In our previous study, we found that 53BP1 was a tumor suppressor and was associated with prognosis in breast cancer. However, little is known about its role in angiogenesis. In the present study, we aimed to reveal the role of 53BP1 in angiogenesis of breast cancer. With RNA interference and ectopic expression strategies to elucidate the detailed function of 53BP1 in angiogenesis, we observed that ectopic expression of 53BP1 inhibited cellular angiogenesis and 53BP1 RNA interference led to an increase in angiogenesis both in vitro and in vivo. In clinical breast cancer samples, 53BP1 was inversely correlated with CD31, MMP-2 and MMP-9 by immunohistochemistry analysis. Furthermore, we showed that the Akt pathway was involved in the antiangiogenesis function of 53BP1. Overall, our findings demonstrate that 53BP1 plays a vital role in inhibiting angiogenesis. These findings suggest that 53BP1 might provide a viable target therapy for breast cancer.
Breast cancer is the most common cancer and leading cause of cancer deaths for women in both developed and developing countries. It is a multi-step and systematic disease involving activation of an oncogene and/or inactivation of a tumor suppressor gene. As a result, the identification of novel oncogenes and tumor suppressor genes involved in the initiation and progression of tumors could generate targets for the development of new anticancer drugs.
Human 53BP1 (tumor protein p53 binding protein 1) was first identified by Iwabuchi et al.[3, 4] as a p53-binding partner that could enhance the transcriptional activity of p53. The notion of 53BP1 as an emerging candidate tumor suppressor has been supported by more and more studies. We have previously found that 53BP1 could inhibit invasion and metastasis in breast cancer and could suppress tumor growth and promote susceptibility to apoptosis in ovarian cancer. Our groups have also revealed a significant association between 53BP1 status and distant metastasis-free survival, with 53BP1-negative tumors having significantly lower metastasis-free survival. Consistent with these findings, Gorgoulis et al. found the lower expression or depletion of 53BP1 in the progression of non-small-cell lung carcinomas and malignant melanoma. Bartkova et al. reported aberrant reduction or loss of 53BP1 in subsets of human carcinomas including breast and lung cancer while it was expressed in all normal tissues. Squatrito et al. reported that 53BP1 behaves as a haploinsufficient tumor suppressor in glioma. All of these findings imply that 53BP1 is an enhanced therapeutic window for cancers.
Although the death incidence of breast cancer has been improved significantly, many patients are dying as a result of metastasis to distant organs rather than the growth of primary tumors. Metastasis formation is a complicated multistep process including angiogenesis. Angiogenesis plays an important role in the growth of breast cancer by supplying nutrients and oxygen and removing waste products from the tumor.[11, 12] It is a vital element in controlling the metastasis of breast cancer. We found that 53BP1 could inhibit the invasion and metastasis of breast cancer;(5) however, the function of 53BP1 in angiogenesis is still unknown. In the present study we aimed to explore the role of 53BP1 in regulating angiogenesis of breast cancer and the potential therapeutic target of 53BP1 for breast cancer antiangiogenesis.
Materials and Methods
Cell lines and reagents
Breast cancer cell lines MCF-7, MDA-MB-231 and human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA). Antibodies against Akt, p-Akt (Ser 473), small interfering RNA (siRNA) oligonucleotide targeting AKT and a negative control were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-53BP1 antibody was from Bethyl Laboratories (Montgomery, AL, USA). Anti-CD31 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Growth factor-reduced matrigel was obtained from BD Biosciences (Bedford, MA, USA). The other reagents were from Sigma–Aldrich (St Louis, MO, USA) unless specifically described.
MCF-7, MDA-MB-231 and HUVEC cells were routinely cultured in appropriate medium supplemented with 10% FBS and 100 units of penicillin-streptomycin at 37°C with 5% CO2 in a humidified incubator.
Plasmid construction and transfection
The plasmid information is the same as previously described. The expression plasmid vector and the empty vector were used to transfect the MDA-MB-231 cells using lipofectamine 2000 to establish 53BP1 overexpression (53BP1-OVE) and control (Mock) cell lines.
For RNA interference of 53BP1, the sense shRNA target sequence was as follows: GCCAGGUUCUAGAGGAUGA. The pSuper-Neo-GFP shRNA53BP1 vector and the empty vector were used to transfect MCF-7 cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol to establish pSN-shRNA53BP1 (shRNA-53BP1) and control (control) cell lines.
Total RNA were extracted with TRIZOL reagents according to the manufacturer's protocol (TaKaRa, Dalian, China). Briefly, cDNA was synthesized from 0.8 μg of total RNA by PrimerScript RT Reagent Kit. The qRT-PCR was performed using a SYBR green PCR mix in Applied Biosystems (Carlsbad, CA, USA) StepOne and StepOnePlus Real-Time PCR Systems. The samples were loaded in quadruple and the results of each sample were normalized to GAPDH. The experiments were repeated at least in triplicate to confirm the findings.
