INFLUENCE OF SURGICAL RESECTION ON PLASMA ENDOGLIN (CD105) LEVEL IN NON‑SMALL CELL LUNG CANCER PATIENTS

Endoglin (CD105) is a 180-kDa cell membrane glycoprotein which serves as a coreceptor for transforming growth factor (TGF)‑b1 or TGF‑b3, in the presence of the TGF‑b type II receptor (TbRII) [1]. Endoglin is highly expressed by activated endothelial cells (ECs) [2]. Hypoxia and TGF‑b are two main factors that coope­rate to induce its expression [3]. Endoglin promotes angiogenesis mainly by activation of vascular ECs proliferation [4]. Endoglin overexpressed on endothelia of vessels in several human solid malignancies [5] and its overexpression is associated with lower patient survival rates, presence of nodes metastases and distant metastatic disease [6].

Tumor vascular endothelium shows up-regulation of various receptors, including the vascular endothelial growth factor receptor 2 (VEGF R2) and angiopoietin receptor, Tie2 [7]. Two other endothelial receptors for angiogenic factors, the VEGF receptor 1 (VEGF R1) and the orphan receptor Tie 1, are also up regulated by hypoxia [7]. So far, two different mechanisms are known which lead to the formation of soluble receptors [8]. Firstly, soluble receptors can be translated from differentially spliced pre-mRNA molecules lacking the transmembrane domain (e. g., sVEGF R1) [9] and the second mechanism involves limited proteolysis in the extracellular domain of the membrane receptor leaving the ligand-binding domain intact (e. g., sTie2) [8]. The external domain can be cleaved or shed and released into the circulation [7].

Similarly to classical receptors, accessory receptor — endoglin exists in two forms: as a membrane-bound and a soluble (s‑endoglin) in the circulation. Recent findings [10] suggest that the ectodomain of endoglin is released through proteolytic cleavage by membrane-type 1 matrix metalloproteinase (MT1‑MMP) — matrix metalloproteinase‑14 (MMP‑14). Coexpression of endoglin with MMP‑14 on the cell membrane leads to the cleavage of endoglin at the glycine-leucine bond at position 586, releasing the nearly complete endoglin extracellular domain [10]. Hawinkels` et al. [10] study also shows that MMP‑14 is the most abundantly expressed MT‑MMP in ECs and that knockdown of MMP‑14 strongly reduces s‑endoglin levels in the conditioned media of these cells cultures. Local up‑regulation of endothelial MMP‑14 expression may increase s‑endoglin, decrease membrane-localized endoglin, and transform the endothelium to a quiescent state [10]. Similarly to endoglin, MMP‑14 is highly expressed not only by ECs, but also by several other cell types, i. e. by cells of lung tumor [11]. In Atkinson’s et al. [11] studies, among all MT‑MMPs (MMPs 14‑17, 24 and 25), MMP‑14, -15 and –17 displayed higher expression in tumor relative to normal lung specimens. In addition MMP‑14 mRNA expression strongly correlated to MMP‑14 proteolytic activity in tumor models. Therefore, s‑endoglin might be shed not only from endothelial cells, but also from tumor cells. To conclude, the level of s‑endoglin could be an indicator of tumor‑activated endothelium, but it depends on shedding proteases expression, mainly MMP‑14.

The soluble receptor displays biological activity by acting as a specific endogenous antagonist complexing the corresponding ligand and thus preventing the ligand-mediated signal transduction [8]; soluble receptors are capable of scavenging circulating ligands, e. g. VEGF can be bound to sVEGF R1 [9], Ang1 and Ang2 — to sTie2 [8]. The role of s‑endoglin is not yet clear, maybe it competes with TGF‑b for TbRII binding [12]. But it is known that s‑endoglin has antiangiogenic properties; it is capable of reducing spontaneous and VEGF‑induced angiogenesis. Also, s‑endoglin fused with the Fc fragment of human immunoglobulin G strongly reduces microvessel density in a mouse model of invasive ductal breast carcinoma [10]. Experiment of Le et al. [12] showed two diffe­rent oligomeric forms of recombinant s‑endoglin. The dimeric s‑endoglin enhanced TGF‑b signalling in U937 cells, in a dose‑dependent fashion. However, tetrameric s‑endoglin was not active in this system, thus, its biological relevance is not yet clear. This form of s‑endoglin might be a resting inactive form that can undergo conformational changes into dimeric or other active forms under certain activating conditions. They concluded that the recombinant s‑endoglin is capable of modulating TGF‑b signal effectively, thus, can potentially be applied for therapeutic purposes.

The quantification of soluble forms of receptors might be interesting in terms of diagnostic and/or prognostic, but also therapeutic applications. The aim of present study was to analyze the changes of s‑endoglin level in plasma of lung cancer patients and to estimate the correlation of s‑endoglin with other soluble receptors, sTie2 and sVEGF R1.

MATERIALS AND METHODS

Study population. The study included patients with stage I non-small cell lung cancer, who underwent tumor resection without any preoperative therapy. These patients were treated in the University Hospital Department of Thoracic Surgery and Tumors, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, during the years 2008–2010. Three blood samples were taken from each patient: one prior to surgery and others on postoperative days 7 and 30.

