FLNA expression modulates pathological markers of pituitary neuroendocrine tumours.

Due to the current limited knowledge about the role of filamin A (FLNA) in pituitary tumour progression, we aimed to analyse FLNA expression levels and its impact on aggressive markers of pituitary neuroendocrine tumours (PitNETs), using an integrative approach of in vivo and in vitro models and human samples. An increase in the expression levels of FLNA was observed in the advanced tumoural stages of the hyperplastic adenomatous pituitary model, concomitant with a decrease in cell proliferation and with a modification in the subcellular localisation of this protein. Similarly, overexpression of FLNA in the somatolactotropic GH3 cell line induced a decrease in the cell proliferation, promoted a migratory phenotype, increased invasion activity, and decreased the prolactin secretion. Cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4) expression increased in both models in correlation with the increase observed in FLNA levels. When human tissues were analysed a significant increase of FLNA was observed in PitNETs compared to normal pituitary gland, with heterogeneous intracellular localisation. Higher levels of FLNA expression were observed in tumours with invasive characteristics. These results underline the crucial roles of FLNA as a modulator of pathological markers and as a potential prognostic marker in pituitary tumours.

Ultrastructural and Molecular Evidence of Macroautophagy in Functioning PitNETs and Experimental Pituitary Tumors.

INTRODUCTION: Macroautophagy is a lysosome-mediated degradation process that controls the quality of cytoplasmic components and organelles, with its regulation depending on autophagy-related proteins (Atg) and with Beclin1/Atg6 and microtubule-associated protein light chain 3 (LC3/Atg8) being key players in the mammalian autophagy. As reports on this mechanism in the field of pituitary neuropathology and neuroendocrinology are scarce, our study analyzed the ultrastructural signs of macroautophagy and the expression of Beclin1 and LC3 proteins in human functioning PitNETs and in experimental pituitary tumors. METHODS: A group of humans functioning PitNETs and an experimental lactotroph model in rats of the F344 strain stimulated with estradiol benzoate (BE) were used. Ultrastructural and molecular evidence of the macroautophagic process was evaluated using different techniques. RESULTS: In functioning PitNETs cohort, 60% exhibited evidence of macroautophagy, with a significant difference found for Beclin1 and LC3 between macro- and micro-PitNETs (p < 0.05). In the experimental model, the expression of both Beclin1 and LC3 proteins was immunopositive in normal and tumoral glands when analyzed by immunofluorescence, Western blot, and immunohistochemistry. In the experimental model, protein expression was associated with increased glandular size and weight. CONCLUSIONS: Our study revealed evidence of macroautophagy at the pituitary level and the important role of Beclin1 and LC3 in the progression of functioning PitNETs, implying that this mechanism participate in regulating pituitary cell growth.

Oophorectomy improves pituitary activin inhibitory function preventing lactotroph hyperplasia development.

Among pituitary adenomas, prolactinomas are the most frequently diagnosed (about 50%). Dopamine agonists are generally effective in the treatment of prolactinomas. However, a subset of about 25% of patients does not respond to these agents. The management of drug-resistant prolactinomas remains a challenge for endocrinologists and new inhibitory treatments are needed. Pituitary activins inhibit lactotroph function. Its expression and action were found reduced in animal models of lactotroph hyperplasia (female mice overexpressing the B subunit of the human chorionic gonadotrophin and female mice knockout for dopamine receptor type 2). In these models, an oophorectomy avoids prolactinoma development. Hormonal replacement with oestradiol and/or progesterone is not enough to reach the tumor size observed in transgenic females. We postulated that the loss of gonadal inhibins after an oophorectomy contributes to prevent hyperplasia development. Here, we demonstrated that an oophorectomy at 2 months age recovers the following in adulthood: (i) pituitary activin expression, (ii) activin receptor expression specifically in lactotroph population, (iii) activin biological activity in lactotrophs with a concomitant reduction of Pit-1 expression. To summarize, when an oophorectomy is performed, inhibins are lost and the inhibitory action of pituitary activins on lactotroph population is recovered, helping to prevent lactotroph hyperplasia development. These results emphasize the importance of the inhibitory action of activins on lactotroph function, positioning activins as a good therapeutic target for the treatment of resistant prolactinomas.

Changes of stem cell niche during experimental pituitary tumor development.

To investigate the putative stem cell/tumor stem cell (SC/TSC) niche contribution to hyperplasic/adenomatous pituitary lesions, we analyzed variation in the pituitary stem cell population during the development of experimental pituitary tumors. Pituitary tumors were induced in female F344 rats with estradiol benzoate for 5, 10, 20 and 30 days. Cells positive for GFRa2, Sox2, Sox9, Nestin, CD133 and CD44 were identified in the marginal zone and in the adenoparenchyma in both control and 30D groups, with predominant adenoparenchyma localization of GRFa2 and SOX9 found in tumoral pituitaries. GFRa2, Nestin, CD133 and CD44 were upregulated at the initial stages of tumor growth, whereas Sox9 significantly decreased at 5D, with Sox2 remaining invariable during the hyperplasic/adenomatous development. In addition, isolated pituispheres from normal and tumoral pituitary glands enriched in SC/TSC were characterized. Pituispheres from the 30D glands were positive for the above-mentioned markers and showed a significant increase in the proliferation. In conclusion, our data revealed pituitary SC pool fluctuations during hyperplastic/adenomatous development, with differential localization of the SC/TSC niche in this process. These findings may help to provide a better understanding of these cell populations, which is crucial for achieving advancements in the field of pituitary tumor biology.

