Angiopoietins lack of prognostic significance in ductal mammary carcinoma

Angiopoietins (Ang) have been shown to regulate the process of vasculature and angiogenesis in tumour. Different angiopoietins have different roles during the angiogenic process. The current study sought to examine the levels of the expression of Ang-1, Ang-2, Ang-3 and their receptor Tie-2 in mammary ductal carcinoma and to assess their relevance to prognosis. Fresh frozen ductal carcinoma tissues (n = 90) and adjacent non-cancerous breast tissues (n = 32) were used. The expression of Ang-1, Ang-2 and Ang-3 transcripts in cancer and normal breast tissues were examined quantitatively using quantitative RT-PCR. The protein expression of Ang-1, Ang-2 and Tie-2 was assessed by immunohistochemistry on frozen sectioned tissues. Ang-1, Ang-2 and Ang-3 were detected in mammary tissues. Ang-1 was seen in both normal epithelial cells, breast cancer cells as well as in endothelial cells. Ang-2 was seen at a higher level than Ang-1 and it is expressed in epithelial, endothelial as well as stromal cells to certain degree. Ang-1 and Ang-2 transcripts were detected almost equally in cancer and normal breast tissue, and Ang-3 was high in cancer tissue compared to normal breast but not significant (155 ± 123 & 24.1 ± 22.6, P > 0.05). No significant differences were seen between patients with different predicted prognosis (using the Nottingham Prognostic Index as a guide) (Ang-1 p = 0.34, Ang-2 p = 0.26 and Ang-3 p = 0.32, respectively). No significant correlation was seen between Ang-1, Ang-2 and Ang-3 with tumour grade. When the levels of the transcripts were compared against clinical outcome (disease free, developed recurrence and patients who died of breast cancer), levels of Ang-3 transcript was found to be high in breast cancer patient who had bone metastasis 33.8 ± 28.3, although the difference was not significant (p = 0.08). No significant difference was seen with levels of Ang-1 and Ang-2 transcripts and clinical outcomes. Furthermore, no significant trend was observed between Tie-2 receptor and clinical/pathological parameters in the cohort. These data suggest that angiopoietins (Ang-1, Ang-2 and Ang-3) are expressed in mammary tissues, both in normal and tumour. These molecules have limited value in predicting the prognosis and clinical outcome in patients with mammary ductal carcinoma.


Background
Angiogenesis, generation of new microvessels from preexisting blood vessels, is essential for tumour growth and invasion [1]. Cancer cells stimulate angiogenesis by secreting angiogenic factors, such as vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and fibroblast growth factor (FGF) [2]. Angiopoietins, a new family of angiogenic growth factors that are mostly specific for the vascular endothelium, have been identified in recent years [3,4]. Ang-1 plays a role in maintaining and stabilizing mature vessels by promoting the interaction between endothelial cells and surrounding support cells, whereas Ang-2 is expressed at sites of vascular remodelling and is thought to antagonise the stabilising action of Ang-1 [5,6]. Ang-1 and Ang-2 share about 60% amino acid identity and bind with similar affinity to the endothelial cell tyrosine kinase receptor Tie2 [4,7]. Angiopoietins and Tie-2 are related to vascular remodelling and sprouting, which occur in a complementary and coordinated fashion during vascular development, along with vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors (Flk-1 and Flt-1) [8].
Breast cancer is the most common form of malignancies in females in the U.K, and metastasis of breast cancer is common. About 7% of patients with breast cancer present with widespread metastases at the initial presentation [9]. The most common sites of metastasis are bone, lungs, liver, chest wall and central nervous system. Less common sites are the adrenals, ovaries, pericardium, thyroid and bone marrow [10]. Tumour cell dissemination is mediated via a number of mechanisms, including local tissue invasion, haematogenous and/or lymphatic spread as well as direct seeding of surfaces or body cavities. Angiogenesis plays a pivotal role in the vascular spread of breast cancer as well as key to the growth of breast tumours.
Up-regulation of angiopoietins expression has been noted in many malignant cancers such as gastric carcinoma [11,12], colorectal cancer [13], hepatocellular carcinoma [14], renal cell carcinoma [15], ovarian cancer [16] and non-small lung cancer [17]. Although a few reports on the expression of angiopoietins in breast cancer have become available in recent years [18,19], these studies provide little conclusive evidence of the correlation between these molecules and breast cancer progression.
In the present study we examined the expression Ang-1, Ang-2 and for the first time Ang-3 and their receptor Tie-2, at mRNA and protein levels in human ductal mammary carcinomas and investigated the correlation between the level of the expression of these molecules and clinical/ pathological parameters of breast cancer.

Tissue collection and preparation
Ductal cancer tissues of the breast (n = 90) were collected immediately after surgery and stored in a deep freezer until use. 'Normal' (adjacent non-cancerous) (n = 32), which were from the same patients with breast cancer and away from tumour margins were also collected. Patients were routinely followed clinically after surgery as previously described [20]. The median followup of the cohort was 120 months.

