Although DNA methylation is one of the critical ways for silencing tumor suppressor and DNA repair genes during tumor initiation and progression, the mechanisms underlying DNA methylation in cancer remain unclear. Here we show that prostaglandin E2 (PGE2) silences certain tumor suppressor and DNA repair genes via DNA methylation to promote tumor growth. These findings uncover a previously unrecognized role for PGE2 in the promotion of tumor progression.

Regulation of phosphoinositides is important to tumorigenesis. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is a dual specificity phosphatase that dephosphorylates the 3Ј-sites of the phosphoinositides PI(3,4)P 2 2 and PI(3,4,5)P 3 (1). Endogenous PI(3,4,5)P 3 levels are also regulated by phosphatidylinositol 3-kinase (PI3K), which phosphorylates the D3 position of phosphatidylinositol (PI) on PI(4)P and PI(4,5)P 2 to produce PI(3,4)P 2 and PI(3,4,5)P 3 . PI(3,4,5)P 3 and PI(3,4)P 2 recruit the pleckstrin homology domains of specific intracellular proteins to the plasma membrane, an essential event in the activation of PI3K-dependent kinases such as phosphoinositide-dependent kinase-1 and protein kinase B/AKT, which have a key role in cellular survival and transformation (2). PI3K-dependent signaling is frequently activated in a variety of tumor types, including non-small cell lung cancer (NSCLC). Several genetic events previously described in NSCLC activate PI3K, including amplification of PIK3CA and activating mutations in PIK3CA, EGFR,. PTEN gene expression is frequently silenced in NSCLC (7), but the mechanisms contributing to the loss of PTEN expression in NSCLC have not been defined. PTEN genetic deletion is a rare event in NSCLC (8), raising the possibility that PTEN is silenced transcriptionally or post-transcriptionally. Of note, PTEN expression is transcriptionally suppressed by tumor necrosis factor-␣ (TNF␣) through NFB (9, 10), a heterodimeric transcription factor that is constitutively activated in NSCLC (7).NFB consists of the transactivation subunit RelA/p65 and the DNA-binding subunits p50 (NFB1) and p52 (NFB2), which are processed from the precursors p105 and p100, respectively (11). In unstimulated conditions, NFB is sequestered in the cytoplasm by inhibitor of NFB (IB) and remains transcriptionally inactive. Upon stimulation by inflammatory cytokines or peptide growth factors, IB is phosphorylated by IB kinase (IKK), a multiprotein complex consisting of two kinase subunits (IKK␣ and IKK␤) and a regulatory subunit (IKK␥/NEMO), and undergoes proteasome-dependent degradation. The released NFB translocates into the nucleus and regulates the expression of target genes with key roles in the prevention of apoptosis, promotion of tumor growth, and activation of inflammatory responses (12).NSCLC cells undergo apoptosis in response to PI3K pathway inhibition (13,14). We previously found that a stress kinase, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 The abbreviations used are: PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PI(4)P, phosphatidylinositol 4-monophosphate; PI(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; PI, L-␣-phosphat...

Human enhancer of filamentation 1 (HEF1; also known as NEDD9 or Cas-L) is a scaffolding protein that is implicated in regulating diverse cellular processes, such as cellular attachment, motility, cell cycle progression, apoptosis, and inflammation. Here, we identify HEF1 as a novel hypoxia-inducible factor-1α (HIF-1α)-regulated gene and reveal that HEF1 mediates hypoxia-induced migration of colorectal carcinoma cells. HEF1 is highly expressed in cultured colorectal carcinoma cells exposed to hypoxia and in the hypoxic areas of human colorectal cancer (CRC) specimens. Moreover, our data show that HIF-1α mediates the effects of hypoxia on induction of HEF1 expression via binding to a hypoxia-responsive element of the HEF1 promoter. Importantly, the induction of HEF1 expression significantly enhances hypoxia-stimulated HIF-1α transcriptional activity by modulating the interaction between HIF-1α and its transcriptional cofactor p300. Inhibition of HEF1 expression also reduced the levels of hypoxia-inducible genes, including those that regulate cell motility. Cell migration was reduced dramatically following knockdown of HEF1 expression under hypoxic conditions. Thus, this positive feedback loop may contribute to adaptive responses of carcinoma cells encountering hypoxia during cancer progression. Cancer Res; 70(10); 4054-63. ©2010 AACR.

Cancer cells in which theClass I phosphatidylinositol 3-kinase (PI3K) 1 consists of a family of heterodimeric complexes composed of a p110 catalytic subunit and a regulatory subunit that exists predominantly in a p85 form (1-3). The known gene family members for p85 (␣, ␤, and ␥) and p110 (␣, ␤, ␦, and ␥) are expressed in a tissuespecific fashion. p85␣ and -␤ can also exist in smaller forms (p50 and p55). PI3K phosphorylates the D3 position of PI on PI(4)P and PI(4,5)P to produce PI(3,4)P 2 and PI(3,4,5)P 3 . The 3Ј sites of PI(3,4)P 2 and PI(3,4,5)P 3 are dephosphorylated by the PTEN tumor suppressor, whereas the 5Ј site of PI(3,4,5)P 3 is dephosphorylated by SHIP to produce PI(3,4)P 2 (1). These mechanisms tightly regulate the levels of 3-phosphorylated PI in the cell. PI(3,4,5)P 3 and PI(3,4)P 2 recruit the pleckstrin homology domains of specific intracellular proteins to the plasma membrane, an essential event in the activation of PI3K-dependent kinases such as phosphoinositide-dependent kinase-1 and AKT, also known as protein kinase B. In addition, AKT phosphorylation at Thr 308 by phosphoinositide-dependent kinase-1 and Ser 473 by integrin-linked kinase (and possibly other kinases) constitutes an essential event in AKT activation (4, 5).The PI3K pathway clearly has a key role in cellular survival and transformation. AKT phosphorylates several pro-and antiapoptotic proteins, including the Bcl-2 family member BAD, caspase-9, cyclic AMP response element-binding protein,