TGFß1 regulates prolactin secretion during postnatal development: gender differences.

Serum prolactin levels gradually increase from birth to puberty in both male and female rats, with higher levels observed in female since the first days of life. The increase in lactotroph secretion was attributed to the maturation of prolactin-inhibiting and prolactin-releasing factors; however, those mechanisms could not fully explain the gender differences observed. Prolactin secretion from isolated lactotrophs, in the absence of hypothalamic control, also increases during the first weeks of life, suggesting the involvement of intra-pituitary factors. We postulate that pituitary transforming growth factor beta 1 (TGFß1) is involved in the regulation of prolactin secretion as well as in the gender differences observed at early postnatal age. Several components of the local TGFß1 system were evaluated during postnatal development (11, 23, and 45 days) in female and male Sprague-Dawley rats. In vivo assays were performed to study local TGFß1 activation and its impact on prolactin secretion. At day 11, female pituitaries present high levels of active TGFß1, concomitant with the highest expression of TGFß1 target genes and the phospho-Smad3 immunostaining in lactotrophs. The steady increase in prolactin secretion inversely correlates with active TGFß1 levels only in females. Dopamine and estradiol induce TGFß1 activation at day 11, in both genders, but its activation induces the inhibition of prolactin secretion only in females. Our findings demonstrate that: (1) TGFß1 activation is regulated by dopamine and estradiol; (2) the inhibitory regulation of local TGFß1 on prolactin secretion is gender specific; and (3) this mechanism is responsible, at least partially, for the gender differences observed being relevant during postnatal development.

Trastuzumab inhibits pituitary tumor cell growth modulating the TGFB/SMAD2/3 pathway.

In pituitary adenomas, early recurrences and resistance to conventional pharmacotherapies are common, but the mechanisms involved are still not understood. The high expression of epidermal growth factor receptor 2 (HER2)/extracellular signal-regulated kinase (ERK1/2) signal observed in human pituitary adenomas, together with the low levels of the antimitogenic transforming growth factor beta receptor 2 (TBR2), encouraged us to evaluate the effect of the specific HER2 inhibition with trastuzumab on experimental pituitary tumor cell growth and its effect on the antiproliferative response to TGFB1. Trastuzumab decreased the pituitary tumor growth as well as the expression of ERK1/2 and the cell cycle regulators CCND1 and CDK4. The HER2/ERK1/2 pathway is an attractive therapeutic target, but its intricate relations with other signaling modulators still need to be unraveled. Thus, we investigated possible cross-talk with TGFB signaling, which has not yet been studied in pituitary tumors. In tumoral GH3 cells, co-incubation with trastuzumab and TGFB1 significantly decreased cell proliferation, an effect accompanied by a reduction in ERK1/2 phosphorylation, an increase of SMAD2/3 activation. In addition, through immunoprecipitation assays, a diminution of SMAD2/3-ERK1/2 and an increase SMAD2/3-TGFBR1 interactions were observed when cells were co-incubated with trastuzumab and TGFB1. These findings indicate that blocking HER2 by trastuzumab inhibited pituitary tumor growth and modulated HER2/ERK1/2 signaling and consequently the anti-mitogenic TGFB1/TBRs/SMADs cascade. The imbalance between HER2 and TGFBRs expression observed in human adenomas and the response to trastuzumab on experimental tumor growth may make the HER2/ERK1/2 pathway an attractive target for future pituitary adenoma therapy.

Evidence of cellular senescence during the development of estrogen-induced pituitary tumors.

Although pituitary adenomas represent 25% of intracranial tumors, they are usually benign, with the mechanisms by which these tumors usually avoid an invasive profile and metastatic growth development still remaining unclear. In this context, cellular senescence might constitute a plausible explanation for the benign nature of pituitary adenomas. In this study, we investigated the emergence of cellular senescence as a growth control mechanism during the progression of estrogen-induced pituitary tumors. The quantification of Ki67-immunopositive cells in the pituitaries of estrogenized male rats after 10, 20, 40, and 60 days revealed that the mitogenic potential rate was not sustained for the whole period analyzed and successively decreased after 10 days of estrogen exposure. In addition, the expression of cellular senescence features, such as the progressive rise in the enzymatic senescence-associated b-galactosidase (SA-b-gal) activity, IL6, IL1b, and TGFb expression, was observed throughout pituitary tumor development. Furthermore, tumoral pituitary cells also displayed nuclear pATM expression, indicating activated DNA damage signaling, with a significant increase in p21 expression also being detected. The associations among DNA damage signaling activation, SA-b-gal expression, and p21 may provide a reliable combination of senescence-associated markers for in vivo pituitary senescence detection. These results suggest a role for this cellular process in the regulation of pituitary cell growth. Thus, cellular senescence should be conceived as a contributing component to the benign nature of pituitary adenomas, thereby influencing the capability of the pituitary gland to avoid unregulated cell proliferation.