Dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists are effective in inhibiting proliferation of progenitor/stem-like cells isolated from nonfunctioning pituitary tumors.

The role of progenitor/stem cells in pituitary tumorigenesis, resistance to pharmacological treatments and tumor recurrence is still unclear. This study investigated the presence of progenitor/stem cells in non-functioning pituitary tumors (NFPTs) and tested the efficacy of dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists to inhibit in vitro proliferation. They found that 70% of 46 NFPTs formed spheres co-expressing stem cell markers, transcription factors (DAX1, SF1, ERG1) and gonadotropins. Analysis of tumor behavior showed that spheres formation was associated with tumor invasiveness (OR = 3,96; IC: 1.05-14.88, p = 0.036). The in vitro reduction of cell proliferation by DRD2 and SSTR2 agonists (31 ± 17% and 35 ± 13% inhibition, respectively, p < 0.01 vs. basal) occurring in about a half of NFPTs cells was conserved in the corresponding spheres. Accordingly, these drugs increased cyclin-dependent kinase inhibitor p27 and decreased cyclin D3 expression in spheres. In conclusion, they provided further evidence for the existence of cells with a progenitor/stem cells-like phenotype in the majority of NFPTs, particularly in those with invasive behavior, and demonstrated that the antiproliferative effects of dopaminergic and somatostatinergic drugs were maintained in progenitor/stem-like cells.

MicroRNAs in parathyroid physiopathology.

Parathyroid glands regulate calcium homeostasis through synthesis and secretion of parathormone (PTH). They sense the extracellular calcium concentration through the G-protein coupled calcium sensing receptor (CASR) and release PTH in order to preserve calcium concentration in the physiological range. Tumors of the parathyroid glands are common endocrine neoplasia associated with primary or secondary/tertiary hyperparathyroidisms. Small non-coding RNAs are regulators of gene expression able to modulate hormone synthesis, hormone release and endocrine cell proliferation. In this scenario, microRNA (miRNA) expression profiles have been investigated in parathyroid tumors, while miRNAs are involved in hypocalcemia and uremia-induced PTH release from normal parathyroid cells. Here we reviewed data about the role of miRNAs in the regulation of: 1) PTH synthesis and secretion; 2) CASR expression; 3) parathyroid cell tumorigenesis. Though studies about miRNAs in parathyroid gland pathophysiology are limited, they contribute in elucidating regulatory pathways involved in PTH release and parathyroid cell tumorigenesis.

Dopamine receptor type 2 (DRD2) inhibits migration and invasion of human tumorous pituitary cells through ROCK-mediated cofilin inactivation.

Non-functioning pituitary tumors (NFPTs) frequently present local invasiveness. Dopamine receptor 2 (DRD2) agonists are the only medical therapy that induces tumor shrinkage in some patients. Invasion requires cytoskeleton rearrangements that are tightly regulated by cofilin pathway, whose alterations correlate with invasion in different tumors. We investigated the effect of DR2D agonist on NFPT cells migration/invasion and the molecular mechanisms involved. We demonstrated that DRD2 agonist reduced migration (-44 ± 25%, p < 0.01) and invasion (-34 ± 6%, p < 0.001) and increased about 4-fold Ser3-phosphorylated inactive cofilin (P-cofilin) in NFPT cells. These effects were abolished by inhibiting ROCK, a kinase that phosphorylates cofilin. The overexpression of wild-type or phosphodeficient S3A-cofilin increased HP75 cells migration (+49 ± 6% and +57 ± 9% vs empty vector, respectively, p < 0.05), while phosphomimetic mutant had no effect. Interestingly, P-cofilin levels were lower in invasive vs non-invasive tumors by both western blot (mean P-cofilin/total cofilin ratio 0.77 and 1.93, respectively, p < 0.05) and immunohistochemistry (mean percentage of P-cofilin positive cells 17.6 and 45.7, respectively, p < 0.05). In conclusion, we showed that the invasiveness of pituitary tumors is promoted by the activation of cofilin, which can be regulated by DRD2 and might represent a novel biomarker for pituitary tumors' invasive behavior.

Identification of imaging biomarkers for the assessment of tumour response to different treatments in a preclinical glioma model.

