Selumetinib suppresses cell proliferation, migration and trigger apoptosis, G1 arrest in triple-negative breast cancer cells

Selumetinib inhibited the proliferation and migration of TNBC cell.

Breast cancer is one of the most common cancer deaths in female. Estrogen receptor(ER)-negative breast cancer constitutes approximately 30 % of breast cancer cases. Triple-negative is defined as a subgroup with ER, PR(progesterone receptor) and human epidermal growth factor receptor 2 (HER2) all negative. TNBC are assumed importance for its molecular characters, aggressive progress and distinct tranfer ability [1, 2]. Beneficial results of current anti-HER2 or hormonal therapy could not improve the curative effect of chemotherapy. In the absence of proper treatments, TNBC often progresses to metastatic lesions in the brain and lung in three years. Once being with metastasis, the 5-year survival rate of TNBC would be less than 30 %. Newly therapies are urgently needed to improve the prognosis for TNBC patients. Actually, TNBCs exhibit a high level of molecular heterogeneity without high-frequency driver mutations. About 60–70 % of TNBCs has mutations of p53. For PIK3CA mutations, it would be 11 %. No other mutations were believed as highly prevalent driver in TNBCs, which hampered the development of targeted therapy for TNBCs. So far, some new regimes such as anti-androgens, anti-mitotic, PI3KCA pathway inhibitors and so on, had been tested in TNBC. Here, we focus on the key survival pathway, mitogen-activated and extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), which modulated by epidermal growth factor receptor(EGFR) [3, 4]. Over-expression of the EGFR is one of the key pathway regulating the proliferation and survive of cells. Hence, these genes may be good choose as therapeutic targets for TNBC [5]. Infante performed a phase Ib clinical study to determine the safety, tolerability, clinical activity and steady-state pharmacokinetics of trametinib, an oral MEK inhibitor, in combination with gemcitabine on breast cancer [6]. The results showed that trametinib combined with gemcitabine is safe and effective. Selumetinib, the benzimidazole ARRY-142886, has been reported to be highly potent MEK inhibitor, with an IC₅₀ of 12 nmol/L against purified MEK [7]. There were many phase I and phase II clinical studies about Selumetinib on melanoma, colorectal cancer (CRC), non-small-cell lung cancer (NSCLC) and others, for its favorable toxicity [8‐10]. Selumetinib also produces clinically meaningful increases in iodine uptake and retention in a subgroup of patients with radioiodine-refractory thyroid cancer [11]. We considered that Selumetinib might be potential drug for preventing TNBC metastasis and recurrence in a preclinical setting.

In the current study, we first found that Selumetinib inhibited proliferation and migration in two triple-negative breast cancer cell lines. Then we investigated its probable mechanism of action. MicroRNAs (miRNAs) [12] , 20–22 bp non-coding RNA, had been the hot are of cancer research for its post-transcriptional regulation function, which invovled a wide variety of biological processes, such as proliferation, differentiation, apoptosis, cell cycle and so on [13]. So far many miRs had been reported took part in MEK/ERK signaling pathway, including miR-768-3p [14], miR-221 [15], miR-199a/b-3p [16], and so on. Here, we treated TNBC cell lines HCC1937 and MDA-MB-231 with Selumetinib, and our founding indicated miR-302a/CUL1 maybe one significant downstream factors. The miR-302-367 cluster is over-expressed in embryonic stem and some kinds of carcinoma cells [17]. It works as anti-oncogene in many kinds of tumor cells, its overexpression could be of therapeutic value [18]. Kaid show that miR-302a regulated cell proliferation and self-renewal of esophageal cancer stem-like cells [19]. Here we found after treated with Selumetinib miR-302a, an well known ‘bona fide’ tumor suppressor, up-regulated markedly. It is difficult and costly to verified the gene regulatory networks. The bioinformatic methods offered the convenient to predicte possible diagram between miRNAs and their targets. The most publiced programs are TargetScan, miRanda, Tarbase, miRecords, RNAhybrid, and so on [20‐23]. Here we found CUL-1 is the directly target of miR-302a. CUL-1 is a essential components of the p19(SKP1)/p45(SKP2)/CUL-1 complex, named SCF, as the scaffold element [24]. Previous researches suggest that SCF as ubiquitin ligase is the key factor to cell cycle and survival. Aberrant expression of CUL-1 is critical for tumorigenesis, such as lung cancer [25], gastric cancer [26]. In the present study, we found CUL-1 also demonstrate an oncogenic activity of the OS.

