**3. Discussion**

ABCC1 and ABCC4 have previously been implicated as negative prognostic indicators for breast cancer [16–19,22]. This may be due to their ability to export chemotherapeutic drugs, leading to multidrug resistance [13–21]. However, ABCC1 and ABCC4 can also transport a wide range of substrates involved in inflammation and cellular signaling and have been shown in several different cancer types to have a role in cancer development and progression that is independent of drug resistance [39–47,55]. Therefore, in this study, we investigated whether ABCC1 or ABCC4 are involved in cellular proliferation or migration in breast cancer cell lines. We found that both MCF-7 and MDA-MB-231 cells expressed both ABCC1 and ABCC4, with MDA-MB-231 cells having a higher expression of both transporters, which is in agreement with previous studies [56,57]. MDA-MB-231 cells are basal-type triple-negative and often reported as more aggressive than MCF-7 cells, so this would correlate with the expression levels observed. It has been reported that ABCC1 is frequently expressed in lymph node metastases of breast cancer patients and that MRP1 expression is more pronounced in lymph node metastases than in corresponding primary tumors [58].

Using both small molecule inhibitors and siRNA knockdown of ABCC1 and ABCC4, we investigated the impact of these transporters on the proliferation, clonogenic capacity, migration, and invasion of the breast cancer cell lines. MK571, which inhibits both ABCC1 and ABCC4, and Reversan, which inhibits ABCC1, both had significant effects on the proliferation and clonogenic capacity of MCF-7 and MDA-MB-231 cells, suggesting a role for ABCC1 or both transporters in cellular proliferation. Similarly, one of the ABCC1 knockdowns and the double ABCC1/ABCC4 knockdowns decreased the colony formation with MCF-7 cells, although only one of the double knockdowns affected colony formation in the MDA-MB-231 cells. It should be noted that MK571 not only inhibits ABCC1 and ABCC4 but, also, other members of the ABCC family, and it is a leukotriene antagonist, inhibiting the binding of LTD<sup>4</sup> to the cysteinyl leukotriene receptor 1 [50], which could also impact cell signaling. However, the similar results obtained with the knockdowns would argue that the effects observed are due to ABCC1/ABCC4. Similarly, Reversan is also able to inhibit the related protein ABCB1, but given that Reversan affected both cell lines, and only the MCF-7 cells showed a significant expression of ABCB1 (Figure S1), it is unlikely this is the cause. That knockdown of ABCC1 alone did not affect the colony formation of MDA-MB-231 cells, and only one of the double knockdowns affected it, maybe because of the higher expression level of the transporters in MDA-MB-231 cells. The siRNA method only results in knockdown, not knockout, as seen in Figure 5. Although the protein levels are decreased in MDA-MB-231 cells, they were higher to start with, so it is possible that sufficient protein remains even after knockdown. Furthermore, siRNA knockdown is only transient, and it is not known if all cells were affected or just a proportion of them. We chose to use siRNA, because with complete knockdowns, the overexpression of other ABC transporters often occurs to compensate. In the future, perhaps the use of lentiviral shRNA could be investigated [42,43,46].

When investigating cellular migration, only MK571 had a significant effect, and only with the MDA-MB-231 cells, suggesting perhaps that the dual inhibition of both ABCC1 and ABCC4 was important. However, the siRNA knockdowns showed that the ABCC4 knockdown affected migration in both cell lines. Why the reportedly specific ABCC4 inhibitors, Ceefourin 1 and Ceefourin 2 [52], did not cause any effect on the cellular migration, when ABCC4 knockdown did, is not clear. The cellular invasion assays were more variable but, again, showed an effect of ABCC4 knockdown in MDA-MB-231 cells.