Western blot analysis
Cells were washed twice with cold phosphate-buffered saline (PBS) and lysed on ice in RadioImmuno precipitation assay (RIPA) buffer (1 × PBS, 1% NP40, 0.1% sodium dodecyl sulfate [SDS], 5 mM EDTA, 0.5% sodium deoxycholate and 1 mM sodium orthovanadate) with protease and phosphorase inhibitors and quantified using the BCA Protein Assay Kit (Merck, Darmstadt, Germany). Equal amounts of protein were separated using SDS polyacrylamide gel, electrotransferred to polyvinylidene fluoride membranes (ImmobilonP; Millipore, Bedford, MA, USA) and blocked in 5% non-fat dry milk in Tris-buffered saline, pH 7.5 (100 mM NaCl, 50 mM Tris and 0.1% Tween-20). Membranes were immunoblotted overnight at 4°C with primary antibodies, followed by their respective horseradish peroxidase conjugated secondary antibodies. Signals were detected using enhanced chemiluminescence. β-actin was used as the loading control.
Capillary tube formation assay
Forty-eight-well plates were coated with 200 μL growth factor-reduced matrigel and incubated at 37°C for 1 h to allow gelling. Tumor cell conditioned medium (TCM) was prepared as previously described. The HUVEC were resuspended using TCM collected from cultured cells and then seeded on matrigel-coated plates. Then HUVEC were incubated for 6 h. The branch points of the formed tubes, which represent the degree of angiogenesis in vitro, were scanned under a light microscope and quantities in at least 10 microscopic fields. The experiments were repeated at least in triplicate.
Chick embryo chorioallantoic membrane (CAM) assay
A CAM assay was performed as previously described.[15, 16] Fertilized chick eggs were incubated for 3 days at 37°C and relative humidity of 80%. On the third day of incubation, eggs were opened on the air sac side. Various TCM were then placed on the CAM. The cavity was covered with parafilm and eggs were incubated for an additional 5 days. Angiogenesis was quantified by counting the number of branching blood vessels. Each experiment was performed three times.
Mouse aortic ring assay
As previously described, aortas were harvested from 2-month-old BALB/c mice. The 48-well plates were coated with 120 uL of matrigel. After gelling, the rings were placed in the wells and sealed in place with an overlay of 80 uL matrigel. Last, 200 uL TCM from cultured cells was added into each well. On day 7, the fields covered by sprouting from the aortic rings were measured under a light microscope. Six independent experiments were performed. All animal studies were performed with the approval of Shandong University Animal Care and Use Committee.
The transwell assay was performed as previously described.(5) Briefly, an equal number of cells were added to the upper compartment of the chamber. After incubation for 18 h, the non-invasive cells in the upper compartment were removed and the cells in the lower compartment of the chamber were counted under a light microscope for at least 10 random visual fields.
In vivo matrigel plug assay
The in vivo matrigel plug assay was performed as previously described.[16, 18] Growth-factor-reduced matrigel premixed with 2 × 106 cells was subcutaneously implanted into either side of the flank of the same BALB/c nude mice. After 21 days the mice were killed. The xenografts were removed for immunohistochemical staining. All animal experiments were performed with the approval of Shandong University Animal Care and Use Committees.
Eighty-three paraffin-embedded breast tissue samples were obtained from the Department of Pathology of Qilu Hospital of Shandong University between 2007 and 2010. The streptavidin–peroxidase–biotin (SP) immunohistochemical method was performed to study altered protein expression paraffin-embedded breast tissues as previously described.[19, 20] For all patients in the present study, written informed consent was obtained and the study was approved by the Ethical Committee of Shandong University.
The results were analyzed using the software spss 18.0 (SPSS Inc., Chicago, IL, USA). The experiments were performed at least three times and the data were expressed as mean ± SD. A two-tailed Student's t-test was used to analyze the statistical difference; P < 0.05 was considered significant.
Establishment of stable 53BP1 transfectants of breast cancer cell lines
Given that MDA-MB-231 cells possess low endogenous levels of 53BP1, they were used to establish stable MDA-MB-231 cells that constitutively overexpressed the 53BP1 protein. Because the 53BP1 level was higher in breast cancer MCF-7 cell lines, we used shRNA to generate 53BP1-knockdown cell models to study the function of 53BP1 in angiogenesis. Transfection efficiency was confirmed using western blot analysis. As shown in Figure 1, the MDA-MB-231 cells transfected with 53BP1 expression vector (53BP1-OVE) showed significantly increased 53BP1 in both mRNA and protein levels compared with the control cell lines. Also, MCF-7 cells transfected with 53BP1 shRNA showed significantly decreased 53BP1 expression compared with the control cells.
