A CD147-targeting siRNA inhibits the proliferation, invasiveness, and VEGF production of human malignant melanoma cells by down-regulating glycolysis
Introduction
Glycolysis, promoted by hypoxic stimuli or injury to the mitochondrial respiration, is the result of an anaerobic enzymatic conversion of glucose to pyruvate or lactate to produce energy stored in the form of adenosine triphosphate (ATP) [1], [2]. It is enhanced in tumor cells and the extracellular pH is lower in tumors than in normal tissue [3]. This is considered to be the consequence of an excessive efflux of lactate, the end product of glycolysis. In 1950’s, Warburg [4] first reported that cancer cells depend on glycolysis for energy and that this results in excessive levels of lactate even in the presence of oxygen. Meienhofer et al. [5] suggested that tumor cell growth and survival depend on the switch from anaerobic to aerobic glycolysis. Tumor cells become addicted to aerobic glycolysis for several reasons including the increased demand for rapid energy production and for the metabolites of glycolysis to support malignant behavior. Lactate is excreted from cells through an H+/lactate co-transporter, the monocarboxylate transporter (MCT), leading to a decrease in the extracellular pH of tumors [6]. Tumor acidity produces a more aggressive phenotype of cancer cells with a number of altered behaviors. Turner [7] first proposed the role of acidic pH in tumor metastasis. Low pH treatments clearly induced experimental metastasis [8], [9] and the invasive behavior of tumor cells [10], [11], [12]. In addition, low pH contributed to the in vivo induction of VEGF expression in brain tumors [13] and human pancreatic adenocarcinoma [14].
CD147/basigin (CD147), an integral plasma membrane protein of the immunoglobulin superfamily, is expressed widely on many cell types and its expression is increased in various tumor cells [15], [16]. Elevated CD147 stimulates matrix metalloproteinase (MMP)-production in stromal fibroblasts and endothelial cells, leading to extracellular matrix degradation, tumor growth promotion, and metastasis [17], [18], [19]. CD147 also stimulates the expression of vascular endothelial growth factor (VEGF) [19], [20]. Interestingly, CD147 associates closely with two members of the proton-coupled MCT family, MCT1 and 4 [21] and acts as an essential chaperone for their correct plasma membrane expression and catalytic activity [21], [22]. MCT1 and 4 catalyze the proton-linked transport of monocarboxylates, of which lactate is metabolically the most important. The rapid transport of lactate by MCTs is of critical importance for almost all cells, especially tumor cells with elevated glycolysis, resulting in a decrease in extracellular pH.
Malignant melanoma (MM) is highly malignant because of its rapid growth, advanced stage, and metastatic properties [23]. We previously showed that CD147 expression was enhanced in MM cells and that CD147 played an important role in tumor proliferation, the production of MMPs, angiogenesis, invasiveness, and metastatic activity in vitro and in vivo [18], [19]. Because the role of CD147 on tumor progression is similar to that of low extracellular Ph and CD147 is closely associated with MCT1 and 4, we hypothesized that it is involved in MM glycolysis and in the transport of lactate across the plasma membrane as well as the changes in the behavior of MM cells. Therefore, after knocking down the expression of CD147 in an MM cell line with siRNA we examined the co-localization of CD147 with MCT1 or 4 on the MM cell membrane by confocal microscopy and assayed the glycolysis rate, extracellular pH, ATP synthesis, and the tumor cellular activities. We found that the association between CD147 and MCT1 or 4 on the plasma membrane of MM cells was remarkably weakened and that the level of lactate and ATP were decreased. The environmental pH and cellular activities were restored after inhibition of CD147 in A375 cells. Our findings suggest that its ability to lower the pH of the microenvironment, resulting in the enhanced proliferation, invasiveness, metastasis, and angiogenesis of tumor cells, may be the fundamental property of CD147 that affects tumorigenesis and tumor development.
Section snippets
Cell culture
The human MM cell line A375, normal human fibroblasts (NHFBs), and normal human melanocytes (NHMCs) were preserved in our laboratory (Kagoshima University). A375 cells and NHFBs were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution (Invitrogen) and incubated at 37 °C in a humidified atmosphere containing 5% CO2. NHMCs were maintained in Ham’s F-10 (Sigma), supplemented with 5% FBS, 20 μg/mL bovine pituitary extract (Sigma), 50 ng/mL TPA
Enhanced expression of CD147 and MCT1 and -4 in human MM cell line A375
As shown in Fig. 1, Western blotting demonstrated that CD147 expression, not detected in NHMCs, was enhanced in A375 cells. The expression of MCT1 and 4 was also up-regulated in A375 cells compared with NHMC.
