Overexpression of seven in absentia homolog 2 protein in human breast cancer tissues is associated with the promotion of tumor cell malignant behavior in in vitro

  • Authors:
    • Jie Sun
    • Xiaojuan Zhang
    • Yanchun Han
    • Juan Zhen
    • Yuan Meng
    • Min Song
  • View Affiliations

  • Published online on: July 25, 2016     https://doi.org/10.3892/or.2016.4976
  • Pages: 1301-1312
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Abstract

Seven in absentia homolog 2 (SIAH2), a homologue of Drosophila seven in absentia (Sina), has emerged as an oncogene and plays important roles in cancer development and progression. This study further assessed the role of SIAH2 in breast cancer and the underlying molecular events. The data showed that SIAH2 protein was overexpressed in invasive breast cancer (IBC) compared to the expression noted in normal or ductal carcinoma in situ (DCIS) tissues, expression of which is associated with malignant behaviors. SIAH2 may function differently in different molecular subtypes (e.g., luminal- vs. basal-like type) of breast cancer. Manipulation of SIAH2 expression led to a ‘cross-talk’ of the ERK and PI3K pathway, which could be one of the mechanisms by which SIAH2 regulates viability, apoptosis, and invasion capacity in these breast cancer cell lines.

Introduction

Breast cancer is a major health problem and the leading cause of cancer-related mortality in women. Clinically, breast cancer is a highly heterogeneous adenocarcinoma with various molecular and genetic alterations. For example, Perou et al (1), were the first to carry out a gene expression profile analysis of human breast tissue specimens using gene microarray technology and identified a number of differentially expressed genes associated with breast cancer development. Subsequent studies using cDNA microarray were able to classify breast cancer into four different subtypes: luminal-like, normal breast-like Her-2 overexpression, and basal-like subtype (2,3). Each subtype has unique gene expression characteristics, biological behavior, and clinical prognosis (4). For example, luminal-like breast cancer is characterized by a high level of ER expression and better prognosis, whereas the other three subtypes have negative ER expression. The basal-like breast cancer subtype has a unique phenotype, poorer prognosis, and insensitivity to endocrine and targeted therapy. Thus, research attention has focused on the basal-like subtype of breast cancer.

Basal-like breast cancer accounts for 8−20% of all invasive breast cancers or ~25% of advanced and difficult-to-treat breast cancer (5). Clinically, basal-like breast cancer occurs at a relatively young age and often expresses basal-cell cytokeratin subtypes (CK5/6, CK14, and CK17), but does not express hormone receptors (ER or PR) or Her-2. Eighty-five percent of tumor tissues have mutated p53 protein and 60% of cases express epidermal growth factor receptor (EGFR) (6,7), indicating that this subtype of breast cancer has a highly proliferative and invasive clinical behavior with poor survival rate. At the gene level, multiple gene signaling pathways are involved in growth and metastasis of breast cancer, such as the mitogen-activated protein kinase (MEK)-extracellular regulated protein kinase (ERK) pathway and phosphatidylinositol 3-kinase (PI3K)-serine/threonine protein kinase (AKT) pathway (8). The MEK-ERK pathway is closely related to tumor cell proliferation and resistance to apoptosis and, thus, contributes to tumor development and progression, while the PI3K/AKT pathway is one of the main anti-apoptotic mechanisms utilized by tumor cells to create a survival advantage (9,10).

