Downregulation of MAP3K1 attenuates cellular proliferation, migration, and invasion of HR-positive, HER2-negative breast cancer cell lines
As shown in Supplemental Fig. S1 (shMAP3K1-targeted mRNA sequences) and Supplemental Fig. S2 (shMAP3K1-targeted amino acid sequences), we assessed the efficiencies of shMAP3K1-mediated inhibition in both MCF7 and T-47D cells. For that, we assessed mRNA levels of MAP3K1 in control MCF7 and T-47D cells, and the cells transfected with shMAP3K1 using RT-qPCR. We found that mRNA expression levels of MAP3K1 were significantly downregulated in shMAP3K1-transfected MCF7 cells and shMAP3K1-transfected T-47D cells when compared with scrambled MCF7 cells and scrambled T-47D cells, respectively (Supplemental Fig. S3). To approve the specificity of MAP3K1, we used immunofluorescence, immunohistochemical analysis, and western blotting to detect MAP3K1 expression in control MCF7 and control T-47D cells (primary antibody alone, secondary antibody alone, and combination of primary and secondary antibodies), and in shMAP3K1-transfected MCF7 cells and in shMAP3K1-transfected T-47D cells (combination of primary and secondary antibodies). The results are illustrated in Supplemental Fig. S3.
These breast cancer cell lines expressing higher levels of MAP3K1 were transfected with shMAP3K1 to downregulate the MAP3K1 protein expression (Fig. 1a). When compared with the scrambled group, shMAP3K treatment significantly reduced the expression level of MAP3K1 by 84% in MCF7 cells (p < 0.001), and by 75% in T-47D cells (p < 0.001) (Fig. 1a). Proliferation assay showed that the downregulation of MAP3K1 decreased cell number in both breast cancer cell lines (Fig. 1b ). The cell number was significantly inhibited at Day 3, Day 5, and Day 7 of shMAP3K1-transfected MCF7 and T-47D cells. The results of the migration assay showed that the number of migrated cells was reduced by 48.1% in shMAP3K1-transfected MCF7 and by 82.3% in shMAP3K1-transfected T-47D cells when compared with scramble-transfected MCF7 and T-47D cells (Fig. 1c). The results of the invasion assay showed that the number of invaded cells was reduced by 73.7% in MCF7 cells and by 63.1% in T-47D cells when compared with scrambled MCF7 and T-47D cells (Fig. 1d). These findings indicated that inhibition of MAP3K1 suppressed cell number, cell migration, and cell invasion in these two HR-positive, HER2-negative breast cancer cell lines.
Downregulation of MAP3K1 induces G2/M phase arrest and apoptosis and enhances drug sensitivity in HR-positive, HER2-negative breast cancer cell lines
Hu et al. reported that transfection with MAP3K1 small interfering RNA leads to the downregulation of expression of CDC25C and cyclin B1 (key molecules for G2/M transition during the cell cycle) in MCF7 cells and MCF-12F cells (normal mammary epithelial cell line) [34]. In this study, we sought to assess whether shMAP3K1 could inhibit the cell number in both breast cancer cell lines by blocking programmed G2/M phase and downregulating cyclin B1.
After 48 h, we determined the distribution of the cell cycle phases in each cell line. As shown in Fig. 2a, in shMAP3K1-transfected MCF7 cells, there was a significant increase in the number of cells in the G2/M phase of cell cycle, and concomitantly, a significant decrease in the number of cells in the G0/G1 phase was observed. On the contrary, in T-47D cells, shMAP3K1 treatment led to an arrest of a significant number of cells in the G2/M phase of cell cycle, and there was a significant decrease in the number of cells in the S phase. These findings indicated that shMAP3K1 blocked cell cycle progression through the G2/M phase. Annexin V staining revealed that shMAP3K1 treatment resulted in increased apoptotic events (early and late apoptosis level) in both MCF7 cells (16.34% ± 0.77% vs. 1.84 % ± 0.42%, p = 0.000633) and T-47D cells (13.68% ± 0.31% vs. 4.87% ± 0.16%, p = 0.0000517) when compared with scrambled MCF7 and T-47D cells, respectively (Fig. 2b).
