Canonical Wnt signaling regulates the fate and activities of cells through the actions of ?-catenin, a nuclear-localizing mediator that can activate the transcription of Wnt target genes important in cell growth and proliferation. A chronic increase in the cellular levels of ?-catenin can occur through oncogenic activation of the Wnt signal transduction pathway, a condition that leads to aberrant and elevated expression of Wnt target genes. Constitutive Wnt target gene expression is an abnormal condition that can transform cells and cause cancer, including colon cancer, a disease defined by epithelial cell transformation within the intestine. Colon cancer most commonly derives from chronic activation of the canonical Wnt signaling pathway through mutations of components in the destruction complex, a multi-subunit regulator in the cytoplasm that degrades ?-catenin to maintain appropriate, physiological levels [1–4]. Nuclear-localized ?-catenin activates target gene expression via direct binding to LEF/TCF transcription factors, a family of four DNA binding proteins that occupy distinct gene targets (i.e., gene programs) and direct specific phenotypes and functions of cells. Gene programs identified to be altered by oncogenic Wnt signaling include cell cycle progression, epithelial-mesenchymal transition (EMT), angiogenesis, migration, cell survival, and most recently discovered by our group, metabolism [5–8].
Many groups have used overexpression of dominant-negative isoforms of LEF/TCF transcription factors (dnLEF/TCFs) in multiple contexts and model systems to identify Wnt target genes [5, 6, 9, 10]. These shorter forms retain the capabilities of full length LEF/TCFs to occupy Wnt response elements (WREs) throughout the genome, but they lack the ability to recruit ?-catenin. Interference by these dominant-negative isoforms represses target gene transcription, and thus, genome-wide expression analysis of downregulated transcription can reveal candidate target genes and the gene programs with which they are associated. We used this type of analysis in colon cancer cells to discover that Wnt signaling promotes tumor cell preferences for aerobic glycolysis/Warburg metabolism, with the Wnt target gene pyruvate dehydrogenase kinase 1 (PDK1) playing a significant role in this metabolic fate [8]. In that study, we also observed additional metabolism-linked genes to be sensitive to dnLEF/TCF expression, suggesting that Wnt signaling coordinately regulates PDK1 within a larger gene program. One of the additional genes affected was monocarboxylate transporter 1 (SLC16A1, encoding the protein MCT-1), a known lactate transporter observed to be upregulated in many cancers [11, 12]. qRT-PCR analysis of xenograft tumors from a colon cancer cell line showed MCT-1 downregulation in the presence of dnLEF/TCFs, and ChIP-seq ENCODE data shows TCF-4 occupancy of SLC16A1 in HCT116 colon cancer cells [8]. These preliminary findings strongly implicate MCT-1 as a direct Wnt target gene that might be coordinately regulated with PDK1. Here, we investigate this possibility and show that MCT-1/SLC16A1 is a direct target gene of ?-catenin-LEF/TCF complexes in colon cancer cells.
MCT-1 is one of 14 members of the SLC16 family of transporters [13]. While the functions of many MCT family members remain uncharacterized, MCT-1 through MCT-4 is confirmed proton-linked monocarboxylic acid transporters [14]. These four family members have been shown to transport monocarboxylates including acetoacetate, ?-hydroxybutyrate, short chain fatty acids, pyruvate, and lactate. In a normal setting, MCTs are necessary for lactate efflux from highly glycolytic/hypoxic muscle fibers during exercise, and also reabsorption or uptake of monocarboxylates from the gut, liver, and kidney for gluconeogenesis or lipogenesis—activities tightly linked to aerobic and anaerobic glycolysis [14]. MCT-1 has a reasonably strong affinity for lactate compared to the other MCTs (Km of 2.5–4.5 mM, compared to MCT-2 Km?=?0.7 mM; MCT-3 Km?=?6 mM; MCT-4 Km?=?17–34 mM), and it is broadly expressed, while other MCT family members are localized to specific regions of the body at varying levels of expression [13, 15].
