Noxin promotes proliferation of breast cancer cells via P38-ATF2 signaling pathway

Abstract

Noxin (also called chromosome 11 open reading frame 82 or DNA damage-induced apoptosis suppressor) is associated with anti-apoptosis and cell proliferation in response to stress signals. However, to our knowledge, the role of Noxin in regulating cell proliferation is still controversial and there are no reports of the function and clinicopathological association in breast cancer. In this study, immunohistochemistry results showed that Noxin expression was significantly correlated with advanced tumor–node–metastasis stage (p = 0.027), positive regional lymph node metastasis (p = 0.002), and poor overall survival (p = 0.002). Proliferation assay results showed that Noxin obviously promoted the ability of proliferation of normal breast cells. Subsequent western blot results revealed that Cyclin D1 and Cyclin E1 were upregulated by overexpressing Noxin, whereas Cyclin D1 and Cyclin E1 were downregulated after depleting Noxin. The levels of phosphorylated P38 and activating transcription factor 2 were obviously increased after overexpressing Noxin, and their expression was downregulated accordingly by transfecting Noxin–small interfering RNA. Moreover, P38 inhibitor counteracted the elevating expression of phosphorylated activating transcription factor 2, Cyclin D1, and Cyclin E1 induced by Noxin overexpression and thereby reversed the effect of Noxin overexpression on facilitating cell growth. Taken together, our studies indicated that Noxin was overexpressed in breast cancer and its positive expression was significantly correlated with advance tumor–node–metastasis stage, positive lymph node metastasis, and poor prognosis. Noxin facilitated the expression of Cyclin D1 and Cyclin E1 through activating P38-activating transcription factor 2 signaling pathway, thus enhanced cell growth of breast cancer.

Introduction

Noxin, also called C11orf82 (chromosome 11 open reading frame 82) or Ddias (DNA damage-induced apoptosis suppressor), is an anti-apoptotic protein in response to a wide range of stress signals, such as γ- and ultraviolet (UV) irradiation, hydrogen peroxide, adriamycin, and cytokines.^1–4 The functional role of Noxin is still controversial to date. Nakaya et al. ¹ had confirmed that Noxin may inhibited cell cycle and directly participate in the repair process in mouse model. Other studies indicated that Noxin may enhance cell cycle in lung and liver carcinoma.^2,5 Zhang et al. ⁵ had proved that Noxin was highly expressed in hepatocellular carcinoma and significantly correlated with the clinicopathological factors. Thus, we speculated that the role of Noxin in regulating cell cycle and proliferation may be tissue specific. However, there are no literatures of Noxin expression in breast cancer.

The aim of this study is to investigate the clinicopathological association of Noxin expression and the impact of Noxin on cell cycle and proliferation in human breast cancer. In this study, we performed immunohistochemistry (IHC) to assess Noxin expression in breast cancer and to evaluate its clinicopathological association. By overexpressing or depressing of Noxin in breast cancer cell lines, we evaluated the effect of Noxin on the regulation of cell cycle and proliferation and investigated its downstream signaling pathway.

Materials and methods

Patients and clinical specimens

The study protocol was approved by the Institutional Review Board of China Medical University. All participants provided written informed consent, and the study was conducted according to the principles expressed in the Declaration of Helsinki. Primary tumor specimens were obtained from 140 patients with breast cancer, including 77 cases of triple-negative breast cancer (TNBC, deficient in the expression of estrogen receptor, progesterone receptor, and Her2/neu) and 63 cases of non-triple-negative breast cancer (NTNBC). In all, 53 cases of paired normal breast tissues were also obtained. All patients diagnosed with invasive ductal carcinoma (IDC) underwent complete surgical resection at the Affiliated Cancer Hospital of China Medical University between 2001 and 2003. Complete follow-up data were available for all 140 analyzed cases. Patient survival was defined as the time from the day of diagnosis to the end of the follow-up period or the day of death due to recurrence or metastasis. None of the patients had received radiotherapy or chemotherapy before undergoing surgical resection, and all patients were treated with routine chemotherapy after surgery. The median age was 51 years (range from 26 to 84 years). Of the 140 patients, 66 patients were equal to or older than 51 years. Lymph node metastases were present in 55 of the 140 cases. The tumors included 95 stages I–II cases and 45 stages III–IV cases.

Cell lines

MCF-10A, MCF-7, T-47D, BT-549, MDA-MB-231, and MDA-MB-468 cell lines were obtained from Shanghai Cell Bank (Shanghai, China). All cells were cultured in RPMI 1640 medium (Invitrogen, Waltham, MA, USA) containing 10% fetal calf serum (Invitrogen) and 100 IU/mL penicillin plus 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The cells were grown in sterile culture dishes at 37°C in a 5% CO₂ atmosphere and subcultured every 2 days, using 0.25% trypsin (Invitrogen) for cell detachment.

