AR loss in prostate cancer stroma mediated by NF-κB and p38-MAPK signaling disrupts stromal morphogen production.

Androgen Receptor (AR) activity in prostate stroma is required to maintain prostate homeostasis. This is mediated through androgen-dependent induction and secretion of morphogenic factors that drive epithelial cell differentiation. However, stromal AR expression is lost in aggressive prostate cancer. The mechanisms leading to stromal AR loss and morphogen production are unknown. We identified TGFß1 and TNFα as tumor-secreted factors capable of suppressing AR mRNA and protein expression in prostate stromal fibroblasts. Pharmacological and RNAi approaches identified NF-κB as the major signaling pathway involved in suppressing AR expression by TNFα. In addition, p38α- and p38δ-MAPK were identified as suppressors of AR expression independent of TNFα. Two regions of the AR promoter were responsible for AR suppression through TNFα. FGF10 and Wnt16 were identified as androgen-induced morphogens, whose expression was lost upon TNFα treatment and enhanced upon p38-MAPK inhibition. Wnt16, through non-canonical Jnk signaling, was required for prostate basal epithelial cell survival. These findings indicate that stromal AR loss is mediated by secreted factors within the TME. We identified TNFα/TGFß as two possible factors, with TNFα mediating its effects through NF-κB or p38-MAPK to suppress AR mRNA transcription. This leads to loss of androgen-regulated stromal morphogens necessary to maintain normal epithelial homeostasis.

Microfluidic-based human prostate-cancer-on-chip.

Lack of adequate models significantly hinders advances in prostate cancer treatment, where resistance to androgen-deprivation therapies and bone metastasis remain as major challenges. Current in vitro models fail to faithfully mimic the complex prostate physiology. In vivo animal models can shed light on the oncogenes involved in prostate cancer development and progression; however, the animal prostate gland is fundamentally different from that of human, and the underlying genetic mechanisms are different. To address this problem, we developed the first in vitro microfluidic human Prostate-Cancer-on-Chip (PCoC) model, where human prostate cancer and stromal fibroblast cells were co-cultivated in two channels separated by a porous membrane under culture medium flow. The established microenvironment enables soluble signaling factors secreted by each culture to locally diffuse through the membrane pores affecting the neighboring culture. We particularly explored the conversion of the stromal fibroblasts into cancer-associated fibroblasts (CAFs) due to the interaction between the 2 cell types. Immunofluorescence microscopy revealed that tumor cells induced CAF biomarkers, αSMA and COL1A1, in stromal fibroblasts. The stromal CAF conversion level was observed to increase along the flow direction in response to diffusion agents, consistent with simulations of solute concentration gradients. The tumor cells also downregulated androgen receptor (AR) expression in stromal fibroblasts, while an adequate level of stromal AR expression is maintained in normal prostate homeostasis. We further investigated tumor invasion into the stroma, an early step in the metastatic cascade, in devices featuring a serpentine channel with orthogonal channel segments overlaying a straight channel and separated by an 8 µm-pore membrane. Both tumor cells and stromal CAFs were observed to cross over into their neighboring channel, and the stroma's role seemed to be proactive in promoting cell invasion. As control, normal epithelial cells neither induced CAF conversion nor promoted cell invasion. In summary, the developed PCoC model allows spatiotemporal analysis of the tumor-stroma dynamic interactions, due to bi-directional signaling and physical contact, recapitulating tissue-level multicellular responses associated with prostate cancer in vivo. Hence, it can serve as an in vitro model to dissect mechanisms in human prostate cancer development and seek advanced therapeutic strategies.

Gut Commensal Segmented Filamentous Bacteria Fine-Tune T Follicular Regulatory Cells to Modify the Severity of Systemic Autoimmune Arthritis.

