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Human breast cancers metastasize early in tumorigenesis and distant lesions, though dormant are very likely extant at the time of diagnosis and treatment in the majority of cases. Removal of primary tumors by surgeons as an imperative of the current treatment approach, also removes inhibitory factors secreted by the primary tumor that had maintained the dormancy of the metastases. We have identified a factor secreted by human breast cancer cells that supports the formation of blood vessels and may be a principal early factor supporting the growth and development of metastases in human disease. Here we demonstrate for the first time that this factor, secreted (s) human (h) nucleoside diphosphate kinase type B (shNDPK-B), product of the nm23-h2 gene, can be detected specifically with high sensitivity (50 pg/ml; 2.5 pM) in an ELISA assay of our own design. We further demonstrate that shNDPK-B is released into the circulation in immunocompromized mice carrying the human breast carcinoma cell MDA-MB-231. These data support the hypothesis that shNDPK-B may be responsible for the early events in angiogenesis supporting both primary and metastatic tumor growth and development.
The most common cancer among women in the United States is cancer of the breast . Typical treatment involves either mastectomy or lumpectomy plus postoperative (adjuvant) treatment. Nonetheless, more than forty-thousand women will die of the disease this year . Breast cancer specific mortality is almost exclusively a function of a metastatic process  and the presence of distant metastases is associated with the least favorable outcome. Those whose breast cancer reoccurs at distant sites in the body have only a 9% chance of living an additional 10 years, compared to a 56% survival rate for women with a reoccurrence of breast cancer which is isolated to the breast . Thus the imperative in breast cancer research is to understand and treat the metastatic process.
Evidence now strongly suggests that metastases present at the time of mastectomy have existed in a dormant state and have proliferated only once the anti-angiogenic effects of the primary tumor have been removed [4,5]. Animal studies confirm this [6,7]. The notion of metastases as extant, dormant lesions awaiting events that trigger their conversion to rapidly growing tumors that attract a blood supply is supported by recent genomic studies that implicate nucleotide metabolism among other critical changes in gene transcription . Development of metastases after removal of a primary tumor is a likely clinical course even among node negative patients that will eventually develop distant metastases . Based on the temporal association of metastasis development to removal of a primary tumor, a model of tumor homeostasis has been described in which the primary tumor exerts an anti-proliferative effect on extant subclinical micrometastases . Evidence indicates that dormancy is a function of the primary tumor preventing the initiation of angiogenesis , e.g., angiostatin . Thus, mechanisms that orchestrate primary tumor growth and development are distinct, if related, to those that support the growth of metastases, but metastases are uniquely suppressed by their primary tumor. This inconvenient truth suggests that women that have a primary breast lesion removed may be placed at an accelerated risk for growth and development of their metastases.
The events that permit intravasation and extravasation in the passage of tumor cells to distant sites may preceded early on and be unopposed by the primary tumor whose influence is that which suppresses angiogenesis. With the importance of angiogenesis increasingly apparent, the formation and extracellular actions of nucleotides have been suggested by our group to participate via P2 nucleotide receptors [11–14]. Thus, we have suggested that breast cancer cell-secreted shNDPK-B participates in intravasation, extravasation of tumor cells in their passage to distant sites as well as attraction of a blood supply at the metastatic site.
Nucleoside diphosphate kinase (NDPK) domains are present in a large family of structurally and functionally conserved proteins from bacteria to humans that generally catalyze the transfer of γ-phosphates from a nucleoside triphosphate (NTP) such as GTP, to a nucleoside diphosphate acceptor (NDP; e.g., ADP). NDPK has been shown to act as a histidine kinase, a transcription activator and an exonuclease . The gene is located at chromosome 17q21.3 near BRCA1 locus encoding a 686 bp mRNA . Aliases include puf, NDPK-B, NM23B, NM23-H2, MGC111212 and NME2. There are eight isoforms of NDPK containing 152 amino acids.
