1. miRNA-drug resistance mechanisms Summary Hypothesis: The interplay between miRNAs, signaling pathways and epigenetic and genetic alterations are responsible for the development of drug resistance Signaling pathways Copy Number Variations (CNVs) Copy Number Variations (CNVs) Epigenetics MicroRNAs

2. Tamoxifen resistance miRNA mRNA protein Methylation ChIP-seq Trastuzumab sensitive ERBB2+ SKBr3 and BT474 Trastuzumab sensitive ERBB2+ SKBr3 and BT474 Trastuzumab resistant 0 6 12 18 24 30 weeks DNA, RNA, miRNA, protein isolation miRNA, mRNA, protein, methylation analysis ER+ Trastuzumab resistance: Dynamic analysis

3. Aim 1: Classify miRNA, mRNA, protein expression and methylation status profile of tamoxifen and trastuzumab resistance phenotype to identify key molecular signatures as markers for resistance and treatment response.

4. Ward et al, Oncogene 2012 Unpublished data: Ward et al Previous work:

5. Aim 1: Classify miRNA, mRNA, protein expression and methylation status profile of tamoxifen and trastuzumab resistance phenotype to identify key molecular signatures as markers for resistance and treatment response. Aim 2: Identify early and late epigenetic and/or genetic changes during the process of trastuzumab resistance which can be used as a predictive marker for resistance development. Aim 3: Classify miRNA, mRNA, protein expression and methylation status profile of tamoxifen and trastuzumab resistance phenotype in metastasis compared to primary tumor in vivo (xenograft mouse models and patient samples?)

6. Aim 1: Classify miRNA, mRNA, protein expression and methylation status profile of tamoxifen and trastuzumab resistance phenotype to identify key molecular signatures as markers for resistance and treatment response. Aim 2: Identify early and late epigenetic and/or genetic changes during the process of trastuzumab resistance which can be used as a predictive marker for resistance development. Aim 3: Classify miRNA, mRNA, protein expression and methylation status profile of tamoxifen and trastuzumab resistance phenotype in metastasis compared to primary tumor in vivo (xenograft mouse models and patient samples?) Aim 4: Identify biomarkers (e.g. circulating miRNAs) in the plasma of xenograft mouse models and breast cancer patients (?) as an early onset marker for resistance development

7. Aim 5: Understand the molecular mechanisms what causes the alterations in the miRNA expression. Aim 6: Identify the molecular mechanisms how genetic and epigenetic changes are responsible for the development of tamoxifen and trastuzumab resistance phenotype in vitro and in vivo.

8. Previous work: Ward et al, Oncogene 2012 EMT: epithelial to mesenchymal transitionMTDH: metadherin

9. Aim 5: Understand the molecular mechanisms what causes the alterations in the miRNA expression. Aim 6: Identify the molecular mechanisms how genetic and epigenetic changes are responsible for the development of tamoxifen and trastuzumab resistance phenotype in vitro and in vivo. Aim 7: Identify and test new potential targets to overcome tamoxifen and trastuzumab resistance and find new strategies for breast cancer therapies. e.g. combination with -signaling pathways inhibitor -miRNAs inhibitors -HDAC inhibitors…

10. miRNA-drug resistance mechanisms Internal Networking Aim 1-3+5 Aim 3+7 Aim 6 Aim 3,4,6,7 Aim 3+4 Aim 6 Aim 2 Aim 7