ORIGINAL ARTICLE
Int J Biol Markers 2014; 29 ( 3): e239-e245 DOI: 10.5301/jbm.5000080
Serum ANGPTL2 levels reflect clinical features of breast cancer patients: implications for the pathogenesis of breast cancer metastasis Motoyoshi Endo1, Yutaka Yamamoto2, Masahiro Nakano3, Tetsuro Masuda1, Haruki Odagiri1, Haruki Horiguchi1, Keishi Miyata1, Tsuyoshi Kadomatsu1, Ikuyo Motokawa1, Seiji Okada4, Hirotaka Iwase3, Yuichi Oike1,5 Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto - Japan Department of Molecular-Targeting Therapy for Breast Cancer, Kumamoto University Hospital, Kumamoto - Japan 3 Department of Breast and Endocrine Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto - Japan 4 Center for AIDS Research, Kumamoto University, Kumamoto - Japan 5 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo - Japan 1 2
Abstract Introduction: Breast cancer is a leading cause of cancer-related death in women worldwide, and its metastasis is a major cause of disease mortality. Therefore, identification of the mechanisms underlying breast cancer metastasis is crucial for the development of therapeutic and diagnostic strategies. Our recent study of immunodeficient female mice transplanted with MDA-MB231 breast cancer cells demonstrated that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) accelerates metastasis through both increasing tumor cell migration in an autocrine/paracrine manner, and enhancing tumor angiogenesis. To determine whether ANGPTL2 contributes to its clinical pathogenesis, we asked whether serum ANGPTL2 levels reflect the clinical features of breast cancer progression. Methods: We monitored the levels of secreted ANGPTL2 in supernatants of cultured proliferating MDA-MB231 cells. We also determined whether the circulating ANGPTL2 levels were positively correlated with cancer progression in an in vivo breast cancer xenograft model using MDA-MB231 cells. Finally, we investigated whether serum ANGPTL2 levels were associated with clinical features in breast cancer patients. Results: Both in vitro and in vivo experiments showed that the levels of ANGPTL2 secreted from breast cancer cells increased with cell proliferation and cancer progression. Serum ANGPTL2 levels in patients with metastatic breast cancer were significantly higher than those in healthy subjects or in patients with ductal carcinoma in situ or non-metastatic invasive ductal carcinoma. Serum ANGPTL2 levels in patients negative for estrogen receptors and progesterone receptors, particularly triple-negative cases, reflected histological grades. Conclusions: These findings suggest that serum ANGPTL2 levels in breast cancer patients could represent a potential marker of breast cancer metastasis. Keywords: ANGPTL2, Metastasis, Breast cancer, Triple-negative, Nuclear grade Received: September 19, 2013; Accepted: February 18, 2014
INTRODUCTION Breast cancer is the most frequently diagnosed malignant neoplasm in women and is a leading cause of cancer-related death worldwide (1). Breast cancer mortality after therapeutic intervention is primarily due to metastasis of tumor cells from the primary lesion (2, 3). Therefore, identification of the molecular mechanisms driving breast cancer metastasis is important in order to develop effec-
tive therapeutic or early diagnostic strategies to treat this condition. Recently, chronic inflammation has received much attention as playing a key role at various stages of tumor development, including initiation, growth, invasion, and metastasis (4). In this regard, we recently reported that angiopoietin-like protein 2 (ANGPTL2) increases inflammatory carcinogenesis (5). Furthermore, our xenograft studies in female mice transplanted with MDA-MB231
© 2014 Wichtig Publishing - eISSN 1724-6008
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Serum ANGPTL2 reflects clinical features of breast cancer
breast cancer cells demonstrated that tumor cell-derived ANGPTL2 accelerates tumor metastasis by increasing tumor cell migration and enhancing tumor angiogenesis (6). However, the role of ANGPTL2 in the clinical pathogenesis of breast cancer remains uncharacterized. To investigate this question, we asked whether serum ANGPTL2 levels paralleled clinical features of breast cancer progression. We found that circulating ANGPTL2 levels in xenograft mouse models increased according to cancer progression, particularly in the presence of metastasis. Serum ANGPTL2 levels in patients with advancedstage breast cancer were significantly higher than those observed in healthy subjects or patients with ductal carcinoma in situ (DCIS) or non-metastatic invasive ductal carcinoma (IDC). In patients negative for estrogen receptors and progesterone receptors, serum ANGPTL2 levels were positively correlated with nuclear grade status. Our findings show that serum ANGPTL2 levels in hormone receptor (HR)-negative patients, and especially triple-negative (TN) ones, might reflect the clinical features of breast cancer.
centrifuged at 3,000 rpm for 10 minutes, and supernatants were stored at -80°C prior to assay. This study was approved by the ethics committees of the Kumamoto University. Written informed consent was obtained from each subject.
MATERIALS AND METHODS
Total RNA from cultured cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). DNase-treated RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara Bio, Otsu, Japan). PCR products were amplified using a Thermal Cycler Dice Real Time system (Takara Bio, Otsu, Japan), and the relative transcript abundance was normalized to that of the 18S mRNA. PCR oligonucleotides were as follows: human ANGPTL2, forward, 5’- GCCACCAAGTGTCAGCCTCA-3’, reverse, 5’- TGGACAGTACCAAACATCCAACATC-3’: human 18S, forward, 5’- TTTGCGAGTACTCAACACCAACATC-3’, reverse, 5’- GAGCATATCTTCGGCCCACAC-3’. The data was analyzed with the 2-tailed Student’s t-test using Excel software (Microsoft, Redmond, WA). A p value of less than 0.05 was considered statistically significant.