Tissue processing, RNA extraction and cDNA synthesis
Total RNA was isolated from frozen-sectioned breast tissues ('normal' (adjacent non-cancerous) and cancer). Multiple sections were homogenised to extract RNA, using RNA-Zol reagent (ABgene, Epsom, Surrey, UK), according to manufacturer's instructions, as we previously reported [21]. cDNA was generated from 1 μg RNA using an AMVreverse transcription kit (ABgene, Epsom, Surrey, UK), and the standard gunanidine isothiocyanate method as described in the manufacturer's protocol. The purity and concentration of RNA was determined by spectrophotometry at 260 nm and 280 nm.
Conventional PCR primers were designed using Beacon Designer software (Palo Alto, California, USA), to allow amplification of regions that have no overlap with known genes and span at least one intron. Primers were synthesized by Life Technologies (Paisley, Scotland, UK) ( Table  1).

Quantitative analysis of the Ang-1, Ang-2, Ang-3 and Tie-2 transcripts in tumour and normal breast Tissues
We employed the iCycler IQ system (BioRad, Camberley, UK), to quantify the transcript level (shows as copies/μl calculated from internal standard) of the Ang-1, Ang-2, Ang-3 and Tie-2, as we previously reported [20]. The system used a universal probe (UniPrimer™), which recognised a specific sequence (z sequence), which had been

Immunohistochemistry
This was as we recently reported [22]. Briefly, immunohistochemical staining was performed on paired frozen-sectioned tissues (cancer tissue paired with normal background tissue from the same patient). Frozen sections were cut, air dried and fixed in 50% methanol and 50% acetone for 15 minutes. The sections were then air dried once more for 10 minutes and stored at -20°C in foilwrapped slide trays. Immediately before staining specimens were then placed in PBS (Optimax wash buffer) for 5 minutes. The slides were incubated with primary rabbit polyclonal antibodies against Ang-1, Ang-2 and Tie-2 (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA) or positive control at 1:50 dilution for 1 hour. After 4 washes with PBS, the slides were placed in universal multi-link biotinylated (Dako). Secondary antibody at (1:100) dilution and incubated for 30 minutes. This was followed by 4 washes with PBS. Slides were then placed in avidin biotin complex (ABC -Vector Labs) for 30 minutes. The bound antibody complex was detected using diaminobenzidine tetrahydrochloride (3,3'-diaminobenzidine)-DAB (Sigma), chromogen for 5 minutes. The slides were washed with H 2 O for 5 minutes and placed in Mayer's haematoxylin for 1 minute, followed by differentiation in H 2 O for 10 minutes. This was followed by dehydration in methanol (3 times) and clearing in 2 changes of xylene before mounting under cover slip and examined on the microscope negative controls (using PBS buffer instead of the primary antibody) and positive controls were used in this study. The complete procedure was carried out at room temperature.

Statistical analysis
Statistical analysis was carried out using Minitab version using a Macro written for the study. Statistical analysis was performed using a Student's t-test.
The level of the Tie-2 receptor showed no significant difference between tumour and background (tumour: 7.1 ± 4.7 vs background: 7.1 ± 7.4, P = 0.87). Although there were marginally higher levels of the Tie-2 receptor in node positive tumours, this did not reach significance (median value node positive: 11.5 vs median value node negative: 2.9 P = 0.37). Again, the differences were not significant p > 0.05 ( Figure  1C).

Correlation of Ang-1, Ang-2, Ang-3 and their receptor Tie-2 with prognosis and staging
The Nottingham Prognostic Index (NPI) was used as one parameter to assess the prognosis of the patients, in that patients with NPI value less than 3.4 (NPI-1) were regarded as with good prognosis, 3.  (Figure 2A). The levels of the molecules were also analyzed against TNM status. No significant difference was seen between any groups for all three Angs ( Figure 2B).

Expression of Ang-1, Ang2, Ang-3, Tie-2 and clinical outcome following 10-year follow-up
When we combined the three groups (with metastasis, recurrence, and mortality) to form a poor prognostic group (referred to as Poor Prog. in Figure 4D) and compared this group with those who remained diseasefree ( Figure 4D), higher levels of all Angiopoietins molecules were found in the patients with poor prognosis (Ang-1 422 ± 421, Ang-2 416 ± 268, Ang-3 508 ± 502) although this did not reached a significance. Figure 5 shows weak staining of Ang-1, Ang-2 and Tie-2 compared with breast cancer. While the normal breast tissues duct showed strong staining of Ang-1, Ang-2 and Tie-2 compared with breast cancer duct epithelial cells.

