The online version of this article (doi:10.1186/1476-4598-11-76) contains supplementary material, which is available to authorized users.
Jonhan Ho, Michelle Barbi de Moura, Bennett Van Houten contributed equally to this work.
The authors declare that they have no competing interests.
JH, SJM, and LMD performed immunohistochemical analysis of the nevus-melanoma TMAs. MBM and GV performed Seahorse analysis of melanoma cell lines and tumor tissues. GV established melanocytes and melanoma cell cultures and immunoblot analysis for each of the antibodies used for immunohistochemical analysis. ST, GV, MBM performed in vivo imaging of glycolysis of human melanoma xenografts that supported the notion of metabolic symbiosis (data not shown). YL and LHM performed analysis of serum LDH and immunohistochemical data. SJM and JMK provided patient samples (sera and tumor tissues). ST, JMK, DB, BVH and SJM analyzed all experiments and wrote the manuscript. All authors read and approved the final manuscript.
Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.
Additional file 1: Figure S1. Schematic presentation of LDH1-5 and their involvement in OXPHOS and glycolysis. Red circles indicate LDHA subunits and blue circles indicate LDHB subunits. (PPTX 52 KB)12943_2012_1060_MOESM1_ESM.pptx
Additional file 2: Figure S2. Validation of antibodies used in the nevus-melanoma TMA analyses. Immunoblot analysis of whole cell lysates, prepared from HEMs and different melanoma cell lines were probed with antibody specific for MCT4, MCT1, HIF-1α, LDHB, LDHA. α-tubulin served as loading control. (PPTX 412 KB)12943_2012_1060_MOESM2_ESM.pptx
Additional file 3: Figure S3. LDHA and HIF-1α expression in nevi and melanomas. (A-B, panels a) TMA cores comprised of nevi, and primary and metastatic melanoma tissue core, probed with antibody to LDHA or HIF-1α, and counterstained with hematoxylin. (A-B, panels b) 10X magnification of select TMA cores. (PPTX 4 MB)12943_2012_1060_MOESM3_ESM.pptx
Additional file 4: Figure S4. ATP5A1 and LDHB expression in nevi and melanomas. (A-B, panels a) TMA cores comprised of nevi, primary melanoma, and metastatic melanoma, probed with antibody to ATP5A1 or LDHB, and counterstained with hematoxylin. (A-B, panels b) 10X magnification of select TMA cores. (PPTX 5 MB)12943_2012_1060_MOESM4_ESM.pptx
Additional file 5: Figure S5. MCT1 and MCT4 expression in the nevus>melanoma TMA. The TMA study was performed as described in the legends to Additional files 3 and 4: Figures S3 and S4. (PPTX 7 MB)
Additional file 6: Figure S6. Pairwise correlation matrix analysis of expression of the various molecules in the nevus>melanoma TMA. Depicted in the lower left corner are dot plots of the H-scores between each permutation pair of the dataset of the six proteins whose expression was determined in the TMA. Shown in the upper right corner are Spearman rank correlation coefficients with corresponding p-values for the respective permutation pair of the six proteins. Rho and p values showing significant association are highlighted, and/or presented in a larger font size. Green-colored boxes in the matrix depict expected and known significant associations, blue-colored boxes show known insignificant associations, and red-colored boxes denote novel and significant associations. (PPTX 122 KB)12943_2012_1060_MOESM6_ESM.pptx
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- Importance of glycolysis and oxidative phosphorylation in advanced melanoma
Michelle Barbi de Moura
Lyn M Duncan
John M Kirkwood
Bennett Van Houten
Stergios J Moschos
- BioMed Central
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