Purpose: Signal transducer and activator of transcription 3 (Stat3) protein is persistently activated in breast cancer and promotes tumor cell survival. To gain a better understanding of the role of constitutive Stat3 signaling in breast cancer progression, we evaluated the expression profile of potential Stat3-regulated genes that may confer resistance to apoptosis. Experimental Design: Stat3 signaling was blocked with antisense oligonucleotides in human MDA-MB-435s breast cancer cells and Affymetrix GeneChip microarray analysis was done. The candidate Stat3 target gene Survivin was further evaluated in molecular assays using cultured breast cancer cells and immunohistochemistry of breast tumor specimens. Results: Survivin, a member of the inhibitor of apoptosis protein family, was identified as a potential Stat3-regulated gene by microarray analysis. This was confirmed in Survivin gene promoter studies and chromatin immunoprecipitation assays showing that Stat3 directly binds to and regulates the Survivin promoter. Furthermore, direct inhibition of Stat3 signaling blocked the expression of Survivin protein and induced apoptosis in breast cancer cells. Direct inhibition of Survivin expression also induced apoptosis. Increased Survivin protein expression correlates significantly (P = 0.001) with elevated Stat3 activity in primary breast tumor specimens from high-risk patients who were resistant to chemotherapy treatment. Conclusions: We identify Survivin as a direct downstream target gene of Stat3 in human breast cancer cells that is critical for their survival in culture. Our findings suggest that activated Stat3 signaling contributes to breast cancer progression and resistance to chemotherapy by, at least in part, inducing expression of the antiapoptotic protein, Survivin.

Akt negatively regulates apoptotic pathways at a premitochondrial level through phosphorylation and modulation of proteins such as Bad, Forkhead proteins, and GSK-3␤. Akt has also been shown to protect cell death at a post-mitochondrial level, although its downstream targets have not been well documented. Here, we demonstrate that Akt, including AKT1 and AKT2, interacts with and phosphorylates X-linked inhibitor of apoptosis protein (XIAP) at residue serine-87 in vitro and in vivo. Phosphorylation of XIAP by Akt protects XIAP from ubiquitination and degradation in response to cisplatin. Moreover, autoubiquitination of XIAP is also inhibited by Akt. Consistent with this, an XIAP mutant introduced into cells which mimics the Akt-phosphorylated form (i.e. XIAP-S87D) displays reduced ubiquitination and degradation as compared with wild type XIAP. The greater stability of XIAP-S87D in cells translated to increased cell survival after cisplatin treatment. Conversely, a mutant that could not be phosphorylated by Akt (XIAP-S87A) was more rapidly degraded and showed increased cisplatin-induced apoptosis. Furthermore, suppression of XIAP by either siRNA or adenovirus of antisense of XIAP induced programmed cell death and inhibited Akt-stimulated cell survival in ovarian cancer cells. These data identify XIAP as a new downstream target of Akt and a potentially important mediator of the effect of Akt on cell survival.Akt, also named protein kinase B (PKB) 1 or RAC kinase, is a family of phosphatidylinositol 3-OH-kinase-regulated serine/ threonine kinase (1-3). Three isoforms of Akt have been identified: Akt1/PKB␣, Akt2/PKB␤, and Akt3/PKB␥, all of which are activated by growth factors in a phosphatidylinositol 3-OHkinase-dependent manner (4 -6). Accumulated evidence shows that Akt and its downstream targets constitute a major cell survival pathway. Akt promotes cell survival and suppresses apoptotic death in a number of cell types induced by a variety of stimuli, including growth factor withdrawal, cell cycle discordance, and loss of cell adhesion (7). Several downstream targets containing the Akt phosphorylation consensus sequence (R-X-R-X-X-S/T) have been identified which shed light on the mechanisms by which Akt promotes cell survival and blocks apoptosis. The first anti-apoptotic Akt target identified was the pro-apoptotic protein BAD. BAD is a pro-death member of the Bcl-2 family that initiates apoptosis by binding to Bcl-x L on the outer mitochondrial membrane, causing the release of cytochrome c into the cytosol. Akt phosphorylates BAD on Ser 136 , promoting the association of BAD with 14-3-3 proteins in the cytosol and inactivating its proapoptotic function (7). The execution of cellular apoptosis also involves changes in the transcriptional program (7). Akt decreases the transcription of a subset of death genes by phosphorylation of the Forkhead family of transcription factors, which causes their nuclear exclusion and inactivation (7). Akt also phosphorylates and activates the cyclic AMP-response element-binding pr...

The tumor suppressor p53 is important in the decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. Cip/WAF1 and PTEN, were inhibited by Aurora-A in a Ser-215 phosphorylation-dependent manner (i.e. phosphomimic p53-S215D lost and nonphosphorylatable p53-S215A retained normal p53 function). As a result, Aurora-A overrides the apoptosis and cell cycle arrest induced by cisplatin and ␥-irradiation, respectively. However, the effect of Aurora-A on p53 DNA binding and transactivation activity was not affected by phosphorylation of Ser-315, a recently identified Aurora-A phosphorylation site of p53

