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Introduction

In recent years, monoclonal antibodies (mAbs) have been widely used in the treatment of malignant tumors including breast cancer, lymphoma and colorectal cancer (1,2). The HER2-targeted humanized mAb trastuzumab has been recommended for patients with HER2-positive early and metastatic breast cancer. Compared with conventional chemotherapy, trastuzumab can significantly improve disease-free survival (DFS) and overall survival (OS) of patients (3–5). However, 30–50% of HER2-positive breast cancer patients are not sensitive to trastuzumab. The mechanism of trastuzumab resistance has not been fully elucidated. There are few biological predictors of the efficacy of trastuzumab apart from HER2 (6,7).

Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are important immune mechanisms of the antitumor effects of mAbs, in addition to suppression of the HER2 signaling pathway, tumor cell apoptosis and inhibition of angiogenesis (6–8). Several IgG1 mAbs (e.g. rituximab, alemtuzumab and cetuximab) have been approved for the treatment of malignant tumors. Their antitumor effect via CDC has been clearly demonstrated in in vitro studies (9–12). Trastuzumab is a humanized IgG1 antibody. It binds to Fc receptors (FcRs) on immune effector cells or complements through its Fc fragment and induces ADCC or CDC. However, many studies have shown that trastuzumab induces minor or even no CDC (9–12,13).

Activation of the complement system is an important mechanism by which to kill tumor cells. The complement system can be activated by antigen-antibody complexes via the classical pathway and by tumor cells via the alternative pathway. Ultimately, C5b, C6, C7, C8 and C9 bind together to form the membrane attack complex (MAC). The MAC attaches to the surface of tumor cells, leading to the lysis of cells, which is the process of CDC (8). Membrane-bound complement regulatory proteins (mCRPs) can hinder complement pathway activation at different points and thus reduces CDC. mCRPs protect normal cells from complement-mediated cytolytic effects. Therefore, it is conceivable that this may be the mechanism of the tolerance of tumor cells to mAb-induced CDC. CD55, CD59 and CD46 are the most extensively studied mCRPs in tumor cells (14–16). Overexpression of one or more mCRPs has been found in different types of cancers, including colorectal, lung, prostate and breast cancer (17–21). Highly expressed mCRPs may block the complement pathway and thus protect tumor cells from mAb-induced CDC. Terui et al (22) detected CD55 and CD20 expression on the surface of B lymphoma tumor cells and observed the CDC effect induced by rituximab. They found that CD55 overexpression decreased the susceptibility to rituximab-induced CDC of B lymphoma tumor cells. Golay et al (23) reported that downregulation of CD55 and CD59 enhanced rituximab-mediated CDC in chronic lymphoma cells.

However, the relevance of mCRP expression in breast cancer tissue with the prognosis of trastuzumab in a clinical setting remains unknown. In this study, we detected expression levels of CD55, CD59 and CD46 in formalin-fixed paraffin-embedded (FFPE) tumor specimens from HER2-positive breast cancer patients treated with trastuzumab as postoperative adjuvant therapy by immunohistochemical staining. The relationship between mCRP expression and prognosis following postoperative adjuvant therapy containing trastuzumab was retrospectively analyzed. Previous studies reported that indomethacin (Indo), 4-hydroxy tamoxifen (Tam), levamisole (LMS), cisplatin (DDP) and sodium butyrate can downregulate mCRP expression (21,24–27). Here, we treated breast cancer cells with the aforementioned drugs and detected their inhibitory effect on mCRP expression and subsequently enhanced trastuzumab-mediated CDC.

Materials and methods

Patients and tumor specimens

Through search of the Breast Cancer Information Management System of West China Hospital, Sichuan University, a total of 65 breast cancer patients who had undergone radical mastectomy during the period from 2007 to 2010 were included in the present study. The inclusion criteria included: i) pathological diagnosis of breast cancer; ii) HER2-positive breast cancer [3+ by immunohistochemical analysis (IHC) or with gene amplification by fluorescence in situ hybridization (FISH)]; iii) availability of standard treatment containing trastuzumab at least for 1 year; and iv) >3 years of complete follow-up record upon radical mastectomy. Exclusion criteria were: i) diagnosis of metastatic breast cancer before treatment; and ii) administration of any preoperative treatment. We collected the clinical information of patients and performed telephone follow-up periodically through the Breast Cancer Information Management System (once every 6 months during the initial 1–5 years and once yearly after 5 years). The study met the recommendations of the ‘Declaration of Helsinki’ and was approved by the Ethics Committee of West China Hospital.

