61-induced Taxol-resistant phe- Introduction notype into a hypersensitive one. Thus, the augmentation of Taxol-induced apoptotic cell death in the presence of Increasing efforts have been devoted to characterize avb3 antagonists demonstrated a strong synergism as cellular proteins that play an active role in breast cancer verified by the terminal transferase-mediated dUTP nick- progression. In this regard, we recently isolated and end labeling (TUNEL) assay and by flow cytometric identified CYR61 (CCN1; the human homolog of a analysis for DNA content. Indeed, functional blockade of mouse immediate early response gene, Cyr61), an avb3, similarly to the pharmacological MAPKinhibitor angiogenic regulator that is differentially expressed in U0126, synergistically increased both the proportion of invasive and metastatic human breast cancer cells CYR61-overexpressing breast cancer cells in the G2 phase (Tsai et al., 2000). CYR61, a cysteine-rich, heparin- of the cell cycle and the appearance of sub-G1 hypodiploid binding protein that is secreted and associated with the (apoptotic) cells caused by Taxol. Strikingly, CYR61 cell surface and the extracellular matrix (Yang and Lau, overexpression impaired the accumulation of wild-type 1991), belongs to the Cysteine-rich 61/Connective tissue p53 following Taxol exposure, while inhibition of avb3 growth factor/Nephroblastoma-overexpressed (CCN) or ERK1/ERK2 MAPK signalings completely restored gene family of angiogenic and growth regulators, which Taxol-induced upregulation of p53. Moreover, antisense consists of CCN1 (CYR61), CCN2 (CTFG), CCN3 downregulation of CYR61 expression abolished the (NOV), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 anchorage-independent growth of breast cancer cells (WISP-3) (Brigstock, 1999; Lau and Lam, 1999; Perbal, engineered to overexpress HRG, and significantly 2001; Brigstock et al., 2003; Planque and Perbal, 2003; Menendez et al., 2003; Perbal, 2004). All CCN proteins, including CYR61, have been shown to mediate *Correspondence: R Lupu, Evanston Northwestern Healthcare functions as diverse as cell proliferation, migration, Research Institute, 1001 University Place, Evanston, IL 60201, USA; adhesion, cell survival, differentiation, and extracellular E-mail: [email protected] Received 31 May 2004; revised 2 September 2004; accepted 2 September matrix formation. Remarkably, CYR61 also regulates 2004; published online 13 December 2004 more complex processes, such as angiogenesis and A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 762 tumorigenesis (Babic et al., 1998, 1999; Xie et al., 2001a; cancer cells overexpressing HRG. Moreover, it is Tsai et al., 2002; Planque and Perbal, 2003). reasonable to hypothesize that the aggressive pheno- In the breast cancer scenario, we have previously types induced by HRGare mediated, in part if not shown that CYR61 is a downstream effector of Here- entirely, through the interaction of CYR61 with its gulin (HRG), a member of the epidermal growth factor integrin receptor avb3. Taken into account that the (EGF)-like growth factor family closely involved in aggressiveness of a tumor depends, at least in part, on its breast cancer progression and metastasis (Lupu et al., ability to proliferate in a deregulated manner and its 1996; Tsai et al., 2000, 2003; Atlas et al., 2003; sensitivity to chemotherapy-induced cell damage, we Menendez et al., 2003) Moreover, we established that here examined the effects of CYR61 overexpression in forced expression of CYR61, in the absence of HRG breast cancer cell survival and chemosensitivity, and the and Her-2/neu (erbB-2) oncogene overexpression, is signaling pathways involved. Interestingly, our data sufficient to promote acquisition of hormone indepen- provide evidence that CYR61 is sufficient to regulate dence and anti-estrogen resistance in human breast breast cancer cell proliferation, cell survival, and cancer cells (Tsai et al., 2002). Indeed, CYR61 enhances chemosensitivity through its ability to strongly activate a metastatic phenotype by promoting cell proliferation a avb3-ERK1/ERK2 MAPK signaling. The identifica- in soft agar, cell migration and invasion, and Matrigel tion of a previously unrecognized CYR61-triggered outgrowth of breast cancer cells (Tsai et al., 2000, 2002). ‘CYR61-avb3 autocrine loop’ in the epithelial compart- In our evaluation of CYR61 in breast tumor biopsies, a ment of breast carcinoma strongly suggests that target- 40% of estrogen receptor (ER)-negative invasive breast ing avb3 may simultaneously prevent breast cancer carcinomas were found to express high levels of CYR61 angiogenesis, growth, and chemoresistance. protein (Tsai et al., 2000). More recently, CYR61 overexpression was associated with advance stage, size, and lymph node involvement of primary breast tumors Results (Xie et al., 2001b). Although these results strongly indicate that CYR61 may play a key role in the process CYR61 overexpression upregulates avb3 integrin of breast cancer progression and might serve as valuable in breast cancer cells tool for monitoring the tumor status, the ultimate molecular mechanisms by which CYR61 promotes an We envisioned that high levels of CYR61 would be a aggressive breast cancer phenotype are still largely prerequisite for the high levels of avb3 integrin found in unknown. HRG-overexpressing breast cancer cells. To assess this Depending on the biological context and cell model hypothesis, we took advantage of a recent cellular model system, CYR61 is thought to act in an autocrine– of breast cancer CYR61 overexpression developed in paracrine manner to promote cell growth, migration, our laboratory, in which nontumorigenic and estrogen- and angiogenesis, through the interaction with its dependent MCF-7 cells, which express very low to multiple integrin receptors (avb3, avb5, aIIbb3, and a6b1; undetectable levels of CYR61, HRG, and erbB-2, were Kireeva et al., 1998; Jedsadayanmata et al., 1999; Chen stably transfected with the full-length cDNA of CYR61 et al., 2000; Grzeszkiewicz et al., 2001). However, most (Tsai et al., 2002). To determine if overexpression of of the CYR61-promoted effects are mediated via its CYR61 in breast cancer cells altered the levels of its direct binding with the integrin receptor avb3, which has integrin receptor avb3 in the absence of HRGover- been implicated in the pathophysiology of malignant expression, the cell surface expression of avb3 in two tumors. In addition to its expression on the surface of representative CYR61 derivatives (MCF-7/C2-6 and angiogenic endothelial cells, avb3 is also expressed on the MCF-7/C2-9 clones) was assessed by flow cytometry of surface of tumor cells in a variety of cancers. In breast cells stained with the anti-avb3 monoclonal antibody cancer, avb3 characterizes the metastatic phenotype, and LM609. As control, the cells were stained with the its expression is upregulated in invasive tumors and appropriate antibody isotype. In MCF-7 breast cancer distant metastases (Gasparini et al., 1998; Meyer et al., cells transfected with an empty vector (MCF-7/V), 1998). We recently postulated that CYR61-enhanced which expresses very low levels of CYR61, avb3 integrin breast cancer progression may require a concomitant was almost not detectable by flow cytometry (Figure 1a). overexpression of avb3 (Tsai et al., 2000; Menendez et al., Interestingly, an equivalent flow cytometric analysis 2003). Indeed, we observed that avb3 levels were demonstrated a very significant increase of avb3 levels in significantly augmented in MCF-7 cells engineered to CYR61-transfected MCF-7/C2-6 and MCF-7/C2-9 overexpress HRGwhen compared with control cells clones (Figure 1a). (Tsai et al., 2000). In addition, we determined that To confirm the results obtained by flow cytometry we functional blockade of avb3 using LM609, a monoclonal next analysed the impact of CYR61 overexpression in antibody which binds to a conformational epitope the subcellular localization of avb3. To address this resulting from the post-translational association of the question, CYR61-overexpressing MCF-7/C2-6 and av and b3 integrin subunits, completely blocked the MCF-7/C2-9 clones and matched control MCF-7/V Matrigel outgrowth of HRG-overexpressing breast cells were permeabilized with Triton X-100 for the cancer cells (Tsai et al., 2000). These results strongly intracellular delivery of an anti-avb3 monoclonal anti- suggested that a functional avb3-dependent signaling is body, and then the cellular pattern of avb3 was assessed required for maintaining the invasive capacity of breast by immunofluorescence microscopy. The membrane

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 763

a b α β c GM = 3 ± 1 FITC- v 3 M1 = 1.5 ± 1% MCF-7/V MDA-MB-231 MCF-7/C2-6 MCF-7 MCF-7/C2-9 MCF-7/V

CYR61 Cell number M1 MCF-7/V α v β 3 α β GM = 8 ± 2 FITC- v 3

M1 MCF-7/C2-6 = 32 ± 5% MCF-7/C2-9 700 ** 600

500

Cell number M1

MCF-7/C2-9 MCF-7 400 **

300 versus α β ** GM = 11 ± 3 FITC- v 3

M1 = 31 ± 4% Expression Index 3 200 β v α 100 ** P < 0.005

Cell number 0 M1 MCF-7/C2-6 MCF-7

α β MCF-7/V FITC- v 3 MCF-7/C2-9 MCF-7/C2-6 MDA-MB-231

Figure 1 CYR61 overexpression upregulates avb3 integrin expression in MCF-7 cells. (a) Flow cytometric quantification of avb3 integrin expression in CYR61 derivatives MCF-7/C2-6 and C2-9 clones, and matched control MCF-7/V cells. The specific surface expression of avb3 in CYR61-overexpressing MCF-7/C2-6 and C2-9 clones, and matched control MCF-7/V cells was determined by flow cytometry by measuring the binding of a mouse anti-avb3 antibody (clone LM609; Chemicon) as described in ‘Materials and methods’. The data presented summarize the mean of the Geo Mean (GM) fluorescence parameter (7s.d.) from three independent experiments. (b) In situ immunofluorescent staining of avb3 integrin in CYR61 derivatives MCF-7/C2-6 and C2-9 clones, and matched control MCF-7/V cells. (c) Correlation between the expression of CYR61 and avb3 in breast cancer cells. Top: Subconfluent wild-type MCF-7, MCF-7/V, MCF-7/C2-9, MCF-7/C2-6, and MDA-MB-231 breast cancer cells were maintained in serum-free media for 48 h in 100-mm plates. The conditioned media were collected, concentrated 20 Â , resolved by 10% Tris-glycine SDS–PAGE, and assayed for CYR61 expression using a rabbit anti-CYR61 polyclonal antibody (ab2026; 1 : 2000 dilution). In all, 50 mg weight of whole-cell lysates from the same experimental 100-mm plate were resolved by 10% Tris-glycine SDS–PAGE, transferred to nitrocellulose membranes, and then probed with a mouse monoclonal anti-avb3 antibody (LM609 clone). Bottom: A avb3 expression index was calculated using the formula GM (Geo Mean Fluoresence) Â %ofavb3-positive cells. Data presented summarize the mean (columns)7s.d. (bars) of three independent experiments

staining of avb3 was almost undetectable in MCF-7/V increased (>200 times) the basal level of avb3 expression control cells (Figure 1b). Conversely, CYR61-over- in MCF-7 breast cancer cells (Figure 1c, bottom). expressing MCF-7/C2-6 and MCF/C2-9 transfectants CYR61 was highly expressed in the CYR61 transfec- showed a prominent cell-surface staining of avb3 tants as determined by Western blotting analysis using (Figure 1b). In fact, avb3 integrin in CYR61-over- conditioned media concentrated from the C2-6 and C2-9 expressing MCF-7 cells was to some extent distributed clones or a vector control clone (Figure 1c, top). Indeed, throughout the cytoplasm. Although direct quantitative the expression level of CYR61 in MCF-7/C2-6 and interpretation of immunofluorescence is not possible, MCF-7/C2-9 clones was comparable to that in MDA- comparison of the intensity of avb3 dots observed with MB-231 cells (an aggressive breast cancer cell line the avb3-specific antibody revealed an obvious and naturally overexpressing HRGand CYR61), as com- highly reproducible difference between CYR61 transfec- pared with the wild-type MCF-7 or MCF-7/V cells, in tants and control cells. Upregulation of avb3 by CYR61 which CYR61 was nearly undetectable. This is the first overexpression was also evident when avb3 protein indication showing that upregulation of avb3 integrin accumulation was monitored by immunoblotting expression in human breast epithelial cells can be (Figure 1c, top). When the expression levels of avb3 achieved solely by CYR61 overexpression, irrespective integrin were quantitatively evaluated using an expres- of HRGstatus. Importantly, the basal level of avb3 sion index (Geo Mean (GM) Fluoresence Â %ofavb3- expression was significantly higher in MDA-MB-231 positive cells-M1; Figure 1a), this approach clearly cells as compared with MCF-7 cells engineered to demonstrated that CYR61 overexpression dramatically overexpress CYR61. Therefore, in breast cancer cells,