Quantitation of the ANGPTL2 protein by ELISA The ANGPTL2 concentrations in culture medium from tumor cells or in serum from humans or mice were estimated using the ANGPTL2 assay kit (IBL, Gunma, Japan) as previously described (7, 8). Human studies We collected blood samples from breast cancer patients (n=625) at Kumamoto University between May 2003 and July 2011, as well as from healthy female volunteers (n=19) aged 20-54 years working at Kumamoto University. All patients were examined to evaluate potential tumor spread in the whole body using mammography, ultrasound sonography, and computed tomography (CT) at the time of initial diagnosis. Stage IV patients and subjects with relapse after initial treatment (such as surgery and adjuvant treatment) also received bone scintigraphy or positron emission tomography to search for additional lesions. Diagnosis of solitary lesions resulting from potential relapse and/or metastasis was undertaken using fine needle aspiration biopsy and core needle biopsy to confirm metastasis. Blood samples from breast cancer patients without distant metastasis were obtained immediately before surgery or at the time of initial systemic treatment with chemotherapy, anti-HER2 therapy, or endocrine therapy. Serum samples of patients with distant metastasis were obtained before initial treatment. Blood samples were e240
Cell lines and culture Human breast adenocarcinoma cell lines MCF-7, T47D, SK-BR-3, MDA-MB453, and MDA-MB231 were purchased from the American Type Culture Collection (ATCC) and cultured in conditions recommended by the supplier. Luciferase-expressing MDA-MB231 cells (MB231/ luc) were generated and maintained as described (6). To detect ANGPTL2 concentrations in culture medium, 5,000 cells were plated in each well of a 96-well plate (Iwaki, Tokyo, Japan) with 100 μL medium; after the first plating, the medium was then assayed at specific time points using an ANGPTL2 assay kit (IBL, Gunma, Japan). Real-time quantitative RT-PCR
Proliferation assay A total of 5,000 cells per well were plated in 96-well plates (Iwaki, Tokyo, Japan), and cell number was determined at specific time points using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. The growth rate was expressed as the number of cells at a specific time point relative to that determined at 0 hours. Mouse xenograft model For the breast cancer xenograft model, we used MB231/luc cells implanted in the mammary fat pad of an 8-week-old female JAK3-deficient NOD-SCID (NOJ) mice (6), and we monitored bioluminescence images
© 2014 Wichtig Publishing - eISSN 1724-6008
Endo et al
as previously described (6). All experiments were performed according to the guidelines of the Institutional Animal Committee of Kumamoto University. Statistics Cell proliferation and ANGPTL2 levels in the culture medium were analyzed using the Kruskal-Wallis test. Serum ANGPTL2 levels were analyzed using the SteelDwass test. All calculations were performed using the JMP10 software (SAS Institute, Cary, NC, USA). A p value of less than 0.05 was considered statistically significant. RESULTS Proliferating ANGPTL2-expressing cells secrete ANGPTL2 protein in vitro To examine whether tumor cell-secreted ANGPTL2 concentrations increase with tumor cell proliferation, we employed an enzyme-linked immunosorbent assay (ELISA) to measure ANGPTL2 protein levels in the medium of cultured human breast cancer MDA-MB231 cells, which abundantly express ANGPTL2 (6). As expected, MDAMB231 cells proliferated over time (Fig. 1A) and ANGPTL2 concentrations in the medium increased with proliferation (Fig. 1B). Next, we calculated the ratio between ANGPTL2 concentration and proliferation rate and found that ANGPTL2 levels increased from day 1 to day 2 but remained unchanged from day 2 to day 3 (Fig. 1C). We also examined ANGPTL2 mRNA induction and observed a trend similar to that of the secreted ANGPTL2 protein (Fig. 1D). Thus, cultured breast cancer cells secrete ANGPTL2 as they proliferate, and induction is more apparent at early proliferative stages. Serum ANGPTL2 levels increase in parallel with metastasis in a xenograft mouse model Next, we analyzed whether circulating tumor-cell secreted ANGPTL2 levels reflect cancer progression in vivo in a breast cancer mouse model. To do so, we first examined the ANGPTL2 mRNA levels in various breast cancer cell lines, including MCF-7 (ER+, HER2-), T47D (ER+, HER2-), SK-BR-3 (ER-, HER2+), MDA-MB453 (ER-, HER2+), and MDA-MB231 (ER-, HER2-) (Fig. 2A). As we previously reported (6), ANGPTL2 induction is more robust in MDA-MB231 cells than in other breast cancer cell lines. Therefore, we implanted MDA-MB231 cells harboring a luciferase expression vector (MB231/luc) into the mouse mammary fat pad of JAK3-deficient NOD-SCID (NOJ) mice (7) and then monitored bioluminescence images and serum ANGPTL2 concentrations. Tumors in the mammary fat pad grew gradually, based on biolumi-