We have described the ability of human breast cancer cells grown in vitro to secrete NDPK (shNDPK-B) into the growth media in a fashion that suggests that this enzyme would be present in the microenvironment of the primary tumor as well as metastases . Moreover, the conditioned media from these cultures as well as its purified component, shNDPK-B, promotes angiogenesis of human endothelial cells in culture . Since breast tumor cells that metastasize to sites such as bone, lung and brain in women must attract a blood supply if they are to grow and kill the host, we have proposed that it is in part on the basis of their secretion of shNDPK-B, that breast tumor cells are able to stimulate angiogenesis in vivo and that this may be a primal event that precedes the actions of vascular endothelial growth factor (VEGF).
Here we further investigate our hypothesis by determining if human breast tumors carried by mice secrete NDPK-B in amounts sufficient to circulate in the blood stream of the host. The presence of human sNDPK-B in the mouse during tumor growth would be consistent with its action in stimulating angiogenesis. Measurement of secreted shNDPK-B has never before been investigated as no specific assay has been available. We have developed a sensitive, selective and reliable assay for NDPK-B described here for the first time.
The luciferase-tagged human carcinoma MDA-MB-231 cell line, 231-Luc2 (pGl4 Luc lentrivirus) was purchased from Caliper Sciences (Hopkinton, MA), MDA-MB-231 cells were cultured in Eagle’s Minimum Essential Medium (MEM) containing 10% FBS at 37°C in 5% CO2. Cells were harvested using trypsin (0.05%) and 4×106 cells were mixed 50:50 (v/v) with Matrigel (Sigma, MO) and injected s.c into the mammary fat of 4- to 6-week-old female CB-17 SCID mice.
At two week intervals, the jugular vein was lanced and 100–125 μl of blood collected. The blood was clotted in the tube at room temperature for 1–2 hr. Serum was separated by centrifugation at 6,000 × g for 10 min. Serum was separated and used in experiments or stored at −80°C.
Moue anti-Human Nm23 H2 H00004831-M0 was purchased from Abnova, (Taiwan), rabbit anti-mouse IgG-HRP conjugate was purchased from Southern Biotech (Birmingham, AL). The primary antibody was used at 1:750 dilution and a 1:2000 dilution was employed for the secondary antibody. Diethanolamine (OPD) Peroxidase substrate was purchased from (Sigma, MO), Immulon microtiter plates (96 well) were from Fisher Scientific. Recombinant Nm23-h2 protein from was from Abnova (Taiwan). Ninety-six-well plates were coated with standard NDPK-B and serum test protein. The standard NDPK-B samples were employed over a range of 400 ng, 60 ng, 40 ng, 6 ng, 4 ng, 0.6 ng, 0.4 ng, and 0.06 ng/mL in PBS containing 0.05% sodium azide. Serum test protein was diluted 2-fold in PBS containing azide and 50 μl aliquots were added to wells and the plate incubated for 2 hr at RT and then overnight at 4°C on a rocker. Following incubation overnight, the plate was tapped upside down on adsorbent toweling to remove the sample and blocking buffer (100 μl) containing 5% BSA and Tween 20 (0.05%) in phosphate-buffered saline (PBST) added for 1.5 hr at room temperature. Wells were then washed once with 150 μl of PBST. Primary anti-nm23-H2 antibody diluted with PBST buffer containing 1 mM EDTA and 0.25% BSA (100 μl) was added and the plate incubated for 2 hr at RT with slow rocking. Plates were then washed ×3 with 150 μl PBST. Specific secondary antibody-HRP conjugate was then added (100 μl) for 2 hr at 37°C followed by washing × 3 with PBST. Plates were developed by addition of 100 μl of OPD substrate (o-phenylenediamine dihydrochloride) and then incubated for 45 min s at 37°C. Absorbance was measured at 450 nm using a microplate reader.
All graphs were prepared using Prism Graphing Software (V4.03; GraphPad Software, San Diego, CA, USA) and statistical analyses were performed using InStat Statistical Software (V3.0; GraphPad Software). A P≤0.05 was considered significant. All experiments were tested for statistical significance using ANOVA and Kruskal–Wallis multiple comparisons post-test unless otherwise stated. Data points and error bars represent means ± S.E.M.
