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Role of laminin 332 in lymph node metastasis of papillary thyroid carcinoma
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Role of laminin 332 in lymph node metastasis of papillary thyroid carcinoma Kyoung Ho Oh a, June Choi a, Jeong-Soo Woo a, Seung Kuk Baek a, Kwang Yoon Jung a, Min Ji Koh b, Young-Sik Kim c, Soon Young Kwon a,* a
Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, Republic of Korea Phillips Academy, Andover, MA, USA c Department of pathology, College of Medicine, Korea University, Ansan Hospital, Ansan, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 September 2016 Accepted 26 January 2017 Available online xxx
Objective: The invasiveness of papillary thyroid carcinoma (PTC), including the occurrence of cervical lymph node metastasis, is the main determining factors contributing to recurrence and poor prognosis. Laminin 332 is a glycoprotein involved in cell migration and cancer cell invasion into surrounding tissues and is therefore related to poor prognosis in many cancers. Here, we investigated the expression and role of laminin 332 in PTC and examine the possibility that laminin 332 could be involved in the invasiveness of PTC. Methods: Laminin 332 expression was determined by immunohistochemical staining in all 40 patients. The correlations between laminin 332 expression and clinical factors were investigated. We examined the expression of the laminin 332 g2 chain using reverse transcription polymerase chain reaction and western blotting in PTC cells and determined the relationship between the expression of laminin 332 and the invasiveness of these cell lines using cell invasion assays. Results: Laminin 332 was expressed specifically within tumor tissue. The frequency of laminin 332 g2 chain expression was significantly correlated with cervical lymph node metastasis (p = 0.003). Invasiveness increased as the expression of laminin 332 g2 increased in the tested PTC cell lines. Conclusion: Laminin 332 expression may be a useful marker for predicting lymph node metastasis in papillary thyroid carcinoma, and could increase the ability of cancer cells to invade, which would influence the prognosis of patients with PTC. © 2017 Elsevier B.V. All rights reserved.
Keywords: Glycoprotein Thyroid cancer Invasion Prognosis
1. Introduction Thyroid carcinoma is the most common endocrine cancer and has a high incidence relative to other types of cancer in humans [1]. Among thyroid malignancies, papillary thyroid carcinoma (PTC) is the most common, accounting for 70–90%
* Corresponding author at: Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, 123 Jeokgeum-ro (Gojan-dong), Danwon-gu, Ansan-si, Gyeonggi-do 425-707, Republic of Korea. Fax: +82 31 4125174. E-mail address: [email protected] (S.Y. Kwon).
of all well-differentiated thyroid malignancies [2]. Patients with PTC generally have a good prognosis, and the majority of patients who undergo appropriate treatment display excellent outcomes [1,3]. Thus, there is controversy regarding potential overdiagnosis and excessive interventions in patients with PTC [4]. However, clinician agree that PTC should be aggressively treated when the prognosis is poor. The factors associated with poor prognosis include age, extracapsular extension, lymph node metastasis (LNM), and poorly differentiated cell type [2]. In addition, various molecular, biological, and genetic factors are being studied for their possible association with prognosis in patients with PTC. Primary LNM occurs in
http://dx.doi.org/10.1016/j.anl.2017.01.010 0385-8146/© 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: Oh KH, et al. Role of laminin 332 in lymph node metastasis of papillary thyroid carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2017.01.010
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approximately 30–40% of adult patients [5]. Additionally, LNM, a risk factor for local and regional recurrence, affects the treatment modality employed; ensuring optimal combined treatment for LNM requires a better understanding of the molecular mechanisms underlying its development. However, no definite predictor of cervical LNM in patients with PTC has been determined [1]. Laminin is a glycoprotein produced by endothelial and epithelial cells and localized in the basement membrane. Laminin plays roles in numerous cellular functions, including cell migration, adhesion, and differentiation [6]. Structurally, laminins consist of three chains (a, b, and g) and are named according to their chain composition [7]. Thus far, 16 laminins have been characterized. Laminin 322 (previously known as laminin 5) is a 460-kDa molecule that is composed of a3, b3, and g2 chains. LAMC2, the human gene encoding the g2 chain, is located on chromosome 1q5-q31. Laminin 332 functions as an adhesive molecule in mature normal tissue and mediates the migration of epithelial cells during wound repair [8]. Additionally, laminin 332 appears to play an important role in tumor invasion, and recent studies have supported the hypothesis that laminin 332 expression in cancer cells promotes cells growth, invasion, and metastasis [9]. Moreover, expression of laminin 332 is related to poor prognosis in breast, colon, and pancreatic cancer [10–12]. In the present study, we aimed to identify the relationship between the expression of laminin 332 and LNM in PTC. Furthermore, we investigated the association between laminin 322 expression and invasiveness in PTC using PTC cell lines.
