Shuya Zhang

Leptin and leptin receptor expression in rat and mouse pituitary cells.

Leptin is a circulating hormone secreted mainly by adipose tissue. Recent studies have shown leptin production by other tissues, including the placenta, stomach, and mammary tissues. Various reports have suggested that the anterior pituitary may have a role in the regulatory effects of leptin. We recently localized leptin in the human anterior pituitary, but analysis of leptin in rodent pituitary has not been previously reported. In this study we examined rat and mouse pituitary tissues and various cell lines for leptin by RT-PCR, immunohistochemistry, and Western blotting. Leptin receptor messenger RNA was also examined in these tissues by RT-PCR. Leptin was present in a small percentage of rat (4.8 ± 0.7%) and mouse (7 ± 2%) pituitary cells. Colocalization studies with leptin and pituitary hormones showed leptin expression mainly in TSH cells (24 ± 2% of TSH cells in the rat pituitary and 31 ± 1% of TSH cells in the mouse pituitary). A folliculo-stellate (FS) cell line, TtT/GF, also expressed leptin. Th...

MicroRNA expression in ileal carcinoid tumors: downregulation of microRNA-133a with tumor progression

MicroRNAs (miRNAs) are involved in cell proliferation, differentiation, and apoptosis and can function as tumor suppressor genes or oncogenes. The role of miRNAs in neuroendocrine tumors such as ileal carcinoids is largely unknown. We examined the differential expression of 95 miRNAs by RT–PCR using the QuantiMir System in eight matching primary and metastatic carcinoid tumors from the ileum. All miRNAs chosen for the QuantiMir System array were based on their potential functions related to cancer biology, cell development, and apoptosis. The expression of miRNAs for the samples was normalized to miRNA-197, and the matching primary and metastatic tumors were compared. There was downregulation of miRNA-133a, -145, -146, -222, and -10b in all samples between the primary and matching metastatic tumors and upregulation of miRNA-183, -488, and -19a+b in six of eight metastatic carcinoids compared to the primary tumors. miRNA-133a was further analyzed by TaqMan real-time RT–PCR and northern hybridization using six additional matching primary and metastatic samples, which supported the PCR array findings. There were significant differences in miRNA-133a expression with downregulation in the metastasis compared to the primary in the eight original cases (P<0.009) and in the six additional cases used for validation (P<0.014). Laser capture microdissection and real-time RT–PCR analysis using normal ileum found miRNA-133a expression in normal enterochromaffin cells. In situ hybridization in normal ileum showed that some of the mucosal endocrine cells expressed miRNA-133a. Both primary and metastatic ileal carcinoid tumors expressed miRNA-133a by in situ hybridization. These results provide information about novel marker miRNAs that may be used as biomarkers and/or therapeutic targets in intestinal carcinoid tumors.

MicroRNA expression in ACTH-producing pituitary tumors: up-regulation of microRNA-122 and -493 in pituitary carcinomas

MicroRNAs (miRNAs) are involved in cell proliferation, differentiation, and apoptosis, and can function as tumor suppressor genes or oncogenes. The expression of miRNAs in pituitary carcinomas has not been previously examined. We used miRNA profiling with 1,145 probes to study miRNA expression in normal anterior pituitary (6 cases), adrenocorticotropin (ACTH)-producing adenomas (8 cases), and ACTH-producing pituitary carcinomas (two cases). Real-time RT-PCR and in situ hybridization were used to confirm and independently validate miRNAs that were significantly up-regulated or down-regulated between the pituitary tissues. There were more miRNAs up- (188) or down-regulated (160) between adenomas and normal pituitaries compared to carcinomas and normal pituitaries (92 up- and 91 down-regulated) or between carcinomas and adenomas (46 up- and 52 down-regulated). Both real-time RT-PCR and in situ hybridization showed significant up-regulation of miRNA-122 between pituitary carcinomas and adenomas. MiRNA-493 was also up-regulated in carcinomas compared to ACTH adenomas. Analysis of genes that miRNA-493 interacts with included LGALS3 and RUNX2 (http://microrna.sanger.ac.uk) both of which have been shown to have roles in pituitary tumor cell growth. These results provide information about marker miRNAs that may lead to further insights into the regulation of pituitary tumor growth and development.

KIT is an independent prognostic marker for pancreatic endocrine tumors: a finding derived from analysis of islet cell differentiation markers.