A method for the detection of NDPK-B (NM23-H2) is not available commercially and has not been described elsewhere. We tested six Nm23-H2 antibodies available commercially. Four of the six were able to detect hNM23-H2 with an r2 between 0.95 to 0.98 (data not shown). Mouse monoclonal antibody (Abnova, Taiwan) was found to be the most reliable and was employed in our experiments (Fig. 1). Previous data from our laboratory has examined the presence of NDPK/NM23 in the conditioned media from cultures of breast cancer cells growing in vitro. Detection of NDPK was based on measuring ATP generation in the luciferin-luciferase assay, Western blot analysis, protein sequence analysis and inhibitor studies [13,17,18].
In order to measure NDPK-B secretion in the SCID immunocompromized mouse model of human breast cancer, it is necessary to find a specific assay that can distinguish this protein among a host of competing activities that may appear in mouse serum. Moreover, we chose to employ the MDA-MB-231Luc+ carcinoma cell line transfected with luciferase. This cell can be imaged in the whole animal when injected with luciferin. In order to determine whether the MDA-MB-231Luc+ cells expressed shNDPK-B, we compared the conditioned media from the 231 cells with that of the MDA-MB-435 cells that we have shown previously secrete shNDPK-B  which stimulates angiogenesis in vitro . Both cell lines were found to secrete significant amounts of shNDPK-B by ELISA; 5 × 106 MDA-MB-435 or 231Luc+ cells were grown to 80% confluence in 100 mm dishes and changed to a Krebs buffer containing 1% BSA for 90 minutes. The concentrations of shNDPK-B (shNM23-H2) were 421.7 and 429.6 ng/ml respectively.
We next followed SCID mice injected with 2.5×106 MDA-MB-231Luc+ cells. Tumors developed at the site of injection and blood was collected at the time of tumor cell placement and at 2-week intervals thereafter until week 10. shNDPK-B was elevated at the first time point in the mice and remained elevated throughout the duration of the study (Fig. 2).
Our hypothesis that breast tumor cells secrete sNDPK-B to generate nucleotides extracellularly to act as purinergic receptor agonists at endothelial cells is supported by considerable in vitro evidence and with data presented here for the first time that human sNDPK-B appears in the blood stream of mice carrying human tumors. If primary tumors secrete both pro- and anti-metastatic factors as is currently thought [6,19–21], then the actions of sNDPK-B, produced at distant sites by metastatic cells, on the local vasculature would be a signal unopposed after removal of a primary tumor and thus capable of stimulating the development of a blood supply for the cells now able to proliferate.
It is clear that in breast cancer, metastatic lesions are extant at the time of diagnosis in a large number of cases. It is also apparent that mammography screening identifies a large number of women who do not have cancer but are nonetheless given a putative diagnosis and suffer emotional and physical stress as a result of a false positive diagnosis. Even in cases where small tumors are detected in the breast, it is likely that cells have moved from the site of the primary and are already extant as dormant micrometastases.
Combining what we know from animal studies with the current treatment of women with cancer of the breast, suggests that removal of a primary tumor activates and or accelerates the development of metastatic lesions [8–10] that are the cause, all too often, of death of the patient. Efforts to maintain dormancy of metastases in patients when first seen by the oncologist might offer a new strategy for adjuvant treatments to prevent their growth. If a non-invasive treatment were available that were known to suppress the development of a blood supply to these lesions, administration of this treatment prior to removal of a primary breast lesion might prevent death in those cases where activation of distant metastases has yet to occur.
Because a role for purine nucleotides has been firmly established in angiogenesis as suggested here and elsewhere  and our lab has identified safe and effective antagonists of shNDPK-B , a benefit can be realized now for women at high risk for developing disease. We suggest that adjuvant therapy with inhibitors of shNDPK-B should be instituted prior to surgical procedures in women with clear evidence of epithelial carcinoma of the breast with or without positive sentinel nodes and family history of breast cancer. A placebo controlled trial of one or more polyphenol inhibitors, available as non-toxic natural compounds, should be instituted.
The authors wish to thank Deanna Milton for technical assistance. This work was supported by NIH T32 CA09563.