Christoph Zenzmaier

ROS Signaling by NOX4 Drives Fibroblast-to-Myofibroblast Differentiation in the Diseased Prostatic Stroma

Stromal remodeling, in particular fibroblast-to-myofibroblast differentiation, is a hallmark of benign prostatic hyperplasia (BPH) and solid tumors, including prostate cancer (PCa). Increased local production of TGFβ1 is considered the inducing stimulus. Given that stromal remodeling actively promotes BPH/PCa development, there is considerable interest in developing stromal-targeted therapies. Microarray and quantitative PCR analysis of primary human prostatic stromal cells induced to undergo fibroblast-to-myofibroblast differentiation with TGFβ1 revealed up-regulation of the reactive oxygen species (ROS) producer reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and down-regulation of the selenium-containing ROS-scavenging enzymes glutathione peroxidase 3, thioredoxin reductase 1 (TXNRD1), and the selenium transporter selenoprotein P plasma 1. Consistently, NOX4 expression correlated specifically with the myofibroblast phenotype in vivo, and loss of selenoprotein P plasma 1 was observed in tumor-associated stroma of human PCa biopsies. Using lentiviral NOX4 short hairpin RNA-mediated knockdown, pharmacological inhibitors, antioxidants, and selenium, we demonstrate that TGFβ1 induction of NOX4-derived ROS is required for TGFβ1-mediated phosphorylation of c-jun N-terminal kinase, which in turn is essential for subsequent downstream cytoskeletal remodeling. Significantly, selenium supplementation inhibited differentiation by increasing ROS-scavenging selenoenzyme biosynthesis because glutathione peroxidase 3 and TXNRD1 expression and TXNRD1 enzyme activity were restored. Consistently, selenium depleted ROS levels downstream of NOX4 induction. Collectively, this work demonstrates that dysregulated redox homeostasis driven by elevated NOX4-derived ROS signaling underlies fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma. Further, these data indicate the potential clinical value of selenium and/or NOX4 inhibitors in preventing the functional pathogenic changes of stromal cells in BPH and PCa.

GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib.

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomibs inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts, thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy, we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. Indeed, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data, we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib.

Dysregulation of Dkk‐3 expression in benign and malignant prostatic tissue

The Dickkopf (Dkk) family comprises four members Dkk‐1, ‐2, ‐3, and ‐4. Dkk‐3, the most divergent family member, unlike the others does not modulate Wnt signaling. Dkk‐3 is proposed to function as a secreted tumor suppressor since it is downregulated in a number of cancer cells and prostate cancer tissue and thus may be a promising candidate molecule for therapeutic interference.

Attenuated Proliferation and Trans-Differentiation of Prostatic Stromal Cells Indicate Suitability of Phosphodiesterase Type 5 Inhibitors for Prevention and Treatment of Benign Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is characterized by tissue overgrowth and stromal reorganization primarily due to cellular proliferation and fibroblast-to-myofibroblast trans-differentiation. To evaluate the potential of phosphodiesterase type 5 (PDE5) inhibitors like tadalafil for prevention and treatment of BPH, we analyzed the role of the nitric oxide/cyclic GMP (cGMP)/PDE5 pathway for cellular proliferation and TGFbeta1-induced fibroblast-to-myofibroblast trans-differentiation in primary prostate stromal cells. Inhibition by tadalafil of PDE5, which is mainly expressed in the stromal compartment of the prostate, reduced proliferation of primary prostate stromal cells and to a lesser extent of primary prostate basal epithelial cells. Attenuated proliferation due to elevated intracellular cGMP levels was confirmed by inhibition of the cGMP-dependent protein kinase G by its inhibitor KT2358. Moreover, tadalafil strongly attenuated TGFbeta1-induced fibroblast-to-myofibroblast trans-differentiation. The inhibitory effect on trans-differentiation was also observed after small interfering RNA-mediated PDE5 knockdown. As confirmed by the MAPK kinase 1 inhibitor PD98059, this effect was mediated via MAPK kinase 1 signaling. We conclude that BPH patients might benefit from adjuvant therapies with PDE5 inhibitors that inhibit stromal enlargement due to cell proliferation, as well as TGFbeta1-induced trans-differentiation processes.

Identification of evolutionarily conserved genetic regulators of cellular aging

To identify new genetic regulators of cellular aging and senescence, we performed genome‐wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress‐induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.

Therapeutic Targeting of Redox Signaling in Myofibroblast Differentiation and Age-Related Fibrotic Disease

Myofibroblast activation plays a central role during normal wound healing. Whereas insufficient myofibroblast activation impairs wound healing, excessive myofibroblast activation promotes fibrosis in diverse tissues (including benign prostatic hyperplasia, BPH) leading to organ dysfunction and also promotes a stromal response that supports tumor progression. The incidence of impaired wound healing, tissue fibrosis, BPH, and certain cancers strongly increases with age. This paper summarizes findings from in vitro fibroblast-to-myofibroblast differentiation systems that serve as cellular models to study fibrogenesis of diverse tissues. Supported by substantial in vivo data, a large body of evidence indicates that myofibroblast differentiation induced by the profibrotic cytokine transforming growth factor beta is driven by a prooxidant shift in redox homeostasis due to elevated production of NADPH oxidase 4 (NOX4)-derived hydrogen peroxide and supported by concomitant decreases in nitric oxide/cGMP signaling and reactive oxygen species (ROS) scavenging enzymes. Fibroblast-to-myofibroblast differentiation can be inhibited and reversed by restoring redox homeostasis using antioxidants or NOX4 inactivation as well as enhancing nitric oxide/cGMP signaling via activation of soluble guanylyl cyclases or inhibition of phosphodiesterases. Current evidence indicates the therapeutic potential of targeting the prooxidant shift in redox homeostasis for the treatment of age-related diseases associated with myofibroblast dysregulation.

