Angiogenic markers have been associated with tumor growth and invasiveness in almost every type of cancer and their study provides useful insights of their role in determining tumor behavior. Among these angiogenic and growth factors, the vascular endothelial growth factor (VEGF) is the most important usually studied in oncological malignances, as well as in pituitary tumors (28–30).
It has been demonstrated that VEGF regulates the synthesis and secretion of the endothelial cell-specific molecule-1, endocan or ESM1 (13). Endocan is a secreted proteoglycan considered a biomarker of both neovascularization and tumor progression in hepatocarcinoma and renal carcinoma among other cancers (11, 31). Furthermore, not only the pro-angiogenic molecule VEGF but also FGF2 modulate endocan expression (32). Importantly, the dermatan chain of endocan can bind to different growth factors and enhance their biological activity (33), positioning this molecule as an important player in the complex network which regulates angiogenesis and proliferation.
In this context, we evaluated ESM1 mRNA expression in human pituitary tumors and its relation to sinus invasion and angiogenic markers. We determined that not all tumors analyzed expressed this gene, but interestingly, in ESM1- positive samples FGF2 mRNA levels were significantly augmented, suggesting an association of these two angiogenic factors in the tumoral pituitary, as previously described in mesotheliomas (32).
There is growing evidence that under VEGF and FGF2 influence, endocan may promote pituitary tumor cell proliferation as well as endothelial cell growth. Indeed, silencing the endocan gene, Esm1, in GH3 and MMQ prolactin secreting cells downregulated FGF2 and VEGFR2 among other angiogenic proteins causing reduced cell viability (34). Moreover, the knockdown of Esm1 in HUVEC endothelial cells inhibited tube formation and increased sensitivity to the anti-angiogenic Avastin treatment in vitro, indicating endocan could be a molecular target for pituitary adenoma treatment (34). Additionally, in human pituitary adenoma cells in vitro, silencing FGF2 inhibited cell proliferation and invasion capacity (35). Taken together the present and other results point to endocan and FGF2 as potential biomarkers of pituitary tumor progression.
Opposed to the higher FGF2 levels found in ESM1 + samples, PDGF mRNA levels were slightly lower in endocan expressing tumors of our cohort, yielding a strong negative correlation between FGF2 and PDGF expression. There is a scarcity of information on the role of PDGF in the pituitary, and little evidence of PDGF involvement on pituitary adenoma progression. Some studies detected mRNA expression of PDGF and its receptor in human pituitary tissue (36), as well as in the murine folliculo-stellate pituitary cell line TtT/GF cells. In this cell line, PDGF increased VEGF secretion via PDFG receptor (19). Our data show an inverse expression of FGF2 and PDGF, which may probably reflect a complex but precise balance of the four angiogenic factors in pituitary tumorigenesis. No previous data in the literature analyzed the relation between endocan and VEGF, FGF2 or PDGF angiogenic factors, and FGF2 and PDGF expression levels were not compared before in pituitary tumor samples. This pattern, showing positive correlation of endocan and FGF2 and inverse correlation of FGF2 and PDGF expression in the pituitary tumor context could be related to the peculiarities that the angiogenic process has in these tumors, as we and other authors already demonstrated (5, 37, 38). It may be associated to the low grade of malignancy of these tumors in which not all angiogenic factors are simultaneously upregulated.
In turn, endocan association with pituitary tumor invasiveness evidences inconsistencies in the literature. Determined by immunohistochemistry, some authors found that endocan expression in tumor cells but not in endothelial cells, correlated with tumor grade invasion (18). Instead, in another study only endothelial endocan expression correlated with tumor size and recurrence, both signs of aggressiveness, and no correlation with invasion was found (17). Similarly, no relation of endocan and cavernous sinus invasion or resistance to treatment with first-generation somatostatin analogues was found in acromegaly patients (39), though endocan may participate in the resistance of prolactinomas to dopamine therapy (34).
It is worth mentioning, that we evaluated by qRT-PCR the mRNA expression levels of endocan, which identifies the intratumoral ESM1 gene being transcribed. Many reports correlate high ESM1 mRNA levels with poor prognosis and also with metastasis in different cancers such as breast, colon, renal, bladder and others (40–42), but no studies correlating mRNA and aggressiveness in pituitary adenomas have been performed. When we classified tumors by the Knosp grade system (0–4 grade), a correlation between ESM1 and tumor invasion was observed when invasion was considered in the first stages of tumor development, from 0 to 3 Knosp grade in the pituitary tumor cohort (r = 0.464 and p = 0.0526). We do not discard an effect of the small number of invasive tumors in the cohort or heterogeneity within the tumors. Even so, it is probable that ESM1 (probably by its orchestrated relation with VEGF, FGF2 and PDGF) could be implicated in the angiogenic and proliferative process within these tumors in the first period of invasion (eg. passing through 2 to 3 Knosp grade) and endocan would not increase further in the truly invasive phase of pituitary adenomas (Knosp grade 4). Interestingly, Almong et al. described endocan was markedly expressed in concordance with the switch between dormant to fast growing phenotype in experimental angiogenic tumors (15). Moreover, the enhanced FGF2 expression we found in ESM1 positive tumors could be supporting the fast-growing state in these tumors as FGF2 has been linked to invasion in cancer and in pituitary adenomas as well (35, 43).
Antiangiogenic therapy has been used to treat aggressive pituitary tumors in humans (22, 23), and many studies were performed in experimental models, mainly with anti-VEGF therapies (5, 44, 45). The study of the expression and action of different angiogenic biomarkers such as endocan, FGF2 and PDGF, together with VEGF, will contribute in designing targeted treatment of aggressive and resistant pituitary tumors. Importantly, many compensatory mechanisms in tumors may uncover when VEGF pathway is being blocked (46). Treatment of patients with aggressive tumors is therefore challenging since conventional pharmacological agents often fail. Moreover, invasion into the cavernous sinus is frequently cause of incomplete resection, which in turn results in higher recurrence rate and therefore, determining biomarkers of invasion is paramount.