Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2′,7′-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

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Cancer cells often express tumor antigens that are recognized by host cytolytic T lymphocytes. Such antigens include GAGE (G antigen), MAGE (melanoma specific antigen), PAGE (prostate specific antigen), and CT45 (cancer/testis antigen). Repression of tumor antigens is a mechanism by which cancer cells may evade recognition by the immune system. Methylation of CpG sites in promoter regions of specific genes leads to recruitment of methylated DNA binding proteins and co‐repressors that cause chromatin compaction and gene repression. This is a key mechanism contributing to abnormal gene expression patterns observed in cancer cells. We are investigating the effect on gene expression of 5‐aza‐2′‐deoxycytidine (ADC), an inhibitor of DNA methyltransferase 1 (DNMT1), in the human breast cancer cell line, MCF7. Cells were treated in vitro for 72 hours with 1 µM ADC in DMSO. RNA was then isolated for microarray analysis (Affymetrix). Compared with cells treated with DMSO alone, ADC‐treated cells exhibited increased expression (≥2‐fold) of 38 genes. Ten of these genes were members of the GAGE, MAGE, PAGE, or CT45 families, with all 10 exhibiting >5‐fold increased expression. These results suggest that tumor antigens are repressed epigenetically in MCF7 cells. De‐repression of these antigens using drugs that target epigenetic factors may provide an effective strategy for cancer treatment. American Cancer Society

Cancer cell DNA is globally hypomethylated, but has regions of hypermethylation. Such regional hypermethylation, mediated by methylated DNA binding (MBD) proteins, inhibits gene expression. Demethylation of DNA or inhibition of MBD proteins may prove beneficial in treating cancers. We are investigating the effects on gene expression of 5‐aza‐2′‐deoxycytidine (ADC), an inhibitor of DNA methyltransferase 1 (DNMT1), and siRNA directed against MBD protein 2 (MBD2) in the human liver cancer cell lines, Hep3B and HepG2. Cells are treated in vitro for 72h with 1 µM ADC in dimethylsulfoxide (DMSO) or with DMSO alone, or are transfected for 72h with siRNA directed against MBD2 or with scrambled siRNA. RNA is then isolated for microarray analysis (Affymetrix). ADC‐treated Hep3B and HepG2 cells exhibited increased expression (≥2‐fold) of 168 and 166 genes, respectively. Only 10 of these genes were the same in both cell lines. There was no overlap between the two cell lines in classes of genes that were induced by ADC. MBD2 siRNA induced in Hep3B cells increased expression (≥2‐fold) of 351 genes. Only 5 of these genes were also induced by ADC in Hep3B cells. Inhibition of DNMT1 by ADC and knockdown of MBD2 by siRNA lead to unique molecular responses in liver cancer cells. The ramifications of these findings with respect to therapies that target epigenetic factors remain to be determined. American Cancer Society