incubated overnight with monoclonal anti-laminin 332 g2 antibodies (D4B5; 1:200; Chemicon Int., Germany) at 4 C, washed with Tris-buffered saline (TBS), and incubated with secondary antibodies and streptavidin-horseradish peroxidase (HRP) from the Cap-plus Detection Kit. The sections were then processed with 3,3-diaminobenzidine (DAB) from the Cap-plus Detection Kit. Thereafter, sections were rinsed briefly in water, counterstained with Meyer’s hematoxylin (Sigma), and mounted. Scoring and interpretation of immunohistochemical results was independently performed by two pathologists and two otolaryngology-head and neck surgeons blinded to the clinical outcomes. The immunohistochemical score for the expression of laminin 332 was assigned based on the percentage of stained tumor cells, as follows: 0, <5%; 1+, 5–25%; 2+, 26–50%; and 3 +, >50%. The correlations of laminin 332 expression with clinical factors, such as tumor size, multiplicity, and LNM, were also investigated. 2.3. Cell culture
2. Patients and methods
SNU-790 cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea), and BHP10-3 and BCPAP cells were kindly provided by Professor Soon Hyun Ahn (Seoul National University, Korea); all three of these cell lines are PTC cell lines. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and L-glutamine (300 mg/L) [13]. All cell lines were grown in plastic culture flasks (VWR, Canada) incubated at 37 C in a humidified atmosphere containing 5% CO2. Cells were subcultured at 72-h intervals using 0.25% trypsin-0.02% EDTA and seeded into fresh medium at a density of 2.5–3.5 105 cells/mL.
2.1. Patients
2.4. Western blot analysis
PTC samples were obtained randomly from the surgical pathology archive of Ansan Hospital, Korea University Medical Center between May 2006 and December 2008. Study procedures were approved by the Institutional Review Board of Korea University Medical Center. The present study involved 40 patients with well-differentiated papillary thyroid carcinoma (seven men and 33 women) who underwent thyroidectomy with central compartment neck dissection at the Department of ENT, Ansan Hospital, Korea University Medical Center. The patients’ ages ranged from 25 to 82 years (mean, 48.3 years). Among the 40 patients, 20 had LNM. LNM was evaluated by pathologic finding. None of the patients received pre-operative chemotherapy or radiotherapy, and none had synchronous or metachronous cancers in other organs.
Cells were seeded in six-well plates at a density of 2 105 cells/well and cultured for 48 h. The cells were then lysed with cell lysis buffer (RIPA buffer, 100 mL) and quantified using a BCA array. Equal amounts of quantified total protein (20 mg, 10%) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to membranes and incubated with blocking buffer for 1 h. The blocked membrane was probed with an antibody against the laminin 332 g2 chain (1:1000; clone D4B5; cat. no. MAB19562; Merck Millipore, Darmstadt, Germany) overnight at 4 C. The blot was probed for glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal control with anti-GAPDH antibodies (1:200; cat. no. sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blot was washed three times with TBS-T (0.1%) for 10 min each. The immunoreactive bands were detected by incubation with secondary antibodies (1:5000; anti-mouse IgG) for 1 h and then washed three times with TBS-T (0.1%) for 10 min each.
2.2. Immunohistochemistry Tumor samples were fixed with 10% formaldehyde solution, embedded in paraffin, and sectioned into 4-mm-thick slices. Tissue sections were deparaffinized with xylene, incubated with 0.3% hydrogen peroxide in methanol, and treated with protease XXIV (Sigma, St. Louis, MO, USA). The sections were then treated with blocking solution from a Cap-plus Detection Kit (Invitrogen, Carlsbad, CA, USA). Blocked sections were
2.5. Real-time reverse transcription polymerase chain reaction (qRT-PCR) For gene expression analysis, 2 105 cells were cultured for 48 h on different days (n = 3). Cells were washed with PBS, and
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RNA was isolated with TRIzol reagent. The isolated RNA (1 mg) was used to synthesize cDNA with a Roche First-strand RNA Synthesis Kit. RT-PCR was conducted to detect the expression of laminin 332 g2. 2.6. Cell invasion assay To analyze cell invasion, we used Boyden chamber invasion assays. Transwells (8-mm-pore-size Corning Costar Transwell Permeable Supports; Fisher Scientific, Waltham, MA, USA) were coated with Matrigel (BD Matrigel Basement Membrane Matrix; diluted 1:50 in RPMI1640 serum-free medium) and incubated overnight at 37 C in 5% CO2. Cells were suspended in culture medium (5 104 cells/mL), added to 24-well transwells, and incubated in a humidified tissue culture incubator at 37 C in 5% CO2. After 24 h of incubation, nonmigrated cells were removed from the upper surface of the membrane. A cotton-tipped swab was inserted into the Matrigel-coated transwell and moved gently and firmly over the membrane surface. Transwells were removed from the 24well plates and stained with a Hemacolor rapid staining kit (Merck Millipore). The number of migrated cells was counted at three random regions using light microscopy (Axiocam HRc; Zeiss, Jena, Germany). To exam the effects of laminin 332, we treated SNU-790 cells with 5 mL IgG (ChromePure Mouse IgG; Jackson ImmunoResearch Inc., West Grove, PA, USA) as a control group or 5 mL of antibodies against laminin 332 g2 (1:1000; clone D4B5, cat. no. MAB19562; Merck Millipore) as an experimental group. The methods were the same as those described above. Experiments were performed three times. 2.7. Statistical analysis For analysis of immunohistochemical data, Pearson Chisquared tests and one-way analysis of variance (ANOVA) were performed with IBM SPSS Software Version 20.0 for Windows (IBM, Armonk, NY, USA). Invasion assay results were analyzed by the Kruskal–Wallis test. Differences with p values of less than 0.05 were considered statistically significant.