Fig. 1 - MDA-MB231 breast cancer cells secrete ANGPTL2 in vitro. (A) Relative number of proliferating MDA-MB231 cells at the indicated time points of the in vitro culture (4 independent experiments, each containing 4 replicates). On day 1 of cell culture the number of proliferating cells was set at 1. (B) ANGPTL2 protein concentration in the culture medium at the indicated days (4 independent experiments, each containing 4 replicates). (C) The ratio between ANGPTL2 concentration and relative cell number. (D) Comparative ANGPTL2 expression levels of MDAMB231 cells at the indicated time points (4 independent experiments, each containing 2 replicates). mRNA levels on day 1 of cell culture were set at 1. Error bars show SD. **p20 mm
42
2.5 (1.71-3.49)
149
2.42 (1.71-3.16)
n (-)
282
2.27 (1.56-3.30)
299
2.28 (1.71-2.98)
n (+)
111
2.78 (2.04-3.61)
114
2.31 (1.66-3.25)
NG 1, 2
45
2.27 (1.58-2.94)
371
2.28 (1.48-2.64)
NG 3
42
2.94 (2.03-3.74)
42
2.62 (1.70-3.42)
0.072
0.0096
© 2014 Wichtig Publishing - eISSN 1724-6008
0.993
0.971
e243
Serum ANGPTL2 reflects clinical features of breast cancer
TABLE III - A SSOCIATION BETWEEN SERUM ANGPTL2 LEVELS AND CLINICOPATHOLOGICAL FACTORS ACCORDING TO HORMONE RECEPTOR STATUS HR+
HER2+
TN
n
ANGPTL2 levels Median (25th, 75th percentiles)
P value
n
ANGPTL2 levels Median (25th, 75th percentiles)
P value
n
ANGPTL2 levels Median (25th, 75th percentiles)
P value
T≤20 mm
251
2.28 (1.72-2.95)
0.269
27
2.27 (1.61-3.23)
0.674
31
2.29 (1.56-3.32)
0.407
T>20 mm
142
2.42 (1.71-3.18)
28
2.40 (1.50-3.03)
21
2.62 (1.75-3.60)
n (-)
282
2.30 (1.73-2.98)
37
2.18 (1.44-3.05)
36
2.29 (1.58-3.44)
n (+)
111
2.38 (1.67-3.27)
18
2.59 (2.02-3.12)
16
2.72 (1.62-3.85)
NG 1, 2
359
2.29 (1.73-3.03)
31
2.28 (1.48-2.64)
26
1.85 (1.59-3.12)
NG 3
34
2.54 (1.60-3.11)
24
2.62 (1.70-3.42)
26
2.87 (2.06-3.75)
0.952
0.727
0.123
0.249
0.362
0.024
Luminal: HR+ and HER2-, HER2+: HER2+, irrespective of HR status, TN: HR- and HER2-.
and ANGPTL2 are induced by the hypoxic microenvironment of cancer cells (6, 13). ANGPTL4 is also induced by hypoxia and inhibits endothelial cells interactions to promote tumor metastasis (14). A common theme of these studies is that ANGPTL2 and 4 and Ang2 are induced by hypoxia, a state created by many tumor cells. Recently, we showed that ANGPTL2 induction is regulated by DNA methylation, which is seen in hypoxic cancer microenvironments (15). In this study, we did not examine whether ANGPTL2 and other Ang cooperate with each other in the cancer cell microenvironment. That relationship should be investigated in future studies. We recently reported studies of immunodeficient female mice transplanted with MDA-MB231 breast cancer cells showing that tumor cell-secreted ANGPTL2 shortened survival time and promoted cancer metastasis by accelerating motility, invasion, and tumor angiogenesis in an autocrine/paracrine manner (6). Taken together with the clinical findings reported here, we suggest that inhibiting ANGPTL2 activity or secretion from breast cancer cells represents a novel potential therapy to antagonize advanced breast cancer. Recently, Zheng et al reported that the immune inhibitory receptor human leukocyte immunoglobulin-like receptor B2 (LILRB2), and its mouse orthologue paired immunoglobulin-like receptor B2 (PIRB2), are ANGPTL2 receptors (16). The authors also reported that LILRB2 and PIRB2 are respectively expressed on human and mouse hematopoietic stem cells (HSCs), and that ANGPTL2 binding to these receptors supports ex vivo HSC expansion. Furthermore, in mouse models of human acute myeloid leukemia, impaired PIRB signaling promotes leukemia cell differentiation (16). Since we reported that ANGPTL2 contributes to tumor invasion, metastasis, and e244
recurrence (6), it will be of interest to investigate whether breast cancer stem cells express ANGPTL2 and whether ANGPTL2 signaling contributes to the invasive phenotypes of those cells. Currently, tumor markers such as CEA and CA15-3 are used to detect breast cancer recurrence and are thought, in some cases, to be potentially more sensitive than radiographic imaging to detect metastatic disease. However, the use of these markers alone is not recommended to detect relapse or to monitor the therapeutic effect (17), partly because these markers are not always sensitive enough to detect cancer recurrence (18, 19), and partly because their serum levels can vary with liver function or lifestyle (20). Also, successful systemic therapy often increases serum concentrations of these factors (an occurrence known as marker “flare”) due to the release of antigens by cytolysis (20, 21). Thus, additional tools enabling earlier diagnosis are needed to decrease the number of deaths from tumor metastasis. Here we demonstrated that serum ANGPTL2 levels in patients with metastatic breast cancer were significantly higher than those in healthy subjects or in patients with DCIS or non-metastatic IDC. In addition, serum ANGPTL2 levels in HR-negative patients, especially TN patients, reflected the nuclear grade status. These findings suggest that serum ANGPTL2 levels in breast cancer patients could serve as a potential diagnostic tool of histological grades. ACKNOWLEDGEMENTS We thank Mss. S. Iwaki, O. Takahashi, K. Tabu, and M. Nakata for technical assistance.
