Arnold I. Chin

Involvement of receptor-interacting protein 2 in innate and adaptive immune responses

Host defences to microorganisms rely on a coordinated interplay between the innate and adaptive responses of immunity. Infection with intracellular bacteria triggers an immediate innate response requiring macrophages, neutrophils and natural killer cells, whereas subsequent activation of an adaptive response through development of T-helper subtype 1 cells (TH1) proceeds during persistent infection. To understand the physiological role of receptor-interacting protein 2 (Rip2), also known as RICK and CARDIAK, we generated mice with a targeted disruption of the gene coding for Rip2. Here we show that Rip2-deficient mice exhibit a profoundly decreased ability to defend against infection by the intracellular pathogen Listeria monocytogenes. Rip2-deficient macrophages infected with L. monocytogenes or treated with lipopolysaccharide (LPS) have decreased activation of NF-κB, whereas dominant negative Rip2 inhibited NF-κB activation mediated by Toll-like receptor 4 and Nod1. In vivo, Rip2-deficient mice were resistant to the lethal effects of LPS-induced endotoxic shock. Furthermore, Rip2 deficiency results in impaired interferon-γ production in both TH1 and natural killer cells, attributed in part to defective interleukin-12-induced Stat4 activation. Our data reflect requirements for Rip2 in multiple pathways regulating immune and inflammatory responses.

Toll-like Receptor 3-Mediated Suppression of TRAMP Prostate Cancer Shows the Critical Role of Type I Interferons in Tumor Immune Surveillance

Inflammation has increasingly been recognized as a critical component influencing tumor growth. Recent reports have revealed conflicting evidence for the role of Toll-like receptors (TLR) in modulating tumorigenesis. In our study, we implicate TLR3 in mediating immune surveillance with increased growth of implanted transgenic adenocarcinoma of the mouse prostate (TRAMP) tumors in TLR3(-/-) compared with TLR3(+/+) mice. Activation of TLR3 by polyinosinic-polycytidylic acid (polyI:C) leads to induction of multiple inflammatory pathways, including NF-kappaB, mitogen-activated protein kinases, and interferon (IFN) regulatory factors. We explored the potential of TLR3 stimulation in prostate cancer immunotherapy and showed that treatment with polyI:C can strongly suppress both s.c. implanted TRAMP tumors in syngenic mice as well as orthotopic prostate cancers in TRAMP C57Bl6 x FvB F1 Tg(+/-) transgenic mice. Treated tumors remained well differentiated to moderately differentiated with increased infiltration of T lymphocytes and natural killer (NK) cells compared with poorly differentiated adenocarcinoma observed in untreated tumors. Like TLR3(-/-) mice, IFN-alpha receptor 1 (IFNAR1)(-/-) mice exhibited reduced tumor surveillance and impaired tumor suppression following polyI:C treatment. We observed that type I IFN-dependent induction of cytokines was responsible for NK activation, with depletion of NK cells leading to increased tumor growth as well as expansion of CD4(+)CD25(+)Foxp3(+) T regulatory lymphocytes. Our study therefore delineates the importance of IFNAR-dependent functions in TLR3-mediated tumor suppression and supports the use of TLR3 agonists for prostate cancer immune-based therapies.

TANK Potentiates Tumor Necrosis Factor Receptor-Associated Factor-Mediated c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase Activation through the Germinal Center Kinase Pathway

ABSTRACT Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are mediators of many members of the TNF receptor superfamily and can activate both the nuclear factor κB (NF-κB) and stress-activated protein kinase (SAPK; also known as c-Jun N-terminal kinase) signal transduction pathways. We previously described the involvement of a TRAF-interacting molecule, TRAF-associated NF-κB activator (TANK), in TRAF2-mediated NF-κB activation. Here we show that TANK synergized with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in SAPK activation. TRAF2 and TANK individually formed weak interactions with germinal center kinase (GCK)-related kinase (GCKR). However, when coexpressed, they formed a strong complex with GCKR, thereby providing a potential mechanism for TRAF and TANK synergy in GCKR-mediated SAPK activation, which is important in TNF family receptor signaling. Our results also suggest that TANK can form potential intermolecular as well as intramolecular interactions between its amino terminus and carboxyl terminus. This study suggests that TANK is a regulatory molecule controlling the threshold of NF-κB and SAPK activities in response to activation of TNF receptors. In addition, CD40 activated endogenous GCKR in primary B cells, implicating GCK family proteins in CD40-mediated B-cell functions.