The SIAH family of proteins are homologues of the Drosophila SIAH protein, and humans have two highly conserved SIAHs: SIAH1 and SIAH2 (11). SIAH2, localized at chromosome 3q25, contains two exons and a 9.8-kb intron and encodes a 324-amino-acid protein (12) that is only expressed in proliferating cells. Schmidt et al (13) showed that the SIAH2 protein is expressed in pancreatic, breast, lung, and cervical cancers and the level of SIAH2 protein is associated with increased tumor malignancy. Thus, SIAH2 is considered to be an oncogene. Recent studies revealed that SIAH2-regulated cell proliferation and apoptosis occur through activation of the ERK pathway (13). Inhibition of SIAH2 activity led to upregulation of Spry2 expression, one of its substrates, and subsequently a decrease in ERK activity (14,15) and inhibition of tumor growth and metastasis. For example, Ahmed et al (15) found that inhibition of SIAH2 expression using SIAH2 shRNA downregulated the level of p-ERK and, thus, inhibited tumor growth and proliferation and promoted apoptosis. Schmidt et al (13) found that inhibition of SIAH2 expression using RNAi inhibited Ras-mediated malignant transformation of fibroblasts and xenograft growth of pancreatic tumor cells in nude mice, and that knockdown of SIAH2 expression in K-Ras-transformed pancreatic tumor cells led to downregulation of the p-ERK level. Thus, in this study, it was hypothesized that SIAH2 or its substrates may be part of the feedback loop of the Ras/Raf/MEK/ERK pathway in breast cancer cells. Therefore, immunohistochemistry, western blotting, and gene knockdown studies were performed to detect SIAH2 expression in basal cell-like and non-basal cell-like breast cancer tissue samples. The effects of SIAH2 knockdown on the regulation of the biological behavior of breast cancer cells and the underlying mechanism were then investigated.

Materials and methods

Patients and specimens

In this study, a total of 200 cases of breast tissues were retrospectively collected between 2000 and 2009 from the Central Hospital, Shenyang Medical College (Shenyang, China). The study was in compliance with the published Helsinki Declaration and was approved by the Medical Ethics Committee of the Chinese Medical University. Informed consent was obtained prior to surgery from all enrolled patients. The formalin-fixed and paraffin-embedded surgical tissue blocks were retrieved, sectioned, and stained routinely with hematoxylin and eosin (H&E) and then reviewed by two senior pathologists to carry out a histological diagnosis according to the World Health Organization breast carcinoma histological classification criteria (2012). In addition, 30 cases out of these 155 invasive breast cancer tissues had paired tumor and normal tissues (at least 5 cm away from the primary tumor lesion) and were flash-frozen in liquid nitrogen and stored in a −80°C freezer until protein extraction.

Immunohistochemistry

Four-micron-thick sections were prepared for immunostaining with a streptavidin-peroxidase (S-P) method (Ultrasensitive; Maixin, Fuzhou, China) according to the manufacturer's instructions. The primary antibody used was a monoclonal mouse anti-SIAH2 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The colorimetric reaction was performed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Maixin). For the negative control, the primary antibody was replaced with a non-immunized normal goat serum. The stained tissue sections were reviewed and scored by two pathologists independently; brown particles appearing in the nucleus were regarded as positive staining of the cell. The intensity of immunostaining was scored as 1, weak; 2, moderate; and 3, intense and the percentage of positive cells as 0%, negative; 1−50%, 1; 51−75%, 2; ≥76%, 3. Scoring was determined from at least five high power fields (x400 magnification). The scores of each sample were multiplied to reach a final score of 0, 1, 2, 3, 4, 6, or 9, and the tissues were determined as negative if the final score was <4 and positive if the score was ≥4.

Cell lines and culture

Human breast cancer MDA-MB-231 and MCF-7 cell lines and normal human mammary epithelial cell line MCF-10A were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). MDA-MB-231 cells were maintained in Leibovitz L-15 medium with 10% fetal bovine serum (FBS), and MCF-7 cells in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, while MCF-10A cells were maintained in DMEM with 5% FBS, 10 µg/ml insulin, and 20 ng/ml EGF in a humidified incubator with 5% CO2 at 37°C.

According to previous studies of gene expression profiling and gene copy number (1,16−20), MDA-MB-231 cells belong to the basal-like subtype of breast cancer cells, while MCF-7 cells represent luminal breast cancer cells.

Construction of vectors, transient gene transfection, and treatment with ERK and PI3K inhibitors

The SIAH2 expression vector pcDNA3.1-flag-SIAH2 and pcDNA3.1-flag empty vector were kindly provided by Dr Ze'ev Ronai of The Burnham Institute (La Jolla, CA, USA). SIAH2 shRNA and the negative control shRNA were purchased from Santa Cruz Biotechnology. For transient gene transfections, cells were grown and transfected with pcDNA3.1-flag-SIAH2, pcDNA3.1-flag, SIAH2 shRNA, or control shRNA using Attractene or HiperFect (both from Qiagen) following the manufacturer's instructions. Afterwards, the cells were subjected to western blotting and the transfected cells were also subjected to treatment with ERK (PD98059) or PI3K (LY294002) (both from Sigma, St. Louis, MO, USA) inhibitor at 10 µmol/l for 24 h.

