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Tafur et al. J Cancer Metastasis Treat 2018;5:xx I http://dx.doi.org/10.20517/2394-4722.2018.102 Page 7 of 14

A GPR81 C Kaplan-Meier Survival Curve
With number of subjects at risk
1.0
P-value (low vs. high) = 0.0011
0.8
Survival Probability 0.6 0.4

B 0.2
GPR81 GPR81 expression
low (n=409)
0.0 high (n=217)
0 50 100 150
Months
Subjects at risk
low 270 95 13
409
high 217 184 86 12

Figure 2. GPR81 is highly expressed in human hormone-receptor-positive breast cancer tissues. A: relative mRNA expression of GPR81 in
+
+
ER (n = 32), HER2 (n = 17) and TNBC (n = 12) breast cancer tissues to normal tissue (n = 12); B: relative mRNA expression of GPR81 in
stage I (n = 21), II (n = 32), III (n = 25) and IV (n = 4) of breast cancer; C: Kaplan-Meier plot of estimated overall survival in breast cancer
patients (n = 626) with low expression of GPR81, below the median (black line) or high expression of GPR81, above the median (red line).
Log-rank test P-value = 0.0011. Results are presented as mean ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001 (two-way ANOVA)
positive breast cancer samples (ER and HER2 ) and significantly lower levels were found in the NT and
+
+
TNBC samples [Figure 2A]. We also analyzed the relative gene expression of GPR81 by the stage of human
breast cancer. Breast cancer stage II-III had significantly higher GPR81 expression than stage IV [Figure 2B].
Additionally, the Kaplan-Meir survival plot showed that high expression levels GPR81 was strongly associated
with better prognosis and overall survival in breast cancer patients [Figure 2C] (P = 0.0011) (n = 626) . We
[32]
also tested GPR81 expression in other breast cancer cell lines. Hormone-receptor positive HC1500 cells had
higher expression of the lactate receptor and lactate importer compared to the TNBC, MDA-MB231 cells
[Supplementary Figure 1A-C]. These results demonstrate that GPR81 is mainly expressed in hormone
positive and less aggressive types of human breast cancer.

GPR81 regulates expression of the gene involved in lactate import in MCF-7 epithelial breast
cancer cell line
A previous study reported that GPR81 regulates the expression of lactate transport related genes MCT1
and MCT4 in pancreatic cancer cells through an as yet unknown mechanism . To determine the role of
[26]
the endogenous lactate receptor GPR81 in the lactate metabolism of estrogen positive breast cancer cells,
we silenced GPR81 (~60%) in MCF-7 cancer cells using siRNA (siGPR81) up to four days in MPM culture
[Figure 3A]. GPR81 knockdown in MCF-7 cells led to a 2-fold reduction mRNA and protein expression of
the lactate importer, MCT1, but had no effect on the lactate exporter, MCT4 [Figure 3B and C]. In addition,
GPR81 did not significantly alter the expression of other lactate metabolism-related genes (LDHA, LDHB,
MCT2) [Supplementary Figure 2].

GPR81 regulates lactate uptake in MCF-7 epithelial breast cancer cell line
We found that the two epithelial breast cancer cell lines had higher steady state levels of intracellular lactate
and pyruvate than the corresponding post-EMT mesenchymal cell lines . Given that MCT1 is primarily a
[33]