Date of Award

11-15-2006

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Irvin M Modlin

Abstract

Background: Carcinoid tumors are rare tumors that, despite advances in medical research, have remained enigmatic. Carcinoid tumors can be categorized using gross pathological, histological, and biochemical profiles. While of some utility, this classification method fails to enable accurate determination of malignant potential. Incorporation of molecular analysis of carcinoids will create a paradigm shift in classification. To this end, high throughput technologies such as Quantitative RT PCR, Tissue Microarray, and cDNA Microarray will facilitate such changes. In our investigations, cDNA microarray analysis identified Metastasis-associated protein-1 (MTA1), Histone Deacetylase 2 (HDAC2), and aberrant transcriptional regulation as factors involved in the molecular pathogenesis of SICs. HDAC2 mediates histone deacetylation, resulting in chromatin remodeling and transcriptional repression while MTA1, found in complex with HDAC2, is not only a mediator of transcriptional repression, but also of cell growth and motility. These proteins, as mediators of epigenetic processes, are markers of malignancy in gastric, breast, colorectal, and hematological malignancies. HER2 has been implicated in MTA1 upregulation in the breast cancer model. Analysis of HDAC2 and MTA1 expression and their roles in SIC malignancy could potentially alter SIC classification and lead to the investigation of anti-epigenetic cancer strategies such as HDAC inhibitors. Hypothesis: MTA1 and HDAC2 (MTA1/HDAC2 complex), involved in transcriptional regulation are both markers of SIC malignancy and function in SIC pathogenesis. HER2 mediates MTA1 upregulation in SICs, as in breast tumors. HER2, ER, and CpG island methylation may be involved in SIC tumorigenesis by regulating MTA1 expression. Methods: The gene expression profile of eight (8) GI carcinoid samples was analyzed using Afffymetrix ™ Gene Chip ® technology. Transcript expression of HDAC2, MTA1, HER2, ER2, and Trefoil Factor 1 (TFF1) was examined in normal mucosa and SIC samples using Quantitative RT-PCR. Protein expression of HDAC2, MTA1, HER2, ER2, and TFF1 was measured on a carcinoid tissue microarray containing 55 SIC tumor samples with matched normal mucosa. HDAC activity was quantified directly through an enzymatic assay and Trichostatin A (TSA) inhibition and indirectly by analysis of histone acetylation on TMA. MTA1 upregulation in SICs was explored through analysis of HER2 gene and protein expression patterns and of the MTA1 promoter region. Results: Affymetrix GeneChip and Ingenuity Pathway analysis identified aberrant transcriptional repression mediated by MTA1 and HDAC2 as a principal pathway in SICs. Q-RT-PCR: MTA1: nml-1.68±0.16, primary- 3.58±1.5*, mets-5.96±2.4#; HDAC2: nml-0.07±0.02, primary-2.0±0.8&, mets-4.1±0.6&; HER2: nml-2.28±0.51, primary-1.02±0.23*, mets-0.8±0.39#; ERα±: nml-0.49±0.19, primary- 5.71±3.1#, mets-3.01±1.4#; TFF1: nml-0.86±0.21, primary- 0.005±0.002*, mets-0.0059±0.0018*. TMA analysis: MTA1: nml-3±1 local-75±12#, disseminated-78±1#; HDAC2: nml-524± 81. local-712±101#, disseminated-762±105#; HER2: nml- 10±1, local-9±1NS, disseminated-6±0.5NS; ER±: nml- 11±0.5, local-10.1±1NS, disseminated-12±0.5NS; TFF1: nml- 98, local-83±5NS, disseminated-75±4NS.HDAC activity in SICs: nml- 0.08, nml with TSA- 0.04, SIC- 0.32, SIC with TSA- 0.081. Indirect HDAC activity assessment through TMA: nml- 757±116, local- 502±121#, disseminated- 523±112#.Ratio of acetylated to unacetylated H2B: nml- 1.74, SIC-1.43. (*p=0.05, #p<0.05, NS=not significant) Analysis of the MTA1 promoter region revealed c-Myc, c-Fos/c-Jun, c-Myc, SP1 responsive elements as well as CpG islands. Attempts to amplify the MTA1 promoter region were unsuccessful. Conclusion: HDAC2 and MTA1 are functional markers of SIC malignancy. Both transcript and protein expression increased as tissue progressed from normal SI mucosa to primary SIC to metastatic SIC tissue. Involved in epigenetic processes, these proteins and their respective pathways may be potential therapeutic targets of HDAC inhibitors. Low HER2 expression and a lack of correlation with MTA1 expression suggests other mechanisms of MTA1 upregulation in SIC tissue. These may include c-Myc, c-Fos/c-Jun, c-Myb, SP1, and CpG island (de)methylation. Further investigations will provide more information regarding modes of MTA1 upregulation in SIC. Overall, epigenetic modulation via the MTA1/HDAC2 complex may be of importance in SIC tumorgenesis and molecular analysis may be a means of enhancing SIC classification with a view to facilitating improved diagnosis, prognostication, and therapeutic strategy.

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