1113

CHGA

chromogranin A;CHGA;chromogranin A

SP-I|betagranin (N-terminal fragment of chromogranin A)|catestatin|chromofungin|chromogranin-A|parathyroid secretory protein 1|pituitary secretory protein I

14q32

protein-coding

PURPOSE: The use of replication-selective oncolytic adenoviruses is an emerging therapeutic approach for cancer, which thus far has not been employed for carcinoids. We therefore constructed Ad[CgA-E1A], a novel replication-selective oncolytic adenovirus, where the chromogranin A (CgA) promoter controls expression of the adenoviral E1A gene. EXPERIMENTAL DESIGN: The Ad[CgA-E1A] virus was evaluated for E1A protein expression, replication ability, and cytolytic activity in various cell lines. It was also evaluated for treatment of xenografted human carcinoid tumors in nude mice. To use Ad[CgA-E1A] for the treatment of carcinoid liver metastases, it is important that normal hepatocytes do not support virus replication to minimize hepatotoxicity. We therefore evaluated CgA protein expression in normal hepatocytes. We also evaluated CgA gene expression in normal hepatocytes and microdissected tumor cells from carcinoid metastases. RESULTS: We found that Ad[CgA-E1A] replicates similarly to wild-type virus in tumor cells with neuroendocrine features, including the BON carcinoid cell line and the SH-SY-5Y neuroblastoma cell lines, whereas it is attenuated in other cell types. Thus, cells where the CgA promoter is active are selectively killed. We also found that Ad[CgA-E1A] is able to suppress fast-growing human BON carcinoid tumors in nude mice. Furthermore, CgA is highly expressed in microdissected cells from carcinoid metastases, whereas it is not expressed in normal hepatocytes. CONCLUSION: Ad[CgA-E1A] is an interesting agent for the treatment of carcinoid liver metastases in conjunction with standard therapy for these malignancies.

BACKGROUND: A proportion of men with prostate cancer will progress to develop metastatic disease involving the lymph-nodes and bone. To identify novel candidates associated with metastatic progression, we compared the proteomic profiles of LNCaP (lymph-node metastatic, androgen-dependant) and PC-3 (bone metastatic, androgen-independent), human prostate cancer cells. METHODS: Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS), was used to identify differentially expressed proteins. Western blotting was used to validate the identity of any candidates. Immunohistochemistry was used to assess tissue expression. RESULTS: 2D-PAGE followed by ESI-MS/MS analyses identified the expression of glutathione S-transferase-pi (GST-pi) and protein gene product 9.5 (PGP 9.5) in PC-3 cells, but absent expression in LNCaP cells. PGP 9.5 expression in PC-3 cells was confirmed by Western blotting, in addition to expression in DU145 cells. Analysis of cell conditioned media showed that PGP 9.5 was secreted. Sequencing of the PGP 9.5 gene promoter region in bisulfite modified DNA, suggested that the regulation of expression involves promoter hypermethylation. RT-PCR analysis for Chromogranin A (ChA) mRNA (a marker of neuroendocrine cells), showed expression in PC-3 and DU145 cells but was undetectable in LNCaP cells. Immunohistochemistry localised PGP 9.5 expression exclusively within neuroendocrine cells and nerve fibres. CONCLUSIONS: Our unexpected finding that the neuroendocrine cell markers PGP 9.5 and ChA are expressed by PC-3 and DU145 cells, suggests that these cells may have been derived from metastatic adenocarcinomas which had undergone neuroendocrine differentiation or alternatively the expression occurred ectopically as a result of cell culture.

BACKGROUND: A proportion of men with prostate cancer will progress to develop metastatic disease involving the lymph-nodes and bone. To identify novel candidates associated with metastatic progression, we compared the proteomic profiles of LNCaP (lymph-node metastatic, androgen-dependant) and PC-3 (bone metastatic, androgen-independent), human prostate cancer cells. METHODS: Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS), was used to identify differentially expressed proteins. Western blotting was used to validate the identity of any candidates. Immunohistochemistry was used to assess tissue expression. RESULTS: 2D-PAGE followed by ESI-MS/MS analyses identified the expression of glutathione S-transferase-pi (GST-pi) and protein gene product 9.5 (PGP 9.5) in PC-3 cells, but absent expression in LNCaP cells. PGP 9.5 expression in PC-3 cells was confirmed by Western blotting, in addition to expression in DU145 cells. Analysis of cell conditioned media showed that PGP 9.5 was secreted. Sequencing of the PGP 9.5 gene promoter region in bisulfite modified DNA, suggested that the regulation of expression involves promoter hypermethylation. RT-PCR analysis for Chromogranin A (ChA) mRNA (a marker of neuroendocrine cells), showed expression in PC-3 and DU145 cells but was undetectable in LNCaP cells. Immunohistochemistry localised PGP 9.5 expression exclusively within neuroendocrine cells and nerve fibres. CONCLUSIONS: Our unexpected finding that the neuroendocrine cell markers PGP 9.5 and ChA are expressed by PC-3 and DU145 cells, suggests that these cells may have been derived from metastatic adenocarcinomas which had undergone neuroendocrine differentiation or alternatively the expression occurred ectopically as a result of cell culture.

