Thesis

Blockade of EIF5A hypusination/activation reverses TGFbeta-induced EMT and potently kills metastatic breast cancer cells in combination with HDAC6 inhibition

Metastatic breast cancers carry a 5-year survival prognosis of less than 20%. Thus, it is highly desirable to identify therapeutic strategies that specifically target both primary and metastatic tumors. It is generally accepted that epithelial-mesenchymal transition (EMT) is an important component of the metastatic cascade in solid tumor types such as breast cancer. In this regard, we have previously established that PEAK1 promotes breast cancer metastasis by switching Transforming Growth Factor β (TGFβ) signaling toward its EMT-promoting functions. Eukaryotic Initiation Factor 5A 1/2 (eIF5A1/2) are unique translation factors in that they are the only known protein substrates for the post-translational hypusine modification - a key modification required for eIF5A translation activity. Since eIF5A is required for Pseudopodium-Enriched Atypical Kinases 1 (PEAK1) translation, we hypothesized that TGFβ may induce PEAK1 upregulation during EMT by directly activating the eIF5A hypusination pathway. We show evidence of an active eIF5A/PEAK1 pathway in undifferentiated, mesenchymal breast cancer tissue. Notably, inhibition of eIF5A hypusination blocks PEAK1 translation, cell viability and TGFβ-induced EMT in breast cancer cells. In this regard, we demonstrate that TGFβ induces post-translational hypusination/activation of eIF5A in metastatic breast cancer cells. While it remains to be determined whether canonical TGFβ signaling factors directly activate hypusination pathway enzymes, TGFβ is known to activate other eIF5A regulatory enzymes that have previously been reported to mediate EMT in breast cancer. For example, TGFβ-induced EMT requires Activin Receptor Type-1B (ACVR1B/ALK4)-dependent Histone Deacetylase 6 (HDAC6) activation and HDAC6 promotes eIF5A deacetylation leading to its rapid nuclear export and hypusination; therefore, we hypothesize that cytoplasmic localization of eIF5A and eIF5A hypusination are required for cell proliferation/survival and TGFβ-induced EMT in breast cancer. Since HDAC inhibitors are promising new anti-cancer agents being evaluated in clinical trials, we designed experiments to test whether blockade of eIF5A hypusination could increase the potency or efficacy of HDAC6 inhibitors. Most notably, we demonstrate that dual treatment with non-cytotoxic doses of HDAC6 and eIF5A hypusination inhibitors synergize to potently and selectively kill metastatic breast cancer cells and block TGFβ-induced EMT. This also resulted in a further accumulation of eIF5A in the nucleus regardless of TGFβ treatment. In this regard, we have formulated a pathway in which we believe TGFβ stimulates HDAC6 and DHPS function to export eIF5A into the cytoplasm and promotes PEAK1 translation to result in EMT, invasion and metastasis in breast cancer cells. Taken together, these results pave the way for pre-clinical trials designed to selectively target breast cancer metastasis.

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