Annexin 2–CXCL12 Interactions Regulate Metastatic Cell Targeting

Published OnlineFirst August 19, 2014; DOI: 10.1158/1541-7786.MCR-14-0118

Signal Transduction Molecular Cancer Research Annexin 2–CXCL12 Interactions Regulate Metastatic Cell Targeting and Growth in the Bone Marrow Younghun Jung1, Jingcheng Wang1, Eunsohl Lee1, Samantha McGee1, Janice E. Berry1, Kenji Yumoto1, Jinlu Dai2, Evan T. Keller2, Yusuke Shiozawa1, and Russell S. Taichman1

Abstract

Anxa2þ þ Anxa2 Annexin 2 (ANXA2) plays a critical role in hematopoietic stem BMSC / or BMSC / . Significantly larger tumors grew Anxa2þ þ cell (HSC) localization to the marrow niche. In part, ANXA2 in the mice when the tumors were established with BMSC / Anxa2 supports HSCs by serving as an anchor for stromal-derived factor- compared with the tumors established with BMSC / .In 1 (CXCL12/SDF-1). Recently, it was demonstrated that prostate addition, fewer prostate cancer cells underwent apoptosis when Anxa2þ þ Anxa2 cancer cells, like HSCs, use ANXA2 to establish metastases cocultured with BMSC / compared with BMSC / , in marrow. The present study determined the capacity of and similar results were obtained in tumors grown in vivo. Finally, ANXA2 expression by bone marrow stromal cells (BMSC) to significantly more vascular structures were observed in the tumors Anxa2þ þ facilitate tumor recruitment and growth through ANXA2– established with the BMSC / compared with the tumors Anxa2 CXCL12 interactions. Significantly more CXCL12 was expressed established with BMSC / . Thus, ANXA2–CXCL12 interac- Anxa2þ þ Anxa2 by BMSC / than by BMSC / resulting in more pros- tions play a crucial role in the recruitment, growth, and survival of Anxa2þ þ tate cancer cells migrating and binding to BMSC / than prostate cancer cells in the marrow. Anxa2 BMSC / , and these activities were reduced when CXCL12 interactions were blocked. To further confirm that BMSC Implications: The tumor microenvironment interaction between signaling through ANXA2–CXCL12 plays a critical role in tumor ANXA2–CXCL12 is critical for metastatic phenotypes and may growth, immunocompromised SCID mice were subcutaneously impact chemotherapeutic potential. Mol Cancer Res; 13(1); 197–207. implanted with human prostate cancer cells mixed with 2014 AACR.

Introduction osteoclasts, osteomacs, and endothelial cells aid in the development and organization of the HSC niche (7–9). Prostate cancer is the second leading cause of cancer deaths in Bone marrow stromal cells (BMSC) are a heterogeneous pop- American males (1, 2). The high mortality rate is due mainly to the ulation of cells, which also are known to contribute to the spread of malignant cells to many tissues including bone (3, 4). At generation of the HSC niche. There is growing evidence that a the single cell level, the multistep process of metastasis is very subpopulation of BMSCs has progenitor and/or stem-like activ- inefﬁcient, where less than 2% of disseminated tumor cells ities that are capable of locally generating multiple connective (DTC) are estimated to form micrometastasis and fewer still tissue lineages under conditions of stress or wound repair (e.g., (0.02%) develop into macrometastasis (5). In part, a barrier endothelial, adipocytic, osteoblastic, cartilage, and muscle cells; for DTCs to form clinically relevant metastases is not only the ref. 7–9). Although still controversial, if BMSCs are mobilized ability to survive in the circulation during dissemination but also or injected into the circulation, BMSCs may participate in tissue the ability to home to and engage in a safe environment, which repair at distant organs (9). In the context of tumors, when can immediately support their survival. We recently reported that BMSCs are localized in tumor beds, cytokine cross-talk leads to DTCs target and engage the hematopoietic stem cell (HSC) niche the emergence of an increasingly aggressive tumor phenotype to establish metastases in marrow (6). While the nature and the (10–17). components of the HSC niche remain controversial, signiﬁcant Two important signal transducers in the bone marrow niche are evidence suggests that cells of the osteoblastic lineage, adipocytes, stromal-derived factor-1 (SDF-1 or CXCL12; refs. 18–29) and annexin 2 (ANXA2; ref. 30). CXCL12 is a central regulator of HSC homing and recruitment into marrow and HSC mobilization into 1Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan. 2Department of Urology and the circulation. Indeed, blockage of CXCL12 or its receptor pre- Pathology, University of Michigan School of Medicine, Ann Arbor, vents HSC homing or mobilizes niche-bound HSC into the Michigan. circulation (18–24). ANXA2, on the other hand, likely serves as Corresponding Author: Russell S. Taichman, Department of Periodontics and an adhesion molecule, facilitating HSC binding to the osteoblas- Oral Medicine, University of Michigan School of Dentistry, 1011 North University tic niche (25). Critically, it has been shown that CXCL12 and Avenue, Ann Arbor, MI 48109. Phone: 1-734-764-9952; Fax: 1-734-763-5503; ANXA2 directly bind to one another, and this interaction facil- E-mail: [email protected] itates hematopoietic progenitor cell migration in response to doi: 10.1158/1541-7786.MCR-14-0118 CXCL12 (26). In fact, loss of ANXA2 is associated with decreased 2014 American Association for Cancer Research. levels of CXCL12 in marrow. Likewise, CXCL12 and ANXA2 play