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specifically expressed in cells adjacent to the tumor stroma (Fig. 1). Statistical analysis showed that the frequency of laminin 332 expression was significantly correlated with the presence of cervical LNMs (p = 0.003). Cases displaying larger tumor sizes tended to show higher expression of laminin 332, although the correlation was not statistically significant ( p = 0.082). Additionally, no association between multiplicity of cancer and the expression of laminin 332 was identified. The results of multivariate logistic regression analysis demonstrated that only expression of laminin 332 (p = 0.031) significantly associated with lymph node metastasis (Table 2). 3.2. Expression of the laminin 332 g2 chain was correlated with cell invasion in PTC cells The protein and mRNA expression levels of the laminin 332 g2 in PTC cells are shown in Fig. 2A and 2B. In PTC cells, the expression levels of the g2 chain followed the order of SNU790 > BHP10–3 > BCPAP. Using a Boyden chamber invasion assay, we investigated whether laminin 332 promoted the invasion of PTC cells. This assay was repeated three times, and the mean number of migrated cells from three independent experiments differed significantly (p < 0.01). As shown in Fig. 2C, among the PTC cell lines, SNU-790 cells exhibited the greatest number of invaded cells, and the trend in the tested cells lines followed the order of SNU790 > BCPAP > BHP10-3. Based on the level of g2 chain expression, increased cell invasion was observed in cells with higher levels of g2, except in BHP10-3 cells. 3.3. Expression of the laminin 332 g2 chain affected cell invasion in PTC cells To examine the effects of laminin 332 on cell invasion, we performed cell invasion assays using Boyden chambers. We used SNU-790 cells, which exhibited the most invasive activity. Cell invasion was significantly reduced in SNU-790 cells treated with anti-laminin 332 g2 chain antibodies compared with that in cells treated IgG as a control group (p < 0.05) (Fig. 3).
3. Results 3.1. Expression of laminin 332 was correlated with lymph node metastasis in patients with PTC Of the 40 patients analyzed in this study, laminin 332 expression was observed in 37 cases (92.5%; Table 1). All cases in the LNM-positive group showed laminin 332 expression (100%). Laminin 332 was localized to the cytoplasm and cell membranes of tumor cells and was
4. Discussion Due to the indolent nature of PTC, there is a tendency for patients to fail to seek proper treatment. However, if a patient with PTC shows poor prognostic factors, such as extracapsular invasion and LNM, his/her quality of life is negatively impacted. Therefore, it is important to accurately predict the prognosis of PTC in order to ensure proper treatment. To this
Table 1 Relationship between the immunoreactivity of laminin 332 and clinical features in patients. IHC score
Laminin 332
Total
Number of additional tumors
pT3, pT4
LN metastasis
Mean size (cm)
0 1 2 3
>5% 5–25% 26–50% >50%
3 8 19 10
1 0 6 5
2 (67%) 6 (75%) 17 (89%) 10 (100%)
0 (0%) 1 (13%) 11 (58%) 8 (80%)
1.23 1.40 1.94 2.49
(33%) (0%) (32%) (50%)
IHC = immunohistochemistry, LN = lymph node, pT = pathologic T stage.
