Prognostic significance of monocarboxylate transporter expression in oral cavity tumors

ABSTRACT

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.

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 pre-neoplastic fields with genetically altered cells clonally related to the carcinoma (field cancerization) that results in high loco-regional 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.

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, São 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-μm were used for immunohistochemical analysis. For MCT1 IHQ was performed according with avidin–biotin-peroxidase 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 (ab15309-500, 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 (sc-71038, 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 (χ²) 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 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 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.

Table 1.

Clinicopathological data for oral cavity tumor patients.

Variable	n	(%)
Age
<60 years	69	50.4
>60 years	66	48.2
Gender
Male	103	75.2
Female	32	23.4
Tumor Location
Tonsil	4	2.9
Retromolar trigone	11	8.0
Gingiva	12	8.8
Buccal mucosa	22	16.1
Floor of the mouth	37	27.0
Tongue	49	35.8
Diferentiation
Well	79	57.7
Moderate	38	27.7
Poor	17	35.8
T stage
T1+T2	71	51.8
T3+T4	64	46.7
N stage
N0	102	74.5
N1+N2+N3	33	24.1
Stage
I/II	55	40.1
III/IV	80	58.4
Tobacco use
No	35	25.5
Yes	99	72.3
Alcohol use
No	39	28.5
Yes	87	63.5
Recurrence
No	73	53.3
Yes	62	45.3

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 = 0.050) and CAIX was found in 62.9% (p = 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 co-expression of MCTs with the other proteins studied, we observed that only MCT1 (p = 0.014) expression was significantly associated with CD147 expression (Table 3).

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 (%)	p	n	MCT2 overall positive (%)	p	n	MCT4 PM positive (%)	p
			<0.001			0.322			<0.001
NT	24	0 (0)		20	13 (65.0)		22	1 (4.5)
T	128	120 (93.8)		89	47 (52.8)		125	89 (71.2)
	n	CD147 PM positive (%)	p	n	GLUT1 PM positive (%)	p	n	CAIX PM positive (%)	p
			<0.001			0.050			0.003
NT	24	6 (25.0)		21	18 (87.7)		19	5 (26.3)
T	136	106 (77.9)		128	123 (96.1)		124	78 (62.9)

PM: Plasma membrane expression; NT: non-tumoral tissue; T: tumoral tissue

Figure 1.

Representative images of the immunohistochemical staining. MCT1 (A), MCT4 (C), CD147 (D), GLUT1 (E), CAIX (F) were expressed at the plasma membrane while MCT2 (B) was present in the cytoplasm. (x200 magnification).

Table 3.

Association of MCT1, MCT2 and MCT4 with CD147, CD44, GLUT1 and CAIX expression in oral cavity tumors.

	n	CD147 PM Positive (%)	p	n	GLUT1 PM Positive (%)	p	n	CAIX PM Positive (%)	p
MCT1			0.014			0.284			0.435
Negative	8	3 (37.5)		8	7 (87.5)		8	4 (50.0)
Positive	120	92 (76.7)		118	114 (96.6)		116	74 (63.8)
MCT2			0.711			1.000			0.871
Negative	41	30 (73.2)		40	40 (100.0)		40	24 (60.0)
Positive	47	36 (76.6)		47	47 (97.9)		47	29 (61.7)
MCT4			0.087			0.306			0.282
Negative	36	23 (63.9)		34	32 (94.1)		33	18 (54.5)
Positive	89	70 (78.7)		89	87 (97.8)		89	58 (65.2)

PM: Plasma membrane expression.

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 = 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 T3+T4 (p = 0.015) and with stage III/IV (p = 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 = 0.005, respectively), while GLUT1 was also associated with absence of recurrence (p = 0.020). Finally, CAIX was significantly associated with age ( > 60 years; p = 0.043).

Table 4.

Association between MCT1, MCT2, MCT4, CD147, GLUT1 and CAIX expression with the clinicopathological parameters.