The 65 FFPE breast tumor tissues and 14 FFPE normal breast tissues from 65 breast cancer patients were retrospectively collected from the Pathology Department of West China Hospital. Disease-free survival (DFS) was defined as the time from radical mastectomy to tumor recurrence (28). Relapse rate was defined as the proportion of the patients with recurrent disease during the follow-up period.

Immunohistochemical staining

The streptavidin-biotin complex (SABC) method was used to detect the expression levels of CD55, CD59 and CD46 in the FFPE specimens. The specimens were heated using a microwave at low power for 5 min for antigen retrieval. Three percent H2O2 deionized water solution was used to block endogenous peroxidase. Anti-CD55 (clone SM3030P; Acris Co., Gemany; 1:200), anti-CD59 (clone p282; Abcam Co., USA; 1:100) or anti-CD46 (clone H-294; Santa Cruz Biotechnology Co., USA; 1:100) antibodies were added to the samples and incubated at 4°C overnight. After PBS washes, the biotinylated secondary antibody (ZB-2010 or ZB-2020; Zhongshan Golden Bridge Co., China) was added. The samples were incubated for 30 min, and subjected to PBS washes. Afterwards, the samples were incubated with streptavidin-biotin-peroxidase labeled tertiary antibody (ZB-2404; Zhongshan) for 30 min. Color was developed using diaminobenzidine (DAB) chromogenic color development reagent. PBS instead of the primary antibody was used in the negative control group. Staining intensity of the tumor cells was classified as: −, +, + + and + + + (Fig. 1). At least 1,000 tumor cells were assessed and high CD55, CD59 or CD46 expression was defined as +++ expression of CD55, CD59 or CD46 in more than 1% of breast cancer cells. Otherwise, the breast cancer specimens were identified as having low CD55, CD59 or CD46 expression (29)

Figure 1 Expression levels of mCRPs (CD55, CD59 and CD46) in the breast cancer samples (SP, ×400) as detected by immunohistochemistry. The expression of (A) CD55, (B) CD59 and (C) CD46 in breast cancer samples.

Cell culture

The ER−/HER2+ breast cancer cell line SK-Br-3 and the ER+/HER2+ BT-474 cell line (The Cell Bank, Chinese Academy of Sciences) (30,31), were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS; Gibco, USA) and MEM (Gibco) containing 10% FBS, respectively.

Cell cytotoxicity as determined by MTT assay

SK-Br-3 or BT-474 cells (1×104 cells/well) were seeded in 96-well plates and incubated overnight to allow cells to adhere. The cells were pretreated with the following drugs: Indo (20, 40, 80, 160 and 320 μM), Tam (1, 2, 4, 8 and 16 μM), LMS (0.16, 0.32, 0.64, 1.28 and 2.56 mM), sodium butyrate (0.3, 0.6, 1.2, 2.4 and 4.8 mM) (all from Sigma USA) or DDP (0.25, 0.5, 1, 2 and 4 μM; Shandong Qilu Pharmaceutical Factory, China) for 72 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) was then added and incubated for 4 h. Absorbance at 490 nm was measured using a Multiskan Spectrum (Thermo Scientific Co., USA). The maximum non-toxic doses of the drugs in the SK-Br-3 or BT-474 cells were determined.

Detection of trastuzumab-mediated CDC effects

The SK-Br-3 and BT-474 cell lines were used to study trastu-zumab-induced CDC. Normal human serum (NHS) obtained from healthy volunteers (Blood Bank of West China Hospital, Sichuan University, China) was provided voluntarily for this study. Cells were incubated with heat-inactivated NHS (56°C, 60 min) as the negative control. Phosphatidylinositol-specific phospholipase C (PI-PLC; Invitrogen Co., USA) specifically hydrolyzes and inactivates anchor proteins CD55 and CD59 (32). Cells were incubated with PI-PLC as the positive control. Breast cancer cells were pretreated with maximum non-toxic doses of Indo (40 μM), Tam (2 μM), LMS (0.16 mM), sodium butyrate (0.5 μM) and DDP (0.32 mM) for 72 h, and subsequently washed with PBS. Cells were detached by 0.125% trypsin and washed with PBS. A total of 1×105 cells were then cultured with FBS-free medium containing 50% human serum (v/v) and 50 μg/ml trastuzumab for 1 h (32). Afterwards, trypan blue staining was performed to count the dead and viable cell number in a hemocytometer under a microscope. Experiments were repeated at least three times. The cell death rate was expressed as the percentage of dead cells relative to the total number of cells (viable cells and dead cells included).

