Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation.

BACKGROUND: Thrombospondin-4 (THBS4) is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of THBS4 expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter. METHODS: Fifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP) were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of THBS4 transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon. RESULTS: THBS4 expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and THBS4 methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high THBS4 methylation, but the correlation was not significant. THBS4 methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and negatively with the occurrence of adenomas elsewhere in the colon. CONCLUSIONS: THBS4 shows increased methylation in colorectal cancer, but this is not strongly associated with altered gene expression, either because methylation has not always reached a critical level or because other factors influence THBS4 expression. THBS4 may act as a tumour suppressor gene, demonstrated by its suppression of tumour colony formation in vitro. THBS4 methylation is detectable in normal colonic mucosa and its level may be a biomarker for the occurrence of adenomas and carcinoma.

Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system.

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of alpha-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to alpha-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates alpha-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited alpha-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in alpha-synuclein phosphorylation in central nervous system.

Proneural and proneuroendocrine transcription factor expression in cutaneous mechanoreceptor (Merkel) cells and Merkel cell carcinoma.

Merkel cells form part of the peripheral neuroendocrine system of the skin and act as mechanoreceptors in touch response. Merkel cell carcinoma (MCC) is a rare, aggressive disease with similarities to small cell lung cancer (SCLC), which is also of neuroendocrine origin. We previously identified a novel DNA binding protein complex specific for MCC suspension cell lines, termed Merkel nuclear factor (MNF) by its binding to the POU-IV family DNA binding consensus sequence. Here we report that MNF contains the POU-IV family member Brn-3c and that Brn-3c is expressed in normal Merkel cells. Additionally, Brn-3c protein reactivity is restricted to a subset of MCC biopsies and is not seen in biopsies revealing adherent, variant cell lines lacking neuroendocrine markers. Recently, proper development of murine Merkel cells was shown to require the proneural basic helix-loop-helix transcription factor, atonal family member, MATH1. We demonstrate a correlation between Brn-3c and HATH1 reactivity in MCC biopsies and cell lines with retention of neuroendocrine phenotype. In SCLC, the related basic helix-loop-helix transcription factor HASH1 is responsible for neuroendocrine phenotype, but HASH1 transcripts were not detected in MCC cell lines. We propose that HATH1 and Brn-3c may form a transcriptional hierarchy responsible for determining neuroendocrine phenotype in Merkel cells and that lack of Brn-3c and/or HATH1 in MCC may indicate a more aggressive disease requiring closer patient follow-up.