Nuclear exclusion of the forkhead transcription factor FOXO3a by protein kinase Akt contributes to cell survival. We investigated the pathological relationship between phosphoylated-Akt (Akt-p) and FOXO3a in primary tumors. Surprisingly, FOXO3a was found to be excluded from the nuclei of some tumors lacking Akt-p, suggesting an Akt-independent mechanism of regulating FOXO3a localization. We provide evidence for such a mechanism by showing that IkappaB kinase (IKK) physically interacts with, phosphorylates, and inhibits FOXO3a independent of Akt and causes proteolysis of FOXO3a via the Ub-dependent proteasome pathway. Cytoplasmic FOXO3a correlates with expression of IKKbeta or Akt-p in many tumors and associates with poor survival in breast cancer. Further, constitutive expression of IKKbeta promotes cell proliferation and tumorigenesis that can be overridden by FOXO3a. These results suggest the negative regulation of FOXO factors by IKK as a key mechanism for promoting cell growth and tumorigenesis.

The RAS-ERK pathway is known to play a pivotal role in differentiation, proliferation and tumour progression. Here, we show that ERK downregulates Forkhead box O 3a (FOXO3a) by directly interacting with and phosphorylating FOXO3a at Ser 294, Ser 344 and Ser 425, which NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript consequently promotes cell proliferation and tumorigenesis. The ERK-phosphorylated FOXO3a degrades via an MDM2-mediated ubiquitin-proteasome pathway. However, the nonphosphorylated FOXO3a mutant is resistant to the interaction and degradation by murine double minute 2 (MDM2), thereby resulting in a strong inhibition of cell proliferation and tumorigenicity. Taken together, our study elucidates a novel pathway in cell growth and tumorigenesis through negative regulation of FOXO3a by RAS-ERK and MDM2.The constitutive activation of certain signal transduction cascades leads to the development of tumours and the resistance of tumours to clinical therapy 1,2 . The RAS-ERK pathway triggers one of these cascades and governs many important functions, such as cell fate, differentiation, proliferation and survival in invertebrate and mammalian cells 3,4 . Human tumours frequently overexpress RAS or harbour activated RAS with a point mutation, which contributes substantially to tumour cell growth, invasion and angiogenesis 1, 2 , 5 -8. Cell plasma membrane receptor tyrosine kinases activate RAS GTPases, and GTP-bound RAS activates A-RAF, B-RAF and RAF-1 (ref. 10,17,18,21 , but the E3 ubiquitin ligase responsible for FOXO3a degradation has yet to be identified. MDM2, an E3 ubiquitin ligase plays an important role in the development of multiple human cancers through degrading tumour suppressor proteins, such as p53, RB and E-cadherin [22][23][24][25] . In addition, MDM2 has been shown to be regulated by the RAS-ERK signalling pathway 26 and blocking ERK activity with an MEK1 inhibitor, U0126, reduces MDM2 expression in breast cancer cells 27 .Here, we identify a novel pathway involving the downregulation of FOXO3a expression by RAS-ERK and MDM2, which leads to promotion of cell growth and tumorigenesis. We show that ERK interacts with and phosphorylates FOXO3a at Ser 294, Ser 344 and Ser 425; phosphorylation of FOXO3a at these residues increases FOXO3a-MDM2 interaction and enhances FOXO3a degradation via an MDM2-dependent ubiquitin-proteasome pathway. The non-phosphorylated FOXO3a-mimic mutant, compared to the phosphorylated FOXO3a-mimic mutant, exhibits more resistance to the interaction and degradation by MDM2, resulting in a strong inhibition of cell proliferation in vitro and tumorigenesis in vivo. RESULTS ERK suppresses FOXO3a stability and induces its nuclear exclusionThe RAS-ERK is an essential oncogenic signalling cascade that promotes tumour cell growth and development 8,28 . It was known that other oncogenic kinases, AKT and IKK, (Fig. 1b, c). Similarly, using Erk small interference RNA (siRNA) to knockdown ERK protein expression level in HeLa cells (Fig. 1d), or trea...

TNFalpha has recently emerged as a regulator linking inflammation to cancer pathogenesis, but the detailed cellular and molecular mechanisms underlying this link remain to be elucidated. The tuberous sclerosis 1 (TSC1)/TSC2 tumor suppressor complex serves as a repressor of the mTOR pathway, and disruption of TSC1/TSC2 complex function may contribute to tumorigenesis. Here we show that IKKbeta, a major downstream kinase in the TNFalpha signaling pathway, physically interacts with and phosphorylates TSC1 at Ser487 and Ser511, resulting in suppression of TSC1. The IKKbeta-mediated TSC1 suppression activates the mTOR pathway, enhances angiogenesis, and results in tumor development. We further find that expression of activated IKKbeta is associated with TSC1 Ser511 phosphorylation and VEGF production in multiple tumor types and correlates with poor clinical outcome of breast cancer patients. Our findings identify a pathway that is critical for inflammation-mediated tumor angiogenesis and may provide a target for clinical intervention in human cancer.