OBJECTIVES: Ascites secondary to neuroendocrine tumor metastases may arise from a variety of mechanisms. Our aim was to measure serum and ascitic chromogranin-A (CgA) to help determine whether ascites resulted from intraperitoneal/retroperitoneal disease burden or from other carcinoid complications such as congestive heart failure or portal hypertension. METHODS: Patients with metastatic neuroendocrine tumors and ascites were identified. Chromogranin-A was obtained and measured from both serum and ascites. The causes of carcinoid ascites was categorized into 2 groups: high intraperitoneal or retroperitoneal disease burden (ie, peritoneal metastases and/or lymphatic obstruction; n = 12, group 1) or other organ-specific carcinoid complications such as CHF or portal hypertension (n = 12, group 2). RESULTS: An ascites CgA/serum CgA ratio greater than 1 was more likely to be found in group 1 (P = 0.01). This ratio produced 100% sensitivity and 75% specificity for ascites secondary to peritoneal metastases and/or lymphatic obstruction. CONCLUSIONS: An ascites CgA/serum CgA ratio greater than 1 produces excellent accuracy in predicting peritoneal metastases and/or retroperitoneal disease as the cause of ascites in the setting of metastatic carcinoid. This test may play a role in the earlier identification of those patients who may be well served by aggressive management.

INTRODUCTION: Chromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN de-differentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis and played a role in the regulation of NEN proliferation. METHODS: Chromogranin A mRNA (PCR) and protein (ELISA/western blot) were studied in 10 normal human mucosa, 5 enterochromaffin cell preparations, 26 small intestinal NEN primaries and 9 liver metastases. Cell viability (WST-1 assay), proliferation (bromodeoxyuridine ELISA) and expression of AKT/AKT-P (CASE ELISA/western blot) in response to chromogranin A silencing, inhibition of prohormone convertase and mTOR inhibition (RAD001/AKT antisense) as well as different chromogranin A fragments were examined in 4 SI-NEN cell lines. RESULTS: Chromogranin A mRNA and protein levels were increased (37-340 fold, p<0.0001) in small intestinal NENs compared to normal enterochromaffin cells. Western blot identified chromogranin A-associated processing bands including vasostatin in small intestinal NENs as well as up-regulated expression of prohormone convertase in metastases. Proliferation in small intestinal NEN cell lines was decreased by silencing chromogranin A as well as by inhibition of prohormone convertase (p<0.05). This inhibition also decreased secretion of chromogranin A (p<0.05) and 5-HT (p<0.05) as well as expression of vasostatin. metastatic small intestinal NEN cell lines were stimulated (50-80%, p<0.05) and AKT phosphorylated (Ser473: p<0.05) by vasostatin I, which was completely reversed by RAD001 (p<0.01) and AKT antisense (p<0.05) while chromostatin inhibited proliferation (~50%, p<0.05). CONCLUSION: Chromogranin A was differentially regulated in primary and metastatic small intestinal NENs and cell lines. Chromogranin A fragments regulated metastatic small intestinal NEN proliferation via the AKT pathway indicating that CgA plays a far more complex role in the biology of these tumors than previously considered.

OBJECTIVES: We analyzed the incidence of elevated serum levels of chromogranin A (CgA) (as marker of neuroendocrine activity) in nonmetastatic and metastatic prostate cancer populations. MATERIAL AND METHODS: 264 consecutive men with nonmetastatic prostate adenocarcinoma considered for radical prostatectomy (group 1) and 89 consecutive men with metastatic prostate adenocarcinoma (group 2) represented our population. In all 353 cases a blood sample for the determination of serum total PSA and CgA levels was obtained (RIA). Two different cut-off for elevated serum CgA levels were used: >60 and >90 ng/ml. RESULTS: In group 1, 35.0% of cases presented CgA levels >60 ng/ml and 6.4% >90 ng/ml. In group 2, 100% of cases presented CgA levels >60 ng/ml and 69.7% >90 ng/ml. The OR for CgA level >60 and >90 ng/ml significantly increased from nonmetastatic to metastatic cases (p = 0.0001). In group 1 the percentage of cases with CgA >60 ng/ml was 29.6% in Gleason score or=7 (4 + 3) (p = 0.0001). In group 2, the percentage of cases with CgA >90 ng/ml was 51.8% in Gleason score or=7 (4 + 3) (p = 0.0028). CONCLUSIONS: We describe a significant incidence of elevated serum levels of CgA either in nonmetastatic (using 60 ng/ml as cut-off) or in metastatic (using 90 ng/ml as cut-off) prostate adenocarcinoma cases.

