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Page 12 of 14 Tafur et al. J Cancer Metastasis Treat 2018;5:xx I http://dx.doi.org/10.20517/2394-4722.2018.102

transformation of epithelial breast cancer cells into triple negative mesenchymal cancer cells was associated
with significantly reduced expression of GPR81. Lee et al. reported that GPR81 expression is significantly
[25]
increased in breast cancer patients compared to normal mammary tissues. We also observed a significant
association between GPR81 overexpression with ERα-positive and human epidermal growth HER2 positive
human breast cancer tissues as opposed to triple negative breast cancer. This is consistent with previous
observations that ERα-positive breast cancer tissues overexpressed GPR81 . In addition, GPR81 was
[25]
expressed at higher levels in the first three stages of breast cancer (Stage I, II and III) as compared to Stage
IV, and GPR81 expression correlated with better overall survival of breast cancer patients. These findings
suggest that GPR81 may be an important regulator in hormone-positive breast cancer and could be used as
a prognostic marker in the progression of breast cancer.

GPR81 was reported to regulate the expression of genes involved in lactate metabolism, including lactate
transporters, MCT1 and MCT4, in pancreatic cancer cells . In partial agreement, our study revealed
[26]
that GPR81 specifically regulates the lactate importer MCT1, but not lactate exporter MCT4, in epithelial
breast cancer cells. Oxidative breast tumor cells with high expression of the lactate importer, MCT1, have
been reported to import and oxidize extracellular lactate, a mechanism that is essential for cell viability
under glucose deprivation [14,34,49] . In fact, Lee et al. showed that epithelial breast cancer cells imported and
[25]
utilized 14C-lactate for mitochondrial respiration. We previously showed that inhibition of MCT1 reduced
cell proliferation in epithelial but not mesenchymal cancer cells when lactate was used as the primary
extracellular fuel source. Here we report that GPR81 silencing caused downregulation of MCT1 and reduced
cell growth in epithelial breast cancer cells grown in complete MPM medium. We reported that the two
epithelial breast cancer cell lines contained significantly higher intracellular lactate and pyruvate that the
two corresponding post-EMT mesenchymal cell lines (submitted manuscript). In the present study, we
observed that silencing of GPR81 in MCF7 cells led to lower intracellular lactate and higher levels of lactate
in the culture media, indicating reduction in lactate uptake. Furthermore, we found a protective effect of
GPR81 against apoptosis, which was previously described in epithelial MCF-7 breast cancer cells, in which
GPR81 activation triggered the PI3K/Akt signaling pathway to inhibit apoptosis . Together these results
[25]
suggest a specific regulation of GPR81 on lactate importer MCT1, which affects cell proliferation, apoptosis
and survival of hormone-positive epithelial breast cancer cells.

In adipocytes, GPR81 is coupled to Gi/q which inhibits adenylate cyclase activity and decreases the production
of cAMP. However, a previous study did not find a significant change in cAMP levels when GPR81 was
silenced in epithelial MCF-7 breast cancer cells , suggesting that GPR81 regulates MCT1 expression by
[25]
another signaling pathway in this cells. Alternate GPR81-associated signaling that is independent of the
PKA/cAMP pathway, but involves the GPR81-binding protein β-arrestin, has been reported in monocyte/
macrophage . Further studies are needed to examine whether the β-arrestin pathway or other pathways
[48]
are linked to GPR81 signaling in epithelial breast cancer, as these pathways may provide targets for future
development of adjuvant therapies for Luminal A breast cancer.

DECLARATIONS
Acknowledgments
We thank Dr. Kevin Claffey, Department of Cell Biology, University of Connecticut for providing us the
Cleaved PARP antibody and the cell line HC1500. This work supported by the Fund for Biology from the Cell
Biology Department at the University of Connecticut Health Center.

Authors’ contributions
Made substantial contributions to conception and design of the study and performed data analysis and
interpretation: Tafur D, White B
Performed NMR studies, data acquisition, analysis and interpretation: Svrcek P