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similar roles in recruiting and localizing tumor cells into the HSC were labeled with 2.5 mg/mL of the lipophilic dye carboxyfluor- niche (27–30). escein diacetate (CFDA, cat. V12883; Molecular Probes) in RPMI In the present study, we explored whether ANXA2 facilitates for 30 minutes at 37C and washed in PBS. Thereafter, prostate metastasis and growth of prostate cancer through its interactions cancer cells (1 104 cells per well) were added to each well and with CXCL12. We demonstrate that ANXA2-expressing BMSCs were left for 30 minutes at room temperature to allow binding to express more CXCL12 than do ANXA2-deficient BMSCs, and the BMSCs. In some cases, adhesion assays of prostate cancer cells to production of these 2 proteins increases the recruitment of pros- BMSCs were done in the presence or absence of function blocking tate cancer cells into the marrow niche. In addition, CXCL12 antibody to ANXA2 (cat. 610069; BD Pharmingen), with matched produced by ANXA2-expressing BMSCs promotes prostate cancer IgG as an antibody control. Total fluorescence per well was proliferation, protects prostate cancer from chemotherapy- determined initially, followed by washing wells with PBS to induced apoptosis, and increases the development of vascular quantify fluorescence for prostate cancer cells adherent to BMSCs. structures. Our results suggest that ANXA2 and CXCL12 interactions facilitate the recruitment, growth, and survival of prostate Transwell chemotaxis assay Anxa2þ þ Anxa2 cancer cells in marrow. BMSC / or BMSC / (1 105 cells per well) were seeded onto 24-well culture plates. Prostate cancer cells or Materials and Methods HBMECs were labeled with 2.5 mg/mL of CFDA as described Cell culture above. Prostate cancer cells or HBMECs were resuspended in The human prostate cancer cell line PC3 was obtained from the serum-free RPMI-1640 and equilibrated for 10 minutes at 37 C. ATCC. C4-2B, the metastatic subline of the prostate cancer cell line CFDA-labeled prostate cancer cells or HBMECs were loaded into LNCaP, was originally isolated from a lymph node of a patient the top chambers of 8-mm Transwell microporous membrane 24- with disseminated bony and lymph node involvement. Lucifer- well plates (cat. 3422; Costar Corp.). A total of 650 mLof Luc Luc ase-expressing prostate cancer cell lines (PC3 and C4-2B conditioned medium (CM) from 3-day cultures of GFP Anxa2þ/þ Anxa2/ 5 cells) and GFP-expressing prostate cancer cell lines (PC3 and BMSC or BMSC (1 10 cells per well) was GFP C4-2B cells) were established by lentiviral transduction. added into the bottom well. The plates were incubated at 37 C Human bone marrow endothelial cells (HBMEC) were isolated for 3 hours. At the termination of the experiments, the intensity of from a normal Caucasian male and immortalized with SV40 large fluorescence in the lower chamber, indicating the number of cells T-antigen (31). Prostate cancer cells and HBMEC were grown in that had migrated, was determined by plate reader (Molecular RPMI-1640 (Invitrogen) supplemented with 10% FBS and 1% Probes). In some cases, migration of prostate cancer cells to CM penicillin/streptomycin (P/S). isolated from BMSCs was analyzed in the presence of neutralizing þ þ BMSCs from Anxa2 / or Anxa2 / mice (5- to 7-week-old) anti-CXCL12 monoclonal antibodies (120 mg/mL, cat. MAB310; were used for this study. Dr. K.A. Hajjar (Weill Medical College of R&D Systems) or AMD3100, a selective CXCR4 antagonist (4 ng/ Cornell University, New York, NY) graciously provided our lab- mL, cat. A5602; Sigma). oratory with a pair of the homozygous Anxa2 / mice for breed- ing (32). After sacrifice, marrow was flushed from femurs and Cell death assay þ/þ / Anxa2þ þ Anxa2 tibias of both Anxa2 and Anxa2 mice with a-MEM (Invi- BMSC / or BMSC / (1 105 cells per well) were trogen) containing 2% FBS and cultured in a-MEM containing seeded onto 12-well culture plates for 24 hours. GFP-expressing 15% heat-inactivated FBS and 1% P/S to generate primary BMSCs. prostate cancer cells (1 105 cells per well) were added to the Once confluent, the cells were passaged 2 to 3 times with trypsin to wells and cultured together with the BMSCs for 48 hours before minimize macrophage contamination. Subsequently, repopu- the addition of the anticancer drug, taxotere (1 mg/mL, cat. lated BMSCs were obtained, and the cells were cultured in a-MEM NDC0409-0201-10; Hospira) for an additional 48 hours. In containing 10% heat-inactivated FBS and 1% P/S. These isolated addition, HBMECs (1 105 cells per well) were seeded onto Anxa2þ/þ Anxa2/ cells were termed as BMSC and BMSC . For dif- 12-well culture plates for 24 hours and the cells were treated with ferentiation assays, mouse primary BMSCs were cultured in ANXA2 (1 mg/mL, cat. 11-511-248-344; Genway Biotech) or adipogenic, osteogenic, or chondrogenic conditions for 2 weeks, CXCL12 (200 ng/mL, cat. 350-NS; R&D Systems) for 48 hours. fixed and stained for identification with Alizarin Red S, Oil Red O, Apoptosis in these cultures was measured by flow cytometry and Alcian Blue, respectively. (FACSAria dual laser flow-cytometer, Becton Dickinson) using PE Annexin V Apoptosis Detection Kit I (cat. 559763; BD Bios- Proliferation assay ciences). Assays were performed in triplicate and the results are Anxa2þ þ Anxa2 BMSC / or BMSC / (1 105 cells per well) were representative of 3 independent experiments. In tissue sections Luc seeded onto 12-well culture plates for 24 hours and PC3 cells (1 from mice inoculated with human prostate cancer, apoptosis of Anxa2þ þ 105 cells per well) were directly cocultured with BMSC / prostate cancer cells and endothelial cells in the tumors was Anxa2 or BMSC / for 48 hours. The amount of luciferase in these measured by terminal deoxynucleotidyl transferase–mediated cultures was measured by Dual-Luciferase Reporter Assay kit (cat. dUTP nick end labeling (TUNEL) staining (cat. 11684817910; E1910; Promega) and detected using a luminometer (BD Mono- Roche). light 2010, BD Biosciences). Quantitative RT-PCR Adhesion assay Total RNA was extracted using the RNeasy mini or micro kit Anxa2þ þ Anxa2 BMSC / or BMSC / were plated onto 96-well plates (Qiagen). cDNA was established using a First-Strand synthesis kit at a concentration of 1 104 cells/100 mL/well in growth medium, (Invitrogen). Quantitative PCR was performed on an ABI 7700 and the cultures were incubated for 2 days. Prostate cancer cells sequence detector (Applied Biosystems) using TaqMan Universal

