Promoter DNA Methylation of Oncostatin M receptor-β as a Novel Diagnostic and Therapeutic Marker in Colon Cancer

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging “cancer methylome”. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-β (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients.

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening

Immunocytochemistry of cell lines Schwannomatosis cell lines express the Schwann cell marker S100B and do not express the fibroblast marker Thy-1.

<p>Fibroblast cells stain positive for Thy-1. Green is a positive antibody test; blue is a DAPI nuclear stain.</p

Expression of cell cycle regulators in SWN cell lines and primary human Schwann cells in culture.

<p>Several cell cycle related genes were differentially expressed in a SWN cell line compared with parent tumor by microarray. qPCR comparison of Primary Human Schwann Cells and Immortalized Hp-SWN-14F cell line suggest that the alterations in expression are due to active growth during cell culturing.</p

Schwannomatosis cell lines maintain Schwann cell characteristics after passaging and freezing.

<p>Primary Human Schwann cells and immortalized Hp-SWN-14F (at passage 6 exhibit similar growth rates and expression of S100B. Hnp-SWN-14O retains S100B staining and Schwann cell properties at passage 11.</p

Schwannomatosis Cell line and Parent Tumor characteristics.

<p>Schwannomatosis cell line Hp-SWN-14F and its parent tumor s express Schwann cell markers, p75/NGFR, S100B, and NGF as determined by RT-PCR.</p