Midline2 is overexpressed and a prognostic indicator in human breast cancer and promotes breast cancer cell proliferation in vitro and in vivo

Abstract

Midline2 (MID2) is an ubiquitin-conjugating E2 enzyme linked to tumor progression and a novel interacting partner of breast cancer 1, early-onset (BRCA1). However, the role of MID2 in breast cancer remains unknown. This study investigated the expression, prognostic value, and role of MID2 in breast cancer. The expression of MID2 mRNA and protein was significantly upregulated in breast cancer tissue and established cell lines compared with that in normal breast epithelial cells and paired adjacent non-tumor tissue (P<0.001). Immunohistochemical analysis demonstrated that MID2 was overexpressed in 272 of 284 (95.8%) paraffin-embedded, archived breast cancer tissue. Moreover, MID2 expression increased with advanced clinical stage (P<0.001). High MID2 expression was significantly associated with advanced clinical stages and T, N, and M staging (all P<0.05). Univariate and multivariate analyses indicated that high MID2 expression was an independent prognostic factor for poor overall survival in the entire cohort (93.73 vs. 172.1 months; P<0.001, log-rank test) and in subgroups with stages Tis+ I+ II and III+ IV. Furthermore, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colony formation, and anchorage-independent growth ability assays were conducted. Results showed that siRNA silencing of MID2 expression significantly reduced MCF-7 and MDA-MB-231 cell proliferation in vitro and blocked the growth of MDA-MB-231 cell xenograft tumors in vivo (P<0.05). This study indicated that MID2 may be a novel prognostic marker and interventional target in breast cancer.

Keywords

breast cancer / MID2 / proliferation / overall survival / xenograft

Introduction

At least one-fifth of deaths worldwide are related to cancer [ 1]. Cancer is predicted to remain the leading cause of mortality over the next 20 years [ 2, 3]. Individualized treatment approaches are needed for cancer, even among patients with the same tumor type; treatment strategies are formed based on the ability to precisely categorize and identify patients who may benefit from specific treatments. Given the advances in research on genes and molecules involved in cancer development and progression, researchers reveal that increased number of cell signaling pathway components can be used as a novel treatment target or potential biomarker for individualized therapy. Cancer treatment guidelines recommend routine assessment of several genes, including epidermal growth factor receptor, Kirsten rat sarcoma viral oncogene homolog, and erb-b2 receptor tyrosine kinase 2, for clinical diagnosis of cancer patients [ 4– 6]. Furthermore, various tumor-specific genes are screened in numerous tumor types, including lung, breast, and gastric cancers [ 7– 9]. However, additional biomarkers and therapeutic targets must be identified to achieve early diagnosis and improve treatment outcomes in breast cancer patients.

Dysregulated gene and protein expression is a major mechanism leading to tumorigenesis [ 10, 11]. Alterations in translation, processing, and degradation of specific proteins are related to cancer progression [ 12– 14]. Ubiquitin-mediated protein degradation is the main pathway for protein degradation. Ubiquitylation is a common form of post-translational modification and is mediated by ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin protein ligases (E3) [ 15]. Covalent attachment of ubiquitin to protein substrates marks the protein for degradation and affects transcriptional regulation and cell signaling [ 16]. Numerous genes involved in ubiquitination are associated with tumor progression; these genes include STIP1 homology and U-box containing protein 1, ubiquitin specific peptidase 26, and phosphatase and tensin homolog [ 17– 19].

Tripartite motif (TRIM) proteins contain a RING finger motif, one or two B-box motifs, and a coiled-coil region; these proteins comprise the largest subfamily of ubiquitin E3 ligases and ubiquitin-conjugating E2 enzymes [ 20]. Previous studies demonstrated the function of E3 for several TRIM family members, such as TRIM23, TRIM11, TRIM18, TRIM21, TRIM25, and TRIM32 [ 21– 26]. A functional genomics approach based on systematic data collection revealed that the TRIM proteins share common functions, that is, these proteins can identify specific cell compartments via homo-multimerization [ 27]. Research also revealed the biological roles of the TRIM family proteins in cellular proliferation, invasion, and chemosensitivity to drugs [ 28, 29].