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PCR Master Mix Kit (Applied Biosystems) according to the direc- Intratibial tumor growth luc tions of manufacturer. TaqMan MGB probes (Applied Biosys- To evaluate tumor growth in bone marrow, PC3 (1 105 luc tems) were as follows: human Anxa2r (Hs01588662_s1) and cells) or C4-2B (3 105 cells) were inoculated intratibially into human Cxcr4 (Hs00237052_m1). b-Actin (Hs99999903_m1) 5- to 7-week-old male SCID mice. For intratibial injection, mice was used as an internal control for the normalization of target were anesthetized with 2.5% isofluorane/air, and both legs were gene expression. cleaned with betadine and 70% ethanol. The knee was flexed and a 27-G3/8-inch needle was inserted into the proximal end of right ELISAs tibia followed by injection of 20 mL single-cell suspensions of Anxa2þ/þ Anxa2/ 5 BMSC or BMSC (1 10 cells per well) were prostate cancer cells. Tumors were allowed to become established cultured in 12-well culture plates for 48 hours. An antibody for 5 weeks. sandwich ELISA was used to evaluate CXCL12 levels in the CM All animals were sacrificed at week 5 and tibiae with tumors fi by the modi cation of directions of manufacturer (R&D Systems). were collected for histology. CXCL12 secretion levels were normalized to total cell numbers. Statistical analyses Immunostaining Results are presented as mean SD. Significance of the differ- Cells and tumor sections were used for immunostaining. Cells ence between 2 measurements was determined by unpaired fi were xed with 10% neutral-buffered formalin (cat. HT501320; Student t test, and multiple comparisons were evaluated by the Sigma) and permeabilized with PBT (1:500 dilution of Triton- Newman–Keuls multiple comparison test. Values of P < 0.05 were fi X100 in PBS). Tumor sections were deparaf nized, hydrated, and considered significant. then blocked with Image-iT FX signal enhancer (cat. I36933; Life Technology) for 30 minutes and incubated for 2 hours at room Results temperature with primary antibodies combined with reagents of Zenon Alexa Fluor 488 (green) or 555 (red) labeling kit (Life ANXA2 and CXCL12 expression are correlated in the bone Technology). Polyclonal anti-ANXA2 (cat. 610069; 1:25 dilution; marrow environment BD Pharmingen), polyclonal anti-CXCL12 (cat. ab25117; 1:100 We examined the extent to which ANXA2 expression is dilution; Abcam), monoclonal anti-HLA (cat. 311402; 1:50 dilu- correlated with CXCL12 expression in the long bones of tion; BioLegend), CD31 (cat. ab32457; 1:100 dilution; Abcam), wild-type and Anxa2 knocked-out mice. CXCL12 expression monoclonal p-AKT (cat. ab81283; 1:100 dilution; Abcam), and was robustly expressed in marrow of wild-type mice compared monoclonal anti-AKT (cat. ab32505; 1:100 dilution; Abcam) with ANXA2-deficient mice (Fig. 1A). In vitro, more CXCL12- were used as primary antibodies. After washing with PBS, tumor expressing cells were identified in BMSCs derived from wild- Anxa2þ/þ sections were mounted with ProLong Gold antifade reagent with type mice (BMSC ) compared with BMSCs derived from Anxa2/ DAPI (cat. P36931; Invitrogen). Images were taken with Olympus Anxa2 knocked-out mice (BMSC ;Fig.1B).CXCL12 Anxa2þ/þ FV-500 confocal microscope. In some cases, the tumor sections protein levels in the CM of BMSC were significantly Anxa2/ were stained by TUNEL staining (cat. 11684817910; Roche). greater than those generated by BMSC cultures (Fig. 1C). The findings suggest that ANXA2 expression by Western blotting BMSCs is correlated with CXCL12 expression in bone marrow PC3 cells (89%–90% confluent) were cultured in 6-well microenvironment. plates in RPMI (1 mL) without FBS for 18 hours. After serum starvation, the cells were treated with ANXA2 (1 mg/mL), CXCL12 Interactions between ANXA2 and CXCL12 by BMSCs contribute (200 ng/mL), or a combination of ANXA2 (1 mg/mL) and CXCL12 to prostate cancer cell adhesion and migration (200 ng/mL) for 30 minutes at 37 C. Whole-cell lysates were To test whether expression of the ANXA2 receptor (Anxa2r) prepared from cells, separated on 10% SDS-PAGE, and transferred and the receptor for CXCL12, Cxcr4, are correlated, mRNA levels to polyvinylidene difluoride (PVDF) membranes. The mem- were quantified using qRT-PCR. Significantly higher levels of branes were first incubated with 5% milk for 1 hour before the Anxa2r or Cxcr4 expression were observed in the bone-homing addition of antibodies to p-AKT (cat. 9271; 1:1,000 dilution; Cell prostate cancer cell lines PC3 and C4-2B, compared with human Signaling) and AKT (cat. 9272; 1:1,000 dilution; Cell Signaling) BMSCs (Fig. 2A and B). Studies were next performed to deter- followed by overnight at 4 C. Subsequently the blots were incu- mine whether ANXA2 serves as an adhesion molecule for bated with peroxidase-coupled anti-rabbit IgG secondary anti- prostate cancer cell binding to BMSCs. As shown in Fig. 2C, Anxa2þ þ body (cat. 7074; 1:2,000 dilution; Cell Signaling) for 1 hour, the binding of prostate cancer cells to BMSC / was sig- and protein expression was detected with SuperSignal West nificantly greater than the binding of prostate cancer cells to Anxa2 Dura Chemiluminescent Substrate (cat. Prod 34075; Thermo BMSC / . When the experiments were repeated in the pre- Scientific). sence of a function blocking antibody targeting ANXA2, signi- Anxa2þ þ ficant reduction of prostate cancer binding to BMSC / Subcutaneous tumor growth were observed (Fig. 2D). To evaluate tumor growth, subcutaneous tumors were estab- CXCL12 is a key regulator of hematopoietic stem cell migration luc Anxa2þ þ lished. The PC3 cells (2 105 cells) mixed with BMSC / and tumor metastasis into the marrow. As ANXA2 expression is Anxa2 or BMSC / (2 105 cells) in Collagen I (cat. 354236; BD linked to expression of CXCL12, we next performed migration Anxa2þ þ Bioscience) were injected subcutaneously into 5- to 7-week-old assays of prostate cancer cells in response to BMSC / or Anxa2 male SCID mice. Mice were imaged weekly by bioluminescence BMSC / . For these studies, prostate cancer cells were imaging (BLI). After 5 weeks, the animals were sacrificed and added to the upper chamber of a migration assay dish and CM Anxa2þ þ Anxa2 tumors were prepared for histology. derived from BMSC / or BMSC / was added to the