Prediction of the biologic behavior of pancreatic endocrine tumor (PET) without local invasion or metastasis is often difficult. The 2004 World Health Organization (WHO) classification uses size, angioinvasion, mitotic activity, and Ki-67 index as prognostic criteria. Recently, cytokeratin 19 (CK19) was shown to be another prognostic marker, but the mechanism by which CK19 predicts prognosis is unknown. As CK19 is the first cytokeratin expressed in all epithelial cells in fetal pancreas, we sought to test expression of other markers of islet cell differentiation including KIT, Pdx-1, Pax4, and Pax6 in PET and correlation of these markers with clinical behavior. Clinical information and histology was reviewed in 97 PETs. All tumors were classified according to WHO criteria and a tumor, node, and metastases stage system. Immunohistochemistry was performed using antibodies to Ki-67, KIT, CK19, Pdx-1, Pax4, and Pax6. Associations of clinicopathologic and immunohistochemical features with prognosis were evaluated using Cox proportional hazards regression models. WHO and tumor, node, and metastases classifications, mitotic counts and Ki-67 labeling, infiltrative border, necrosis, perineural invasion, extrapancreatic extension, tumor size, and positive CK19 and KIT expression were significantly associated with death from disease in a univariate setting. In multivariate analysis, only WHO criteria and KIT expression were shown to be independent. An immunohistochemical classification system was derived from a combination of KIT and CK19 expression: low risk (KIT–/CK19–), intermediate risk (KIT–/CK19+), and high risk (KIT+/CK19+). Survival, metastases, and recurrence of PET were significantly different among the 3 groups. These results indicate that KIT is a new and independent prognostic marker for PETs. The classification system derived from KIT and CK19 was able to predict clinical behavior of PET.

Analysis of epidermal growth factor receptor and activated epidermal growth factor receptor expression in pituitary adenomas and carcinomas.

Epidermal growth factor receptor plays an important role in the pathogenesis of many malignancies. Various growth factors, including epidermal growth factor receptor, have been shown to influence pituitary tumor growth and differentiation. To analyze the role of epidermal growth factor receptor in pituitary tumor development, we examined normal pituitaries (n=8), pituitary adenomas (n=158), and pituitary carcinomas (n=7) for expression of epidermal growth factor receptor protein and messenger RNA using tissue microarrays and RT-PCR. We also examined (a) the expression of phospho-epidermal growth factor receptor, the activated form of epidermal growth factor receptor, in pituitary tumors and normal pituitaries by immunohistochemistry and (b) the effects on epidermal growth factor receptor expression of treating pituitary cells (HP75 cell line) with epidermal growth factor. Epidermal growth factor receptor and the phosphorylated variant expression were present in normal pituitary cells. Epidermal growth factor receptor messenger RNA was also detected in normal pituitaries, pituitary adenomas, and carcinomas by in situ hybridization and RT-PCR. Most pituitary adenomas showed expression of epidermal growth factor receptor and the phosphorylated variant. Nonfunctional adenomas showed higher levels of expression of epidermal growth factor receptor (76 vs 34%) and of phospho-epidermal growth factor receptor (26 vs 8%) as compared to functional adenomas. Five of seven pituitary carcinomas showed strong expression of both epidermal growth factor receptor and phospho-epidermal growth factor receptor. When a human pituitary cell line (HP75) was cultured in the presence of epidermal growth factor receptor, there was an increase in the levels of both epidermal growth factor receptor and phospho-epidermal growth factor receptor after 5 h of treatment, thus confirming that epidermal growth factor receptor signaling was active in pituitary tumors. These results indicate that activated epidermal growth factor receptor is expressed in pituitary adenomas and carcinomas. Higher levels in pituitary carcinomas suggest a role in pituitary tumor progression.

Inactivation of the p16 gene in human pituitary nonfunctioning tumors by hypermethylation is more common in null cell adenomas.

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A/MTS1 (p16) gene is associated with loss of expression of p16 protein in pituitary tumors. We analyzed a series of 21 pituitary adenomas and three normal pituitaries along with a human pituitary cell line (HP75) for methylation of exon 1 by methylation-specific PCR, immunohistochemistry, and Western blotting. PCR analysis showed that 5/7 (71%) of null cell adenomas, but only 2/7 (29%) gonadotroph tumors were hypermethylated. In addition, 1 of 2 ACTH tumors but no GH (n=4) or PRL (n=1) adenoma examined were hypermethylated. Immunostaining and Western blot analysis of protein expression supported the methylation-specific PCR analyses.These results show that p16 gene silencing by hypermethylation is more common in null cell adenomas compared to other nonfunctioning adenomas such as gonadotroph tumors and that the role of p16 in the pathogenesis of pituitary adenomas is restricted to specific tumor subtypes.

Raf-1 oncogenic signaling is linked to activation of mesenchymal to epithelial transition pathway in metastatic breast cancer cells

Aberrant activation of the Raf/MEK/MAPK pathway plays a key role in breast cancer development and progression. Dysregulation of Raf/MEK/MAPK oncogenic signaling often results from overexpression of the HER-2/Neu tyrosine kinase receptor leading to chemoendocrine resistance, development of distant metastases and ultimately poor prognosis in breast cancer patients. HER-2/Neu overexpression is also linked to activation of the epithelial to mesenchymal transition (EMT) pathway, loss of adhesion molecules and metastasis. Recently, it has been demonstrated that cancer cells that undergo EMT acquire a CD44+/CD24-/low basal cancer stem cell-like phenotype and are characterized by activation of HER-2/Neu and TGFβ oncogenic signaling pathways with increased capacity of self-renewal, drug resistance, invasion and distant metastases. Following metastatic dissemination, cancer cells re-activate certain epithelial properties through mesenchymal to epithelial transition (MET) to establish neoplastic lesions at secondary sites, although the molecular mechanisms regulating MET remain elusive. In this study we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces HER-2/Neu overexpression leading to the development of distant metastases in ERα+ MCF-7 breast cancer xenografts. Importantly, development of distant metastases in xenograft models was linked to activation of the MET pathway characterized by reduced expression of EMT inducer genes (TGFB2, TWIST1 and FOXC1) and overexpression of BMB7, CXCR7 and EGR family of transcription factors. In summary, our results demonstrate for the first time that amplification of Raf/MEK/MAPK oncogenic signaling during tumor growth promotes the genesis of metastatic lesions from primary tumors by activating the mesenchymal epithelial transition.