While increased expression of MCT-1 in response to the physiological stresses of exercise and physical stimulation has been well defined, the molecular mechanisms that govern its expression are still poorly understood. At the transcriptional level, the SLC16A1 promoter contains nuclear factor of activated T-cells (NFAT)-binding sequences [14], but the significance of these elements is unknown. In rat skeletal muscle tissues, PGC? (a transcriptional co-activator linked to regulation of genes involved in energy metabolism) has been associated with MCT-1 upregulation in response to muscle activity [16]. However, no follow-up studies have been conducted to determine whether the SLC16A1 promoter is subject to direct activation. The ribonucleotide metabolite and AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide-1-?-d-ribonucleoside (AICAR), has been shown to upregulate or downregulate SLC16A1 promoter activity depending on the study and tissue context [17]. Likewise, butyrate, another metabolite and energy source for the colon epithelium has been identified to enhance transcription and transcript stability of SLC16A1 mRNA [18], but the mechanisms and responsive genomic regions behind these effects are not known. Finally, hypoxia was shown to upregulate MCT-1 in human adipocytes [19], but this is a singular example. In most tissues and cell lines studied, MCT-1 expression is not affected by hypoxia [20]. Instead, MCT-4 is considered to be the main transcriptional responder to hypoxia as multiple, high affinity HIF response elements (HREs) have been identified in its promoter and hypoxic expression has been demonstrated in many tissues [20].
The observation that MCT-1 expression is increased in cancer has led to studies focused on its regulation in cancer cells. For example, the tumor suppressor p53 directly binds to the MCT-1 promoter for transcription repression, and therefore, the loss of p53 in cancer cells enables MCT-1 mRNA production [21]. c-Myc also directly regulates MCT-1 transcription, especially in cancer cells where high levels of c-Myc drive metabolic pathways [22]. A common theme among cancer cells is the use of elevated MCT-1 expression to support the glycolytic preference of cells via its ability to export lactate. This export minimizes the cellular stresses from acid buildup and maintains proper intracellular pH, activities crucial to cancer cell survival [23]. Alternatively, a recent study found that MCT-1 primarily exports pyruvate, where co-expressed MCT-4 plays the dominant role in exporting lactate [24]. This function appears to promote glycolysis as inhibition of MCT-1 transporter activity or downregulation of its protein levels leads to increased oxidative phosphorylation and decreased proliferation [24]. Taken together, no matter the precise actions of its transporter functions, the use of MCT-1-specific inhibitors has shown this transporter to be a key player in cancer cell metabolism, survival, and proliferation, making it a potentially important candidate target in glycolytic cancer cells [25].
Recent findings highlight how MCT-1 overexpression may be an exploitable feature for cancer therapy. Birsoy et al. have shown that breast cancer cells expressing MCT-1 are sensitive to 3-bromopyruvate (3-BP), a molecule that can have anti-proliferative effects by targeting glycolytic enzymes and other metabolic pathways [26]. Like its parent molecule pyruvate, 3-BP must be transported across the plasma membrane. Birsoy et al. used genome-wide screening to discover that 3-BP is imported into cells strictly through MCT-1 and no other transporter or alternative pathway. Whether this makes MCT-1 expression the single most important biomarker for determining tumor sensitivity to 3-BP is not yet known, as its precise mode of action has not been defined and only breast cancer cells were used in the study. Nevertheless, there are several case reports documenting the use of this compound in cancer patients, underscoring the importance of understanding how SLC16A1 gene expression is regulated [27, 28]. Here, we show that MCT-1/SLC16A1 is a direct Wnt target gene coordinately regulated with other genes that promote glycolysis in colon cancer cells. We define a region in the upstream promoter with at least two WREs and show that the endogenous gene is sensitive to dnLEF/TCF inhibition in multiple colon cancer cell lines. We show that transcriptional regulation by ?-catenin/LEF/TCFs is separate and additive with c-Myc action. We demonstrate that colon cancer cells are sensitive to 3-BP and that the sensitivity tracks partially, but not completely, with the strength of oncogenic Wnt signaling. Finally, we show that Wnt signaling inhibitors do not synergize with 3-BP to suppress proliferation, but instead interfere with the anti-proliferative effects of 3-BP and provide a resistance mechanism for colon cancer cells.