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Expression of ANXA2 and CXCL12 by BMSCs contributes to prostate cancer cell growth Next, we evaluated whether or not the interaction of ANXA2 and CXCL12 plays a role in prostate cancer tumor growth in vitro. Luc Here, prostate cancer cells (PC3 ;1 105) were cocultured with Anxa2þ þ Anxa2 BMSC / or BMSC / (1 105) for 48 hours in 12-well culture plates. Thereafter, luciferase activity was measured by Dual-Luciferase Reporter Assay kit to monitor prostate cancer growth. More luciferase activity was observed when the tumor Anxa2þ þ Anxa2 cells were cocultured with BMSC / than with BMSC / (Fig. 3A). On the basis of the in vitro studies, we examined whether there are differences in the ability of BMSCs to support prostate cancer growth in vivo linked to expression of ANXA2 and CXCL12. For these studies, tumors were established by subcutaneously inject- Luc Anxa2þ þ ing PC3 cells (2 105) mixed with BMSC / or Anxa2 BMSC / (2 105) in type I collagen into 5- to 7-week-old male SCID mice. Bioluminescent imaging at 5 weeks was used to monitor tumor growth. Significantly larger tumor growth Anxa2þ þ occurred when the tumor cells were mixed with BMSC / Anxa2 compared with tumors established with BMSC / (Fig. 3B and C). To further test the growth of prostate cancer cells in the luc bone marrow, tumors were established by injecting PC3 (1 luc 105 cells) or C4-2B (3 105 cells) intratibially into 5- to 7-week- old male SCID mice. We found that prostate cancer cells are able to grow in bone marrow and these tumor cells interact with ANXA2 or CXCL12-expressing osteoblast cells on the bone surface in marrow (Fig. 3D). To explore the molecular mechanisms whereby ANXA2 and CXCL12 regulate tumor growth, we examined whether ANXA2 alone, CXCL12 alone, or the combination of ANXA2 and CXCL12 activates AKT in prostate cancer cells. For these investigations, prostate cancer cells were treated with ANXA2 and/or CXCL12 and phosphorylation of AKT was evaluated by Western blotting. The data demonstrate that ANXA2 or CXCL12 alone highly induces AKT phosphorylation in PC3 cells, and the combination of ANXA2 and CXCL12 significantly enhances activation of AKT phosphorylation (Fig. 3E). To validate the in vitro results, we examined induction of AKT phosphorylation on the tumors generated in Fig. 3B. The immunofluorescence data demonstrate fi Figure 1. that the expression levels of p-AKT was signi cantly enhanced in Anxa2þ/þ ANXA2 expression is colocalized with CXCL12 expression in BMSCs in the tumor cells mixed with BMSC compared with tumors Anxa2 marrow. A, expression of ANXA2 (red arrows) and CXCL12 (green arrows) in established with BMSC / or tumors alone (Fig. 3F). These osteoblasts on the long bones of wild-type and Anxa2 knocked-out mice as data suggested that ANXA2 and CXCL12 facilitate prostate cancer fl detected by immuno uorescent staining. Yellow arrows, colocalization of growth via the AKT pathway. ANXA2 and CXCL12 on osteoblasts; blue, DAPI nuclear stain. Bar ¼ 50 mm. B, expression of ANXA2 (red) and CXCL12 (green) by BMSCAnxa2þ/þ or BMSCAnxa2/ as detected by immunofluorescent staining. Blue, DAPI nuclear Expression of ANXA2 and CXCL12 by BMSCs contributes to stain. Bar, ¼ 20 mm. C, secretion of CXCL12 by BMSCAnxa2þ/þ or BMSCAnxa2/ protection from chemotherapy-induced apoptosis as determined by ELISA. Data are representative of mean with SD of 3 Prostate cancer cells are known to develop resistance to che- independent experiments (Student t test). motherapies, particularly in marrow. To explore the role that ANXA2 and CXCL12 play in this process, cocultures of BMSCs and bottom chambers. More prostate cancer cells migrated toward prostate cancer cells were evaluated by FACS for Annexin V Anxa2þ þ the CM derived from BMSC / than they did toward staining (Fig. 4). Apoptotic levels for prostate cancer cells cocul- Anxa2 Anxa2þ þ BMSC / CM (Fig. 2E). When the experiments were per- tured with BMSC / were lower than when prostate cancer Anxa2 formed in the presence of neutralizing anti-CXCL12 antibody cells were cultured alone or with BMSC / . Further protec- or AMD3100, a selective CXCR4 antagonist, significant reduction from apoptosis was noted when the microtubule-inhibiting Anxa2þ þ tion of prostate cancer migrating to BMSC / CM were drug taxotere was included in the cultures (Fig. 4A and B). To observed (Fig. 2F). These data suggest that prostate cancer evaluate whether these in vitro studies are also relevant to our in moves toward CXCL12 secreted by BMSCs. These observations vivo studies, TUNEL staining was next performed on the tumors suggest that ANXA2 and CXCL12 participate in tissue locali- generated in Fig. 3B. Fewer apoptotic tumor cells were found in Luc Anxa2þ þ zation of prostate cancer. the tumors established with PC3 and BMSC / compared

