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Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is an aggressive subtype of this disease. Targeted treatment has improved outcome, but there is still a need for new therapeutic strategies as some patients respond poorly to treatment. Our aim was to identify compounds that substantially affect viability in HER2+ breast cancer cells in response to combinatorial treatment. We performed a high-throughput drug screen of 278 compounds in combination with trastuzumab and lapatinib using two HER2+ breast cancer cell lines (KPL4 and SUM190PT). The most promising drugs were validated in vitro and in vivo, and downstream molecular changes of the treatments were analyzed. The screen revealed multiple drugs that could be used in combination with lapatinib and/or trastuzumab. The Src-inhibitor dasatinib showed the largest combinatorial effect together with lapatinib in the KPL4 cell line compared to treatment with dasatinib alone (p < 0.01). In vivo, only lapatinib significantly reduced tumor growth (p < 0.05), whereas dasatinib alone, or in combination with lapatinib, did not show significant effects. Protein analyses of the treated xenografts showed significant alterations in protein levels compared to untreated controls, suggesting that all drugs reached the tumor and exerted a measurable effect. In silico analyses suggested activation of apoptosis and reduced activity of survival pathways by all treatments, but the opposite pattern was observed for the combinatorial treatment compared to lapatinib alone.

Transforming growth factor-β (TGF-β) promotes extracellular matrix deposition by down-regulating the expression of matrix degrading proteinases and upregulating their inhibitors. Tissue inhibitor of metalloproteinases (TIMP)-3 is an ECM-associated specific inhibitor of matrix degrading metalloproteinases. Here, we have characterized the signaling pathways mediating TGF-β-induced expression of TIMP-3. Basal and TGF-β-induced TIMP-3 mRNA expression was abolished in Smad4-deficient mouse embryonic fibroblasts and restoring Smad4 expression rescued the response. Inhibition of Smad signaling by expression of Smad7 and dominant negative Smad3 completely abolished TGF-β-elicited expression of TIMP-3 in human fibroblasts, whereas overexpression of Smad3 enhanced it. Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activation with PD98059 and p38 mitogen-activated protein kinase activity by SB203580 resulted in suppression of TGF-β-induced TIMP-3 expression, indicating that ERK1/2 and p38 MAPK mediate the effect of TGF-β on TIMP-3 expression. Specific activation of p38α and ERK1/2 by constitutively active mutants of MKK3b or MEK1, respectively, and simultaneous co-expression of Smad3 resulted in induction of TIMP-3 expression in the absence of TGF-β indicating that Smad3 co-operates with p38 and ERK1/2 in the induction of TIMP-3 expression. These results demonstrate the complex interplay between Smad3, p38α, and ERK1/2 signaling in the regulation of TIMP-3 gene expression in fibroblasts, which may play a role in inflammation, tissue repair, and fibrosis.

Development of bone metastases is dependent on the cancer cell-bone cell interactions in the bone microenvironment. Transforming growth factor β (TGF-β) is released from bone during osteoclastic bone resorption and induces production of osteolytic factors, such as interleukin 11 (IL-11), in breast cancer cells. IL-11 in turn increases osteolysis by stimulating osteoclast function, launching a vicious cycle of cancer growth and bone destruction. We aimed to identify and functionally characterize microRNAs (miRNAs) that mediate the bone metastatic process, focusing on miRNAs that regulate the TGF-β induction of IL-11. First, we profiled the expression of 455 miRNAs in a highly bone metastatic MDA-MB-231(SA) variant as compared to the parental MDA-MB-231 breast cancer cell line and found 16 miRNAs (3.5%) having a >3-fold expression difference between the two cell types. We then applied a cell-based overexpression screen with Pre-miRNA constructs to functionally identify miRNAs regulating TGF-β-induced IL-11 production. This analysis pinpointed miR-204, miR-211, and miR-379 as such key regulators. These miRNAs were shown to directly target IL11 by binding to its 3' UTR. MiR-379 also inhibited Smad2/3/4-mediated transcriptional activity. Gene expression analysis of miR-204 and miR-379-transfected cells indicated that these miRNAs downregulated the expression of several genes involved in TGF-β signaling, including prostaglandin-endoperoxide synthase 2 (PTGS2). In addition, there was a significant correlation between the genes downregulated by miR-379 and a set of genes upregulated in basal subtype of breast cancer. Taken together, the functional evidence and clinical correlations imply novel mechanistic links between miRNAs and the key steps in the bone metastatic process in breast cancer, with potential clinical relevance.

IntroductionFew studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information.MethodsWe investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods.ResultsWe identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process.ConclusionThis study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer.

Human papillomavirus (HPV) infection is a prerequisite of developing cervical cancer, approximately half of which are associated with HPV type 16. HPV 16 encodes three oncogenes, E5, E6, and E7, of which E5 is the least studied so far. Its roles in regulating replication and pathogenesis of HPV are not fully understood. Here we utilize high-throughput screening to coordinately investigate the effect of E5 on the expression of host protein-coding and microRNA genes. MicroRNAs form a class of 22nt long noncoding RNAs with regulatory activity. Among the altered cellular microRNAs we focus on the alteration in the expression of miR-146a, miR-203 and miR-324-5p and their target genes in a time interval of 96 hours of E5 induction. Our results indicate that HPV infection and subsequent transformation take place through complex regulatory patterns of gene expression in the host cells, part of which are regulated by the E5 protein.