BackgroundIn breast cancer, overexpression of the transmembrane tyrosine kinase ERBB2 is an adverse prognostic marker, and occurs in almost 30% of the patients. For therapeutic intervention, ERBB2 is targeted by monoclonal antibody trastuzumab in adjuvant settings; however, de novo resistance to this antibody is still a serious issue, requiring the identification of additional targets to overcome resistance. In this study, we have combined computational simulations, experimental testing of simulation results, and finally reverse engineering of a protein interaction network to define potential therapeutic strategies for de novo trastuzumab resistant breast cancer.ResultsFirst, we employed Boolean logic to model regulatory interactions and simulated single and multiple protein loss-of-functions. Then, our simulation results were tested experimentally by producing single and double knockdowns of the network components and measuring their effects on G1/S transition during cell cycle progression. Combinatorial targeting of ERBB2 and EGFR did not affect the response to trastuzumab in de novo resistant cells, which might be due to decoupling of receptor activation and cell cycle progression. Furthermore, examination of c-MYC in resistant as well as in sensitive cell lines, using a specific chemical inhibitor of c-MYC (alone or in combination with trastuzumab), demonstrated that both trastuzumab sensitive and resistant cells responded to c-MYC perturbation.ConclusionIn this study, we connected ERBB signaling with G1/S transition of the cell cycle via two major cell signaling pathways and two key transcription factors, to model an interaction network that allows for the identification of novel targets in the treatment of trastuzumab resistant breast cancer. Applying this new strategy, we found that, in contrast to trastuzumab sensitive breast cancer cells, combinatorial targeting of ERBB receptors or of key signaling intermediates does not have potential for treatment of de novo trastuzumab resistant cells. Instead, c-MYC was identified as a novel potential target protein in breast cancer cells.
The genes encoding microRNAs of the human miR-200 family map to fragile chromosomal regions and are frequently downregulated upon tumor progression. Although having been reported to regulate epithelial-tomesenchymal transition and transforming growth factorbeta-driven cell invasion, the role of the miR-200 family in EGF-driven breast cancer cell invasion, viability, apoptosis and cell cycle progression is still unknown. In particular, there is no study comparing the roles of the two clusters of this miRNA family. In this study, we show for the first time that miR-200 family members differentially regulate EGF-driven invasion, viability, apoptosis and cell cycle progression of breast cancer cells. We showed that, all miR-200 family members regulate EGFdriven invasion, with the miR-200bc/429 cluster showing stronger effects than the miR-200a/141 cluster. Furthermore, expression of the miR-200a/141 cluster results in G1 arrest supported by increased p27/Kip1 and decreased cyclin dependent kinase 6 expression. In contrast, expression of the 200bc/429 cluster decreases G1 population and increases G2/M phase, in line with the observed reduction of p27/Kip1 and upregulation of the inhibitory phosphorylation of Cdc25C, respectively. To test the hypothesis that phenotypical differences observed between the two clusters are caused by differential targeting spectrums, we performed genome-wide microarray profiling in combination with gain-of-function studies. This identified phospholipase C gamma 1 (PLCG1), which was downregulated only by the miR-200bc/429 cluster, as a potential candidate contributing to these phenotypical differences. Luciferase reporter assays validated PLCG1 as a direct functional target of miR-200bc/429 cluster, but not of miR-200a/141 cluster. Finally, loss of PLCG1 in part mimicked the effect of miR-200bc/429 overexpression in viability, apoptosis and EGF-driven cell invasion of breast cancer cells. Our results suggest that the miR-200 family has a tumor-suppressor function by negatively regulating EGF-driven cell invasion, viability and cell cycle progression in breast cancer.
Elongation growth and a several other phenomena in plant development are controlled by the plant hormone auxin. A number of recent discoveries shed light on one of the classical problems of plant physiology: the perception of the auxin signal. Two types of auxin receptors are currently known: the AFB/TIR family of F box proteins and ABP1. ABP1 appears to control membrane transport processes (H+ secretion, osmotic adjustment) while the TIR/AFBs have a role in auxin-induced gene expression. Models are proposed to explain how membrane transport (e.g., K+ and H+ fluxes) can act as a cross-linker for the control of more complex auxin responses such as the classical stimulation of cell elongation.
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