Aberrant activation of RAS/Raf/MEK/ERK signaling pathways had been reported in many kinds of cancer and been considered as targeted for its oncogenic effect. Not only pre-clinically but also many phase I or II clinical trial had been displayed the obvious therapeutic effects in solid tumor [27‐29]. Some research reported that BRAF mutation was closely related to the sensitivity of Selumetinib [30]. Chen found that Selumetinib selectively rescued primary glial progenitors from TMX toxicity, such as cognitive dysfunction and changes in CNS metabolism, hippocampal volume, and brain structure, in vitro while enhancing TMX effects on MCF7 [31]. MEK pathway also plays key role in TNBC. Here, we found MAP/ERK kinase (MEK) 1/2 inhibitor, Selumetinib, repress the viability and induced apoptosis of HCC1937 and MDA-MB-231 in a dose-dependent manner. The G1 arrest and mobility declined were also linked to dose of Selumetinib (Fig. 1a to d). Then we goes deeply into the mechanism. We screened miRNA profile of MDA-MB-231 with or without Selumetinib (Fig. 2a). miR-302a was significantly and gradually up-regulated miRNAs with the concentration of Selumetinib in both MDA-MB-231 and HCC-1937 (Fig. 2b). We focus on microRNA (miR)-302 family for its tumor supperessor function in many kinds of tumor [32, 33]. Yan reported miR-302 was important to miRNA-induced pluripotent stem cells (mirPS) of endometrial cancer cell lines, take part in the inhibition of cell proliferation and tumorigenicity [34]. Previous papers revealed that miR-302a regulated the expression of AKT1 [35], NR2F2 [36], CDK2 [37] and so on targeted genes. By targetscan, we predicted there were two complementary sequences in the miR-302a and 3′UTR-CUL1 (Fig. 2c). Luciferase reporter assay showed miR-302a negative regulation of CUL1 directly (Fig. 2d). According to Fig. 2e and f, it was confirmed that CUL1 level was closed related with Selumetinib concertration. CUL1 is a key component of SCF ubiquitin ligases [38]. SCF promotes the ubiquitination and degradation of a broad range of proteins involved in cell cycle progression, signal transduction and transcription. As a key member of SCF, CUL1 is over-expressed in many kinds of cancer [39‐41] and represent as target molecular for therapy [42‐44]. In this paper, we found Selumetinib could inhibit both proliferation and migration in TNBC cells, and miR-302a/CUL maybe the key factor in this process. So we assume that it is equal to Selumetinib that we knocked down the CUL1 in TNBC cells. We also choose tow substrate, TIMP1 and TRAF2 of CUL1 to clarify this hypothesis. As expected, after silencing the CUL1, the viability and migration ability of TNBC cell were reduced markedly. For further prove miR-302a/CUL-1 is the operator nodes of Selumetinib onTNBCs. We regulated the miR-302 or CUL-1 level using miR-302a-AMO or CUL1 over-expression plasmid respectively in HCC1937 and MDA-MB-231 cells. Figure 4 indicated the effect of Selumetinib reversed accompany with raising of CUL-1 and silencing of miR-302a.

MEK pathway has been shown over-activated in TNBC. Based on our results, MEK1/2 inhibitor, Selumetinib, reduced viability through inducing apoptosis and G1 arrest, meanwhile the inhibition of mobility by Selumetinib was also be found in TNBCs. In these processes, we indicated miR-302a/CUL1 work as critical pathway in Selumetinib on TNBC.