Protein extraction and western blotting

Total cellular protein was extracted using lysis buffer and the supernatants were collected by centrifugation of the cell lysis at 15,000 rpm for 30 min at 4°C. The protein lysis was boiled for 5 min and 60 µg from each protein sample each was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto PVDF membranes. After blocking with 5% skim milk solution in PBS, the membranes were incubated with a primary antibody against SIAH2 (at a dilution of 1:200), p-ERK (1:400), p-AKT (1:400), or β-actin (1:200) overnight at 4°C. Afterwards, membranes were incubated with each corresponding secondary antibody at room temperature for 2 h. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit and then quantified using EC3 imaging system (UVP, LLC). The gray scale values of each protein band were normalized to the values of the corresponding β-actin band. The experiments were repeated once independently.

Cell viability MTT assay

The transfected cells were seeded in 96-well plates at 1×104 cells/well and the cell viability was evaluated using the MTT assay. The absorbance, which was directly proportional to the number of living cells in culture, was measured at 490 nm using a microplate reader. A blank with dimethyl sulfoxide alone was measured and subtracted from all values. The experiments were performed in triplicate and repeated at least three times independently.

Tumor cell Matrigel invasion assay

Cell invasive ability was assessed using a 24-well Transwell with 8-µm pore polycarbonate membrane inserts. Matrigel (100 µg/ml at dilution of 1:7 with cell culture medium) was applied to the upper surface of the membranes. After transfection for 24 h, cells were seeded in the upper chamber at a density of 5×104 cells/well and the bottom of the chamber was filled with cell culture medium with 20% FBS. Next the plates were incubated for 24 h. At the end of the experiments, cells that had invaded into the surface of the membrane were fixed with methanol and stained with hematoxylin. Five high-magnification microscope fields were randomly selected per filter to count the number of invaded cells.

Flow cytometric apoptosis assay

Annexin V/FITC double staining was carried out as described by the manufacturer (Invitrogen, Carlsbad, CA, USA). Cells were immediately analyzed by flow cytometry and quantified using CellQuest software (both from BD Biosciences) for the percentage of apoptotic cells in each experimental group.

Statistical analysis

All statistical calculations were performed by SPSS version 13.0 for Windows software (SPSS, Chicago, IL, USA). The χ2 test and one-way analysis of variance (ANOVA) were performed to assess statistically significant differences between groups of tissues or cell treatments. A P<0.05 was considered statistically significant.

Results

Characteristics of the patients

The 200 breast cancer cases included 10 normal breast tissues (NBTs), 35 ductal carcinoma in situ (DCIS) tissues, and 155 invasive breast carcinoma (IBC) tissues. Among the 155 IBCs, 74% (115/155) were invasive ductal carcinomas, 12% (18/155) were invasive lobular carcinomas, 10% (15/155) were medullary carcinomas, and 4% (7/155) were of other histological types. Clinicopathological data and expression of p53 and Ki67 proteins were also collected. In brief, the median age of the patients was 50 years old (ranging between 28 and 84 years) and the median tumor size was 25 mm (ranging between 6 and 90 mm) (Table I). Moreover, 28 patients were identified as basal-like breast cancers by immunohistochemistry on the basis of Nielsen's (21) method. Specifically, the basal-like breast cancers often express high molecular weight basal cytokeratin (CK5/6, CK14, and CK17) and/or EGFR, while they do not express hormone receptors [estrogen receptor (ER) and progesterone receptor (PR)] or Her-2/neu. However, according to Nielsen's criteria, the 155 breast cancer cases were categorized into five intrinsic subgroups: the basal-like group (ER, HER-2, CK5/6+ and/or EGFR+), the luminal A group (ER+ and HER-2), the luminal B group (ER+ and Her-2+), the Her-2 group (ER and Her-2+) and the negative (null) group (ER, Her-2, CK5/6 and EGFR).

Table I

Association of SIAH2 expression with clinicopathological factors in 155 invasive breast carcinomas.

Table I

Association of SIAH2 expression with clinicopathological factors in 155 invasive breast carcinomas.