Discussion
Tumour metastasis and rapid growth is critically dependent on blood or lymphatic vessels routes. Prognosis of solid tumours are closely linked to angiogenic or lymphangiogenic factors. Despite numerous studies examining the angiopoietins in breast tumours, to our best knowledge the current study is the first study reporting the relationship of all three angiopoietins and Tie-2 with clinical/pathological of ductal carcinoma of the breast. The current study has reported that the expression of Angs and Tie-2 were almost similar in ductal carcinoma and normal background tissues, with the exception of Ang-3 which showed higher levels of expression in breast cancer compared with normal breast tissues.
In the current study, we failed to detect a significant difference when a number of clinical and pathological factors Angs and Tie2 transcript and tumour grade Figure 3 Angs and Tie2 transcript and tumour grade. Grades 3 breast tumours demonstrate higher levels of Ang-1, Ang-3 and Tie-2 compared to Grade 1. Ang-2 transcripts level raised in grade 2 breast tumours.
were considered. Furthermore, all three angiopoietins showed a marginal higher level of the respective transcripts in advanced breast cancer (NPI3 & Grade-3), although this did not reach statistical significance. Our findings are consistent with Currie et al [18] who showed no significant correlation between Tie2 (or the Angs) with most clinic/pathological indices in breast cancer.
Although their study demonstrated a significant correlation between oestrogen receptor (ER) status and both Tie2 and Ang-4 expression, our results did not show this correlation which is in agreement with Tautsui et al [19].
It is noteworthy from the present study that low levels of Angs and their receptor Tie-2 were found in patients who Q PCR analysis shows association between Angiopoietins and clinical outcome in breast cancer Figure 4 Q PCR analysis shows association between Angiopoietins and clinical outcome in breast cancer. (A&B): Patients who developed metastatic disease and local recurrence have low levels of Angs and Tie-2 transcripts than who diseases free. (C): Patients who died from breast cancer showed higher levels of Ang-1and Ang-3 copies than who diseases free (p > 0.05), whereas Ang-2 and Tie-2 had higher levels of expression in diseases free compared who died from breast cancer (p > 0.05). (D): Comparison between patients who remained disease free and those who developed breast cancer related progression (recurrence, metastasis and mortality). Ang-1 and Ang-3 transcripts had higher levels in poor prognosis patients in relation to diseases free patients, and again Ang-2 and Tie-2 transcripts had higher levels of expression in diseases free compared to poor prognosis. Neither reached a statistical significance.
had cancer metastasis and local recurrence compared to diseases free after 10 years fellow up. Patients who died from breast cancer had higher levels of only Ang-1 and Ang-3. These findings are in contrast with Sfiligoi et al. [23] who reported that Ang-2 associates with tumour aggressiveness, whereas Ang-1 does not. Moreover, previous studies have demonstrated that the expression of Angs has a strong prognostic significance in breast cancer and correlates with MVD and VEGF expression [19,23], while Currie et al. [18] found no significant relation between Ang-2 expression and MVD in breast cancer. One of the reasons for these discrepancies may be the difference in methodologies and the sample size used in the respective studies. The current study was limited by its sample number. A study with bigger cohort will certainly help to further clarify the observations.
Despite some studies show a significant correlation between Angs expression and clinic/pathological parameters in cancers other than breast cancer including gastric, liver and colorectal cancer [11,12,14]. It is clear from the present study and indeed other limited reports [18] that the case in ductal carcinoma of the breast is far from being clear. A number of possibilities exist. Firstly, different organs may have a difference vasculature network. In ear-lier studies examining the mechanism of human breast cancer angiogenesis, it has been demonstrated that endothelial cell proliferation is a relatively rare event in breast cancer [24,25]. Secondly, breast cancer is a heterogeneous disease whose clinical outcome encompasses a wide spectrum of possibilities from definitive cure to early death. Axillary nodal status has been shown to be one of the most important prognostic factors in the patients, with up to 30% of node-negative patients eventually relapse.
Taken together, although angiopoietins have been shown to be expressed at higher levels in GI tumours, the same has not been seen in breast cancer as reported here. It is possible therefore that angiopoietins and Tie2 play a lesser role in the progression of breast, compared with other angiogenic factors and indeed lymphangiogenic factors as widely reported. In summary, although our study shows high levels of Angs mRNA in advanced breast tumour such as in Grade-3 and NPI3 compared with early stage of cancer (Grade-1&NPI1), no significant difference is obtained. We conclude therefore that these molecules may not be significant prognostic factors in ductal carcinoma of the breast. The observation warrant further studies in larger cohorts.
Immunostaining of normal background breast tissue (left Panel) and breast cancer tissue (Right Panel) for Ang-1, Ang-2 and Tie-2 (original magnification, 40×, 100×; 200× and 400×) Figure 5 Immunostaining of normal background breast tissue (left Panel) and breast cancer tissue (Right Panel) for Ang-1, Ang-2 and Tie-2 (original magnification, 40×, 100×; 200× and 400×). A higher intensity of staining was noted in epithelial cells of breast cancer compared with non-cancerous mammary tissue. Strong staining of Ang-1, Ang-2 and Tie-2 is shown in normal breast duct compared to breast cancer duct.
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