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Original Article the Expressions of Autotaxin-Lysophosphatidate Signaling-Related Proteins in Metastatic Breast Cancer

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Original Article the Expressions of Autotaxin-Lysophosphatidate Signaling-Related Proteins in Metastatic Breast Cancer Int J Clin Exp Pathol 2019;12(8):2920-2930 www.ijcep.com /ISSN:1936-2625/IJCEP0095924 Original Article The expressions of autotaxin-lysophosphatidate signaling-related proteins in metastatic breast cancer Su Jung Shim1, Eunah Shin2, Choong-Sik Lee3, Ja Seung Koo4 1Department of Radiation Oncology, Eulji Hospital, Eulji University School of Medicine, Seoul, Korea; 2Department of Pathology, CHA Gangnam Medical Center, CHA University School of Medicine, Seoul, Korea; 3Department of Pathology, Chungnam National University College of Medicine, Daejun, Korea; 4Department of Pathology, Yonsei University College of Medicine, Seoul, South Korea Received April 23, 2019; Accepted May 23, 2019; Epub August 1, 2019; Published August 15, 2019 Abstract: Purpose: We evaluated the expression of autotaxin-lysophosphatidate signaling-related proteins and the clinical implications for metastatic breast cancer. Methods: We constructed tissue microarrays (TMA) with 126 cases of metastatic breast cancer [31 (24.6%) bone metastases, 36 (28.6%) brain metastases, 11 (8.7%) liver metastases, and 48 (38.1%) lung metastasis], and we conducted immunohistochemical staining for the autotoxin- lysophosphatidate signaling-related proteins ATX, LPA1, LPA2, and LPA3. Results: Stromal ATX (P = 0.006) and LPA1 (P < 0.001) were differently expressed according to their metastatic organ; stromal ATX showed high expression in bone metastasis, and LPA1 showed high expression in liver and lung metastases. Stromal ATX positivity was higher than others in luminal A type tumors (P = 0.035), and stromal LPA3 positivity was correlated with a high Ki-67 labeling index (LI) (P = 0.005). In univariate analysis, tumoral LPA3 negativity was correlated with shorter overall survival (OS) ( P = 0.015) in metastatic breast cancer. When analyzed according to the metastatic sites, tumoral LPA3 negativity was correlated with shorter OS (P = 0.010) in lung metastasis, whereas stromal LPA3 negativity was correlated with shorter OS (P = 0.026) in brain metastasis. In multivariate Cox analysis, tumoral LPA3 negativ- ity was an independent poor prognostic factor (HR = 2.311, 95% CI: 1.029-5.191, P = 0.043). Conclusion: Among autotoxin-lysophosphatidate signaling-related proteins, stromal ATX was highly expressed in bone metastases, and LPA1 was highly expressed in liver and lung metastases. Tumoral LPA3 might be a prognostic factor in metastatic breast cancer. Keywords: Autotaxin, breast cancer, lysophosphatidate receptor, metastasis Introduction in breast cancer, and ATX, LPA, and LAP recep- tors in breast cancer are involved in angiogen- Autotaxin (ATX) is a glycoprotein transcribed by esis, tumor cell invasion, and migration [7]. the ENPP2 gene on chromosome 8 [1]. ATX is the same molecule as lysophospholipase D, so Breast cancer has both high morbidity and mor- it converts lysophosphatidylcholine (LPC) to tality, which is mainly attributed to distant the bioactive lipid mediator lysophosphatidate metastasis. The main metastatic sites of breast (LPA). LPA, after binding to the appropriate cancer are the lung, brain, liver, and bones [8, receptor, activates phospholipase C, the MAPK 9]; however, most current studies have involved pathway, the PI3K pathway, and the PhoA path- brain and bone metastases [10-15]. The gen- way, and it is involved in various cellular pro- eral pathogenesis of tumor metastasis involves cesses [2, 3]. The LPA receptor is a G-protein a reciprocal interaction between tumor cells coupled receptor. There are at least 6 LPA and host tissue, consisting of adhesion, prote- receptors, LPA1-LPA6. LPA1-LPA3 belong to the olysis, invasion, and angiogenesis [9, 16]. EGD family (LPA1-EDG2, LPA2-EDG4, LPA3- As has been reported previously, metastatic EDG7), and LPA4-LPA6 are similar to the P2Y breast cancer shows specific tumor character- nucleotide receptor [4]. ATX-LPA signaling is istics according to the metastatic site, young involved in tumor formation, progression, and age, ER negativity, prior lung metastasis, HER-2 metastasis [5, 6]. The expression of ATX and overexpression, EGFR overexpression, and ba- LPA 1-3 has been reported to be especially high sal subtype [12-14]. Lower histologic grade, ER Autotaxin-lysophosphatidate in metastatic breast cancer Table 1. Source, clone, and dilution of antibodies Antibody Company Clone Dilution Autotaxin-related proteins ATX Abcam, Cambridge, UK Polyclonal 1:1000 LPA1 Abcam, Cambridge, UK EPR9710 1:100 LPA2 Abcam, Cambridge, UK Polyclonal 1:100 LPA3 Abcam, Cambridge, UK Polyclonal 1:250 Molecular subtype-related proteins Estrogen receptor Thermo Scientific, San Diego, CA, USA SP1 1:100 Progesterone receptor DAKO, Glostrup, Denmark PgR 1:50 Human epidermal growth factor receptor-2 DAKO, Glostrup, Denmark Polyclonal 1:1500 Ki-67 Abcam, Cambridge, UK SP6 1:100 Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphhodesterase 2 (ENPP2), LPA1-EDG2, LPA2-EDG4, and LPA3-EDG7. positivity, ER positivity/PR