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 764 naturally overexpressing both HRGand CYR61 significantly higher in CYR61 derivatives MCF-7/C2-6 (i.e. MDA-MB-231 cells), it is likely that HRGfurther and MCF-7/C2-9 (Figure 2, left panel). On the other enhances CYR61-stimulated avb3 expression in a syn- hand, the content of total MAPK was similar in ergistic manner. We are currently investigating the CYR61-overexpressing and control MCF-7/V cells, ultimate mechanism responsible for the exacerbated whereas the activation status of ERK1/ERK2 MAPK, levels of avb3 in MDA-MB-231 breast cancer cells. as monitored by immunoblotting using polyclonal antibodies recognizing the activated form of the ERK1 and ERK2 enzymes (P-ERK1/ERK2 MAPK), was CYR61 overexpression upregulates PI-30K-AKT significantly higher in MCF-7/C2-6 and MCF-7/C2-9 and MEK1/MEK2-ERK1/ERK2 signaling cascades transfectants in comparison to matched control MCF-7/ in breast cancer cells V cells (Figure 2, left panel). LY294002, a specific 0 The CYR61-driven avb3 signaling network activates inhibitor of the p110 catalytic subunit of PI-3 K, several signaling pathways leading to enhanced en- particularly affected PI-30K/AKT signaling as indicated dothelial cell survival and proliferation (Grzeszkiewicz by the complete inhibition of P-AKTSer473. As previously et al., 2001; Leu et al., 2002). It has been previously demonstrated by Zimmermann and Moelling (1999) in shown that some of the phenotypic changes dictated by MCF-7 breast cancer cells, a very significant activation avb3 are dependent upon activation of phosphatidylino- of ERK1/ERK2 was promoted by LY294002-induced sitol 30-kinase (PI-30K)/protein kinase B (AKT) and inhibition of PI-30K and AKT in MCF-7/V control cells, ERK1/ERK2 MAPK transduction cascades (King et al., thus evidencing an active crosstalk between the Raf- 1997; Gilmore et al., 2000; Lee and Juliano, 2000; Zhang MEK-ERK and the PI-30K-AKT pathways. Interest- et al., 2002; Roberts et al., 2003), which play a critical ingly, inhibition of AKT activation by LY294002 role in apoptosis and cell proliferation, respectively. slightly altered the hyperactivation status of ERK1/ Therefore, we examined whether the antiapoptotic PI- ERK2 MAPK in CYR61-derivatives MCF-7/C2-6 and 30K/AKT cascade as well as the cell cycle regulatory MCF-7/C2-9 (Figure 2, right panel). U0126, a non- ERK1/ERK2 MAPK signaling pathway were altered competitive inhibitor of the dual specificity MAPK in CYR61-overexpressing MCF-7 breast cancer cells. kinases MEK1 and MEK2, the enzymes that activate While downstream of PI-30K, the content of total MAPK, completely eliminated P-MAPK without affect- AKT was similar in CYR61-overexpressing and ing active AKT (Figure 2, right panel). The levels of control MCF-7/V cells, active AKT (P-AKTSer473) was total AKT and total MAPK were not altered by any of MCF-7/C2-9 MCF-7/C2-6 MCF-7/V Untreated LY294002 U0126 U0126 + LY MCF-7 Activated AKT P -AKT AKT AKT

P -ERK1 Activated AKT P -AKT Activated MAPK P -ERK1 AKT AKT ERK1 MAPK ERK2 P -ERK1 Activated MAPK P -ERK1 Activated AKT P -AKT ERK1 AKT MAPK ERK2 AKT β Activated MAPK P -ERK1 -actin P -ERK1

MCF-7/C2-6 MCF-7/V MAPK ERK1 ERK2 MCF-7

MCF-7/V Activated AKT P -AKT

MCF-7/C2-9 MCF-7/C2-6 AKT AKT Activated MAPK P -ERK1 P -ERK1 MCF-7/C2-9 MAPK ERK1 ERK2

Figure 2 CYR61 overexpression activates PI-30K-AKT and MEK1/MEK2-ERK1/ERK2 signaling cascades in MCF-7 breast cancer cells. Left: Total and activated (phosphorylated) AKT and ERK1/ERK2 MAPK protein levels in CYR61-overexpressing MCF-7/C2-6 and MCF-7/C2-9 and matched control MCF-7/V cells. Overnight serum-starved cells at 75–80% conﬂuence were washed two times with PBS, lysed in buffer, subjected to electrophoresis on 10% SDS–PAGE, and transferred to nitrocellulose membranes. Immnunoblots with anti-AKT, anti-P-AKT, anti-ERK1/ERK2 MAPK, and anti-P-ERK1/ERK2 MAPK show equivalent amounts of the proteins in each extract (25 mg). Blots were reprobed with an antibody for b-actin to control for protein loading and transfer. Results are representative of three independent experiments. Right: Overnight serum-starved cells at 75–80% conﬂuence were treated for 2 h with vehicle (v/v), LY294002 (40 mM), U0126 (20 mM), or both LY294002 and U0126. Total protein (25 mg) was resolved by SDS–PAGE and subjected to immunoblot analyses for AKT, P-AKT, ERK1/ERK2 MAPK, and P-ERK1/ERK2 MAPK as described above. Blots were reprobed with an antibody for b-actin to control for protein loading and transfer. Results are representative of three independent experiments

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 765 the inhibitors. These results strongly indicate that, avb3-negative MCF-7/V cells did not display signs of downstream of avb3, CYR61 overexpression concomi- cytotoxicity even at S-247 concentrations as high as tantly activates PI-30K-AKT and MEK1/MEK2- 10 mM. Of interest, CYR61-overexpressing MCF-7/C2-6 ERK1/ERK2 signaling cascades in breast cancer cells. and MCF-7/C2-6 were insensitive to very high concen- These findings not only support earlier studies eviden- trations of S-205, a highly potent and selective cing the ability of CYR61 to strongly activate AKT in antagonist of the b3 integrin aIIbb3, further demonstrat- other cellular systems (Chen et al., 2001; Xie et al., ing the specificity of the molecular target (i.e. avb3) 2004), but suggest further that CYR61 overexpression through which these avb3 antagonists promote cytotoxi- may molecularly disrupt the crosstalk between these two city towards breast cancer cells. When the cell growth signaling cascades at the level of Raf and AKT as the was measured by counting the cells after a 48 h 0 inhibition of PI-3 K does not lead to activation of the treatment with avb3 integrin antagonists, a dose- Raf-MEK-ERK cascade in CYR61-overexpressing dependent inhibitory effect on cell proliferation as well MCF-7/C2-6 and MCF-7/C2-9 clones. as cell adhesion was observed in CYR61-overexpressing MCF-7/C2-6 and MCF-7/C2-9 clones treated with SC68448, S-247, or S-197. Of note, at SC68448, S-247, avb3 signaling mediates cell survival and proliferation and S-196 concentrations higher than 1 mM, the cells in CYR61-overexpressing breast cancer cells were rounded and assembled in clusters either floating in We next evaluated whether cell survival and prolifera- the medium or weakly adherent on the culture support, tion in MCF-7 cells engineered to overexpress CYR61 whereas in the presence of S-205, the peptidomimetic was associated with an increased signaling through agonist with the lowest affinity to avb3, they formed a typical epithelioid monolayer (data not shown). These CYR61 integrin receptor avb3. To evaluate this issue, we examined the metabolic effects of a novel group of small results, altogether, demonstrate that an active avb3 signaling is necessary to maintain cell viability in peptidomimetic antagonists of avb3 (SC56631, SC68448, S-247, S-197, and S-205; Oncology Pharmacology, CYR61-overexpressing breast cancer cells. Discovery Research, Pharmacia Corporation, St Louis, MO, USA) towards CYR61 transfectants and control CYR61 overexpression induces breast cancer cell MCF-7/V cells. Table 1 provides the potency (IC50 resistance to paclitaxel (Taxolt) value) of these antagonists against human placenta- Integrin signals are involved in diverse biological derived avb3 or human platelet-derived aIIbb3, the two responses, including angiogenesis and tumor progres- known b3 integrins. Figure 3 shows the dose-dependent effects of SC56631, SC68448, S-247, S-197, and S-205 on sion as well as in a variety of cellular activities, including the metabolic status of CYR61-overexpressing MCF-7/ cell migration, proliferation, and survival. Of interest, C2-6 and MCF-7/C2-9 clones and MCF-7/V control integrin signaling has recently been shown to modulate cells as judged by the mitochondrial conversion of the cancer cell responses to chemotherapeutic agents tetrazolium salt, MTT, to its formazan product (MTT (Aoudjit and Vuori, 2001). This integrin-dependent assay). A marked decrease in cell viability was observed phenomenon of innate chemoresistance has been termed cell adhesion-mediated drug resistance (Damiano et al., in CYR61- and avb3-overexpressing MCF-7/C2-6 and MCF-7/C2-9 clones cultured in the presence of 1999; Gilmore et al., 2000). Here, we evaluated whether CYR61-induced upregulation of a b would modulate SC68448, S-247, and S-196. When the efficacy of avb3 v 3 breast cancer cell response to paclitaxel (Taxolt), an antagonists was evaluated by calculating the IC30 and antimitotic drug commonly used in the treatment of IC50 values (the dose of agents producing a 30 and 50% reduction in cell viability, respectively), we observed that advanced or metastatic breast cancer (Hortobagyi et al., CYR61-overexpressing MCF-7/C2-6 and MCF-7/C2-9 1997; Perez, 1998; Wiseman and Spencer, 1998). Using clones were exquisitely sensitive to S-247, the most the MTT assay, which is a crude measure of cell viability, we observed that CYR61-overexpressing potent and selective avb3 antagonist, with IC50 values lower than 3 mM (Table 2). Conversely, CYR61- and MCF-7/C2-6 and MCF-7/C2-9 clones were between nine- and 12-fold more resistant to Taxol-induced cytotoxicity when compared with matched control MCF-7/V cells (Figure 4a). Table 1 Potency of avb3 antagonists against human placenta-derived We speculated that the reduced sensitivity to Taxol avb3 or human platelet-derived aIIbb3, the two known b3 integrins seen in CYR61-overexpressing MCF-7 cells was not avb3 antagonist IC50 (avb3)IC50 (aIIbb3) simply the result of changes in cell proliferation, but might actually be due to a CYR61-promoted decrease SC56631 10 9 (1) SC68448 0.8 407 (509) in apoptotic cell death following Taxol-induced cell S-247 0.6 1290 (2150) damage. To address this question, cells were exposed to S-196 1.19 54 (45) 10 nM Taxol, cell death was measured by an ELISA that S-205 >10 000 1.36 (À) detects DNA-histone fragmentation, and the x-fold increase in apoptosis-related cell death was calculated Comparison of IC50 values for each receptor (values in parentheses) by comparing the ELISA optical density readings of determines compound selectivity between b3 integrins, and further predicts the effect of a compound in cell-based in vitro and in vivo treated samples, with the values of the untreated cells as efficacy models 1.0. Likewise, CYR61-overexpressing MCF-7/C2-6 and