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Figure 2. ANXA2 and CXCL12 interact to regulate prostate cancer cell adhesion and migration. A, expression of mRNA for the ANXA2 receptor (Anxa2r)by prostate cancer cells (PC3 or C4-2B) determined by real-time RT-PCR. hBMSCs were used as negative controls. B, expression of mRNA for the CXCL12 receptor, Cxcr4 by prostate cancer cells (PC3 or C4-2B). hBMSCs were used as negative controls. C, adhesion of prostate cancer cells to BMSCs expressing different levels of ANXA2. Adhesion was determined by quantifying the number of CFDA-stained prostate cancer cells bound to BMSC, with þ þ binding to BMSCAnxa2 / set at 100%. D, adhesion of prostate cancer cells to BMSCs with the presence or absence of a functional blocking antibody to ANXA2. IgG matched control was used as an antibody control. E, migration of prostate cancer cells (PC3 or C4-2B) toward CM derived from BMSCAnxa2þ/þ or BMSCAnxa2/ was performed in Transwell plates, where percentage of migration was set at 100% for the initial cell numbers in top chamber. F, migration of prostate cancer cells to BMSCs in the presence of neutralizing anti-CXCL12 antibody or AMD3100, a selective CXCR4 antagonist. Data are representative of mean with SD of 3 independent experiments (Student t test).