Colorectal cancer (CRC) is now the second-leading cause of cancer deaths in the USA. Colorectal cancer progression and metastasis depends on the orchestration of the aberrant signaling pathways that control tumor cell proliferation, survival and migration/invasion. Epidemiological, clinical, and animal studies have demonstrated that prostaglandin-endoperoxide synthase 2 (PTGS2) and epithelial growth factor (EGF) signaling pathways play key roles in promoting colorectal cancer growth and metastasis. In this review, we highlight major advances in our understanding of the roles of PTGS2 and EGF signaling in colorectal cancer.

The transactivation of nuclear receptors is regulated by both ligand binding and phosphorylation. We previously showed that RARalpha (retinoic acid receptor alpha) phosphorylation by c-Jun N-terminal kinase contributes to retinoid resistance in a subset of NSCLC cells (non-small cell lung cancer cells), but the aetiology of this resistance in the remainder has not been fully elucidated [Srinivas, Juroske, Kalyankrishna, Cody, Price, Xu, Narayanan, Weigel and Kurie (2005) Mol. Cell. Biol. 25, 1054-1069]. In the present study, we report that Akt, which is constitutively activated in NSCLC cells, phosphorylates RARalpha and inhibits its transactivation. Biochemical and functional analyses showed that Akt interacts with RARalpha and phosphorylates the Ser96 residue of its DNA-binding domain. Mutation of Ser96 to alanine abrogated the suppressive effect of Akt. Overexpression of a dominant-negative form of Akt in an NSCLC cell line decreased RAR phosphorylation, increased RAR transactivation and enhanced the growth-inhibitory effects of an RAR ligand. The findings presented here show that Akt inhibits RAR transactivation and contributes to retinoid resistance in a subset of NSCLC cells.

Prostaglandin E 2 (PGE 2 ), one of the downstream products of cyclooxygenase-2 enzymatic activity, promotes colorectal carcinogenesis in part by stimulating cell division. In this study, we define a critical mechanism in this process by showing that the prometastatic adapter protein human enhancer of filamentation 1 (HEF1; NEDD9) links PGE 2 to the cell cycle machinery in colorectal cancer cells. PGE 2 rapidly induced expression of HEF1 mRNA and protein in colorectal cancer cells. HEF1 overexpression elicited the same effects as PGE 2 treatment on cell proliferation, cell cycle progression, and tumor growth. Conversely, HEF1 knockdown suppressed PGE 2 -driven cell proliferation and cell cycle progression. Cell cycle alterations involved HEF1 fragmentation as well as co-distribution of HEF1 and cell cycle kinase Aurora A along spindle asters during cell division. Moreover, Aurora A co-immunoprecipitated with HEF1 and was activated by HEF1. Consistent with a role for HEF1 in colorectal carcinogenesis, we found elevated expression of HEF1 expression in 50% of human colorectal cancers examined, relative to paired normal tissues. These findings establish that PGE 2 induces HEF1 expression, which in turn promotes cell cycle progression through its interaction with and activation of Aurora A. Further, they establish that HEF1 is a crucial downstream mediator of PGE 2 action during colorectal carcinogenesis. Cancer Res; 70(2); 824-31. ©2010 AACR.

The gene encoding Migration and Invasion Inhibitory Protein (MIIP), located on 1p36.22, is a potential tumour suppressor gene in glioma. In this study, we aimed to explore the role and mechanism of action of MIIP in colorectal cancer (CRC). MIIP protein expression gradually decreased along the colorectal adenoma-carcinoma sequence and was negatively correlated with lymph node and distant metastasis in 526 colorectal tissue samples (P<0.05 for all). Analysis of The Cancer Genome Atlas (TCGA) data showed that decreased MIIP expression was significantly associated with MIIP hemizygous deletion (P=0.0005) that was detected in 27.7% (52/188) of CRC cases, and associated with lymph node and distant metastasis (P<0.05 for both). We deleted one copy of the MIIP gene in HCT-116 CRC cells using zinc finger nuclease technology and demonstrated that MIIP haploinsufficiency resulted in increased colony formation and cell migration and invasion, which was consistent with the results from siRNA-mediated MIIP knockdown in two CRC cell lines (P<0.05 for all). Moreover, MIIP haploinsufficiency promoted CRC progression in vivo (P<0.05). Genomic instability and spectral karyotyping assays manifested that MIIP haploinsufficiency induced chromosomal instability (CIN). Besides modulating the downstream proteins of APC/CCdc20, securin and cyclin B1, MIIP haploinsufficiency inhibited topoisomerase II (Topo II) activity and induced chromosomal missegregation. Therefore, we report that MIIP is a novel potential tumour suppressor gene in CRC. Moreover, we characterized the MIIP gene as a novel CIN suppressor gene, through altering the stability of mitotic checkpoint proteins and disturbing Topo II activity.