Differentiell exprimierte Isoenzyme des Zellmetabolismus als neues therapeutisches Ziel beim tripelnegativen Mammakarzinom
Zusammenfassung der Projektergebnisse
During my DFG-supported research fellowship at the Yale Cancer Center Breast Cancer Research Program, New Haven, CT, US, I investigated whether isoenzyme expression shifts in metabolic enzymes could be exploited therapeutically in breast cancer. Initial results of gene expression analysis supported the hypothesis of PPAP2C as a potential target in breast cancer, because cancer cells compared to normal cells cancer cells showed an overexpression and shift towards the PPAP2C isoform. Knockdown experiments showed successful inhibition of the PPAP2A, B and C isoform mRNA expression and the siRNA’s used were specific. Protein analysis failed due to lack of a validated specific antibody. Initial growth assay experiments showed growth inhibition after PPAP2C knockdown at 96hours not only in cancer cells but also normal breast cells, HMEC, although PPAP2C expression was low. This result was contradicting to our hypothesis that in normal cells the isoform that is expressed at the highest concentration, in this case PPAP2A, could compensate the loss of the other isoform. We concluded, that the most likely explanation would be a unique function of PPAP2C. Other groups have shown that Phosphatidic Acid Phosphatase C regulates cell cycle progression and therefore we performed cell cycle analysis. Despite using a very similar protocol compared to what had been published we did not see an effect on cell cycle distribution 48 hours after PPAP2C knockdown in HCC-1428 cells and subsequently the priory detected growth inhibition effect could not be reproduced or be detected in other cell lines although various siRNA concentrations, cell densities and DharmaFect concentrations were used. A possible explanation is the low proliferation rate of the cells and also the long phase of contamination issues so that the validity of the results is in question. Generally speaking, I experienced different problems with the initially suggested working program and methods mainly due to the previously mentioned long contamination period that was really stressful. We were not able to fulfill the proposed time plan. However, this made us trouble-shoot a lot, which helped me to thoroughly understand the techniques and problems scientists in lab have to face regularly. We think, that despite our conflicting results PPAP2C could still be a target in breast and possibly other cancers where its differential overexpression and its effect on cell growth has been described. As future experiments we propose cell growth assays with adequately proliferating cells at different siRNA concentrations, further investigate the role of PPAP2C on cell cycle progression with flow cytometry in normal breast cell lines and cancer cell lines at different time points, elucidate the mechanism(s) of anticancer of targeting specific isoenzymes by assessing changes in phosphorylated proteins with a Human Phospho-Kinase Array, evaluate global metabolic alterations after targeting isoenzymes via metabolomic profiling using mass-spectrometry and ultimately identify small molecular inhibitors for therapeutic drug development and lead compound optimization.
