Influence of lymphatic invasion on locoregional recurrence following mastectomy: indication for postmastectomy radiotherapy for breast cancer patients with one to three positive nodes.

PURPOSE: The indication for postmastectomy radiotherapy (PMRT) in breast cancer patients with one to three positive lymph nodes has been in discussion. The purpose of this study was to identify patient groups for whom PMRT may be indicated, focusing on varied locoregional recurrence rates depending on lymphatic invasion (ly) status. METHODS AND MATERIALS: Retrospective analysis of 1,994 node-positive patients who had undergone mastectomy without postoperative radiotherapy between January 1990 and December 2000 at our hospital was performed. Patient groups for whom PMRT should be indicated were assessed using statistical tests based on the relationship between locoregional recurrence rate and ly status. RESULTS: Multivariate analysis showed that the ly status affected the locoregional recurrence rate to as great a degree as the number of positive lymph nodes (p < 0.001). Especially for patients with one to three positive nodes, extensive ly was a more significant factor than stage T3 in the TNM staging system for locoregional recurrence (p < 0.001 vs. p = 0.295). CONCLUSION: Among postmastectomy patients with one to three positive lymph nodes, patients with extensive ly seem to require local therapy regimens similar to those used for patients with four or more positive nodes and also seem to require consideration of the use of PMRT.

Critical roles of mucin 1 glycosylation by transactivated polypeptide N-acetylgalactosaminyltransferase 6 in mammary carcinogenesis.

The structure of O-glycosylated proteins is altered in breast cancer cells, but the mechanisms of such an aberrant modification have been largely unknown. We here report critical roles of a novel druggable target, polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which is upregulated in a great majority of breast cancers and encodes a glycosyltransferase responsible for initiating mucin-type O-glycosylation. Knockdown of GALNT6 by small interfering RNA significantly enhanced cell adhesion function and suppressed the growth of breast cancer cells. Western blot and immunostaining analyses indicated that wild-type GALNT6 protein could glycosylate and stabilize an oncoprotein mucin 1 (MUC1), which was upregulated with GALNT6 in breast cancer specimens. Furthermore, knockdown of GALNT6 or MUC1 led to similar morphologic changes of cancer cells accompanied by the increase of cell adhesion molecules beta-catenin and E-cadherin. Our findings implied that overexpression of GALNT6 might contribute to mammary carcinogenesis through aberrant glycosylation and stabilization of MUC1 and that screening of GALNT6 inhibitors would be valuable for the development of novel therapeutic modalities against breast cancer.

Genomic screening for genes upregulated by demethylation revealed novel targets of epigenetic silencing in breast cancer.

Breast cancer arises through the accumulation of multiple genetic alterations and epigenetic changes such as methylation, which silences gene expression in a variety of cancers. In the present study, we applied genomic screening to identify genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine (DAC) in a human breast cancer cell line (MCF7). We identified 288 genes upregulated and 29 genes downregulated more than fivefold after treatment with DAC, and gene ontology analyses revealed the genes to be involved in immune responses, apoptosis, and cell differentiation. In addition, real-time PCR analysis of ten genes silenced in MCF7 cells confirmed that they are upregulated by DAC, while bisulfite-pyrosequencing analysis confirmed that nine of those genes were silenced by methylation. We also found that treating MCF7 cells with DAC restored induction of DFNA5 by p53, as well as by two other p53 family genes, p63gamma and p73beta. Introduction of NTN4 into MCF7 cells suppressed cell growth, indicating that NTN4 has tumor suppressive activity. In primary breast cancers, we detected cancer-specific methylation of NTN4, PGP9.5, and DKK3, suggesting that methylation of these genes could be useful markers for diagnosis of breast cancer. Thus, DNA methylation appears to be a common event in breast cancer, and the genes silenced by methylation could be useful targets for both diagnosis and therapy.

Cytoplasmic RASSF2A is a proapoptotic mediator whose expression is epigenetically silenced in gastric cancer.

Gastric cancer cells often show altered Ras signaling, though the underlying molecular mechanism is not fully understood. We examined the expression profile of eight ras-association domain family (RASSF) genes plus MST1/2 and found that RASSF2A is the most frequently downregulated in gastric cancer. RASSF2A was completely silenced in 6 of 10 gastric cancer cell lines as a result of promoter methylation, and expression was restored by treating the cells with 5-aza-2'-deoxycytidine. Introduction of RASSF2A into non-expressing cell lines suppressed colony formation and induced apoptosis. These effects were associated with the cytoplasmic localization of RASSF2A and morphological changes to the cells. Complementary DNA microarray analysis revealed that RASSF2A suppresses the expression of inflammatory cytokines, which may in turn suppress angiogenesis and invasion. In primary gastric cancers, aberrant methylation of RASSF2A was detected in 23 of 78 (29.5%) cases, and methylation correlated significantly with an absence of the lymphatic invasion, absence of venous invasion, absence of lymph node metastasis, less advanced stages, Epstein-Barr virus, absence of p53 mutations and the presence of the CpG island methylator phenotype-high. These results suggest that epigenetic inactivation of RASSF2A is required for tumorigenesis in a subset of gastric cancers.

Gene amplification and overexpression of PRDM14 in breast cancers.

Several genes that encode PR (PRDI-BF1 and RIZ) domain proteins (PRDM) have been linked to human cancers. To explore the role of the PR domain family genes in breast carcinogenesis, we examined the expression profiles of 16 members of the PRDM gene family in a panel of breast cancer cell lines and primary breast cancer specimens using semiquantitative real-time PCR. We found that PRDM14 mRNA is overexpressed in about two thirds of breast cancers; moreover, immunohistochemical analysis showed that expression of PRDM14 protein is also up-regulated. Analysis of the gene copy number revealed that PRDM14 is a target of gene amplification on chromosome 8q13, which is a region where gene amplification has frequently been detected in various human tumors. Introduction of PRDM14 into cancer cells enhanced cell growth and reduced their sensitivity to chemotherapeutic drugs. Conversely, knockdown of PRDM14 by siRNA induced apoptosis in breast cancer cells and increased their sensitivity to chemotherapeutic drugs, suggesting that up-regulated expression of PRDM14 may play an important role in the proliferation of breast cancer cells. That little or no expression of PRDM14 is seen in noncancerous tissues suggests that PRDM14 could be an ideal therapeutic target for the treatment of breast cancer.