**1. Introduction**

Breast cancer is the most common cancer in women, affecting more than two million women worldwide per year [1]. The development of new targeted therapeutics and the encouragement of women to carry out early screening programs have significantly improved survival rates in the Western world [2–5]. Breast cancers that express the estrogen receptor and/or the progesterone receptor can be treated with receptor-blocking hormone therapy or with aromatase inhibitors to decrease the levels of estrogen produced [6,7]. Cancers that have high expression of human epidermal growth factor receptor 2 (HER2) can be treated with monoclonal antibodies, which bind to the receptor and block it [7]. However, some breast cancers, termed triple-negative, do not express any of these receptors, and thus, the only treatment available is conventional chemotherapy. Triple-negative breast cancers make up around 10–15% of all breast cancer cases [8], are typically aggressive, and show high levels of metastases and mortality [9,10]. The development of metastasis and/or cancers becoming resistant to therapeutic agents is a growing problem. Women diagnosed at an early stage of breast cancer may have recurrent disease, and at least a quarter of all cases may develop a resistance to therapeutic treatments [2]. Moreover, with the increase in disease progression, the incidence of therapeutic resistance becomes more alarming.

One common cause of therapeutic resistance in breast cancer is the efflux of drugs by membrane proteins of the ATP-binding cassette (ABC) transporter family of proteins [11,12]. ABCC1 (multidrug resistance protein 1/MRP1) and ABCC4 (multidrug resistance protein 4/MRP4) are two members of the C subfamily of ABC transporters that are capable of effluxing several different chemotherapeutic drugs out of cancer cells [13–15]. Previous studies have shown that ABCC1 expression is a negative prognostic marker, associated with a decreased survival rate in breast cancer patients [16–18] and an increased risk of relapse [19]. Following chemotherapeutic treatment, the expression of ABCC1 in breast cancer tumors was found to increase [20], and its expression level was shown to be highest in the most aggressive subtypes of breast cancers [18]. ABCC4 expression is similarly upregulated in chemotherapy-treated breast tumors compared to noncancerous tissue [21], and ABCC4 polymorphisms are linked with the response to aromatase inhibitors for estrogen-receptor-positive breast cancer [22].

However, it has been proposed that ABCC1 and ABCC4 may play a role in the hallmarks of cancer development and progression, independent of their drug efflux capabilities [23]. This is because ABCC1 and ABCC4 are capable of transporting numerous physiological substrates and have roles in metabolism and inflammation [13,24,25]. For example, ABCC1 can transport glutathione and inflammatory mediators such as leukotrienes and prostaglandins, as well as the bioactive lipids sphingosine-1-phosphate (S1P) and lysophosphatidyl inositol (LPI), which are implicated in cell proliferation, migration, and invasion [26–31]. ABCC4 can efflux the cyclic nucleotides cAMP and cGMP involved in cellular signaling, as well as leukotrienes, prostaglandins, and thromboxanes and S1P [32–38]. Studies on neuroblastoma (a rare childhood cancer) have confirmed that both ABCC1 and ABCC4 play an important physiological role in its development, independent of their role in multidrug resistance, affecting cellular proliferation, migration, and differentiation [39]. ABCC4 has also been implicated in cancer cell proliferation in leukemia [40,41], gastric cancer [42], lung cancer [43], renal cancer [44], ovarian cancer [45], and pancreatic cancer [46,47]. However, less is known about whether ABCC1 and ABCC4 have a role in breast cancer development and/or progression.

In this study, the role of ABCC1 and ABCC4 in breast cancer progression was investigated. The breast cancer cell lines MDA-MB-231 and MCF-7 were used. Both are considered to be aggressive, but MCF-7 is a luminal-type breast cancer with the presence of progesterone, estrogen, and human epidermal growth factor 2 (HER2) receptors, whereas MDA-MB-231 is a basal-type triple-negative cell line. The effect of small molecules inhibitors or siRNA knockdown of ABCC1 and ABCC4 on cellular proliferation, clonogenic capacity, cell migration, and invasion were investigated.
