The DNA cytosine deaminase APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer.

Breast tumors often display extreme genetic heterogeneity characterized by hundreds of gross chromosomal aberrations and tens of thousands of somatic mutations. Tumor evolution is thought to be ongoing and driven by multiple mutagenic processes. A major outstanding question is whether primary tumors have preexisting mutations for therapy resistance or whether additional DNA damage and mutagenesis are necessary. Drug resistance is a key measure of tumor evolvability. If a resistance mutation preexists at the time of primary tumor presentation, then the intended therapy is likely to fail. However, if resistance does not preexist, then ongoing mutational processes still have the potential to undermine therapeutic efficacy. The antiviral enzyme APOBEC3B (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3B) preferentially deaminates DNA C-to-U, which results in signature C-to-T and C-to-G mutations commonly observed in breast tumors. We use clinical data and xenograft experiments to ask whether APOBEC3B contributes to ongoing breast tumor evolution and resistance to the selective estrogen receptor modulator, tamoxifen. First, APOBEC3B levels in primary estrogen receptor-positive (ER+) breast tumors inversely correlate with the clinical benefit of tamoxifen in the treatment of metastatic ER+ disease. Second, APOBEC3B depletion in an ER+ breast cancer cell line results in prolonged tamoxifen responses in murine xenograft experiments. Third, APOBEC3B overexpression accelerates the development of tamoxifen resistance in murine xenograft experiments by a mechanism that requires the enzyme's catalytic activity. These studies combine to indicate that APOBEC3B promotes drug resistance in breast cancer and that inhibiting APOBEC3B-dependent tumor evolvability may be an effective strategy to improve efficacies of targeted cancer therapies.

MicroRNAs link estrogen receptor alpha status and Dicer levels in breast cancer.

To identify microRNAs (miRNAs) associated with estrogen receptor (ESR1) status, we profiled luminal A, ESR1+ breast cancer cell lines versus triple negative (TN), which lack ERα, progesterone receptor and Her2/neu. Although two thirds of the differentially expressed miRNAs are higher in ESR1+ breast cancer cells, some miRNAs, such as miR-222/221 and miR-29a, are dramatically higher in ESR1- cells (∼100- and 16-fold higher, respectively). MiR-222/221 (which target ESR1 itself) and miR-29a are predicted to target the 3' UTR of Dicer1. Addition of these miRNAs to ESR1+ cells reduces Dicer protein, whereas antagonizing miR-222 in ESR1- cells increases Dicer protein. We demonstrate via luciferase reporter assays that these miRNAs directly target the Dicer1 3' UTR. In contrast, miR-200c, which promotes an epithelial phenotype, is 58-fold higher in the more well-differentiated ERα+ cells, and restoration of miR-200c to ERα- cells causes increased Dicer protein, resulting in increased levels of other mature miRNAs typically low in ESR1- cells. Together, our findings explain why Dicer is low in ERα negative breast cancers, since such cells express high miR-221/222 and miR-29a levels (which repress Dicer) and low miR-200c (which positively affect Dicer levels). Furthermore, we find that miR-7, which is more abundant in ERα+ cells and is estrogen regulated, targets growth factor receptors and signaling intermediates such as EGFR, IGF1R, and IRS-2. In summary, miRNAs differentially expressed in ERα+ versus ERα- breast cancers actively control some of the most distinguishing characteristics of the luminal A and TN subtypes, such as ERα itself, Dicer, and growth factor receptor levels.

AAV-2-mediated expression of IGF-1 in skeletal myoblasts stimulates angiogenesis and cell survival.

The transplantation of skeletal myoblasts is being tested in various organ systems to facilitate tissue repair and regeneration. Previous studies have indicated that transplanted cells for varied reasons were not surviving in sufficient numbers following transplantation, thus negatively affecting overall therapeutic efficacy of the approach. We hypothesize that the genetic modification of myoblasts to express insulin-like growth factor 1 (IGF-1) locally may enhance the survival of transplanted cells by stimulating neo-vascularization, decreasing apoptosis, and promoting cell proliferation. Using an adeno-associated virus (adeno-associated virus type 2) vector system, the IGF-1 gene was introduced into canine skeletal myoblasts. As a negative control, myoblasts transduced with the green fluorescence protein (GFP) was used. Relative angiogenic response induced by IGF-1 myoblast was compared to VEGF165-induced neo-vascularization using Matrigel plugs under similar conditions. In vitro evaluation and characterization revealed that the secreted IGF-1 protein was biologically and functionally active in promoting endothelial cell proliferation, migration and assembly into vessel-like structures. Matrigel plugs containing the three test groups were implanted subcutaneously in nude mice (n = 5). After 3 weeks, analysis of explanted samples revealed an enhanced neo-vascularization with an average microvessel density per field for IGF-1 at 55.9 versus 33.4 for vascular endothelial growth factor and 24 for GFP. Additionally, apoptosis was significantly reduced (p