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Aurora kinases play a key role in mitosis and are frequently overexpressed in a variety of tumor cells. Inhibition of aurora kinases results in mitotic arrest and death of cancer cells, and has been explored as an anticancer strategy. However, how aurora inhibition kills cancer cells is poorly understood. In this study, we found that inhibition of aurora kinases by siRNA or small-molecule inhibitors led to induction of p53 upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family protein, in colorectal cancer cells irrespective of p53 status. Deficiency in PUMA increased polyploidy, improved cell survival, and abrogated mitochondria-mediated apoptosis induced by aurora kinase inhibitors. In response to aurora kinase inhibition, PUMA was directly activated by p65 through the canonical NF-κB pathway following AKT inhibition. Furthermore, PUMA was necessary for the chemosensitization and in vivo antitumor effects of aurora kinase inhibitors in colon cancer cells. These results suggest that PUMA induction mediates the apoptotic response to mitotic arrest imposed by aurora kinase inhibition, and may be a useful indicator for the anticancer activity of aurora kinase inhibitors.

Aberrant Ras/Raf/MEK/ERK signaling is one of the most prevalent oncogenic alterations and confers survival advantage to tumor cells. Inhibition of this pathway can effectively suppress tumor cell growth. For example, sorafenib, a multi-kinase inhibitor targeting c-Raf and other oncogenic kinases, has been used clinically for treating advanced liver and kidney tumors, and also has shown efficacy against other malignancies. However, how inhibition of oncogenic signaling by sorafenib and other drugs suppresses tumor cell growth remains unclear. In this study, we found that sorafenib kills cancer cells by activating PUMA (p53-upregulated modulator of apoptosis), a p53 target and a BH3-only Bcl-2 family protein. Sorafenib treatment induces PUMA in a variety of cancer cells irrespective of their p53 status. Surprisingly, the induction of PUMA by sorafenib is mediated by IκB-independent activation of nuclear factor (NF)-κB, which directly binds to the PUMA promoter to activate its transcription. NF-κB activation by sorafenib requires glycogen synthase kinase 3β activation, subsequent to ERK inhibition. Deficiency in PUMA abrogates sorafenib-induced apoptosis and caspase activation, and renders sorafenib resistance in colony formation and xenograft tumor assays. Furthermore, the chemosensitization effect of sorafenib is dependent on PUMA, and involves concurrent PUMA induction through different pathways. BH3 mimetics potentiate the anti-cancer effects of sorafenib, and restore sorafenib sensitivity in resistant cells. Together, these results demonstrate a key role of PUMA-dependent apoptosis in therapeutic inhibition of Ras/Raf/MEK/ERK signaling. They provide a rationale for manipulating the apoptotic machinery to improve sensitivity and overcome resistance to the therapies that target oncogenic kinase signaling.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that has an important role in immunity and inflammation by inducing cellular responses such as apoptosis. The transcription factor nuclear factor-kappaB (NF-kappaB) can paradoxically suppress and promote apoptosis in response to TNF-alpha. In this study, we found that p53 upregulated modulator of apoptosis (PUMA), a p53 downstream target and a BH3-only Bcl-2 family member, is directly regulated by NF-kappaB in response to TNF-alpha. TNF-alpha treatment led to increases in PUMA mRNA and protein levels in human colon cancer cells. The induction of PUMA was p53 independent, and mediated by the p65 component of NF-kappaB through a kappaB site in the PUMA promoter. The apoptotic effect of PUMA induction by TNF-alpha was unmasked by depleting the antiapoptotic protein Bcl-X(L). In mice, PUMA was also induced by TNF-alpha in an NF-kappaB-dependent manner. TNF-alpha-induced apoptosis in a variety of tissues and cell types, including small intestinal epithelial cells, hepatocytes, and thymocytes, was markedly reduced in PUMA-deficient mice. Collectively, these results demonstrated that PUMA is a direct target of NF-kappaB and mediates TNF-alpha-induced apoptosis in vitro and in vivo.