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Fig. 1. Laminin-5 expression in papillary thyroid carcinoma. Examples of laminin-5 g2 chain expression scoring and interpretation. The immunohistochemical score for the expression of laminin-5 was assigned based on the percentage of stained tumor cells. (a. Grade 0, <5%; b. Grade 1, 5–25% (red circle); c. Grade 2, 26–50% (red arrows); and d. Grade 3, >50%). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
end, the relationships between prognosis and various molecular and genetic markers, including BRAF, RAS, RET/PTC, and galectin-3, have been reported [14]. Laminin 332 is a heterotrimeric protein that consists of a, b, and g chains and plays a major role in normal tissue anchoring between epithelial and mesenchymal tissues [15]. Various chains, including 5a, 4b, and 4g chains, are present in different combinations to generate at least 16 laminin isoforms. These laminin isoforms show distinct tissue distributions, and their expression is often precisely regulated during development [16]. Laminin 332 is composed of a3, b3, and g2 chains, and the g2 chain is a unique component of this protein [17]. Laminin 332 is involved in several biological pathways, including self-polymerization, extracellular matrix (ECM) binding, and cellular interactions [18]. Moreover, laminin 332 is known to be related to poor prognosis in several cancers due to its influence on cellular invasion and metastasis [18]. The expression of laminin 332, particularly the g2 chain, is correlated with invasiveness in adenocarcinoma of the colon [17]. In hepatocellular cancers, detection of the laminin 332 g2 chain is strongly associated with metastasis [19]. In this study, a monoclonal anti-laminin 332 g2 antibody (D4B5) was used for immunohistochemical analysis, and we sought to test the feasibility of using laminin 332 as a marker of LNM in patients with PTC. This study is the first
report to suggest a relationship between laminin 332 expression in PTC and LNM as all cases in the LNM-positive group expressed laminin 332 (100%). There was a significant difference in laminin 332 expression between LNM-positive and LNM-negative groups; laminin 332 was more significantly expressed in LNM-positive patients (p = 0.003). These results suggested that overexpression of laminin 332 in PTC may be useful as a biomarker for predicting cervical LNM. As a follow up, we analyzed the expression and function of laminin 332 in PTC. We observed that increased expression of LAMC2 was related to increased cell invasion, which consistent with the previous findings. The invasiveness of BHP10-3 did not follow the tendency of laminin 332 expression level in experiment of cell lines. We assumed that the other factors could influence the invasiveness of BHP10-3. However, the other cell lines followed the tendency that more expression level of laminin 332 increase more invasiveness of PTC cells, and cell invasion was significantly reduced in SNU-790 cells treated with antilaminin 332 g2 chain antibodies compared with that in cells treated IgG as a control group. Therefore, we suppose that laminin 332 is the important factor of cell invasion in PTC. In situ carcinoma cells often deposit laminin 332 onto underlying BM structures. Previous studies have shown that when BM structures are synthesized without laminin 332,
Table 2 Factors associating with lymph node metasatsis in patients with papillary thyroid carcinoma. Factor
Comparison
Odds ratio
95% CI
p value
Multifocality T stage Mean size Lamin 332
Absent vs. present pT1, T2 vs. T3, T4 <2 cm vs. 2 cm IHC score 0, 1 vs. 2, 3
0.78 4.54 7.45 15.78
[0.32–3.99] [1.45–25.98] [1.21–40.87] [1.40–103.99]
0.187 0.071 0.051 0.031
IHC = immunohistochemistry.
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Fig. 2. mRNA and protein expression levels of the laminin 332 g2 chain were correlated with cell invasion in papillary thyroid carcinoma (PTC). (A) Western blot analysis of LAMC2. LAMC2 protein expression in the tested cell lines was consistent with mRNA levels of the laminin 332 chains. (B) RT-PCR analysis of laminin 332 g2 chain expression in PTC cell lines. (C) Cell invasion assay. Stained cells indicate cells that transmigrated across the Matrigel membrane. The graph shows the number of migrated cells for each cell line. Among the PTC cell lines examined in this study, the number of migrated cells showed the following trend: SNU790 > BCPAP > BHP10-3.
tumor cells are better able to migrate into the interstitial stroma [9]. Tumor cells at the invasion front often overexpress the laminin 332 g2 chain, and proteolytic fragments of the laminin 332 g2 monomer may promote tumor cell invasion by binding to epidermal growth factor receptor and other unidentified receptors [9,20]. Expression of the laminin 332 g2 chain was primarily observed in the cytoplasm of carcinoma cells in this study, with the strongest expression found in the periphery of PTCs (Fig. 1). Conversely, only a few g2 chain-expressing cells were observed in the center and papillary portions of the tumor. More importantly, however, laminin 332 was strongly
expressed at the invasive front in the majority of PTC cases with LNM (Fig. 3). These findings suggested that laminin 332 expression played an important role in cancer migration and invasion. 5. Conclusion The expression of laminin 332 may facilitate the diagnosis of PTC, and high expression can serve as a useful biological marker for predicting LNM. The importance of laminin 332 in the metastasis of thyroid cancer makes it an attractive target for cancer therapeutics. Although further studies are needed to clarify the regulatory mechanisms of laminin 332 in PTC, our current results may be useful for the development of new therapeutic agents for preventing LNM in patients with PTC. Financial disclosure This research was supported by a Korea University Grant. Acknowledgments The authors thank Ph. D. Bok Kee Eun in Core-Laboratory for Convergent Translational Research, College of Medicine, ***** University, for helpful advice on experiments. References
Fig. 3. Cell invasion assay. Stained cells indicate cells that transmigrated across the Matrigel membrane. The graph shows the number of migrated cells for each cell line. In SNU-790 cells, the number of migrated cells was lower after treatment with anti-LAMC2 antibodies than that in the control group (IgG).