IHC

All tissue specimens were fixed in neutral formaldehyde, embedded in paraffin, and sectioned (thickness, 4 µm). The streptavidin–peroxidase immunohistochemical method was used. The tissue sections were incubated in 4°C overnight with Noxin rabbit polyclonal antibody (1:100, ab-199279; Abcam, Cambridge, UK); phosphate-buffered saline was used instead in the case of the blank control. The sections were then incubated with biotin-labeled secondary antibodies (Ultrasensitive; MaiXin, Fuzhou, China) at 37°C for 30 min and then with diaminobenzidine for coloration. The staining intensity was scored as 0 (no signal), 1 (weak), 2 (moderate), or 3 (high). The percentage of cells stained was scored as 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (76%–100%). The scores of each tumor sample were multiplied to yield a final score of 0–12. As most of the noncancerous tissues were scored lower than 4, the expression of Noxin was considered positive in tumors with final scores ≥4 and negative in those with final scores <4.

Western blotting analysis

Total protein was extracted using a lysis buffer (Pierce, Rockford, IL, USA) and quantified with the Bradford method. ⁶ A volume of 50 µg of the total protein samples was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4°C with the following primary antibodies: Noxin (1:100, ab-199279; Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000, Sigma, St. Louis, MO, USA), Myc-tag, Cyclin A2, Cyclin B1, Cyclin D1, Cyclin D2, Cyclin D3, Cyclin E1, Cyclin E2, Cyclin H, p-P38, P38, phosphorylated activating transcription factor 2 (p-ATF2), ATF2, phosphorylated extracellular signal-regulated kinase (p-ERK), ERK, p-AKT, and AKT (1:1000; Cell Signaling Technology, Danvers, MA, USA). Membranes were washed and subsequently incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology, CA, USA) at 37°C for 2 h. Bound proteins were visualized using electrochemiluminescence (Pierce) and detected with a bio-imaging system (DNR Bio-Imaging Systems, Jerusalem, Israel), each carried out in triplicate.

Plasmid transfection and small interfering RNA treatment

Plasmids pCMV6-ddk-myc and pCMV6-ddk-myc-Noxin were purchased from Origene (Rockville, MD, USA). Noxin–small interfering RNA (siRNA; sc-96450) and negative control (NC)-siRNA (sc-37007) were purchased from Santa Cruz Biotechnology. Transfection was carried out using the Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions, each carried out in triplicate.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Cells were plated in 96-well plates in medium containing 10% fetal bovine serum at about 3000 cells per well 24 h after transfection. For quantitation of cell viability, cultures were stained after 4 days using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 20 µL of 5 mg/mL MTT (thiazolyl blue) solution was added to each well and incubated for 4 h at 37°C, and then, the media was removed from each well, and the resultant MTT formazan was solubilized in 150 µL of dimethyl sulfoxide (DMSO). The results were quantitated spectrophotometrically using a test wavelength of 490 nm, each carried out in triplicate.

Colony formation assay

The MCF-7 and MDA-MB-468 cells were transfected with pCMV6 or pCMV6-Noxin plasmids, NC, or Noxin-siRNA for 48 h. Thereafter, cells were planted into 6-cm cell culture dishes (1000 per dish for MCF-7 and MDA-MB-468 cell lines) and incubated for 12 days. Plates were washed with phosphate-buffered saline and stained with Giemsa. The number of colonies with more than 50 cells was counted. The colonies were manually counted by microscope, each carried out in triplicate.

Statistical analysis

SPSS version 22.0 for windows (SPSS, Chicago, IL, USA) was used for all analyses. Pearson’s chi-squared test was used to assess possible correlations between Noxin and clinicopathological factors. Kaplan–Meier survival analyses were carried out in 140 cases of specimens and compared using the log-rank test. Mann–Whitney U test was used for the image analysis of MTT assay and colony formation assay results; p < 0.05 was considered to indicate statistically significant differences.

