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Endothelial ALK1 Is a Therapeutic Target to Block Metastatic Dissemination of Breast Cancer Sara I

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Endothelial ALK1 Is a Therapeutic Target to Block Metastatic Dissemination of Breast Cancer Sara I Cancer Microenvironment and Immunology Research Endothelial ALK1 Is a Therapeutic Target to Block Metastatic Dissemination of Breast Cancer Sara I. Cunha1,2, Matteo Bocci3, John Lovrot€ 4, Nikolas Eleftheriou3, Pernilla Roswall2, Eugenia Cordero3, Linda Lindstrom€ 4, Michael Bartoschek3, B. Kristian Haller2, R. Scott Pearsall5, Aaron W. Mulivor5, Ravindra Kumar5, Christer Larsson3, Jonas Bergh4, and Kristian Pietras2,3 Abstract Exploration of new strategies for the prevention of breast can- combinations with chemotherapy. Gene-expression analysis of cer metastasis is justifiably at the center of clinical attention. In this breast cancer specimens from a population-based nested case– study, we combined a computational biology approach with control study encompassing 768 subjects defined endothelial mechanism-based preclinical trials to identify inhibitors of acti- expression of ALK1 as an independent and highly specificprog- vin-like receptor kinase (ALK) 1 as effective agents for blocking nostic factor for metastatic manifestation, a finding that was angiogenesis and metastasis in breast cancer. Pharmacologic tar- corroborated in an independent clinical cohort. Overall, our results geting of ALK1 provided long-term therapeutic benefitinmouse suggest that pharmacologic inhibition of endothelial ALK1 con- models of mammary carcinoma, accompanied by strikingly stitutes a tractable strategy for interfering with metastatic dissem- reduced metastatic colonization as a monotherapy or part of ination of breast cancer. Cancer Res; 75(12); 2445–56. Ó2015 AACR. Introduction disseminated breast cancer, both for the neoadjuvant and the adjuvant setting, are sorely needed. Exploration of new strategies for the prevention of breast cancer The angiogenic process is required for tumor progression from metastasis is justifiably at the center of clinical attention (1). an indolent state (5). The vascular tree provides a tumor with its The haematogenous dissemination of tumor cells is a multistep metabolic requirements, while simultaneously providing an process requiring: (i) detachment of malignant cells from the escape route by which malignant cells can leave the primary primary tumor, (ii) intravasation into and extravasation from the tumor bulk. Indeed, in particular cases, high vascular density was blood stream, and (iii) colonization of the distant organ (2). demonstrated to be a prognostic factor for poor outcome in breast However, we currently have limited knowledge on the molecular cancer, as well as in other malignancies (6, 7). The introduction of drivers contributing to each of the steps in the metastatic cascade multitargeted agents incorporating antiangiogenic activity in in breast cancer, and thus efforts to target-specific signaling path- clinical practice has led to improved disease control in terms of ways involved in the systemic spread of the disease have largely prolonged progression-free survival (PFS). Consequently, drugs been unsuccessful so far (3). In cases where breast tumors are targeting the vascular endothelial growth factor (VEGF) pathway found in early stages, the prognosis following adequate therapy is are now included in the first line therapy for metastatic disease for good with a 3-year overall survival (OS) rate reaching above 90% a range of malignancies (8–11). However, attempts to target (4). Nevertheless, many women are not diagnosed until the tumor tumor angiogenesis in breast cancer has met with ambiguous has reached advanced stages; tumor stage is an established factor success (12, 13). Although providing initial relief for metastatic for poor prognosis (4). Thus, novel treatment strategies to combat breast cancer patients by improving response rates and prolong- ing PFS, no conclusive evidence for long-term benefit in OS has been provided to date (13). Consistent with this clinical reality, recent preclinical studies indicate that tumors in mice treated 1Ludwig Institute for Cancer Research, Uppsala, Sweden. 2Division of systemically with anti-VEGF therapy rapidly acquire resistance, Vascular Biology, Department of Medical Biochemistry and Biophys- coupled to recurring tumors that appear to be more locally ics, Karolinska Institutet, Stockholm, Sweden. 3Division of Translation- al Cancer Research, Department of Laboratory Medicine, Medicon invasive and have a higher propensity to seed distant metastases Village, Lund University, Lund, Sweden. 