INTRODUCTION Prolactinomas are the most frequent pituitary tumor subtype. Despite most of them respond to medical treatment, a proportion are resistant and become a challenge in clinical management. Wnt/β-Catenin pathway has been implicated in several cancers including pituitary tumors and other sellar region malignancies. Interestingly, Wnt/β-Catenin inhibition augments the cytotoxicity of the chemotherapeutic agent Temozolomide (TMZ) in different cancers. TMZ is now being implemented as rescue therapy for aggressive pituitary adenoma treatment. However, the molecular mechanisms associated with TMZ action in pituitary tumors remain unclear. OBJECTIVES Our aims in the present study were to evaluate differential β-Catenin expression in human resistant prolactinomas and Wnt/β-Catenin signaling activation and involvement in Prolactin (PRL) secreting experimental models treated with TMZ. RESULTS We first evaluated by immunohistochemistry β-Catenin localization in human resistant prolactinomas in which we demonstrated reduced membrane β-Catenin in prolactinoma cells compared to normal pituitaries, independently of the Ki-67 proliferation indexes. In turn, in vivo 15 mg/kg of orally administered TMZ markedly reduced PRL production and increased prolactinoma cell apoptosis in mice bearing xenografted prolactinomas. Intratumoral β-Catenin strongly correlated with Prl and Cyclin D1, and importantly, TMZ downregulated both β-Catenin and Cyclin D1, supporting their significance in prolactinoma growth and as candidates of therapeutic targets. When tested in vitro, TMZ directly reduced MMQ cell viability, increased apoptosis and produced G2/M cell cycle arrest. Remarkably, β-Catenin activation and VEGF secretion were inhibited by TMZ in vitro. CONCLUSIONS We concluded that dopamine resistant prolactinomas undergo a β-Catenin relocalization in relation to normal pituitaries and that TMZ restrains experimental prolactinoma tumorigenicity by reducing PRL production and β-Catenin activation. Together, our findings contribute to the understanding of Wnt/β-Catenin implication in prolactinoma maintenance and TMZ therapy, opening the opportunity of new treatment strategies for aggressive and resistant pituitary tumors.

Clinically silent corticotroph tumors are usually macroadenomas that comprise 20% of ACTH tumors. They frequently progress to aggressive tumors with high recurrence, invasiveness, and on rare occasions, they may become hormonally active causing Cushing’s disease. Trustable biomarkers that can predict their aggressive course, as well as their response to traditional or new therapies, are paramount. Aberrant β-Catenin expression and localization have been proposed as responsible for several malignancies including pituitary tumors. Nevertheless, the role of β-Catenin in the aggressive transformation of silent corticotropinomas and their response to Temozolomide salvage treatment have not been explored yet. In this work, we present a case of a silent corticotroph tumor that invaded cavernous sinus and compressed optic chiasm and, after a first total resection and tumor remission it recurred six years later as an aggressive ACTH-secreting tumor. This lesion grew with carotid compromise and caused Cushing’s signs. It required multiple medical treatments including Cabergoline, Ketoconazole, TMZ, and radiotherapy. Besides, other two surgeries were needed until it could be controlled. Interestingly, we found α-SMA vascular area reduction and differential β-Catenin cell localization in the more aggressive tumor stages characterized by high Ki-67 indexes and p53 expression. Our results may indicate a role of angiogenesis and β-Catenin trigged events in the pituitary tumor progression, which could in turn affect the response to TMZ and/or conventional treatments. These molecular findings in this unusual case could be useful for future management of aggressive pituitary tumors.