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 766 110 120 110 100 100 90 90 80 80 70 IC 70 60 30 IC 60 30 50 IC 50 40 50 40 IC50 30 30 MCF-7/V MCF-7/V 20 20

Metabolic status (% of control) MCF-7/C2-9

MCF-7/C2-9 Metabolic status (% of control) 10 10 MCF-7/C2-6 MCF-7/C2-6 0 0 110110 SC68448 (µM) S-196 (µM)

120 110 110 110 100 100 100 90 90 90 80 80 80 70 70 70 IC30 IC30 IC 60 60 60 30 50 50 50 IC50 IC50 IC 40 40 40 50 30 30 30 MCF-7/V 20 MCF-7/V 20 MCF-7/V 20 Metabolic status (% of control) Metabolic status (% of control) MCF-7/C2-9 Metabolic status (% of control) MCF-7/C2-9 MCF-7/C2-9 10 10 10 MCF-7/C2-6 MCF-7/C2-6 MCF-7/C2-6 0 0 0 1 10 1 10 1 10 SC56631 (µM) S-247 (µM) S-205 (µM)

Figure 3 CYR61 overexpression generates a CYR61-avb3 autocrine prosurvival loop in MCF-7 breast cancer cells. avb3 integrin antagonists specifically reduce cell viability of CYR61-overexpressing breast cancer cells. The cell viability effects from exposure of cells to each avb3 antagonist was determined using a modified MTT reduction assay and analysed generating concentration–effect curves as a plot of the fraction of unaffected (surviving) cells versus drug concentration. Dashed lines represent the IC30 and IC50 values, which were defined as the concentration of antagonist producing 30 and 50% reduction in control absorbance (by interpolation), respectively. Data presented summarize the mean (7s.d.) of five independent experiments made in triplicate

Table 2 CYR61 overexpression sensitizes breast cancer cells to avb3 antagonists MCF-7/V MCF-7/C2-6 MCF-7/C2-9

avb3 antagonist IC30 IC50 IC30 IC50 IC30 IC50

SC56631 >10 >10 B10 >10 B10 >10 SC68448 >10 >10 2.3 6.1 8.1 >10 S-247 >10 >10 2.0 4.3 2.8 5.5 S-196 >10 >10 2.6 6.4 4.2 8.2 S-205 >10 >10 >10 >10 >10 >10

Breast cancer cell sensitivity to avb3 integrin antagonists was expressed in terms of the concentration (mM) of antagonist required for either 30 or 50% (IC30 and IC50, respectively) reduction of cell viability. Since the percentage of control absorbance was considered to be the surviving fraction of cells, the IC30 and IC50 values were deﬁned as the concentration of drug that produced 30 and 50% reduction in control absorbance (by interpolation), respectively. Bold values indicates p10mM

MCF-7/C2-9 clones treated with Taxol exhibited a ability of cancer cells to grow in vivo, CYR61-over- negligible degree of cell death compared with that expressing MCF-7 transfectants and MCF-7/V control observed in control MCF-7/V cells (Figure 4b). cells were further analysed for their anchorage-indepen- Since soft-agar colony formation of cancer cells is dent growth properties in the presence of Taxol. Very thought to be one of the best in vitro correlates of the low concentrations of Taxol (o10 nM) dramatically

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 767 abc ∗ P < 0.05 versus MCF-7/V ∗∗ P < 0.005 versus MCF-7/V ∗∗P < 0.005 versus MCF-7/V 120 100 MCF-7/V 10 ∗ 110 ∗∗ MCF-7/C2-6 90 9 ∗ 100 ∗∗ MCF-7/C2-9 80 8 90 ∗∗ ∗∗ 70 7 80 ∗∗ 60 6 70 ∗∗ ∗∗ IC50 50 5 60 50 40 4 ∗∗ 40 30 3 ∗∗ ∗ 30 20 2 Cell viability (% of control) MCF-7/V 20 10 MCF-7/C2-9 1 10 Number of Colonies (% Control) MCF-7/C2-6 Apoptotic cell death (fold increase) 0 0 0 1 10 100 2.55 10 50 100 TAXOL (nM) TAXOL (nM) MCF-7/V MCF-7/C2-6 MCF-7/C2-9 Figure 4 CYR61 overexpression induces breast cancer cell resistance to Taxol. (a) MCF-7 cells engineered to overexpress CYR61 are less sensitive to Taxol-induced cell toxicity. CYR61-overexpressing MCF-7 cells (C2-6 and C2-9 clones) and matched control MCF-7/ V cells were cultured in 96-well plates in the presence or absence of increasing concentrations of Taxol until untreated control cells reached confluence. Cell viability and IC50 values were then determined using a modified MTT reduction assay as described above. The data presented summarize the mean (7s.d.) of five independent experiments made in triplicate. (b) CYR61 overexpression reduces Taxol-induced cell death. The induction of cell death by exposure of CYR61-overexpressing MCF-7/C2-6 and MCF-7/C2-9 clones, and matched control MCF-7/V cells to 10 nM Taxol (24 h), was assessed using the Cell Death Detection ELISAPLUS kit as per the manufacturer’s instructions. The enrichment of histone-DNA fragments in treated cells was expressed as fold increase in absorbance as compared with control (vehicle-treated) cells. Data presented summarize the mean (columns)7s.d. (bars) of three independent experiments made in duplicate. (c) CYR61 overexpression increases breast cancer cell resistance to Taxol upon anchorage-independent conditions. Data presented summarize the mean percentages (columns)7s.d. (bars) of five independent experiments made in triplicate (colony number obtained in untreated samples ¼ 100%)

suppressed colony formation of empty-vector control cells to Taxol are shown in Table 3. In order to measure cells growing in semisolid agar. In contrast, significant the increase in Taxol sensitivity, a ‘sensitization factor’ higher concentrations of Taxol (>50 nM) were required was determined by dividing Taxol IC50 values in the to inhibit the anchorage-independent soft agar clono- absence of avb3 antagonists by those in their presence. genic growth of CYR61-overexpressing MCF-7/C2-6 SC68448, S-196, and S-247 enhanced the cytotoxic and MCF-7/C2-6 clones (Figure 4c). Thus, under activity of Taxol in a dose-dependent manner. Thus, anchorage-independent growth conditions, CYR61 as the concentration of avb3 antagonists increased, the overexpression increased between 5 and 10 times breast efficacy of Taxol was significantly increased. Impor- cancer cell resistance to Taxol. Altogether, these findings tantly, the sensitization effects were dependent on the support the recent observations by Lin et al. (2004) ability of the peptidomimetic antagonists to specifically suggesting that CYR61 plays an important role in interact with avb3. Thus, CYR61-overexpressing MCF- resistance to chemotherapeutic agent-induced apoptosis 7/C2-6 and MCF-7/C2-9 clones incubated in the and clearly demonstrate that CYR61 overexpression presence of 0.5 mM SC68448 (the avb3 antagonist with dramatically decreases the cytotoxic activity of Taxol the lowest affinity and specificity for avb3) showed a towards breast cancer cells. 5–9-fold increase in Taxol sensitivity, while the addition of S-247 (the avb3 antagonist with the highest affinity and specificity for avb3) spectacularly increased Taxol- induced cytotoxicity up to 80 times. In agreement with Functional blockade of avb3 reverses CYR61-induced breast cancer cell resistance to Taxol-induced cytotoxicity the ability of the peptidomimetic antagonists to specifically work through its avb3 target, there was no To investigate whether CYR61-induced resistance to significant enhancement of Taxol-induced cytotoxicity Taxol was specifically mediated through avb3, CYR61- when avb3-negative MCF-7/V cells were coexposed to overexpressing and matched control cells were treated SC68448, S-196, or S-247. with Taxol in the absence or presence of avb3 SC68448, S-196, and S-247 as single agents signifi- antagonists. The effects of coexposure to suboptimal cantly decreased cell viability of CYR61-overexpressing doses (lower than the IC50) of SC68448, S-196, and MCF-7 cells at the concentrations examined. This S-247 on the response of CYR61-overexpressing MCF-7 indicated the presence of a potentially significant

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 768

Table 3 Effects of avb3 antagonists, U0126, and LY294002 on breast cancer cell sensitivity to Taxol +SC68448 +S-196 +S-247 +U0126 +LY294002

a Cells Taxol IC50 (nM) Resistance IC50 (nM)IC50 (nM)IC50 (nM)IC50 (nM)IC50 (nM)

MCF-7/V 5 — 5.5 (1) 4.0 (1) 3.5 (1) 3.4 (1) 3.0 (1) MCF-7/C2-6 45 9 9.0 (5) 3.5 (13) 0.6 (80) 5.5 (8) 3.7 (12) MCF-7/C2-9 60 12 7.5 (8) 5.0 (12) 0.75 (80) 10 (6) 7.5 (8)

