The α2β1 binding domain of chondroadherin inhibits breast cancer-induced bone metastases and impairs primary tumour growth: A preclinical study

Cancer Letters 358 (2015) 67–75

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

The α2β1 binding domain of chondroadherin inhibits breast cancerinduced bone metastases and impairs primary tumour growth: A preclinical study Nadia Rucci a,*,1, Mattia Capulli a,1, Ole K. Olstad b, Patrik Önnerfjord c, Viveka Tillgren c, Kaare M. Gautvik b, Dick Heinegård c,2, Anna Teti a a

Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy Department of Clinical Chemistry, Ullevaal University Hospital, Institute of Medical Biochemistry, University of Oslo, Oslo, Norway c Department of Clinical Sciences, Section of Rheumatology, Lund University, Lund, Sweden b

A R T I C L E

I N F O

Article history: Received 3 July 2014 Received in revised form 9 December 2014 Accepted 12 December 2014 Keywords:: cyclicCHAD Doxorubicin Breast cancer Bone metastases

A B S T R A C T

cyclicCHAD is a peptide representing the α 2 β 1 integrin binding sequence of the matrix protein chondroadherin (CHAD), which in our hands proved effective at counteracting bone loss in ovariectomised mice by inhibiting osteoclastogenesis. Given that bone metastases are characterised by exacerbated osteoclast activity as well, we tested this therapy in mice intracardiacally injected with the osteotropic human breast cancer cell line MDA-MB-231. Treatment with cyclicCHAD significantly decreased cachexia and incidence of bone metastases, and induced a trend of reduction of visceral metastasis volume, while in orthotopically injected mice cyclicCHAD reduced tumour volume. In vitro studies showed its ability to impair tumour cell motility and invasion, suggesting a direct effect not only on osteoclasts but also on the tumour cell phenotype. Interestingly, when administered together with a suboptimal, poorly effective, dose of doxorubicin (DXR), cyclicCHAD improved survival and reduced visceral metastases volume to a level similar to that of the optimal dose of DXR alone. Taken together, these preclinical data suggest that cyclicCHAD is a new inhibitor of bone metastases, with an appreciable direct effect also on tumour growth and a synergistic activity in combination with low dose chemotherapy, underscoring an important translational impact. © 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction Metastasis formation is an inefficient event requiring a proper microenvironment to be colonised, as proposed more than one hundred years ago by Stephen Paget [1]. Indeed, once in the bloodstream, tumour cells are attracted to favoured sites of metastasis through site-specific interactions with cells and matrices in the target tissue [2], which support their proliferation and expansion in the host organ. In bone metastases, tumour cells cooperate with bone cells promoting a vicious cycle of bone destruction and tumour growth that disrupts the physiologic cycle orchestrating bone cell activities. Indeed, tumour cells do not destroy the bone, but they secrete factors that stimulate osteoclast-mediated bone resorption and the

* Corresponding author. Tel.:+39 0862 433525; fax: +39 0862 433523. E-mail address: [email protected] (N. Rucci). 1 Equal contributors. 2 This article is dedicated to Professor Dick Heinegård, who prematurely died after having largely contributed to this work and approved the manuscript. http://dx.doi.org/10.1016/j.canlet.2014.12.032 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.

consequent release of numerous molecules, stored in the bone matrix or secreted by the cell, that potentiate cancer spread and bone destruction [3,4]. Therefore, osteolysis fulfils two functions: i) creates the physical space for tumour expansion and 2) stimulates tumour growth [3]. As a matter of fact, the use of antiresorptive drugs is a fruitful approach for the treatment of bone metastases, since the block of osteoclast resorbing activity impairs osteolysis as well as the release of pro-tumour factors. However, although these treatments significantly reduce morbidity, they do not improve patient survival, likely because they are poorly effective in directly counteracting tumour cell expansion. Therefore, there is a need for further experimentation to better antagonise cancer-induced skeletal-related events. In a recent work, we demonstrated that chondroadherin (CHAD), an extracellular protein expressed in the cartilage territorial matrix and in bone, was downregulated in bone biopsies of osteoporotic patients as well as in ovariectomised mice, two conditions characterised by exacerbated osteoclast activity [5]. The analysis of the active domains of CHAD led us to hypothesise that its C-terminal integrin binding domain could elicit a bone matrix–bone cell signalling. Therefore, we synthesised a stable cyclic peptide representing the integrin binding domain of CHAD (i.e. cyclicCHAD), which proved