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[14] Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Wartofsky L, et al. 2015 American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: the American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer. Thyroid 2016;26:1–133. [15] Rousselle P, Beck K. Laminin 332 processing impacts cellular behavior. Cell Adh Migr 2013;7:122–34. [16] Tsuruta D, Kobayashi H, Imanishi H, Sugawara K, Ishii M, Jones JC. Laminin-332-integrin interaction: a target for cancer therapy. Curr Med Chem 2008;15:1968–75. [17] Pyke C, Salo S, Ralfkiaer E, Romer J, Dano K, Tryggvason K. Laminin-5 is a marker of invading cancer cells in some human carcinomas and is coexpressed with the receptor for urokinase plasminogen activator in budding cancer cells in colon adenocarcinomas. Cancer Res 1995;55:4132–9. [18] Domogatskaya A, Rodin S, Tryggvason K. Functional diversity of laminins. Annu Rev Cell Dev Biol 2012;28:523–53. [19] Giannelli G, Falk-Marzillier J, Schiraldi O, Stetler-Stevenson WG, Quaranta V. Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science 1997;277:225–8. [20] Schenk S, Hintermann E, Bilban M, Koshikawa N, Hojilla C, Quaranta V, et al. Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution. J Cell Biol 2003;161:197–209.
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Role of laminin 332 in lymph node metastasis of papillary thyroid carcinoma Kyoung Ho Oh a, June Choi a, Jeong-Soo Woo a, Seung Kuk Baek a, Kwang Yoon Jung a, Min Ji Koh b, Young-Sik Kim c, Soon Young Kwon a,* a
Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, Republic of Korea Phillips Academy, Andover, MA, USA c Department of pathology, College of Medicine, Korea University, Ansan Hospital, Ansan, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 September 2016 Accepted 26 January 2017 Available online xxx
Objective: The invasiveness of papillary thyroid carcinoma (PTC), including the occurrence of cervical lymph node metastasis, is the main determining factors contributing to recurrence and poor prognosis. Laminin 332 is a glycoprotein involved in cell migration and cancer cell invasion into surrounding tissues and is therefore related to poor prognosis in many cancers. Here, we investigated the expression and role of laminin 332 in PTC and examine the possibility that laminin 332 could be involved in the invasiveness of PTC. Methods: Laminin 332 expression was determined by immunohistochemical staining in all 40 patients. The correlations between laminin 332 expression and clinical factors were investigated. We examined the expression of the laminin 332 g2 chain using reverse transcription polymerase chain reaction and western blotting in PTC cells and determined the relationship between the expression of laminin 332 and the invasiveness of these cell lines using cell invasion assays. Results: Laminin 332 was expressed specifically within tumor tissue. The frequency of laminin 332 g2 chain expression was significantly correlated with cervical lymph node metastasis (p = 0.003). Invasiveness increased as the expression of laminin 332 g2 increased in the tested PTC cell lines. Conclusion: Laminin 332 expression may be a useful marker for predicting lymph node metastasis in papillary thyroid carcinoma, and could increase the ability of cancer cells to invade, which would influence the prognosis of patients with PTC. © 2017 Elsevier B.V. All rights reserved.
Keywords: Glycoprotein Thyroid cancer Invasion Prognosis
1. Introduction Thyroid carcinoma is the most common endocrine cancer and has a high incidence relative to other types of cancer in humans [1]. Among thyroid malignancies, papillary thyroid carcinoma (PTC) is the most common, accounting for 70–90%
* Corresponding author at: Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, 123 Jeokgeum-ro (Gojan-dong), Danwon-gu, Ansan-si, Gyeonggi-do 425-707, Republic of Korea. Fax: +82 31 4125174. E-mail address: [email protected] (S.Y. Kwon).
of all well-differentiated thyroid malignancies [2]. Patients with PTC generally have a good prognosis, and the majority of patients who undergo appropriate treatment display excellent outcomes [1,3]. Thus, there is controversy regarding potential overdiagnosis and excessive interventions in patients with PTC [4]. However, clinician agree that PTC should be aggressively treated when the prognosis is poor. The factors associated with poor prognosis include age, extracapsular extension, lymph node metastasis (LNM), and poorly differentiated cell type [2]. In addition, various molecular, biological, and genetic factors are being studied for their possible association with prognosis in patients with PTC. Primary LNM occurs in
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Please cite this article in press as: Oh KH, et al. Role of laminin 332 in lymph node metastasis of papillary thyroid carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2017.01.010
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approximately 30–40% of adult patients [5]. Additionally, LNM, a risk factor for local and regional recurrence, affects the treatment modality employed; ensuring optimal combined treatment for LNM requires a better understanding of the molecular mechanisms underlying its development. However, no definite predictor of cervical LNM in patients with PTC has been determined [1]. Laminin is a glycoprotein produced by endothelial and epithelial cells and localized in the basement membrane. Laminin plays roles in numerous cellular functions, including cell migration, adhesion, and differentiation [6]. Structurally, laminins consist of three chains (a, b, and g) and are named according to their chain composition [7]. Thus far, 16 laminins have been characterized. Laminin 322 (previously known as laminin 5) is a 460-kDa molecule that is composed of a3, b3, and g2 chains. LAMC2, the human gene encoding the g2 chain, is located on chromosome 1q5-q31. Laminin 332 functions as an adhesive molecule in mature normal tissue and mediates the migration of epithelial cells during wound repair [8]. Additionally, laminin 332 appears to play an important role in tumor invasion, and recent studies have supported the hypothesis that laminin 332 expression in cancer cells promotes cells growth, invasion, and metastasis [9]. Moreover, expression of laminin 332 is related to poor prognosis in breast, colon, and pancreatic cancer [10–12]. In the present study, we aimed to identify the relationship between the expression of laminin 332 and LNM in PTC. Furthermore, we investigated the association between laminin 322 expression and invasiveness in PTC using PTC cell lines.