© 2014 Wichtig Publishing - eISSN 1724-6008
Endo et al
Informed Consent: Written informed consent was obtained from each participating subject.
Financial Support: This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Japan Society for the Promotion of Science (JSPS) through its Funding Program for Next Generation World-Leading Researchers (NEXT Program), a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Core Research for Evolutional Science and Technology (CREST) program of Japan Science and Technology Agency
REFERENCES 1. 2.
3. 4. 5.
6.
7.
8.
9.
10. 11.
Boyle P, Levin B. World Cancer Report 2008. Lyon: IARC press; 2008. Eckhardt BL, Francis PA, Parker BS, Anderson RL. Strategies for the discovery and development of therapies for metastatic breast cancer. Nat Rev Drug Discov. 2011; 11(6): 479-497. Weigelt B, Peterse JL, van ’t Veer LJ. Breast cancer metastasis: markers and models. Nat Rev Cancer. 2005; 5(8): 591-602. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010; 140(6): 883-899. Aoi J, Endo M, Kadomatsu T, et al. Angiopoietin-like protein 2 is an important facilitator of inflammatory carcinogenesis and metastasis. Cancer Res. 2011; 71(24): 7502-7512. Endo M, Nakano M, Kadomatsu T, et al. Tumor cell-derived angiopoietin-like protein ANGPTL2 is a critical driver of metastasis. Cancer Res. 2012; 72(7): 1784-1794. Okada T, Tsukano H, Endo M, et al. Synoviocyte-derived angiopoietin-like protein 2 contributes to synovial chronic inflammation in rheumatoid arthritis. Am J Pathol. 2010; 176(5): 2309-2319. Tabata M, Kadomatsu T, Fukuhara S, et al. Angiopoietinlike protein 2 promotes chronic adipose tissue inflammation and obesity-related systemic insulin resistance. Cell Metab. 2009; 10(3): 178-188. Kadomatsu T, Tabata M, Oike Y. Angiopoietin-like proteins: emerging targets for treatment of obesity and related metabolic diseases. FEBS J. 2011; 278(4): 559-564. Rigamonti N, De Palma M. A role for angiopoietin-2 in organ-specific metastasis. Cell Rep. 2013; 4(4): 621-623. Minami T, Jiang S, Schadler K, et al. The calcineurin-NFATangiopoietin-2 signaling axis in lung endothelium is critical for the establishment of lung metastases. Cell Rep. 2013; 4(4): 709-723.
(JST), and by grants from the Takeda Science Foundation and the Yasuda Medical Foundation. Conflict of Interest Statements: The authors have declared that no conflict of interest exists. Address for correspondence: Yuichi Oike, M.D., Ph.D. or Motoyoshi Endo, M.D., Ph.D. Department of Molecular Genetics Kumamoto University 1-1-1 Honjo, Kumamoto 860-8556, Japan [email protected] [email protected]
12. Holopainen T, Saharinen P, D’Amico G, et al. Effects of angiopoietin-2-blocking antibody on endothelial cell-cell junctions and lung metastasis. J Natl Cancer Inst. 2012; 104(6): 461-475. 13. Semenza GL. Cancer-stromal cell interactions mediated by hypoxia-inducible factors promote angiogenesis, lymphangiogenesis, and metastasis. Oncogene. 2013; 32(35): 4057-4063. 14. Zhang H, Wong CC, Wei H, et al. HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs. Oncogene. 2012; 31(14): 1757-1770. 15. Odagiri H, Kadomatsu T, Endo M, et al. The secreted protein ANGPTL2 promotes metastasis of osteosarcoma cells through integrin α5β1, p38 MAPK, and matrix metalloproteinases. Sci Signal. 2014; 7(309): 2004612. 16. Zheng J, Umikawa M, Cui C, et al. Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development. Nature. 2012; 485(7400): 656-660. 17. Khatcheressian JL, Wolff AC, Smith TJ, et al; American Society of Clinical Oncology. American Society of Clinical Oncology 2006 update of the breast cancer follow-up and management guidelines in the adjuvant setting. J Clin Oncol. 2006; 24(31): 5091-5097. 18. Emens LA, Davidson NE. The follow-up of breast cancer. Semin Oncol. 2003; 30(3): 338-348. 19. Kokko R, Holli K, Hakama M. Ca 15-3 in the follow-up of localised breast cancer: a prospective study. Eur J Cancer. 2002; 38(9): 1189-1193. 20. Stearns V, Yamauchi H, Hayes DF. Circulating tumor markers in breast cancer: accepted utilities and novel prospects. Breast Cancer Res Treat. 1998; 52(1-3): 239-259. 21. Kiang DT, Greenberg LJ, Kennedy BJ. Tumor marker kinetics in the monitoring of breast cancer. Cancer. 1990; 65(2): 193-199.