Synergy of Histone-Deacetylase Inhibitor AR-42 with Cisplatin in Bladder Cancer

PURPOSE Cisplatin based chemotherapy regimens form the basis of systemic bladder cancer treatment, although they show limited response rates and efficacy. Recent molecular analysis of bladder cancer revealed a high incidence of mutations in chromatin regulatory genes, suggesting a therapeutic avenue for histone deacetylase inhibitors. We investigated the ability of the novel histone deacetylase inhibitor AR-42 to synergize with cisplatin in preclinical models of bladder cancer. MATERIALS AND METHODS We assessed the ability of the pan-histone deacetylase inhibitor AR-42 with and without cisplatin to destroy bladder cancer cells by survival and apoptosis assays in vitro, and by growth and differentiation in an in vivo xenograft model. We also assessed the response to the bladder cancer stem cell population by examining the effect of AR-42 on the CD44(+)CD49f(+) population with and without cisplatin. Synergy was calculated using combination indexes. RESULTS The AR-42 and cisplatin combination synergistically destroyed bladder cancer cells via apoptosis and it influenced tumor growth and differentiation in vivo. When tested in the CD44(+)CD49f(+) bladder cancer stem cell population, AR-42 showed greater efficacy with and without cisplatin. CONCLUSIONS AR-42 may be an attractive novel histone deacetylase inhibitor with activity against bladder cancer. Its efficacy in bladder cancer stem cells and synergy with cisplatin warrant further clinical investigation. Our in vitro and animal model studies provide preclinical evidence that AR-42 may be administered in conjunction with cisplatin based chemotherapy to improve the treatment of bladder cancer in patients.

Ultrasound-based combination therapy: potential in urologic cancer

Immune-sensitive urologic malignancies include prostate, kidney and bladder cancers. To date, most immunotherapeutic treatments have been applied to advanced metastatic disease. Limited efficacy in this setting is likely due to an excessive disease burden, which overwhelms the capacity of the immune system. Immunotherapy has not been widely utilized in a low-disease-burden state – a setting in which the immune system may be best suited to effectively mount a clinically meaningful response. The emergence of high-intensity focused ultrasound, and more recently, low-intensity focused ultrasound technologies, have demonstrated not only immune-stimulatory effects but also an interesting capacity to alter tissue architecture and cell membrane properties, which may be exploited to increase tumoral uptake of drugs and vaccines. In this article, we review the literature supporting the novel use of ultrasound combination therapy with adjunctive agents in the treatment of urologic malignancy.

Role of Rip2 in development of tumor-infiltrating MDSCs and bladder cancer metastasis.

Tumor invasion and metastases represent a complex series of molecular events that portends a poor prognosis. The contribution of inflammatory pathways mediating this process is not well understood. Nod-like receptors (NLRs) of innate immunity function as intracellular sensors of pathogen motifs and danger molecules. We propose a role of NLRs in tumor surveillance and in programming tumor-infiltrating lymphocytes (TILs). In this study, we examined the downstream serine/threonine and tyrosine kinase Rip2 in a murine model of bladder cancer. In Rip2-deficient C57Bl6 mice, larger orthotopic MB49 tumors developed with more numerous and higher incidence of metastases compared to wild-type controls. As such, increased tumor infiltration of CD11b+Gr1hi myeloid-derived suppressor cells (MDSCs) with concomitant decrease in T cells and NK cells were observed in Rip2-deficient tumor bearing animals using orthotopic and subcutaneous tumor models. Rip2-deficient tumors showed enhanced epithelial-to-mesenchymal transition, with elevated expression of zeb1, zeb2, twist, and snail in the tumor microenvironment. We found that the absence of Rip2 plays an intrinsic role in fostering the development of granulocytic MDSCs by an autocrine and paracrine effect of granulocytic colony stimulating factor (G-CSF) expression. Our findings suggest that NLR pathways may be a novel modality to program TILs and influence tumor metastases.

Biased Expression of the FOXP3Δ3 Isoform in Aggressive Bladder Cancer Mediates Differentiation and Cisplatin Chemotherapy Resistance