incubated overnight with monoclonal anti-laminin 332 g2 antibodies (D4B5; 1:200; Chemicon Int., Germany) at 4 C, washed with Tris-buffered saline (TBS), and incubated with secondary antibodies and streptavidin-horseradish peroxidase (HRP) from the Cap-plus Detection Kit. The sections were then processed with 3,3-diaminobenzidine (DAB) from the Cap-plus Detection Kit. Thereafter, sections were rinsed briefly in water, counterstained with Meyer’s hematoxylin (Sigma), and mounted. Scoring and interpretation of immunohistochemical results was independently performed by two pathologists and two otolaryngology-head and neck surgeons blinded to the clinical outcomes. The immunohistochemical score for the expression of laminin 332 was assigned based on the percentage of stained tumor cells, as follows: 0, <5%; 1+, 5–25%; 2+, 26–50%; and 3 +, >50%. The correlations of laminin 332 expression with clinical factors, such as tumor size, multiplicity, and LNM, were also investigated. 2.3. Cell culture
2. Patients and methods
SNU-790 cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea), and BHP10-3 and BCPAP cells were kindly provided by Professor Soon Hyun Ahn (Seoul National University, Korea); all three of these cell lines are PTC cell lines. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and L-glutamine (300 mg/L) [13]. All cell lines were grown in plastic culture flasks (VWR, Canada) incubated at 37 C in a humidified atmosphere containing 5% CO2. Cells were subcultured at 72-h intervals using 0.25% trypsin-0.02% EDTA and seeded into fresh medium at a density of 2.5–3.5 105 cells/mL.
2.1. Patients
2.4. Western blot analysis
PTC samples were obtained randomly from the surgical pathology archive of Ansan Hospital, Korea University Medical Center between May 2006 and December 2008. Study procedures were approved by the Institutional Review Board of Korea University Medical Center. The present study involved 40 patients with well-differentiated papillary thyroid carcinoma (seven men and 33 women) who underwent thyroidectomy with central compartment neck dissection at the Department of ENT, Ansan Hospital, Korea University Medical Center. The patients’ ages ranged from 25 to 82 years (mean, 48.3 years). Among the 40 patients, 20 had LNM. LNM was evaluated by pathologic finding. None of the patients received pre-operative chemotherapy or radiotherapy, and none had synchronous or metachronous cancers in other organs.
Cells were seeded in six-well plates at a density of 2 105 cells/well and cultured for 48 h. The cells were then lysed with cell lysis buffer (RIPA buffer, 100 mL) and quantified using a BCA array. Equal amounts of quantified total protein (20 mg, 10%) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to membranes and incubated with blocking buffer for 1 h. The blocked membrane was probed with an antibody against the laminin 332 g2 chain (1:1000; clone D4B5; cat. no. MAB19562; Merck Millipore, Darmstadt, Germany) overnight at 4 C. The blot was probed for glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal control with anti-GAPDH antibodies (1:200; cat. no. sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blot was washed three times with TBS-T (0.1%) for 10 min each. The immunoreactive bands were detected by incubation with secondary antibodies (1:5000; anti-mouse IgG) for 1 h and then washed three times with TBS-T (0.1%) for 10 min each.
2.2. Immunohistochemistry Tumor samples were fixed with 10% formaldehyde solution, embedded in paraffin, and sectioned into 4-mm-thick slices. Tissue sections were deparaffinized with xylene, incubated with 0.3% hydrogen peroxide in methanol, and treated with protease XXIV (Sigma, St. Louis, MO, USA). The sections were then treated with blocking solution from a Cap-plus Detection Kit (Invitrogen, Carlsbad, CA, USA). Blocked sections were
2.5. Real-time reverse transcription polymerase chain reaction (qRT-PCR) For gene expression analysis, 2 105 cells were cultured for 48 h on different days (n = 3). Cells were washed with PBS, and
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RNA was isolated with TRIzol reagent. The isolated RNA (1 mg) was used to synthesize cDNA with a Roche First-strand RNA Synthesis Kit. RT-PCR was conducted to detect the expression of laminin 332 g2. 2.6. Cell invasion assay To analyze cell invasion, we used Boyden chamber invasion assays. Transwells (8-mm-pore-size Corning Costar Transwell Permeable Supports; Fisher Scientific, Waltham, MA, USA) were coated with Matrigel (BD Matrigel Basement Membrane Matrix; diluted 1:50 in RPMI1640 serum-free medium) and incubated overnight at 37 C in 5% CO2. Cells were suspended in culture medium (5 104 cells/mL), added to 24-well transwells, and incubated in a humidified tissue culture incubator at 37 C in 5% CO2. After 24 h of incubation, nonmigrated cells were removed from the upper surface of the membrane. A cotton-tipped swab was inserted into the Matrigel-coated transwell and moved gently and firmly over the membrane surface. Transwells were removed from the 24well plates and stained with a Hemacolor rapid staining kit (Merck Millipore). The number of migrated cells was counted at three random regions using light microscopy (Axiocam HRc; Zeiss, Jena, Germany). To exam the effects of laminin 332, we treated SNU-790 cells with 5 mL IgG (ChromePure Mouse IgG; Jackson ImmunoResearch Inc., West Grove, PA, USA) as a control group or 5 mL of antibodies against laminin 332 g2 (1:1000; clone D4B5, cat. no. MAB19562; Merck Millipore) as an experimental group. The methods were the same as those described above. Experiments were performed three times. 2.7. Statistical analysis For analysis of immunohistochemical data, Pearson Chisquared tests and one-way analysis of variance (ANOVA) were performed with IBM SPSS Software Version 20.0 for Windows (IBM, Armonk, NY, USA). Invasion assay results were analyzed by the Kruskal–Wallis test. Differences with p values of less than 0.05 were considered statistically significant.