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Int J Biol Markers 2014; 29 ( 3): e239-e245 DOI: 10.5301/jbm.5000080
Serum ANGPTL2 levels reflect clinical features of breast cancer patients: implications for the pathogenesis of breast cancer metastasis Motoyoshi Endo1, Yutaka Yamamoto2, Masahiro Nakano3, Tetsuro Masuda1, Haruki Odagiri1, Haruki Horiguchi1, Keishi Miyata1, Tsuyoshi Kadomatsu1, Ikuyo Motokawa1, Seiji Okada4, Hirotaka Iwase3, Yuichi Oike1,5 Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto - Japan Department of Molecular-Targeting Therapy for Breast Cancer, Kumamoto University Hospital, Kumamoto - Japan 3 Department of Breast and Endocrine Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto - Japan 4 Center for AIDS Research, Kumamoto University, Kumamoto - Japan 5 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo - Japan 1 2
Abstract Introduction: Breast cancer is a leading cause of cancer-related death in women worldwide, and its metastasis is a major cause of disease mortality. Therefore, identification of the mechanisms underlying breast cancer metastasis is crucial for the development of therapeutic and diagnostic strategies. Our recent study of immunodeficient female mice transplanted with MDA-MB231 breast cancer cells demonstrated that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) accelerates metastasis through both increasing tumor cell migration in an autocrine/paracrine manner, and enhancing tumor angiogenesis. To determine whether ANGPTL2 contributes to its clinical pathogenesis, we asked whether serum ANGPTL2 levels reflect the clinical features of breast cancer progression. Methods: We monitored the levels of secreted ANGPTL2 in supernatants of cultured proliferating MDA-MB231 cells. We also determined whether the circulating ANGPTL2 levels were positively correlated with cancer progression in an in vivo breast cancer xenograft model using MDA-MB231 cells. Finally, we investigated whether serum ANGPTL2 levels were associated with clinical features in breast cancer patients. Results: Both in vitro and in vivo experiments showed that the levels of ANGPTL2 secreted from breast cancer cells increased with cell proliferation and cancer progression. Serum ANGPTL2 levels in patients with metastatic breast cancer were significantly higher than those in healthy subjects or in patients with ductal carcinoma in situ or non-metastatic invasive ductal carcinoma. Serum ANGPTL2 levels in patients negative for estrogen receptors and progesterone receptors, particularly triple-negative cases, reflected histological grades. Conclusions: These findings suggest that serum ANGPTL2 levels in breast cancer patients could represent a potential marker of breast cancer metastasis. Keywords: ANGPTL2, Metastasis, Breast cancer, Triple-negative, Nuclear grade Received: September 19, 2013; Accepted: February 18, 2014
INTRODUCTION Breast cancer is the most frequently diagnosed malignant neoplasm in women and is a leading cause of cancer-related death worldwide (1). Breast cancer mortality after therapeutic intervention is primarily due to metastasis of tumor cells from the primary lesion (2, 3). Therefore, identification of the molecular mechanisms driving breast cancer metastasis is important in order to develop effec-
tive therapeutic or early diagnostic strategies to treat this condition. Recently, chronic inflammation has received much attention as playing a key role at various stages of tumor development, including initiation, growth, invasion, and metastasis (4). In this regard, we recently reported that angiopoietin-like protein 2 (ANGPTL2) increases inflammatory carcinogenesis (5). Furthermore, our xenograft studies in female mice transplanted with MDA-MB231
© 2014 Wichtig Publishing - eISSN 1724-6008
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Serum ANGPTL2 reflects clinical features of breast cancer
breast cancer cells demonstrated that tumor cell-derived ANGPTL2 accelerates tumor metastasis by increasing tumor cell migration and enhancing tumor angiogenesis (6). However, the role of ANGPTL2 in the clinical pathogenesis of breast cancer remains uncharacterized. To investigate this question, we asked whether serum ANGPTL2 levels paralleled clinical features of breast cancer progression. We found that circulating ANGPTL2 levels in xenograft mouse models increased according to cancer progression, particularly in the presence of metastasis. Serum ANGPTL2 levels in patients with advancedstage breast cancer were significantly higher than those observed in healthy subjects or patients with ductal carcinoma in situ (DCIS) or non-metastatic invasive ductal carcinoma (IDC). In patients negative for estrogen receptors and progesterone receptors, serum ANGPTL2 levels were positively correlated with nuclear grade status. Our findings show that serum ANGPTL2 levels in hormone receptor (HR)-negative patients, and especially triple-negative (TN) ones, might reflect the clinical features of breast cancer.
centrifuged at 3,000 rpm for 10 minutes, and supernatants were stored at -80°C prior to assay. This study was approved by the ethics committees of the Kumamoto University. Written informed consent was obtained from each subject.
MATERIALS AND METHODS
Total RNA from cultured cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). DNase-treated RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara Bio, Otsu, Japan). PCR products were amplified using a Thermal Cycler Dice Real Time system (Takara Bio, Otsu, Japan), and the relative transcript abundance was normalized to that of the 18S mRNA. PCR oligonucleotides were as follows: human ANGPTL2, forward, 5’- GCCACCAAGTGTCAGCCTCA-3’, reverse, 5’- TGGACAGTACCAAACATCCAACATC-3’: human 18S, forward, 5’- TTTGCGAGTACTCAACACCAACATC-3’, reverse, 5’- GAGCATATCTTCGGCCCACAC-3’. The data was analyzed with the 2-tailed Student’s t-test using Excel software (Microsoft, Redmond, WA). A p value of less than 0.05 was considered statistically significant.