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effective at inhibiting osteoclast formation as well as at counteracting bone loss in a mouse model of osteoporosis [5]. Based on this finding, in this work we tested the cyclicCHAD as a new experimental therapeutic agent for bone metastases. We observed that, along with its strong anti-resorptive activity, the peptide exhibited an anti-tumour effect, reducing cancer cell migration and invasion ability in vitro and slowing tumour growth in vivo. Most importantly, in a combination therapy, cyclicCHAD strongly potentiated the efficacy of a low dose of the highly toxic chemotherapeutics doxorubicin (DXR) in vivo, and reduced tumour cell migration in vitro with an efficacy comparable to that of the highest effective dose of DXR, thus providing robust translational prospective to improve the treatment of bone metastases in patients. Materials and methods Materials DMEM (Dulbecco’s modified minimum essential medium), FBS (foetal bovine serum), penicillin, streptomycin and trypsin were from GIBCO (Uxbridge, UK). Sterile plastic ware was from Falcon Becton-Dickinson (Cowley, Oxford, UK) or Costar (Cambridge, MA, USA). Trizol reagent, primers and reagents for real-time RT-PCR and semiquantitative RT-PCR were from Invitrogen (Carlsbad, CA). The Brilliant® SYBR® Green QPCR master mix was from Stratagene (La Jolla, CA). The Human Cell Motility PCR Array (#PAHS-128A2) was from SA Biosciences a Qiagen company (Valencia, CA). All the other reagents were of the purest grade from Sigma Aldrich Co. (St. Louis, MO). Synthesis and purification of the peptides A peptide representing the C-terminal disulphide loop that contains the cell binding sequence of CHAD (CQLRGLRRWLEAKASRPDATC) was synthesised and made cyclic via the cysteine residues in both termini (cyclicCHAD) [5]. A scrambledCHAD (CQLRGLRRWEKLAASRPDATC) with no activity was also synthesised and used as a control peptide [5]. All synthesis and purification of peptides were made by Schafer-N (Copenhagen, Denmark). The correct structure and purity of peptides was checked in-house by MALDI-TOF mass spectrometry.

Tumour immunohistochemistry Orthotopic tumours were excised, fixed in 4% paraformaldehyde and embedded in paraffin. Slide-mounted tissue sections (4 μm thick) were deparaffinised in xylene and hydrated serially in 100%, 95%, and 80% ethanol. Endogenous peroxidases were quenched in 3% H2O2 in PBS for 1 hour, then sections were incubated with the anti-Ki67 primary antibody for 1 hour at room temperature. Sections were washed in PBS and antibody binding was revealed using the Ultra-Vision Detection System anti-Polyvalent HRP/DAB kit according to the manufacturer’s instructions (Lab Vision; Scaffold, UK). Micro computed tomography (μCT) analysis Images from tibias fixed in 4% formaldehyde were acquired in a SkyScan 1174 with a resolution of 6 μm (X-ray voltage 50 kV). 3D and 2D morphometric parameters were calculated, for the trabecular bone, 150 slides (4 μm tick) from the growth plate. Threshold values were applied for segmenting trabecular bone corresponding to bone mineral density values of 0.6/cm3 calcium hydroxyapatite. 3D parameters were based on analysis of a Marching Cubes type model with a rendered surface [8]. Calculation of all of 2D areas and perimeters was based on the Pratt algorithm [9]. Bone structural variables and nomenclature were those suggested in Bouxsein et al. [10]. Bone histomorphometry Tibias fixed in 4% paraformaldehyde were dehydrated in acetone and processed for glycol–methacrylate embedding without decalcification. Histomorphometric measurements were carried out on 5 μm-thick sections with an interactive image analysis system (IAS 2000; Delta Sistemi, Rome, Italy) [5] and with the suggested nomenclature [11]. Osteoclast number/bone surface (number/mm2) and osteoclast surface/bone surface (%) were evaluated after histochemically staining the sections for Tartrate-Resistant Acid Phosphatase (TRAcP) activity. Osteoblast surface/ bone surface (%) was evaluated after staining the sections with methylene blue/ azure II. Proliferation, migration and invasion assays

Procedures involving animals and their care were conducted in conformity with national and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, Dec. 12, 1987; Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica Italiana n. 40, Feb. 18, 1992; NIH guide for the Care and Use of Laboratory Animals, NIH Publication No. 85–23, 1985), and obtained institutional approval.

Cell proliferation was evaluated using the Promega CellTiter 96® MTS kit, according to the manufacturer’s protocol. To test motility, MDA-MB-231 and 4T1 cells were grown in standard conditions until 70% confluence. Monolayers were then scratched with the tip of a 20-μl pipette, and after 24 hours ten representative fields were marked in the previously scratched area and the numbers of cells migrated in these fields were counted. Migration was also assessed by adding cells (1 × 106) to 12 μm polycarbonate filters coated with 4.5 μg/cm2 gelatine in the upper compartment of transwell chambers. After 6 hours of incubation in the presence of NIH3T3 cell-conditioned media used as chemoattractant, filters were stained with haematoxylin/eosin. For cell invasion assay, procedure was the same as for migration except that the polycarbonate filters were coated with 35 μg/cm2 matrigel and the experiment was carried on for 12 hours.