siRNA-mediated silencing of CD147 expression
Specific siRNA targeting CD147 mRNA was used to knock down CD147 expression. We established two new clones C1 and C2 that effectively and stably inhibited the CD147 expression using pSUPER/CD147 siRNA1, but not pSUPER/CD147 siRNA2 that were accordant to our previous report (data not shown)
Discussion
This study showed that the expression of CD147, MCT1, and 4 was enhanced and that CD147 and MCT1 or 4 co-localized on the plasma membrane of A375 MM cells. Silencing of CD147 by means of siRNA technology down-regulated the expression of MCT1 and 4 and abrogated the co-localization of CD147 with these MCTs. It also demonstrated that glycolysis was activated and extracellular pH was decreased in A375 cells compared with NHMC and that the glycolysis rate, extracellular pH and the production of ATP
Acknowledgments
This study was supported by grants from the National Natural Science Foundation of China; Program for New Century Excellent Talents at University; The Hunan Science Fund for Distinguished Young Scholars. J. Su was the recipient of a grant from Jiaikai Foundation, Kagoshima, Japan.
References (50)
- et al.
Mitochondrial DNA in human malignancy
Mutat. Res.
(2001) - et al.
Interaction of the erythrocyte lactate transporter (monocarboxylate transporter 1) with an integral 70-kDa membrane glycoprotein of the immunoglobulin superfamily
J. Biol. Chem.
(1997) - et al.
Cancer metabolism: facts, fantasy, and fiction
Biochem. Biophys. Res. Commun.
(2004) - et al.
Glycolysis in cancer: a potential target for therapy
Int. J. Biochem. Cell Biol.
(2007) - et al.
Effect of extracellular AMP on cell proliferation and metabolism of breast cancer cell lines with high and low glycolytic rates
J. Biol. Chem.
(1997) - et al.
Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance
Cancer Cell
(2006) - et al.
The relevance of tumor pH to the treatment of malignant disease
Radiother. Oncol.
(1984) - et al.
Fluorescence resonance energy transfer studies on the interaction between the lactate transporter MCT1 and CD147 provide information on the topology and stoichiometry of the complex in situ
J. Biol. Chem.
(2002) - et al.
Distribution of monocarboxylate transporter MCT1 and MCT2 in rat retina
Neuroscience
(1999) - et al.
Mitochondrial defects in cancer
Mol. Cancer
(2002)
MR imaging of the tumor microenvironment
J. Magn. Reson. Imag.
On respiratory impairment in cancer cells
Science
Regulation of genes for glycolytic enzymes in cultured rat hepatoma cell lines
Eur. J. Biochem.
The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis
Nat. Rev. Cancer
Increased release of tumour cells by collagenase at acidic pH: a possible mechanism for metastasis
Experientia
Glucose starvation and acidosis: effect on experimental metastasic potential, DNA content and MTX resistance of murine tumour cells
Br. J. Cancer
Experimental metastasis and differentiation of murine melanoma cells: actions and interactions of factors affecting different intracellular signalling pathways
Clin. Exp. Metast.
Acidic pH enhances the invasive behavior of human melanoma cells
Clin. Exp. Metast.
Pericellular pH affects distribution and secretion of cathepsin B in malignant cells
Cancer Res.
Exposure to hypoxia, glucose starvation and acidosis: Effect on the invasive capacity of murine tumor cells and correlation with cathepsin (L+B)
Clin. Exp. Metast.
Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo
Cancer Res.
Regulation of vascular endothelial growth factor expression by acidosis in human cancer cells
Oncogene
Basigin, a new, broadly distributed member of the immunoglobulin superfamily, has strong homology with both the immunoglobulin V domain and the H-chain of major histocompatibility complex class II antigen
J. Biochem.
Basigin, a new member of the immunoglobulin superfamily: genes in different mammalian species, glycosylation changes in the molecule from adult organs and possible variation in the N-terminal sequences
Cell Struct. Funct.
The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily
Cancer Res.
Cited by (117)
Basigin binds bacteria and activates Dorsal signaling to promote antibacterial defense in Penaeus vannamei
2023, Fish and Shellfish ImmunologyMetabolic tricks of cancer cells
2022, Biochimica et Biophysica Acta - Reviews on CancerSLC16 Family: From Atomic Structure to Human Disease
2021, Trends in Biochemical SciencesCitation Excerpt :It has been shown that carbonic anhydrases interact with MCT-bound CD147 and thus modulate the L-lactate transport activity of the SLC16 transporters [22]. In cancer cells, CD147 promotes tumor cell proliferation and invasiveness and the production of vascular endothelial growth factor, and it plays a key role in regulating glycolysis and ATP synthesis in tumor cells [23]. Changes in the expression level and transport function of certain family members are associated with severe health disorders (e.g., cancer, diabetes, neurological disorders, eye diseases).
Targeted degradation of CD147 proteins in melanoma
2020, Bioorganic ChemistryThe tortuous path of lactate shuttle discovery: From cinders and boards to the lab and ICU
2020, Journal of Sport and Health ScienceS100 Soil Sensor Receptors and Molecular Targeting Therapy Against Them in Cancer Metastasis
2020, Translational Oncology