Quantitation of the ANGPTL2 protein by ELISA The ANGPTL2 concentrations in culture medium from tumor cells or in serum from humans or mice were estimated using the ANGPTL2 assay kit (IBL, Gunma, Japan) as previously described (7, 8). Human studies We collected blood samples from breast cancer patients (n=625) at Kumamoto University between May 2003 and July 2011, as well as from healthy female volunteers (n=19) aged 20-54 years working at Kumamoto University. All patients were examined to evaluate potential tumor spread in the whole body using mammography, ultrasound sonography, and computed tomography (CT) at the time of initial diagnosis. Stage IV patients and subjects with relapse after initial treatment (such as surgery and adjuvant treatment) also received bone scintigraphy or positron emission tomography to search for additional lesions. Diagnosis of solitary lesions resulting from potential relapse and/or metastasis was undertaken using fine needle aspiration biopsy and core needle biopsy to confirm metastasis. Blood samples from breast cancer patients without distant metastasis were obtained immediately before surgery or at the time of initial systemic treatment with chemotherapy, anti-HER2 therapy, or endocrine therapy. Serum samples of patients with distant metastasis were obtained before initial treatment. Blood samples were e240
Cell lines and culture Human breast adenocarcinoma cell lines MCF-7, T47D, SK-BR-3, MDA-MB453, and MDA-MB231 were purchased from the American Type Culture Collection (ATCC) and cultured in conditions recommended by the supplier. Luciferase-expressing MDA-MB231 cells (MB231/ luc) were generated and maintained as described (6). To detect ANGPTL2 concentrations in culture medium, 5,000 cells were plated in each well of a 96-well plate (Iwaki, Tokyo, Japan) with 100 μL medium; after the first plating, the medium was then assayed at specific time points using an ANGPTL2 assay kit (IBL, Gunma, Japan). Real-time quantitative RT-PCR
Proliferation assay A total of 5,000 cells per well were plated in 96-well plates (Iwaki, Tokyo, Japan), and cell number was determined at specific time points using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. The growth rate was expressed as the number of cells at a specific time point relative to that determined at 0 hours. Mouse xenograft model For the breast cancer xenograft model, we used MB231/luc cells implanted in the mammary fat pad of an 8-week-old female JAK3-deficient NOD-SCID (NOJ) mice (6), and we monitored bioluminescence images
© 2014 Wichtig Publishing - eISSN 1724-6008
Endo et al
as previously described (6). All experiments were performed according to the guidelines of the Institutional Animal Committee of Kumamoto University. Statistics Cell proliferation and ANGPTL2 levels in the culture medium were analyzed using the Kruskal-Wallis test. Serum ANGPTL2 levels were analyzed using the SteelDwass test. All calculations were performed using the JMP10 software (SAS Institute, Cary, NC, USA). A p value of less than 0.05 was considered statistically significant. RESULTS Proliferating ANGPTL2-expressing cells secrete ANGPTL2 protein in vitro To examine whether tumor cell-secreted ANGPTL2 concentrations increase with tumor cell proliferation, we employed an enzyme-linked immunosorbent assay (ELISA) to measure ANGPTL2 protein levels in the medium of cultured human breast cancer MDA-MB231 cells, which abundantly express ANGPTL2 (6). As expected, MDAMB231 cells proliferated over time (Fig. 1A) and ANGPTL2 concentrations in the medium increased with proliferation (Fig. 1B). Next, we calculated the ratio between ANGPTL2 concentration and proliferation rate and found that ANGPTL2 levels increased from day 1 to day 2 but remained unchanged from day 2 to day 3 (Fig. 1C). We also examined ANGPTL2 mRNA induction and observed a trend similar to that of the secreted ANGPTL2 protein (Fig. 1D). Thus, cultured breast cancer cells secrete ANGPTL2 as they proliferate, and induction is more apparent at early proliferative stages. Serum ANGPTL2 levels increase in parallel with metastasis in a xenograft mouse model Next, we analyzed whether circulating tumor-cell secreted ANGPTL2 levels reflect cancer progression in vivo in a breast cancer mouse model. To do so, we first examined the ANGPTL2 mRNA levels in various breast cancer cell lines, including MCF-7 (ER+, HER2-), T47D (ER+, HER2-), SK-BR-3 (ER-, HER2+), MDA-MB453 (ER-, HER2+), and MDA-MB231 (ER-, HER2-) (Fig. 2A). As we previously reported (6), ANGPTL2 induction is more robust in MDA-MB231 cells than in other breast cancer cell lines. Therefore, we implanted MDA-MB231 cells harboring a luciferase expression vector (MB231/luc) into the mouse mammary fat pad of JAK3-deficient NOD-SCID (NOJ) mice (7) and then monitored bioluminescence images and serum ANGPTL2 concentrations. Tumors in the mammary fat pad grew gradually, based on biolumi-