Clinicopathological factorsNSIAH2 expression
P-value
PositiveNegative
Age (years)0.883
 <50562729
 ≥50995148
Tumor size (mm)0.693
 <25874542
 ≥25683335
Nuclear grade0.000
 I17314
 II602139
 III785424
Tumor stage0.572
 I401822
 II814437
 III341618
Lymph node metastasis0.284
 No904248
 Yes653629
Genotyping0.000
 Basal-like28253
 Luminal A49940
 Luminal B1055
 Her-222157
 Null462422
Differential expression of SIAH2 protein in breast cancer tissues and cell lines

SIAH2 protein was expressed in the nuclei of occasional cells within the luminal layer of ducts and acini in normal mammary glands in 4 out of the 10 cases (40%). The level of SIAH2 expression was usually of mild to moderate intensity and, if used as a cut-off point for the tumor tissues, SIAH2 expression was considered negative in normal tissues (Fig. 1A). SIAH2 expression was positive and of moderate to strong intensity with a homogeneous distribution in the nuclei of 5 out of 35 (14.29%) DCIS cases (Fig. 1B and C). In contrast, the SIAH2 protein was expressed in 50.32% (78/155) of invasive breast carcinoma tissues with the majority of a strong intensity (Fig. 1C and D). SIAH2 expression was significantly upregulated in IBCs compared to normal or DCIS tissues (P=0.000; Table II and Fig. 1E), whereas there was no significance between normal and DCIS tissues (P=0.102). Our western blot data also confirmed upregulation of SIAH2 expression in IBCs compared to the paired distant normal tissues (t=6.558, P=0.000; Fig. 2A).

Table II

Differential expression of the SIAH2 protein in breast tissues (n=200).

Table II

Differential expression of the SIAH2 protein in breast tissues (n=200).

DiagnosisNMean of IS staining scoreP-valueSIAH2 expression
P-value
Low (IS<4) n/total (%)High (IS≥4) n/total (%)
NBT101.10±1.450.00010/10 (100)0/10 (0)0.000
DCIS352.03±1.5230/35 (85.71)5/35 (14.29)
IBC1554.01±1.6077/155 (49.68)78/155 (50.32)
Association of SIAH2 expression with clinicopathological data and p53 and Ki67 expression

Next, the association of SIAH2 expression with clinicopathological data and p53 and Ki67 expression was investigated in the 155 IBC cases. The data showed that SIAH2 expression was associated with a higher tumor nuclear grade and molecular classification of breast cancer subtypes. Moreover, SIAH2 expression was associated with p53 and Ki67 expression and inversely associated with lymph node metastasis in basal-like IBC. In the luminal A and null subtype of IBC, SIAH2 expression was associated with Ki67 expression (Tables I, III and V). However, there was no association found between SIAH2 expression and other clinicopathological data.

Table III

Association of SIAH2 expression with p53 level in each subtype of breast cancer.

Table III

Association of SIAH2 expression with p53 level in each subtype of breast cancer.

SIAH2 positiveSIAH2 negativeSpearman's correlation (rs)P-value
Basal-like
 p53+2000.548
 p53530.017
Luminal A
 p53+250.108
 p537350.598
Luminal B
 p53+210.218
 p53341.00
Her-2
 p53+620.111
 p53950.671
Null
 p53+1090.008
 p5314131.00

Table V

Association of SIAH2 expression with tumor lymph node metastasis in each subtype of breast cancer.

Table V

Association of SIAH2 expression with tumor lymph node metastasis in each subtype of breast cancer.

SIAH2 positiveSIAH2 negativeSpearman's correlation (rs)P-value
Basal-like
 Lymph node metastasis positive63−0.503
 Lymph node metastasis negative1900.026
Luminal A
 Lymph node metastasis positive314−0.014
 Lymph node metastasis negative6261.00
Luminal B
 Lymph node metastasis positive320.200
 Lymph node metastasis negative231.00
Her-2
 Lymph node metastasis positive1240.239
 Lymph node metastasis negative330.334
Null
 Lymph node metastasis positive1260.233
 Lymph node metastasis negative12160.14
Change in breast cancer cell malignant behaviors after knockdown of SIAH2 expression

According to the genotyping and gene expression analysis by Neve et al (18), MCF-7 breast cancer cells were chosen as a luminal breast cancer cell line and MDA-MB-231 cells as a basal-like type of breast cancer in the current study. A MCF-10A normal mammary epithelial cell line was used as a control. It was found that the SIAH2 protein was highly expressed in the MDA-MB-231 and MCF-7 cells, whereas the MCF-10A cell line had a very low expression level of SIAH2 protein (P<0.05; Fig. 2B). MDA-MB-231 cells expressed a much higher level of SIAH2 protein than MCF-7 cells (P<0.05, Fig. 2B).

