Purpose Multiple-gene sequencing is entering practice, but its clinical value is unknown. We evaluated the performance of a customized germline-DNA sequencing panel for cancer-risk assessment in a representative clinical sample. Methods Patients referred for clinical BRCA1/2 testing from 2002 to 2012 were invited to donate a research blood sample. Samples were frozen at −80° C, and DNA was extracted from them after 1 to 10 years. The entire coding region, exon-intron boundaries, and all known pathogenic variants in other regions were sequenced for 42 genes that had cancer risk associations. Potentially actionable results were disclosed to participants. Results In total, 198 women participated in the study: 174 had breast cancer and 57 carried germline BRCA1/2 mutations. BRCA1/2 analysis was fully concordant with prior testing. Sixteen pathogenic variants were identified in ATM, BLM, CDH1, CDKN2A, MUTYH, MLH1, NBN, PRSS1, and SLX4 among 141 women without BRCA1/2 mutations. Fourteen participants carried 15 pathogenic variants, warranting a possible change in care; they were invited for targeted screening recommendations, enabling early detection and removal of a tubular adenoma by colonoscopy. Participants carried an average of 2.1 variants of uncertain significance among 42 genes. Conclusion Among women testing negative for BRCA1/2 mutations, multiple-gene sequencing identified 16 potentially pathogenic mutations in other genes (11.4%; 95% CI, 7.0% to 17.7%), of which 15 (10.6%; 95% CI, 6.5% to 16.9%) prompted consideration of a change in care, enabling early detection of a precancerous colon polyp. Additional studies are required to quantify the penetrance of identified mutations and determine clinical utility. However, these results suggest that multiple-gene sequencing may benefit appropriately selected patients.

An important transcriptional regulatory element of the estrogen receptor (ER) gene that binds a protein expressed in ER-positive breast carcinomas has been identified. Using a transient expression assay, we identified a 75-bp region of the 5' untranslated leader of the ER gene which augments expression from the ER promoter. This region contains two binding sites for a protein, estrogen receptor factor 1 (ERF-1), which is expressed in ER-positive breast carcinomas. A concatenated ERF-1 binding site probe identified a 30,000-Da protein. Low-level ERF-1 expression was detected in normal human mammary epithelial cells. Abundant ERF-1 expression was also found in endometrial carcinoma cell lines that express the ER-positive phenotype. These results indicate that ERF-1 expression represents a common mechanism of ER regulation in hormonally responsive carcinomas.

The ERF-1 transcription factor was previously shown to be involved in the regulation of estrogen receptor (ER) gene transcription in hormonally responsive breast and endometrial carcinomas. In this study we sought to identify the gene for ERF-1. ERF-1 activates ER gene transcription by binding to the imperfect palindrome CCCT-GCGGGG within the promoter of the ER gene. ERF-1 protein was purified from the ER-positive breast carcinoma cell line, MCF7, utilizing ion exchange and DNA affinity chromatography. Peptide sequence analysis was used to isolate a 2.7 kb cDNA clone from an MCF7 cDNA library. This cDNA encodes a protein of 48 kDa previously identified as the AP2␥ transcription factor. By gel-shift analysis, in vitro synthesized ERF-1 comigrates with MCF7 native ERF-1 complex and demonstrates identical sequence binding specificity as native ERF-1. In addition, AP2 polyclonal antisera supershifts both in vitro synthesized and native ERF-1 complexes. These results show that ERF-1 is a member of the AP2 family of developmentally regulated transcription factors. Given the central role of ER expression in breast carcinoma biology, ERF-1 is likely to regulate expression of a set of genes characteristic of the hormonally-responsive breast cancer phenotype.

The AP2 transcription factor family is a set of developmentally regulated, retinoic acid inducible genes composed of four related factors, AP2␣, AP2␤, AP2␥, and AP2␦. AP2 factors orchestrate a variety of cell processes including apoptosis, cell growth, and tissue differentiation during embryogenesis. In studies of primary malignancies, AP2␣ has been shown to function as a tumor suppressor in breast cancer, colon cancer, and malignant melanoma. In cell culture models, overexpression of AP2␣ inhibits cell division and stable colony formation, whereas, a dominant-negative AP2␣ mutant increases invasiveness and tumorigenicity. Here we show that AP2␣ targets the p53 tumor suppressor protein.Studies with chromatin immunoprecipitation demonstrate that AP2␣ is brought to p53 binding sites in p53-regulated promoters. The interaction between AP2␣ and p53 augments p53-mediated transcriptional activation, which results in up-regulation of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1 . AP2␣ is able to induce G 1 and G 2 cell cycle arrest only in the presence of wild-type p53. Thus, we conclude that the tumor suppressor activity of AP2␣ is mediated through a direct interaction with p53. These results also provide a mechanism to explain patterns of gene expression in cancers where AP2␣ is known to function as a tumor suppressor.The AP2 transcription factor family is a set of developmentally regulated, retinoic acid inducible genes composed of four related factors-AP2␣, AP2␤, AP2␥, and AP2␦ (1-4). AP2 factors orchestrate a variety of cell processes including apoptosis, cell growth, and tissue differentiation during embryogenesis (5, 6). Studies in AP2 knock-out mice have indicated an important role for AP2 factors in the development of neural crest, epidermal, and urogenital tissues (5-9). AP2 factors have been shown to regulate the expression of genes in various tissues and tumors. In melanoma cells, AP2 has been shown to regulate the MUC18 and c-KIT promoters (10). AP2␣ and AP2␥ appear to be coordinately involved in regulating the basal and cAMP-induced expression of human chorionic gonadotropin in the placenta (11). In breast cancer, the AP2 factors have been implicated in the regulation of the ErbB2 (3, 12) and ER␣ genes (13,14).Several lines of investigation have led to the conclusion that AP2␣ is a tumor suppressor gene. Studies of primary breast tumors using immunohistochemistry demonstrated a progressive loss of AP2␣ expression with tumor progression from normal mammary epithelium, to DCIS, and to invasive cancer (15). Similar findings have been reported for colon carcinomas evaluated by immunohistochemistry, which demonstrated a decrease in AP2␣ expression in advanced Duke's stage carcinomas (16). Interestingly, there was an increase in AP2␣ mRNA in advanced stage colon tumors as determined by in situ hybridization indicating that the loss of AP2␣ was at the level of protein synthesis or stability. Loss of AP2␣ expression has also been reported to occur with malignant transformation and tumor progression in cuta...

The AP2 transcription factors exhibit a high degree of homology in the DNA binding and dimerization domains. In this study, we methodically compared the binding specificity of AP2alpha and AP2gamma using PCR-assisted binding site selection and competitive gel shift assay and determined that the consensus binding site for both factors is(G)/(C)CCNN(A/)C(/G)(G)/(A)G(G/)C(/T.)The use of single site promoter constructs with either a high or low affinity site demonstrated a direct relationship between site affinity and transcriptional activation. Overexpression of AP2alpha and AP2gamma resulted in the activation of a low affinity binding site construct to levels comparable to those seen with a high affinity site construct at lower amounts of protein expression. Both AP2alpha and AP2gamma were able to trans-activate the cloned human estrogen receptor alpha promoter in ER-negative MDA-MB-231 cells through high affinity AP2 sites in the untranslated leader sequence. This provides a functional mechanism to explain the correlation between AP2 activity and estrogen receptor expression in breast cancer. Since there is overexpression of AP2 factors in breast cancer compared to normal breast epithelium, our results suggest that increased factor expression may activate a set of target genes containing lower affinity binding sites that would normally not be expressed in normal breast epithelium.