	MCT1PM expression			MCT2 overall expression			MCT4 PM expression
Variable	n	Positive (%)	p	n	Positive (%)	p	n	Positive (%)	p
Age			0.554			0.548			0.433
<60 years	66	61 (92.4)		51	25 (49.0)		65	44 (67.7)
>60 years	60	57 (95.5)		36	20 (55.6)		58	43 (74.1)
Gender			0.935			0.658			0.704
Male	96	90 (93.8)		73	37 (50.7)		95	68 (71.6)
Female	30	28 (93.3)		14	8 (57.1)		28	19 (67.9)
Tumor Location			0.072			0.882			0.752
Tonsil	4	4 (100.0)		0	0		4	2 (50.0)
Retromolar trigone	11	10 (90.9)		3	1 (33.3)		10	7 (70.0)
Gingiva	11	8 (72.7)		7	4 (57.1)		11	7 (63.6)
Buccal mucosa	19	19 (100.0)		5	3 (60.0)		18	15 (83.3)
Floor of the mouth	35	33 (94.1)		32	18 (56.3)		34	23 (67.6)
Tongue	46	44 (95.7)		40	19 (47.5)		46	33 (71.7)
Diferentiation			0.304			0.029			0.073
Well	75	72 (96.0)		62	38 (61.3)		73	57 (78.1)
Moderate	34	30 (88.2)		14	7 (50.0)		34	24 (70.6)
Poor	17	16 (94.1)		11	2 (18.2)		16	8 (50.0)
T stage			0.494			0.918			0.015
T1+T2	64	59 (92.2)		43	22 (51.2)		61	37 (60.7)
T3+T4	62	59 (95.2)		44	23 (52.3)		62	50 (80.6)
N stage			0.979			0.160			0.285
N0	94	88 (93.6)		60	34 (56.7)		91	62 (68.1)
N1+N2+N3	32	30 (93.8)		27	11 (40.7)		32	25 (78.1)
Stage			0.806			0.976			0.032
I/II	50	47 (94.0)		33	17 (51.5)		47	28 (59.6)
III/IV	76	71 (93.4)		54	28 (51.9)		76	50 (65.8)
Tobacco use			0.962			0.384			0.707
No	33	31 (93.9)		14	9 (64.3)		31	23 (74.2)
Yes	92	86 (93.5)		72	35 (48.6)		91	63 (69.2)
Alcohol use			0.819			0.350			0.114
No	35	35 (100.0)		14	9 (64.3)		35	28 (80.0)
Yes	80	75 (93.8)		72	35 (48.6)		79	51 (64.6)
Recurrence			0.725			0.116			0.580
No	68	63 (92.6)		57	26 (45.6)		67	46 (68.7)
Yes	58	55 (94.8)		30	19 (63.3)		56	41 (73.2)

	CD147 PM expression			GLUT1 PM expression			CAIX PM expression
Variable	n	Positive (%)	p	n	Positive (%)	p	n	Positive (%)	p
Age			0.654			0.677			0.043
<60 years	69	53 (76.8)		64	62 (96.9)		64	35 (54.7)
>60 years	65	52 (80.0)		62	59 (95.2)		58	42 (72.4)
Gender			0.343			0.596			0.764
Male	102	78 (76.5)		95	92 (96.8)		94	60 (63.8)
Female	32	27 (84.4)		31	29 (93.5)		28	17 (60.7)
Tumor Location			0.536			0.428			0.966
Tonsil	4	3 (75.0)		4	4 (100.0)		4	3 (75.0)
Retromolar trigone	11	8 (72.7)		11	11 (100.0)		11	7 (63.6)
Gingiva	12	8 (66.7)		10	9 (90.0)		10	6 (60.0)
Buccal mucosa	22	17 (77.3)		20	18 (20.0)		17	12 (70.6)
Floor of the mouth	37	33 (89.2)		34	34 (100.0)		34	20 (58.8)
Tongue	48	36 (75.0)		47	45 (95.7)		46	29 (63.0)
Diferentiation			<0.001			0.005			0.412
Well	78	66 (85.0)		75	74 (98.7)		74	49 (66.2)
Moderate	38	26 (68.4)		35	34 (97.1)		34	18 (52.9)
Poor	17	6 (35.3)		16	13 (81.25)		14	9 (64.3)
T stage			0.437			0.365			0.146
T1+T2	70	53 (75.7)		63	59 (93.6)		60	34 (56.7)
T3+T4	64	52 (81.3)		63	62 (98.4)		62	43 (69.4)
N stage			0.578			1.000			0.056
N0	101	78 (77.2)		95	91 (95.8)		91	53 (58.2)
N1+N2+N3	33	27 (81.8)		31	30 (96.8)		31	24 (77.4)
Stage			0.156			0.080			0.158
I/II	54	39 (72.1)		50	46 (92.0)		47	26 (55.3)
III/IV	80	66 (82.5)		76	75 (98.7)		75	51 (68.0)
Tobacco use			0.212			0.124			0.287
No	35	23 (71.4)		34	31 (91.2)		31	17 (54.8)
Yes	98	75 (80.6)		91	89 (97.8)		90	59 (65.6)
Alcohol use			0.799			0.334			0.177
No	39	32 (82.1)		39	36 (92.3)		36	28 (77.7)
Yes	86	66 (76.7)		78	76 (97.4)		77	49 (63.6)
Recurrence			0.807			0.020			0.263
No	72	57 (79.2)		67	67 (100.0)		65	44 (67.7)
Yes	62	48 (77.4)		59	54 (91.5)		57	33 (57.8)

