MAB5268 Sigma-AldrichAnti-Chromogranin A Antibody, clone LK2H10

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase β-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

Genomic analyses of hundreds of prostate tumors have defined a diverse landscape of mutations and genome rearrangements, but the transcriptomic effect of this complexity is less well understood, particularly at the individual tumor level. We selected a cohort of 25 high-risk prostate tumors, representing the lethal phenotype, and applied deep RNA-sequencing and matched whole genome sequencing, followed by detailed molecular characterization.Ten tumors were exposed to neo-adjuvant hormone therapy and expressed marked evidence of therapy response in all except one extreme case, which demonstrated early resistance via apparent neuroendocrine transdifferentiation. We observe high inter-tumor heterogeneity, including unique sets of outlier transcripts in each tumor. Interestingly, outlier expression converged on druggable cellular pathways associated with cell cycle progression, translational control or immune regulation, suggesting distinct contemporary pathway affinity and a mechanism of tumor stratification. We characterize hundreds of novel fusion transcripts, including a high frequency of ETS fusions associated with complex genome rearrangements and the disruption of tumor suppressors. Remarkably, several tumors express unique but potentially-oncogenic non-ETS fusions, which may contribute to the phenotype of individual tumors, and have significance for disease progression. Finally, one ETS-negative tumor has a striking tandem duplication genotype which appears to be highly aggressive and present at low recurrence in ETS-negative prostate cancer, suggestive of a novel molecular subtype.The multitude of rare genomic and transcriptomic events detected in a high-risk tumor cohort offer novel opportunities for personalized oncology and their convergence on key pathways and functions has broad implications for precision medicine.

Spindle cell oncocytoma of the adenohypophysis (SCO) is a non-endocrine neoplasm with few recurrent forms described. It arises from the folliculo-stellate cells of the adenohypophysis.

It has proved to be impossible to culture epithelial cells from the gastrointestinal tract of adult animals. Researchers have had to use either cell lines derived from newborn rat small intestine or colon carcinoma cell lines that have retained some of the properties of the gastrointestinal mucosa. We have described a method for establishing conditionally immortalized cell lines from the stomach, small intestine, colon, pancreas, and liver from tissue obtained from a transgenic mouse strain carrying a temperature-sensitive mutant of the SV40 large T gene (the "Immortomouse"). This immortalizing gene has proved to be useful for establishing cell lines from a number of transgenic mice following crossbreeding of the Immortomouse with the transgenic mouse of interest. These cell lines are being used in numerous studies. In this review we describe the methods for developing such lines and list the range of cell lines that have been developed from colon, small intestine, stomach, liver, and pancreas of a number of transgenic mice.

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the beta1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to beta1 integrin. Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin. Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor alpha2beta1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.

Hereditary medullary thyroid carcinoma (MTC) is caused by germline mutations in the RET proto-oncogene, resulting in constitutive activation of the RET tyrosine kinase. A substantial proportion of sporadic MTCs also have RET mutations, making the RET tyrosine kinase a potential therapeutic target in MTC. We have established a transplantable MTC in nude mice from a sporadic human MTC carrying a RET C634R mutation. Transplanted tumors had an exponential growth rate with an approximate doubling time of about 3 weeks, and expressed a neuroendocrine phenotype characteristic of MTC, e.g., expression of calcitonin, chromogranin A (CgA), synaptophysin, synaptic vesicle protein 2 (SV2), vesicular monoamine transporter-1 and -2, carcinoembryonic antigen, cytokeratin 8/18, epithelial cadherin, and neural cell adhesion molecule. Plasma calcitonin and CgA levels were elevated in tumor-bearing mice and correlated with tumor size. Cytogenetic analysis, including spectral karyotyping, confirmed the human origin of the xenografted tumors and demonstrated an abnormal, near triploid karyotype. Treatment of tumor-bearing nude mice with the tyrosine kinase inhibitor ZD6474, which specifically inhibits RET, epidermal growth factor receptor (EGFR), and vascular endothelium growth factor receptor (VEGFR) tyrosine kinases, resulted in a dose-dependent inhibition of tumor growth. Oral ZD6474 given once daily (250 mg/kg, 5 days/week) reduced tumor volume to 11% when compared with controls after 4 weeks. Our results show that this transplantable MTC, designated GOT2, represents a novel and useful model for studies of MTC and RET tyrosine kinase-dependent tumor growth.

A monoclonal antibody ( LK2H10 ) produced against a human pheochromocytoma reacted immunohistochemically with 126 normal and neoplastic endocrine tissues with secretory granules which were formalin-fixed and paraffin-embedded. Antibody LK2H10 did not react with 46 other endocrine tissues or tumors without secretory granules nor with 113 normal and neoplastic nonendocrine cells and tumors. Tumors with abundant secretory granules showed intense and diffuse staining, and tumors with few granules, such as Merkel cell carcinomas, neuroblastomas, and small cell carcinomas of lung, showed focal staining. Antibody LK2H10 did not react with melanomas, nevi, posterior pituitary, peripheral nerve tissues, or neurons. The target structure of LK2H10 was identified as human chromogranin, of which the major fraction was chromogranin A (mol wt 68,000 daltons). Preabsorption with purified chromogranin A blocked immunoperoxidase staining by LK2H10 in normal adrenal medulla, in the anterior pituitary, and in a pheochromocytoma. Ultrastructural immunohistochemistry with LK2H10 showed that chromogranin was present in cytoplasmic secretory granules. These results indicate that chromogranin is widely distributed in the secretory granules of most polypeptide-producing endocrine tissues, and it is readily detected with the use of monoclonal antibody LK2H10 . The detection of this marker can be very helpful as a diagnostic aid for neuroendocrine cells and tumors.

One of two mouse monoclonal antibodies (LK2H10) produced by hybridoma technology against a human endocrine tumor (pheochromocytoma) demonstrated specific immunoreactivity for 69 normal and neoplastic endocrine and tissues known to contain secretory granules. This immunoreactivity was specific, since other normal tissues, tumors from endocrine cells without granules, and tumors from other nonendocrine tissues were negative when tested with antibody LK2H10. The antibody reacted with human fetal adrenal medulla and human pancreatic endocrine cells and with adrenal medullary cells from monkeys and pigs. The antigen detected by antibody LK2H10 is associated with cytoplasmic secretory granules, has an estimated molecular weight of 68,000, and may be related to human chromogranin.