CELL CYCLE 2016, VOL. 15, NO. 14, 1865–1873 http://dx.doi.org/10.1080/15384101.2016.1188239

REPORT

Prognostic significance of monocarboxylate transporter expression in oral cavity tumors ~es-Sousaa,b,*,#, Sara Granjaa,b,#, Celine Pinheiroa,b,c,d, Daniela Fernandesa,b, Adhemar Longatto-Filhoa,b,d,e, Susana Simo ~arandag, Mario Perez-Sayansh, Andre Lopes Carvalhod,f, Ana Carolina Lausd, Cira Danielle Casado Alvesf, J. M. Suarez-Pen i,j,k h Fernando C. Schmitt , Abel Garcıa-Garcıa , and Fatima Baltazara,b a Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; bICVS/3B’s-PT Government Associate Laboratory, Braga/Guimar~aes, Portugal; cBarretos School of Health Sciences Dr. Paulo Prata – FACISB, Barretos, Sao Paulo, Brazil; dMolecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Sao Paulo, Brazil; eLaboratory of Medical Investigation (LIM-14), Faculdade de Medicina da Universidade de, S~ao Paulo, Brazil; fHead and Neck Surgery Department, Barretos Cancer Hospital, Barretos, Sao Paulo, Brazil; gDepartment of Pathology and Forensic Sciences, University Hospital and School of Medicine of Santiago de Compostela, Santiago de Compostela, A Coru~ na, Spain; h Oral Medicine, Oral Surgery and Implantology Unit, Faculty of Medicine and Dentistry, Health Research Institute of Santiago (IDIS), Santiago de Compostela, A Coru~na, Spain; iIPATIMUP - Institute of Molecular Pathology and Immunology of University of Porto, Porto, Portugal; jMedical Faculty of the University of Porto, Porto, Portugal; kDepartment of Pathology and Medicine, Laboratoire National de Sante, Dudelange, Luxembourg

ABSTRACT

ARTICLE HISTORY

Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer. The majority of patients present advanced stage disease and has poor survival. Therefore, it is imperative to search for new biomarkers and new alternative and effective treatment options. Most cancer cells rely on aerobic glycolysis to generate energy and metabolic intermediates. This phenotype is a hallmark of cancer, characterized by an increase in glucose consumption and production of high amounts of lactate. Consequently, cancer cells need to up-regulate many proteins and enzymes related with the glycolytic metabolism. Thus, the aim of this study was to characterize metabolic phenotype of oral cavity cancers (OCC) by assessing the expression pattern of monocarboxylate transporters (MCTs) 1, 2 and 4 and other proteins related with the glycolytic phenotype. Material and Methods: We evaluated the immunohistochemical expression of MCT1, MCT4, CD147, GLUT1 and CAIX in 135 human samples of OCC and investigated the correlation with clinicopathological parameters and the possible association with prognosis. Results: We observed that all proteins analyzed presented significantly higher plasma membrane expression in neoplastic compared to non-neoplastic samples. MCT4 was significantly associated with T-stage and advanced tumoral stage, while CD147 was significantly correlated with histologic differentiation. Interestingly, tumors expressing both MCT1 and MCT4 but negative for MCT2 were associated with shorter overall survival. Conclusion: Overexpression of MCT1/4, CD147, GLUT1 and CAIX, supports previous findings of metabolic reprograming in OCC, warranting future studies to explore the hyper-glycolytic phenotype of these tumors. Importantly, MCT expression revealed to have a prognostic value in OCC survival.

Received 11 March 2016 Revised 28 April 2016 Accepted 5 May 2016 KEYWORDS

aerobic glycolysis; head and neck cancer; lactate transport; monocarboxylate transporters

Introduction Head and neck cancers are broad and heterogeneous, including malignancies that arise in the craniofacial bones, soft tissues, salivary glands, skin and mucosal membranes. The majority (more than 90%) are squamous cell carcinomas (SCC) arising from the epithelia of the sinonasal tract, oral cavity, pharynx and larynx.1 Head and neck squamous cell carcinoma (HNSCC) accounts for nearly 4% of all diagnosed cancers 2 with an incidence of approximately 650000 new cases and 350000 cancer deaths worldwide annually. The median age for diagnosis is 60 years, with a male predominance.3 Carcinogen exposure, like tobacco or alcohol, and human papillomavirus (HPV) infection are the main causes of HNSCC.4 Despite advances in

treatment that led to an increased quality of life, survival has not improved in the recent decades and the 5-year survival is 40-50%. Additionally, it has been recognized that there are preneoplastic fields with genetically altered cells clonally related to the carcinoma (field cancerization) that results in high locoregional recurrence (about 60%). Oral cavity carcinomas (OCC) are one of the most common of HNSCC. Besides the progress of treatment options, the 5-year survival rate has only increased a little during the past years, largely due to the advanced stages of disease at diagnosis and the frequent development of relapse and second primary tumors.5,6 Therefore is urgent to identify new OCC biomarkers.

CONTACT Fatima Baltazar, Phd [email protected] Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy. # These authors equally contributed to this work. *Present affiliation: Manchester Cancer Research Center, The University of Manchester, Manchester, United Kingdom. © 2016 Taylor & Francis

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e S. SIMOES-SOUSA ET AL.

