*2.6. Investigation into Potential Mechanisms by Which ABCC Transporters A*ff*ect Cellular Proliferation or Migration*

ABCC1 and ABCC4 both transport a wide array of different molecules with the potential to impact cellular proliferation and migration, including cyclic nucleotides, eicosanoids, and lipid mediators such as S1P and LPI. It has previously been proposed that ABCC1 is involved in creating a feedback signaling loop with the G protein-coupled receptor, GPR55, whereby ABCC1 exports LPI, which binds to GPR55 and activates it, leading to downstream signaling and increased proliferation [31]. Therefore, we measured the expression of GPR55 in the breast cancer cell lines by Western blot (Figure 8a). GPR55 is indeed expressed in the MDA-MB-231 cells; however, there was little, if any, expression in the MCF-7

cells. Since the effects we observed with ABCC1 and ABCC4 inhibitors and knockdown were with both cell lines, this would argue against it being due to LPI export and GPR55 activation.

≥ ≥ ≥ ANOVA with a Dunnett's post **Figure 8.** Efflux of cAMP or S1P are possible mechanisms by which ABCC transporters affect breast cancer cells. (**a**) Western blot analysis of the expression of GPR55 in breast cancer cell lines. The blue arrow indicates the band corresponding to GPR55 (**b**) Efflux of cAMP from MDA-MB-231 breast cancer cells was assayed using an ELISA. Data are mean ± sem, n ≥ 2, each in duplicate. (**c**) Efflux of Prostaglandin E<sup>2</sup> (PGE<sup>2</sup> ) from MCF-7 breast cancer cells was assayed using an ELISA. Data are mean ± sem, n ≥ 2, each in duplicate. (**d**) Efflux of S1P from MDA-MB-231 breast cancer cells was assayed using an ELISA. Data are mean ± sem, n ≥ 3, each in duplicate. Data were analyzed by a one-way ANOVA with a Dunnett's post hoc test. \* *p* < 0.05 and \*\* *p* < 0.01, significantly lower than untreated or negative siRNA treatment.

Next, the efflux of cAMP was investigated as a potential mechanism, as ABCC4 is capable of effluxing cyclic nucleotides [32]. An ELISA was used to assay cAMP in the cell medium (Figure 8b). The concentration of cAMP in the medium from MCF-7 cells was outside the standard range and, therefore, could not be reliably measured. For MDA-MB-231, the knockdown of ABCC1 had no significant effect on the concentration of cAMP in the medium, which is unsurprising, as ABCC1 is not reported to transport cyclic nucleotides. However, the knockdown with ABCC4 siRNA #35 did significantly decrease the concentration of cAMP in the medium, as did the treatment with MK571.

An efflux of eicosanoids such as prostaglandins could be another possible way in which ABCC4 mediates an effect [33]. An ELISA was used to measure Prostaglandin E<sup>2</sup> (PGE2) levels in the cell medium. The concentration of PGE<sup>2</sup> in the medium from MDA-MB-231 cells was outside the range of standards used and, thus, could not be reliably measured. The concentration of PGE<sup>2</sup> in the medium from MCF-7 cells can be seen in Figure 8c. Neither the knockdown of ABCC proteins nor the treatment with MK571 had any effect on the levels of PGE<sup>2</sup> measured.

Finally, S1P was investigated, as there are reports that both ABCC1 and ABCC4 are capable of transporting S1P [29,38]. Figure 8d shows the results of the S1P ELISA. Only the MDA-MB-231 cells effluxed sufficient S1P for reliable measurement. Although none of the siRNA knockdown samples showed any significant difference in S1P concentrations, the treatment with MK571, which inhibits both ABCC1 and ABCC4, did significantly decrease the amount of S1P in the medium.