Luc Anxa2 Anxa2 with tumors generated from PC3 mixed with BMSCs / pare the tumors established with PC3 and BMSC / (Fig. 5B (Fig. 4C and D). Together the data suggest that ANXA2 and and C). CXCL12 expression by BMSCs are likely to play a role in the One possible mechanism that may account for the observation resistance of prostate cancer cells to chemotherapy. that more CD31 endothelial cells were present in tumors gener- Anxa2þ þ ated with BMSC / is that BMSCs enhance endothelial cell Expression of ANXA2 and CXCL12 by BMSCs contributes to the survival. To test this possibility, endothelial cells were treated with generation of vascular structures in tumors ANXA2 and CXCL12 alone or in combination, and survival was To determine whether ANXA2 and CXCL12 contribute to determined by FACS for Annexin V staining as a measurement of endothelial cell recruitment into tumor beds, migration by endo- apoptosis. We found that fewer HBMECs underwent apoptosis in thelial cells in response to CM isolated from BMSCs was evalu- the presence of ANXA2 and/or CXCL12 (Fig. 5D). TUNEL staining ated. When HBMEC were added to the upper chamber of the was next used to examine endothelial cell survival in tumors. Luc Anxa2þ þ culture dish with BMSCs cell CM present in the bottom, fewer Tumors established with PC3 and BMSC / had fewer Anxa2 Luc HBMEC migrated toward BMSC / CM compared with apoptotic endothelial cells than tumors established with PC3 Anxa2þ þ Anxa2 BMSC / CM. When the experiments were performed in the mixed with BMSC / (Fig. 5E and F). Together the data presence of AMD3100, signiﬁcant reduction of HBMECs migrat- suggest that ANXA2-mediated CXCL12 in BMSCS plays an impor- Anxa2þ þ Anxa2 ing to BMSC / or BMSC / CM was observed (Fig. 5A). tant role in sustaining vascular structures. These data suggest that endothelial cells move toward CXCL12 secreted by ANXA2 expressing BMSCs and further suggest that ANXA2 and CXCL12 participate in tissue localization of endo- Discussion thelial cells. Next, immunostaining for CD31, a marker of endo- Recent studies suggest that niche cells in the bone marrow thelial cells, was performed on tumors generated by PC3 cells in participate in the cellular and molecular events for the tumor Anxa2þ þ Anxa2 combination with BMSC / or BMSC / . Tumors estab- progression and metastasis (5–9).Inthisstudy,weshow Luc Anxa2þ þ lished with PC3 and BMSC / demonstrated signiﬁcantly ANXA2 and CXCL12 interactions produced by BMSCs facilitate larger and more abundant CD31-expressing blood vessels com- prostate cancer recruitment and proliferation, aid in protection

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Figure 3. þ þ ANXA2 and CXCL12 interact to regulate prostate cancer cell growth. A, PC3Luc cells were placed in coculture with BMSCAnxa2 / or BMSCAnxa2 / , and proliferation was evaluated at 48 hours by determining the change in luciferase levels using a Dual-Luciferase Reporter Assay kit. Data are representative of mean with SD of 3 independent experiments (Student t test). B and C, SCID mice were implanted subcutaneously PC3luc cells alone or mixed with BMSCAnxa2þ/þ or BMSCAnxa2/. Tumor growth was evaluated by BLI at 5 weeks (n ¼ 5 animals per group, mean SD, Student t test). D, SCID mice were implanted intratibially with PC3luc or C4- 2Bluc to directly test tumor growth in bone marrow. H&E staining demonstrates tumor growth in marrow (left). Bar, ¼ 50 mm. (Continued on the following page.)

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Figure 4. ANXA2 and CXCL12 in the marrow environment enhance prostate cancer survival and drug resistance. PC3GFP or C4-2BGFP cells were directly cultured þ þ with BMSCAnxa2 / or BMSCAnxa2 / for 48 hours and taxotere was introduced into the culture for an additional 48 hours. Annexin V staining was performed on the recovered (A) PC3 or (B) C4-2B cells and quantiﬁed by FACS. Data in A and B are representative of mean with SD of 3 independent experiments (Student t test). C, TUNEL staining of apoptotic prostate cancer cells (black arrows) of tumors grown from PC3luc cells alone or mixed with þ þ BMSCAnxa2 / or BMSCAnxa2 / implanted into SCID mice. Bar, ¼ 50 mm. D, quantiﬁcation of percentage of TUNEL-positive cells in C (mean SD, Student t test).

of prostate cancer cells from chemotherapy-induced apoptosis, late HSC homing and localization to the bone marrow (27– and facilitate the generation of vascular structures in tumors. 30). Subsequently, we reported that ANXA2 and CXCL12 bind Together these data suggest that ANXA2 and CXCL12 interac- directly to each other, and these interactions facilitate hemato- tions may be integral to tumor development in the marrow poietic progenitor cell migration in response to CXCL12 (26). microenvironment. We therefore hypothesized that ANXA2-expressing bone mar- The engagement of DTCs to the bone marrow niche and the row stromal cells facilitate prostate cancer metastasis and maintenance of these cells in the niche are essential steps to growth by inducing CXCL12. We found that ANXA2-expressing establish metastases. In this context, ANXA2, an osteoblast- BMSCs express elevated levels of CXCL12, which increases the expressed adhesion molecule, may be a crucial docking signal binding and recruitment of prostate cancer cells into the mar- (30). CXCL12 is a chemokine, which in addition to functioning row niche and promotes prostate cancer proliferation. Thus, a as a secreted molecule is known to bind to extracellular matrix model can be envisioned in which CXCL12 facilitates the proteins (27–29). Previously, we and other groups have iden- recruitmentofDTCstotheniche,ANXA2facilitatesprostate tiﬁed several factors that may be involved in establishing a cancer engagement into the niche, whereupon CXCL12 pro- tumor niche. We have shown that CXCL12 produced by the duced by the niche cells stimulates prostate cancer growth. niche participates in the regulation of prostate cancer metas- CXCL12 is known to play a central role in promoting the tasis (27–29), and we have also demonstrated that ANXA2 and growth of tumor cells including ovarian carcinoma (33), small its receptor ANXA2R regulate the localization and maintenance cell lung cancer (34), head and neck squamous cell carcinoma of prostate cancer cells in the niche (30). More importantly, (35), pancreatic cancer (36), and prostate cancer (37). It has tumor cell metastases share molecular mechanisms that regu- also been demonstrated that secreted CXCL12 induces prostate

