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 Anxa2À À 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 / Anxa2À À 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 Anxa2À À 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 Anxa2À À CXCL12 interactions. Significantly more CXCL12 was expressed established with BMSC / . Thus, ANXA2–CXCL12 interac- Anxa2þ þ Anxa2À À 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. Anxa2À À 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 Ó2014 AACR. Introduction osteoclasts, osteomacs, and endothelial cells aid in the develop- ment 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 Ó2014 American Association for Cancer Research. levels of CXCL12 in marrow. Likewise, CXCL12 and ANXA2 play www.aacrjournals.org 197 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst August 19, 2014; DOI: 10.1158/1541-7786.MCR-14-0118 Jung et al. 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 Â 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 interac- tions facilitate the recruitment, growth, and survival of prostate Transwell chemotaxis assay Anxa2þ þ Anxa2À À cancer cells in marrow. BMSC / or BMSC / (1 Â 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 Anxa2À/À 5 cells) and GFP-expressing prostate cancer cell lines (PC3 and BMSC or BMSC (1 Â 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 À À 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). À À 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 À/À Anxa2þ þ Anxa2À À tibias of both Anxa2 and Anxa2 mice with a-MEM (Invi- BMSC / or BMSC / (1 Â 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 Â 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.