MID2 (also known as TRIM1) can restrict N-tropic murine leukemia virus infection [ 30]. MID2 is a candidate gene for Opitz-Kaveggia syndrome and Opitz G/BBB syndrome [ 31]. MID2 also participates in the activated NF-kB/AP-1 signaling pathway, thereby suggesting that this protein may play an important role in tumor progression [ 32]. Furthermore, MID2 is a novel protein-interacting partner for breast cancer 1, early-onset (BRCA1), a nuclear phosphoprotein that functions as a major tumor suppressor in breast cancer [ 33]. Therefore, MID2 may regulate protein degradation and play an important role in breast cancer. However, the expression profile and biological roles of MID2 in breast cancer remain poorly characterized.

In this study, we demonstrated that MID2 mRNA and protein expression was upregulated in breast cancer cells and human breast cancer tissue. High-level MID2 expression was significantly associated with the progression of human breast cancer. MID2 was also identified as an independent prognostic factor for overall survival in patients with breast cancer. Furthermore, silencing MID2 by using specific RNAi inhibited the proliferation of breast cancer cells in vitro and xenograft tumor growth in vivo. These findings suggest that MID2 plays an important role in the proliferation and tumorigenicity of human breast cancer cells and may be a valuable therapeutic target for breast cancer.

Materials and methods

Cell lines and real-time PCR

Primary normal breast epithelial cells (NBECs) were collected from the mammoplasty material of a 30-year-old woman at the Department of Plastic Surgery, First Affiliated Hospital of Sun Yat-sen University (P. R. China). The cells were cultured in the keratinocyte serum-free medium (Invitrogen) supplemented with epithelial growth factor, bovine pituitary extract, and antibiotics (120 μg/ml streptomycin and 120 μg/ml penicillin). Breast cancer cell lines (MCF-7, MDA-MB-453, ZR-75-30, T47D, and MDA-MB-231) were cultured as previously described [ 34].

Total RNA was extracted from cultured cells by using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The extracted RNA was reversely transcribed and subjected to real-time PCR by using a previously described method [ 34] with the following primers: MID2 forward 5′-TTTGCCTCATGCATAGCAGT-3′ and MID2 reverse 5′-TTAAGCGCAACAGCGAACTA-3′. Expression data were normalized to the geometric mean of the housekeeping gene GAPDH to control variability in expression levels (forward: 5′-ACCACAGTCCATGCCATCAC-3′ and reverse: 5′-TCCACCACCCTGTTGCTGTA-3′) and calculated as 2^{−[(C_Tof MID2 or p21 or p27) – (C_Tof GAPDH)]}, where C_T represents the threshold cycle for each transcript.

Western blot analysis

Western blot analysis was performed in accordance with previously described standard methods [ 35] by using anti-MID2 antibody and anti-Ki67 antibody (dilution 1: 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were then stripped and re-probed with an anti-β-actin mouse monoclonal antibody (Sigma, Saint Louis, MI, USA) as loading control. Each sample was detected and analyzed three times.

SiRNA silencing of MID2

Retroviral transduction technology was applied to permanently deplete MID2, and two human siRNA sequences were cloned into pSuper-retro-puro to generate pSuper-retro-MID2-RNAi. The siRNA sequences were as follows: RNAi#1 5′-GAAGGCCAAACACGTGGCCACTATA-3′ and RNAi#2 5′-CCAAAGACGTGGCAATGCTACTGCA-3′ (synthesized by Invitrogen). Recombinant retrovirus production and infection were performed as described previously [ 34].

Immunohistochemistry

A total of 284 paraffin-embedded breast cancer samples stored in our laboratory were analyzed. Clinicopathological classification and staging were determined according to the criteria established by the American Joint Committee on Cancer [ 36]. The clinicopathological features of the patients are summarized in Table 1. Two normal breast tissue specimens were also collected through breast plastic surgery and stored in our laboratory [ 34]. Immunohistochemistry (IHC) procedure and scoring of MID2 expression were performed as previously described [ 37]. Briefly, staining intensity was scored on a scale of 0 to 3 (where 3 indicates strong brown staining, 2 indicates moderate yellowish brown staining, 1 indicates weak light yellow staining, and 0 indicates no staining). Tumors with a staining intensity of≥2 or with at least 50% MID2-positive malignant cells were classified as having high expression. Meanwhile, tumors with a staining intensity of<2 or with at least 50% MID-2 positive malignant cells were classified as having low expression. All the procedures were adopted in accordance with the ethical standards of the concerned committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975 as revised in 2000. Informed consent was obtained from all the patients prior to inclusion in the study.