PM: Plasma membrane.

Overall and disease free survivals were not associated with the presence or absence of any protein individually. Interestingly, the presence of both MCT1 and MCT4 combined with the absence of MCT2 was significantly associated with shorter overall survival (p = 0.044, Fig. 2, Table 5). To evaluate whether the presence MCT1 and MCT4 combined with the absence of MCT2 is an independent prognostic factor, we carried out a multivariate Cox regression analysis and found that this factor is independently associated with patients' poor survival, with a hazard ratio of 2.152 (p = 0.032) (Table 5). Additionally, advanced tumor stage was found to be an independent prognostic factor in our series (Table 5).

Figure 2.

Kaplan–Meier curves showing overall survival rates for MCT1 positive, MCT4 positive and MCT2 negative tumors. Breslow tests were used to determine p values. Green line: (MCT1+MCT4+MCT2-) positive cases; blue line: (MCT1+MCT4+MCT2-) negative cases.

Table 5.

Prognostic factors for overall survival in oral cavity carcinomas.

		Univariate analysis			Multivariate analysis
		Overall survival			Overall survival
Variable	n	months ± SD	p*	HR	95% CI	p#
Age
< 60	67	53.377 ± 4.323	0.051
≥ 60	66	52.184 ± 6.074		1.006	0.973-1.040	0.724
Gender
Male	101	50.641 ± 3.703	0.230
Female	32	58.660 ± 8.661		0.779	0.262_2.315	0.654
Tumor stage
I/II	54	69.548 ± 6.493	0.004
III/IV	79	44.395 ± 4.236		2.121	1.010_4.455	0.047
Radiotherapy
No	30	64.149 ± 5.704	0.023
Yes	57	47.601 ± 5.864		1.252	0.538-2.915	0.602
Chemotherapy
No	69	64.790 ± 5.824	0.018
Yes	65	44.929 ± 4.796		2.177	1.020-4.644	0.044
MCT1positive_MCT4positive_MCT2negative
Negative	107	58.874 ± 4.79	0.044
Positive	27	38.529 ± 6.32		2.152	1.069–l4.332	0.032

^*pLog-rank test;

^#pmultivariate Cox proportional of Hazards model; SD, standard deviation; CI, confidence interval.

Discussion

Metabolic reprograming of cancer cells has been recently recognized as a hallmark of cancer,³³ responsible for tumor aggressiveness and drug resistance. The expression pattern of the hyperglycolytic phenotype has been widely described in different tumors being characterized by the expression of several metabolism-related proteins such as GLUT1, CAIX, MCTs and CD147, among others.^20,34,35