The study protocol was approved by the Ethical Committee of the Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń (Poland). All patients gave consent.

Blood sampling and processing. s‑Endoglin, sTie2, sVEGF R1 concentrations were evaluated in plasma. Two millilitres of blood were taken from elbow vein. EDTA was used as an anticoagulant. Within 30 min after the collection, the blood samples were centrifuged at 2–8°C for 15 min at 1000 x g. The plasma was stored at ‑70°C.

s‑Endoglin determination. s‑Endoglin, sTie2, sVEGF R1 concentrations were assayed by commercially available sandwich enzyme-linked immunosorbent assay kits from R&D Systems (Quantikine Human Endoglin/CD105, sTie2, sVEGF R1 Immunoassay, R&D Systems Inc., Minneapolis, USA). Kit is designed to measure human endoglin/CD105, sTie2, sVEGF R1 in cell culture supernates, serum, and plasma.

Statistical analysis was done using Wilcoxon signed-rank test and Pearson`s linear correlation. The results were considered statistically significant for p < 0.05.

RESULTS

In Table 1 the comparison of s‑endoglin concentration before surgical treatment of lung cancer patients and on 7^th day and 30^th day after tumor resection is presented. The median of s‑endoglin concentration decreased on 7^th day when compared with preoperative level (3212.0 vs. 4112.0 pg/ml; p < 0.0001) and then it increased on 30^th day to reach greater values than on 7^th day (4447.0 vs. 3212.0 pg/ml; p < 0.01), but it was comparable with pretreatment level (4447.0 vs. 4112.0 pg/ml; p = 0.478).

In this study the estimation of correlation between s‑endoglin and other soluble receptors was accomplished (Table 2). The correlation between s-endoglin and sTie2, both before surgery (r=0.44) and on postoperative day 7 (r=0.52) and on 30^th day (r=0.58) was high. However, s‑endoglin was correlated with sVEGF R1 only on postoperative day 7, this correlation was very high (r=0.75).

DISCUSSION

Endoglin is primarily expressed in proliferating vascular endothelial cells, and its expression increases during tumor angiogenesis. Such properties have made endoglin a reliable marker of various solid tumors vasculature [13–21].

Soluble endoglin in serum is also elevated in various cancers, including breast, colorectal and liver cancers, and it correlates with the presence of metastatic disease [22–25].

Serum s‑endoglin has also prognostic value. It is an indicator of prostate cancer metastasis to the pelvic lymph nodes and of biochemical recurrence after medical prostatectomy. In multivariate analysis, only endoglin and Gleason score, but not PSA or clinical stage, were predictive of lymph node metastases [26–28]. Elevated pretreatment plasma s‑endoglin level is predictive for decreased clinical benefit and a shorter overall survival in metastatic breast cancer patients treated with 2^nd-line hormone therapy [29].

Besides that, the high level of s‑endoglin decreased in patients receiving chemotherapy. Conventional chemotherapy regimens suppress endothelial cells in tumor vasculature and consequently inhibit the release of s‑endoglin from endothelial cells [23].

In our study, after surgical resection of lung cancer the level of plasma s‑endoglin decreased on 7^th day when compared with preoperative level, and next on 30^th day — increased and it was comparable with that before surgery intervention. This problem can be explained in the follo­wing way. The decrease of s‑endoglin probably is a consequence of resection of tumor mass, highly vasculated and expressing both endoglin and shedding enzyme, MMP-14. However, increase of s‑endoglin on 30^th day in comparison with postoperative day 7 might be the result of stimulation of ECs and circulating progenitor endothelial cells (EPCs) by various tumor-derived angiogenic factors (e.g., VEGF, Ang2), which levels in circulation after surgical treatment are increased [30, unpublished own data].

Myśliwiec et al. [24] received similar results: after surgical treatment of colorectal cancer s-endoglin level on postoperative day 3 decreased when compared with preoperative level, then it increased on day 10 to reach greater values than on postoperative day 3, but lower than preoperative point. They explain these changes as follows: decrease of s‑endoglin level after surgery might be at least in part due to the action of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). TNF‑alpha has been reported to down-regulate endoglin at post-transcriptional level [31].

In our study there were interesting correlations between plasma s‑endoglin levels with other soluble form of receptors. s‑Endoglin correlated with sTie2 both before and after surgery (BEFORE: r=0.44, AFTER (day 7): r=0.52, AFTER (day 30): r=0.58) and with sVEGF R1 — only on postoperative day 7 (r=0.75). These two soluble receptors are formed in different manner: sTie2, similarly to s‑endoglin is produced by proteolytic proces­sing, however, sVEGF R1 derived predominantly from alternative splicing. This could explain above correlations of s‑endoglin: with sTie2 — constant, and with sVEGF R1 — only in one investigated point.

The increased level of serum endoglin in various cancers compared to controls, prognostic value of this angiogenic factor and changes of its level after chemotherapy or surgical treatment suggest potential application of soluble form of endoglin as tumor marker in the future.

ACKNOWLEDGEMENT

The work was supported by the subsidy for financing the statutory activity of Nicolaus Copernicus University in Toruń, number 461/2010.

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