Background: Pituitary adenomas can become invasive and aggressive, and in turn produce tumor recurrence. Endocan or endothelial cell specific molecule-1 (ESM1) has been associated with angiogenesis and tumor growth in gliomas, and lung, kidney, liver among other cancers, but there is a scarcity of information regarding its role in pituitary adenomas. In the search for biomarkers of tumor behavior our objective was to determine the relationship between endocan (ESM1) mRNA expression and cavernous sinus invasion, and its association with gene expression of VEGF , FGF2 and PDGF angiogenic factors in human pituitary adenomas. Methods : ESM1 , VEGF , FGF2 and PDGF expression was determined by qRT-PCR in 28 pituitary tumor samples and tumor invasion was determined by the Knosp grade classification. Results were associated with clinical data. Results : We found that ESM1 was expressed in 75% of the tumors. FGF2 expression was higher in ESM1 -positive compared to ESM1 -negative tumors, and a significant negative correlation in the expression of FGF2 and PDGF was found. Although ESM1 expression did not show any differences in invasive and non-invasive tumors, a positive correlation between ESM1 and Knosp grade was reached when grade 0-3 tumors were considered, suggesting its participation in the initial periods of tumor invasion. Finally, no difference in ESM1 expression was found between functioning and non-functioning adenomas, or patients age. Conclusions: Our study points to a precise pattern of angiogenic factor expression in pituitary tumors, and its relation to invasive behavior, which should be considered in the tailoring of drug treatments for aggressive and resistant pituitary adenomas.

The role of angiogenesis in pituitary tumor development has been questioned, as pituitary tumors have been usually found to be less vascularized than the normal pituitary tissue. Nevertheless, a significantly higher degree of vasculature has been shown in invasive or macropituitary prolactinomas when compared to noninvasive and microprolactinomas. Many growth factors and their receptors are involved in pituitary tumor development. For example, VEGF, FGF-2, FGFR1, and PTTG, which give a particular vascular phenotype, are modified in human and experimental pituitary adenomas of different histotypes. In particular, vascular endothelial growth factor, VEGF, the central mediator of angiogenesis in endocrine glands, was encountered in experimental and human pituitary tumors at different levels of expression and, in particular, was higher in dopamine agonist resistant prolactinomas. Furthermore, several anti-VEGF techniques lowered tumor burden in human and experimental pituitary adenomas. Therefore, even though the role of angiogenesis in pituitary adenomas is contentious, VEGF, making permeable pituitary endothelia, might contribute to adequate temporal vascular supply and mechanisms other than endothelial cell proliferation. The study of angiogenic factor expression in aggressive prolactinomas with resistance to dopamine agonists will yield important data in the search of therapeutical alternatives.

Preclinical and clinical studies support that Notch signaling may play an important oncogenic role in cancer, but there is scarce information for pituitary tumors. We therefore undertook a functional study to evaluate Notch participation in pituitary adenoma growth. Tumors generated in Nude mice by subcutaneous GH3 somatolactotrope cell injection were treated in vivo with DAPT, a γ-secretase inhibitor, thus inactivating Notch signaling. This treatment led to pituitary tumor reduction, lower prolactin and GH tumor content and a decrease in angiogenesis. Furthermore, in silico transcriptomic and epigenomic analyses uncovered several tumor suppressor genes related to Notch signaling in pituitary tissue, namely Btg2, Nr4a1, Men1, Zfp36 and Cnot1. Gene evaluation suggested that Btg2, Nr4a1 and Cnot1 may be possible players in GH3 xenograft growth. Btg2 mRNA expression was lower in GH3 tumors compared to the parental line, and DAPT increased its expression levels in the tumor in parallel with the inhibition of its volume. Cnot1 mRNA levels were also increased in the pituitary xenografts by DAPT treatment. And the Nr4a1 gene was lower in tumors compared to the parental line, though not modified by DAPT. Finally, because DAPT in vivo may also be acting on tumor microenvironment, we determined the direct effect of DAPT on GH3 cells in vitro. We found that DAPT decreases the proliferative, secretory and migration potential of GH3 cells. These results position selective interruption of Notch signaling as a potential therapeutic tool in adjuvant treatments for aggressive or resistant pituitary tumors.