PURPOSE: Hypoxia-inducible factor 1α (HIF-1α) activity is one of the major players in hypoxia-mediated glioma progression and resistance to therapies, and therefore the focus of this study was the evaluation of HIF-1α modulation in relation to tumour response with the purpose of identifying imaging biomarkers able to document tumour response to treatment in a murine glioma model. METHODS: U251-HRE-mCherry cells expressing Luciferase under the control of a hypoxia responsive element (HRE) and mCherry under the control of a constitutive promoter were used to assess HIF-1α activity and cell survival after treatment, both in vitro and in vivo, by optical, MRI and positron emission tomography imaging. RESULTS: This cell model can be used to monitor HIF-1α activity after treatment with different drugs modulating transduction pathways involved in its regulation. After temozolomide (TMZ) treatment, HIF-1α activity is early reduced, preceding cell cytotoxicity. Optical imaging allowed monitoring of this process in vivo, and carbonic anhydrase IX (CAIX) expression was identified as a translatable non-invasive biomarker with potential clinical significance. A preliminary in vitro evaluation showed that reduction of HIF-1α activity after TMZ treatment was comparable to the effect of an Hsp90 inhibitor, opening the way for further elucidation of its mechanism of action. CONCLUSION: The results of this study suggest that the U251-HRE-mCherry cell model can be used for the monitoring of HIF-1α activity through luciferase and CAIX expression. These cells can become a useful tool for the assessment and improvement of new targeted tracers for potential theranostic procedures.

MicroRNA deregulation in parathyroid tumours suggests an embryonic signature.

Primary hyperparathyroidism is a common endocrine disorder caused by abnormal tumour parathyroid cell proliferation. Parathyroid tumours show a great variability both in clinical features, such as the severity of PTH secretion, the rate and the pattern of cell proliferation, and genetic background. Studies aiming to develop new diagnostic markers and therapeutic approaches need a deeper definition of this variability. Dysregulation of microRNAs (miRNAs) has been shown to play an essential role in the development and progression of cancer. MiRNAs are small noncoding RNAs that inhibit the translation and stability of messenger RNAs (mRNAs). Here, data about the miRNA expression pattern in parathyroid normal and tumour glands were reviewed. Though available data in parathyroid tumours are very limited, the expression pattern of a subset of specific miRNAs clearly discriminated parathyroid carcinomas from normal parathyroid glands and, more clinically relevant, from parathyroid adenomas. Investigation showed that parathyroid tumours were characterized by an embryonic expression pattern of miRNAs such as miR-296, or the miRNA clusters C19MC and miR-371-3, typically in stem cells committed to differentiation or during human embryonic development, respectively. Further, miRNA profiles were correlated with tumour aggressive behaviour. Moreover, the interaction with the oncosuppressor menin suggests that miRNAs might modulate the function of the known oncosuppressors or oncogenes involved in parathyroid tumourigenesis and thus overseeing the tumour phenotype. In conclusion, miRNAs might provide new diagnostic markers and new therapeutic approaches by developing molecular miRNA-targeted therapies for the cure of parathyroid tumours, whose unique option is surgery.

The expression of Axl receptor tyrosine kinase influences the tumour phenotype and clinical outcome of patients with malignant pleural mesothelioma.

BACKGROUND: Recent preclinical studies identified Axl, a tyrosine kinase receptor implicated in tumour progression and epithelial-to-mesenchymal transition, as a putative therapeutic target in malignant pleural mesothelioma (MPM), an invariably fatal malignancy with limited treatment options. Here, we studied the expression of Axl and its ligand Gas-6 (growth arrest signal-6) in primary specimens of MPM, correlating their expression levels with tumour phenotype and clinical outcomes. METHODS: Two independent cohorts of consecutive patients diagnosed with MPM were studied: a derivation cohort composed of 63 cases and a validation set of 35 cases. Clinical variables including patients' demographics, tumour stage, histotype, performance status (PS), Axl and Gas-6 staining were tested for predicting overall survival (OS) using univariate and multivariate analyses. RESULTS: In the derivation cohort, Axl (P=0.001) but not Gas-6 overexpression (P=0.35) emerged as a univariate prognostic factor for OS, together with stage (P=0.05), PS (P<0.001) hypoalbuminaemia (P<0.001) and anaemia (P<0.001). Multivariate analyses confirmed Axl overexpression (P=0.01), PS (P=0.01), hypoalbuminaemia (P<0.001) and anaemia (P=0.04) as independent predictors of OS. The prognostic role of Axl overexpression was externally validated in an independent cohort (P=0.03). CONCLUSION: Overexpression of Axl is found in the majority of MPM specimens and influences patient's survival independently from other established prognostic factors. Such information may support patient selection for future trials.

miR-296 regulation of a cell polarity-cell plasticity module controls tumor progression.

The expression of small, non-coding RNA or microRNAs (miR), is frequently deregulated in human cancer, but how these pathways affect disease progression is still largely elusive. Here, we report on a miR, miR-296, which is progressively lost during tumor progression and correlates with metastatic disease in colorectal, breast, lung, gastric, parathyroid, liver and bile ducts cancers. Functionally, miR-296 controls a global cell motility gene signature in epithelial cells by transcriptionally repressing the cell polarity-cell plasticity module, Scribble (Scrib). In turn, loss of miR-296 causes aberrantly increased and mislocalized Scrib in human tumors, resulting in exaggerated random cell migration and tumor cell invasiveness. Re-expression of miR-296 in MDA-MB231 cells inhibits tumor growth in vivo. Finally, miR-296 or Scrib levels predict tumor relapse in hepatocellular carcinoma patients. These data identify miR-296 as a global repressor of tumorigenicity and uncover a previously unexplored exploitation of Scrib in tumor progression in humans.