MTT assay

Cells at an initial density of 0.2 × 10⁴ cells/well were seeded in 96-well plates in triplicate as previously described [ 38]. MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide; Sigma) was used to treat cells with a designated concentration of 0.5 mg/ml for 4 h at 37 °C for various durations. Briefly, 150 μl of dimethyl sulfoxide (Sigma) was employed to treat cells after the medium was removed. Absorbance was then determined.

Anchorage-independent growth assay

Five hundred cells were trypsinized and suspended in 2 ml of complete medium containing 0.3% agar (Sigma). The agar-cell mixture was placed on top of a layer of solidified 1% complete medium/agar. The number of viable colonies that contained more than 50 cells or larger than 0.5 mm was counted after culture for 10 d. The experiment was performed independently three times for each cell line.

Colony formation assay

Cells were plated in 60 mm plates (0.5×10³ cells per plate), cultured for 10 d, fixed with 10% formaldehyde for 5 min, and stained with 1% crystal violet for 30 s. The number of colonies was counted.

Xenograft tumor model

Non-obese diabetic/server combined immuno-deficiency (NOD/SCID) mice (4–5 weeks old, 18–20 g) were purchased from Hunan SJA Laboratory Animal Co. Ltd. (Changsha, Hunan, China). The Institutional Animal Care and Use Committee of Sun Yat-sen University approved all experimental procedures. Each mouse was injected on the mammary pads with MDA-MB-231-vector cells (5 ×10⁶) on the left side and with MDA-MB-231-MID2-RNAi1 cells (5×10⁶) on the right side. Tumors were examined every 5 d. Tumor length (L) and width (W) were measured using calipers, and tumor volumes were calculated using the equation (L ×W²)/2. The animals were euthanized on day 35, and tumors were excised and weighed. All institutional and national guidelines for the care and use of laboratory animals were followed.

Statistical analysis

All statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA) [ 34]. Associations between MID2 expression and patients’ clinicopathological characteristics were analyzed using Chi-square test. Bivariate correlations among study variables were calculated using the Spearman’s rank correlation coefficient. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Survival data were evaluated using the univariate and multivariate Cox regression analyses [ 39]. A two-tailed P-value of less than 0.05 was considered statistically significant in all tests.

Results

MID2 is upregulated in breast cancer cell lines

Western blot and real-time PCR analysis revealed that MID2 protein and mRNA expression was almost undetected in cultured NBECs (NBEC1 and NBEC2) isolated from two patients (Fig. 1A and 1B). However, MID2 protein expression was upregulated in all five breast cancer cell lines tested compared with that in NBECs. Real-time PCR analysis confirmed that MID2 mRNA expression was significantly upregulated (by at least 5.2-fold) in breast cancer cell lines compared with that in NBEC1 cells (Fig. 1B).

MID2 is upregulated in human breast cancer

To examine MID2 expression in clinical breast cancer specimens, we determined MID2 expression levels in six paired breast tumor tissue (T) and matched adjacent non-tumor breast tissue (ANT). Western blot and real-time PCR analysis revealed that MID2 protein and mRNA expression was upregulated in human primary breast tumor tissue compared with that in adjacent non-tumor breast tissue (Fig. 2A and 2B). MID2 protein expression was also analyzed in the same specimens by IHC. MID2 was overexpressed in breast cancer specimens but was undetected or underexpressed in adjacent non-tumor breast tissue. These results strongly indicate that MID2 is upregulated in human breast cancer.

To confirm whether MID2 is upregulated in breast cancer, we examined MID-2 expression through IHC of 284 paraffin-embedded, archived human breast cancer tissue specimens. These specimens included the following: 23 cases of non-invasive carcinoma in situ (Tis), 25 cases of clinical stage I, 126 cases of clinical stage II, 79 cases of clinical stage III, and 31 cases of clinical stage IV. These tissue sections were stained using MID2 antibodies and subsequently scored. The summarized staining scores are shown in Table 1. MID2 expression was observed in the tumor cells of 272/284 (95.8%) specimens (Fig. 3A). MID2 was expressed at relatively low levels in the early-stage tumors (stages Tis, I, and II) and at higher levels in advanced stage tumors (stages III and IV). Quantitative analysis revealed that MID2 expression was significantly higher in breast tumors than that in normal breast tissue (Fig. 3B). These results indicate that MID2 overexpression is a common feature of breast cancer.