negativity, strand histological grade was assessed using the growth pattern, and the presence of fibrotic foci Nottingham grading system [21]. in invasive ductal carcinoma are associated with bone metastasis [11, 17, 18]. Consequently, Tissue microarray (TMA) it is easily presumed that metastatic breast cancer can have different tumor characteristics A representative area showing tumor and tumor according to the metastatic site. ATX-LPA sig- stroma was selected on an H&E-stained slide, naling in breast cancer has been reported to be and a corresponding spot was marked on the involved in metastasis, and the expression of surface of the paraffin block. Using a biopsy ATX and LPAR in breast cancer is especially needle, the selected area was punched out, associated with advanced metastatic disease and a 3-mm tissue core was transferred to a 6 x 5 recipient block. Two tissue cores from each [19]. The overexpression of ATX or LPA1-3 in transgenic mice results in lung metastasis [20]. invasive tumor were extracted to minimize However, studies on autotaxin-lysophosphati- extraction bias. Each tissue core was assigned date signaling-related proteins in metastatic a unique tissue microarray location number breast cancer according to the metastatic sites that was linked to a database containing other have not yet been reported, so we aimed to clinicopathologic data. assess the expression of autotaxin-lysophos- phatidate signaling-related proteins and relat- Immunohistochemistry ed clinical implications in metastatic breast The antibodies used for immunohistochemistry cancer according to the metastatic site. are shown in Table 1. Formalin-fixed, paraffin- Materials and methods embedded tissue sections were used for immu- nohistochemistry, and 3-μm-thick tissue sec- Patient selection and histologic evaluation tions were deparaffinized and rehydrated in xylene and graded alcohol. We used a Venta- Cases of metastatic breast cancer to the liver, na Discovery XT automated stainer (Ventana lung, brain, and bone were selected from the Medical System, Tucson, AZ, USA). CC1 buffer data files of the Department of Pathology, (Cell Conditioning 1; citrate buffer Ph 6.0, Severance Hospital. Only patients who were Ventana Medical System) was used for antigen diagnosed with invasive ductal carcinoma were retrieval. Appropriate positive and negative included. This study was approved by the controls were used for each antibody. The posi- Institutional Review Board (IRB) of Severance tive controls were used according to the manu- Hospital. A total of 126 cases were retrieved facturer’s instructions (ATX; human tonsil tis- and all the slides that were prepared per case sue, LPA1; human thyroid tissue, LPA2; Breast were reviewed. Pathologic diagnoses were con- cancer tissue, LPA3; Human prostate carcino- firmed by 2 pathologists (JSK and WJ), and the ma). A negative control was used with a sec- 2921 Int J Clin Exp Pathol 2019;12(8):2920-2930 Autotaxin-lysophosphatidate in metastatic breast cancer ondary antibody alone and the primary anti- saline (TBS) with 0.1% Tween-20 and probed body omitted. with antibody against ATX (1:1000, Abcam, ab140915), LPA1 (1:2000, Abcam, ab166903), Interpretation of immunohistochemical stain- and LPA2 (1:1000, Abcam ab38322) diluted in ing 1% BSA in TBS, 0.1% Tween 20, and 0.02% NaN3. The membranes were washed and then All immunohistochemical markers were asse- incubated with secondary antibodies (HRP con- ssed by light microscopy. A cut-off value of 1% jugated anti-mouse IgG, or anti-rabbit IgG) or more positively stained nuclei was used to (1:20000, Santa Cruz) for 1 h at room tem- define ER and PR positivity [22]. HER-2 staining perature. The bands were visualized using was analyzed according to the American Society WesternBright ECL (advansata, K-12045-D50) of Clinical Oncology (ASCO)/College of American after washing the membrane and exposed to Pathologists (CAP) guidelines using the follow- x-ray film. ing categories: 0 = no immunostaining; 1+ = weak incomplete membranous staining, less Tumor phenotype classification than 10% of tumor cells; 2+ = complete mem- branous staining, either uniform or weak in at In this study, we classified breast cancer phe- least 10% of tumor cells; and 3+ = uniform notypes according to the immunohistochemis- intense membranous staining in at least 30% try results for ER, PR, HER-2, Ki-67, and FISH of tumor cells [23]. HER-2 immunostaining was results for HER-2 as follows [25]: luminal A considered positive when strong (3+) membra- type, ER or/and PR positive, HER-2 negative nous staining was observed, but cases with 0 and Ki-67 LI < 14%; Luminal B type, (HER-2 to 1+ were regarded as negative. Cases show- negative) ER or/and PR positive, HER-2 nega- ing 2+ HER-2 expression were evaluated for tive and Ki-67 LI ≥ 14%; (HER-2 positive) ER or/ HER-2 amplification by fluorescent in situ hy- and PR positive and HER-2 overexpressed or/ bridization (FISH). The Ki-67 labeling index (LI) and amplified; HER-2 overexpression type, ER was defined as the percentage of tumor cells and PR negative and HER-2 overexpressed or/ showing positive nuclear staining. and amplified; TNBC type: ER, PR, and HER-2 negative. All stained slides were semi-quantitatively eval- uated [24].
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