Screening MT1-MMP Activity and Inhibition in Three-Dimensional Tumor Spheroids.

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be crucial for tumor angiogenesis, invasion, and metastasis, and thus MT1-MMP is a high priority target for potential cancer therapies. To properly evaluate MT1-MMP inhibitors, a screening protocol is desired by which enzyme activity can be quantified in a tumor microenvironment-like model system. In the present study, we applied a fluorogenic, collagen model triple-helical substrate to quantify MT1-MMP activity for tumor spheroids embedded in a collagen hydrogel. The substrate was designed to be MT1-MMP selective and to possess fluorescent properties compatible with cell-based assays. The proteolysis of the substrate correlated to glioma spheroid invasion. In turn, the application of either small molecule or protein-based MMP inhibitors reduced proteolytic activity and glioma spheroid invasion. The presence of MT1-MMP in glioma spheroids was confirmed by western blotting. Thus, spheroid invasion was dependent on MT1-MMP activity, and inhibitors of MT1-MMP and invasion could be conveniently screened in a high-throughput format. The combination of the fluorogenic, triple-helical substrate, the three-dimensional tumor spheroids embedded in collagen, and Hit-Pick software resulted in an easily adaptable in vivo-like tumor microenvironment for rapidly processing inhibitor potential for anti-cancer use.

PRL-R Variants Are Not Only Associated With Prolactinomas But Also With Dopamine Agonist Resistance.

CONTEXT: Knockout prolactin receptor gene (PRL-R) mice are animal models for prolactinomas and PRL acts via autocrine/paracrine inhibiting lactotroph proliferation. Recently, variants of the PRL-R were identified in prolactinoma patients and their frequency was higher compared to individuals from the genomic database. OBJECTIVE: We analyzed PRL-R variants frequency in an extensive cohort of prolactinoma patients and evaluated their association with clinical, laboratorial, and imaging characteristics and hormonal response to cabergoline. DESIGN: Observational, retrospective, and cross-sectional study. SETTING: This study took place at the Neuroendocrinology Unit of Clinics Hospital, Medical School of University of São Paulo, Brazil, a tertiary referral center. PATIENTS AND METHODS: Study participants included adults with sporadic prolactinomas treated with cabergoline, where response to therapy was defined by prolactin normalization with up to 3 mg/week doses. DNA was extracted from blood samples and the PRL-R was analyzed by polymerase chain reaction techniques and automatic sequencing. The association of PRL-R variants with serum prolactin levels, maximal tumor diameter, tumor parasellar invasiveness, and response to cabergoline was analyzed. RESULTS: We found 6 PRL-R variants: p.Ile100(76)Val, p.Ile170(146)Leu, p.Glu400(376)Gln/p.Asn516(492)Ile, p.Glu470Asp e p.Ala591Pro; the last 2 are newly described in prolactinomas' patients. The variants p.Glu400(376)Gln/p.Asn516(492)Ile and p.Ala591Pro were more frequent amongst patients compared to genomic databases, and the p.Asn516(492)Ile showed pathogenic potential using in silico analysis as previously described. PRL-R variants were associated with male sex (P = 0.015), higher serum PRL levels (P = 0.007), larger tumors (P = 0.001), and cabergoline resistance (P < 0.001). CONCLUSIONS: The prolactin/prolactin receptor system seems to be related to prolactinoma tumorigenesis and cabergoline resistance. Additional studies are needed to better understand the PRL-R variants' role and their potential as therapeutic targets.

Survivin: A Potential Marker of Resistance to Somatostatin Receptor Ligands.

CONTEXT: Invasive and somatostatin receptor ligand (SRL)-resistant pituitary tumors represent a challenge in the clinical practice of endocrinologists. Efforts have been made to elucidate reliable makers for both. Survivin and eukaryotic translation initiation factor-binding protein 1 (4EBP1) are upregulated in several cancers and involved in apoptosis and cell proliferation. OBJECTIVE: We explored the role of these markers in somatotropinomas. METHODS: Immunostains for survivin and 4EBP1, and also for somatostatin receptor type 2 (SSTR2), Ki-67, and cytokeratin 18, were analyzed in tissue microarrays containing 52 somatotropinoma samples. Tumor invasiveness was evaluated in all samples while drug resistance was evaluated in 34 patients who received SRL treatment. All these parameters were correlated with first-generation SRL (fg-SRL) responsiveness and tumor invasiveness. RESULTS: Low survivin expression (P = 0.04), hyperintense signal on T2 weighted image (T2WI) (P = 0.01), younger age (P = 0.01), sparsely granular adenomas (SGA) (P = 0.04), high postoperative growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels (P = 0.049 and P < 0.001, respectively), and large postoperative tumor size (P = 0.02) were associated with resistance to fg-SRL. Low survivin and SSTR2 expression and high 4EBP1 expression were associated with SGA (P = 0.04, P = 0.01, and P = 0.001, respectively). Younger age (P = 0.03), large tumor pre- and postoperative (P = 0.04 and P = 0.006, respectively), low SSTR2 expression (P = 0.03), and high baseline GH and IGF-1 (P = 0.01 and P = 0.02, respectively) were associated with tumor invasiveness. However, survivin, 4EBP1, Ki-67, and granulation patterns were not associated with tumor invasion. CONCLUSION: This study suggests that low survivin expression is predictive of resistance to fg-SRL in somatotropinomas, but not of tumor invasiveness.