Intracardiac injection of tumour cells

Mammosphere cultures

The human breast cancer cell line MDA-MB-231 or the mouse mammary carcinoma 4T1 cell line were injected (1 × 105/100 μl PBS) into the left ventricle of 4-weekold female BALB/c-nu/nu mice anaesthetised with pentobarbital (60 mg/kg b.w.) [6,7]. Animals were treated intraperitoneally (i.p.), 5 days/week for 5 weeks with vehicle (PBS), 10 mg/kg body weight (b.w.) cyclicCHAD or scrambledCHAD, 1 mg/kg b.w. alendronate as reference drug, 0.1 mg/kg and 0.2 mg/kg b.w. doxorubicin (DXR) or with combined 10 mg/kg b.w. cyclicCHAD plus 0.1 mg/kg b.w. DXR, starting the day after tumour cell injection. Animals were monitored daily for cachexia, evaluated by body weight waste, and for behaviour and survival. At the end of the experiment, mice were sacrificed and subjected to X-ray analysis and to anatomical dissection for evaluation of bone and visceral metastases, respectively. Bone metastases were quantified as percent of hindlimbs carrying a metastasis (evaluated, by X ray analysis, as an osteolytic area) over the total number of hindlimbs of injected mice. To evaluate visceral metastases, at necroscopy they were classified according to the diameter in the following diameter categories: small (<2 mm), medium (2–5 mm) and large (>5 mm). The metastasis volume was then calculated according to the the formula of the volume of a sphere [4/3XπXr2].

MDA-MB-231 and 4T1 cells were cultured as monolayers up to 80–90% of confluence. Cells were then detached and mammospheres were allowed to grow by plating cells in low attachment 24 well plates, at a density of 130 cells/cm2, in DMEM without serum and with 1% N2 (1 mM human transferrin, 0.0861 mM insulin recombinant full chain, 0.002 mM progesterone, 10 mM putrescine, 0.003 mM selenite) and 1% Invitrogen B27 supplement for 7 days. At the end of the experiment, the number of mammospheres was counted, while their size was calculated as volume according to the formula of a sphere (4/3x π x r3).

Animals

Orthotopic injection of tumour cells Four-week-old female BALB/c and BALB/c-nu/nu mice were anaesthetised as described above, and 4T1 (1 × 106/50 μl PBS) or MDA-MB-231 cells (1.5 × 106/50 μl PBS), respectively, were injected into the fat pad of the inguinal left breast using a tuberculin syringe with a 27½G needle. Animals were treated i.p., 5 days/week with vehicle (PBS) or 10 mg/kg b.w. cyclicCHAD starting the day after tumour cell injection or when tumours reached a volume of 1 cm3. Tumour volume was calculated twice a week by measuring their three axes using a calliper and applying the formula of the volume of an ellipse [4/3XπX(axbxc)] [7].

cDNA real-time array cDNA obtained from RNA extracted from MDA-MB-231 cells treated with vehicle or cyclicCHAD was mixed with a SYBR® Green master mix (RT2 SYBR® Green qPCR master mix) and then dispensed in the wells of the Human Cell Motility PCR Array (#PAHS-128A2, Superarray Biosciences) including 84 genes involved in cell motility. Wells were subjected to real-time RT-PCR (Stratagene MX 3000), following the manufacturer’s instructions. Array data were automatically analysed by the dedicated software, RT2 Profiler PCR Array data analysis template v3.2 (Superarray Biosciences). Briefly, each array was normalised by shifting the global minimum value to 0 to reduce the differences in fluorescence intensity in different arrays. The gene expression profiles from each array were then normalised versus a set of 6 housekeeping genes, and genes were filtered using as cut-off parameter the statistical significance (P-value, obtained from a t-test performed on all the arrays, <0.05). Comparative real-time RT-PCR Total RNA was extracted from cultured cells using the Trizol® procedure. RNA (1 μg) was reverse transcribed in cDNA using M-MLV reverse transcriptase and the

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Table 1 Primer sequences and real-time RT-PCR conditions employed. Gene

