The rapid growth and poor vascularization of solid tumors expose cancer cells to hypoxia, which promotes the metastatic phenotype by reducing intercellular adhesion and increasing cell motility and invasiveness. In this study, we found that hypoxia increased free NADH levels in cancer cells, promoting CtBP recruitment to the E-cadherin promoter. This effect was blocked by pyruvate, which prevents the NADH increase. Furthermore, hypoxia repressed Ecadherin gene expression and increased tumor cell migration, effects that were blocked by CtBP knockdown. We propose that CtBP senses levels of free NADH to control expression of cell adhesion genes, thereby promoting tumor cell migration under hypoxic stress.NADH ͉ E-cadherin ͉ metastasis ͉ HIF1␣ ͉ adhesion T he ability of tumors to metastasize is a hallmark of malignancy. A critical event during metastasis is the reduction of adhesion, which facilitates tumor cell invasion into surrounding tissues and vascular channels, ultimately leading to the development of new sites of cancer progression. E-cadherin-mediated cell-cell adhesion is essential for maintaining the homeostasis and architecture of epithelial tissues. Down-regulation of Ecadherin expression occurs concomitantly with dedifferentiation and invasion of epithelial cells during tumorigenesis (1, 2). Consequently, E-cadherin and its associated complex are thought to be key mediators of tumor cell invasion (3).Highly aggressive, rapidly growing tumors are exposed to hypoxia, which occurs as a consequence of high metabolic activity and inadequate blood supply (4). It has been proposed that hypoxia is the initiating event that sets tumors on the road to metastasis (5). We have shown that the transcriptional corepressor CtBP has the unique ability to sense levels of free nuclear NADH and transmit this information to complexes that regulate gene expression (6). Transcription of E-cadherin and several other cell adhesion proteins is known to be repressed by CtBP (7) via ZEB and other factors believed to interact with the E-cadherin promoter (8,9). CtBP binding to these transcriptional repressors is induced by elevations in free NADH (6). We propose that the redox-sensing property of CtBP provides a regulatory switch for E-cadherin expression under hypoxic conditions.In this study, we show that the transcriptional corepressor CtBP has a central role in cancer cell migration in response to hypoxia. Hypoxia increases free NADH levels, which promotes CtBP recruitment to the E-cadherin promoter, repressing Ecadherin gene expression and increasing tumor cell migration. Thus, the pathway we have described links tumor hypoxia to cell migration through NADH regulation of CtBP function.

The Sir2 histone deacetylases are important for gene regulation, metabolism, and longevity. A unique feature of these enzymes is their utilization of NAD + as a cosubstrate, which has led to the suggestion that Sir2 activity reflects the cellular energy state. We show that SIRT1, a mammalian Sir2 homologue, is also controlled at the transcriptional level through a mechanism that is specific for this isoform. Treatment with the glycolytic blocker 2-deoxyglucose (2-DG) decreases association of the redox sensor CtBP with HIC1, an inhibitor of SIRT1 transcription. We propose that the reduction in transcriptional repression mediated by HIC1, due to the decrease of CtBP binding, increases SIRT1 expression. This mechanism allows the specific regulation of SIRT1 in response to nutrient deprivation.

Genetic knock out of the transcriptional co-repressor carboxyl-terminal-binding protein (CtBP) in mouse embryonic fibroblasts results in up-regulation of several genes involved in apoptosis. We predicted, therefore, that a propensity toward apoptosis might be regulated through changes in cellular CtBP levels. Previously, we have identified the homeodomain-interacting protein kinase 2 as such a regulator and demonstrated that HIPK2 activation causes Ser-422 phosphorylation and degradation of CtBP. In this study, we found that c-Jun NH 2 -terminal kinase 1 activation triggered CtBP phosphorylation on Ser-422 and subsequent degradation, inducing p53-independent apoptosis in human lung cancer cells. JNK1 has previously been linked to UV-directed apoptosis. Expression of MKK7-JNK1 or exposure to UV irradiation reduced cellular levels of CtBP via a proteasome-mediated pathway. This effect was prevented by JNK1 deficiency. In addition, sustained activation of the JNK1 pathway by cisplatin similarly triggered CtBP degradation. These findings provide a novel target for chemotherapy in cancers lacking p53.Carboxyl-terminal-binding protein (CtBP) 2 was originally identified by virtue of its ability to interact with the carboxyl terminus of the adenoviral protein E1A (1). Like other E1A-binding proteins, CtBP also interacts with a wide variety of cellular factors, many of which have been characterized as DNA binding transcriptional repressors (2, 3). Thus, CtBP has been categorized as a co-repressor. Genetic and biochemical studies in Drosophila have shown that CtBP is required for the functions of several developmentally important transcription factors, including snail, kruppel, knirps, and Tramtrack69 (4 -6). In mammals, CtBP has two isoforms, CtBP1 and CtBP2, which have been shown to participate in multiple developmental pathways as well (2). A human disease, holoprosencephaly, results from a mutation in the CtBP-interacting domain of TGIF, a three amino acid loop extension (TALE) homeodomain protein (7). The embryonic lethality of CtBP1 and two genetic knock-outs in mice supports the idea that these factors are essential for mammalian development (8). Additionally, Evi-1, an oncoprotein responsible for acute myelogenous leukemia, requires interaction with CtBP to mediate transformation (9, 10).Initially, CtBP was thought to negatively modulate the oncogenic transformation activity of the E1A protein (1, 11). Later, Grooteclaes and Frisch (12) demonstrated that this occurs by sequestering CtBP from its cellular targets. Genes regulated by CtBP have been identified serendipitously, for the most part, by identifying certain CtBP binding transcription factor binding sites in gene promoters. In this manner, for example, Grooteclaes and Frisch (12) determined that the cell adhesion gene E-cadherin was regulated by CtBP through the transcriptional repressor ZEB. Subsequent experiments showed that reducing cellular CtBP levels via small interference RNA up-regulates E-cadherin transcription (13). A more comprehensive analysi...