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specifically expressed in cells adjacent to the tumor stroma (Fig. 1). Statistical analysis showed that the frequency of laminin 332 expression was significantly correlated with the presence of cervical LNMs (p = 0.003). Cases displaying larger tumor sizes tended to show higher expression of laminin 332, although the correlation was not statistically significant ( p = 0.082). Additionally, no association between multiplicity of cancer and the expression of laminin 332 was identified. The results of multivariate logistic regression analysis demonstrated that only expression of laminin 332 (p = 0.031) significantly associated with lymph node metastasis (Table 2). 3.2. Expression of the laminin 332 g2 chain was correlated with cell invasion in PTC cells The protein and mRNA expression levels of the laminin 332 g2 in PTC cells are shown in Fig. 2A and 2B. In PTC cells, the expression levels of the g2 chain followed the order of SNU790 > BHP10–3 > BCPAP. Using a Boyden chamber invasion assay, we investigated whether laminin 332 promoted the invasion of PTC cells. This assay was repeated three times, and the mean number of migrated cells from three independent experiments differed significantly (p < 0.01). As shown in Fig. 2C, among the PTC cell lines, SNU-790 cells exhibited the greatest number of invaded cells, and the trend in the tested cells lines followed the order of SNU790 > BCPAP > BHP10-3. Based on the level of g2 chain expression, increased cell invasion was observed in cells with higher levels of g2, except in BHP10-3 cells. 3.3. Expression of the laminin 332 g2 chain affected cell invasion in PTC cells To examine the effects of laminin 332 on cell invasion, we performed cell invasion assays using Boyden chambers. We used SNU-790 cells, which exhibited the most invasive activity. Cell invasion was significantly reduced in SNU-790 cells treated with anti-laminin 332 g2 chain antibodies compared with that in cells treated IgG as a control group (p < 0.05) (Fig. 3).
3. Results 3.1. Expression of laminin 332 was correlated with lymph node metastasis in patients with PTC Of the 40 patients analyzed in this study, laminin 332 expression was observed in 37 cases (92.5%; Table 1). All cases in the LNM-positive group showed laminin 332 expression (100%). Laminin 332 was localized to the cytoplasm and cell membranes of tumor cells and was
4. Discussion Due to the indolent nature of PTC, there is a tendency for patients to fail to seek proper treatment. However, if a patient with PTC shows poor prognostic factors, such as extracapsular invasion and LNM, his/her quality of life is negatively impacted. Therefore, it is important to accurately predict the prognosis of PTC in order to ensure proper treatment. To this
Table 1 Relationship between the immunoreactivity of laminin 332 and clinical features in patients. IHC score
Laminin 332
Total
Number of additional tumors
pT3, pT4
LN metastasis
Mean size (cm)
0 1 2 3
>5% 5–25% 26–50% >50%
3 8 19 10
1 0 6 5
2 (67%) 6 (75%) 17 (89%) 10 (100%)
0 (0%) 1 (13%) 11 (58%) 8 (80%)
1.23 1.40 1.94 2.49
(33%) (0%) (32%) (50%)
IHC = immunohistochemistry, LN = lymph node, pT = pathologic T stage.