Genetics, clinical features and outcomes of non-syndromic pituitary gigantism: experience of a single center from Sao Paulo, Brazil.

PURPOSE: Non-syndromic pituitary gigantism (PG) is a very rare disease. Aryl hydrocarbon receptor-interacting protein (AIP) and G protein-coupled receptor 101 (GPR101) genetic abnormalities represent important etiologic causes of PG and may account for up to 40% of these cases. Here, we aimed to characterize the clinical and molecular findings and long-term outcomes in 18 patients (15 males, three females) with PG followed at a single tertiary center in Sao Paulo, Brazil. METHODS: Genetic testing for AIP and GPR101 were performed by DNA sequencing, droplet digital PCR and array comparative genomic hybridization (aCGH). RESULTS: Pathogenic variants in the AIP gene were detected in 25% of patients, including a novel variant in splicing regulatory sequences which was present in a sporadic male case. X-LAG due to GPR101 microduplication was diagnosed in two female patients (12.5%). Of interest, these patients had symptoms onset by age 5 and 9 years old and diagnosis at 5 and 15 years, respectively. X-LAG, but not AIP, patients had a significantly lower age of symptoms onset and diagnosis and a higher height Z-score when compared to non-X-LAG. No other differences in clinical features and/or treatment outcomes were observed among PG based on their genetic background. CONCLUSION: We characterize the clinical and molecular findings and long-term outcome of the largest single-center PG cohort described so far.

Association between KISS1 rs5780218 promoter polymorphism and onset of growth hormone secreting pituitary adenoma.

OBJECTIVES: This study analyzed the KISS1 c.-145delA (rs5780218) promoter polymorphism in a cohort of patients with growth hormone secreting pituitary adenoma (somatotropinoma) and controls, to investigate its role in the incidence of acromegaly and to assess patient/tumor characteristics. Material and methods rs5780218 allelic and genotypic distributions were compared between 49 somatotropinoma patients and 167 healthy controls. rs5780218 was also assessed in relation to patient characteristics and tumor aggressiveness, as characterized by tumor invasion and resistance to conventional therapy. The relationship between KISS1 mRNA expression and the rs5780218 genotype was also assessed in available pituitary tumor samples. RESULTS: The homozygous -/- variant genotype was associated with high rates of somatotropinoma (P<0.01), but not with tumor invasiveness, patient characteristics or hormonal remission. KISS1 mRNA expression was much lower in somatotropinomas carrying the deleted allele than in homozygous wild type AA. CONCLUSIONS: In this pilot study, the rs5780218 promoter polymorphism was evaluated in pituitary adenoma, and showed a possible association with the incidence of somatotropinoma but not with tumor progression.

Differential gene expression of sirtuins between somatotropinomas and nonfunctioning pituitary adenomas.

Sirtuins 1-7 (SIRT) are a highly conserved family of histone deacetylases involved in the regulation of longevity that have a considerable impact in transcription, DNA repair regulation, telomeric stability, cell senescence and apoptosis. In the present study, SIRT1-7 mRNA levels were evaluated in 37 somatotropinomas and 31 nonfunctioning pituitary adenomas (NFPAs) using qPCR and relation to tumor size, invasiveness and Ki-67 proliferative index was made. Overexpression of SIRT1 was observed in 86.5% of somatotropinomas versus 41.9% of NFPAs (P < 0.01). SIRT3 was more underexpressed in NFPAs than somatotropinomas (77.4 and 40.5%, respectively, P < 0.01) as well as SIRT4 and SIRT7. Despite the lack of association between sirtuins and invasiveness or Ki-67 index, SIRT1 and SIRT3 expressions were related to tumor size. Mean of the largest diameter was smaller in adenomas with SIRT1 overexpression than with normal expression (P < 0.01) and SIRT3 underexpression was associated with larger tumors (P < 0.01). In conclusion, a pronounced difference in sirtuins expression was identified between pituitary adenomas, suggesting that these genes are potential markers of pituitary adenomas and could be employed in the characterization of somatotropinomas and NFPAs. The role of sirtuins in pathogenesis of pituitary tumors merits further investigation and possibly will provide new molecular insight about their progression.