(Continued.) Colocalization of ANXA2 and CXCL12 on osteoblasts (yellow arrows) associated with the bone surface was detected by immunofluorescent staining (top). Blue, DAPI nuclear stain. Bar ¼ 50 mm. HLA-positive tumor cells (red arrows) are associated with ANXA2 (green arrows)-expressing osteoblasts (middle) or CXCL12 (green arrows)-expressing osteoblasts (bottom) as detected by immunofluorescent staining. Bar, ¼ 50 mm. E, ANXA2, CXCL12, or a combination of ANXA2 and CXCL12 were treated to PC3 cells for 30 minutes. Western blotting was performed to detect p-AKT or AKT levels. F, immunofluorescent staining of p-AKT or AKT on the tumors generated in B.

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Figure 5. ANXA2 and CXCL12 expression by BMSCs increases vascular formation. A, migration of HBMECs toward CM isolated from BMSCAnxa2þ/þ or BMSCAnxa2/. Migration assays of HBMECs were performed in Transwell plates using CM from BMSCAnxa2þ/þ or BMSCAnxa2/ with and without AMD3100, a selective CXCR4 antagonist. Data are representative of mean with SD of 3 independent experiments (Student t test). B, CD31 staining (red) for blood vessel cells in tumors grown from PC3luc cells alone or mixed with BMSCAnxa2þ/þ or BMSCAnxa2/ implanted into SCID mice. Blue, DAPI nuclear stain. Bar, ¼ 50 mm. C, quantification of percentage of CD31-positive cells in B (mean SD, Student t test). D, apoptotic HBMECs in the presence of ANXA2, CXCL12, or combination of ANXA2 and CXCL12 were quantified by FACS analysis using Annexin V staining. Data are representative of mean with SD of 3 independent þ þ experiments (Student t test). E, TUNEL staining of apoptotic endothelial cells (black arrows) in tumors grown from PC3luc cells mixed with BMSCAnxa2 / or BMSCAnxa2/ implanted into SCID mice. Bar, ¼ 50 mm. F, quantification of percentage of TUNEL-positive cells in E (mean SD, Student t test).