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Fig. 1. Laminin-5 expression in papillary thyroid carcinoma. Examples of laminin-5 g2 chain expression scoring and interpretation. The immunohistochemical score for the expression of laminin-5 was assigned based on the percentage of stained tumor cells. (a. Grade 0, <5%; b. Grade 1, 5–25% (red circle); c. Grade 2, 26–50% (red arrows); and d. Grade 3, >50%). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
end, the relationships between prognosis and various molecular and genetic markers, including BRAF, RAS, RET/PTC, and galectin-3, have been reported [14]. Laminin 332 is a heterotrimeric protein that consists of a, b, and g chains and plays a major role in normal tissue anchoring between epithelial and mesenchymal tissues [15]. Various chains, including 5a, 4b, and 4g chains, are present in different combinations to generate at least 16 laminin isoforms. These laminin isoforms show distinct tissue distributions, and their expression is often precisely regulated during development [16]. Laminin 332 is composed of a3, b3, and g2 chains, and the g2 chain is a unique component of this protein [17]. Laminin 332 is involved in several biological pathways, including self-polymerization, extracellular matrix (ECM) binding, and cellular interactions [18]. Moreover, laminin 332 is known to be related to poor prognosis in several cancers due to its influence on cellular invasion and metastasis [18]. The expression of laminin 332, particularly the g2 chain, is correlated with invasiveness in adenocarcinoma of the colon [17]. In hepatocellular cancers, detection of the laminin 332 g2 chain is strongly associated with metastasis [19]. In this study, a monoclonal anti-laminin 332 g2 antibody (D4B5) was used for immunohistochemical analysis, and we sought to test the feasibility of using laminin 332 as a marker of LNM in patients with PTC. This study is the first
report to suggest a relationship between laminin 332 expression in PTC and LNM as all cases in the LNM-positive group expressed laminin 332 (100%). There was a significant difference in laminin 332 expression between LNM-positive and LNM-negative groups; laminin 332 was more significantly expressed in LNM-positive patients (p = 0.003). These results suggested that overexpression of laminin 332 in PTC may be useful as a biomarker for predicting cervical LNM. As a follow up, we analyzed the expression and function of laminin 332 in PTC. We observed that increased expression of LAMC2 was related to increased cell invasion, which consistent with the previous findings. The invasiveness of BHP10-3 did not follow the tendency of laminin 332 expression level in experiment of cell lines. We assumed that the other factors could influence the invasiveness of BHP10-3. However, the other cell lines followed the tendency that more expression level of laminin 332 increase more invasiveness of PTC cells, and cell invasion was significantly reduced in SNU-790 cells treated with antilaminin 332 g2 chain antibodies compared with that in cells treated IgG as a control group. Therefore, we suppose that laminin 332 is the important factor of cell invasion in PTC. In situ carcinoma cells often deposit laminin 332 onto underlying BM structures. Previous studies have shown that when BM structures are synthesized without laminin 332,
Table 2 Factors associating with lymph node metasatsis in patients with papillary thyroid carcinoma. Factor
Comparison
Odds ratio
95% CI
p value
Multifocality T stage Mean size Lamin 332
Absent vs. present pT1, T2 vs. T3, T4 <2 cm vs. 2 cm IHC score 0, 1 vs. 2, 3
0.78 4.54 7.45 15.78
[0.32–3.99] [1.45–25.98] [1.21–40.87] [1.40–103.99]
0.187 0.071 0.051 0.031
IHC = immunohistochemistry.
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Fig. 2. mRNA and protein expression levels of the laminin 332 g2 chain were correlated with cell invasion in papillary thyroid carcinoma (PTC). (A) Western blot analysis of LAMC2. LAMC2 protein expression in the tested cell lines was consistent with mRNA levels of the laminin 332 chains. (B) RT-PCR analysis of laminin 332 g2 chain expression in PTC cell lines. (C) Cell invasion assay. Stained cells indicate cells that transmigrated across the Matrigel membrane. The graph shows the number of migrated cells for each cell line. Among the PTC cell lines examined in this study, the number of migrated cells showed the following trend: SNU790 > BCPAP > BHP10-3.
tumor cells are better able to migrate into the interstitial stroma [9]. Tumor cells at the invasion front often overexpress the laminin 332 g2 chain, and proteolytic fragments of the laminin 332 g2 monomer may promote tumor cell invasion by binding to epidermal growth factor receptor and other unidentified receptors [9,20]. Expression of the laminin 332 g2 chain was primarily observed in the cytoplasm of carcinoma cells in this study, with the strongest expression found in the periphery of PTCs (Fig. 1). Conversely, only a few g2 chain-expressing cells were observed in the center and papillary portions of the tumor. More importantly, however, laminin 332 was strongly
expressed at the invasive front in the majority of PTC cases with LNM (Fig. 3). These findings suggested that laminin 332 expression played an important role in cancer migration and invasion. 5. Conclusion The expression of laminin 332 may facilitate the diagnosis of PTC, and high expression can serve as a useful biological marker for predicting LNM. The importance of laminin 332 in the metastasis of thyroid cancer makes it an attractive target for cancer therapeutics. Although further studies are needed to clarify the regulatory mechanisms of laminin 332 in PTC, our current results may be useful for the development of new therapeutic agents for preventing LNM in patients with PTC. Financial disclosure This research was supported by a Korea University Grant. Acknowledgments The authors thank Ph. D. Bok Kee Eun in Core-Laboratory for Convergent Translational Research, College of Medicine, ***** University, for helpful advice on experiments. References
Fig. 3. Cell invasion assay. Stained cells indicate cells that transmigrated across the Matrigel membrane. The graph shows the number of migrated cells for each cell line. In SNU-790 cells, the number of migrated cells was lower after treatment with anti-LAMC2 antibodies than that in the control group (IgG).
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