53BP1 inhibited angiogenesis in vitro
We found that 53BP1 significantly inhibited the cell growth and migration of breast cancer cells.(5) To further investigate whether 53BP1 inhibited tumor growth and migration by suppressing angiogenesis, we first performed a vascular network formation assay. As HUVECs that were cultured on matrigel could rapidly align, then extend processes into the matrix, and finally form capillary-like structures surrounding a central lumen. As shown in Figure 2(a), HUVEC cultured with tumor cell-conditioned medium from 53BP1 knockdown MCF-7 cells caused a significant increase in tube formation compared with the control group, while that of ectopic 53BP1 in MDA-MB-231 cells showed fewer number of tube formation than the control MDA-MB-231 cells (P = 0.026 and P = 0.004, respectively). These data suggest that 53BP1 could inhibit angiogenesis in vitro.
We further used a CAM angiogenesis model to study whether knockdown/overexpression of 53BP1 would be a feasible approach to suppress tumor-induced angiogenesis in vivo. Consistent with the data above, upregulation of 53BP1 in MDA-MB-231 cells led to a significant reduction in angiogenesis (P = 0.006), while CAM seeded with TCM from 53BP1 shRNA-treated MCF-7 cells exhibited neovascularization and the control CAM exhibited less neovascularization (P = 0.025) (Fig. 2b). These results confirmed the role of 53BP1 in angiogenesis.
53BP1 inhibited angiogenesis ex vivo
We further explored the antiangiogenic activity of 53BP1 using ex vivo and in vivo angiogenesis models. First, we examined the sprouting of vessels from aortic rings ex vivo. Collagen gel cultures of rat aorta were treated with TCM from the transfected breast cancer cell lines. As shown in Figure 2(c), we found that overexpression of 53BP1 in MDA-MB-231 significantly inhibited microvessel sprouting, leading to significant inhibition of the formation of a meshwork of vessels around the aortic rings. Meanwhile, 53BP1 knockdown significantly promoted the formation of microvessel structures compared with MCF-7 control cells (P = 0.002 and P = 0.008, respectively).
53BP1 inhibited angiogenesis in vivo
To investigate the effects of 53BP1 on angiogenesis in vivo, we used a mouse xenograft breast tumor model. Our previous data demonstrated that 53BP1 significantly inhibited tumor growth in vivo. To further investigate whether 53BP1 inhibited tumor growth by suppressing angiogenesis, we used an anti-CD31 antibody to stain xenograft sections. As shown in Figure 3, the microvessel density (MVD) in 53BP1-OVE tumors was obviously less than that in the control group (5.1 ± 3.7 vs 21.4 ± 7.8; P < 0.001). A marked increase in the expression of CD31 was observed in the shRNA 53BP1 group compared with the control MCF-7 group (27.3 ± 5.9 vs 11.2 ± 4.3; P < 0.001). These results imply that 53BP1 might inhibit breast tumor growth by suppressing tumor angiogenesis.
Relationship between 53BP1 and angiogenesis markers in breast cancer
The above results demonstrate high expression of 53BP1 with decreased angiogenic ability. However, the relationship between 53BP1 expression and MVD in breast cancer tissues has not been previously characterized. We used the anti-53BP1, anti-CD31 antibody to stain 83 paraffin-embedded breast tissue samples. As shown in Figure 4, in the 41 cases of 53BP1-positive staining cases, there were 13 cases that were CD31 positive. However, in the cases of 44 53BP1-negative staining cases, 25 cases were CD31 positive. Therefore, we found that overexpression of 53BP1 was associated with lower microvessel density compared with the 53BP1 low-expressing tissues using the Chi-squared test (P = 0.012), suggesting that expression of 53BP1 is inversely correlated with MVD in clinical breast cancer samples.
To investigate the role of 53BP1 in angiogenesis, we also assessed the relationship between 53BP1, MMP-2 and MMP-9, which are commonly associated with angiogenesis and metastasis. Using immunohistochemistry staining, we found that in the above 53BP1-positive (41 cases) and 53BP1-negative (44 cases) groups, positive staining of MMP-2 was 14 and 26 cases, respectively. Also, there were 12 and 28 cases of MMP-9-positive staining, respectively. Using the Chi-squared test, MMP-2 and MMP-9 was significantly correlated with the expression of 53BP1 in tumor tissue, that is, the stronger the expression of 53BP1, the weaker the expression of MMP-2 and MMP-9 (P = 0.037 and P = 0.003, respectively) (Fig. 4).