Detection of CD55 mRNA expression levels using qRT-PCR

Cells were pretreated with 2 μM Tam for 6, 12 and 24 h. Total cellular RNA was extracted using the BIOzol total RNA extraction kit (BioFlux Co., Japan) according to the manufacturer’s instructions. First-strand cDNAs were synthesized from total RNA using M-MLV transcriptase and oligo-dT primer. Quantitative RT-PCR (qRT-PCR) was performed using duplicate SsoFast™ Eva Green® Supermix kit (Bio-Rad, USA) in Chromo4 real-time PCR detector (Bio-Rad) following the manufacturer’s instructions. The primer sequences used are presented in Table I (33). The relative quantitation of gene expression was performed using the comparative Ct (ΔΔCt) method with GAPDH as a housekeeping gene. Experiments were repeated at least twice.

Table I Primer sequences.

Table I

Primer sequences.

Sequence detected	Primer sequence
CD59	F: 5′-TAACCCAACTGCTGACTGCAA-3′ R: 5′-TTTGGTAATGAGACACGCATCAA-3′
CD55	F: 5′-TTCCCCCAGATGTACCTAATGC-3′ R: 5′-TTACAGTATCCTCGGGAAAACTTGT-3′
GAPDH	F: 5′-GAAGGTGAAGGTCGGAGTC-3′ R: 5′-GAAGATGGTGATGGGATTTC-3′

[i] F, forward; R, reverse.

Western blot assays

After pretreatment with 2 μM (maximum non-toxic doses) Tam for 72 h, SK-Br-3 and BT-474 cells were lysed at 4°C in RIPA lysis buffer (Biyuntian Co., China). Total protein concentration was determined using the Bradford protein assay (Bio-Rad). Equal amounts of cell lysates were separated on 12% SDS-PAGE and transferred to PVDF membranes. PVDF membranes were blocked with 5% nonfat milk in PBS-T (0.5% of Tween-20 in PBS). The membranes were incubated at 4°C overnight with the primary antibodies against CD55 (BRIC216), CD59 (YTH53.1) or β-actin (all from Santa Cruz Biotechnology, USA). After PBS-T washing, horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnogy) were added. The membranes were then incubated at room temperature for 1 h. Individual protein bands were visualized using chemiluminescence reagent (Millipore, USA). The film was exposed in a dark room and scanned with HP LaserJet 3055 scanner. Volume densitometry of the immunoreactive bands was quantitated with One Quantity software (Bio-Rad, USA).

Detection of mCRP expression levels by flow cytometry

SK-Br-3 and BT-474 cells were pretreated with the maximum non-toxic doses of Indo, Tam, LMS, DDP or sodium butyrate for 72 h and washed with PBS. Cells were incubated on ice for 30 min with the FITC-conjugated anti-CD55 antibody, FITC-conjugated anti-CD59 antibody (Beijing Jingmei Gene Valley Technology Co., China) or FITC-conjugated anti-CD46 antibody (Biolegend Co., USA). IgG1 isotype control/FITC (Beijing Jingmei Gene Valley Technology Co., China) was used as the negative control antibody. Then, the cells were washed, and resuspended in formaldehyde fixing solution (1% BSA-PBS + 1% polyphosphate formaldehyde), and subjected to flow cytometry (FCM).

Statistical analysis

Statistical significance of the correlation between the clinicopathologic characteristics or prognosis and the patients with mCRP high expression and low expression was determined with a χ2 test comparison. Kaplan-Meier survival analysis was used for estimation of DFS, and log-rank test was used for inter-group comparison. Cox proportional hazards model analysis was used for estimation of univariate hazard ratios (HRs) for DFS and multivariate analysis. Menopausal status, tumor size (cm), tumor grade, stage, lymph node metastasis status, hormone receptor status and mCRP expression were included in the model. The CDC of trastuzumab was expressed as mean ± SEM. Independent samples t-test was used for comparison between two groups. P<0.05 was considered to indicate a statistically significant difference. SPSS software version 16.0 (SPSS Inc., USA) was used for the statistical analysis.