Next, the effects of SIAH2 expression and knockdown on the regulation of breast cancer cell proliferation and invasion capacity was investigated by transient transfection of pcDNA3.1-flag-SIAH2 and SIAH2 siRNA, respectively, into breast cancer cell lines. Western blotting data showed SIAH2 expression or knockdown in breast cancer cell lines (Fig. 3A). A tumor cell invasion assay showed that knockdown of SIAH2 expression in the MDA-MB-231 cells with high SIAH2 expression resulted in induction of tumor cell invasion capacity, whereas restoration of SIAH2 expression in the MCF-7 cells reduced tumor cell invasion (Fig. 3B).

Furthermore, knockdown of SIAH2 expression reduced the viability of breast cancer MDA-MB-231 cells, whereas overexpression of SIAH2 protein also reduced cell viability in the MCF-7 luminal-like breast cancer cell line (Fig. 3C).

Additionally, the underlying mechanism for the different roles of the SIAH2 protein in the various breast cancer cells was explored. Our data showed that the MDA-MB-231 cells had a higher level of p-ERK, EGFR and HIF-1α expression, but a lower level of p-AKT than the MCF-7 cells (Fig. 4).

'Cross-talk' between the ERK and PI3K pathways in breast cancer cell lines

To investigate the effect of SIAH2 on regulation of p-ERK, p-AKT and HIF-1α expression, SIAH2 shRNA or pcDNA3.1-flag-SIAH2 was transfected into the breast cancer cell lines. Overexpression of SIAH2 in the MDA-MB-231 cell line had no effect on p-ERK, p-AKT or HIF-1α expression, whereas knockdown of SIAH2 expression was able to reduce the level of p-ERK, but induced the levels of p-AKT and HIF-1α. In contrast, overexpression of SIAH2 protein in the MCF-7 cell line increased the p-ERK level, but decreased the p-AKT and HIF-1α levels, whereas knockdown of SIAH2 expression slightly decreased p-ERK expression, but had no effects on the p-AKT level (Fig. 5A).

To determine the relationship between the ERK and PI3K pathways in the breast cancer cells, the cells were transfected with SIAH2 shRNA and then treated with p-ERK and/or PI3K inhibitor (PD98059 and/or LY294002, respectively). The results showed that, in the MDA-MB-231 basal-like breast cancer cell line, inhibition of SIAH2 expression or blocking the activity of the ERK pathway caused negative feedback activation of the PI3K pathway (Fig. 5B). Similarly, in the MCF-7 luminal breast cancer cell line, blockage of the PI3K pathway caused negative feedback activation of the ERK pathway (Fig. 5B).

Effects of SIAH2 on regulation of breast cancer cell invasion capacity through feedback activation/inhibition of the PI3K pathway

Next, the effects of SIAH2 on regulation of breast cancer cell invasion capacity and the underlying molecular events were assessed. An invasion assay showed that the invasion capacity of the MDA-MB-231 cells was increased after knockdown of SIAH2 expression, whereas the invasion capacity of the MCF-7 cells was decreased after transfection with SIAH2 cDNA. We speculate that these results can be partially explained by the feedback activation of the PI3K pathway and increased expression of HIF-1α after SIAH2 silencing, or blockage of the PI3K pathway and a decrease in HIF-1α expression due to overexpression of SIAH2.

To prove whether the increase in tumor cell invasion ability after knockdown of SIAH2 expression was caused by feedback activation of the PI3K pathway in the MDA-MB-231 cell line, the cells were treated with SIAH2 siRNA and PI3K inhibitor (LY294002) simultaneously. The data showed that the induced tumor cell invasion capacity after SIAH2 knock down was reversed by treatment with the PI3K inhibitor in the MDA-MB-231 cells (Fig. 6A). In contrast, treatment with LY294002 alone had no effect on MDA-MB-231 cell invasion capacity (Fig. 6B).