Taxol-induced cytotoxicity in the absence or presence of avb3 integrin antagonists SC68448, S-196, and S-247; MEK1/MEK2 blocker U0126; or PI-30K inhibitor LY294002 was determined using a modiﬁed MTT reduction assay as described in ‘Materials and methods’. Agents were studied in combination concurrently and were not renewed during the entire period of cell exposure. The degree of sensitization to Taxol by avb3 integrin antagonists, U0126 and LY294002 (values in parentheses), was evaluated by dividing IC50 values of control cells (absence of avb3 integrin antagonists, U0126 or LY294002) by those obtained when cells were exposed to avb3 antagonists, U0126, or LY294002 during exposure to a Taxol. Resistance to Taxol-induced cytotoxicity was expressed as a x-fold factor by dividing IC50 values in CYR61-overexpressing MCF-7/C2-6 and MCF-7/C2-9 clones by that found in MCF-7/V-matched control cells

synergic/antagonist component. Thus, possible syner- We also examined whether, downstream of avb3, 0 gistic interactions between avb3 antagonists and Taxol inhibition of PI-3 K-AKT or MEK1/MEK2-ERK1/ could not be accurately discriminated from additive or ERK2 pathways altered the cytoprotective actions of antagonistic effects in the basis of the above data alone. CYR61 overexpression in MCF-7 cells. To link CYR61- Although there is still controversy over which method is induced activation of PI-30K or MAPK cascades to best for detecting true in vitro synergy between drug Taxol resistance, we used nontoxic concentrations of combinations, we performed a series of isobologram LY294002 and U0126, specific inhibitors of PI-30K and transformations of multiple dose-response analyses of MEK1/MEK2 enzymatic activities, respectively. (Berenbaum, 1989). Representative transformations are After coexposure to 20 mM LY294002, CYR61-over- presented graphically (isobolograms) in Figure 5a. The expressing MCF-7/C2-6 and MCF-7/C2-9 clones dashed line drawn between the IC50 value for each avb3 showed up to 12-fold increase in sensitivity to Taxol. antagonist alone and the IC50 for Taxol alone indicates The cotreatment with 10 mM of the MEK inhibitor the alignment of theoretical isoeffect data points for U0126 enhanced up to eightfold Taxol-induced cyto- additive interactions between avb3 antagonists and toxicity in CYR61-overexpressing MCF-7 cells (Table 3). Taxol. The true IC50 points (the experimental concen- Conversely, unchanged Taxol IC50 values were observed trations of avb3 antagonists and Taxol which com- for matched control MCF-7/V cells in the presence of binedly produced 50% reduction in cell survival) were LY294002 or U0126. These results strongly suggest that, plotted and compared with the additive line. Data downstream of avb3, CYR61-induced activation of PI- points above the dashed diagonal line of the additive 30K-AKT or MEK1/MEK2-ERK1/ERK2 transduc- effects in the isobole suggest antagonism and those tion cascades actively contribute to CYR61-promoted below the diagonal suggest synergism. While these breast cancer cell resistance to Taxol. figures provide a graphical representation of avb3 antagonists–Taxol interactions, the values of the mean Functional blockade of a b synergistically enhances combination index (CI) for a particular cell line, drug v 3 Taxol-induced apoptosis in CYR61-overexpressing combination, and effect level are also labeled. In breast cancer cells addition, Student t tests were computed to evaluate whether significant differences in the CI means values Since apoptosis is the predominant mechanism of occurred as compared to a null hypothesized CI of 1 cytotoxicity induced by chemotherapeutic agents, we (additivity) and to formally evaluate whether antagon- analysed whether the failure of CYR61-overexpressing ism or synergism was evident. At the 50% effect level, MCF-7 cells to activate apoptosis may account for concurrent administration of avb3 antagonists and Taxol CYR61-promoted resistance to Taxol. In addition, we resulted in a strong synergism (CIso0.3) in CYR61- evaluated the possibility that the synergistic decrease in overexpressing MCF-7 C2-6 and MCF-7/C2-9 clones. cell viability of CYR61-overexpressing MCF-7 breast In other words, the amount of the two agents together cancer cells cotreated with avb3 antagonists and Taxol necessary to reduce MCF-7/C2-6 and MCF-7/C2-9 cell would represent an enhancement of Taxol-induced viability by 50% was only B0.2 times as much as would apoptotic cell death. In these experiments, we employed be required if they demonstrated purely additive the lowest clinically relevant concentration of Taxol (i.e. behavior (Po0.001 versus a null hypothesized inter- 10 nM) that blocks normal cell cycle progression at the action index of 1 – additivity). The above results imply G2–M of the cell cycle and induces ERK1/ERK2 that CYR61 regulates breast cancer cell sensitivity to MAPK activation (Torres and Horwitz, 1998; MacK- chemotherapeutic agents such as Taxol mainly through eigan et al., 2000, 2002). To evaluate Taxol-related its interaction with the avb3 integrin, and that therapies apoptosis, we used a fluorometric terminal transferase- depriving CYR61-overexpressing breast cancer cells of mediated dUTP nick-end labeling (TUNEL) assay, their avb3 proliferative signaling dramatically increase which measure the fragmented DNA of apoptotic cells Taxol killing. by catalytically incorporating fluorescein-12-dUTP at

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 769 a 1.2 1.2 ∗∗ 1.2 ∗∗ ∗∗ MCF-7/C2-9; CI = 0.200 MCF-7/C2-9; CI50= 0.195 MCF-7/C2-9; CI50= 0.277 50 ∗∗ ∗∗ ∗∗ MCF-7/C2-6; CI = 0.289 MCF-7/C2-6; CI50= 0.239 MCF-7/C2-6; CI50= 0.245 1 50 1 1 ANTAGONISM (CI >1) ANTAGONISM (CI >1) ANTAGONISM (CI >1) 0.8 ADDITIVISM (CI = 1) 0.8 ADDITIVISM (CI = 1) 0.8 ADDITIVISM (CI = 1) units) units) units) 50 50 50 0.6 0.6 0.6

0.4 0.4 0.4 TAXOL (IC TAXOL (IC TAXOL (IC 0.2 0.2 SYNERGISM 0.2 SYNERGISM (CI < 1) SYNERGISM (CI < 1) (CI < 1) 0 0 0 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2

SC68448 (IC50 units) S-196 (IC50 units) S-247 (IC50 units)

b + 0 nM Taxol + 10 nM Taxol + 0 µM S-247 + 0.5 µM S-247 + 0 µM S-247 + 0.5 µM S-247 FITC-TUNEL

2% 2% 32% 35% DAPI MCF-7/V FITC-TUNEL

2% 4% 5% 30% DAPI MCF-7/C2-6

+ 0 µM S-247 + 0.5 µM S-247 + 0 µM S-247 + 0.5 µM S-247 + 0 nM Taxol + 10 nM Taxol

Figure 5 Functional blockade of avb3 synergistically enhances Taxol killing in CYR61-overexpressing MCF-7 breast cancer cells. (a) The nature of the cytotoxic interaction between avb3 integrin antagonists and Taxol was evaluated by the isobologram technique. The straight – dashed – line represents zero interaction (additivity) between two agents. The experimental isoeffect points are the concentrations (expressed relative to the IC50 concentrations) of the two agents which when combined killed 50% of the cells. When the experimental isoeffect points fall below that line, the combination effect of the two drugs is considered to be supraadditive or synergistic, whereas antagonism occurs if the point lies above it. CIx values of o1 indicate synergy, a value of 1 represents addition, and values of >1 indicate antagonism. (b) Functional blockade of avb3 synergistically enhances Taxol-induced apoptotic cell death in CYR61-overexpressing MCF-7 breast cancer cells. Detection of apoptosis in cells treated with 10 nM Taxol in the absence or presence of 0.5 mM S-247 for 24 h was assessed by TUNEL analysis, performed using the DeadEndTM Fluorometric TUNEL System as described in ‘Material and methods’. The immunoﬂuorescence photomicrographs of cells undergoing apoptosis (green staining) and the corresponding DAPI counterstained photomicrographs are shown. The number in the lower right of each panel represents the percentage of TUNEL-positive cells from one representative experiment (n ¼ 3)

30-OH DNA ends using the enzyme terminal deoxynu- after Taxol exposure as assessed by TUNEL labeling cleotidyl transferase (TdT). In our experiments, fluor- (Figure 5b, bottom panels). Single treatment with the escein-12-dUTP-labeled DNA in Taxol-treated cells was avb3 antagonist S-247 of CYR61-overexpressing MCF- visualized directly by fluorescence microscopy. With 7/C2-6 and matched control MCF-7/V cells did induce a this protocol, Taxol by itself was found to induce negligible increase in apoptotic cell death. In agreement a significant increase in basal apoptosis (e.g. versus with Cruet-Hennequart et al. (2003), who demonstrated untreated cells) in CYR61-negative MCF-7/V cells that blockade of av integrins in ovarian cancer cells by (Figure 5b, top panels). Conversely, the CYR61 deri- neutralizing antibody as well as cyclic-RGD peptides is vative MCF-7/C2-6 did not undergo major apoptosis linked to G1–S cell cycle arrest in the absence of

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 770 apoptosis, we recently observed that S-247 significantly 2002; McDaid and Horwitz, 2001; Seidman et al., 2001; reduces S phase cell subpopulation without promoting Yu et al., 2001). Considering the ability of avb3 apoptotic cell death of HRG- and CYR61-overexpres- antagonists to specifically block ERK1/ERK2 MAPK sing MDA-MB-231 breast cancer cells (Vellon et al., hyperactivation in CYR61-overexpressing breast cancer 2004). These results, altogether, strongly suggest that cells, we reasoned that a functional blockade of avb3 avb3 antagonists such as S-247 may specifically regulate integrin may mimic the enhancing effects of pharmaco- breast cancer cell cycle progression without affecting logical blockers of MEK on Taxol-induced apoptotic breast cancer cell death. Interestingly, while the con- cell death. To examine the mechanism of enhanced current administration of S-247 and Taxol exerted little apoptosis observed with Taxol and avb3 antagonists, effects on Taxol-mediated apoptosis in CYR61-negative their effects on cell cycle progression were studied in MCF-7/V cells, a significant enhancement of Taxol- parallel to those induced by the pharmacological induced apoptosis towards CYR61-overexpressing MERK/ERK inhibitor U0126 (Figure 6b; Table 4). MCF-7/C2-6 cells was detected in the presence of the First, the CYR61-overexpressing MCF-7/C2-9 clone avb3 antagonist S-247 (Figure 5b, bottom panels). was treated with Taxol, S-247, and U0126 as single Indeed, the combination treatment in MCF-7/C2-6 cells agents. As expected, U0126-induced inhibition of produced an B3-fold enhancement of apoptosis over ERK1/ERK2 activity arrested cells in G1, while 45 nM the expected additive effect. This trend was also Taxol (the IC50 value for MCF-7/C2-9 cells) produced a observed following coexposure of CYR61 transfectants significant G2–M block. In total, 13% of the cells to Taxol and avb3 antagonists SC68448 and S-196 underwent apoptosis in the presence of Taxol, whereas (data not shown). Therefore, it is tempting to postulate a negligible increase in cell death was detected in the that CYR61 overexpression in breast cancer cells may presence of U0126 when compared with the untreated drive breast cancer cell survival and chemoresistance by controls. As previously reported by MacKeigan et al. emitting a proliferative and/or survival input via the (2000), the combination of Taxol and U0126 substan- integrin receptor avb3, which might integrate signals tially increased cell death as evidenced by accumulation from CYR61 to proliferative and/or antiapoptotic of a sub-G1 population that has o2 N DNA and signaling pathways. represents dead cells. In addition, a significant increase in the percentage of cells in G2–M was observed when MCF-7/C2-9 cells were concurrently treated with U0126 avb3 antagonists regulate CYR61-overexpressing breast cancer cell survival and chemoresistance through and Taxol. Similarly to U0126, a suboptimal concentra- modulation of the MEK1/MEK2-ERK1/ERK2 tion of the avb3 antagonist S-247 (0.25 mM) caused little MAPK pathway apoptosis (8% compared with 5% in untreated control cells), while it significantly decreased S phase cell To assess whether avb3-dependent activation of PI- subpopulation from 31 to 13%. A more dramatic 30K-AKT and/or MEK1/MEK2-ERK1/ERK2 reduction in the S phase proliferating fraction of MAPK pathways was related to CYR61-enhanced MCF-7/C2-9 cells (up to 75% reduction at 1 mM S- breast cancer cell survival and chemoresistance, the 247) with a modest increase in sub-G1 apoptotic cells activation status of these transduction cascades was was observed in the presence of higher concentrations evaluated in the presence of cell growth inhibitory of S-247 (data not shown). Remarkably, there was a concentrations of avb3 antagonists. Interestingly, opti- synergistic increase in the proportion of sub-G1 cells (up mal concentrations of avb3 antagonists (1 mM) comple- to 27%), while the percentage of Taxol-treated MCF-7/ tely abolished hyperactivation of ERK1/ERK2 MAPK C2-9 cells in G2–M dramatically increased from 30 to in MCF-7 cells engineered to overexpress CYR61, 57% following coexposure to S-247 (Figure 6b; Table 4). whereas AKT activation remained unaffected following These results not only confirm that avb3 antagonists functional blockade of avb3 (Figure 6a). These results such as S-247 specifically regulate cell cycle progression demonstrate that avb3 antagonists such as S-247 and without affecting apoptotic cell death but also further S-196 specifically block the CYR61-triggered ‘CYR61- demonstrate that functional blockade of avb3 signaling avb3 autocrine loop’ by inactivating the MEK1/ in CYR61-overexpressing breast cancer cells mimics the MEK2-ERK1/ERK2 signaling cascade without affec- molecular effects of MEK1/MEK2 inhibitors by syner- ting the antiapoptotic activity of AKT. gistically promoting both the proportion of cells in the Taxol binds to b-tubulin, stabilizes the microtubule, G2–M phase of the cell cycle and the appearance of sub- prevents its depolymerization, leads to arrest of cells in G1 hypodiploid cells caused by Taxol (Figure 6c). G2–M and ultimately, triggers apoptosis (Schiff et al., 1979; Parness and Horwitz, 1981; Blagosklonny et al., CYR61-avb3 loop modulates Taxol-induced accumulation 1995, 1996; Blagosklonny and Fojo, 1999). Interestingly, of p53 through the MEK1/MEK2-ERK1/ERK2 MAPK Taxol also induces the activation of the Raf-MEK-ERK cascade pathway, which is considered a proliferation and cell survival pathway (Blagosklonny et al., 1995; McDaid Data accumulated in recent years have shown that and Horwitz, 2001; Seidman et al., 2001). Accordingly, Taxol can activate a number of signal transduction it has been shown that inhibition of MEK combined pathways implicated in different, often opposite, cell with Taxol induces a dramatic enhancement of apopto- activities such as proliferation, differentiation, stress sis in various tumor cell lines (MacKeigan et al., 2000, response, and apoptosis. Thus, it has been shown that