Purpose: The transcriptional regulation mediating cancer cell differentiation into distinct molecular subtypes and modulating sensitivity to existing treatments is an enticing therapeutic target. Our objective was to characterize the ability of the forkhead/winged transcription factor FOXP3 to modulate the differentiation of bladder cancer. Experimental Design: Expression of FOXP3 was analyzed by immunohistochemistry in a tumor microarray of 587 samples and overall survival in a subset of 187 patients following radical cystectomy. Functional assays were performed in SW780 and HT1376 cell lines in vitro and in vivo and gene expression profiling performed by RNA-Seq. Validation was undertaken using gene expression profiles of 131 patients from The Cancer Genome Atlas (TCGA) consortium in bladder cancer. Results: FOXP3 expression correlates with bladder cancer stage and inversely with overall survival, with biased expression of the FOXP3Δ3 isoform. Functional assays of FOXP3Δ3 demonstrated resistance to chemotherapy in vitro, whereas subcutaneous xenografts overexpressing FOXP3Δ3 developed larger and more poorly differentiated bladder cancers. RNA expression profiling revealed a unique FOXP3Δ3 gene signature supporting a role in chemotherapy resistance. Accordingly, knockdown of Foxp3 by siRNA in HT1376 cells conferred sensitivity to cisplatin- and gemcitabine-induced cytotoxicity. Validation in TCGA dataset demonstrated increased expression of FOXP3 in subtypes II to IV and skewing of molecular subtypes based on FOXP3Δ3-specific gene expression. Conclusions: (i) Biased expression of the FOXP3Δ3 isoform in bladder cancer inversely correlates with overall survival, (ii) FOXP3Δ3 induces a unique gene program that mediates cancer differentiation, and (iii) FOXP3Δ3 may augment chemotherapy resistance. Clin Cancer Res; 22(21); 5349–61. ©2016 AACR.

Loss of MyD88 leads to more aggressive TRAMP prostate cancer and influences tumor infiltrating lymphocytes

The influence of pattern recognition receptor (PRR) signaling in the prostate tumor microenvironment remains unclear. Although there may be a role for PRR agonists as adjuvants to therapy, prior evidence suggests tumor promoting as well as tumor inhibiting mechanisms. The purpose of this study is to examine the role of the key Toll‐like receptor (TLR) signaling adaptor protein myeloid differentiation primary response gene 88 (MyD88) in prostate cancer development.

RARP in high‑risk prostate cancer: use of multi‑parametric MRI and nerve sparing techniques

To examine the outcomes of patients with high-risk prostate cancer (PCa) treated by robot-assisted radical prostatectomy (RARP) and evaluate the value of multi-parametric magnetic resonance imaging (MRI) in estimating tumor stage, extracapsular extension, and grade, and the application of nerve sparing (NS) techniques. Patient demographics, preoperative imaging, surgical parameters, pathological features, functional and recurrence outcomes were collected retrospectively in patients with high-risk PCa who underwent RARP between December 2009 and October 2013. Pathological whole mount slides to assess NS were compared with potency, recovery of continence, and surgical margins (SM). Forty-four cases of high-risk PCa were identified with a median followup of 24 months and positive surgical margins (PSM) rate of 14%. Continence returned in 86%, with potency rate of 58%. Of the 25 cases with a preoperative multi-parametric MRI, MRI improved clinical staging from 28% to 88%, respectively. Following risk stratification of NS by microscopic analysis of whole mount pathology, patients with Group A (bilateral NS), Group B (unilateral NS), Group C (partial NS), and Group D (non-NS) had 100%, 92%, 91%, and 50% continence rates, and 100%, 80%, 45%, and 0% potency rates, respectively, with an inverse correlation to PSM. RARP in men with high-risk PCa can achieve favorable oncologic and functional outcomes. Preoperative MRI may localize high-grade tumors and improve clinical staging. Extent of NS is influenced by clinical staging and may balance potency and continence with PSMs.

Rip2: A Key Molecule that Regulates both Innate and Acquired Immunity

The Receptor interacting protein-2 (Rip2, also called RICK, CARDIAK) is an intracellular serine-threonine kinase that contains a carboxy-termin al caspase activation and recruitment domain (CARD). The initial biochemical analysis emphasized a role for Rip2 in the activation of nuclear factor-kappaB (NF-κB) and apoptosis when overexpressed. The subsequent generation of mice with a targeted deletion of the gene for Rip2 and the description of a possible target for Rip2 kinase activity has clarified the role of Rip2. Following infectious challenges, the activation of a protective immune response relies on the coordinated interplay of contextual stimulation and inflammatory processes. All mammals must balance the need to combat dangerous pathogens from the destructive potential for mistaking autologous cells or proteins as appropriate targets for response. Rip2 has carved out an evolutionary niche serving as a regulator of inflammatory responses. Rip2 helps to direct or propagate signals towards cell-mediated immune responses and resolution of infection by modifying signals from pathogen recognition receptors (PRRs) such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (Nod) family members of innate immunity, the T cell receptor (TCR) complex of acquired immunity, and cytokine signaling of the interleukin (IL)-1 receptor family and IL-12 signaling pathways. Here we wish to outline the progress made in describing the biological significance of Rip2 and the mode of regulation of this kinase. Further studies considering Rip2 as a target of intervention have the potential to be of great clinical value.