In this retrospective study, we evaluated the chromogranin A (CgA) baseline value as a predictor of clinical outcome in patients with metastatic castration-resistant prostate cancer (CRPC) treated with abiraterone 1000 mg per day, whose disease progressed after docetaxel chemotherapy. In the 48 evaluable patients, serum CgA level was normal when <120 ng/ml (group A, n=16), within three times the upper normal value (UNV) when between 120 and 360 (group B, n=16), more than three times the UNV when >/=360 ng/ml (group C, n=16). Decline in PSA level >/=50% or more (PSA RR) was observed in 26 of 48 (54%) patients. PSA response rate did not correlate with the CgA groups. CgA levels more than three times the UNV predicted an early radiological progressive disease in eight of 11 cases (73%). The median progression-free survival (PFS) among the CgA groups A, B, and C was 9.2, 9.2, and 4.8 months respectively, P=0.0459. The median overall survival (OS) among the CgA groups was 19.0, 18.8, and 10.8 months respectively, P=0.2092. In the multivariate analysis, PSA RR (nonresponsive vs responsive) and CgA levels (group 3 vs groups 1+2) were predictors of PFS (P=0.0002 and P=0.0047 respectively), whereas PSA RR only was significantly associated with OS (P=0.0024), while CgA levels remained of borderline significance (P=0.0919). A serum CGA level more than three times the UNV predicted PFS and showed a trend vs OS prediction, independently from PSA response, in CRPC patients treated with abiraterone.

BACKGROUND: Accurate neuroendocrine neoplasia (NEN) staging is vital for determining prognosis and therapeutic strategy. The great majority of NENs express chromogranin A (CgA) which can be detected at a protein or transcript level. The current standards for lymph node metastasis detection are histological examination after Hematoxylin and Eosin (H&E) and CgA immunohistochemical (IHC) staining. We hypothesized that detection of CgA mRNA transcripts would be a more sensitive method of detecting these metastases. FINDINGS: We compared these traditional methods with PCR for CgA mRNA extracted from formalin fixed paraffin embedded slides of lymph nodes (n = 196) from small intestinal NENs, other gastrointestinal cancers and benign gastrointestinal disease. CgA PCR detected significantly more NEN lymph nodes (75%) than H&E (53%) or CgA IHC (57%) (p = 0.02). PCR detected CgA mRNA in 50% (14 of the 28) of SI-NEN lymph nodes previously considered negative. The false positive rate for detection of CgA mRNA was 19% in non-neuroendocrine cancers, and appeared to be due to occult neuroendocrine differentiation or contamination by normal epithelium during histological processing. CONCLUSIONS: Molecular pathological analysis demonstrates the limitations of observer-dependent histopathology. CgA PCR analysis detected the presence of CgA transcripts in lymph nodes without histological evidence of tumor metastasis. Molecular node positivity (stage molN1) of SI-NEN lymph nodes could confer greater staging accuracy and facilitate early and accurate therapeutic intervention. This technique warrants investigation using clinically annotated tumor samples with follow-up data.

INTRODUCTION: Chromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN de-differentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis and played a role in the regulation of NEN proliferation. METHODS: Chromogranin A mRNA (PCR) and protein (ELISA/western blot) were studied in 10 normal human mucosa, 5 enterochromaffin cell preparations, 26 small intestinal NEN primaries and 9 liver metastases. Cell viability (WST-1 assay), proliferation (bromodeoxyuridine ELISA) and expression of AKT/AKT-P (CASE ELISA/western blot) in response to chromogranin A silencing, inhibition of prohormone convertase and mTOR inhibition (RAD001/AKT antisense) as well as different chromogranin A fragments were examined in 4 SI-NEN cell lines. RESULTS: Chromogranin A mRNA and protein levels were increased (37-340 fold, p<0.0001) in small intestinal NENs compared to normal enterochromaffin cells. Western blot identified chromogranin A-associated processing bands including vasostatin in small intestinal NENs as well as up-regulated expression of prohormone convertase in metastases. Proliferation in small intestinal NEN cell lines was decreased by silencing chromogranin A as well as by inhibition of prohormone convertase (p<0.05). This inhibition also decreased secretion of chromogranin A (p<0.05) and 5-HT (p<0.05) as well as expression of vasostatin. metastatic small intestinal NEN cell lines were stimulated (50-80%, p<0.05) and AKT phosphorylated (Ser473: p<0.05) by vasostatin I, which was completely reversed by RAD001 (p<0.01) and AKT antisense (p<0.05) while chromostatin inhibited proliferation (~50%, p<0.05). CONCLUSION: Chromogranin A was differentially regulated in primary and metastatic small intestinal NENs and cell lines. Chromogranin A fragments regulated metastatic small intestinal NEN proliferation via the AKT pathway indicating that CgA plays a far more complex role in the biology of these tumors than previously considered.