Similarly, in the MCF-7 cells, SIAH2 overexpression and treatment with the ERK inhibitor (PD98059) simultaneously recovered p-AKT expression and activity of the PI3K pathway activity (Fig. 6A); thus, tumor cell invasion capacity was also recovered (Fig. 6B). These data indicate that the effects of SIAH2 on the regulation of breast cancer cell invasion capacity occurred through feedback activation/inhibition of the PI3K pathway.

Effect of SIAH2 shRNA and ERK and PI3K inhibitors on regulation of cell viability

Next, the effect of SIAH2 on the regulation of cell viability in the MDA-MB-231 and MCF-7 cells was assessed. MTT data showed that knockdown of SIAH2 expression suppressed MDA-MB-231 cell viability, whereas overexpression of SIAH2 protein did not affect cell viability. In contrast, knockdown of SIAH2 expression did not significantly affect MCF-7 cell viability, but overexpression of SIAH2 protein decreased MCF-7 cell viability. Furthermore, MDA-MB-231 cell viability was decreased after treatment with the p-ERK inhibitor (PD98059) and addition of SIAH2 siRNA, while addition of the PI3K inhibitor (LY294002) had no effect on MDA-MB-231 cell viability. Upon combined treatment with these two inhibitors (PD98059 and LY294002), MDA-MB-231 cell viability was significantly suppressed (Fig. 7). This result could be due to feedback activation of the PI3K pathway after knockdown of SIAH2 expression or inhibition of the ERK pathway by PD98059 treatment. The PI3K pathway could mediate the biological effects on inhibition of apoptosis, which partly counteracted the effects on inhibition of cell proliferation caused by SIAH2 knockdown or PD98059 treatment. Addition of these two inhibitors (PD98059 and LY294002) together terminated the decreased apoptosis by activation of the PI3K pathway. Therefore, MDA-MB-231 cell viability was slightly reduced after addition of SIAH2 siRNA (0.93±0.017) or PD98059 (1.10±0.018 in LY294002-treated cells vs. PD98059-only-treated cells (0.9487±0.025) and control cells (1.133±0.021). Cell viability was significantly reduced after addition of these two inhibitors (PD98059 and LY294002) together (1.10±0.018 in LY294002-treated cells or 0.14±0.014 in LY294002- and PD98059-treated cells vs. 1.133±0.025 in control cells). However, in the MCF-7 cells, addition of PD98059 had no significant effect on cell viability, but addition of LY294002 significantly decreased cell viability. The combination of these two inhibitors (PD98059 and LY294002) caused the same degree of cell proliferation reduction as LY294002 alone (Fig. 7), indicating that there was no effect on cell viability after activation of the ERK pathway plus treatment with LY294002 in the MCF-7 cells. All in all, these results suggest that MDA-MB-231 cells are sensitive to the ERK inhibitor, whereas MCF-7 cells are sensitive to the PI3K inhibitor.

To prove whether the slight decrease in cell viability after knockdown of SIAH2 expression was caused by feedback activation of the PI3K pathway in the MDA-MB-231 cells, the cells were transfected with SIAH2 siRNA and then treated with the PI3K inhibitor (LY294002). It was found that cell viability was reduced significantly (Fig. 7). Similarly, the MCF-7 cells were transfected with SIAH2 cDNA and then treated with the ERK inhibitor (PD98059); it was found that with the recovery of p-AKT expression, cell viability was also recovered (Fig. 7).

Similar data were obtained in SIAH2 knockdown as well as treatment with the ERK and PI3K inhibitors on regulation of tumor cell apoptosis between MDA-MB-231 and MCF-7 cells (Fig. 8).

Discussion

In the present study, the expression of the SIAH2 protein in normal, premalignant, and malignant breast tissues was analyzed for association with clinicopathological data from IBC patients. The effects of SIAH2 expression or knockdown on the regulation of breast cancer cell biology behavior and the underlying molecular events were then explored. It was found that expression of the SIAH2 protein was upregulated in IBCs compared to normal or DCIS tissues. SIAH2 expression was associated with a higher tumor nuclear grade and molecular classification of breast cancer subtypes. The in vitro data showed that manipulation of SIAH2 expression (overexpression or knockdown) led to 'cross-talk' of the ERK and PI3K pathways in different subtypes of breast cancer cell lines (luminal- vs. basal-like), which could be one of the mechanisms by which SIAH2 regulates viability, apoptosis, and invasion capacity in breast cancer cells.

