Department of Pharmacology & Chemical Biology at the University of Pittsburgh
Neumann Lab Magee-Womens Research Institute, A408
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SCIENCE

The main interest of the Neumann laboratory is to expand our knowledge of cell signaling that is in part mediated by oxidation and reducing (redox) reactions. Reactive oxygen species (ROS) deregulate the redox homeostasis and promote tumor formation by initiating an aberrant induction of signaling networks that cause tumorigenenesis, including breast cancer. To investigate the specific mechanisms underlying redox-induced tumorigenesis, the Neumann laboratory focuses on redox-induced posttranslational modifications (PTM) of protein cysteines, which play an important part in cell signaling. Peroxiredoxin 1 (PRDX1) is a peroxidase that has emerged as an important protein in cell signaling as it scavenges the second messenger H2O2, binds to and regulates signaling proteins and when knocked out in mice causes a variety of cancers, including breast cancer. Therefore, studying PRDX1 is an ideal model to further our understanding in redox-regulated cell-signaling pathways in breast cancer with the goal to utilize redox-induced PTMs as novel drug-targets in breast cancer therapy. All of these studies include many aspects of translational breast cancer research utilizing basic biochemistry, molecular and cell biology, cell lines, mouse models and clinical samples.

PRDX1 regulates cell signaling in redox reactions through protein interactions

Mice lacking PRDX1 are viable and fertile but have a shortened lifespan owing to the development beginning at about 9 months of severe hemolytic anemia and several malignant cancers, both of which are also observed at increased frequency in heterozygotes. The hemolytic anemia is characterized by an increase in erythrocyte reactive oxygen species, leading to protein oxidation, hemoglobin instability, Heinz body formation and decreased erythrocyte lifespan. The malignancies include lymphomas, sarcomas and carcinomas, and are frequently associated with loss of PRDX1 expression in heterozygotes, which suggests that this protein functions as a tumor suppressor. PRDX1-deficient fibroblasts show decreased proliferation and increased sensitivity to oxidative DNA damage, whereas PRDX1- null mice have abnormalities in numbers, phenotype and function of natural killer cells. Our results implicate PRDX1 as an important defense against oxidants in ageing mice.

 


 

Fig. 1 Premature death in aging PRDX1-/- mice. Kaplan–Meier survival curve of cohorts of wild-type (n = 34), PRDX1 +/- (n = 88) and Prdx1 +/+ (n = 64) littermates on a mixed B6 X 129SvEv background. Mutant lines generated from three independently targeted ES cell clones were studied with similar results. The ages of surviving mice are indicated by tick marks. The difference in survival between wild-type and PRDX1 -/- mice is statistically significant (P=0.05, Mantel–Cox test). Inset, percentage of mice in these cohorts that developed hemolytic anemia (red), malignancy (green) or both (blue). The x-axis is identical to the main graph.

PRDX1 ablation increases the susceptibility to Ras-induced breast cancer. We found Akt hyperactive in fibroblasts and mammary epithelial cells lacking PRDX1. Investigating the nature of such elevated Akt activation established a novel role for PRDX1 as a safeguard for the lipid phosphatase activity of PTEN, which is essential for its tumor suppressive function. We found binding of PRDX1 to PTEN essential for protecting PTEN from oxidation-induced inactivation. Along those lines, PRDX1 tumor suppression of Ras- or ErbB-2-induced transformation was mediated mainly via PTEN.

 
 

Fig. 2 PRDX1 prevents Akt-driven tumorigenesis through protecting PTEN lipid phosphatase activity from oxidation-induced inactivation. (A) PRDX1 regulates PTEN phosphatase activity during oxidative stress, since binding of Prdx1 and PTEN occurs in conditions of mild or nil cellular stress. This constitutes a setting in which H2O2 is scavenged by PRDX1, which itself becomes in turn reversibly oxidized, in a controlled fashion. (B) However, under conditions of elevated oxidative stress, PRDXs are known to become irreversibly over-oxidized and dissociate from PTEN. Thereby, PTEN is inactivated by H2O2 resulting in hyperactivation of Akt. Hyperactive Akt then in turn can promote oncogenic signaling via ErbB-2- and Ras.

