Monocarboxylate transporters (MCTs) are transmembrane proteins and essential regulators of lactate metabolism in cancer.¹⁾ MCT4 is encoded by SLC16A3 and facilitates lactic acid efflux from cells based on high glycolysis rates.²⁾ MCT4 overexpression has been found in aggressive cancer types, including breast, colorectal, head and neck, and lung cancer, because of their increased need for lactate production in cancer cells.^3-9) MCT4 expression is significantly correlated with aggressive pathological characteristics, advanced staging, and prognosis in colorectal and head and neck cancer.^4,5) In clear renal cell carcinoma, high MCT4 expression is associated with reduced progression-free survival.⁷⁾

Lactate is a product of the glycolytic mechanism. High levels of lactate in thyroid cancer are reported to be involved in the pathway of tumorigenesis and immune system inhibition.^10,11) A recent in vitro study suggests that inhibition of lactate shuttles by MCT4 decreased cell proliferation of anaplastic thyroid carcinoma (ATC).¹²⁾ It has been previously indicated that the tissue lactate level of papillary thyroid carcinoma (PTC) is significantly higher than that of the matched normal thyroid or benign tissues.¹³⁾ One study suggested higher MCT4 expression in fibroblasts of PTC tissues and a potential association of higher MCT4 expression with advanced PTC.¹⁴⁾ Another study discovered patients with MCT4-expressed PTCs had shorter survivals than those with MCT4-negative PTCs.¹⁵⁾ To our knowledge, no study has focused on the potential association of MCT4 expression and de-differentiation of thyroid cancer.

In this study, the mRNA expression of SLC16A3, which encodes the MCT4 protein, was evaluated using a public database. Subsequently, the protein expression of MCT4 in ATCs, PTCs, and normal thyroid tissues was evaluated.

Three different transcriptome datasets of PTC, ATC, and their matched or unmatched normal thyroid tissues were collected from the Gene Expression Omnibus (ncbi.nlm.nih.gov/geo; GSE65144, GSE29265, and GSE33630). Using these datasets, mRNA expression of SLC16A3 encoding MCT4 protein between normal, PTC, and ATC was compared to determine whether this gene is an oncogene.

Archival formalin-fixed, paraffin-embedded (FFPE) tissues were selected from surgically removed thyroid samples collected between 1997 and 2013 at the Asan Medical Center in Korea. A total of 138 fresh frozen PTC samples with 120 matched normal thyroid samples and 21 ATC samples were used for immunohistochemistry (IHC) analysis. The FFPE tissue samples were arrayed using a tissue-arraying instrument (MTAII; Beecher Instruments, Silver Spring, Sun Prairie, WI, USA). The MCT4 protein expression was assessed via IHC staining with an anti-MCT4 antibody (Invitrogen, Waltham, MA, USA). IHC staining was conducted on the tissue microarray sections using a BenchMark XT automated immunostaining device (Ventana Medical Systems, Tucson, AZ, USA) with the opt iView DAB IHC Detection Kit (Ventana Medical Systems) according to the manufacturer’s instructions, as previously described.^13,16) The MCT4 expression was graded semi-quantitatively by an experienced endocrine pathologist (D.E.S.) as follows: 0, negative; 1+ (<10% positive), weak; 2+ (10-50% positive), moderate; 3+ (>50% positive), strong. The samples with higher intensity scores (2+ or 3+) were classified as positive for MCT4 expression. The study protocol was approved by the Institutional Review Board of the Asan Medical Center (IRB no: 2013-0539).

R (version 3.5.1, R Foundation for Statistical Computing, Vienna, Austria; https://www.r-project.org/) was used for statistical analysis. Continuous and categorical variables are presented as mean (standard deviation) and number (percentage). Fisher’s least significant difference was used for post hoc analysis. Continuous variables were compared using the Student’s t-test. All p-values were two-sided, and p<0.05 was considered statistically significant.

Using three different public datasets, first, the clinical importance of SLC16A3 encoding MCT4 protein was explored by tracing the gene expression during thyroid cancer progression from normal to ATC. The mRNA expression of SLC16A3 was significantly higher in ATCs compared with PTCs and normal thyroid tissues (p<0.01, and p<0.001, respectively; Fig. 1A, C). To exclude individual gene expression variation, matched analysis of normal and ATC tissues was performed. As a result, SLC16A3 expression was consistently upregulated in ATCs compared with their matched normal tissues (p<0.05; Fig. 1B). Next, the other two datasets, including normal tissues, PTCs, and ATCs, were analyzed. Interestingly, SLC16A3 expression in both datasets progressively increased as human thyroid cancer advanced from normal and PTC to ATC (Fig. 1C, D). In summary, SLC16A3 is an oncogene that promotes human thyroid progression.

Increased glucose metabolism by glycolysis and lactate production is a hallmark of cancer cells.¹⁷⁾ There are 14 members of MCTs, and the first four MCTs (MCTs 1, 2, 3, and 4) are metabolically essential transporters for monocarboxylates, such as lactate pyruvate and ketone bodies.¹⁸⁾ MCT4 is predominantly expressed in cells with an increased glycolytic pathway to facilitate lactate export.¹⁹⁾ Increased lactate secretion by tumor and stromal cells acidifies the tumor microenvironment and contributes to cancer cell proliferation, survival, angiogenesis, and altered immune responses.²⁰⁾ An experimental approach to knock down SLC16A3 showed that MCT4 contributes to cancer cell aggressiveness by facilitating cell migration and invasion.^8,9) Another experimental study discovered that inhibiting MCT4 and lactate export reduces ATC cell growth, indicating a potential treatment strategy.¹²⁾ These findings suggested the importance of MCT4 in the tumor microenvironment and indicated that targeting MCT4 can be implemented as a therapeutic strategy.

In this study, ATC was found to have significantly increased the expression of SLC16A3 and MCT4 protein compared with normal tissues and PTCs following cancer progression and de-differentiation. Recently, several studies have suggested an association between increased MCT4 expression and PTC aggressiveness.^14,15,21) A study by Nahm et al.¹⁵⁾ evaluated the expression of glycolysis-related proteins, including glucose transporter 1, hexokinase II, carbonic anhydrase IX, and MCT4. In univariate analysis, MCT4 expression was significantly associated with the survival of patients with PTC and poorly differentiated thyroid carcinoma.¹⁵⁾ However, no specific analysis has focused on MCT4 expression in ATCs and other pathological subtypes. Our study has shown that MCT4 is highly expressed in thyroid cancers, especially ATCs, and is associated with thyroid de-differentiation. These results suggested that MCT4 is helpful as a diagnostic and prognostic biomarker for thyroid cancer. Furthermore, MCT4 is a potential therapeutic target for re-differentiation therapy and treatment of patients with ATC.

A part of this study was presented as an abstract at a meeting of the Korean Thyroid Association in 2022.

No potential conflict of interest relevant to this article was reported.