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 771 a b Activated AKT P -AKT U0126 S-247 1 1 G G AKT AKT

P -ERK1 500 Activated MAPK 320

MCF-7/V P -ERK2 400 MAPK ERK1 240 ERK2 1 Control Taxol 300 Number 1 1

160 -M Number 2 Sub-G 1 G 200 G G -M

Activated AKT P -AKT 2 S G

80 S 500 100 Sub-G

AKT AKT 160 -M 2

G 0 0 P -ERK1 400 Activated MAPK 0 20 40 60 80 100 120 0 20 40 60 80 100 120 120 1 P -ERK2 Channels (FL3-A) Channels (FL3-A)

MCF-7/C2-6 300 ERK1 1 MAPK Number Taxol + U0126 Taxol + S-247 ERK2 Number 80 Sub-G S -M 2 200 -M 1 2 G Sub-G G G 100 S 40 P -AKT Activated AKT -M 200 1

240 2 0 0 AKT AKT 0 20 40 60 80 100 120 0 30 60 90 120 150 G 150

Channels (FL3-A) 180 1 Channels (FL3-A) Sub-G

P -ERK1 1 Activated MAPK P -ERK2 G MCF-7/C2-9 Number Number 120 100 MAPK ERK1 Sub-G ERK2 S

60 S 50 S-247 S-196 S-205 0 0 SC68448

Untreated 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Channels (FL3-A) Channels (FL3-A)

c ∗ P < 0.05 ∗∗ P < 0.005 ∗∗ ∗∗ 6 5 ∗∗

5 ∗ 4

4 3

2 2 Fold change in G2-M phase 1 1 Fold change in Sub-G1 (apoptosis) percentage

0 0 Taxol Taxol S-247 S-247 U0126 U0126 Untreated Untreated axol + U0126 axol + U0126 Taxol + S-247 Taxol + S-247 T T

Figure 6 Functional blockade of avb3 sensitizes CYR61-overexpressing breast cancer cells to Taxol through the MEK1/MEK2- ERK1/ERK2 pathway. (a) Functional blockade of avb3 integrin specifically inactivates CYR61-induced ERK1/ERK2 MAPK transduction cascade without affecting AKT activity. Overnight serum-starved cells at 75–80% confluence were treated for 2 h with either vehicle (v/v) or 1 mM of specific avb3 antagonists (SC68448, S-247, S-196, or S-205). Total protein (25 mg) was resolved by SDS– PAGE and subjected to immunoblot analyses for AKT, P-AKT, ERK1/ERK2 MAPK, and P-ERK1/ERK2 MAPK as described above. Results are representative of three independent experiments. (b) Blockade of avb0 integrin signaling by S-247 and blockade of ERK1/ERK2 MAPK activity by U0126 similarly modulate Taxol-induced effects on cell cycle progression in CYR61-overexpressing MCF-7/C2-9 cells. Distribution of MCF-7/C2-9 cells in the different cell cycle compartments was analysed by flow cytometry after 24 h in presence of Taxol (45 nM), U0126 (20 mM), and S-247 (0.25 mM) as single agents or following concurrent combinations of Taxol plus U0126 or Taxol plus S-247. Representative cell cycle profiles are shown for each treatment. (c) The fold-change in the percentage of sub-G1 (left panel) and G2–M (right panel) phase MCF-7/C2-9 cells relative to untreated cells is shown on the Y-axis. Cell cycle analyses were repeated at least three times. Standard deviations were less 0.1-fold for each experimental condition

Taxol-induced apoptosis involves a dose- and time- 24 h exposure to increasing concentrations of Taxol (up dependent accumulation of the tumor suppressor to 100 nM). Interestingly, CYR61-overexpressing MCF- protein p53 (Blagosklonny et al., 1995; Giannakakau 7 cells showed a markedly reduced ability to upregulate et al., 2001). Here, we evaluated the effects of Taxol on p53 expression in response to Taxol (Figure 7, left p53 expression and ERK1/ERK2 activity in CYR61- panels). In agreement with previous studies, basal ERK1 overexpressing and matched control MCF-7/V cells. As and ERK2 activities in MCF-7/V cells were notably expected, a dramatic dose-dependent increase of p53 enhanced by treatment with nanomolar doses of Taxol, expression was observed in MCF-7/V control cells after whereas high endogenous activity of ERK1/ERK2

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 772 MAPK in CYR61-overexpressing MCF-7 cells was inhibition of MEK1/MEK2-ERK1/ERK2 cascade by enhanced to a lesser extent following Taxol exposure U0126 signiﬁcantly reduced Taxol-induced activation of (Figure 7, right panels). Based on these results, we p53 in matched control MCF-7/V cells, in agreement decided to study whether the functional blockade of avb3 with earlier studies demonstrating that the ability of would restore the ability of Taxol to upregulate p53 in Taxol to both induce wild-type p53 in MCF-7 cells and CYR61-overexpressing breast cancer cells in an ERK1/ activate ERK1/ERK2 requires a functional Raf-MEK1/ ERK2 MAPK-related manner. Remarkably, coadding MEK2-ERK1/ERK2 cascade (Blagosklonny et al., avb3 integrin antagonist S-247 for 24 h elicited a Taxol- 1995). Taken together, these ﬁndings suggest that induced upregulation of p53 in CYR61-overexpressing CYR61 could suppress Taxol-induced apoptosis in an MCF-7 cells comparable to that found in matched avb3-ERK1/ERK2 MAPK dependent manner. Intrigu- control cells (Figure 7, right panels). Moreover, this ingly, CYR61, also through its integrin receptor avb3, effect was concurrent with a complete inactivation of seems to inhibit the function of wild-type p53 following ERK1/ERK2 MAPK. Similarly, MEK inhibitor U0126 Taxol-induced cell damage. We are currently investigat- completely blocked ERK1/ERK2 activation by Taxol in ing whether CYR61-activated avb3-ERK1/ERK2 CYR61 transfectants, and restored the ability of MAPK signaling may contribute to the lack of CYR61-overexpressing MCF-7 cells to upregulate p53 functional p53 by modulating either protein stability following Taxol exposure. Conversely, pharmacological and/or cellular localization of p53 in CYR61-overexpressing MCF-7 cells.

Table 4 Cell cycle percentages for CYR61-overexpressing MCF-7/ Blockade of CYR61 expression decreases cell survival C2-9 cells treated with Taxol, U0126, S-247, and their combinations and chemoresistance in HRG-overexpressing breast Sub-G1 (apoptosis) G1 SG2–M cancer cells Control 5 56 32 12 In a final scenario, we characterized breast cancer cell Taxol 13 28 42 30 survival and chemosensitivity following blockade of U0126 6 79 13 8 CYR61 expression in MCF-7 cells engineered to over- S-247 7 60 24 16 Taxol+U0126 24 37 20 43 express HRG(MCF-7/T7 cells). When the conditioned Taxol+S-247 27 12 31 57 media from T7/CYR61-AS4 and T7/CYR61-AS7 clones were collected and the expression levels of CYR61 were Untreated MCF-7/C2-9 cells and MCF-7/C2-9 cells treated with Taxol determined by Western blotting, very low levels of in the absence or presence of U0126 or S-247 for 24 h were fixed, CYR61 were observed when compared to those found in stained with propidium iodide, and analysed for DNA content by flow cytometry. The percentage of cells in each cell cycle phase is shown. HRG-overexpressing MCF-7/T7 cells (Figure 8a). In Standard deviation in each experimental condition was less than 1% addition, blockade of CYR61 expression in MCF-7 cells after three independent experiments engineered to overexpress HRGwas able to reduce avb3

p53

p53 P -ERK1 P -ERK2 β -actin β-actin MCF-7/V 01 10 100 Taxol (nM) 05010 0 10 50 0 10 50 S-247 (0.5 µM) - - - + + + - - - Taxol (nM) U0126 (20 µM) ------+ + +

p53

p53 P -ERK1 P -ERK2 β -actin β-actin 0110100

MCF-7/C2-6 Taxol (nM) 05010 0 10 50 0 10 50 S-247 (0.5 µM) - - - + + + - - - Taxol (nM) U0126 (20 µM) ------+ + +