Results

Noxin expression in breast cancer specimens and cell lines

Initially, IHC was performed in 140 cases of breast cancer samples. We found that Noxin was negatively or dimly expressed in normal breast tissues (Figure 1(a)); however, Noxin was overexpressed (score ≥ 4) in the cytoplasm of breast cancer specimens (Figure 1(b) and (c)). The positive ratio of Noxin expression in normal breast tissues was 17.0% (9/53), which was obviously lower than that in breast cancer tissues (41.4%, 58/140; p < 0.001, Figure 1(d)). However, the positive ratio of Noxin expression in TNBC was 42.9% (33/77), which showed no obvious differences with the expression of Noxin in NTNBC (25/63, 39.7%, p > 0.05). Statistical analysis results showed that Noxin expression was significantly correlated with advanced tumor–node–metastasis (TNM) stage (p = 0.027) and positive lymph node metastasis (p = 0.002). There were no correlations between Noxin expression with age and TNBC (p > 0.05, Table 1). Kaplan–Meier analysis indicated that the Noxin-positive patients (135.34 ± 5.10 months) had a significantly shorter survival compared to Noxin-negative patients (148.78 ± 1.22 months, p = 0.002, Figure 1(e)). Subsequent western blot results indicated the expression of Noxin in two of five breast cancer cells was obviously higher than that in normal breast cell lines MCF-10A (Figure 1(f)).

Figure 1.

Noxin was highly expressed in breast cancer specimens and cell lines. (a) Immunohistochemistry analysis results showed that Noxin was negatively or weakly expressed in normal breast tissues; (b and c) however, Noxin was overexpressed in the cytoplasm of breast cancer specimens. (d) In the same samples, we can observe that Noxin presented higher cytosolic expression in breast cancer than that in normal breast tissues. (e) Kaplan–Meier analysis indicated that the survival of patients with overexpression of Noxin was significantly shorter than those with negative expression of Noxin. (f) The expression of Noxin was higher in two of five breast cancer cell lines than that in normal breast cell lines MCF-10A, each carried out in triplicate.

Table 1.

Correlation of Noxin overexpression with clinicopathological features in 140 cases of breast cancer.

Clinicopathological factors	N	positive	negative	χ 2	p
Age (years)
<51	74	33	41	0.648	0.493
≥51	66	25	41
TNM classification
I + II	95	33	62	5.454	0.027
III–IV	45	25	20
Lymph node metastasis
Positive	55	32	23	10.478	0.002
Negative	85	26	59
Triple negative (ER, PR, and Her-2)
Yes	77	33	44	0.144	0.733
No	63	25	38

TNM: tumor–node–metastasis; ER: estrogen receptor; PR: progesterone receptor; Her-2: human epidermal growth factor receptor 2.

Noxin promoted proliferation of breast cancer cells

Next, we overexpressed Noxin in breast cancer cells by Noxin–cDNA transfection in MCF-7 cells or depleted Noxin in MDA-MB-468 cells by RNAi (Figure 2(a)). MTT assays revealed that overexpression of Noxin enhanced tumor proliferation in MCF-7 cell lines, and knockdown of Noxin by RNAi depressed cell growth in MDA-MB-468 cells (Figure 2(b)). Similarly, overexpression of Noxin increased the formation of colony numbers of MCF-7 cells, and depletion of Noxin decreased the formation of colony of MDA-MB-468 cells (Figure 2(c)).

Figure 2.

Noxin promoted proliferation of breast cancer cells. (a) After overexpressing Noxin in MCF-7 or depleting Noxin in MDA-MB-468 cells, protein level of Myc-tag or Noxin was examined 72 h after transfection. (b) Results of MTT assay: the cell growth was increased after overexpressing Noxin in MCF-7 cells but decreased in MDA-MB-468 cells after depleting Noxin, each carried out in triplicate (bars represent SD; *p < 0.05 and **p < 0.001). (c) Representative images of colony formation assay after overexpressing Noxin in MCF-7 or depleting Noxin in MDA-MB-468, each carried out in triplicate (bars represent SD; *p < 0.05 and **p < 0.01).

Noxin increased the expression of Cyclin D1 and Cyclin E1

After transfecting pCMV6-Noxin in MCF-7 cells or Noxin-siRNA in MDA-MB-468 cells, we used western blot to investigate the effect of Noxin expression on the protein levels of multiple cyclins. The protein levels of Cyclin D1 and Cyclin E1 were obviously upregulated by overexpressing Noxin in MCF-7 cells. However, the protein levels of Cyclin D1 and Cyclin E1 were visibly downregulated by depleting Noxin in MDA-MB-468 cells. The protein levels of other cyclins showed no changes after modulating Noxin expression (Figure 3).

Figure 3.

Noxin increased expression of Cyclin D1 and Cyclin E1. Protein levels of Myc-tag, Noxin, Cyclin A2, Cyclin B1, Cyclin D1, Cyclin D2, Cyclin D3, Cyclin E1, Cyclin H, and GAPDH were examined by western blot after overexpressing Noxin in MCF-7 and depleting Noxin in MDA-MB-468, each carried out in triplicate.