Cancer is not only a complex genetic disease, but also a disease of deregulated bioenergetic metabolism. As vastly described, most of solid tumors exhibit a preference for glycolysis for energy production, even in the presence of oxygen, a phenomenon named Warburg effect.7-9 In this way, cancer cells consume high amounts of glucose and consequently produce lactate. HNSCC cells also feature high levels of aerobic glycolysis.10,11 In fact, some reports have already described that lactate, the end product of glycolysis, can be used as a prognostic tool in this type of tumor. Indeed, high levels of tumor lactate concentrations predict an increased metastasis formation12 and a reduced overall and disease-free patient survival.13 Moreover, most tumor cells generate significantly greater levels of lactate when compared to that of normal human oral keratinocytes.14 However, lactate accumulation in cancer cells induces apoptosis 15,16 and, therefore, it must be transported out of the cells.17 It is widely accepted that lactate is transported by monocarboxylate transporters (MCTs).18 MCTs constitute a family of 14 plasma membrane transporters, however, only the first 4 isoforms are responsible for the proton-linked lactate transport.19 Taking into account the tumor microenvironmental scenario and the molecular events that occur in carcinogenesis, it is possible to predict the importance of these lactate transporters in cancer. In fact, since MCT1/4 isoforms are vital for intracellular pH homeostasis, by exporting lactate coupled with a proton, these isoforms are described as up-regulated in highly glycolytic tumor cells,20,21 playing a crucial role on the hyper glycolytic phenotype of cancer cells.20 Importantly, these transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function.22-24 The expression pattern of MCTs in OCC and the correlation with clinical-pathological data remain poorly explored and unclear. In fact, there is only one report showing overexpression of MCT4 in OCC and its association with different clinico-pathological and poor survival.25 Regarding the expression of other proteins related to the metabolic phenotype, such as the glucose transporter 1 - GLUT1,26 and the endogenous hypoxia-related marker carbonic anhydrase IX (CAIX),27 the literature is still limited, with little evidence for their expression in OCC 27 and association with poor survival parameters.28 Despite these results, the expression pattern of MCTs and other proteins related with the glycolytic phenotype of a tumor is not fully explored. Thus, in the present study we examined the expression of MCT1, MCT2 and MCT4, MCT1/MCT4 chaperone CD147, GLUT1 and CAIX in a series of patient derived-tumors with further correlation with the clinico-pathological data. In this way, we believe to provide evidence for the characterization of the glycolytic phenotype of OCC. To the best of our knowledge, this is the first study describing the expression pattern of the above glycolytic metabolism-related proteins in OCC.

Material and methods Tissue samples A total of 136 formalin-fixed paraffin-embedded samples of oral cavity tumors were retrieved from the archives of Barretos Cancer Hospital, S~ao Paulo, Brazil (90 cases) and from the Department of Oral Medicine, Oral Surgery and Implantology,

University of Santiago de Compostela, Spain (46 cases). Also, 25 surgical margin samples of normal epithelium where obtained from Barretos Cancer Hospital tumor samples. Samples obtained from the University of Santiago de Compostela were organized in tissue microarrays (TMAs). To achieve representative sampling and minimize sample loss, sample duplicates were included in the TMAs. Clinicopathological data included patient’s age, gender, tobacco and alcohol history, tumor location and differentiation, TNM staging, disease recurrence and follow-up. Immunohistochemistry Sections of 3-mm were used for immunohistochemical analysis. For MCT1 IHQ was performed according with avidin–biotinperoxidase complex method (R.T.U. VECTASTAIN Elite ABC Kit (Universal), Vector Laboratories), with primary antibody for MCT1 diluted 1:200 (AB3538P, Chemicon International) as previously described.29 Immunohistochemistry for MCT2, MCT4, GLUT1 and CAIX was performed according to the streptavidin-biotin-peroxidase complex principle (Ultravision Detection System Anti-polyvalent, HRP, Lab Vision Corporation), using primary antibodies raised against MCT2 diluted 1:200 (sc-50322 Santa Cruz Biotechnology), MCT4 (sc-50329, Santa Cruz Biotechnology) diluted 1:500, GLUT1 (ab15309500, Abcam) diluted 1:500 and against CAIX (ab15086, Abcam) diluted 1:2000 as previously described.30,31 CD147 immunostaining was performed using a polymer system (UltraVision ONE Detection System: HRP Polymer Lab Vision Corporation) with primary antibody raised against CD147 (sc71038, Santa Cruz Biotechnology), diluted 1:500, as previously described.32 Colon carcinoma tissue was used as positive control for MCT1, MCT4, GLUT1 and CD147, while stomach was used for CAIX and kidney for MCT2. Tissue sections were counterstained with hematoxylin and permanently mounted. Immunohistochemical evaluation Sections were scored semi-quantitatively for extension of staining as follows: 0: 0% of immunoreactive cells; 1: < 5% of immunoreactive cells; 2: 5-50% of immunoreactive cells; and 3: > 50% of immunoreactive cells and for intensity of staining as follows: 0: negative; 1: weak; 2: intermediate; and 3: strong. The final score was defined as the sum of both parameters (extension and intensity), and grouped as negative (score 0, 2 and 3) and positive (score 4, 5 and 6). Protein localization was also assessed (plasma membrane versus cytoplasm). Immunohistochemical evaluation was performed by 2 independent pathologists and discordant results were discussed in a double-head microscope to determine the final score. Statistical analysis Data was stored and analyzed using the IBM SPSS Statistics software (version 22, IBM Company, Armonk, NY). All comparisons were examined for statistical significance using Pearson’s chi-square test (x2) or Fisher’s exact test, according to sample characteristic. Overall survival was defined as the time between the date of first consultation and date of last