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cancer proliferation in in vitro (37–39), following activation of Finally, migration, expansion, and survival of vascular ERK1/2 and AKT pathways (34, 36, 37). We also found that endothelial cells are essential for establishment of a functional ANXA2 and CXCL12 interactions effectively activate through network of angiogenesis. Ling and colleagues demonstrated AKT signaling pathways that, in many respects, have been that Anxa2-deficient mice have markedly impaired neoangio- linked to tumor growth. genesis. This contributes to a failure to localize plasmin In marrow, osteoblasts play important roles in the produc- activity to the endothelial cell surface and a failure to activate tion of a mineralized matrix that serves as the basis of selected matrix metalloproteinase (32). It has also been dem- bone remodeling. Osteoblasts are also known to express many onstrated that ANXA2-dependent plasmin generation in cell adhesion molecules, including ANXA2, which may facil- breast tumor is necessary to trigger the switch to neoangiogen- itate bone activities. Recently, it was demonstrated that esis, thereby stimulating tumor growth (46, 47). We found Luc Anxa2þ þ ANXA2 regulates osteogenic differentiation, yet the molecular that PC3 cells implanted with BMSC / showed sig- mechanisms remain unclear (40–42). Hypoxia and VEGF both nificantly more and larger CD31-expressing blood vessels Luc Anxa2 induce ANXA2 expression by osteoblasts and endothelial cells than PC3 mixed BMSC / cells. It has also been dem- through SRC and MEK kinase–dependent pathway (40). onstrated that CXCL12 stimulates the formation of capillary- ANXA2 increases the mineralization of osteoblastic cells and like structures with human vascular endothelial cells (48–51). alkaline phosphatase activity (41). ANXA2 also supports oste- Recently, we demonstrated that prostate cancer cells use ogenic differentiation of mesenchymal stem cells (MSC) and CXCL12 and its receptors (CXCR4 and CXCR7) as key ele- further regulates both intramembrane and endochondral ossi- ments in metastasis and growth in bone, where CXCR4 sig- fication in adolescent idiopathic scoliosis (AIS; ref. 42). We naling leads to an angiogenic switch (48–50). We have have also reported that fewer osteoblasts are found in the long further demonstrated that CXCR4 signaling results in the / bone of Anxa2 knocked-out (Anxa2 ) mice compared with activation of MAP and AKT pathways, which ultimately þ þ wild-type (Anxa2 / ) mice and, likewise, a decrease of bone increases the secretion of VEGF and IL8 (48–50). More impor- phenotype markers (Runx2, osteocalcin, Collagen 1a) is seen tantly, CXCR4 directly regulates blood vessel formation (48), in bones from these knock-out mice (data not shown). Critical due to inhibiting the secretion of the vascular inhibitor pho- to this report, lower CXCL12-expressing cells in BMSCs phoglycerate kinase 1 (PGK1; ref. 50). Thus, there are both Anxa2 derived from Anxa2 knocked-out mice (BMSC / )were direct and indirect mechanisms whereby ANXA2 and CXCL12 identified compared with cells isolated from wild-type mice interactions may regulate tumor growth. Clearly, more studies Anxa2þ þ (BMSC / ). As ANXA2 mediates osteogenic activities in are warranted. marrow, the release of tumor-supporting activities from either In summary, this work provides further evidence for the osteoblasts or bone matrix may provide some clues to how roles that ANXA2 and CXCL12 play in the tumor micro- ANXA2 regulates metastasis and CXCL12 signaling simply by environment and specifically in prostate cancer migration, providing lower levels of substrates or adhesive surfaces for growth, and survival in bone marrow. Our data also provide DTCs to lodge. Alternatively, while the molecular mechanisms evidence that ANXA2 and CXCL12 regulate vascular formation need to be further investigated, our data also suggest that an in tumors. Together these studies suggest that ANXA2 and ANXA2-deficient environment may lead to less osteoblastic CXCL12 interactions facilitate a number of activities critical regulation of bone remodeling, which ultimately results in a for tumor growth. decrease of prostate cancer tumor growth and survival in bone marrow. Both of these possibilities are worthy of further Disclosure of Potential Conflicts of Interest study. No potential conflicts of interest were disclosed. CXCL12 is known to regulate tumor cell apoptosis. CXCL12 may protect tumor cells from drug-induced apoptosis directly through the activation of antiapoptotic pathways but also Authors' Contributions indirectly by modulating the adherence of cancer cells. CXCL12 Conception and design: Y. Jung, R.S. Taichman mediates adhesion of small cell lung cancer cells (SCLC) to Development of methodology: J. Wang, J. Dai, R.S. Taichman marrow stromal cells and protects SCLC against drug-induced Acquisition of data (provided animals, acquired and managed patients, apoptosis (34). CXCL12 activates NF-kB, which inhibits radi- provided facilities, etc.): Y. Jung, J. Wang, E. Lee, J.E. Berry, K. Yumoto, J. Dai, a E.T. Keller, R.S. Taichman ation-induced TNF production and tumor apoptosis (43, 44). Analysis and interpretation of data (e.g., statistical analysis, biostatistics, k Moreover, NF- B signaling is involved in the expression of computational analysis): Y. Jung, J. Wang, K. Yumoto, Y. Shiozawa, CXCR4, thus promoting tumor cell migration and metastasis R.S. Taichman (43–45). In this study, we found that more prostate cancer Writing, review, and/or revision of the manuscript: Y. Jung, J.E. Berry, Anxa2þ þ cells survived when cocultured with BMSC / vs. cocul- E.T. Keller, Y. Shiozawa, R.S. Taichman Anxa2 ture with BMSC / in in vitro. Similar results were obtain- Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. McGee, R.S. Taichman ed in in vivo studies, as fewer apoptotic cells were found Anxa2þ þ Study supervision: R.S. Taichman in tumors established with BMSC / versus those with Anxa2 BMSC / cells. In part, these results may be due to regulation of CXCL12 expression by ANXA2 or enhanced ability Grant Support to localize CXCL12 to cell surfaces, which may be more This work is directly supported by the NIH (CA163124, CA093900, efficient in activating cell functions than in a soluble form. CA166307, CA069568, DK082481, MH095589, Y. Shiozawa and R.S. Taichman), the Department of Defense (W81XWH-11-1-0636, W81XWH- Whether either of these 2 possibilities is responsible for how 14-1-0403, Y. Shiozawa and R.S. Taichman), the Prostate Cancer Foundation ANXA2 interacts with CXCL12 remains unclear and will require (Y. Shiozawa and R.S. Taichman). This research was supported in part by the additional investigation. NIH through the University of Michigan's Cancer Center Support Grant (P30

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CA046592) by the use of the following Cancer Center Core(s): Flow advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Cytometry, Microscopy and Imaging, Tissue & Molecular Pathology Cores. this fact. Logistical support was also received from the Dental School's Molecular Biology Core. The costs of publication of this article were defrayed in part by the Received March 6, 2014; revised July 14, 2014; accepted July 30, 2014; payment of page charges. This article must therefore be hereby marked published OnlineFirst August 19, 2014.

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Annexin 2−CXCL12 Interactions Regulate Metastatic Cell Targeting and Growth in the Bone Marrow

Younghun Jung, Jingcheng Wang, Eunsohl Lee, et al.

Mol Cancer Res 2015;13:197-207. Published OnlineFirst August 19, 2014.

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