Detachment of epithelial cells from matrix or attachment to an inappropriate matrix engages an apoptotic response known as anoikis, which prevents metastasis. Cellular sensitivity to anoikis is compromised during the oncogenic epithelial-to-mesenchymal transition (EMT), through unknown mechanisms. We report here a pathway through which EMT confers anoikis resistance. NRAGE (neurotrophin receptor-interacting melanoma antigen) interacted with a component of the E-cadherin complex, ankyrin-G, maintaining NRAGE in the cytoplasm. Oncogenic EMT downregulated ankyrin-G, enhancing the nuclear localization of NRAGE. The oncogenic transcriptional repressor protein TBX2 interacted with NRAGE, repressing the tumor suppressor gene p14ARF. P14ARF sensitized cells to anoikis; conversely, the TBX2/NRAGE complex protected cells against anoikis by downregulating this gene. This represents a novel pathway for the regulation of anoikis by EMT and E-cadherin.Metastatic tumor cells survive detachment from their extracellular matrix of origin and/or attachment to inappropriate matrices for extended periods of time, conditions that engage an apoptotic response known as anoikis in normal cells. Tumor cell resistance to anoikis is driven by (epi)genetic alterations or aberrant signaling responses that occur uniquely in the tumor microenvironment, leading to constitutive activation of integrin/growth factor signaling and inactivation of the core apoptotic machinery (23, 27, 28, 30-32, 76, 87).The oncogenic epithelial-to-mesenchymal transition (EMT) is thought to play an important role in tumor progression (46,88,91). The focus of the present study was to understand the molecular basis of the tight correlation between anoikis resistance and the oncogenic EMT (31,51,54,68,89). A common hallmark of EMT is the breakdown of E-cadherin expression or function (103), which suffices to circumvent anoikis in some contexts. For example, the targeted knockout of the E-cadherin gene in a mouse mammary tumor model or the stable knockdown of E-cadherin in a mammary epithelial cell line confers anoikis resistance (19,68). This implies that EMTpromoting transcription factors such as ZEB1/2, Snail1/2 and Twist can abrogate anoikis both by directly regulating apoptosis control genes and by suppressing E-cadherin expression, the latter triggering signaling events-to be addressed here-that control other apoptosis regulatory genes (46,53,75,85,89,92).Ankyrin-G plays a critical role in linking the actin cytoskeleton to the cell membrane and in the biogenesis of the lateral membrane domain of epithelial cells. The N-terminal ankyrin repeat domain interacts with E-cadherin, linking the latter to the cytoskeleton via interaction with spectrin complexes. Accordingly, ankyrin-G localizes primarily to adherens junctions (50,65,73). In addition, ankyrin-G contains two domains, a death domain and a ZU-5 domain, whose homologues in other proteins regulate apoptosis (99, 101). The downregulation of the ankyrin-G gene (ank3) correlates with poor prognosis in diverse human...

Purpose: We investigate the roles of DNA mismatch repair (MMR) and p53 in mediating the induction of autophagy in human tumor cells after exposure to 6-thioguanine (6-TG), a chemotherapy drug recognized by MMR.We also examine how activation of autophagy affects apoptosis (type I cell death) after MMR processing of 6-TG. Experimental Design: Using isogenic pairs of MLH1+ human colorectal cancer cells (HCT116) and MSH2 À /MSH2 + human endometrial cancer cells (HEC59), we initially measure activation of autophagy for up to 3 days after 6-TG treatment using LC3, a specific marker of autophagy. We then assess the role of p53 in autophagic signaling of 6-TG MMR processing using both pifithrin-a cotreatment to chemically inhibit p53 transcription and small hairpin RNA inhibition of p53 expression. Finally, we use Atg5 small hairpin RNA inhibition of autophagy to assess the effect on apoptosis after MMR processing of 6-TG. Results: We find that MMR is required for mediating autophagy in response to 6-TG treatment in these human tumor cells.We also show that p53 plays an essential role in signaling from MMR to the autophagic pathway. Finally, our results indicate that 6-TG^induced autophagy inhibits apoptosis after MMR processing of 6-TG. Conclusions: These data suggest a novel function of MMR in mediating autophagy after a chemical (6-TG) DNA mismatch damage through p53 activation.The resulting autophagy inhibits apoptosis after MMR processing of 6-TG.

The primary functions of the proteasome are driven by a highly allosteric ATPase complex. ATP-binding to only two subunits in this hexameric complex triggers substrate binding, ATPase-20S association, and 20S gate-opening. However, it is unclear how ATP-binding and hydrolysis spatially and temporally coordinates these allosteric effects to drive substrate translocation into the 20S. Here, we use FRET to show that the proteasomal ATPases from eukaryotes (RPTs) and archaea (PAN) bind ATP with high affinity at neighboring subunits, which complements the well-established spiral-staircase topology of the 26S ATPases. We further show that two conserved arginine fingers in PAN located at the subunit interface work together as a single allosteric unit to mediate the allosteric effects of ATP-binding without altering the nucleotide-binding pattern. Rapid kinetics analysis also shows that ring resetting of a sequential hydrolysis mechanism can be explained by thermodynamic equilibrium binding of ATP. These data support a model whereby these two functionally distinct allosteric networks cooperate to translocate polypeptides into the 20S for degradation.

The base excision repair protein MED1 (also known as MBD4), an interactor with the mismatch repair protein MLH1, has a central role in the maintenance of genomic stability with dual functions in DNA damage response and repair. MED1 acts as a thymine and uracil DNA N-glycosylase on T:G and U:G mismatches that occur at cytosine-phosphate-guanine (CpG) methylation sites due to spontaneous deamination of 5-methylcytosine and cytosine, respectively. To elucidate the mechanisms that underlie sequence discrimination by MED1, we did single-turnover kinetics with the isolated, recombinant glycosylase domain of MED1. Quantification of MED1 substrate hierarchy confirmed MED1 preference for mismatches within a CpG context and showed preference for hemimethylated base mismatches. Furthermore, the k st values obtained with the uracil analogues 5-fluorouracil and 5-iodouracil were over 20-to 30-fold higher than those obtained with uracil, indicating substantially higher affinity for halogenated bases. A 5-iodouracil precursor is the halogenated nucleotide 5-iododeoxyuridine (5IdU), a cytotoxic and radiosensitizing agent. Cultures of mouse embryo fibroblasts (MEF) with different Med1 genotype derived from mice with targeted inactivation of the gene were evaluated for sensitivity to 5IdU. The results revealed that Med1-null MEFs are more sensitive to 5IdU than wild-type MEFs in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Furthermore, high-performance liquid chromatography analyses revealed that Med1-null cells exhibit increased levels of 5IdU in their DNA due to increased incorporation or reduced removal. These findings establish MED1 as a bona fide repair activity for the removal of halogenated bases and indicate that MED1 may play a significant role in 5IdU cytotoxicity. (Cancer Res 2006; 66(15): 7686-93)