The shMAP3K1 resulted in the cell cycle arrest at the G2/M phase, which indicated that the downregulation of MAP3K1 might increase drug sensitivity. As shown in Fig. 2c, we found that inhibition of MAP3K1 promoted decrease in the cell viability in tamoxifen-treated MCF7 (at 0.01, 0.1, 1, 10, and 50 μM) and T-47D (at 0.01, 0.1, 1, 10, and 100 μM) cells when compared with cells transfected with scrambled shRNA. Furthermore, inhibition of MAP3K1 increased the cellular drug sensitivity for doxorubicin (at 0.01, 0.1, 1, and 2 μM) in MCF7 cells, for doxorubicin (at 0.01, 0.1, 1, 10, and 50 μM) in T47-D cells, and for docetaxel (at 0.01, 0.1, 1, 10, and 20 μM) in both MCF7 and T-47D cells (Fig. 2d and Fig. 2e).
Downregulation of MAP3K1 significantly reduces the expression of cyclin B1 and anti-apoptosis-related factors, and NF-κBactivity
We found that the expression of cyclin B1 was essentially reduced by shMAP3K1 transfection, while the expression of cyclin D1 (key molecule for G1 arrest during the cell cycle) was not affected (Fig. 3a). Considering that ERK1/2 is a downstream molecule of MAP3K1-signaling pathway [20], we assessed whether ERK1/ERK2 can be inhibited by MAP3K1 silencing. We found that shMAP3K1 transfection downregulated p-ERK expression by 83% in MCF7 cells and by 42% in T-47D cells when compared with the scrambled group (Fig. 3a).
Previous studies have revealed that the deletion of MAP3K1 results in apoptosis when mouse embryonic stem cells are subjected to hyperosmolarity and microtubule disruption or cardiomyocytes are subjected to oxidative stress [35, 36]. Zang et al. reported that MAP3K1 silencing inhibits cell proliferation and increases apoptosis of esophageal squamous cell carcinoma cells [37]. However, the role of MAP3K1 in regulating anti-apoptotic function in breast cancer cells remains unclear. The members of Bcl-2-related anti-apoptotic protein family, including Bcl-2 and Bcl-xL, play critical roles in the pathogenesis of ER-positive breast cancer cells [38-40]. Therefore, we assessed whether shMAP3K1 transfection led to the downregulation of expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in both MCF7 and T-47D cells. We found that shMAP3K1 transfection downregulated Bcl-2 expression in MCF7 cells, and Bcl-xL expression in both MCF7 and T-47D cells, whereas c-Myc expression remained unaltered (Fig. 3b). Transfection with shMAP3K1 also led to increased expression of cleaved poly (ADP-ribose) polymerase (PARP) and decreased matrix metalloproteinase (MMP)-9 expression (Fig. 3b). These results indicate that using shMAP3K1 to inhibit MAP3K1 can promote apoptosis and attenuate migration and invasion in these breast cancer cells.
NF-κB, acting as a downstream factor in the MAP3K1 signaling pathway, is involved in the pathogenesis of HR-positive breast cancer cells [41]. As shown in Fig. 3c, transfection with shMAP3K1 inhibited the expression of p-IκBα, an essential regulatory molecule of NF-κB, in both breast cancer cell lines. Also, transfection with shMAP3K1 downregulated the nuclear expression of NF-κB (p65) in both breast cancer cell lines. Furthermore, NF-κB-dependent expression of genes, such as nuclear p52 and BCL3, was downregulated in MCF7 and T-47D cells after transfection with shMAP3K1 (Fig. 3c). As indicated by the results from the NF-κB-Luc promoter activity assay, the transcription of NF-κB-dependent genes, induced by the nuclear translocation and DNA-binding activity of NF-κB, was decreased in both breast cancer cell lines after transfection with shMAP3K1 (Fig. 3d).
Taken together, our findings indicated that MAP3K1 might play a role in promoting cell proliferation, anti-apoptotic function, migration, invasion, and NF-κB transcriptional activity, and thus, contributes to malignant progression and poor prognosis of HR-positive, HER2-negative breast cancer patients.
Expression of MAP3K1 and p-ERK in tumor cells of patients with early-stage HR-positive, HER2-negativebreast cancer
To further validate the biological significance of involvement of MAP3K1 in proliferation, local recurrence, and metastases of HR-positive, HER2-negative breast cancer, we assessed the relationship between expression of MAP3K1 in tumor cells, and the DFS and OS of 161 patients with either T1 or T2 status and negative or 1 to 3 nodal metastases of HR-positive, HER2-negative breast cancer. As shown in Table 1, the median age was 49 years (range 23–81 years). The clinicopathological characteristics and treatments are listed in Table 1. All ER-positive and/or PR-positive patients received hormonal therapy; sixteen patients (9.9%) received ovarian ablation or a luteinizing hormone-releasing hormone agonist with or without tamoxifen, and 145 (90.1%) patients received tamoxifen. None of the patients received an aromatase inhibitor (which was not reimbursed by national health insurance at that time). One hundred and twenty-two patients (75.8%) were LN-negative, whereas 39 patients (24.2%) had 1 to 3 LN metastases. Seventy-four patients (46.0%) did not receive any chemotherapy, and 87 patients (54.0%) received standard adjuvant chemotherapy (Table 1). Furthermore, 121 patients (75.2 %) were positive for both ER and PR.