SIAH2 protein is a ring finger E3 ubiquitin ligase involved in hypoxia response and regulation of the Ras/Raf and p38/JNK/NF-κB signaling pathway. SIAH2 substrates include DCC (22), OBF-1 (23,24), TRAF2 (25), PHD1/3 (26,27), Vav1 (28), and Spry2 (29). SIAH2 substrates can interact with the SIAH2 protein in an AXVXP motif-dependent or independent way to affect multiple aspects of cell functions, such as cell survival, mitochondrial biogenesis, and hypoxia and stress response. Thus, overexpression of the SIAH2 protein may lead to breast cancer development, which is consistent with our current data. Furthermore, our current study also demonstrated that SIAH2 expression was associated with tumor nuclear grade and molecular classification of breast cancer subtypes. Higher expression of SIAH2 protein led to higher IBC nuclear grade and contributed to basal-like breast cancer. A previous study also showed that SIAH2 was expressed in pancreatic, breast, lung, and cervical cancers and different tumor cell lines and that the level of SIAH2 expression increased with tumor cell malignant behavior (13).

Furthermore, our current data also showed that SIAH2 expression or knockdown led to 'cross-talk' of the ERK and PI3K pathways, which may be one of the mechanisms by which SIAH2 regulates viability, apoptosis, and invasion capacity in breast cancer cells. Indeed, alteration of multiple signaling pathways has been shown to occur in breast cancer, including the commonly observed MAPK and PI3K pathways (30). Previous studies revealed that SIAH2-regulated cell proliferation and inhibition of apoptosis occurred through activation of the ERK pathway (13,16,17). SIAH2 was also closely related to HIF-1α; an increase in SIAH2 activity under a hypoxic condition is able to upregulate HIF-1α level and, in turn, promote expression of its downstream target genes, including VEGF, c-Met, CXCR4 and lysine oxidase, for tumor growth and metastasis (31).

Moreover, our current study showed differential effects of SIAH2 expression or knockdown on the regulation of cell viability, apoptosis, and invasion capacity of luminal-like and basal-like breast cancer cells (MDA-MB-231 and MCF-7 cells, respectively). Furthermore, it was also found that overexpression of SIAH2 in the MDA-MB-231 cell line had no effect on p-ERK, p-AKT or HIF-1α expression, whereas knockdown of SIAH2 expression was able to reduce the level of p-ERK, but induce p-AKT and HIF-1α levels. In contrast, overexpression of the SIAH2 protein in the MCF-7 cell line increased the p-ERK level, but decreased the p-AKT and HIF-1α levels, whereas knockdown of SIAH2 expression slightly decreased p-ERK expression, but had no effects on the p-AKT level. These data suggest that the role of SIAH2 in different breast cancer cell lines may be based on differential gene expression profiles in the given cell lines. In addition, our ex vivo data showed that SIAH2 may function differently in different molecular subtypes (e.g., luminal- vs. basal-like type of breast cancer) of breast cancer.

In addition, our current study assessed that the effect of SIAH2 on breast cancer cell proliferation occurring through activation of the ERK and PI3K pathways by transfecting breast cancer cells with SIAH2 shRNA and then treating them with the ERK and/or PI3K inhibitors (PD98059 and/or LY294002, respectively). Our results showed that SIAH2 regulated the MCF-7 luminal-like breast cancer cell line growth mainly through apoptosis in a PI3K-dependent manner, whereas suppression of the MDA-MB-231 basal-like tumor cell line growth was mainly regulated by the ERK pathway with little or no involvement of apoptosis. Indeed, ERK is a member of the MAPK family and plays a crucial role in the regulation of cell growth and division. In the ERK pathway, Ras acts as an upstream activation protein to form the Ras-Raf-MEK-ERK pathway. Spry2, an inhibitor of the Ras/Raf signaling pathway, is a SIAH2 substrate and plays an important role in cancer development. A previous study showed that changes in Spry2 expression are associated with downregulation of Ras signaling and that knockdown of SIAH2 expression upregulated Spry2 and weakened Ras signaling (14). Furthermore, PI3K/AKT is an important pathway in mediating the survival signal in cells and inhibition of AKT may trigger feedback that activates other signaling pathways to maintain cell survival. It has been confirmed that after inhibition of AKT signaling, expression of the tyrosine kinase receptor is upregulated and mTORC2 is activated in a feedback manner to activate MAPK activity, while inhibition of mTOR downstream of AKT by rapamycin or temsirolimus can also activate the PI3K/AKT pathway in a feedback manner. Our current study showed cross-reaction or a competitive relationship between the ERK and PI3K pathways and SIAH2 regulation of breast cancer invasion mainly depended on activation of the PI3K pathway. Thus, an unsatisfactory efficacy of a single MEK or AKT inhibitor on breast cancer may be associated with feedback activation, which may be the major obstacle in the development of anticancer drugs.