Fig. 1 - MDA-MB231 breast cancer cells secrete ANGPTL2 in vitro. (A) Relative number of proliferating MDA-MB231 cells at the indicated time points of the in vitro culture (4 independent experiments, each containing 4 replicates). On day 1 of cell culture the number of proliferating cells was set at 1. (B) ANGPTL2 protein concentration in the culture medium at the indicated days (4 independent experiments, each containing 4 replicates). (C) The ratio between ANGPTL2 concentration and relative cell number. (D) Comparative ANGPTL2 expression levels of MDAMB231 cells at the indicated time points (4 independent experiments, each containing 2 replicates). mRNA levels on day 1 of cell culture were set at 1. Error bars show SD. **p20 mm
42
2.5 (1.71-3.49)
149
2.42 (1.71-3.16)
n (-)
282
2.27 (1.56-3.30)
299
2.28 (1.71-2.98)
n (+)
111
2.78 (2.04-3.61)
114
2.31 (1.66-3.25)
NG 1, 2
45
2.27 (1.58-2.94)
371
2.28 (1.48-2.64)
NG 3
42
2.94 (2.03-3.74)
42
2.62 (1.70-3.42)
0.072
0.0096
© 2014 Wichtig Publishing - eISSN 1724-6008
0.993
0.971
e243
Serum ANGPTL2 reflects clinical features of breast cancer
TABLE III - A SSOCIATION BETWEEN SERUM ANGPTL2 LEVELS AND CLINICOPATHOLOGICAL FACTORS ACCORDING TO HORMONE RECEPTOR STATUS HR+
HER2+
TN
n
ANGPTL2 levels Median (25th, 75th percentiles)
P value
n
ANGPTL2 levels Median (25th, 75th percentiles)
P value
n
ANGPTL2 levels Median (25th, 75th percentiles)
P value
T≤20 mm
251
2.28 (1.72-2.95)
0.269
27
2.27 (1.61-3.23)
0.674
31
2.29 (1.56-3.32)
0.407
T>20 mm
142
2.42 (1.71-3.18)
28
2.40 (1.50-3.03)
21
2.62 (1.75-3.60)
n (-)
282
2.30 (1.73-2.98)
37
2.18 (1.44-3.05)
36
2.29 (1.58-3.44)
n (+)
111
2.38 (1.67-3.27)
18
2.59 (2.02-3.12)
16
2.72 (1.62-3.85)
NG 1, 2
359
2.29 (1.73-3.03)
31
2.28 (1.48-2.64)
26
1.85 (1.59-3.12)
NG 3
34
2.54 (1.60-3.11)
24
2.62 (1.70-3.42)
26
2.87 (2.06-3.75)
0.952
0.727
0.123
0.249
0.362
0.024
Luminal: HR+ and HER2-, HER2+: HER2+, irrespective of HR status, TN: HR- and HER2-.
and ANGPTL2 are induced by the hypoxic microenvironment of cancer cells (6, 13). ANGPTL4 is also induced by hypoxia and inhibits endothelial cells interactions to promote tumor metastasis (14). A common theme of these studies is that ANGPTL2 and 4 and Ang2 are induced by hypoxia, a state created by many tumor cells. Recently, we showed that ANGPTL2 induction is regulated by DNA methylation, which is seen in hypoxic cancer microenvironments (15). In this study, we did not examine whether ANGPTL2 and other Ang cooperate with each other in the cancer cell microenvironment. That relationship should be investigated in future studies. We recently reported studies of immunodeficient female mice transplanted with MDA-MB231 breast cancer cells showing that tumor cell-secreted ANGPTL2 shortened survival time and promoted cancer metastasis by accelerating motility, invasion, and tumor angiogenesis in an autocrine/paracrine manner (6). Taken together with the clinical findings reported here, we suggest that inhibiting ANGPTL2 activity or secretion from breast cancer cells represents a novel potential therapy to antagonize advanced breast cancer. Recently, Zheng et al reported that the immune inhibitory receptor human leukocyte immunoglobulin-like receptor B2 (LILRB2), and its mouse orthologue paired immunoglobulin-like receptor B2 (PIRB2), are ANGPTL2 receptors (16). The authors also reported that LILRB2 and PIRB2 are respectively expressed on human and mouse hematopoietic stem cells (HSCs), and that ANGPTL2 binding to these receptors supports ex vivo HSC expansion. Furthermore, in mouse models of human acute myeloid leukemia, impaired PIRB signaling promotes leukemia cell differentiation (16). Since we reported that ANGPTL2 contributes to tumor invasion, metastasis, and e244
recurrence (6), it will be of interest to investigate whether breast cancer stem cells express ANGPTL2 and whether ANGPTL2 signaling contributes to the invasive phenotypes of those cells. Currently, tumor markers such as CEA and CA15-3 are used to detect breast cancer recurrence and are thought, in some cases, to be potentially more sensitive than radiographic imaging to detect metastatic disease. However, the use of these markers alone is not recommended to detect relapse or to monitor the therapeutic effect (17), partly because these markers are not always sensitive enough to detect cancer recurrence (18, 19), and partly because their serum levels can vary with liver function or lifestyle (20). Also, successful systemic therapy often increases serum concentrations of these factors (an occurrence known as marker “flare”) due to the release of antigens by cytolysis (20, 21). Thus, additional tools enabling earlier diagnosis are needed to decrease the number of deaths from tumor metastasis. Here we demonstrated that serum ANGPTL2 levels in patients with metastatic breast cancer were significantly higher than those in healthy subjects or in patients with DCIS or non-metastatic IDC. In addition, serum ANGPTL2 levels in HR-negative patients, especially TN patients, reflected the nuclear grade status. These findings suggest that serum ANGPTL2 levels in breast cancer patients could serve as a potential diagnostic tool of histological grades. ACKNOWLEDGEMENTS We thank Mss. S. Iwaki, O. Takahashi, K. Tabu, and M. Nakata for technical assistance.