Upregulation of MID2 is associated with advanced clinicopathological features and poor overall survival in breast cancer

We then analyzed the association between MID2 and the clinicopathological features of breast cancer. As shown in Table 1, MID2 expression was strongly associated with clinical stage (P<0.001), as well as T (P<0.001), N (P<0.001), and M (P = 0.032) classifications. By contrast, MID2 expression was not correlated with ER (P = 0.34), PR (P = 0.268), and ErbB-2 (P = 0.562) status.

The correlation of clinicopathological features and MID2 expression with the overall survival of 284 patients with breast cancer was analyzed using univariate survival analysis (log-rank test). As shown in Table 2, high MID2 expression was associated with poor overall survival (P<0.001). The mean overall survival time for patients whose tumors expressed high levels of MID2 was only 93.73 months, whereas the overall survival time for those with low MID2 expression was 172.1 months. Similarly, the disease-free survival time was shorter for patients with high MID2 levels than those with low MID2 levels. The cumulative overall survival rate for the group with low MID2 expression was significantly higher than that with high MID2 expression (P<0.001).

The association between MID2 and overall survival in subgroups of patients with different stages of breast cancer was further examined. Patients with tumors exhibiting high MID2 expression showed significantly shorter overall survival than those with low MID2 expression in subgroups with stages Tis+ I+ II (n = 174; P<0.001, log-rank; Fig. 3D) and stages III+ IV (n = 110; P<0.001, log-rank; Fig. 3D). Multivariate survival analysis (Table 3) indicated that MID2 expression level was an independent prognostic factor for overall survival. This finding suggests that MID2 may serve as potential prognostic factor for patients with early and late stages of breast cancer.

Knockdown of endogenous MID2 inhibits breast cancer cell proliferation in vitro

To investigate the biological function of MID2 in breast cancer, we constructed MID2-knockdown cells by using two MID2-specific shRNAs (Fig. 4A). We detected the protein level of Ki67, a recognized marker of proliferation, in stable cell lines. The expression of the Ki67 protein significantly decreased in MID2-downregulated cells (Fig. 4A). Furthermore, MTT assays revealed that MID2 knockdown significantly reduced the proliferation of MCF-7 and MDA-MB-231 breast cancer cells. Moreover, the cell numbers were reduced by approximately 1.5-fold compared with the number of vector control cells on day 5 after plating (Fig. 4B). The colony formation assay indicated that MID2 knockdown also significantly decreased the mean colony numbers (Fig. 4C) and significantly reduced anchorage-independent growth ability (P<0.05; Fig. 4D) compared with those in vector-transfected cells. These results indicate that MID2 may enhance the proliferation and tumorigenicity of breast cancer cells.

Knockdown of endogenous MID2 reduces the growth of breast cancer xenograft tumors in vivo

To validate the results of in vitro cell proliferation assays, we performed a xenograft model in BALB/C nude mice by using MDA-MB-231 cells to evaluate the effect of knocking down MID2 in vivo. MID2-RNAi1-transfected cells exhibited significantly reduced ability to form tumors in nude mice compared with vector-transfected cells (Fig. 5A), as indicated by the endpoint xenograft tumor weights, volumes, and tumor growth curves (Fig. 5B and 5C). Western blot analysis confirmed that MID2 expression was significantly lower in tumors formed by RNAi-transfected cells than that in vector control cells (Fig. 5D). The excised tumors initially stored in 10% formaldehyde solution were further embedded in paraffin, sliced, and immunohistochemically stained with MID2 and Ki67 antibodies. Representative images indicate that the expression levels of MID2 and Ki67 proteins decreased in tumors derived from MID2-RNAi1 cells (Fig. 5E). Overall, these results demonstrate that MID2 may play an important role in the tumorigenicity of breast cancer cells in vivo.

Discussion

This study showed that MID2 upregulation is associated with the progression of human breast cancer. This paper also presents the first evidence that MID2 mRNA and protein expression is upregulated in all stages of human breast cancer; moreover, MID2 can promote the proliferation of breast cancer cells.