PRDX1 specifically coordinates p38MAPK-induced signaling through regulating p38MAPKa phosphatases in a H2O2-dose dependent manner. MAPK phosphatases (MKP-1 and/or MKP-5), which are known to dephosphorylate and deactivate the senescence-inducing MAPK p38a, belong to a group of redox-sensitive phosphatases (protein tyrosine phosphatases: PTPs) characterized by a low pKa cysteine in their active sites. PRDX1 associates with both, MKP-1 and MKP-5, but dissociates from MKP-1 when the PRDX1 peroxidatic cysteine Cys52 is over-oxidized to sulfonic acid. This in turn results in MKP-1 oxidation-induced oligomerization and inactivity towards p38MAPKa. Conversely, over-oxidation of PRDX1-Cys-52 enhances the PRDX1::MKP-5 complex with increasing H2O2 concentrations. This correlates with a protection from oxidation-induced oligomerization and inactivation of MKP-5 so that activation towards p38MAPK is maintained. Further examination of this PRDX1-specific mechanism in a model of ROS-induced senescence of human breast epithelial cells reveals the specific activation of MKP-5, resulting in decreased p38MAPKa activity. Taken together, our data suggest that PRDX1 orchestrates redox-signaling in a H2O2-dose dependent manner through the oxidation-status of its peroxidatic cysteine Cys52.

 
 

 

 

Fig. 3 The peroxidatic cysteine Cys52 of PRDX1 is a sensor in ROS signaling. Under normal ROS homeostasis, Prdx1 promotes both MKP-1 and MKP-5 activity. However, under increased ROS, Prdx1- Cys52-SO3 forms less PRDX1/MKP-1 complexes leading to MKP-1 inactivation. This is comparable to data we obtained for PTEN. Conversely, in the presence of MKP-5, PRDX1-Cys52-SO3 binds to MKP-5 and preserves MKP-5 activity. We therefore speculate that MKP-1, which in some instances prefers JNK over p38MAPK phosphorylation in H2O2 as a substrate, is inactivated under high oxidative stress (because of the dissociation from PRDX1) to allow JNK activity. MKP-5 on the other hand favors, depending on the cellular context, p38MAPK phosphorylation in H2O2 over JNK as a substrate. This way, MKP-5 activation by PRDX1 is thereby preventing p38MAPK phosphorylation in H2O2 signaling in H2O2- induced senescence. The net outcome of all this may be that PRDX1 in an H2O2 dose-dependent manner prevents oxidative p38MAPK phosphorylation in H2O2-mediated stress-induced senescence to promote JNK-mediated signaling.

Another example of PRDX1 regulating the PI3K signaling pathway comes from findings describing that heightened oxidative stress evokes formation of reducible disulfide-bound heterotrimers linking dimeric PRDX1 to monomeric FOXO3. Two cysteines in FOXO3 are found to be essential for PRDX1 binding. One of these, C31, is neighboring the AKT-phosphorylation site T32 and the other, C150, is located adjacent to the FOXO3 DNA-binding site. Notably, in contrast to wild-type FOXO3, FOXO3-cysteine mutants show higher and constitutive T32 phosphorylation and display cytoplasmic retention under H2O2-stress that is reverted by PI3K inhibition. Furthermore, the tumor suppressive miRNA let-7c was discovered downstream of the PRDX1-FOXO3 axis as a novel FOXO3 substrate that, together with let-7b, is regulated by FOXO3 and PRDX1 expression levels. Conjointly, inhibition of let-7 microRNAs increases let-7-phenotypes in PRDX1-deficient breast cancer cells. These data ascertain the existence of an H2O2-sensitive PRDX1-FOXO3 signaling-axis that fine-tunes FOXO3 activity toward the transcription of the novel FOXO3 substrate let-7c in response to oxidative stress. Because redox stress is fundamental in driving the aging process and the development of cancers, this new described mechanism adds to our understanding of how FOXO3 signaling may be instrumental in the regulation of aging and tumor suppression.

 
 

 

 

 

Fig. 4 PRDX1 and FOXO3 interact in an oxidative stress-dependent way. This involves the catalytic/peroxidatic cysteine and C71 of PRDX1 and C31 and C150 of FOXO3. Our data strongly suggest disulfide bonding between PRDX1 and FOXO3 involving PRDX1 C52, C71 and C173 and FOXO3 C31 and C150 regulating the AKT-induced phosphorylation of T32 on FOXO3 and 14-3-3 binding and dissociation. Nuclear localization and 14-3-3 dissociation of FOXO3 may be promoted by mono-ubiquitination or phosphorylation by over-oxidized PRDX1 activating MST1 or oxidative stress activation of JNK or p38. This results in expression in let-7b and let-7c and inhibition of migration.

RESEARCH GROUP

Nadine Ryan, Administrator
Alp Asan, GRS
John Skoko, PhD
Carola Neumann, MD
Barbara Hopkins, GRS
Shireen Attaran, GRS

 



Fellows
John J. Skoko, PhD

Graduate Students
Shireen Attaran

Alparslan Asan




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