53BP1 inhibited angiogenesis through the Akt pathway
In our previous study, we found that 53BP1 could suppress tumor growth of ovarian cancer cells through modulation of the Akt pathway.(6) Therefore, first we detected Akt and p-Akt in the 53BP1 transfected breast cancer cells. As shown in Figure 5(a), using western blot analysis, overexpression of 53BP1 could inhibit phosphorylation of Akt and knockdown of 53BP1 could activate the Akt pathway. Because Akt has been reported to regulate angiogenesis via MMP-2 and MMP-9[23, 24] and we found 53BP1 had a significant relationship with MMP-2 as well as MMP-9 in breast cancer tissues, we investigated the mRNA levels of the two markers in 53BP1 transfected cells. The levels of MMP-2 and MMP-9 were decreased in 53BP1-OVE cells and increased in 53BP1 knockdown MCF-7 cells (Fig. 5b).
To confirm the potential importance of Akt-mediated MMP-2 and MMP-9 signaling in 53BP1-mediated antiangiogenesis, we successfully transfected shRNA-53BP1 MCF-7 cells with siRNA Akt and control siRNA (Fig. 5c). The levels of MMP-2 and MMP-9 were reversed after knockdown of Akt in 53BP1 knockdown MCF-7 cells (Fig. 5d). 53BP1-mediated anti-endothelial cell tube formation and aortic microvessel sprouting was significantly inhibited by siRNA Akt in both shRNA-53BP1 MCF-7 and the control MCF-7 cells (Fig. 6).
53BP1 could inhibit the invasiveness of breast cancer and angiogenesis is one important element in the process. So to further investigate whether 53BP1 inhibited tumor invasion by suppressing angiogenesis, the transwell assay was used after knockdown of Akt. As shown in Figure 7, the migration ability of these cells was inhibited after transfecting the siRNA Akt. In addition, we also detected the effect of NF-κB in the antiangiogenesis of 53BP1. Although the invasion ability was changed, no statistical result was observed in the angiogenesis assay (data not shown). Taken together, these data suggest 53BP1 suppressed the angiogenesis and invasiveness of breast cancer, at least partially, though Akt-mediated MMP-9 and MMP-2.
Understanding the mechanisms leading to invasiveness and metastatic dissemination of cancer cells is crucial to the development of new therapeutic strategies. It is now considered that growth of both primary and secondary metastatic tumors requires a well developed set of blood vessels, that is, angiogenesis.[25, 26] Angiogenesis is a complex process. During angiogenesis, several steps are involved including degradation of the extracellular matrix, migration, proliferation, sprouting, elongation and tube formation of endothelial cells.[25, 27] Thus, inhibition of the steps of angiogenesis by blocking angiogenesis-related markers could be a strategy for cancer therapy.
Our previous studies have shown that 53BP1 is associated with prognosis in breast cancer. We also found that 53BP1 could inhibit the invasion of breast cancer. To further explore the detailed tumor suppressor function of 53BP1 in angiogenesis, the expression of 53BP1 in breast cancer cells was manipulated with ectopic expression or RNA interference. Our findings showed that transfection of 53BP1 could inhibit tube formation of HUVEC and vasculature architecture on the chorioallantoic membrane, as well as sprouting of the aortic ring, which resulted in the reduced migrative ability of MDA-MB-231 cells. Conversely, we found knockdown of 53BP1 in MCF-7 cells could significantly promote angiogenesis both in vitro and in vivo. To confirm this conclusion, we detected angiogenic markers including CD31, MMP-2 and MMP-9[28-31] in breast cancer samples using immunohistochemistry and showed statistically that 53BP1 was inversely correlated with CD31, MMP-2 and MMP-9.
To elucidate the molecular mechanism underlying 53BP1 in angiogenesis, we focused on the Akt signaling pathway, which is critical for cell proliferation, growth and angiogenesis of endothelial cells.[23, 32, 33] We found that 53BP1 inhibited the phosphorylation of Akt in 53BP1-overexpressed MDA-MB-231 cells and activated Akt in 53BP1 knockdown MCF-7 cells. Also, compared with siRNA control cells, tube formation and sprouting of the aortic ring were significantly inhibited in siRNA Akt cells. Taken together, these results suggest that 53BP1 is a potent inhibitor of angiogenesis and Akt signaling is a necessary event in 53BP1-induced antiangiogenesis. In addition, the enhanced migration ability of 53BP1 knockdown MCF-7 cells was decreased after transfection of siRNA Akt; this implied that 53BP1 could inhibit the invasion of breast cancer partially though antiangiogenesis.
To the best of our knowledge, this is the first study to demonstrate the potential role of 53BP1 in the angiogenesis of breast cancer. Our findings provide a new clue to developing novel therapeutic strategies that target 53BP1 by a genetic (antisense or siRNA) or pharmacological (small molecule) inhibitor. Further investigation is needed to determine whether targeting 53BP1 might be an effective novel target in breast cancer treatment.
This work was supported by the National Natural Science Foundation of China (No. 81072150; No. 81172529; and No. 81272903) and the Shandong Science and Technology Development Plan (No. 2012GZC22115).