Novel Mouse Models of Bladder Cancer Identify a Prognostic Signature Associated with Risk of Disease Progression.

To study the progression of bladder cancer from non-muscle-invasive to muscle-invasive disease, we have developed a novel toolkit that uses complementary approaches to achieve gene recombination in specific cell populations in the bladder urothelium in vivo, thereby allowing us to generate a new series of genetically engineered mouse models (GEMM) of bladder cancer. One method is based on the delivery of adenoviruses that express Cre recombinase in selected cell types in the urothelium, and a second uses transgenic drivers in which activation of inducible Cre alleles can be limited to the bladder urothelium by intravesicular delivery of tamoxifen. Using both approaches, targeted deletion of the Pten and p53 tumor suppressor genes specifically in basal urothelial cells gave rise to muscle-invasive bladder tumors. Furthermore, preinvasive lesions arising in basal cells displayed upregulation of molecular pathways related to bladder tumorigenesis, including proinflammatory pathways. Cross-species analyses comparing a mouse gene signature of early bladder cancer with a human signature of bladder cancer progression identified a conserved 28-gene signature of early bladder cancer that is associated with poor prognosis for human bladder cancer and that outperforms comparable gene signatures. These findings demonstrate the relevance of these GEMMs for studying the biology of human bladder cancer and introduce a prognostic gene signature that may help to stratify patients at risk for progression to potentially lethal muscle-invasive disease. SIGNIFICANCE: Analyses of bladder cancer progression in a new series of genetically engineered mouse models has identified a gene signature of poor prognosis in human bladder cancer.

Evaluation of Nonviral piggyBac and lentiviral Vector in Functions of CD19chimeric Antigen Receptor T Cells and Their Antitumor Activity for CD19+ Tumor Cells.

Nonviral transposon piggyBac (PB) and lentiviral (LV) vectors have been used to deliver chimeric antigen receptor (CAR) to T cells. To understand the differences in the effects of PB and LV on CAR T-cell functions, a CAR targeting CD19 was cloned into PB and LV vectors, and the resulting pbCAR and lvCAR were delivered to T cells to generate CD19pbCAR and CD19lvCAR T cells. Both CD19CAR T-cell types were strongly cytotoxic and secreted high IFN-γ levels when incubated with Raji cells. TNF-α increased in CD19pbCAR T cells, whereas IL-10 increased in CD19lvCAR T cells. CD19pbCAR and CD19lvCAR T cells showed similar strong anti-tumor activity in Raji cell-induced mouse models, slightly reducing mouse weight while enhancing mouse survival. High, but not low or moderate, concentrations of CD19pbCAR T cells significantly inhibited Raji cell-induced tumor growth in vivo. These CD19pbCAR T cells were distributed mostly in mesenteric lymph nodes, bone marrow of the femur, spleen, kidneys, and lungs, specifically accumulating at CD19-rich sites and CD19-positive tumors, with CAR copy number being increased on day 7. These results indicate that pbCAR has its specific activities and functions in pbCAR T cells, making it a valuable tool for CAR T-cell immunotherapy.

ARID1A promotes genomic stability through protecting telomere cohesion.

ARID1A inactivation causes mitotic defects. Paradoxically, cancers with high ARID1A mutation rates typically lack copy number alterations (CNAs). Here, we show that ARID1A inactivation causes defects in telomere cohesion, which selectively eliminates gross chromosome aberrations during mitosis. ARID1A promotes the expression of cohesin subunit STAG1 that is specifically required for telomere cohesion. ARID1A inactivation causes telomere damage that can be rescued by STAG1 expression. Colony formation capability of single cells in G2/M, but not G1 phase, is significantly reduced by ARID1A inactivation. This correlates with an increase in apoptosis and a reduction in tumor growth. Compared with ARID1A wild-type tumors, ARID1A-mutated tumors display significantly less CNAs across multiple cancer types. Together, these results show that ARID1A inactivation is selective against gross chromosome aberrations through causing defects in telomere cohesion, which reconciles the long-standing paradox between the role of ARID1A in maintaining mitotic integrity and the lack of genomic instability in ARID1A-mutated cancers.

Identifying tumor promoting genomic alterations in tumor-associated fibroblasts via retrovirus-insertional mutagenesis.

Tumor-associated fibroblasts (TAFs) are often essential for solid tumor growth. However, few genetic or epigenetic alterations have been found in TAFs during the progression of solid tumors. Employing a tumor-stromal cell co-injection model, we adapted here retroviral-insertional mutagenesis to stromal cells to identify novel tumor-associated genes in TAFs. We successfully identified 20 gene candidates that might modulate tumor growth if altered in TAFs at genomic level. To validate our finding, the function of one of the candidate genes, tubulin tyrosine ligase (Ttl), was further studied in TAFs from fibrosarcoma, colon, breast and hepatocarcinoma. We demonstrated that down-regulated TTL expression in TAFs indeed promoted tumor growth in mice. Interestingly, decreased expression of TTL in tumor stromal cells also correlated with poor outcome in human colon carcinoma. Thus, the co-injection model of tumor cells with retrovirus-modified fibroblasts proved a valid method to identify tumor-modulating genes in TAFs, allowing for a deeper insight into the role of the stroma for tumor development.

ARF Confers a Context-Dependent Response to Chemotherapy in Muscle-Invasive Bladder Cancer.