4Department of Oncology (14–16). Thus, the need for a mechanism-based and clinically 5 and Pathology, Karolinska Institutet, Stockholm, Sweden. Acceleron relevant search for alternative angiogenic pathways that may serve Pharma, Cambridge, Massachusetts. as targets for more efficacious drugs without affecting disease stage Note: Supplementary data for this article are available at Cancer Research in breast cancer is highly warranted. Online (http://cancerres.aacrjournals.org/). ALK1 is a type I receptor in the large TGFb family expressed Corresponding Author: Kristian Pietras, Lund University, Division of Transla- selectively by endothelial cells (17). Pharmacologic targeting of tional Cancer Research, Medicon Village, Building 404:A3, SE-22381 Lund, ALK1 has demonstrable therapeutic efficacy in a diverse set of Sweden. Phone: 46-709-209-709; E-mail: [email protected] mouse models of cancer (18–20). Here, we investigate the utility doi: 10.1158/0008-5472.CAN-14-3706 of ALK1 inhibition as an antimetastatic therapy in breast cancer Ó2015 American Association for Cancer Research. using a combined approach of preclinical testing of a clinically www.aacrjournals.org 2445 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2015 American Association for Cancer Research. Cunha et al. 2446 Cancer Res; 75(12) June 15, 2015 Cancer Research Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2015 American Association for Cancer Research. Targeting ALK1 Impairs Distant Breast Cancer Dissemination tractable ALK1 inhibitor and analysis of gene-expression patterns Ag staining, a high pH antigen retrieval buffer (pH 10; DAKO) relating to metastatic spread in breast cancer patient specimens. was used, followed by washes and quenching of endogenous peroxidase activity with 50% methanol/3% H2O2 in PBS for 10 Materials and Methods minutes in room temperature. A wash in PBS preceded blocking with 10% normal goat serum in TNB for 1 hour. The primary Cell culture antibody against T-Ag (1:1,000; a kind gift from Douglas MDA-MB-231 were maintained in culture in DMEM (Invitro- Hanahan, EPFL) was incubated at 4Covernight.Afterwashes gen), supplemented with 10% FCS. EO771 breast cancer cells with PBS containing 0.1% Tween-20, a suitable biotinylated were cultured in DMEM supplemented with 20% FCS. secondary antibody was incubated for 45 minutes in room temperature. Animal care and tumor establishment For cryopreservation, tumors, livers and/or lungs were kept in All animal experiments were approved by the local ethical 30% sucrose at 4 C overnight, followed by embedding in cryo- committee for animal care in Stockholm and Lund (permits sectioning media. Frozen sections were fixed in ice-cold acetone, N96/11 and M142/13). The RIP-TAg2 mice (C57Bl6/J back- followed by blocking using serum free protein block (DAKO) for ground) received water supplemented with 5% sugar to alleviate >90 minutes at room temperature. Primary antibodies directed hypoglycemia from 10 weeks of age. Total tumor burden per against CD31 (dilution 1:100; Pharmingen MEC13.3), podoca- mouse was calculated as the sum of the volume of each individual lyxin (dilution 1:100; R&D Systems, AF1556), and BMP9 (dilu- tumor dissected from a RIP-TAg2 mouse pancreas. The MMTV- tion 1:500; Abcam; ab35088) were incubated overnight at 4 C. PyMT mice (FVB/n strain background) were followed for tumor Appropriate Alexa 594 and Alexa 488–flourochrome-conjugated growth from 8 to 12 weeks in early-stage trials and from 11 to 15 secondary antibodies (Invitrogen) were used and sections were weeks of age in late stage trials. Primary tumor burden was 0 finally mounted using 4 ,6-diamidino-2-phenylindole-contain- determined by caliper measurements on live sedated mice once ing mounting media (Vector Laboratories). a week and the total tumor burden per mouse was calculated as the sum of the volume of each individual mammary tumor. To Quantification of metastases establish EO771 tumors, 5  105 cells were injected orthotopi- The right lateral liver lobe from RIP-TAg2 mice or the left lung cally into the fourth mammary fat pad of isofluran-anesthesized lobes of MMTV-PyMT or EO771-bearing mice were embedded in wildtype C57Bl6/J females. 1  106 cells of the human metastatic paraffin upon tissue fixation. The metastatic burden was assessed breast cancer cell line MDA-MB-231 were transplanted s.c. to by serial sectioning of the entire lung/liver lobe. Following hema- immunocompromised SCID mice. In all cases, tumor volume was toxylin and eosin (H&E) staining on every 25th section, the calculated as length  width2  p/6. number of metastatic foci (>8 cells in diameter) was determined in >15 sections per mouse and >5 mice per group. Therapeutic trials Control IgG or RAP-041 (Acceleron Pharma) was diluted in TBS RNA isolation and quantitative RT-PCR and administered twice weekly by i.p. injection at 12 mg/kg per Total RNA of 12-weeks-old MMTV-PyMT mice mammary tissue injection. Mice carrying orthotopic EO771 mammary carcinomas was isolated using TRIzol extraction (Invitrogen), followed by the were treated with either RAP-041
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