Differential expression of microRNAs in human parathyroid carcinomas compared with normal parathyroid tissue.

Parathyroid carcinoma (PaC) is a rare cause of primary hyperparathyroidism. Though the loss of the oncosuppressor CDC73/HRPT2 gene product, parafibromin, has been involved in the hyperparathyroidism-jaw tumor syndrome and in a consistent set of sporadic PaCs, parathyroid carcinogenesis remains obscure. MicroRNAs are a new class of small, non-coding RNAs implicated in development of cancer, since their deregulation can induce aberrant expression of several target genes. The aim of the present study was to identify differentially expressed microRNAs in parathyroid cancers compared with normal tissues. We performed a TaqMan low-density array profiling of four parathyroid cancers harboring CDC73 inactivating mutations and negative for parafibromin immunostaining. Their microRNA profiling was compared with that of two normal parathyroid biopsies. Out of 362 human microRNAs assayed, 279 (77%) were successfully amplified. Fourteen and three microRNAs were significantly down- and over-expressed in parathyroid cancers respectively. Of these, miR-296 and miR-139 were down-regulated, and miR-503 and miR-222 were over-expressed with a null false discovery rate. Carcinomas could be discriminated from parathyroid adenomas by a computed score based on the expression levels of miR-296, miR-222, and miR-503 as miR-139 was similarly down-regulated in both cancers and adenomas. Finally, miR-296 and miR-222 levels negatively correlated with mRNA levels of the hepatocyte growth factor receptor-regulated tyrosine kinase substrate and p27/kip1 levels respectively. These results suggest the existence of an altered microRNA expression pattern in PaCs together with a potential role of miR-296 as novel oncosuppressor gene in these neoplasia.

Regulation of survivin expression by IGF-1/mTOR signaling.

Survivin is a dual regulator of cell proliferation and cell viability overexpressed in most human tumors. Although strategies to lower survivin levels have been pursued for rational cancer therapy, the molecular circuitries controlling survivin expression in tumors have not been completely elucidated. Here, we show that stimulation with insulin-like growth factor-1 (IGF-1) results in increased survivin expression in prostate cancer cells. This response is independent of de novo gene transcription, changes in mRNA expression or modifications of survivin protein stability. Instead, IGF-1 induced persistence and translation of a pool of survivin mRNA, in a reaction abolished by the mTOR (mammalian target of rapamycin) inhibitor, rapamycin. Forced expression of the mTOR target p70S6K1 reproduced the increase in survivin expression in prostate cancer cells, whereas acute ablation of endogenous p70S6K1 by small interfering RNA downregulated survivin levels. Rapamycin, alone or in combination with suboptimal concentrations of taxol reduced survivin protein levels, and decreased viability of prostate cancer cells. Therefore, IGF-1/mTOR signaling elevates survivin in prostate cancer cells via rapid changes in mRNA translation. Antagonists of this pathway may be beneficial to lower an antiapoptotic threshold maintained by survivin in prostate cancer.

EGFR overexpression in malignant pleural mesothelioma. An immunohistochemical and molecular study with clinico-pathological correlations.

The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial malignancies, against which some antitumoral drugs have been developed. There is a lack of information as to EGFR expression in malignant pleural mesothelioma (MPM), an aggressive and fatal cancer poorly responsive to current oncological treatments. Our aim was to: (a) compare EGFR immunohistochemical expression with mRNA levels measured by real time PCR; (b) assess the relationships between EGFR expression and clinico-pathological data including survival; (c) analyze the EGFR mutations. We developed an immunohistochemical method of EGFR evaluation based on the number of immunoreactive cells and staining intensity in 61 MPMs. EGFR immunoreactivity was documented in 34/61 (55.7%) cases. A significant correlation between EGFR protein and mRNA levels (p = 0.0077) was found, demonstrating the reliability of our quantification method of EGFR membrane expression. Radically resected patients (p = 0.005) and those with epithelial histotype (p = 0.048) showed an increased survival. No statistical correlation between EGFR immunoreactivity and patients survival was observed. No EGFR mutation was documented. This study documents EGFR overexpression in MPM at the protein and the transcriptional levels; it proposes a reliable method for EGFR expression evaluation in MPM. EGFR levels are not associated with clinico-pathological features of patients, including survival.