In this work, we observed a significantly higher plasma membrane expression of all proteins studied, with the exception of MCT2, in the OCC samples when compared with normal oral epithelia, which supports their functional role in the metabolic reprograming of cancer cells. The plasma membrane expression of all proteins studied support their activity. Since most of the molecules here in studied, with the exception of MCT2, are involved in the cancer hyperglycolytic phenotype, one can assume that OCC have a highly glycolytic phenotype, which is supported by previous ^27,36-40 studies and by the applicability of FdG-PET scan in this type of cancer.^41-43 Moreover, since GLUT1 was present in 96.1% of tumor samples, this reinforces the presence of a glycolytic metabolism. This is the first study showing the expression of MCT2 in OCC samples. Even though other reports have already demonstrated MCT2 expression to be associated with non-malignant tissues in other tumor type,³⁴ we showed that MCT2 has no association with non-tumoral or tumoral samples, being expressed in the cytoplasm of the cells in both compartments. This cytoplasmic expression of MCT2 could be related to mitochondrial pyruvate transport, as MCTs were already described to be present in the mitochondria.¹⁸ Interestingly, MCT2 cytoplasmic expression has already been associated with variables related to a less aggressive profile of hepatocellular carcinomas.⁴⁴ In this study, we observed that MCT2 was significantly associated with well-differentiated tumors.

There was a significant and relatively strong association of MCT1 with CD147, which corroborates their inter-dependence for correct trafficking and function,^22,45 but such association was not found for MCT4. This fact was also observed in other types of cancers such as colon and ovary cancer, suggesting that MCT membrane expression in tumors may be depend on another regulatory protein, not yet identified.^46,47 MCT4 overall expression was significantly associated with higher T stage and TNM stages III/IV. A recent study analyzing MCT4 in HNSCC also obtained a significant association of MCT4 overall expression with bigger tumor size (T stage) and higher TNM staging. Importantly, these results pointed out MCT4 as a potential marker for tumor aggressiveness, as suggested in other tumor types.^48-51 Furthermore, the authors suggest that MCT4 may act as a prognosis predictor due to its association with overall survival.²⁵ In the present study, there was no association between MCT4 and overall survival.

Since MCT2 expression was associated with good prognostic variables in other types of cancers ^34,44 and in contrast to MCT1 and MCT4, is mainly responsible for the uptake of monocarboxylates into cells,¹⁸ we analyzed tumors that express both MCT1 and MCT4, as these are the isoforms correlated with the hyperglycolytic phenotype, and were negative for MCT2. Although we did not find any association with the different clinicopatological data, interestingly, we observed that MCT1(+)/MCT4(+)/MCT2(−) tumors have a shorter overall survival.

Herein, CD147 expression significantly decreases as the tumor loses differentiation. In fact, it has been described that CD147 overexpression occurs at an early stage of oral carcinogenesis.⁵² Additionally, its expression levels correlated with the degree of dysplasia.⁵² Some studies also indicate that CD147 is associated with an aggressive phenotype, increasing tumor proliferation,⁵³ migration,⁵⁴ invasion ⁵⁵ and metastization,⁵⁶ as well as with poor survival in HNSCC.^36,57,58 In this study, no associations were found between CD147 and tumor aggressiveness or overall survival.

GLUT1 mediates the important mechanisms involved in cellular glucose influx, allowing cells to proliferate and survive. In HNSCC, GLUT1 expression has been described to increase with the degree of dysplasia and increasing grade.⁵⁹ The results of the present study suggest an important role of GLUT1 in glucose uptake in HNSCC, although this protein does not seem to be involved in the progression of these tumors.

CAIX is a transcriptional target of HIF-1α and, therefore, is used as an endogenous hypoxia marker. Furthermore, CAIX has been described to have an important role in tumor progression and metastasis.⁶⁰ Indeed, CAIX as been already reported to be correlated with HNSCC tumor aggressiveness ⁶⁰ and poor prognosis.^61,62 In the present study, as expected we observed that CAIX was overexpressed in tumoral tissues when compared with the non-tumoral adjacent tissue. This result reinforces that these cells are influenced by a hypoxic microenvironment and therefore, cells up-regulate the glycolytic pathway in order to growth and survive.

In the present study, we found overexpression of the metabolic related proteins in tumor samples, supporting the presence of a hyperglycolytic phenotype in OCC. Further, a combined MCT expression, MCT1/4 positive and MCT2 negative, was associated with poor prognosis.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by the ON.2 SR&TD Integrated Program (NORTE-07-0124-FEDER-000017), co-funded by Programa Operacional Regional do Norte (ON.2- O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through Fundo Europeu de Desenvolvimento Regional (FEDER).