Muscle-invasive bladder cancer (MIBC) generally responds poorly to treatment and tends to exhibit significant mortality. Here we show that expression of the tumor suppressor p14ARF (ARF) is upregulated in aggressive subtypes of MIBC. Accumulation of ARF in the nucleolus is associated with poor outcome and attenuated response to chemotherapy. In both genetically engineered mouse models and murine xenograft models of human MIBC, we demonstrate that tumors expressing ARF failed to respond to treatment with the platinum-based chemotherapy agent cisplatin. Resistance was mediated in part by the integrin-binding protein ITGB3BP (CENPR) and reflected ARF-dependent impairment of protein translation, which was exaggerated by drug treatment. Overall, our results highlight a context-dependent role for ARF in modulating the drug response of bladder cancer. Cancer Res; 77(4); 1035-46. ©2017 AACR.

IFNγ-responsiveness of endothelial cells leads to efficient angiostasis in tumours involving down-regulation of Dll4.

Although IFNγ is regarded as a key cytokine in angiostatic response, our poor understanding of its effective cellular target drastically limits its clinical trials against angiogenesis-related disorders. Here, we investigated the effect of IFNγ on endothelial cells (ECs) and possible molecular mechanisms in angiostasis. By employing Tie2(IFNγR) mice, in which IFNγR expression was reconstituted under the control of Tie2 promoter in IFNγR-deficient mice, we found that the response of ECs to IFNγ was highly effective in inhibiting blood supply and retarding tumour growth. Interestingly, the expression of IFNγR on Tie2(-) cells did not inhibit, but promoted tumour growth in control wild-type mice. Mechanism studies showed that IFNγ reacting on ECs down-regulated the delta-like ligand 4 (Dll4)/Notch signalling pathway. Accordingly, overexpression of Dll4 in human ECs diminished the effect of IFNγ on ECs. This study demonstrates that the action of IFNγ on ECs, but not other cells, is highly effective for tumour angiostasis, which involves down-regulating Dll4. It provides insights for EC-targeted angiostatic therapy in treating angiogenesis-associated disorders in the clinic.

TNFR-1 on tumor cells contributes to the sensitivity of fibrosarcoma to chemotherapy.

Impaired tumor necrosis factor receptor-1 (TNFR-1) signaling has been found in some malignant tumors with poor prognosis. However, the exact role of TNFR-1 signaling in fibrosarcoma remains unclear. Here, we explored the question by comparing the growth of TNFR-1 deficient (Tnfr1 (-)) and TNFR-1 competent (Tnfr1 (+)) fibrosarcoma FB61 cells (FB61-m and FB61-R1) in mice. TNFR-1 expression on fibrosarcoma cells delayed their growth in vivo but not in vitro. Moreover, reduced FB61-R1 tumor growth was also obtained in TNFR-1 knockout mice. The mechanism relies mainly on the TNFR-1-mediated downregulation of vascular endothelial growth factor (VEGF) production by tumor cells. Importantly, treatment of FB61-m tumors with melphalan resulted in a short delay of tumor growth, followed by a quick remission. However, when FB61-R1 tumors were treated with melphalan, tumor growth was similarly delayed at first and then completely rejected. Our results reveal evidence for TNFR-1 on tumor cells as a prerequisite in chemotherapy for fibrosarcoma, and provide novel insight into the therapeutic approach against some types of tumors using TNFR-1 angonist.

TNF signaling drives myeloid-derived suppressor cell accumulation.

TNF, an inflammatory cytokine that is enriched in the tumor microenvironment, promotes tumor growth and subverts innate immune responses to cancer cells. We previously reported that tumors implanted in TNF receptor-deficient (Tnfr-/-) mice are spontaneously rejected; however, the molecular mechanisms underlying this rejection are unclear. Here we report that TNF signaling drives the peripheral accumulation of myeloid-derived suppressor cells (MDSCs). MDSCs expand extensively during inflammation and tumor progression in mice and humans and can enhance tumor growth by repressing T cell-mediated antitumor responses. Peripheral accumulation of MDSCs was drastically impaired in Tnfr-/- mice. Signaling of TNFR-2, but not TNFR-1, promoted MDSC survival through upregulation of cellular FLICE-inhibitory protein (c-FLIP) and inhibition of caspase-8 activity. Loss of TNFRs impaired the induction of MDSCs from bone marrow cells, but this could be reversed by treatment with caspase inhibitors. These results demonstrate that TNFR-2 signaling promotes MDSC survival and accumulation and helps tumor cells evade the immune system.

TNFR2 expression on non-bone marrow-derived cells is crucial for lipopolysaccharide-induced septic shock and downregulation of soluble TNFR2 level in serum.

Persistently high serum levels of soluble tumor-necrosis factor (TNF) receptor 2 (sTNFR2) have been observed in septic shock and many inflammatory diseases. However, its origin and regulation during these pathological processes are still largely unknown. In this study, murine bone marrow (BM) chimeras selectively expressing TNFR2 on either BM-derived or non-BM-derived cells were generated and challenged with lipopolysaccharide (LPS). The results show that TNFR2 expression on non-BM-derived cells is crucial for both the sensitivity of mice to LPS and the downregulation of sTNFR2 in serum. Most importantly, sTNFR2 was released from both BM- and non-BM-derived cells. Non-BM TNFR1 expression influenced the sensitivity of mice to LPS challenge but not the level of serum sTNFR2. These results provide the first in vivo evidence for the origin and regulation of sTNFR2 in serum and could aid in the development of novel anti-TNF strategies against septic shock.