Results

Clinicopathological characteristics of the patients

The clinicopathological characteristics of the 65 breast cancer patients who underwent postoperative adjuvant treatment containing trastuzumab are summarized in Table II. Patient age ranged from 21 to 67 years (with a median age of 49 years). Adjuvant chemotherapy plus trastuzumab was administered to 43 patients and adjuvant endocrine therapy plus adjuvant chemotherapy and trastuzumab to 22 patients. Trastuzumab was given once every three weeks with a starting dose of 8 mg/kg and a maintenance dose of 6 mg/kg, following chemotherapy. Trastuzumab therapy was maintained for at least 1 year. The median follow-up duration was 47 months (range from 24 to 75 months). Relapse occurred in 13 patients during the follow-up period. Two patients relapsed within 1 year, 10 patients within 1–3 years, and 1 patient within 3–5 years. Among them, 5 patients had lymph node metastasis, 4 patients had lung metastasis, 2 patients had bone metastasis, and 2 patients had gastrointestinal metastasis.

Table II Patient characteristics.

Table II

Patient characteristics.

Characteristics	No. of patients (n=65)
Age (years)
Median	49
Range	21–67
Menopausal status
Premenopausal	27
Postmenopausal	38
Histologic type
Invasive ductal carcinoma	65
Tumor size (cm)
≥2	28
>2 - <5	31
≥5	6
Histologic grade
I/II	15
III	50
Tumor stage
I	14
II	31
III	20
Lymph node metastasis
Positive	37
Negative	28
Estrogen receptor status
Positive	20
Negative	45
Progesterone receptor status
Positive	20
Negative	45
Prognosis
Recurrence	13
Non-recurrence	52
Adjuvant therapy
Chemotherapy	43
Chemohormonotherapy	22

Expression levels of mCRPs in the breast tumors are associated with the relapse rate

Levels of CD55, CD59 and CD46 expression in the FFPE breast tumor tissues from 65 patients who underwent postoperative adjuvant therapy containing trastuzumab were detected by immunohistochemistry. CD55, CD59 and CD46 were expressed on the membrane of the breast cancer cells (Fig. 1). Among the patients, 30/65 (46.2%) had high expression levels of CD55, 29 (44.6%) had a high expression level of CD59, and 29 (44.6%) had a high expression level of CD46. In comparison, only 2/14 (14.3%) normal breast tissue specimens had high expression levels of CD55, CD59 or CD46; the remaining 12 (85.7%) specimens had low expression levels.

As shown in Table III, patients with CD55 or CD59 overexpression had significantly higher relapse rates than the patients with low expression of CD55 (33.3 vs. 8.6%; P=0.013) or CD59 (31.0 vs. 11.1%; P=0.046). The relapse rate in the CD46 overexpression group was slightly higher than that in the CD46 low expression group (24.1 vs. 16.7%). However, the difference was not statistically significant (P=0.454). Other clinicopathological characteristics, including menopausal status, histological classification, tumor stage, lymph node metastasis, and hormone receptor status did not differ significantly between patients with high and low expression of mCRPs (P>0.05).

Table III Correlation between CD55, CD59 and CD46 expression and clinicopathological variables of the breast cancer patients.

Table III

Correlation between CD55, CD59 and CD46 expression and clinicopathological variables of the breast cancer patients.

