Mammalian DNA polymerase ␦ (pol ␦), a key enzyme of chromosomal DNA replication, consists of four subunits as follows: the catalytic subunit; p125, which is tightly associated with the p50 subunit; p68, a proliferating cell nuclear antigen (PCNA)-binding protein; and a fourth subunit, p12. In this study, the functional roles of the p12 subunit of pol ␦ were studied. The inter-subunit interactions of the p12 subunit were determined by yeast two-hybrid assays and by pulldown assays. These assays revealed that p12 interacts with p125 as well as p50. This dual interaction of p12 suggests that it may serve to stabilize the p125-p50 interaction. p12 was shown to be a novel PCNA-binding protein. This was confirmed by identification of a PCNA-binding motif at its N terminus by binding assays and by site-directed mutagenesis. The activities and reaction products of recombinant pol ␦ containing a p12 mutant defective in PCNA binding, as well as purified recombinant pol ␦ and its subassemblies, were analyzed. Our results indicate that p12 contributes to PCNA-dependent pol ␦ activity, i.e. the p12-PCNA interaction is functional. Our data indicate that both p12 and p68 are required for optimal pol ␦ activity. This supports the hypothesis that the interaction between pol ␦ and PCNA is a divalent one that involves p12 and p68. We propose a model in which pol ␦ interacts with PCNA via at least two of its subunits, and one in which p12 could play a role in stabilizing the overall pol ␦-PCNA complex as well as pol ␦ itself.Chromosomal DNA replication in eukaryotic cells requires the following three distinct DNA polymerases: polymerase ␣, polymerase ␦ (pol ␦), 3 and polymerase ⑀. DNA pol ␦ is the key enzyme that is thought to play a central role in the elongation of both the leading and the lagging strands of DNA and the maturation of Okazaki fragments (1-3). DNA pol ␦ was originally identified as a new type of DNA polymerase possessing an intrinsic 3Ј-5Ј-exonuclease activity (4). Mammalian pol ␦ holoenzyme consists of the p125 catalytic subunit (which harbors both 5Ј-3Ј DNA polymerase and 3Ј-5Ј-exonuclease activities) and a tightly associated second subunit p50; this core is associated with two other subunits, p68 and p12, that are also referred to as the third and fourth subunits (5-9). The function of pol ␦ as a chromosomal DNA polymerase is dependent on its association with PCNA, which functions as a molecular sliding clamp (10, 11). The third subunits of pol ␦ in both mammalian (p68/p66) and in yeast cells (Cdc27 in Schizosaccharomyces pombe and pol 32 in Saccharomyces cerevisiae) harbor a PCNA-binding motif, and it has been shown that this provides a PCNA interaction site for pol ␦ (12-16). However, the exact nature of the subunit contacts of mammalian pol ␦ with PCNA has yet to be clarified; we (17-20) and others (8) have reported that human pol ␦ p125 binds to PCNA, although other reports have come to the opposite conclusion (14, 21). There is also a report that the p50 subunit of mammalian pol ␦ binds to PCNA (21).The fou...

Despite rapid progress in elucidating the molecular mechanisms of activation of the kinase IKK, the processes that regulate IKK deactivation are still unknown. Here we demonstrate that CUE domain-containing 2 (CUEDC2) interacted with IKKalpha and IKKbeta and repressed activation of the transcription factor NF-kappaB by decreasing phosphorylation and activation of IKK. Notably, CUEDC2 also interacted with GADD34, a regulatory subunit of protein phosphatase 1 (PP1). We found that IKK, CUEDC2 and PP1 existed in a complex and that IKK was released from the complex in response to inflammatory stimuli such as tumor necrosis factor. CUEDC2 deactivated IKK by recruiting PP1 to the complex. Therefore, CUEDC2 acts as an adaptor protein to target IKK for dephosphorylation and inactivation by recruiting PP1.

◥Purpose: Dexamethasone, a uniquely potent corticosteroid, is frequently administered to patients with brain tumors to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in patients with glioblastoma (GBM), particularly in the context of immunotherapy.Experimental Design: We evaluated the dose-dependent effects of dexamethasone when administered with programmed cell death 1 (PD-1) blockade and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A GBM tumors. Clinically, the effect of dexamethasone on survival was evaluated in 181 patients with isocitrate dehydrogenase (IDH) wild-type GBM treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors.Results: Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy reduced survival in a dosedependent manner. Concurrent dexamethasone also abrogated survival following anti-PD-1 therapy with or without radiotherapy in immune-resistant CT-2A models. Dexamethasone decreased T-lymphocyte numbers by increasing apoptosis, in addition to decreasing lymphocyte functional capacity. Myeloid and natural killer cell populations were also generally reduced by dexamethasone. Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive patients with IDH wild-type GBM treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival [reference, no dexamethasone; <2 mg HR, 2.16; 95% confidence interval (CI), 1.30-3.68; P ¼ 0.003 and ≥2 mg HR, 1.97; 95% CI, 1.23-3.16; P ¼ 0.005].Conclusions: Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for patients with GBM.

Endocrine resistance is a major obstacle to hormonal therapy for breast cancers. Although reduced expression of estrogen receptor-α (ER-α) is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. Here we report that CUE domain-containing protein-2 (CUEDC2), a ubiquitin-binding motif-containing protein, is a key factor in endocrine resistance in breast cancer. We show that CUEDC2 modulates ER-α protein stability through the ubiquitin-proteasome pathway. Through the study of specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between CUEDC2 and ER-α protein expression. Notably, subjects with tumors that highly expressed CUEDC2 had poor responsiveness to tamoxifen treatment and high potential for relapse. We further show that ectopic CUEDC2 expression impaired the responsiveness of breast cancer cells to tamoxifen. Therefore, our findings suggest that CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer.

The molecular details linking integrin engagement to downstream cortactin (Ctn) tyrosine phosphorylation are largely unknown. In this report, we show for the first time that Fer and Ctn are potently tyrosine phosphorylated in response to hydrogen peroxide (H 2 O 2 ) in a variety of cell types. Working with catalytically inactive fer and src/yes/fyn-deficient murine embryonic fibroblasts (fer DR/DR and syf MEF, respectively), we observed that H 2 O 2 -induced Ctn tyrosine phosphorylation is primarily dependent on Fer but not Src family kinase (SFK) activity. We also demonstrated for the first time that Fer is activated by fibronectin engagement and, in concert with SFKs, mediates Ctn tyrosine phosphorylation in integrin signaling pathways. Reactive oxygen species (ROS) scavengers or the NADPH oxidase inhibitor, diphenylene iodonium, attenuated integrininduced Fer and Ctn tyrosine phosphorylation. Taken together, these findings provide novel genetic evidence that a ROS-Fer signaling arm contributes to SFK-mediated Ctn tyrosine phosphorylation in integrin signaling. Lastly, a migration defect in fer DR/DR MEF suggests that integrin signaling through the ROS-Fer-Ctn signaling arm may be linked to mechanisms governing cell motility. These data demonstrate for the first time an oxidative link between integrin adhesion and an actin-binding protein involved in actin polymerization.