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...

Myeloid cell leukemia-1 (Mcl-1), a Bcl-2-like antiapoptotic protein, plays a role in cell immortalization and chemoresistance in a number of human malignancies. A peptidylprolyl cis/trans isomerase, Pin1 is involved in many cellular events, such as cell cycle progression, cell proliferation, and differentiation through isomerizing prophosphorylated substrates. It has been reported that down-regulation of Pin1 induces apoptosis, and that Erk phosphorylates and upregulates Mcl-1; however, the underlying mechanisms for the two phenomena are not clear yet. Here, we showed that Pin 1 stabilizes Mcl-1, which is required for Mcl-1 posphorylation by Erk. First, we found expression of Mcl-1 and Pin1 were positively correlated and associated with poor survival in human breast cancer. We then showed that Erk could phosphorylate Mcl-1 at two consensus residues, Thr 92 and 163, which is required for the association of Mcl-1 and Pin1, resulting in stabilization of Mcl-1. Moreover, Pin1 is also required for the up-regulation of Mcl-1 by Erk activation. Based on this newly identified mechanism of Mcl-1 stabilization, two strategies were used to overcome Mcl-1-mediated chemoresistance: inhibiting Erk by Sorafenib, an approved clinical anticancer drug, or knocking down Pin1 by using a SiRNA technique. In conclusion, the current report not only unravels a novel mechanism to link Erk/Pin1 pathway and Mcl-1-mediated chemoresistance but also provides a plausible combination therapy, Taxol (Paclitaxel) plus Sorafenib, which was shown to be effective in killing breast cancer cells. [Cancer Res 2008;68(15):6109-17]

Colorectal cancer is the second leading cause of death from cancer in the United States. Metastases in the liver, the most common metastatic site for colorectal cancer, are found in one-third of the patients who die of colorectal cancer. Currently, the genes and molecular mechanisms that are functionally critical in modulating colorectal cancer hepatic metastasis remain unclear. Here, we report our studies using functional selection in an orthotopic mouse model of colorectal cancer to identify a set of genes that play an important role in mediating colorectal cancer liver metastasis. These genes included APOBEC3G, CD133, LIPC, and S100P. Clinically, we found these genes to be highly expressed in a cohort of human hepatic metastasis and their primary colorectal tumors, suggesting that it might be possible to use these genes to predict the likelihood of hepatic metastasis. We have further revealed what we believe to be a novel mechanism in which APOBEC3G promotes colorectal cancer hepatic metastasis through inhibition of miR-29-mediated suppression of MMP2. Together, our data elucidate key factors and mechanisms involved in colorectal cancer liver metastasis, which could be potential targets for diagnosis and treatment. IntroductionAfter lymph nodes, the liver is the most common site for colorectal cancer metastasis, and liver metastasis is a common cause of cancer-related mortality (1-4). Most colorectal cancer patients with hepatic metastasis are not candidates for surgical treatment, and their 5-year survival rate following diagnosis of hepatic metastasis is below 10% (2, 4). It is well established that 5-year survival rates exceed 90% in patients diagnosed with early stage colorectal cancer (5, 6). It is imperative that we uncover the underlying mechanisms and genetic alterations that predispose to the metastatic phenotype in colorectal cancer. Such an understanding has the potential to improve early detection and prevention in addition to helping with developing novel targeted therapies for late stage disease. Studies reveal that genomic instability in cancer cells leads to cellular heterogeneity, which may guide tumor cell aggression and specific organ colonization during the metastatic process (7,8). Many studies have attempted to identify the metastasis-related genes in

Dysregulation of epigenetic controls is associated with tumorigenesis in response to microenvironmental stimuli; however, the regulatory pathways involved in epigenetic dysfunction are largely unclear. We have determined that a critical epigenetic regulator, microRNA-205 (miR-205), is repressed by the ligand jagged1, which is secreted from the tumor stroma to promote a cancer-associated stem cell phenotype. Knockdown of miR-205 in mammary epithelial cells promoted epithelial-mesenchymal transition (EMT), disrupted epithelial cell polarity, and enhanced symmetric division to expand the stem cell population. Furthermore, miR-205-deficient mice spontaneously developed mammary lesions, while activation of miR-205 markedly diminished breast cancer stemness. These data provide evidence that links tumor microenvironment and microRNA-dependent regulation to disruption of epithelial polarity and aberrant mammary stem cell division, which in turn leads to an expansion of stem cell population and tumorigenesis. This study elucidates an important role for miR-205 in the regulation of mammary stem cell fate, suggesting a potential therapeutic target for limiting breast cancer genesis.

SUMMARY Pro-inflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/ S111, thereby suppressing FOXA2 transactivation activity, leading to decreased NUMB expression and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.