Transforming growth factor ␤ (TGF-␤) 2 plays a complex role in cancer development (1). It is recognized as a tumor suppressor in that genetic lesions affecting TGF-␤ pathway components are found with high incidence particularly in pancreatic and colon cancers (2-4). On the other hand, many clinical and basic studies point to elevated TGF-␤ signaling in late stage cancer, and suggest a pro-invasion/metastasis role for TGF-␤ (5, 6). Indeed, a high TGF-␤ signaling activity is often associated with a poor prognosis for breast and other cancer patients (7-9). Therefore, it is important to delineate the downstream effectors mediating the different TGF-␤ responses at early versus late stages of cancer progression. Such information will be critical for designing strategies targeting specific aspects of TGF-␤ response to combat cancer.The pro-metastasis function of TGF-␤ is directly linked to its ability to initiate epithelial-mesenchymal transition (EMT) in cell culture (10 -12). EMT is a complex transdifferentiation process in which epithelial cells lose junctional adhesion and adopt a mesenchymal phenotype and morphology (13). EMT takes place during embryonic development and wound healing, and is also believed to underlie the change into a more invasive behavior during cancer progression (14). TGF-␤ is a potent inducer of EMT. TGF-␤ directly activates the expression of transcription factors including SNAI1/2, Twist and ZEB1/2 (10 -12). These are master regulators of the EMT program, which suppress the levels of epithelial markers such as E-cadherin and ZO-1, and up-regulate mesenchymal markers including vimentin, fibronectin, and others (15).MicroRNAs (miRNAs) are 20 -22-nucleotide noncoding RNAs that regulate gene expression at post-transcriptional levels (16). Rapidly emerging evidence strongly suggest critical roles of miRNAs in the pathogenesis of cancer (17). Either oncogenic (e.g. miR-155, miR-17-5p) or tumor suppressor (e.g. let-7, miR-15a) functions have been assigned to various miRNAs (18,19). Earlier profiling experiments have identified cohorts of miRNAs whose levels undergo significant changes upon TGF-␤-induced EMT, suggesting possible involvements of miRNAs in this process (20). In particular, independent studies by several laboratories have found the miR-200 family as important suppressors of EMT in a number of different models (21-24). MiR-200 inhibits EMT by directly recognizing complementary sites in the 3Ј-UTR of ZEB1/2 and repressing the translation of these positive regulators of EMT (21-24). MiR-200 itself is repressed by TGF-␤, through an unknown mechanism (24). MiRNAs such as miR-9 and miR-335 promote metastasis by directly suppressing the levels of E-cadherin (miR-9) or SOX4 (miR-335) (25, 26). MiR-10b has also been suggested to facilitate breast cancer metastasis, but this was contradicted by a more recent report in which high miR-10b appeared to suppress motility and invasiveness of breast cancer cells (27,28). MiR-31 acts to repress breast cancer cell migration and invasion, and a low miR-3...

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During the past decade, Akt (also known as protein kinase B, PKB) has been extensively studied. It regulates a variety of cellular processes by mediating extracellular (mitogenic growth factor, insulin and stress) and intracellular (altered tyrosine receptor kinases, Ras and Src) signals. Activation of Akt by these signals is via its pleckstrin homology (PH) domain binding to products of phosphatidylinositol 3-kinase (PI3K). This process is negatively regulated by a dual phosphatase PTEN tumor suppressor. Today, more than 30 Akt substrates have been identified. These phosphorylation events mediate the effects of Akt on cell survival, growth, differentiation, angiogenesis, migration and metabolism. Further, PI3K/PTEN/Akt pathway is frequently altered in many human malignancies and overexpression of Akt induces malignant transformation and chemoresistance. Thus, the Akt pathway is a major target for anti-cancer drug development. This review focuses on Akt signaling mechanism in oncogenesis and chemoresistance, and ongoing translational efforts to therapeutically target Akt.

SUMMARY Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. Here we show that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP.

UDP-sugars, activated forms of monosaccharides, are synthesized through de novo and salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDPsugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDPGal, UDP-glucuronic acid, UDP-L-arabinose, and UDPxylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5-7.5, and at 45°C in the presence of 2 mM Mg 2؉ . The apparent K m values for Glc 1-phosphate and L-arabinose 1-phosphate were 0.34 and 0.96 mM, respectively. PsUSP cDNA was cloned by reverse transcriptase-PCR. PsUSP appears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues from Arabidopsis and rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed in Escherichia coli catalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDPsugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.It is highly probable that the relative amounts and the architecture of cell wall polysaccharides and of the sugar moieties of glycolipids and glycoproteins in higher plants are regulated by the level of nucleotide sugars available, as well as by the levels of glycosyltransferases that incorporate monosaccharide units from respective nucleotide sugars into the polymers. Activated nucleotide sugars that serve as glycosyl donors for these polymers in higher plants are generated through de novo pathways, in which various UDP-and GDP-sugars are produced through sequential interconversions from UDP-Glc and GDP-Man as the starting substrates (1). In the salvage pathways, alternative routes to synthesize nucleotide sugars, free monosaccharides released during the degradation of polysaccharides and glycoconjugates are first phosphorylated by the action of monosaccharide kinases, then converted into nucleotide sugars by the action of pyrophosphorylases in the presence of the respective nucleotide triphosphates as co-substrates. Pyrophosphorylases principally catalyze both the following forward (synthesis of NDP-sugars) and reverse (pyrophospholysis) reactions (1): monosaccharides 1-phosphates ϩ NTP % NDP-sugars ϩ PP i , where NTP and NDP are nucleoside triphosphates and diphosphates, respectively. Var...