Noxin activated P38 signaling pathway

As Noxin was proven to increase the expression of Cyclin D1 and Cyclin E1, we screened the key signaling pathway proteins that may be involved in regulation of cell cycle. The results indicated that phosphorylation of P38 and its downstream factors ATF2 was increased after overexpressing Noxin in MCF-7 cells. The levels of phosphorylation of P38 and ATF2 were decreased when we knockdown Noxin by siRNA in MDA-MB-468. Other signaling pathway proteins presented no obvious changes after overexpressing or depleting Noxin (Figure 4).

Figure 4.

Noxin activated P38 signaling pathway. Protein levels of Myc-tag, Noxin, p-P38, P38, p-ATF2, ATF2, p-ERK, ERK, p-AKT, AKT, and GAPDH were examined by western blot after overexpressing Noxin in MCF-7 and depleting Noxin in MDA-MB-468, each carried out in triplicate.

Noxin upregulated Cyclin D1 and Cyclin E1 via activating P38 signaling pathway

We added P38-specific inhibitor SB203580 (10 µM) into the medium after overexpressing Noxin in MCF-7 cells to validate whether upregulation of Cyclin D1 and Cyclin E1 induced by Noxin was attributed to activation of P38 signaling pathway. As expected, the increase in Cyclin D1 and Cyclin E1 expression followed by overexpression of Noxin was reversed and treated with P38 inhibitor, as well as the phosphorylation level of ATF2 (Figure 5(a)). The effect of Noxin overexpression on upregulation of cell growth was also diminished (Figure 5(b)).

Figure 5.

Noxin upregulated Cyclin D1 and Cyclin E1 expression via activating P38 signaling. SB203508, an inhibitor of P38, was added into the medium of MCF-7 cells with or without Noxin overexpression and DMSO as a negative control. (a) The upregulation of p-ATF2, Cyclin D1, and Cyclin E1 as well as the (b) cell growth induced by overexpressing Noxin was counteracted by adding SB203580 into the medium.

Discussion

In this study, we found that Noxin expression correlated with development of breast cancer patients and predicted poor prognosis. The effect of Noxin on enhancing Cyclin D1 and Cyclin E1 expression was due to activating P38-ATF2 signaling pathway. To our knowledge, there were no reports about the functional role of Noxin in breast cancer. Our results indicated that Noxin may also play a role in regulating cell cycle and proliferation in breast cancer.

We found that Noxin was overexpressed in the cytoplasm of breast cancer tissues. Positive expression of Noxin significantly correlated with advanced TNM stage, positive lymph node metastasis, and poor prognosis. Our results are consistent with the previous studies that Noxin was localized to the periphery of the nucleus and translocated to the nucleus when cells were exposed to the stress. ¹ The anti-apoptotic role and cell-cycle arrest role of Noxin had been extensively addressed in previous studies. However, although using different kind of cells, most of the previous studies investigated the function of Noxin under the induction of stress.^1–4 Only one study evaluated the expression of Noxin without treating with any stresses in human hepatocellular carcinoma and reached similar results with ours that Noxin expression significantly associated with cancer progression. ⁵ These collective data implied that Noxin expression may be involved in cancer progression even under the circumstances without any stresses.

Nakaya et al. ¹ demonstrated that induction of Noxin in response to stress was dependent on P53; however, Noxin did not require P53 to induce cell-cycle arrest. In this study, we found that Noxin enhanced cell growth and colony formation ability in breast cancer cell lines and overexpression of Noxin upregulated the protein levels of Cyclin D1 and Cyclin E1. As we all know that Cyclin D1 and Cyclin E1 are positive regulator of cell cycle, our results suggest a possibility that Noxin may interact with the cell-cycle machinery.^7–10 To clarify the underlying mechanisms, we screened the key proteins involved in regulating Cyclin D1 and Cyclin E1. The results advised us that overexpressing Noxin may enhance the phosphorylation of P38 and its downstream factor ATF2. Adding P38-specific inhibitor after transfecting Noxin reversed the increasing levels of Cyclin D1 and Cyclin E1 as well as cell proliferation. Our data are consistent with the previous studies that the P38-ATF2 signaling pathway participated in promoting proliferation through transcriptional activation of key cell-cycle regulators.^11–14 However, a previous study suggested that Noxin was a negative regulator of P38 signaling pathway in lung cancer cells. ⁸ Either p38 or ATF2 play dual roles in different tumors, and we speculated that the differences between our results and the previous one may attribute to a number of factors, including different type of transfected cells, condition of transfection, different type of plasmid, degree of ectopically introduced gene, and other reasons.^15,16

Overall, our studies revealed that Noxin was overexpressed in breast cancer and its positive expression was significantly correlated with advance TNM stage, positive lymph node metastasis, and poor prognosis. Noxin facilitated the expression of Cyclin D1 and Cyclin E1 through activating P38-ATF2 signaling pathway, thus enhanced breast cancer cell growth.