Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions

Abstract

CST6 is a breast tumor suppressor gene that is expressed in normal breast epithelium, but is epigenetically silenced as a consequence of promoter hypermethylation in metastatic breast cancer cell lines. In the current study, we investigated the expression and methylation status of CST6 in primary breast tumors and lymph node metastases. 25/45 (56%) primary tumors and 17/20 (85%) lymph node metastases expressed significantly lower levels of cystatin M compared to normal breast tissue. Bisulfite sequencing demonstrated CST6 promoter hypermethylation in 11/23 (48%) neoplastic lesions analyzed, including 3/11 (27%) primary tumors and 8/12 (67%) lymph node metastases. In most cases (12/23, 52%), the expression of cystatin M directly reflected CST6 promoter methylation status. In remaining lesions (8/23, 35%) loss of cystatin M was not associated with CST6 promoter hypermethylation, indicating that other mechanisms can account for loss of CST6 expression. These results show that methylation-dependent silencing of CST6 occurs in a subset of primary breast cancers, but more frequently in metastatic lesions, possibly reflecting progressionrelated genomic events. To examine this possibility, primary breast tumors and matched lymph node metastases were analyzed. In 2/3 (67%) patients, primary tumors were positive for cystatin M and negative for CST6 promoter methylation, and matched metastatic lesions lacked cystatin M expression and CST6 was hypermethylated. This observation suggests that progression-related epigenetic alterations in CST6 gene expression can accompany metastatic spread from a primary tumor site. Overall, the results of the current investigation suggest that methylation-dependent epigenetic silencing of CST6 represents an important mechanism for loss of CST6 during breast tumorigenesis and/or progression to metastasis.

Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy

“…This supports the well established concept that the location of the CpG dinucleotides subjected to methylation plays an important role in determining whether genes are silenced or not. Consistent with this, CST6 , which encodes the cysteine protease inhibitor, cystatin M, has been shown to be silenced following DNA methylation of CpG islands located within the promoter region, but not within exon 1 [41]. Of particular note, in MCF-7 and MDA-231 cells where SFRP1 expression is completely abrogated, we observed complete methylation in CpG islands located in the first exon and in the promoter region of SFRP1 in these cells.…”

Exploring the intrinsic differences among breast tumor subtypes defined using immunohistochemistry markers based on the decision tree

“…MAL2, Mal, T-cell differentiation protein 2, has been identified as a molecule predictive of metastases whose increased expression has been validated in ovarian, colorectal and pancreatic cancer41. CST642 is a breast tumor suppressor expressed in normal breast epithelium, but epigenetically silenced as a consequence of promoter hypermethylation in metastatic breast cancer cell lines, which suggests the mechanism of CST6 loss during breast tumorigenesis and/or progression to metastasis. PGAP3 was reported to be specifically expressed in HER2+ tumor cells but not in stroma or HER2 non-amplified breast tumor samples43.…”

Differential secretome analysis reveals CST6 as a suppressor of breast cancer bone metastasis

“…CST6 has been previously noticed to be down-regulated in malignant breast tumor cells [42,43]. However, its functional role in metastasis, especially in bone metastasis, has yet to be fully characterized.…”