Figure 7 CYR61 overexpression impairs Taxol-induced accumulation of p53. Left panels: MCF-7/V (top) and MCF/C2-6 (bottom) cells untreated or treated with increasing concentrations of Taxol for 24 h were lysed as described in ‘Material and methods’. Total protein (50 mg) was immunoblotted for p53 and b-actin. Right panels: MCF-7/V (top) and MCF/C2-6 (bottom) cells untreated or treated with grades concentrations of Taxol in the absence or presence of U0126 or S-247 for 24 h were lysed as described in ‘Material and methods’. Total protein was immunoblotted for p53 (50 mg), MAPK (25 mg), P-MAPK (25 mg), and b-actin (50 mg)

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 773 abc

P -Her-2/neu 700

MCF-7 120 600

** P < 0.005 110

500 100 90 400 MCF-7/T7 versus 80 70 300 ** MCF-7/T7 60 IC50 Number of colonies 200 50 40 T7/CYR61 AS-7 100 30 ** Cell viability (% of control) MCF-7 20 T7 0 10 T7/CYR61 AS-7 CYR61 T7/CYR61 AS-4

T7/CYR61 AS-4 0 1 10 100 TAXOL (nM) MCF-7 MCF-7/T7 T7/CYR61 AS-7 T7/CYR61 AS-4 T7/CYR61 AS-V1 Figure 8 Downregulation of CYR61 expression decreases HRG-enhanced breast cancer cell survival and chemoresistance. (a) Downregulation of CYR61 expression abolishes HRG-promoted estrogen (E2)-independent breast cancer cell growth in anchorage- independent conditions, whereas it does not modify the activation status of Her-2/neu. Top panel: Overnight serum-starved cells at 75–80% confluence were harvested in lysis buffer, 40 mg of total protein was resolved by 3–8% Tris-Acetate NuPAGE, and subjected to immunoblot analyses for P-p185HerÀ2/neu using a Her-2/neu phosphorylation state-specific antibody (clone PN2A). Middle panel: Soft- agar (anchorage-independent) colony formation assays upon E2-depleted conditions for MCF-7, MCF-7/V, MCF-7/T, MCF-7/T7 CYR61 AS-4, and MCF-7/T7 CYR61 AS-7 cells. The data presented are mean of number of colonies (columns)7s.d. (bars). All assays were performed at least three times in triplicate. Bottom panel: CYR61 protein levels in the conditioned media were analysed by Western blotting as described in Figure 1c. (b) Microphotographs of representative soft agar assays are shown (150-fold magnification). (c) Downregulation of CYR61 expression reduces HRG-enhanced breast cancer cell resistance to Taxol-induced cytotoxicity. Taxol sensitivity in HRG-overexpressing MCF-7/T7 cells and their CYR61 antisense derivatives was determined using a standard colorimetric MTT reduction assay as described above. Data presented summarize the mean (7s.d.) of three independent experiments made in triplicate

expression to the basal level observed in the wild-type or agar observed, if any, represent the background level MCF-7/V control cells (data not shown). Moreover, the for the colony formation assay. MCF-7, MCF-7/T7, levels of HRG-induced transactivation of p185HERÀ2/neu T7/CYR61-AS4, and T7/CYR61-AS7 cells were cul- were unchanged in antisense-CYR61 transfectants when tured in E2-depleted media and assayed in a soft-agar compared with those found in T7 and T7/AC-V controls assay in the absence of E2, using phenol red-free (Figure 8a). Thus, this cellular system exhibits an medium containing 10% charcoal-stripped calf serum. intact HRG-Her-2/neu signaling in the absence of The cells were allowed to form colonies for 2 weeks and CYR61 overexpression. Since the colony-forming assay were counted using a colony counter. As expected, in soft agar measures both anchorage-independent MCF-7 cells did not form colonies in the absence proliferation and cell survival, we next focused on an of E2. Interestingly, E2-independent soft agar colony active role of CYR61 on HRG-enhanced breast cancer formation of HRG-overexpressing MCF-7/T7 cells cell survival. Indeed, the acquisition of anchorage- was drastically reduced in the absence of high levels independent growth is generally considered to be one of CYR61 expression. Thus, T7/CYR61-AS4 and of the in vitro properties associated with the malignancy T7/CYR61-AS7 clones were almost unable to grow of cells, and colonization of metastatic tumor cells at a in an anchorage-independent manner (Figure 8a and b). distant site may be partially modeled in soft agar assays. Moreover, downregulation of CYR61 expression in More importantly, it is well established that MCF-7 cells HRG-overexpressing T7 cells signiﬁcantly decreased are not anchorage-independent in the absence of HRG-promoted resistance to Taxol (Figure 8c). These estradiol (E2). Colony formation of MCF-7 cells in soft ﬁndings demonstrate that a functional CYR61-avb3

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 774 loop is necessary for the maintenance of HRG-enhanced resistance to taxanes such as Taxol (Yu et al., 1996, cell survival and chemoresistance in breast cancer 1998; Yu and Hung, 2000; Yu, 2001). Remarkably, our cells. current results reveal that CYR61 can regulate breast cancer cell sensitivity to Taxol-induced cell damage independently of Her-2/neu overexpression and/or HRG-induced Her-2/neu transactivation. During the Discussion preparation of this manuscript, Lin et al. (2004) showed that CYR61 overexpression significantly activates AKT Our early studies demonstrated that CYR61, an and concurrently confers resistance to Taxol-induced angiogenic factor that acts as a downstream effector of apoptosis in breast cancer cells. Since the inhibition HRG-induced metastatic phenotype, is overexpressed in of avb3 using the monoclonal antibody LM609 preven- breast cancers and is actively involved in the acquisition ted this phenotype, the authors suggested that CYR61 of an estrogen-independent breast cancer phenotype can promote breast cancer cell chemoresistance through 0 (Tsai et al., 2000, 2002). CYR61 was found to be activation of an avb3-PI-3 K-AKT signaling pathway. expressed at high levels in HRG-overexpressing invasive Although in our model system CYR61 overexpression breast cancer cell lines and metastatic breast cancers, was found to significantly activate AKT, incubation of whereas expression of CYR61 was undetectable in either CYR61-overexpressing breast cancer cells with small normal breast cancer cell lines or normal breast tissue peptidomimetic antagonists of avb3 did not decrease the (Tsai et al., 2000; Sampath et al., 2001). Moreover, phosphorylation of AKT. In agreement with our forced expression of CYR61 in MCF-7 cells significantly findings, Reinmuth et al. (2003) recently demonstrated enhanced their anchorage-independent cell growth in that the avb3 integrin antagonist S-247 was unable to soft agar and profoundly stimulated their ability to form modulate the activation status of AKT in human large tumors in nude mice (Xie et al., 2001a; Tsai et al., umbilical vein endothelial cells (HUVECs). Considering 2002). These findings motivated us to investigate the that the specific inhibitor of the p110 catalytic subunit of ultimate molecular mechanisms through which CYR61 PI-30K LY294002 suppressed AKT activation and overexpression, independently of HRG, may promote significantly enhanced Taxol killing in CYR61-over- breast cancer aggressiveness. We have previously shown expressing MCF-7 cells, small peptidomimetic antago- that the functional avb3 integrin is required for main- nists of avb3 such as S-247 are likely to inhibit avb3- taining the invasive capacity of HRG-expressing cells driven cellular signaling independently of AKT. For and that the aggressive phenotypes induced by HRGare instance, the integrin-mediated activation of Ras has mediated, in part if not entirely, by CYR61 and its been implicated in preventing anoikis in a manner that is 0 receptor avb3 integrin (Tsai et al., 2000). Here, we independent of PI-3 K, the main upstream activator of further demonstrate that overexpression of CYR61 in AKT (Rosen et al., 2000). In this regard, functional MCF-7 cells is sufficient to dramatically increase the blockade of avb3 using cell growth inhibitory concentra- expression levels of its integrin receptor avb3, which is tions of avb3 integrin antagonists completely prevented known to correlate with the aggressiveness of the breast ERK1/ERK2 MAPK hyperactivation and concurrently cancer disease (Gasparini et al., 1998). Furthermore, sensitized, in a synergistic manner, CYR61-overexpres- functional blockade of avb3 integrin induces a specific sing breast cancer cells to Taxol-induced cell death. cytotoxic effect towards breast cancer cells engineered to Considering that avb3 antagonists such as S-247, when overexpress CYR61, while the specific downregulation used as single agent, showed no apoptotic activity but of CYR61 expression concomitantly reduces both avb3 rather suppressed the S phase cell subpopulation to- expression and HRG-enhanced breast cancer cell wards CYR61- and HRG-overexpressing breast cancer survival. To our best knowledge, this is the first report cells (Vellon et al., 2004), and that the MEK inhibitor showing that CYR61 can promote breast cancer cell U0126 was found to mimic the effects of avb3 proliferation and survival through the upregulation of antagonists on both cell cycle progression and sensitiza- its own integrin receptor avb3, thus generating a tion to Taxol-induced apoptosis and G2–M blockade in CYR61-primed CYR61-avb3 autocrine loop. Interest- CYR61-overexpressing breast cancer cells, it is reason- ingly, Xie et al. (2004) recently reported that CYR61 able to suggest that peptidomimetic avb3 integrin overexpression in glioma cells led to the upregulation of antagonists may modulate Taxol efficacy through distinct integrins. Altogether, these findings strongly specific inhibition of proliferative cascades such as suggest that a prometastatic CYR61-avb3 autocrine loop Ras-Raf-ERK1/ERK2 MAPK, without affecting anti- may occur in a variety of cancers. apoptotic signalings such as PI-30K/AKT. These results, Another important finding of this study is the however, do not exclude the possibility that CYR61 characterization of CYR61 and avb as a survival loop overexpression upregulates AKT in a avb3-dependent protecting human breast cancer cells from cell damage manner. Remarkably, Xie et al. (2004) demonstrated following exposure to Taxol, an antimitotic drug that CYR61, concurrently with its ability to upregulate 0 commonly used in the treatment of advanced or avb3 in glioma cells, significantly activated, in a PI-3 K- metastatic breast cancer. Overexpression and activation dependent manner, the integrin-linked kinase (ILK). of Her-2/neu-coded p185HerÀ2/neu oncoprotein, an HRG- ILK can interact directly with the cytoplasmic domain transactivated orphan receptor, is the major mechanism of the b3 integrin subunit and phosphorylate directly the through which human breast cancer cells acquire serine 473 of AKT (Radeva et al., 1997; Novak et al.,