Primers

PCR conditions

GAPDH

Fw 5′- CTGCACCACCAACTGCTTAG-3′ Rv 5′- AGGTCCACCACTGACACGTT-3′ Fw 5′-ATCACGGTTGGAACGAGAAC-3′ Rv 5′-CACACGGACACCCTTTTCTT-3′ Fw 5′-GGCTGGATGGACGACTATGA-3′ Rv 5′-TGAGGATGACGAACTTGCTG-3′ Fw 5′-CCTGCTTGAGAAGGCCTATG -3′ Rv 5′- GGTCCACGTTGTTCCACTCT-3′ Fw 5′-ACAAGCCTACCCCTCCAGAT-3′ Rv 5′-TCCCGTCAGTTGGTAGGTTC-3′ Fw 5′-GAAGCTCGAGAGAAGGCTGA-3′ Rv 5′-TTGTCTGCCTCAAATGCTTG -3′ Fw 5′-CTGTTCGGAGGCTTCAACTC -3′ Rv 5′-TGAGAGGCAGTAGGCACCTT-3′

35 cycles: 94 °C 30 sec, 60 °C 30 sec, 72 °C 30 sec 35 cycles: 94 °C 30 sec, 60 °C 30 sec, 72 °C 30 sec 35 cycles: 94 °C 30 sec, 60 °C 30 sec, 72 °C 30 sec 35 cycles: 94 °C 30 sec, 60 °C 30 sec, 72 °C 30 sec 35 cycles: 94 °C 30 sec, 60 °C 30 sec, 72 °C 30 sec 35 cycles: 94 °C 30 sec, 60 °C 30 sec, 72 °C 30 sec 35 cycles: 94 °C 30 sec, 60 °C 30 sec, 72 °C 30 sec

ACTR2 BCAR1 CAPN1 NOS2 ROCK1 SRC

equivalent of 0.1 μg was employed for the PCR reactions using the Brilliant® SYBR® Green QPCR master mix. PCR conditions and primer pairs are listed in Table 1. Statistics All data are expressed as the mean + SEM. Statistical analysis was performed by unpaired Student’s t test, ANOVA or Chi-square tests as indicated in the figure legends. A P value <0.05 was considered statistically significant.

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observed that cyclicCHAD significantly inhibited tumour growth over time, up to the 24th day of treatment (Fig. 2A). Moreover, cyclicCHAD induced a trend of reduction of the tumour necrotic area (Fig. 2B), inhibited cell proliferation (Figs 2C, D), and increased apoptosis (Fig. 2E). It has to be noted, however, that at the end of experiments lasting longer (30th day from tumour cell implant), the reduction of tumour volume in cyclicCHAD-treated mice was no longer statistically significant compared to control groups (Fig. 2F). We also observed a beneficial effect of cyclicCHAD on tumour growth starting the treatment after the primary tumours had reached a standardised volume of 1 cm3, more or less after two weeks from tumour cell inoculation (Fig. 3). We also performed a similar experiment injecting 4T1 cells into the mammary fat pad of BALB/c mice, and observed a trend of reduction of tumour growth in cyclicCHAD-treated mice compared to controls (fold increase of tumour growth in 1 week: vehicle = 1.8 ± 0.24, cyclicCHAD = 1.2 ± 0.15, scrambledCHAD = 2.3 ± 0.74; P = 0.12 vs scrambledCHAD and P = 0.08 vs vehicle). Of note, 1 out of 5 and 3 out of 5 mice treated with scrambledCHAD or vehicle, respectively, died within 10 days from injection, while cyclicCHAD injected mice survived until the end of the experiment. Consistently, necroscopy showed a greater propensity of control groups to develop visceral metastases in multiple organs compared to mice treated with cyclicCHAD (Supplementary Table S3).

Results Effect of cyclicCHAD on tumour cells Effect of cyclicCHAD on experimentally induced bone metastases As demonstrated in our previous work, cyclicCHAD strongly inhibited osteoclast formation in vitro [5] and in vivo [5] (Supplementary Table S1). Since osteoclast formation and bone resorption are exacerbated in osteolytic bone tumours, we investigated whether cyclicCHAD could impair osteoclast activation in experimental bone metastases induced by intracardiac injection of the human breast cancer cell line MDA-MB-231 in BALB/c nu/nu immunocompromised female mice. Interestingly, while the survival of the mice was not significantly improved (cyclicCHAD, 18%; vehicle or scrambledCHAD, 15%), at variance with the reference drug alendronate, in mice treated with cyclicCHAD we observed a significant reduction of cachexia (Fig. 1A), together with a significant decrease of bone metastasis incidence (Fig. 1B). Consistent with its anti-osteoclastic effect, we observed a significant lower level of the serum bone resorption marker carboxy-terminal collagen I crosslinks (CTX) (Fig. 1C) along with a higher bone volume over total tissue volume (Fig. 1D) in cyclicCHAD treated mice compared to the vehicle treated group. Osteoclast number (Fig. 1E) and surface (Fig. 1F) over bone surface were also reduced by cyclicCHAD treatment. The incidence of visceral metastases was not influenced by the treatment with the peptide (Fig. 1G), while their size showed a tendency to reduction (Fig. 1H). In contrast, scrambledCHAD was ineffective on all these parameters (Fig. 1). Of note, a similar experiment performed by injecting the highly aggressive mouse mammary carcinoma 4T1 cells in the left ventricle of BALB/c mice showed a trend of reduction of bone and visceral metastasis incidence, and visceral metastasis volume in cyclicCHAD-treated mice compared to controls (Supplementary Table S2). Effect of cyclicCHAD on in vivo tumour growth The observation that cyclicCHAD had an effect, although weak, on visceral metastases prompted us to address whether it could alter the in vivo tumour growth by orthotopic injection of MDAMB-231 cells in the mammary fat pad of BALB/c nu/nu female mice. Treating the mice since the day after tumour cell injection, we