		CD55			CD59			CD46
Characteristics	%	Low (53.8%)	High (46.2%)	P-value	Low (55.4%)	High (44.6%)	P-value	Low (55.4%)	High (44.6%)	P-value
Menopausal status				0.437			0.135			0.981
Premenopausal	41.5	13	14		12	15		15	12
Postmenopausal	58.5	22	16		24	14		21	17
Tumor size (cm)				0.536			0.521			0.324
≤2	43.1	15	13		16	12		18	10
>2 - <5	47.7	18	13		18	13		16	15
≥5	9.2	2	4		2	4		2	4
Histologic grade				0.525			0.682			0.682
I/II	23.1	7	8		9	6		9	6
III	76.9	28	22		27	23		27	23
Stage				0.317			0.246			0.709
I	21.5	8	6		9	5		9	5
II	47.7	19	12		19	12		17	14
III	30.8	8	12		8	12		10	10
Lymph node metastasis				0.643			0.078			0.209
Positive	56.9	19	18		17	20		18	19
Negative	43.1	16	12		19	9		18	10
Estrogen receptor status				0.368			0.465			0.072
Positive	30.8	12	8		13	8		15	6
Negative	69.2	23	22		23	21		21	23
Progesterone receptor status				0.507			0.298			0.114
Positive	30.8	12	8		13	7		14	6
Negative	69.2	23	22		23	22		22	23
Prognosis				0.013			0.046			0.454
Recurrence	20.0	3	10		4	9		6	7
Non-recurrence	80.0	32	20		32	20		30	22
Adjuvant therapy				0.544			0.667			0.338
Chemotherapy	66.2	22	21		23	20		22	21
Chemohormonotherapy	33.8	13	9		13	9		14	8

Expression levels of mCRPs in breast cancer are associated with prognosis

Mean DFS of 65 patients included in our study was 64 months (95% CI, 59–69 months). We found that the mean DFS of patients with CD55 or CD59 high expression was significantly shorter when compared to the patients with low expression of CD55 (56 vs. 70 months; log-rank test, P=0.008) or CD59 (56 vs. 69 months; log-rank test, P=0.033). In contrast, the mean DFS was not significantly different between patients with high and low expression of CD46 (66 vs. 60 months; log-rank test, P=0.516; Fig. 2). Risk factors of DFS including menopausal status, tumor size (cm), tumor grade and stage, lymph node metastasis, hormone receptor status and mCRP expression were analyzed using Cox proportional hazards model. Univariate analysis revealed that overexpression of CD55 (HR=4.822; 95% CI, 1.323–17.57; P=0.02) or CD59 (HR=3.343; 95% CI, 1.027–10.878; P=0.05), but not CD46 (HR=1.43; 95% CI, 0.48–4.257; P=0.52), was associated with a high risk of relapse. Multivariate analysis showed that CD55 overexpression was an independent risk factor of relapse (HR=4.757; 95% CI, 0.985–22.974; P=0.05). However, CD59 overexpression was not significantly associated with risk of relapse (HR=2.378; 95% CI, 0.543–10.414; P=0.25) (Table IV).

Figure 2 Disease-free survival (DFS) curves of breast cancer patients with high and low expression levels of mCRPs. (A and B) A statistically significant difference was observed in DFS between the (A) CD55-low expression and the CD55-high expression group (P<0.05) and the (B) CD59-low expression and CD59-high expression group (P<0.05). (C) No statistically significant difference (P=0.516) was observed between the CD46-low expression and CD46-high expression group in regard to DFS (P>0.05).

Table IV Univariate and multivariate analysis of the prognostic factors.

Table IV

Univariate and multivariate analysis of the prognostic factors.

	Univariate		Multivariate
	HR (95% CI)	P-value	HR (95% CI)	P-value
Menopausal status	0.497 (0.166–1.485)	0.21
Tumor size (cm)	1.032 (0.787–1.352)	0.82
Histologic grade	0.968 (0.266–3.518)	0.96
Stage	1.173 (0.531–2.59)	0.69
Lymph node metastasis	1.291 (0.422–3.947)	0.66
Estrogen receptor status	0.732 (0.239–2.239)	0.59
Progesterone receptor status	0.610 (0.199–1.866)	0.39
CD59	3.343 (1.027–10.878)	0.05	2.378 (0.543–10.414)	0.25
CD55	4.822 (1.323–17.570)	0.02	4.757 (0.985–22.974)	0.05
CD46	1.430 (0.48–4.257)	0.52