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 775 1998; Yoganathan et al., 2000). Taking into considera- antagonists to block the Raf-MEK1/MEK2-ERK1/ tion that the small peptidomimetic avb3 antagonists used ERK2 cascade, which can protect cells against the toxic in this study selectively distinguish between the two effects of Taxol (Rasouli-Nia et al., 1998; MacKeigan known b3 integrins (avb3 and aIIbb3), it is likely that et al., 2000, 2002). Although the ultimate role of p53 in these compounds preferentially block av-driven signal- this working model remains elusive, our observations ing in the avb3 heterodimer, thus unaffecting b3- suggest that overexpression of CYR61, in a avb3- dependent molecular effectors such as PI-30Kand dependent manner, inhibits the function of wild-type ILK. Nevertheless, we are currently investigating p53 in MCF-7 cells, which could contribute to their whether a crosssignaling between CYR61, avb3, ILK, acquired resistance to Taxol. In agreement with this and AKT is occurring in breast cancer cells, and whether hypothesis, O’Kelly et al. (2002) suggested that a lack of anti-avb3 therapies based on either monoclonal antibody functional p53 may be a plausible mechanism through (i.e. LM609) or small peptidomimetic antagonists (i.e. S- which CYR61 overexpression confers resistance to 247) differentially modulate downstream transduction growth inhibition by TNFa. cascades in avb3-overexpressing breast cancer cells. Since CYR61 overexpression by itself activates the Interestingly, av integrin blockade by neutralizing anti- expression of the CYR61 integrin receptor avb3, body as well as cyclic-RGD peptides has been demon- upregulation of CYR61 in the epithelial compartment strated to regulate cell proliferation by causing an of breast carcinoma may coordinate a metastatic inhibition of ILK activity and phosphorylation of AKT phenotype in an autocrine manner by activating in ovarian cancer cells (Cruet-Hennequart et al., 2003). proliferative (i.e. ERK1/ERK2 MAPK) and antiapop- In human breast cancer cells, Taxol treatment at the totic (i.e. AKT) signaling pathways, thus predisposing clinical relevant concentration induces expression of the breast tumor epithelial cells towards deregulated pro- tumor suppressor protein p53 (Blagosklonny et al., liferation and enhanced chemoresistance (Figure 9). 1995; Giannakakau et al., 2001). Our experimental Moreover, given that the CYR61 is also an important observations indicate that CYR61 expression may growth factor regulator in the endothelial compartment protect breast cancer cells from Taxol-induced apoptosis of breast carcinoma, it is reasonable to offer a novel by regulating p53 function. Moreover, our experimental hypothetical model in which CYR61 may drive breast observations strongly suggest that overexpression of tumorigenesis and progression in several concerted CYR61, through its avb3 integrin receptor, may inhibit modes: (1) by regulating endothelial cell survival and the function of wild-type p53 in MCF-7 cells in an recruitment during tumor neovascularization in a ERK1/ERK2 MAPK-related manner. Thus, functional paracrine fashion through an avb3-dependent mechan- blockade of avb3 completely restores the ability of ism; (2) by coordinating breast tumor epithelial cell CYR61-overexpressing breast cancer cells to upregulate migration as a chemokinetic factor; (3) by promoting p53 following Taxol exposure, and concurrently en- breast cancer epithelial cell proliferation in an autocrine/ hances Taxol-induced apoptotic cell death. Interest- paracrine fashion either augmenting the bioactivity of ingly, pharmacological inhibition of ERK1/ERK2 other growth factors (i.e. EGFR); and (4) by enhancing MAPK activity by U0126 recapitulates the effects of breast cancer epithelial cell survival and chemoresistance avb3 integrin antagonists on Taxol-induced p53 accu- through activation of prosurvival signaling pathways mulation and apoptosis in CYR61-overexpressing (ERK1/ERK2 MAPK and/or PI-30K/AKT) down- breast cancer cells. Recent studies have revealed that stream of avb3. From a clinical perspective, our the combination of Taxol, which alters microtubule description of a novel CYR61-triggered ‘CYR61-avb3 polymerization and activates ERK1/ERK2, and U0126, autocrine loop’ in breast epithelial cancer cells, together which inhibits MEK1/MEK-ERK1/ERK2 activity in with the cytotoxic and chemosensitizing effects of small the presence of Taxol, results in an impressive enhance- peptidomimetic antagonists of avb3 towards CYR61- ment of tumor cell killing in several cancer models overexpressing breast cancer epithelial cells, provide a (MacKeigan et al., 2000, 2002; McDaid and Horwitz, starting point to further evaluate the use of agents such 2001; Seidman et al., 2001; Yu et al., 2001). Our current as integrin antagonists that should permit a potentially results point towards the following model for the role of synergistic strike against the breast tumor and its CYR61, avb3, and ERK1/ERK2 MAPK in breast supporting vasculature. cancer chemosensitivity to Taxol. CYR61 overexpression, through its integrin receptor avb3, induces the activation of the Raf-MEK1/MEK2-ERK1/ERK2 cas- Materials and methods cade, which may promote proliferation, growth, and survival. Taxol also induces the stimulation of the Cell culture MEK1/MEK2-ERK1/ERK2 pathway. Alone, avb3 integrin antagonists operate like MEK inhibitors, thus MCF-7 breast cancer cells were obtained from the American blocking CYR61-promoted breast cancer cell prolifera- Type Culture Collection (ATCC) and they were routinely grown in phenol red-containing improved MEM (IMEM, tion and survival by interfering with ERK2/ERK2 Biosource International, Camarillo, CA, USA) supplemented activation. In combination, Taxol and avb3 integrin with 5% (v/v) fetal bovine serum (FBS) and 2 mML-glutamine antagonists leads to enhanced breast cancer cell at 371C in a humidiﬁed atmosphere of 95% air and 5% CO2. apoptosis, and a component of this enhanced apoptosis MCF-7 human breast cancer cells were engineered to over- is attributable to the capacity of avb3 integrin express CYR61 as previously described (Tsai et al., 2002).

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 776 CYR61/ v 3-driven autocrineloop

CYR61 HRG v 3 v 3 v 3 v 3 v 3 antagonists

Plasma membrane

Ras PI-3’K Raf expression

3 v

AKT MEK

Nucleus MAPK CYR61-induced HRG-induced CYR61 expression v 3 v 3 CYR61 CYR61 Cell proliferation Cell survival Chemoresistance Figure 9 HRG-induced upregulation of CYR61 may predispose breast tumor epithelial cells towards deregulated proliferation and chemoresistance: a working model. The functional blocking of avb3 integrin differentially induces cytotoxicity towards HRG- and CYR61-overexpressing breast cancer cells, thus suggesting that a CYR61-activated avb3 integrin signaling is actively involved in breast cancer cell survival. Since CYR61 overexpression by itself is sufﬁcient to activate avb3 integrin expression, upregulation of CYR61 in breast cancer epithelial cells may coordinate a metastatic phenotype in an autocrine manner by activating avb3-downstream signaling cascades such as PI-30K-AKT (Lin et al., 2004; Xie et al., 2004) and MEK1/MEK-ERK1/ERK2 MAPK (as described in this manuscript)

Briefly, MCF-7 cells were stably transfected by electroporation antibody (clone LM609) was from Chemicon International with a eucaryotic expression vector pcDNA3.1/zeocine(À) (Temecula, CA, USA). Anti-ERK1/2 MAPK, P-ERK1/2 containing the full-length cDNA of the human CYR61 gene, MAPK, AKT, and P-AKTSer473 rabbit polyclonal antibodies or with an empty vector as a negative control. Transfected were from Cell Signal Technology (Beverly, MD, USA). Anti- MCF-7 cells were selected in the presence of antibiotic zeocine p53 mouse monoclonal antibody (clone Ab-5) and anti-b-actin (200 mg/ml) for 2 weeks. Since CYR61 mRNA and protein goat polyclonal antibody were from Santa Cruz Biotechnology expression as well as cellular behavior did not vary signifi- (Santa Cruz, CA, USA). The activation status of p185HerÀ2/neu cantly and was similar in most of the clones, a representative was characterized using a Her-2/neu phosphorylation state- vector (V3–2) and two clones (C2-6 and C2-9) were chosen for specific antibody (Ab-18, PN2A clone; Lab Vision-NeoMar- the current studies. They were grown as indicated above, kers, Fremont, CA, USA). except than 200 mg/ml of zeocine was added to the culture Six small peptidomimetics antagonists of avb3 containing the medium. RGD (Arg-Gly-Asp) motif (SC56631, SC68448, S-247, S-196, To block CYR61 expression, a eucaryotic expression vector S-205, and S-106) were developed based on a structure–activity pcDNA3.1/zeocine(À) was constructed with the CYR61 relationship-based rational medicinal chemistry as previously cDNA oriented from 30 to 50 end, that is, in an antisense described (Griggs et al., 2001; Meyer et al., 2001). Briefly, direction, and subsequently transfected into HRG-overexpres- human placenta-derived avb3 or human platelet-derived aIIbb3 sing MCF-7/T7 cells, a breast cancer progression model were bound to 96-well microtiter plates and incubated with previously developed in our laboratory by transfecting the human plasma-derived vitronectin conjugated to biotin for full-length HRG-b2 cDNA into nonmetastatic MCF-7 breast detection purposes. Densitometric determination after coin- cancer cells (Staebler et al., 1994; Harris et al., 1998). Several cubation of the labeled ligand with a competitor provides data CYR61 antisense (T7/CYR61-AS) clones were isolated and that when analysed using four-parameter fitting techniques the presence of antisense CYR61 mRNA was confirmed by the yields the IC50 (Table 1). In all, 2 mM stock solutions in sterile RNAse protection assay (data not shown). Also generated water were stored at 41C until use. were multiple clones of vector-transfected MCF-7/T7 (T7/AC- LY29400, a specific inhibitor of the p110 catalytic subunit of V) cells, all of which behaved similarly to the wild-type MCF- PI-30K, and the MEK1/MEK2 inhibitor U0126 were pur- 7/T7 clone. Two T7/CYR61-AS clones (T7/CYR61-AS4 and chased from Calbiochem (San Diego, CA, USA), dissolved in T7/CYR61-AS7) and one vector clone (T7/AC-V1) were DMSO, and stored in the dark as 10 mM stock solutions at characterized further. They were grown as indicated above, À201C until use. For experimental use, Taxol, SC56631, except that 200 mg/ml of zeocine was added to the culture SC68448, S-247, S-196, S-205, S-106, LY294002, and U0126 medium. were freshly prepared from stock solutions and diluted with growth medium. Control cells were cultured in medium containing the same concentrations of DMSO (v/v) or sterile Materials water (v/v) as the experimental cultures with Taxol, LY294002, The primary antibody for CYR61 immunoblotting was a and U0126 or SC56631, SC68448, S-247, S-196, S-205, and rabbit polyclonal antibody (ab2026) from Novus Biologicals, S-106, respectively. Both DMSO and sterile water had no Inc. (Littleton, CO, USA). Anti-avb3 mouse monoclonal harmful effects on the proliferation or experimental cells.