Analysis of Cox-2 and Thromboxane Synthase Expression in Pituitary Adenomas and Carcinomas*

Recent studies have examined the role of cyclooxygenase-2 (Cox-2) expression in normal pituitaries and pituitary adenomas and have suggested a role for Cox-2 in the regulation of angiogenesis in the pituitary. Thromboxane synthase (TBXAS), which catalyzes the synthesis of thromboxane A2, is one of the downstream enzymes in Cox metabolism and appears to play a role in the regulation of invasiveness and angiogenesis in some neoplasms. To analyze the role of Cox-2 and TBXAS in pituitary tumor progression, we examined normal pituitaries (n=8), pituitary adenomas (n=174), and pituitary carcinomas (n=7) for expression of Cox-2 and TBXAS by immunohistochemistry. Weak Cox-2 and moderate TBXAS expression was present in normal pituitary cells. Most pituitary adenomas showed increased expression of both Cox-2 and TBXAS. Pituitary tumors as a whole, but particularly carcinomas, showed greater Cox-2 expression than did normal pituitaries. Pituitary adenomas and carcinomas also showed greater staining for TBXAS when compared to normal pituitary. Nonfunctional adenomas had significantly higher levels of TBXAS expression compared to functional adenomas (p=0.017). Adenomas and carcinomas showed similar degrees of staining for TBXAS. In summary, TBXAS appears to be one of the up-regulated downstream enzymes of Cox metabolism in pituitary tumors. Both Cox-2 and TBXAS may play an important role in pituitary tumor development and progression.

Expression of cyclooxygenase-2 and thromboxane synthase in non-neoplastic and neoplastic thyroid lesions

Cyclooxygenase-2 (COX-2) and thromboxane synthase (TBXAS) are important enzymes involved in the arachidonic acid pathway and synthesis of prostaglandins. We examined COX-2 and TBXAS immunoreactivity in 150 surgically resected thyroid specimens using immunohistochemistry to determine expression in benign and malignant thyroid lesions and to examine their roles in thyroid tumor progression. Papillary thyroid carcinomas and follicular carcinomas expressed higher levels of COX-2 compared to follicular adenomas and adenomatous nodules. We showed for the first time that TBXAS was expressed in thyroid tissues, with higher levels in papillary carcinomas compared to non-neoplastic and benign thyroid tissues. Western blot was performed on seven thyroid samples.These results indicate that both COX-2 and TBXAS are expressed in benign and malignant thyroid tissues. Although some malignant thyroid tumors showed higher levels of COX-2 expression, COX-2 and TBXAS are probably not useful in the immunohistochemical diagnosis of thyroid malignancies. However, the expression of both COX-2 and TBXAS by thyroid tissue may provide insight into the role of these enzymes in progression from benign to malignant thyroid tumors.

Chromogranin A processing in human pituitary adenomas and carcinomas: Analysis with region-specific antibodies

The expression of various chromogranin A (CgA) peptide fragments was examined with region-specific antisera in benign and malignant pituitary tumors. Analysis of the proconvertases responsible for proteolytic processing of CgA, prohormone convertase 1/3 (PC1/3), and PC2 was also performed. Adenomas were studied using tissue microarrays, and a larger tissue section of a subset of the prolactin (PRL) adenomas was used to compare to the tissue microarray analysis. Carcinomas were analyzed using large tissue sections. There were differences in CgA proteolytic products detected between the functional (PRL, adrenocorticotropic hormone [ACTH], and growth hormone tumors and the nonfunctional (gonadotroph and null cell) tumors, with the former group, expressing lower levels of many peptides. These differences were most notable in the PRL adenomas and carcinomas in which the region-specific antisera against vasostatin I and vasostatin II detected these fragments in the lowest percentage of tumors and/or had the weakest immunoreactivity. The CgA peptide fragment detected by CgA 176–195 (chromacin) antiserum was expressed by the highest percentage of most functional and nonfunctional benign and malignant pituitary tumors. ACTH carcinomas (n+3) were more strongly immunoreactive compared to the ACTH adenomas. These results show that there is differential expression of CgA peptide fragments and PC1/3 among different types of pituitary tumors and that ACTH pituitary carcinomas have higher levels of immunoreactive CgA peptide fragments compared to ACTH adenomas. This study also shows the utility of tissue microarrays in the analysis of a large group of tumors with region-specific antisera.