Aurora Kinase A (Aurora A), a protein canonically known to facilitate mitosis, has emerged as a promising drug target for cancer therapy. Aurora A is amplified in several cancer types, including glioblastoma, breast, and ovarian cancer, and increased expression is correlated to a worse prognosis for patients. In spite of these observations, the impact of increased Aurora A expression on cell signaling and cancer development remains unclear. Aurora A is also mis-localized to the cytoplasm in cancer and engages in oncogenic functions mediated through protein-protein interactions (PPIs). Thus, we hypothesized that through novel PPIs, Aurora A may be promoting oncogenic signaling and cancer cell growth.
The acquired ability of cancer cells to sustain proliferative signaling is a cancer hallmark. One critical pathway through which mutations drive the development of several cancer types is the Ras-Raf-MEK-ERK signaling cascade, which is also known as the mitogen-associated protein kinase (Ras-MAPK) pathway. When we co-expressed Aurora A and H-Ras in both HEK-293T cells and the glioblastoma cancer cell line, 8-MG-BA, ERK phosphorylation increased compared to expression of Aurora A or H-Ras alone. However, pharmacological inhibition of Raf-1 and MEK was able to block the increase in ERK phosphorylation by Aurora A and H-Ras co-expression. In addition, Aurora A was not able to promote ERK phosphorylation when co-expressed with dominant negative H-Ras (S17N mutant). Together, these findings demonstrate that Aurora A requires the MAPK signaling cascade to potentiate ERK activation. Interestingly, we discovered that this effect correlates to a novel interaction between Aurora A and H-Ras. The Aurora A/H-Ras interaction was confirmed using the lysate-based assay, Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET), the affinity-based assay, GST-pull down, and in live cells using the Venus Protein-fragment Complementation Assay (PCA). Through deletion analysis, the binding domains critical to mediate the interaction of Aurora A and H-Ras were characterized. We also determined that H-Ras is not an enzymatic substrate for Aurora A using an in vitro kinase assay and found that cells treated with an Aurora A kinase inhibitor are able to maintain the Aurora A/H-Ras the interaction, suggesting that the Aurora A/H-Ras interaction is kinase-independent.
In conclusion, our studies reveal a role for Aurora A as a positive regulator of Ras-MAPK proliferative signaling. As Aurora A gene expression is downstream of the Ras-MAPK signaling pathway, our data also provides a mechanism by which Aurora A forms a positive feedback loop, linking Aurora A to sustained proliferative signaling in cancer cells. Finally, the kinase-independence of the Aurora A/H-Ras interaction underscores the consideration that Aurora A kinase inhibitors currently in clinical development may not be sufficient to block all oncogenic functions of Aurora A.
Citation Format: MaKendra Umstead, Jinglin Xiong, Zenggang Li, Andrei Ivanov, Yuhong Du, Haian Fu. Investigating the role of Aurora Kinase A as a positive regulator of MAPK signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1892.