Inhibition of mCRP expression enhances trastuzumab-mediated CDC

Our retrospective analysis of clinical samples revealed that CD55 overexpression is an independent risk factor of relapse in breast cancer patients treated with trastuzumab. It is possible that a high CD55 expression level suppresses trastuzumab-mediated CDC, and thus leads to poor prognosis. Therefore, we aimed to ascertain whether downregulation of mCRP expression could sensitize breast cancer cells to trastuzumab-mediated CDC. We confirmed the maximum non-toxic doses of Indo, Tam, LMS, DDP and sodium butyrate (40, 2, 0.16, 0.5 and 0.32 mM, respectively) in SK-Br3 and BT-474 cells by MTT. Breast cancer cells were pretreated with these drugs at their maximum non-toxic doses. Flow cytometric results showed that only Tam inhibited expression levels of CD55 in the SK-BR-3 and BT-474 cells (Fig. 3A). Real-time PCR and western blotting results confirmed the inhibitory effect of Tam on both the mRNA and protein expression levels of CD55 (Fig. 4). In contrast, CD59 expression levels in both breast cancer cell lines did not change significantly after Tam pretreatment.

Figure 3 Effects of the different drug treatments on mCRP expression and trastuzumab-induced CDC in SK-Br-3 and BT-474 cells. (A) (a–c) Representative FACS histograms show the expression levels of CD55, CD59 and CD46 in the SK-Br-3 cell line after Tam treatment. (d–f) Representative FACS histograms show the expression of CD55, CD59 and CD46 in the BT-474 cell line after Tam treatment. (B and C) Compared with the Trastuzumab and NHS group, no statistically significant difference was noted; ▲P>0.05. Significantly different from the trastuzumab and NHS group; ※P<0.05. NHS, normal human serum; Indo, indomethacin; LMS, levamisole; DDP, cisplatin; Tam, 4-hydroxy tamoxifen; PI-PLC, phosphatidylinositol-specific phospholipase C.

Figure 4 mRNA and protein expression levels of CD55 as detected by quantitative RT-PCR and western blot analysis. (A and B) mRNA levels of CD55 in SK-Br-3 and BT-474 cells after pretreatment with Tam for 6, 12 and 24 h. mRNA levels of CD55 were calculated using n-fold change of mRNA (relative to the control). (C) Following pretreatment of SK-Br-3 and BT-474 cells with Tam for 72 h, western blotting was performed to detect the expression of CD55 and CD59. Steady-state β-actin protein levels served as the protein loading control.

To investigate the effect of mCRP inhibition of trastuzumab-mediated CDC, we pretreated breast cancer cells with Tam, DDP, Indo, LMS or sodium butyrate for 72 h, and then incubated the cells with 50% normal human serum (NHS) and trastuzumab for 1 h. Trastuzumab with NHS induced an insignificant cytotoxic response (4.8±0.5 vs. 4.6±0.3%, respectively in the SK-Br3 and BT-474 cells), compared with the negative control group (4.5±0.01 vs. 4.1±0.04%, respectively), while Tam pretreatment significantly enhanced the cell death rate induced by trastuzumab and NHS in the SK-Br3 (14.33±1.7%, P<0.01) and BT-474 cells (9.0±0.6%, P<0.01) (Fig. 3B and C). In contrast, DDP, Indo, LMS, or sodium butyrate pretreatment did not significantly affect trastuzumab-mediated CDC (P>0.05; Fig. 3B and C).

Discussion

Overexpression of mCRPs has been implicated in malignant tumors and is associated with poor prognosis (17–21,34–36). Durrant et al (34) reported that CD55 is highly expressed in colorectal cancer and is an indicator of poor prognosis in colorectal cancer patients. Madjd et al (36) showed that high expression levels of CD46 in breast cancer tissues were positively correlated with the degree of tumor malignancy, poor prognosis and risk of recurrence. Moreover, two studies reported that mCRP overexpression was related to tolerance of lymphoma to rituximab-mediated CDC (22,37). Bannerji et al (38) reported that rituximab-induced CDC was enhanced significantly by reducing CD59 and CD55 expression in leukocytes from patients with chronic lymphocytic leukemia.