Based on these results, we assessed whether cyclicCHAD could directly affect tumour cell behaviour in vitro. CyclicCHAD failed to affect MDA-MB-231 cell proliferation (Supplementary Fig. S1A) and survival (Supplementary Fig. S1B) as well as mammosphere number (Supplementary Fig. S1C) and size (Supplementary Fig. S1D). These two latter parameters were also unchanged in the 4T1 cell line after treatment with cyclicCHAD (Supplementary Fig. S1E,F). However, consistent with what we previously observed in osteoclast precursors [5], cyclicCHAD reduced tumour cell migration (Fig. 4A,B) and invasion (Fig. 4C). As similar effect of cyclicCHAD on motility was observed in 4T1 cells (Fig. 4D). We had previously identified the mechanism of action of cyclicCHAD in pre-osteoclasts being associated with the transcriptional inhibition of the nitric oxide synthase type 2 (nos2), which leads to an impairment of NO-dependent pre-osteoclast migration [5]. Disappointingly, semiquantitative RT-PCR analysis unveiled no basal expression of NOS2 in MDA-MB-231 cells (Fig. 4E), suggesting a different mechanism of action. To identify this alternative mechanism, vehicle- and cyclicCHAD-treated tumour cells were subjected to a real time RT-PCR array containing 84 genes involved in cell motility. Elaboration of data evidenced 13 genes downregulated in cyclicCHAD-treated cells compared to control cells. Six of them were subsequently confirmed to be significantly reduced by comparative real-time RT-PCR (Fig. 4F). Taken together, these data indicate that, at variance with pre-osteoclasts, cyclicCHAD affects MDA-MB231 cell motility by downregulation of genes independent of the NOS2-associated pathway. Combination therapy with doxorubicin Our observations support a weak effect of cyclicCHAD on tumour cells, and this circumstance reduces the interest for the use of cyclicCHAD as anti-tumour agent. However, anti-tumour agents, such as conventional chemotherapeutics, typically induce important, sometime life-threatening, adverse events. Doxorubicin (DXR) is largely used in breast cancer adjuvant therapy and present serious limitation for its high toxicity [12]. Toxicity could be reduced by lower dosages, which, however, are poorly effective at counteracting tumour

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Fig. 1. Effect of cyclicCHAD on experimentally induced metastases. Four-week-old female BALB/c-nu/nu mice were intracardiacally injected with MDA-MB-231 cells. Animals were treated i.p., 5 days/week, with vehicle (PBS, white bar), 10 mg/kg cyclicCHAD (cCHAD, grey bar) or scrambledCHAD (sCHAD, white bar with pattern) and with the reference drug alendronate (dark grey bar), starting the day after injection. Evaluation of (A) cachexia and (B) bone metastasis incidence, (C) serum levels of CTX, (D) bone volume/total tissue volume, osteoclast (E) number and (F) surface, (G) visceral metastases incidence and (H) visceral metastasis volume. In (A,B,G) = Chi square test; in (C– F,H) = Student’s t test; *P < 0.048, **P < 0.01 vs. vehicle or scrambledCHAD. In (G,H) data are statistically not significant, with P > 0.1 vs. vehicle or scrambledCHAD; N. mice/group = 20.