Breast cancer is the most frequently diagnosed non-skin malignancy in 2015 [ 40]. Although diagnostic and treatment technologies for breast cancer have improved, the mortality rate of breast cancer remains high because of distant metastasis. Hence, identifying early diagnostic and prognostic biomarkers for breast cancer is of great clinical importance. This study demonstrates that MID2 mRNA and protein expression is upregulated in breast cancer cell lines and human breast cancer tissue, with MID2 expressed at high levels in 157/284 (55.3%) of human tissue examined by IHC. Moreover, univariate and multivariate analyses show that MID2 is significantly associated with disease staging and can be used as an independent prognostic factor for overall survival in patients with breast cancer. Thus, MID2 is a potential prognostic or diagnostic biomarker in breast cancer.

Previous study demonstrated that endogenous MID1 protein is located in the cytoplasm under different internal transpositions in different cell cycles, such as in microtubules throughout the cell cycle and mitotic spindle and midbodies during mitosis [ 41]. Although MID1 and MID2 share a number of overlapping functions, the current study focused on the effect of MID2 on the proliferation of breast cancer cells. As shown by the MTT, colony formation, and soft agar assays of MID2-overexpressing versus MID2-knockdown cells, MID2 displayed a pro-proliferative function in breast cancer in vitro and in vivo. As such, whether the functional similarity between MID1 and MID2 is evolutionarily intended to provide redundant proliferation-promoting support in human normal cells or perform pathologically selected oncogenic enhancement in breast cancer remains to be determined.

These results raise the question on the precise role of MID2 in breast cancer. MID1 was recently identified to function as an E3 ubiquitin ligase that targets phosphatase 2A (PP2A) degradation by binding to the α4 regulatory domain, a subunit of PP2-type phosphatases, in an embryonic fibroblast cell line [ 22]. Mutation of MID1 leads to marked accumulation of the catalytic subunit of protein phosphatase PP2A [ 22]. α4 strongly interacts with MID1 and MID2, as revealed by yeast two-hybrid assays [ 42]. Basing on this evidence, we speculate that MID2 upregulation in breast cancer enhances the protein degradation of PP2A. PP2A is considered to function as a tumor suppressor and is functionally inactivated in various cancers [ 43]. For example, the expression of PP2A-Aα is reduced by at least 10-fold in approximately 43% of human gliomas [ 44]. PP2A activity is essential in all cell types and participates in numerous cellular pathways [ 43]. Analysis of the KEGG pathway database indicated that PP2A dephosphorylates critical molecules, such as Akt, p53, c-Myc, and β-catenin, which play central roles in various key cellular processes, including cell-cycle regulation, apoptosis, senescence, and differentiation [ 45– 48]. In future studies, we intend to investigate whether a correlation exists between the expression of PP2A and MID2 in human breast cancer. We will also further explore the mechanisms through which MID2 is upregulated and exerts a pro-proliferative effect on breast cancer cells.

MID2 can affect the NF-kB signaling pathway by activating NF-kB/AP-1 [ 32]. NF-kB is a key regulator that participates in cancer cellular processes, including proliferation, metastasis, and epithelial-mesenchymal transition [ 49, 50]. Moreover, MID2 is a novel protein-interacting partner of BRCA1 and plays a critical role in transcription, DNA repair of double-stranded breaks, and recombination [ 33]. Hill et al. [ 51] performed protein-protein interaction screening using two complementary methods to identify new BRCA1-interacting partners. MID2, along with previously known breast cancer-associated genes, such as BRIP1 (BRCA1 interacting protein C-terminal helicase 1), BRCA2 (breast cancer 2, early onset), and DNMT1 (DNA methyltransferase 1), interacts with BRCA1. Several studies demonstrated the significant roles of these genes in different cancers and their association with BRCA1. For example, BRIP1 is involved in the dissociation of BRCA1 from chromatin, inhibition of DNA repair, and promotion of senescence [ 52], as well as in the loss of BRCA2 promoting cancer risk [ 53]. Moreover, depletion of DNMT1 expression in breast cancer has been shown to decrease the proportion of cancer stem cells and subsequently inhibit the tumorigenesis of cells injected into mice [ 54]. These studies imply the significant importance of BRCA1-interacting proteins in breast cancer development and progression. Thus, our findings on the biological functions and clinical significance of MID2 in breast cancer, and perhaps in other cancer types, warrant further exploration. Such investigation would help us understand the unique role of MID2 in the concerted molecular network that initiates tumorigenesis or promotes the malignant progression of cancers. These further studies will also aid in identifying effective cancer diagnostic/prognostic biomarkers and new interventional targets.

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