In the present study, we assessed the expression levels of CD55, CD59 and CD46 in 65 FFPE breast tumor tissues and in 14 FFPE normal breast tissues from 65 HER2-positive breast cancer patients who underwent trastuzumab treatment. Our data demonstrated that breast cancer tissues had higher expression of CD55, CD59 and CD46 than that in the normal tissues. Furthermore, subgroup analysis revealed that high expression levels of CD55 and CD59 were associated with higher recurrence rate, and CD55 overexpression was found to be an independent risk factor of recurrence, suggesting that CD55 overexpression may be an indicator of poor prognosis of operable breast cancer treated with adjuvant trastuzumab. In contrast CD55 expression was not associated with other clinicopathological features, including tumor stage, histological grade, or hormone receptor status in breast cancer. This is consistent with studies by Durrant et al (34) and Ikeda et al (29). This is the first study, to our knowledge, to investigate the predictive role of the expression of three mCRPs (CD55, CD59, and CD46) in the prognosis of breast cancer patients who underwent postoperative adjuvant therapy containing trastuzumab.

Emerging evidence has shown that neutralizing antibodies or siRNAs that downregulate mCRPs can significantly enhance mAb-mediated CDC in vitro (22,23,37). Bellone et al (39) found that trastuzumab-induced CDC was enhanced after silencing of CD55 and CD59 by siRNAs in uterine cancer cells. Zhao et al (21) drew similar conclusions in two non-small cell lung cancer cell lines, A549 and H157. Mamidi et al (10) reported that silencing of membrane regulators (CD46, CD55 and CD59) by anti-mCRP siRNAs enhanced the CDC of trastuzumab and pertuzumab. However, mCRPs are widely expressed throughout the human body, thus neutralizing antibodies and siRNAs of mCRPs may damage normal tissues. Currently, there are no effective neutralizing antibodies to mCRPs that can be applied in clinical practice. Researchers have attempted to solve this challenge by bi-specific antibodies, i.e. engineered antibodies that target tumor cells and mCRPs simultaneously (40). However, specificity remains to be the bottleneck of this therapeutic strategy.

In the present study, we treated breast cancer cells with commonly used clinical drugs that have been reported to reduce mCRP expression levels. We expected that these drugs would effectively reduce mCRP expression levels without increasing toxicity, and improve the efficacy of trastuzumab. We found that 4-hydroxy tamoxifen (Tam) effectively downregulated CD55 expression, but not CD59 and CD46. Pretreatment of Tam enhanced the trastuzumab-mediated CDC in BT-474 and SK-Br3 cells. Several in vitro studies have shown that combined use of Tam and trastuzumab can synergistically inhibit the growth of breast cancer cells (41,42). The authors suggested that suppression of ER and HER-2/neu pathways simultaneously in HER+/ER+ breast cancer may improve the efficacy of breast cancer therapy. Our results suggest that Tam and trastuzumab can synergistically kill breast cancer cells by enhancing trastuzumab-mediated CDC.

In the present study, we found that Tam could not only reduce CD55 expression in HER2+/ER+ BT-474 cells but also in HER2+/ER− SK-Br3 cells. These data raise the possibility that combined Tam and trastuzumab treatment may also enhance the efficacy of trastuzumab in patients with ER-negative breast cancer, besides ER-positive breast cancer. The efficacy and feasibility of combined Tam and trastuzumab for treatment of breast cancer warrants further investigation. In addition, the mechanism of Tam-mediated downregulation of CD55 in SK-Br3 and BT-474 cells remains unclear. CD55 expression is possibly downregulated by modulation of certain cytokines, such as interferon (IFN) (41–43). Our results showed that both CD55 mRNA and protein levels were simultaneously reduced by Tam treatment, suggesting that regulation of CD55 expression could occur at the transcriptional level.

In conclusion, high expression levels of CD55 and CD59 were associated with a higher risk of recurrence, and CD55 overexpression was an independent risk factor of recurrence in breast cancer patients who received postoperative adjuvant therapy containing trastuzumab. In addition, trastuzumab-induced CDC was enhanced in vitro by Tam treatment through CD55 downregulation. These results warrant further investigation in regards to the predictive role of CD55 and CD59 in the efficacy of trastuzumab in postoperative adjuvant therapy of breast cancer. Furthermore, combined use of Tam and trastuzumab may enhance trastuzumab-induced CDC, and thus potentially improve the efficacy of trastuzumab, which requires additional clinical studies to verify.

Acknowledgements

This study was supported by the National Natural Sciences Foundation of China (30972940), West China Hospital and Shanghai Roche Pharmaceuticals Ltd., horizontal cooperation project (H1109114 and H1307060). We would like to thank Shanghai Roche Pharmaceuticals Ltd., for the editorial assistance.

References