growth. We therefore tested whether a cyclicCHAD support therapy in combination with DXR could improve the effects of a low dose of DXR in mice subjected to intracardiac injection of MDA-MB231 cells. Optimal dose of DXR (0.2 mg/kg) was effective at improving cachexia (Fig. 5A), survival (Fig. 5B), incidence of bone metastases (Fig. 5C) and incidence and volume of visceral metastases (Fig. 5D and E, respectively), while half dose was modestly effective (Fig. 5). However, a combination of half dose of DXR (0.1 mg/kg) and optimal dose of cyclicCHAD (10 mg/kg) strongly reduced cachexia, improved survival, decreased incidence of bone and visceral metastases,

and decreased the size of visceral metastases (Fig. 5) compared to half dose of DXR alone or cyclicCHAD alone. Notably, these results were similar to those obtained with the optimal dose of DXR, suggesting additive effects of the two compounds that could have important translational consequences. To assess whether any effect on MDA-MB-231 cells motility was involved in the efficacy of the combination treatment, we performed a scratch assay. We observed that the combination therapy with cyclicCHAD and low DXR concentration significantly reduced tumour cell motility with an efficacy comparable to that of the

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Fig. 2. Effect of cyclicCHAD on primary tumour growth. Four-week-old female BALB/c-nu/nu mice were subjected to orthotopic injection of MDA-MB-231 cells. Starting the day after cell injection, mice were daily treated with vehicle (white diamond or white bar) or 10 mg/kg cyclicCHAD (cCHAD, grey circle or grey bar). (A) Development of tumours was monitored by measuring tumour volume twice a week until the end of the experiment. (B) Haematoxylin/eosin staining of two representative tumours. Black lines underline the necrotic area, quantified in the graph. (C) Evaluation of number of mitotic cells per field of view (FOV) in tumour sections stained with haematoxylin/ eosin. (D) Immunohistochemical detection of the proliferation marker Ki67 (inset pictures) and evaluation of number of Ki67 positive nuclei (graph). (E) Evaluation of apoptotic cells in tumour sections stained with haematoxylin/eosin. (F) Evaluation of tumour volume at the times indicated in abscissa. In (A) *P < 0.05 vs. vehicle (ANOVA); in (C–F) *P = 0.048, **P < 0.02 and ***P < 0.001 vs. vehicle (unpaired Student’s t-test). N. mice/group = 16.

highest concentration of DXR (0.1 μM) alone (Fig. 6A). In contrast, cyclicCHAD had no effect on proliferation, and induced a weak effect in combination with low DXR concentration compared to the highest concentration of DXR alone (Fig. 6B). These results suggest that cyclicCHAD synergy with DXR relies essentially on its capability to affect tumour cell motility. Discussion Bone metastases represent the point of no return for many neoplastic patients, because once they are established the chances of survival dramatically drop [13]. Moreover, they significantly affect the quality of life of patients, causing bone loss, intractable pain, hypercalcaemia, osteolytic fractures and nerve compression syndromes [14]. Exacerbated osteoclast formation and bone resorption are typical of osteolytic bone metastases in breast cancer patients, which justifies the employment of antiresorptive drugs classically

effective in the treatment of osteoporosis. Moreover, these treatments are also beneficial to prevent osteoporosis secondary to anticancer therapy, especially in breast cancer patients subjected to antiestrogen treatment [15]. Antiresorptive drugs available so far to treat patients with bone metastases are the bisphosphonates, which are effective bone resorption inhibitors that bind preferentially at sites of active bone metabolism [16]. Unfortunately, although bisphosphonates significantly reduce morbidity, they do not improve patient survival [17]. Moreover, dosage of bisphosphonates for neoplastic patients is high, severely affecting the quality of bone and increasing the risk of osteonecrosis of the jaw [16]. Among the new antiresorptive drugs under clinical trials, there is denosumab, a fully humanised, function blocking monoclonal antibody to the osteoclastogenic cytokine Receptor Activator of Nuclear Factor-κB Ligand (RANKL) [18]. Although denosumab prolongs the time to skeletal-related events and inhibits the onset of pain via the suppression of osteoclast

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Fig. 3. Effect of cyclicCHAD on primary tumour growth. Four-week-old female BALB/ c-nu/nu mice were subjected to orthotopic injection of MDA-MB-231 cells and treated with vehicle (white diamond) or cyclicCHAD (cCHAD, grey circle), when tumours reached a volume of 1 cm3. Then tumour growth was measured twice a week until the end of the experiment (fold increase vs. tumour volume at the 1st day of treatment, i.e. 14 days after tumour cells inoculation). *P < 0.05 vs. vehicle (ANOVA); N. mice/group = 16.

activation [19], it does not extend survival of metastatic patients as well [20]. It also induces side effects, including osteonecrosis of the jaw and hypocalcemia [21,22], not different from those triggered by bisphosphonates. We demonstrated that the antiresorptive peptide cyclicCHAD reduces the incidence of bone metastases in mice subjected to intracardiac injection of breast cancer cells. Furthermore, besides this predictable result, at variance with our reference drug alendronate, cyclicCHAD induced additional beneficial effects, which included reduced cachexia and, to some extent, decreased size of visceral metastases. Noteworthy, our results demonstrated that, in addition to the well-documented antiosteoclastic effect [5], it also acts as an antitumour agent, although this effect is weak and does not persist over time. It is clear though that we cannot support the use of cyclicCHAD as a full antitumour therapy, although we can confirm an advantage compared to current antiresorptive drugs. Most interestingly, however, cyclicCHAD showed an additive effect over a suboptimal dose of the highly toxic chemotherapeutics DXR. As suggested by the “biological” origin of our peptide, which is part of the bone matrix protein CHAD, in our previous study we did not