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 777 Immunoblotting analysis of CYR61 in cell supernatants equipped with Cell Quest Software (Becton Dickinson). The mean fluorescence signal associated with cells for labeled a b Subconfluent human breast cancer cells were maintained in v 3 was quantified using the GM fluorescence parameter provided serum-free media for 48 h. The conditioned media were with the software. collected, concentrated 20 Â , and resolved by 10% Tris- glycine SDS–PAGE. The separated proteins were electro- blotted onto a Hybond enhanced chemiluminiscence nitrocellulose membrane (Amersham-Pharmacia). The blotted In situ immunofluorescent staining of avb3 integrin membrane was blocked overnight at 41C with 5% (w/v) Cells were seeded at a density of 2 Â 103 cells/well in an eight- bovine serum albumin (BSA) in Tris-buffered saline containing well chamber slide (Nalge Nunc International, Rochester, NY, 0.5% Tween 20 (TBS-T), and then incubated with a rabbit USA). Following an overnight period of serum-starvation, anti-CYR61 polyclonal antibody (1 : 2000 dilution) for 1 h at cells were washed with PBS, fixed with 4% paraformaldehyde room temperature. After three washes with TBS-T, the blot in PBS for 10 min, permeabilized with 0.2% Triton X-100/PBS was incubated with a 1 : 10 000 dilution of horseradish for 15 min, and stored overnight at 41C with 10% horse serum peroxidase-linked donkey anti-rabbit IgGsecondary in PBS. The cells were washed, and then incubated for 1 h with antibody. The CYR61 protein was detected by the enhanced anti-avb3 monoclonal antibody diluted in 0.05% Triton X-100/ chemiluminiscence reaction using Hyperfilm (Amersham- PBS. After extensive washes, the cells were incubated for Pharmacia). 45 min with an FITC-conjugated anti-mouse IgGsecondary antibody (Jackson Immunoresearch Labs, West Grove, PA,

Immunoblotting analysis of avb3, ERK1/ERK2, P-ERK1/ERK2, USA) diluted 1 : 200 in 0.05% Triton X-100/PBS. The cells AKT, P-AKT, p53, and P-p185HerÀ2/neu were washed five times with PBS and mounted with VECTASHIELD þ DAPI (Vector Laboratories, Burlingame, Experimental cells were washed two times with PBS and then CA, USA). As controls, cells were stained with primary or lysed in buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM secondary antibody alone. Control experiments did not display EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium significant fluorescence in any case (data not shown). Indirect pyrophosphate, 1 mM b-glycerolphosphate, 1 mM Na3VO4, immunofluorescence was recorded on a Zeiss microscope. 1 mg/ml Leupeptin, 1 mM phenylmethylsulfonylfluoride) for Images were noise-filtered, corrected for background, and 30 min on ice. The lysates were cleared by centrifugation in an prepared using Adobe Photoshop. eppendorff tube (15 min at 14 000 r.p.m., 41C). Protein content was determined against a standardized control using the Pierce protein assay kit (Rockford, IL, USA). Equal amounts of Cell viability protein were resuspended in 5 Â Laemmli sample buffer for 10 min at 701C, subjected to electrophoresis on 10% SDS– Cell viability was determined using a modified MTT reduction PAGE (avb3, ERK1/ERK2, P-ERK1/ERK2, AKT, P-AKT, assay (Cell Titer 96 Aqueous Non-Radioactive Cell Prolifera- and p53) or 3–8% Tris-Acetate NuPAGE (P-p185HerÀ2/neu) and tion Assay, Promega Inc., Madison, WI, USA). Briefly, cells in transferred to nitrocellulose membranes. Nonspecific binding exponential growth were harvested by trypsinization and on the nitrocellulose filter paper was minimized by blocking seeded at a concentration of 3 Â 103 cells/200 ml/well into 96- for 1 h at room temperature (RT) with TBS-T (25 mM Tris- well plates, and allowed an overnight period for attachment. HCl, 150 mM NaCl (pH 7.5), and 0.05% Tween 20) containing Then the medium was removed and fresh medium along with 5% (w/v) nonfat dry milk. The treated filters were washed in graded concentrations of avb3 integrin antagonists was added TBS-T and then incubated overnight at 41C with specific to cultures. Compounds were not renewed during the entire primary antibodies in TBS-T/5% (w/v) BSA. The membranes period of cell exposure. Following treatments (6–7 days), 96- were washed in TBS-T, horseradish peroxidase-conjugated well plates were centrifugated at 200 g for 10 min and MTS/ secondary antibodies (Jackson Immunoresearch Labs, West PMS solution was added to each well at a 1/5 volume. After Grove, PA, USA) in TBS-T were added for 1 h, and incubation for 3 h at 371C in the dark, absorbances were immunoreactive bands were detected by enhanced chemilumi- measured at 490 nm using a multiwell plate reader. Control nescence reagent (Pierce, Rockford, IL, USA). Blots were cells without agents were cultured using the same conditions reprobed with an antibody for b-actin to control for protein with comparable media changes. Compounds were not loading and transfer. renewed during the entire period of cell exposure. The cell viability effects from exposure of cells to each compound were analysed generating concentration–effect curves as a plot of Flow cytometric quantification of a b integrin v 3 the fraction of unaffected (surviving) cells versus drug The specific surface expression of avb3 was determined by flow concentration. Dose–response curves were plotted as percen- cytometry by measuring the binding of a mouse anti-avb3 tages of the control cell absorbances, which were obtained antibody (clone LM609; Chemicon International). In total, from control wells treated with appropriate concentrations of 60–70% confluent overnight serum-starved cells were washed the compounds vehicles that were processed simultaneously. once with cold PBS and harvested by scrapping in cold PBS. For each treatment, cell viability was evaluated as a percentage The cells were pelleted and resuspended in cold PBS containing using the following equation: (A570 of treated sample/A570 of 1% FBS. The cells were then incubated with anti-avb3 untreated sample) Â 100. Data presented summarize the mean antibody LM609 at 5 mg/ml for 1 h at 41C. After this, the cells (7s.d.) of five independent experiments made in triplicate. were washed twice with cold PBS, resuspended in cold PBS Breast cancer cells sensitivity to avb3 integrin antagonists was containing 1% FBS, and then incubated with a fluorescein expressed in terms of the concentration of antagonist required isothiocyanate (FITC)-conjugated anti-mouse IgGsecondary for either 30 or 50% (IC30 and IC50) reduction of cell viability. antibody (Jackson Immunoresearch Labs, West Grove, PA, Since the percentage of control absorbance was considered to USA) diluted 1 : 200 in cold PBS containing 1% FBS for be the surviving fraction of cells, the IC30 and IC50 values were 45 min at 41C. Finally, the cells were washed once in cold PBS, defined as the concentration of drug that produced 30 and and flow cytometric analysis was performed with a FACScan 50% reduction in control absorbance (by interpolation), flow cytometer (Becton Dickinson, San Diego, CA, USA) respectively.

Oncogene A novel CYR61-avb3 autocrine loop in breast cancer cells JA Menendez et al 778 Synergy analysis determines the formation of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) after apopto- The nature of the cytotoxic interaction between a b integrin v 3 tic cell death. Briefly, cells (7.5 Â 103/well) were grown in 96- antagonists and Taxol was evaluated by the isobologram well plates and treated, in duplicates, for 24 h with 10 nM technique, a dose-oriented geometric method of assessing drug Taxol. The induction of apoptotic cell death was evaluated interactions (Berenbaum, 1989). Briefly, the concentration of using cytosolic fractions obtained from pooled adherent and one agent producing a desired (e.g. 50% inhibitory) effect was floating cells (obtained by centrifugation at 200g for 10 min) by plotted on the horizontal axis (a b integrin antagonist), and v 3 assessing the enrichment of nucleosomes in the cytoplasm the concentration of another agent producing the same degree using anti-histone biotin and anti-DNA peroxidase antibodies of effect was plotted on the vertical axis (Taxol); a straight line (RT for 2 h), and determined exactly as described in the joining these two points represents zero interaction (additivity) manufacturer’s protocol. After three washes, the peroxidase between two agents. The experimental isoeffect points are the substrate was added to each well, and the plates were read at concentrations (expressed relative to the IC concentrations) 50 405 nm at multiple time intervals. The enrichment of histone- of the two agents which when combined killed 50% of the DNA fragments in treated cells was expressed as fold increase cells. When the experimental isoeffect points fall below that in absorbance as compared with control (vehicle-treated) cells. line, the combination effect of the two drugs is considered to be supra-additive or synergistic, whereas antagonism occurs if the point lies above it. A quantitative index of these TUNEL assay interactions was provided by the isobologram equation Detection of apoptosis by analysis was performed using the CI (a/A) (b/B), where, for this study, A and B represent x ¼ þ DeadEndTM Fluorometric TUNEL System (Promega Inc., the respective concentrations of a b antagonist and Taxol v 3 Madison, WI, USA) according to the manufacturer’s instruc- required to produce a fixed level of inhibition (IC ) when 50 tions. Briefly, cells were split at a density of 1 Â 104 cells/well in administered alone, a and b represent the concentrations eight-well chamber slides (Lab-Tek). Following 24 h incuba- required for the same effect when the drugs were administered tion, the cells were treated with 10 nM Taxol in the absence or in combination, and CI represents an index of drug x presence of 0.5 mM S-247 for 24 h. Following treatments, cells interaction (interaction index). CI values of 1 indicate x o were washed twice with PBS and fixed with 4% methanol-free synergy, a value of 1 represents addition, and values of >1 paraformaldehyde for 10 min. Cells were washed twice more indicate antagonism. with PBS and permeabilized with 0.2% Triton X-100 for 5 min. After two more washes, each slide was covered with Soft-agar colony formation assays equilibration buffer for 10 min more. The buffer was then For Taxol sensitivity assays, 1 ml of 0.6% agar in phenol-red- aspirated, and the slides were incubated with TdT buffer at containing IMEM supplemented with 10% FBS was solidified 371C for 1 h. The reaction was stopped with 2 Â SSC, in the bottom of six-well culture dishes. Exponentially growing counterstained with DAPI, and the slides were viewed with cells to be assayed were suspended at 421C in 1 ml of 0.3% an immunofluorescence microscope (Zeiss). agar in complete medium (10% FBS) supplemented with graded concentrations of Taxol. A total of 20 000 cells were Cell cycle analysis seeded in each well. For estrogen-dependent soft agar colony formation assays, cells were grown in phenol-red-free IMEM Adherent and detached cells were collected after trypsin and 5% charcoal-treated calf serum (CCS) for 5 days in T75 detachment, washed in PBS and centrifugated at 1500 r.p.m. 6 flasks. A bottom layer of 1 ml IMEM containing 0.6% agar Cells were resuspended at 2 Â 10 cells/ml in PBS and fixed in and 10% CCS was prepared in six-well culture dishes. After ice-cold 70% ethanol for, at least, 24 h. Fixed cells were the bottom layer solidified, cells (20 000/well) were added in a centrifugated at 1500 r.p.m., and each sample resuspended in propidium iodide (PI) stain buffer (0.1% Triton X-100, 200 mg 1 ml top layer containing E2-depleted 10% CCS. After 2 weeks of DNase-free RNase A, 20 mg of PI) in PBS for 30 min. After of incubation at 371Cin5%CO2 and 95% air, colonies measuring X50 mm were counted after staining with nitroblue staining, samples were analysed using a FACScan (Becton tetrazolium (Sigma, St Louis, MO, USA) using a cell colony Dickinson) and ModFit LT (Verity Software). counter (Ommias 3600, Imaging Products International, Inc., Charley, VA, USA). Statistical analysis All observations were confirmed by at least three, independent Cell death ELISA experiments. The data are presented as means7s.d. The The induction of cell death was assessed using the Cell Death Student’s t test (paired and unpaired) was used to evaluate the Detection ELISAPLUS kit obtained from Roche Molecular statistical significance of mean values. Statistical significance Biochemicals USA (Indianapolis, IN, USA). This kit levels were Po0.05 (denoted as*) and Po0.005 (denoted uses a photometric enzyme immunoassay that quantitatively as**). All Ps are two-tailed.

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