Silencing of CD44 Expression in Prostate Cancer by Hypermethylation of the CD44 Promoter Region

Abstract

SUMMARY:Loss of the CD44 transmembrane glycoprotein in primary prostate cancer has been shown to be associated with unfavorable clinical behavior. Moreover, the majority of prostate cancer metastases lack expression of this molecule. The mechanism of CD44 silencing in prostate cancer was investigated using both patient material and in vivo-propagated human prostate cancer xenografts. In 9 of 11 lymph node metastases of prostate cancer, we demonstrated by methylation-sensitive restriction enzyme digestion that the promoter region of the CD44 gene is methylated, indicating that this represents a major mechanism of CD44 silencing. Similarly, in 6 out of 12 in vivo-growing human prostate carcinoma xenograft models, hypermethylation of the CD44 gene was found. The extent of CpG island methylation was investigated by nucleotide sequencing after bisulphite modification of the CD44 promoter region. In the xenografts displaying hypermethylation, the examined 14 CpG sites in the CD44 transcription regulatory domain, including a Sp1 binding site, were consistently methylated. This correlated with reduced CD44 expression or lack of CD44 expression at mRNA and protein levels. In the xenografts lacking hypermethylation of the CD44 gene, high levels of CD44 mRNA and protein were expressed in some models, whereas in others CD44 mRNA expression was only detectable by RT-PCR and the CD44 protein could hardly be detected or was not detected at all. The results indicate that, in most prostate cancers, loss of CD44 expression is associated with extensive hypermethylation of the CpG island of the CD44 promoter region, but other, posttranscriptional mechanisms may also lead to CD44 loss. (Lab Invest 2000, 80:1291-1298.

SWI/SNF Chromatin-Remodeling Factors Induce Changes in DNA Methylation to Promote Transcriptional Activation

“…These changes were verified by sodium bisulfite sequencing as above, which revealed consistent (100% of clones sequenced) loss of methylation at two sites in the CD44 promoter (Fig. 3D), one of which included an SP1 site that had previously been reported to be hypermethylated in other cell lines (12). Transfection with mutant Brg1 (Fig.…”

“…When these cells were transfected with Brg1 or Brm, endogenous CD44 transcription was induced (7,8). Interestingly, CD44 transcription is silenced in a number of cancer cell lines by hypermethylation of CpG islands within the CD44 promoter that normally remain unmethylated at all times (10)(11)(12)(13)(14). Here, we tested if transcriptional activation by Brg1 and Brm involves loss of methylation of CpG islands in promoter sequences of both the CD44 gene and another gene whose transcription is often silenced by DNA methylation, Ecadherin (15).…”

Epigenetics of prostate cancer: beyond DNA methylation

“…Strikingly, prostate cancers lose the basal cell marker p63 so consistently that its absence can be used for diagnostic purposes [156]. Similarly, the frequently hypermethylated MDR1 [21,157] and CD44 [158][159][160] genes are normally expressed in basal cells. Like GSTP1 hypermethylation (Fig.…”

Molecular biology of prostate cancer

“…255 Loss of CD44 expression is associated with increased grade and stage of PC. 256,257 No metastases express CD44 strongly, and only 14% of metastases expressed even low levels of immunohistochemically detectable CD44. In nine of 11 lymph node metastases of PC, Verkaik et al 257,258 demonstrated by methylation-sensitive restriction enzyme digestion that the promoter region of the CD44 gene is hypermethylated, indicating that this may represent a major mechanism of CD44 silencing.…”

“…256,257 No metastases express CD44 strongly, and only 14% of metastases expressed even low levels of immunohistochemically detectable CD44. In nine of 11 lymph node metastases of PC, Verkaik et al 257,258 demonstrated by methylation-sensitive restriction enzyme digestion that the promoter region of the CD44 gene is hypermethylated, indicating that this may represent a major mechanism of CD44 silencing. Transfection-induced expression of CD44 in the highly metastatic AT3.1 rat PC cell line greatly suppressed its metastatic ability to the lungs, without suppression of in vivo growth rate or primary tumorigenicity.…”