Here we validate the role of BET proteins in the cell cycle and on protein expression in human cancer cells. Given the importance of BET proteins they could be good target candidates for treatment of breast cancer. Currently specific BET inhibitors, such as JQ1, are available; however, their effectiveness in the presence of the environmental tumor cytokines is unknown. In agreement with previous findings , we show that JQ1 reduces cell viability in both MDA-MB231 and MCF-7 breast cancer cell lines. However, further studies will be necessary to determine if JQ1 inhibits proliferation of breast cancer cells or enhances their apoptosis.
It should be noted that cytokines present in the tumor microenvironment not only affect cell fate, but can also alter drug efficacy. Dethlefsen et al. reported that intratumorally IL-6 has a major role in tumor cell growth and metastasis . As chronic inflammation is known to increase progression of breast cancer , it is key to survey the effects of IL-6 after prolonged (14 days) treatment with any therapeutic.
In the present study, we report that a triple-negative breast cancer cell line, MDA-MB-231, is more sensitive to JQ1, a well-known BET inhibitor, than estrogen receptor-positive MCF-7 cells (Fig. 1A, 1B, and 1C). Following 48 hours of JQ1 treatment at 20 µM or 72 hours at 16.5 µM, MDA-MB-231, cells exhibited a significant reduction in proliferation (∼50%). In addition, we demonstrated that stimulation of MDA-MB-231 cells with IL-6 for two weeks reduces the cell viability as compared to untreated cells. In contrast, JQ1 had no effect on the proliferation of untreated MCF-7 cells; however, pretreatment with IL-6 increased the sensitivity of these cells to JQ1 after 48 h. Therefore, in this study we demonstrate that IL-6 may influence the efficacy of JQ1 on the induction of apoptosis in breast cancer cell lines.
Recently, it has been demonstrated that IL-6 stimulation can increase tumor metastasis and the invasiveness of cancers [27, 28]. CXCR4 is the main agent involved in breast cancer metastasis [10, 29], and here we demonstrate that IL-6 increases the proportion of CXCR4-positive MCF-7 cells, but not of MDA-MB-231 cells. Conversely, analysis of CXCR4 surface expression revealed that the inhibition of the BET proteins by JQ1 also inhibited surface expression of the CXCR4, especially in triple-negative breast cancer cells. Furthermore, combinatorial treatment revealed that JQ1 antagonizes the effect of IL-6 on the expression of CXCR4, and in MCF-7 cells JQ1 inhibits the effects of IL-6 effect in a time dependent manner. These findings indicate that the role of BET proteins is more crucial in MDA-MB-231 cells than in MCF-7 cells. In addition, it seems that to IL-6 exerts a strong effect on MCF-7 cells than on MDA-MB-231 cells. Because, the expression of the IL-6R in MCF-7 is higher than MDA-MB-231 .While a previous study demonstrated that JQ1 inhibits the secretion of IL-6 , in the present study we demonstrate for the first time that JQ1 antagonizes the effects of IL-6 in a manner dependent on the cell line and length of exposure.
Many studies have demonstrated the role of cancer stem cells (CSCs) in drug resistance, metastasis, and cancer recurrence [32, 33]. Also, the BET inhibitors were introduced as an effective therapy for different types of cancers [16, 19, 23]. However, our investigation in regard to the effect of the BET inhibitor (JQ1) on the CSC population of two human breast cancer cell lines, MDA-MB-231 and MCF-7, yielded contrasting results. The MDA-MB-231 cell line was found to have the highest percentage of BCSCs (about 90 percent) and treatment with JQ1 did not change this population. However, treatment with JQ1 enriched the BCSC population in MCF-7 cells. In addition, IL-6 pre-treatment decreased the percentage of BCSCs, while combined treatment with IL-6 and JQ1 increased the percentage of BCSCs in both cell lines. In contrast, neither JQ1 nor IL-6 affected the expression of BCSC markers in triple-negative breast cancer cells. In addition, BCSCs are resistant to JQ1 therapy and JQ1 diminished the inhibitory effects of IL-6 on BCSC differentiation . Recently, the induction of phosphorylation of BRD4 by casein kinase II (CK2) was reported to enhance the stability of BRD4 in JQ1-resistant cells . Subsequently, another study revealed that the activator of enzymatic activity against PP2A phosphatase phenothiazine (PTZ) has an opposite function to that of CK2 and can dephosphorylate BRD4, which increases the sensitivity of the resistant cells to JQ1 . Therefore, a combination therapy using JQ1 and a suitable JQ1 sensitizer, such as PTZ, may synergistically increase the therapeutic effects. In addition, treatment of MCF-7 cells with JQ1 causes the expression of the SNAIL gene to increase significantly in a manner that correlates with the increase of the BCSC population in MCF-7 cells (R = 77%, p < 0.05). Therefore, this suggests that increased expression of SNAIL may be responsible for the conversion of non-BCSCs to BCSCs in MCF-7 cells. Another explanation for this occurrence could be survival of BCSCs after treatment with JQ1, since they are resistant to JQ1.
Furthermore, we also found a synergistic interaction between IL-6 and JQ1 in regard to the reduction of c-MYC and SLUG in the MCF-7 and MDA-MB-231 cells, respectively. These findings are consistent with previous results, which demonstrated that BET inhibitors decrease the expression of c-MYC in acute leukemia cells . Further research will be needed to decipher the precise mechanisms through which JQ1 functions for the treatment of breast cancer.