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Page 8 of 16 Tu et al. J Cancer Metastasis Treat 2018;4:58 I http://dx.doi.org/10.20517/2394-4722.2018.67

MDA-MB-231HM.LNm5 cells display enhanced glycolytic reserve, mitochondrial respiration and
ATP synthesis
The increase in ECAR in the presence of oligomycin not only demonstrates the maximum glycolytic rate,
but also shows the glycolytic reserve [Supplementary Figure 1A]. MDA-MB-231HM.LNm5 cells showed
a larger increase in ECAR compared to the MDA-MB-231 parental line following oligomycin treatment
[Figure 2D], revealing higher maximum glycolytic rates and reserves [Figure 2G and H]. Similarly, the dif-
ference between maximum OCR and basal OCR allows calculation of the spare respiratory capacity, which
did not differ between the two cell lines [Supplementary Figure 2B].

Both mitochondrial and non-mitochondrial respiration contributed to the basal and maximum OCR. The
combination of rotenone, a complex I inhibitor, and antimycin A, a complex Ill inhibitor, shut down mi-
tochondrial respiration completely, leaving respiration driven by processes outside the mitochondria only.
MDA-MB-231HM.LNm5 cells showed significantly higher mitochondrial-dependent basal respiration
[Figure 2E] and similar mitochondrial and non-mitachondrial -dependent maximum respiration rates com-
pared to the parental cells [Supplementary Figure 2C and D].

The two processes that control basal mitochondrial respiration, ATP production and proton leak, can be
probed with the blockade of ATP synthase using oligomycin. Measuring the reduction in OCR upon addi-
tion of oligomycin revealed significantly higher mitochondrial ATP synthesis in MDA-MB-231HM.LNm5
cells compared to parental MDA-MB-231 cells [Figure 2F], but unchanged proton leak-driven respiration
[Supplementary Figure 2E].

Gene expression analysis of energy metabolism pathways
In order to associate the observed metabolic changes with specific genetic or epigenetic alterations, we first
selected several genes encoding enzymes that participate in glycolysis and the TCA cycle that were docu-
[38]
mented to contribute to altered metabolism in cancer cells . RT-qPCR analysis of glucose transporter type
1 [solute carrier family 2 member 1 (SLC2A1)], hexokinase 2, fructose-2,6-biphosphatase 3, muscle pyruvate
kinase 2, pyruvate dehydrogenase kinase 1, cytosolic isocitrate dehydrogenase-1 (IDH1), succinate dehydro-
genase complex subunits C and D and fumarate hydratase mRNA showed similar expression levels between
the MDA-MB-231HM.LNm5 cells and parental cells [Figure 3].

To produce an unbiased analysis, the whole transcriptome of each cell line was then deep-sequenced using
RNAseq, and the expression of genes involved in key pathways of energy metabolism was compared, includ-
ing those influencing glycolysis and mitochondrial respiration. Gene expression level was expressed as CPM
and the expression level of gene sets was compared by calculating the ratio between two cell lines using the
MDA-MB-231 parental line as the denominator. Transcript per million was also compared and yields similar
ratio (data not shown). The comparison of all mitochondrial genes showed a symmetrical distribution of ex-
pression around a log-fold change of 0, indicating no predominant direction of effect, although some genes
were dysregulated between the two cell lines [Figure 4A]. Genes encoding enzymes directly involved in gly-
colysis were expressed at lower levels in MDA-MB-231HM.LNm5 cells compared to the parental cells [Figure
4B, Supplementary Table 2]. In particular, hexokinase domain containing 1 (HKDC1), encoding the hexo-
[39]
kinase isoform HKDC1 which catalyzes the rate-limiting and obligatory first step of glucose metabolism ,
was significantly down-regulated (Table 1 log FC = -6.64). However, the majority of genes involved in regu-
2
lating glycolytic processes showed unaltered expression between the two cell lines. The most differentially
expressed genes were those that were down-regulated in metastatic cells [Figure 4B, Table 1, Supplementary
Table 3], including MLX interacting protein-like (MLXIPL, log FC = -6.73), encoding a leucine zipper tran-
2
scription factor of the Myc/Max/Mad superfamily, and FBP1 (log FC = -5.36), encoding the gluconeogenesis
2
regulatory enzyme fructose-1,6-bisphosphatase-1. Reduced expression of these genes in MDA-MB-231HM.
LNm5 was confirmed by RT-qPCR [Figure 5].