Autoantibodies play a major pathogenic role in rheumatoid arthritis. T follicular helper (Tfh) cells promote germinal center B cell and Ab responses. Excessive Tfh cell responses lead to autoimmunity, and therefore, counterregulation is crucial. T follicular regulatory (Tfr) cells, mainly differentiated from T regulatory cells, can negatively regulate Tfh and germinal center B cells. Dysbiosis is involved in rheumatoid arthritis's pathogenesis. We previously demonstrated that the gut microbiota, segmented filamentous bacteria (SFB), promote autoimmune arthritis by inducing Tfh cells. However, little is known regarding whether gut microbiota influence systemic (nongut) Tfr cells, impacting gut-distal autoimmunity. In this study, using SFB in autoimmune arthritic K/BxN mice, we demonstrated that SFB-induced arthritis is linked to the reduction of Tfr cells' CTLA-4, the key regulatory molecule of Tfr cells. This SFB-mediated CTLA-4 reduction is associated with increased Tfr glycolytic activity, and glycolytic inhibition increases Tfr cells' CTLA-4 levels and reduces arthritis. The surface expression of CTLA-4 is tied to TCR signaling strength, and we discovered that SFB-reduced CTLA-4 is associated with a reduction of Nur77, an indicator of TCR signaling strength. Nur77 is known for repressing glycolytic activity. Using a loss-of-function study, we demonstrated that Nur77+/- haplodeficiency increases glycolysis and reduces CTLA-4 on Tfr cells, which is associated with increased arthritis and anti-glucose-6-phosphate isomerase titers. Tfr-specific deletion (KRN.Foxp3CreBcl-6fl/fl) in autoimmune condition reveals that Tfr cells repress arthritis, Tfh cells, and autoantibody responses and that SFB can mitigate this repression. Overall, these findings demonstrated that gut microbiota distally impact systemic autoimmunity by fine-tuning Tfr cells.

Pharmacological TLR4 Antagonism Using Topical Resatorvid Blocks Solar UV-Induced Skin Tumorigenesis in SKH-1 Mice.

An urgent need exists for the development of more efficacious molecular strategies targeting nonmelanoma skin cancer (NMSC), the most common malignancy worldwide. Inflammatory signaling downstream of Toll-like receptor 4 (TLR4) has been implicated in several forms of tumorigenesis, yet its role in solar UV-induced skin carcinogenesis remains undefined. We have previously shown in keratinocyte cell culture and SKH-1 mouse epidermis that topical application of the specific TLR4 antagonist resatorvid (TAK-242) blocks acute UV-induced AP-1 and NF-κB signaling, associated with downregulation of inflammatory mediators and MAP kinase phosphorylation. We therefore explored TLR4 as a novel target for chemoprevention of UV-induced NMSC. We selected the clinical TLR4 antagonist resatorvid based upon target specificity, potency, and physicochemical properties. Here, we confirm using ex vivo permeability assays that topical resatorvid can be effectively delivered to skin, and using in vivo studies that topical resatorvid can block UV-induced AP-1 activation in mouse epidermis. We also report that in a UV-induced skin tumorigenesis model, topical resatorvid displays potent photochemopreventive activity, significantly suppressing tumor area and multiplicity. Tumors harvested from resatorvid-treated mice display reduced activity of UV-associated signaling pathways and a corresponding increase in apoptosis compared with tumors from control animals. Further mechanistic insight on resatorvid-based photochemoprevention was obtained from unsupervised hierarchical clustering analysis of protein readouts via reverse-phase protein microarray revealing a significant attenuation of key UV-induced proteomic changes by resatorvid in chronically treated high-risk SKH-1 skin prior to tumorigenesis. Taken together, our data identify TLR4 as a novel molecular target for topical photochemoprevention of NMSC. Cancer Prev Res; 11(5); 265-78. ©2018 AACRSee related editorial by Sfanos, p. 251.

Host-microbiota interplay in mediating immune disorders.

To maintain health, the immune system must maintain a delicate balance between eliminating invading pathogens and avoiding immune disorders such as autoimmunity and allergies. The gut microbiota provide essential health benefits to the host, particularly by regulating immune homeostasis. Dysbiosis, an alteration and imbalance of the gut microbiota, is associated with the development of several autoimmune diseases in both mice and humans. In this review, we discuss recent advances in understanding how certain factors, such as age and gender, affect the gut microbiota, which in turn can influence the development of autoimmune diseases. The age factor in microbiota-dependent immune disorders indicates a window of opportunity for future diagnostic and therapeutic approaches. We also discuss unique commensal bacteria with strong immunomodulatory activity. Finally, we provide an overview of the potential molecular mechanisms whereby gut microbiota induce autoimmunity, as well as the evidence that gut microbiota trigger extraintestinal diseases by inducing the migration of gut-derived immune cells. Elucidating the interaction of gut microbiota and the host immune system will help us understand the pathogenesis of immune disorders, and provide us with new foundations to develop novel immuno- or microbe-targeted therapies.