Fig. 4. Effect of cyclicCHAD on tumour cell migration and invasion. (A) Wound healing scratch assay, (B) migration and (C) invasion assays of MDA-MB-231 cells treated with vehicle (white bar), 25 μM cyclicCHAD (cCHAD, grey bar) or 25 μM scrambledCHAD (sCHAD, white bar with pattern). (D) Wound healing scratch assay in 4T1 cells treated with vehicle (white bar), 25 μM cyclicCHAD (cCHAD, grey bar), or 25 μM scrambledCHAD (sCHAD, white bar with pattern). (E) RT-PCR evaluation of NOS2 (Nitric Oxide Synthase 2) normalised for the housekeeping gene GAPDH in MDA-MB-231 cells treated as indicated, and in human osteoclasts (hOCs) used as positive control. (F) Real time RT-PCR of mRNAs of the indicated genes in cyclicCHAD-treated MDA-MB-231 relative to vehicle-treated cells (set to 1, dotted line). Results are (A–D, F) the mean ± SEM or (E) representative of three independent experiments. *P < 0.05, **P < 0.015 and ***P < 0.007 vs. vehicle (unpaired Student’s t-test).

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Fig. 5. In vivo combination therapy with doxorubicin. Four-week-old female BALB/c-nu/nu mice were intracardiacally injected with MDA-MB-231 cells. Animals were treated 5 days/week with vehicle (PBS, white bar), 10 mg/kg cyclicCHAD (cCHAD, grey bar), doxorubicin (DXR) [0.1 mg/kg (DXR 0.1, light grey bar) and 0.2 mg/kg (DXR 0.2, dark grey bar)] and with combined cyclicCHAD (10 mg/kg) + DXR (0.1 mg/kg) (cCHAD + DXR 0.1, black bar), starting the day after injection. Evaluation of incidence of (A) cachexia, (B) survival, (C) bone and (D) visceral metastases and (E) visceral metastasis volume. In (A-D) = Chi square test; in E = Student’s t test; *P < 0.045 and **P < 0.01 vs. vehicle or scrambledCHAD, #P < 0.03 vs. DXR 0.1. N. mice/group = 16.

observe any toxic effect of cyclicCHAD in healthy mice even at high dose [5], suggesting that it can be safely used for therapy. These observations could have important translational implications as potential means to affect the metastatic disease by the cyclicCHAD strong antiresorptive effect associated to its weak antitumour effect that, in combination with DXR, could allow reducing DXR dosage and consequent adverse events, keeping a high efficacy of treatment. We have also identified the molecular mechanism whereby cyclicCHAD affects tumour cells. In vitro there is a clear, although weak, inhibition of motility; however, this inhibition is significantly amplified in the combination treatment with suboptimal concentration of DXR, with an effect comparable to that of the highest concentration of DXR alone, thus strengthening our in vivo results.

Motility was also impaired by cyclicCHAD in pre-osteoclasts [5]. However, this latter effect was much stronger and the underlying mechanisms of action was different from MDA-MB-231 cells. In preosteoclasts, the main outcome was the transcriptional inhibition of nos2 and the impairment of the NO-mediated downstream signals that led to the reduced expression of two NO-related motility genes, vasp and migfilin. MDA-MB-231 cells showed no basal expression of NOS2. Nevertheless, they exhibited cyclicCHAD-dependent transcriptional downregulation of a group of other motility genes, including MYH10, ACTR2, CAPN1, SRC, BCAR1, ROCK1, which are independent of the NO pathway. Besides the well-known role of the protoncogene SRC in tumorigenesis and in the development of bone metastases [7], also some of the other downregulated genes are implicated in breast cancer development, such as ROCK1, which codes

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Fig. 6. In vitro combination treatment with doxorubicin. MDA-MB-231 cells were treated with vehicle (PBS, white bar), 12.5 μM cyclicCHAD (cCHAD, grey bar), doxorubicin (DXR) [0.05 μM (DXR 0.05, light grey bar), and 1 μM (DXR 1, dark grey bar)], and with combined cyclicCHAD (12.5 μM) + DXR (0.05 μM) (cCHAD + DXR 0.05, black bar. (A) Motility (scratch assay) and (B) proliferation (MTS) tests were performed after 24 and 48 hours of treatment, respectively. Results are the mean ± SEM of at least three independent experiments. *P < 0.01, **P < 0.008 and ***P < 0.0007 vs. vehicle (Student’s t test).