We detected MAP3K1 expression (51 cases, score 2; 13 cases, score 3) in tumor cells of 64 patients (39.8%), whereas 97 patients exhibited negative MAP3K1 expression (92 cases, score 0; five cases, score 1) (Fig. 4a-c). Table 1 displays the demographic characteristics of the two groups of patients (MAP3K1-positive vs. MAP3K1-negative) and their clinicopathological features. Age, tumor size, histological grade, axillary LN, ER and PR status, and adjuvant chemotherapy were not significantly different between the two groups.
Considering that ERK1/2 is a downstream molecule of MAP3K1-signaling pathway [20], we assessed whether the expression of p-ERK correlates with the expression of MAP3K1 in the same group of patients. In the selected 145 patients who had available tumor samples, we detected MAP3K1 expression (52 cases, score 2; 9 cases, score 3) in tumor cells of 61 patients (42.0%), whereas 84 patients exhibited negative MAP3K1 expression (73 cases, score 0; 11 cases, score 1) (Fig. 4d-f). The p-ERK expression was more frequently detected in MAP3K1-positive tumors (42/59 [71.2%]) than in MAP3K1-negative tumors (19/86 [22.1%]) of patients (p < 0.001, Fig. 4e and Table 1).
Expression of MAP3K1 or p-ERK is associated with poor clinical outcomes of patients with early-stage HR-positive, HER2-negative breast cancer
The median follow-up period for the patients was 8.97 years (95% confidence interval [CI]: 8.57 to 9.37); by the end of the follow-up period, 25 patients (15.5%) exhibited local recurrence and/or distant metastases, 12 patients (7.5%) had died (11 [91.7%] due to breast cancer and one [8.3%] due to causes not related to breast cancer), and 149 remained alive and healthy. The 9-year DFS and OS for all patients was 82.2% (95% CI: 75.3% to 89.1%) and 91.0% (95% CI: 85.9% to 96.1%), respectively.
The tumor stage (p = 0.838), tumor grade (p = 0.553), and axillary LN status (p = 0.763) were not associated with the 9-year DFS (Table 2). Similarly, the tumor stage (p = 0.551), tumor grade (p = 0.679), and axillary LN status (p = 0.943) were not associated with the 9-year OS (Table 2). Furthermore, we found that patients with tumor cells expressing MAP3K1 exhibited a poor 9-year DFS than those without MAP3K1 expression (72.6% [95% CI: 59.5% to 85.7%] vs. 88.5% [95% CI: 81.6% to 95.4%], p = 0.022) (Fig. 5a). Similarly, overexpression of MAP3K1 was significantly associated with poor 9-year OS (MAP3K1-positive group vs. MAP3K1-negative group, 83.8% [95% CI: 73.6% to 94.0%] vs. 96.2% [95% CI: 91.9% to 100%], p = 0.012) (Fig. 5b). Furthermore, p-ERK expression significantly correlated with a poor 9-year DFS (p-ERK-positive group vs. p-ERK-negative group; 74.6% [95% CI: 62.3% to 86.9%] vs. 87.3% [95% CI: 79.9% to 94.7%], p = 0.033) and a poor 9-year OS (86.4% [95% CI: 78.2% to 94.6%] vs. 95.9% [95% CI: 91.2% to 100%], p = 0.023) (Fig. 5c-d).
Using multivariate analyses (Table 2), we found that MAP3K1 expression was still an independent prognostic factor for DFS (hazard ratio = 2.476, 95% CI = 1.112 to 5.517, p = 0.026), whereas tumor stage (p = 0.769), histological grade (p = 0.554), and axillary LN (p = 0.582) were not associated with DFS. Similarly, MAP3K1 expression was still an independent prognostic factor for OS (hazard ratio = 4.489, 95% CI = 1.214 to 16.604, p = 0.024), whereas tumor stage (p = 0.629), histological grade (p = 0.755), and axillary LN status (p = 0.983) did not affect OS (Table 2).