Our results suggest that ABCC1 is important for breast cancer proliferation, whilst ABCC4 has a greater role in cellular migration and invasion (Table 2). This contrasts with a study investigating the role of these transporters in neuroblastoma, where ABCC4 was more associated with proliferation and ABCC1 with migration [39]. Similarly, in pancreatic cancer, ABCC4 was associated with proliferation [47]. However, in epithelial ovarian cancer, ABCC4 was associated with proliferation, migration, and invasion [45]. In agreement with our results, breast cancer cells overexpressing ABCC1 showed an increase in proliferation, which could be inhibited by MK571, and overexpression of ABCC1 enhanced tumor growth in mice [59]. Additionally, mice implanted with breast cancer tumors with varying levels of ABCC4 expression showed no differences in tumor growth, but an increased ABCC4 expression was associated with increased metastases [56]. Therefore, the role(s) played by these transporters is not necessarily the same across different cancers, but within breast cancers, our results appear to agree with other studies in the literature. It should also be noted that, although the current study only investigated ABCC1 and ABCC4, they are not necessarily the only important transporters. In neuroblastoma, ABCC3 was shown to be very important, albeit as a positive prognostic indicator [39]. In contrast, ABCC3 was implicated in breast cancer stem-like features [20]. ABCC11 has also been implicated in aggressive breast cancer and prognosis [18].


**Table 2.** Summary of findings. ↓ significant decrease. — no significant effect. n/d not determined.

Understanding the mechanism by which these transporters mediate their effects is important. Both transporters are able to efflux a wide range of different molecules. It is important to know what molecule is being effluxed that causes the effects observed. It might be that targeting the ABC transporters themselves is not the best approach, as they have historically proven difficult to target, often because they have so many important roles. Instead, understanding the pathway they are involved with and targeting something earlier or later in the signaling pathway might be a more effective approach. Therefore, preliminary investigations into how ABCC1 and/or ABCC4 might elicit their effects on breast cancer cell proliferation and migration were undertaken. ABCC1 has previously been linked to GPR55 in a feedback loop, where ABCC1 exports LPI, which binds to and activates GPR55, leading to downstream signaling, and this was implicated as important for prostate and ovarian cancer cell proliferation [31]. However, we found little/no expression of GPR55 within MCF-7 cells. Given that ABCC1 inhibition or knockdown affected the proliferation and clonogenic capacity of MCF-7 cells, this would argue against LPI export and GPR55 activation being a key feature to explain our findings. The export of cAMP was a second method explored, as ABCC4 is known to be able to efflux cyclic nucleotides [32]. The knockdown of ABCC4 or inhibition with MK571 significantly decreased the amount of cAMP effluxed from MBA-MB231 cells. This agrees well with a previous study [57], and it has been suggested that increasing cellular cAMP levels is a potential method of combatting triple-negative breast cancer [57,60]. The export of PGE<sup>2</sup> has also been suggested as a

potential mechanism by which ABCC4 might be important in breast cancer [61]. In our study, the levels of PGE<sup>2</sup> exported by MDA-MB-231 cells were too high to quantify accurately. With MCF-7 cells, no effect of ABCC1/ABCC4 knockdown or inhibition was observed. Similarly, no effect of ABCC4 overexpression on the PGE<sup>2</sup> efflux from MCF-7 cells was observed previously [56]. However, the same report suggests that the knockdown of ABCC4 in MBA-MB-231 cells does decrease the PGE<sup>2</sup> efflux and that this is important for breast cancer metastasis [56]. Given that we observed the effects of ABCC4 knockdown on the migration of both MCF-7 and MDA-MB-231 cells, this might argue that it is not due simply to PGE<sup>2</sup> efflux; however, further investigation with MDA-MB-231 cells is needed. Finally, we investigated the efflux of S1P, as levels of S1P have previously been implicated in breast cancer [62], and both ABCC1 and ABCC4 are capable of transporting S1P [29,38]. Although the knockdown of ABCC1 or ABCC4 did not show a significant effect on extracellular levels of S1P, the inhibition of both ABCC1 and ABCC4 with MK571 did decrease the amount of S1P effluxed. This result agrees with a previous study that also observed a decreased S1P efflux from MCF-7 cells in the presence of MK571 [63]. The same study also showed that siRNA knockdown of ABCC1 decreased the amount of S1P exported from MCF-7 cells, but a large effect was only seen when the cells were stimulated with estradiol, which we did not do, and this might explain why we see no effects of ABCC1 or ABCC4 knockdown.