for a protein involved in the remodelling of the actin cytoskeleton. It has been recently demonstrated that the Growth and Differentiation Factor-9 (GDF-9) promoted the invasiveness of prostate cancer PC3 cells together with an induction in the expression of ROCK1, which in turn led to epithelial-mesenchymal transition of these cells [23]. As far as the BCAR1 gene (alias p130CAS) is concerned, it is a Src family kinase substrate involved in various cellular events, including migration, survival, transformation, and invasion [24]. In univariate and multivariate survival analyses, high BCAR1 levels were associated with poor relapse-free survival and poor overall survival of breast cancer patients. Moreover, the response to tamoxifen therapy in patients with recurrent disease was reduced if the primary tumour expressed high levels of BCAR1 protein [25]. Relevant in breast cancer is also CAPN1, belonging to the calpain family, a group of neutral cysteine proteases that require calcium for their catalytic activity and whose expression and activity have been correlated with tumour progression [26]. Indeed, it has been recently demonstrated that Calpain-1 expression correlates with grade, oestrogen/progesterone receptor status, and triple-negative disease in breast cancer patients [27]. Although neither MDA-MB-231 cell proliferation nor cell survival were affected by cyclicCHAD in vitro, we observed that in vivo both variables were inhibited in orthotopic tumours. The underlying mechanisms are at present unknown and, at this stage, we can only speculate the involvement of molecular interactions between the peptide and the tumour microenvironment that could generate conditions for additional effects not seen in vitro. Further work is necessary for the complete elucidation of these interesting features. Taken together, these data demonstrated that cyclicCHAD not only counteracts the development of breast cancer-induced bone metastases with an effectiveness similar to that of the antiresorptive drugs, but it also shows further beneficial effects in cancer-induced diseases due to its ability to weakly inhibit tumour expansion and strongly potentiate the efficacy of a suboptimal dose of the highly toxic chemotherapeutics DXR. These results could have important translational implications for future treatments of bone metastases in patients. Acknowledgements This work was supported by European Union grant OSTEOGENE (contract No. LSHM-CT-2003-502941) to AT, DH and KMG, by grants

from the “Associazione Italiana per la Ricerca sul Cancro” (AIRC) to AT (project numbers 4518 and 12713) and NR (project number IG11950), and by a grant from the Swedish Research Council to DH. MC was a recipient of the “1st Mariuccia Borrini” fellowship from the “Associazione Italiana per la Ricerca sul Cancro” (AIRC). We are indebted to Dr. Rita Di Massimo for her excellent contribution in writing this manuscript. Disclosures None. Conflict of interest None. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2014.12.032. References [1] S. Paget, The distribution of secondary growths in cancer of the breast, Lancet 1 (1889) 571–573. [2] K.N. Weilbaecher, T.A. Guise, L.K. McCauley, Cancer to bone: a fatal attraction, Nat Rev Cancer 11 (2011) 411–425. [3] G.D. Roodman, Mechanisms of bone metastasis, N. Engl. J. Med. 350 (2004) 1655–1664. [4] P. Clezardin, A. Teti, Bone metastasis: pathogenesis and therapeutic implications, Clin. Exp. Metastasis 24 (2007) 599–608. [5] M. Capulli, O.K. Olstad, P. Onnerfjord, V. Tillgren, M. Muraca, K.M. Gautvik, et al., The c-terminl domain of chondroadherin: a new regulator of osteoclast motility counteracting bone loss, J. Bone Miner. Res. 29 (2014) 1833–1846. [6] T. Yoneda, A. Sasaki, C. Dunstan, P.J. Williams, F. Bauss, Y.A. De Clerck, et al., Inhibition of osteolytic bone metastasis of breast cancer by combined treatment with bisphophonate ibandronate and tissue inhibitor of the matrix metalloproteinase-2, J. Clin. Invest. 99 (1997) 2509–2517. [7] N. Rucci, I. Recchia, A. Angelucci, M. Alamanou, A. Del Fattore, D. Fortunati, et al., Inhibition of protein kinase c-Src reduces the incidence of breast cancer metastases and increases survival in mice: implications for therapy, J. Pharmacol. Exp. Ther. 318 (2006) 161–172. [8] W.E. Lorensen, H.E. Cline, Marching cubes: a high resolution 3d surface construction algorithm, Comput. Graph. (ACM) 21 (1987) 163–169. [9] W.K. Pratt, Digital Image Processing, 2nd ed., Wiley, New York, 1991.

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