Redox Signaling as a Therapeutic Target to Inhibit Myofibroblast Activation in Degenerative Fibrotic Disease

Degenerative fibrotic diseases encompass numerous systemic and organ-specific disorders. Despite their associated significant morbidity and mortality, there is currently no effective antifibrotic treatment. Fibrosis is characterized by the development and persistence of myofibroblasts, whose unregulated deposition of extracellular matrix components disrupts signaling cascades and normal tissue architecture leading to organ failure and death. The profibrotic cytokine transforming growth factor beta (TGFβ) is considered the foremost inducer of fibrosis, driving myofibroblast differentiation in diverse tissues. This review summarizes recent in vitro and in vivo data demonstrating that TGFβ-induced myofibroblast differentiation is driven by a prooxidant shift in redox homeostasis. Elevated NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) supported by concomitant decreases in nitric oxide (NO) signaling and reactive oxygen species scavengers are central factors in the molecular pathogenesis of fibrosis in numerous tissues and organs. Moreover, complex interplay between NOX4-derived H2O2 and NO signaling regulates myofibroblast differentiation. Restoring redox homeostasis via antioxidants or NOX4 inactivation as well as by enhancing NO signaling via activation of soluble guanylyl cyclases or inhibition of phosphodiesterases can inhibit and reverse myofibroblast differentiation. Thus, dysregulated redox signaling represents a potential therapeutic target for the treatment of wide variety of different degenerative fibrotic disorders.

Aging of the prostate epithelial stem/progenitor cell

Maintenance of the prostatic epithelial cell compartment is ensured by proliferation of adult epithelial progenitor or stem cells. These cells are characterized by an undifferentiated state, high proliferative capacity and long life span. Prostate progenitor/stem cells are localized in their stem cell-niche in the basal cell compartment in close contact to the basement membrane and the stromal cell compartment and are characterized by expression of the basal cytokeratins 5 and 14, high levels of integrins, CD44, the stem cell markers CD133 and ABCG2, and AR negativity. They give rise to secretory luminal (cytokeratins 8/18, CD57, AR, p27, PSA, PAP) and neuroendocrine cells (cytokeratins 8/18, CD57, CgA, NSE, NEPs), the two major cell types observed in the glandular epithelium. A growing body of experimental evidence has identified the amplifying progenitor/stem cell (CD44(+), alpha(2)beta(1)(hi), CD133(+)), as a putative origin of prostate cancer. Differentiation of this cell type can be affected by mutations in the intrinsic genetic program, by age-related changes in stromal-epithelial interactions or in the basement membrane/ECM composition. All these stochastic events occur during aging and can transform a normal prostate progenitor/stem cell into a cancer stem cell, a source of androgen-dependent and independent tumor cell clones. Thus, the heterogeneous and multifocal nature of prostatic cancer with a pleora of different tumor cell clones clearly reflects the differentiation capacity of the prostatic epithelial progenitor cells.

PAGE4 Positivity Is Associated with Attenuated AR Signaling and Predicts Patient Survival in Hormone-Naive Prostate Cancer

Aberrant activation of the androgen receptor (AR) plays a key role during prostate cancer (PCa) development and progression to castration-resistant prostate cancer (CR-PCa) after androgen deprivation therapy, the mainstay systemic treatment for PCa. New strategies to abrogate AR activity and biomarkers that predict aggressive tumor behavior are essential for improved therapeutic intervention. PCa tissue microarrays herein reveal that prostate-associated gene 4 (PAGE4), an X-linked cancer/testis antigen, is highly up-regulated in the epithelium of preneoplastic lesions compared with benign epithelium, but subsequently decreases with tumor progression. We show that AR signaling is attenuated in PAGE4-expressing cells both in vitro and in vivo, most likely via impaired androgen-induced AR nuclear translocation and subsequently reduced AR protein stabilization and phosphorylation at serines 81 and 213. Consistently, epithelial PAGE4 protein levels inversely correlated with AR activation status in hormone-naive and CR-PCa clinical specimens. Moreover, PAGE4 impaired the development of CR-PCa xenografts, and strong PAGE4 immunoreactivity independently predicted favorable patient survival in hormone-naive PCa. Collectively, these data suggest that dysregulation of epithelial PAGE4 modulates AR signaling, thereby promoting progression to advanced lethal PCa and highlight the potential value of PAGE4 as a prognostic and therapeutic target.

Increase of Dkk-3 Blood Plasma Levels in the Elderly

Gene expression of the secreted glycoprotein Dkk-3 is upregulated during cellular senescence in prostate basal epithelial cells and altered in age-related disorders of the human prostate. In order to quantify the influence of such age- and disease-related changes of Dkk-3 levels in body fluids, we established a highly specific and sensitive indirect IEMA. Results revealed a significant increase of Dkk-3 blood plasma levels in the elderly (1.17+/-0.36 vs. 1.61+/-0.61 nmol/L) indicating a non-negligible physiological role and its use as a marker for senescence not only in vitro but also in vivo.