Citations

ABSTRACT Hypoxic stress responses are essential for cellular and organismal survival and drive gene regulation across diverse biological pathways, including cell cycle progression and energy metabolism. Here, we show that topoisomerase IIβ (TOP2B) regulates DNA topology and transcription of hypoxia-inducible genes (HIGs) in a DNA-dependent protein kinase (DNA-PK)-dependent manner. Integrated cellular, biochemical, and genomic analyses reveal an antagonistic yet correlated relationship between TOP2B and DNA-PK activities. Under normoxic conditions, TOP2B associates with HIGs and represses transcription by suppressing DNA negative supercoiling formation and accessibility. Under hypoxic exposure, TOP2B dissociates from HIGs, while DNA-PK and HIF1α are recruited to activate HIGs. Notably, DNA-PK is required for the repressive function of TOP2B, as DNA-PK knockout abrogates TOP2B release and activity, resulting in elevated expression of a number of HIGs. Mechanistically, DNA-PK phosphorylates TOP2B at T1403, stimulating its catalytic activity to restrain DNA unwinding and accessibility, thereby suppressing HIG transcription. Collectively, these findings identify a novel role for TOP2B and DNA-PK-mediated regulation of TOP2B as key transcriptional regulators of HIG expression. We propose that phosphorylation-dependent modulation of TOP2B activity is coordinated with transcriptional states and determines DNA topology to regulate Pol II transcription.

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.

Recent studies indicated that activated transcription of serum‐inducible genes in humans requires topoisomerase IIβ (TOP2B)‐mediated DNA strand break (DSB) and DNA damage response (DDR) signaling. However, the mechanisms by which TOP2B is regulated in this process are unknown. Here, we identified a novel role of BRCA1‐BARD1 complex in transcription of these genes by regulating TOP2B binding to the transcription start site (TSS). Our genomic and in vitro biochemical analyses demonstrated that BRCA1‐BARD1 complex and TOP2B colocalize in a large number of genes and physically interact for TOP2B ubiquitination. Upon transcriptional activation, BRCA1 is phosphorylated at S1524 by ataxia telangiectasia‐mutated (ATM) and ataxia telangiectasia and Rad3‐related protein (ATR). Intriguingly, unphosphorylatable BRCA1 (S1524A)‐BARD1 complex ubiquitinates TOP2B, but not WT or S1524D BRCA1. Using the TSS of a representative serum‐inducible gene, EGR1 , we found that ubiquitinated TOP2B binds to the DNA with much more stability than deubiquitinated one does. Together, our findings suggest that BRCA1‐BARD1 complex stabilizes TOP2B association with serum‐inducible genes through its E3 ligase function in transcription. Transcriptional activation induces TOP2B‐mediated DSB and DDR signaling to activate ATM/ATR that phosphorylates BRCA1 S1524. Phosphorylated BRCA1 is ineffective in ubiquitinating TOP2B, which loosens it from the DNA. We propose that this loosening may contribute to either mobility or removal of TOP2B.

AbstractRecent population studies suggest an intriguing inverse relationship between several types of cancer and neurodegenerative diseases, including Alzheimer's disease. Understanding the intersection of the underlying biology for these two distinct families of diseases with one another may offer novel approaches to identify new therapeutic approaches and possible opportunities to repurpose existing drug candidates. The Alzheimer's Association and the Alzheimer's Drug Discovery Foundation convened a one‐day workshop to delve into this discussion. Workshop participants outlined research focus areas, potential collaborations, and partnerships for future action.

Abstract Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70–Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70–Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70–Bag3 complex may offer an appealing anticancer target. Cancer Res; 74(17); 4731–40. ©2014 AACR.

Abstract We have a long-term interest in the connectivity between autoimmunity and tumor rejection. However, outside of the melanocyte/melanoma paradigm, little is known about whether autoimmune responses to normal tissue can induce rejection of tumors of the same histologic type. Here, we induced direct, pathogen-like cytotoxicity to the normal pancreas in association with the immune adjuvant heat shock protein 70. In sharp contrast to our studies with a similar approach for the treatment of prostate cancer, inflammatory killing of the normal pancreas induced a Th1-like, anti-self-response to pancreatic antigens, which was rapidly suppressed by a concomitant suppressive regulatory T cell (Treg) response. Interestingly, even when Treg cells were depleted, the Th1-like response was insufficient to induce significant ongoing autoimmunity. However, the Th1-like response to antigens expressed in the pancreas at the time of damage was sufficient to induce rejection of tumors expressing either a foreign (ova) antigen or fully syngeneic tumor antigens (on Panc02 tumor cells), provided that Treg were depleted before inflammatory killing of the normal pancreas. Taken together, these data indicate that profound differences exist between the immunoprotective mechanisms in place between different tissues (pancreas and prostate) in their response to pathogen-like damage. Moreover, they also show that, although multiple layers of immunologic safeguards are in place to prevent the development of severe autoimmune consequences in the pancreas (in contrast to the prostate), tumor rejection responses can still be decoupled from pathologic autoimmune responses in vivo, which may provide novel insights into the immunotherapeutic treatment of pancreatic cancer. [Cancer Res 2009;69(19):7767–74]

AbstractA close connectivity between autoimmune and tumor rejection responses is known to exist in the case of melanoma immunotherapy. However, relatively little is known about self-antigens on other types of normal cells, their relation to the development of autoimmune disease, and their possible coexistence as potential tumor rejection antigens on associated tumors. In the current study, we induced inflammatory killing of normal prostate tissue in situ using a fusogenic membrane glycoprotein along with the immune adjuvant hsp70. We show here that, in the prostate, hsp70 induces interleukin (IL)-6, which triggers a CD4- and CD8-dependent progressive autoimmune reactivity, associated with IL-17 expression. This autoimmune response was also able to induce the rejection of established prostate tumors, but not other histologic types of tumors, growing elsewhere in the animal. These data show that the intimate connectivity between autoimmune and tumor rejection responses extends beyond the classic melanoma paradigm and may be clinically valuable for the treatment of established metastatic disease of the prostate. [Cancer Res 2007;67(24):11970–9]