© 2014 Wichtig Publishing - eISSN 1724-6008
Endo et al
Informed Consent: Written informed consent was obtained from each participating subject.
Financial Support: This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Japan Society for the Promotion of Science (JSPS) through its Funding Program for Next Generation World-Leading Researchers (NEXT Program), a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Core Research for Evolutional Science and Technology (CREST) program of Japan Science and Technology Agency
REFERENCES 1. 2.
3. 4. 5.
6.
7.
8.
9.
10. 11.
Boyle P, Levin B. World Cancer Report 2008. Lyon: IARC press; 2008. Eckhardt BL, Francis PA, Parker BS, Anderson RL. Strategies for the discovery and development of therapies for metastatic breast cancer. Nat Rev Drug Discov. 2011; 11(6): 479-497. Weigelt B, Peterse JL, van ’t Veer LJ. Breast cancer metastasis: markers and models. Nat Rev Cancer. 2005; 5(8): 591-602. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010; 140(6): 883-899. Aoi J, Endo M, Kadomatsu T, et al. Angiopoietin-like protein 2 is an important facilitator of inflammatory carcinogenesis and metastasis. Cancer Res. 2011; 71(24): 7502-7512. Endo M, Nakano M, Kadomatsu T, et al. Tumor cell-derived angiopoietin-like protein ANGPTL2 is a critical driver of metastasis. Cancer Res. 2012; 72(7): 1784-1794. Okada T, Tsukano H, Endo M, et al. Synoviocyte-derived angiopoietin-like protein 2 contributes to synovial chronic inflammation in rheumatoid arthritis. Am J Pathol. 2010; 176(5): 2309-2319. Tabata M, Kadomatsu T, Fukuhara S, et al. Angiopoietinlike protein 2 promotes chronic adipose tissue inflammation and obesity-related systemic insulin resistance. Cell Metab. 2009; 10(3): 178-188. Kadomatsu T, Tabata M, Oike Y. Angiopoietin-like proteins: emerging targets for treatment of obesity and related metabolic diseases. FEBS J. 2011; 278(4): 559-564. Rigamonti N, De Palma M. A role for angiopoietin-2 in organ-specific metastasis. Cell Rep. 2013; 4(4): 621-623. Minami T, Jiang S, Schadler K, et al. The calcineurin-NFATangiopoietin-2 signaling axis in lung endothelium is critical for the establishment of lung metastases. Cell Rep. 2013; 4(4): 709-723.
(JST), and by grants from the Takeda Science Foundation and the Yasuda Medical Foundation. Conflict of Interest Statements: The authors have declared that no conflict of interest exists. Address for correspondence: Yuichi Oike, M.D., Ph.D. or Motoyoshi Endo, M.D., Ph.D. Department of Molecular Genetics Kumamoto University 1-1-1 Honjo, Kumamoto 860-8556, Japan [email protected] [email protected]
12. Holopainen T, Saharinen P, D’Amico G, et al. Effects of angiopoietin-2-blocking antibody on endothelial cell-cell junctions and lung metastasis. J Natl Cancer Inst. 2012; 104(6): 461-475. 13. Semenza GL. Cancer-stromal cell interactions mediated by hypoxia-inducible factors promote angiogenesis, lymphangiogenesis, and metastasis. Oncogene. 2013; 32(35): 4057-4063. 14. Zhang H, Wong CC, Wei H, et al. HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs. Oncogene. 2012; 31(14): 1757-1770. 15. Odagiri H, Kadomatsu T, Endo M, et al. The secreted protein ANGPTL2 promotes metastasis of osteosarcoma cells through integrin α5β1, p38 MAPK, and matrix metalloproteinases. Sci Signal. 2014; 7(309): 2004612. 16. Zheng J, Umikawa M, Cui C, et al. Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development. Nature. 2012; 485(7400): 656-660. 17. Khatcheressian JL, Wolff AC, Smith TJ, et al; American Society of Clinical Oncology. American Society of Clinical Oncology 2006 update of the breast cancer follow-up and management guidelines in the adjuvant setting. J Clin Oncol. 2006; 24(31): 5091-5097. 18. Emens LA, Davidson NE. The follow-up of breast cancer. Semin Oncol. 2003; 30(3): 338-348. 19. Kokko R, Holli K, Hakama M. Ca 15-3 in the follow-up of localised breast cancer: a prospective study. Eur J Cancer. 2002; 38(9): 1189-1193. 20. Stearns V, Yamauchi H, Hayes DF. Circulating tumor markers in breast cancer: accepted utilities and novel prospects. Breast Cancer Res Treat. 1998; 52(1-3): 239-259. 21. Kiang DT, Greenberg LJ, Kennedy BJ. Tumor marker kinetics in the monitoring of breast cancer. Cancer. 1990; 65(2): 193-199.
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