Repressive histone tail modifications have been associated with molecular breast cancer subtypes. We investigated whether histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) were associated with tumor features and subtypes while adjusting for prospectively collected reproductive and lifestyle breast cancer risk factors. We have tissue microarray data with immunohistochemical marker information on 804 incident cases of invasive breast cancer diagnosed from 1976-2000 in the Nurses' Health Study. Tissue microarray sections were stained for global H3K9me3 and H3K27me3, and scored into four categories. Multivariate odds ratios (OR) and 95 % confidence intervals (CI) were calculated using logistic regression models for tumor features and subtypes, adjusting for breast cancer risk factors. While there were no significant associations between H3K9me3 and tumor features, H3K27me3 was significantly associated with lower grade tumors compared to high grade tumors in the multivariate model (OR = 1.95, 95 % CI 1.35-2.81, p = 0.0004). H3K27me3 was suggestively associated with estrogen receptor-positive (ER+) tumors (OR = 1.47, 95 % CI 0.97-2.23, p = 0.07). In subtype analyses, H3K27me3 was positively associated with the luminal A subtype compared to all other subtypes (OR = 1.42, 95 % CI 1.14-1.77, p = 0.002), and was inversely associated with HER2-type (OR = 0.58, 95 % CI 0.37-0.91, p = 0.02) and basal-like breast cancer (OR = 0.52, 95 % CI 0.36-0.76, p = 0.0006). In the largest immunohistochemical examination of H3K9me3 and H3K27me3 in breast cancer, we found that H3K27me3 positivity, but not H3K9me3, was associated with lower grade tumors and the luminal A subtype after adjusting for reproductive and lifestyle breast cancer risk factors.

Assessment of Ki67 in breast tumors by DIA was a robust and quantitative method. Results from this large prospective cohort study provide support for the clinical relevance of using Ki67 at the 14% cut point for luminal subtype classification and breast cancer prognosis.

Epigenetic inactivation of tumor-suppressor and other regulatory genes plays a critical role in carcinogenesis. Transcriptional silencing is often maintained by DNA methyl transferase (DNMT)-mediated hypermethylation of CpG islands in promoter DNA. Nucleoside analogs including azacytidine and decitabine have been used to inhibit DNMT and re-activate genes, and are clinically used. Their shortcomings include a short half-life and a slow onset of action due to required nucleotide incorporation during DNA replication, which may limit clinical utility. It might be useful to begin to identify lead compounds having novel properties, specifically distinct and fast-acting gene desilencing. We previously identified chemicals augmenting gene expression in multiple reporter systems. We now report that a subset of these compounds that includes quinacrine re-expresses epigenetically silenced genes implicated in carcinogenesis. p16, TFPI2, the cadherins E-cadherin and CDH13, and the secreted frizzle-related proteins (SFRPs) SFRP1 and SFRP5 were desilenced in cancer cell lines. These lead compounds were fast-acting: re-expression occurred by 12-24 hours. Reactivation of silenced genes was accompanied by depletion of DNMT1 at the promoters of activated genes and demethylation of DNA. A model compound, 5175328, induced changes more rapidly than decitabine. These gene desilencing agents belonged to a class of acridine compounds, intercalated into DNA, and inhibited DNMT1 activity in vitro. Although to define the mechanism would be outside the scope of this initial report, this class may re-activate silenced genes in part by intercalating into DNA and subsequently inhibiting full DNMT1 activity. Rapid mechanisms for chemical desilencing of methylated genes therefore exist.