CELL CYCLE

Table 1. Clinicopathological data for oral cavity tumor patients. Variable Age 60 years Gender Male Female Tumor Location Tonsil Retromolar trigone Gingiva Buccal mucosa Floor of the mouth Tongue Diferentiation Well Moderate Poor T stage T1CT2 T3CT4 N stage N0 N1CN2CN3 Stage I/II III/IV Tobacco use No Yes Alcohol use No Yes Recurrence No Yes

n

(%)

69 66

50.4 48.2

103 32

75.2 23.4

4 11 12 22 37 49

2.9 8.0 8.8 16.1 27.0 35.8

79 38 17

57.7 27.7 35.8

71 64

51.8 46.7

102 33

74.5 24.1

55 80

40.1 58.4

35 99

25.5 72.3

39 87

28.5 63.5

73 62

53.3 45.3

information or patient death. Overall survival curves were estimated by the method of Kaplan-Meier and data compared using the Breslow test. Multivariate survival analyses were done using the Cox proportional hazards model. The threshold for significance was p
0.05. All the reported p values are 2-sided.

Results Expression of MCT1, MCT2, MCT4, CD147, GLUT1 and CAIX in OCC A total of 136 cases where included in this study. Detailed information on the clinical and pathologic data is detailed in Table 1. As observed, the majority of patients were men (76.3%) and the median of age at diagnosis for all patients was 60 years, ranging from 21 to 96 year old. We have selected this

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age since this is the median age used for diagnosis. Regarding the site of the tumor, all are located at oral cavity being the tongue the most frequent primary tumor site (35.8%), followed by the floor of the mouth (27.0%). Other sites included buccal mucosa (16.1%), gingiva (8.8%), retromolar trigone (8.0%) and tonsil (2.9%). The majority of patients (58.4%) presented advanced stage III/IV primary tumors. For all cases, we analyzed the expression of lactate transporters (MCT1, MCT2 and MCT4), MCT1/MCT4 chaperone (CD147), GLUT1 and the hypoxic marker CAIX. The immunohistochemical analysis of these proteins revealed that all the proteins studied, with the exception of MCT2, were significantly overexpressed at the plasma membrane in tumoral samples when compared with normal adjacent epithelium (Table 2). Briefly, plasma membrane staining was observed for MCT1 in 93.8% of the cases (p < 0.001), for MCT4 in 71.2% (p < 0.001), CD147 in 77.9% (p < 0.001), GLUT1 in 96.1% (p D 0.050) and CAIX was found in 62.9% (p D 0.003). Importantly, MCT2 only exhibited cytoplasmic expression (52.8%). In some cases, we observed cytoplasmic expression for GLUT1 and CAIX, as observed in Figure 1. When we analyzed the coexpression of MCTs with the other proteins studied, we observed that only MCT1 (p D 0.014) expression was significantly associated with CD147 expression (Table 3).

Prognostic value of MCT expression The association between protein frequency of expression and various clinical and pathological characteristics was also analyzed. Table 4 (top) shows the analysis of associations between MCT1, MCT2 and MCT4 expression and the clinicopathological parameters. MCT2 cytoplasmic expression was significantly associated with well-differentiated tumors (p D 0.029), while MCT1 plasma membrane expression showed no significant associations with the clinicopathological data. MCT4 plasma membrane expression was significantly associated with an advanced stage T3CT4 (p D 0.015) and with stage III/IV (p D 0.032). Table 4 (bottom) shows the associations of CD147, GLUT1 and CAIX plasma membrane expression with clinicopathological data. Both CD147 and GLUT1 were significantly associated with well-differentiated tumors (p < 0.001 and p D 0.005, respectively), while GLUT1 was also associated with absence of recurrence (p D 0.020). Finally, CAIX was significantly associated with age ( > 60 years; p D 0.043). Overall and disease free survivals were not associated with the presence or absence of any protein individually.

Table 2. Protein expression frequencies for MCT1, MCT2, MCT4, CD147, GLUT1 and CAIX in oral cavity non-tumoral and tumoral tissues. n

MCT1 PM positive (%)

NT T

24 128 n

0 (0) 120 (93.8) CD147 PM positive (%)

NT T

24 136

6 (25.0) 106 (77.9)

p

n

MCT2 overall positive (%)

20 89 n

13 (65.0) 47 (52.8) GLUT1 PM positive (%)

21 128

18 (87.7) 123 (96.1)