Anti-VEGF therapy is being implemented as second line drug for aggressive pituitary adenomas that do not respond to conventional treatments. Although it is approved for treatment of several cancers, many of them acquire resistance. In the present work, we evaluated Bevacizumab (BVZ) effect in an in vivo model of prolactinoma in different stages of tumor progression. Immunodeficient nu/nu mice were injected subcutaneously with MMQ tumoral lactotrophs and once xenografts developed, mice were divided in two groups: ‘Early Onset Treatment (EOT)’ or ‘Late Onset Treatment (LOT)’ depending on tumor size at the start of treatment (smaller than 30 mm3 or larger than 60 mm3, respectively). BVZ (25 mg/kg) or saline solution as control were administered twice a week for 2 weeks. When BVZ was administered in EOT group, treated mice showed a slower tumor growth rate than controls since the second week of treatment, although no significant differences in tumor volume were determined at the moment of sacrifice. On the contrary, this beneficial effect was not observed in LOT mice, which increased significantly tumor size in response to BVZ treatment (N=8 per condition in each group, p<0,05). In order to study the mechanisms of resistance involved in late stages of tumor progression, we analyzed VEGF serum levels, which augmented in LOT mice but were not modulated in EOT mice under BVZ treatment (ELISA, N=8, p<0,05). Furthermore, we detected a significant correlation between serum VEGF levels and tumor growth when all animals were considered (r=0,53, N=32, p<0,05). However, no differences were found when analyzing VEGF and bFGF intratumoral content between BVZ treated and controls animals from neither of both groups (ELISA, N=8, ns). A slight increase in SMA+ vessel size but not in their number was determined in BVZ treated animals from EOT group. On the other side, vessels of LOT treated mice tended to be smaller than controls (IHQ, N=8, ns). PCNA proliferation marker expression was increased in tumors which augmented their size in response to BVZ (WB, N=8, p<0,05) and it correlated with tumor growth when mice were evaluated altogether (r=0,4, N=32, p<0,05). Furthermore, we found that proliferation associated proteins as β-CATENIN and C-MYC were increased in BVZ treated-tumors from LOT but not from EOT group (WB, N=8, p<0,05). Tumor necrotic area showed a trend of reduction in BVZ-treated LOT mice but not in EOT mice regarding to controls (H&E, N=8, ns). Our results suggest that tumor progression stage impacts on BVZ response in prolactinomas and that the resistance mechanisms developed in later stages could be associated, not only with the activation of VEGF autocrine loop as observed, but also with the regulation of other cell processes as proliferation and cell death. Further insight into the mediators involved in these mechanisms could help to predict which patients could respond or not to anti-VEGF treatment.

Pituitary adenomas are among the most frequent intracranial neoplasms and treatment depends on tumor subtype and clinical features. Unfortunately, non responder cases occur, then new molecular targets are needed. Notch system component expression and activation data are scarce in pituitary tumorigenesis, we therefore aimed to characterize Notch system in pituitary tumors of different histotype. In human pituitary adenomas we showed NOTCH1-4 receptors, JAGGED1 ligand and HES1 target gene expression with positive correlations between NOTCH1,2,4 and HES1, and NOTCH3 and JAGGED1 denoting Notch system activation in a subset of tumors. Importantly, NOTCH3 positive cells were higher in corticotropinomas and somatotropinomas compared to non functioning adenomas. In accordance, Notch activation was evidenced in AtT20 tumor corticotropes, with higher levels of NOTCH1-3 active domains, Jagged1 and Hes1 compared to normal pituitary. In the prolactinoma cell lines GH3 and MMQ, in vivo GH3 tumors and normal glands, Notch system activation was lower than in corticotropes. In MMQ cells only the NOTCH2 active domain was increased, whereas NOTCH1 active domain was higher in GH3 tumors. High levels of Jagged1 and Dll1 were found solely in GH3 cells, and Hes1, Hey1 and Hey2 were expressed in a model dependent pattern. Prolactinomas harbored by lacDrd2KO mice expressed high levels of NOTCH1 active domain and reduced Hes1. We show a differential expression of Notch system components in tumoral and normal pituitaries and specific Notch system involvement depending on adenoma histotype, with higher activation in corticotropinomas. These data suggest that targeting Notch pathway may benefit non responder pituitary adenomas.