The Id family of helix-loop-helix transcription factors is upregulated in a variety of human malignancies and has been implicated in promoting tumorigenesis through effects on cell growth, differentiation, and tumor angiogenesis. While expression of Id proteins has been associated with tumorigenesis, the precise mechanistic relationship between Id expression and carcinogenesis has not been clearly delineated. We have previously shown that Id1 delays cellular senescence in primary mammalian cells through inhibition of the cell cycle regulatory protein and familial melanoma gene, p16/INK4a. We have also demonstrated that Id1 expression is upregulated in early stage primary human melanomas and may be an important marker for early malignancy. In order to further define the role of Id1 in human melanoma development, we have evaluated the function of Id1 in primary human melanocytes. Here we show that constitutive expression of Id1 in primary human melanocytes leads to delayed cellular senescence and decreased expression of the familial melanoma gene, p16/INK4a. Although melanocytes constitutively expressing Id1 are shown to possess extended lifespans, this is not associated with an appreciable change in cell growth or telomere length. We conclude that Id1 delays cellular senescence in primary human melanocytes through inhibition of p16/INK4a expression and suggest that Id1 may contribute to the malignant conversion of primary human melanocytes through extension of cellular lifespan.

Id genes have been demonstrated to be upregulated in a wide variety of human malignancies and their expression has been correlated with disease prognosis; however, little is known about the mechanisms of Id gene activation in tumors. We have previously shown that the helix-loop-helix transcription factor, Id1, is highly expressed in primary human melanomas during the radial growth phase and that Id1 is a transcriptional repressor of the familial melanoma gene CDKN2A. Here we use a series of melanoma cell lines that recapitulate the phenotypic characteristics of melanomas at varying stages of malignant progression to evaluate the expression levels of Id1 in this model system and determine the mechanism of Id1 dysregulation in these tumor cells. We find elevated protein levels of Id1 to be present consistently in radial growth phase tumor cells in accordance with our primary tumor data. Id1 transcript levels were also found to be elevated in these radial growth phase melanoma cells without any appreciable evidence of gene amplification and Id1 promoter activity was found to correlate with Id expression levels. We therefore conclude that Id1 expression is primarily regulated at the transcriptional level in radial growth phase melanomas and expect that therapies that target Id1 gene expression may be useful in the treatment of Id-associated malignancies.

No abstract

The full molecular consequences of oncogene activation during tumorigenesis are not well understood, but several studies have recently linked oncogene activation to epigenetic silencing of specific genes.1,2 Transcriptional repressor Id1 is overexpressed in many malignancies including melanoma, and Id1 targets include tumor suppressor genes TSP1, CDKN2A (p16) and CDKN1A (p21), which are frequently epigenetically silenced in cancer. We confirmed that both TSP1 and CDKN2A have abnormal promoter region DNa methylation in primary melanoma, but the mechanism by which this silencing occurs remains unknown. Here we explore the effects of stable lentiviral Id1 overexpression on the expression of these Id1 target genes in human melanoma cell lines. Overexpressed Id1 was functional and bound transcriptional activator E2A, but did not sequester E2A from gene promoters and repress gene expression. Therefore, these Id1 target genes were resistant to Id1-mediated gene silencing. Our results suggest that Id1 activation may need to occur at discrete stages in cooperation with additional gene dysregulation to repress and induce epigenetic silencing of tumor suppressor genes during melanoma progression.

The Ninth Annual AACR Frontiers in Cancer Prevention Research conference was held in Philadelphia in November 7–10, 2010. Its thematic focus was “Prevention: From Basic Science to Public Health Benefit.” Telomere plasticity, the microenvironment, inflammation, transformation to the metastatic phenotype, and pathways to obesity were highlighted as important elements of carcinogenesis amenable to intervention. The integration of information from novel technologies related to physical biology, molecular and genetic profiles, and imaging along with behavioral and clinical parameters have advanced risk stratification and early detection. Cancer prevention represents a powerful testing ground for the development of individually tailored intervention and for increasing the efficiency of drug discovery. Advances in clinical trials relate to more efficient design strategies, have shown first-in-human targeting capabilities, and have developed powerful strategies to overcome accrual barriers. Tailored intervention strategies now show high efficacy on large cohorts across several cancer types. These successes are expected to increase.