However, our current study is a proof-of-principle and additional research needs to be performed to confirm our results. Future studies are required to study additional cell lines to investigate SIAH2 in classifying breast cancer molecularly and developing the protein as a biomarker for early or differential diagnosis of breast cancer.

Abbreviations:

SIAH

seven in absentia homolog

PKB

protein kinase B

PDK

phosphatidylinositol-dependent kinase

Ras

rat sarcoma

Raf

rapidly accelerated fibrosarcoma

MAPK

mitogen-activated protein kinase

ERK

extracellular signal-regulated kinases

MEK

MAPK/ERK kinase

PI3K

phosphatidylinositol-3-kinase

HIF-1α

hypoxia-inducible factor 1α subunit

VEGF

vascular endothelial growth factor

K-Ras

Kirsten rat sarcoma

HIF

hypoxia-inducible factor

VHL

von Hippel-Lindau

IGF

insulin-like growth factor

EGFR

epidermal growth factor receptor

NF-κB

nuclear factor-κB

Spry2

Sprouty2

mTOR

mammalian target of rapamycin

ER

estrogen receptor

PR

progesterone receptor

Her-2

human epidermal growth factor-2

RNAi

ribonucleic acid interference

siRNA

small interference ribonucleic acid

shRNA

short hairpin ribonucleic acid

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium

TBS

Tris-buffered saline

H&E

hematoxylin and eosin

PBS

phosphate-buffered saline

S-P

streptavidin-peroxidase method

DAB

3,3′-diaminobenzidine

PAGE

polyacrylamide gel electrophoresis

SDS

sodium dodecyl sulfate

ECL

enhanced chemiluminescence

PI

propidium iodide

BSA

bovine serum albumin

FCM

flow cytometry

SPSS

Statistical Package for Social Science

NBT

normal breast tissues

DCIS

ductal carcinoma in situ

IBC

invasive breast carcinoma

Acknowledgments

We thank Dr Ze'ev Ronai of The Burnham Institute (La Jolla, CA, USA) for the SIAH2 expression vector (pcDNA3.1-flag-SIAH2).

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September-2016
Volume 36 Issue 3

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Spandidos Publications style
Sun J, Zhang X, Han Y, Zhen J, Meng Y and Song M: Overexpression of seven in absentia homolog 2 protein in human breast cancer tissues is associated with the promotion of tumor cell malignant behavior in in vitro. Oncol Rep 36: 1301-1312, 2016
APA
Sun, J., Zhang, X., Han, Y., Zhen, J., Meng, Y., & Song, M. (2016). Overexpression of seven in absentia homolog 2 protein in human breast cancer tissues is associated with the promotion of tumor cell malignant behavior in in vitro. Oncology Reports, 36, 1301-1312. https://doi.org/10.3892/or.2016.4976
MLA
Sun, J., Zhang, X., Han, Y., Zhen, J., Meng, Y., Song, M."Overexpression of seven in absentia homolog 2 protein in human breast cancer tissues is associated with the promotion of tumor cell malignant behavior in in vitro". Oncology Reports 36.3 (2016): 1301-1312.
Chicago
Sun, J., Zhang, X., Han, Y., Zhen, J., Meng, Y., Song, M."Overexpression of seven in absentia homolog 2 protein in human breast cancer tissues is associated with the promotion of tumor cell malignant behavior in in vitro". Oncology Reports 36, no. 3 (2016): 1301-1312. https://doi.org/10.3892/or.2016.4976