LTßR signalling preferentially accelerates oncogenic AKT-initiated liver tumours.

OBJECTIVES: The relative contributions of inflammatory signalling and sequential oncogenic dysregulation driving liver cancer pathogenesis remain incompletely understood. Lymphotoxin-ß receptor (LTßR) signalling is critically involved in hepatitis and liver tumorigenesis. Therefore, we explored the interdependence of inflammatory lymphotoxin signalling and specific oncogenic pathways in the progression of hepatic cancer. DESIGN: Pathologically distinct liver tumours were initiated by hydrodynamic transfection of oncogenic V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT)/ß-catenin or AKT/Notch expressing plasmids. To investigate the relationship of LTßR signalling and specific oncogenic pathways, LTßR antagonist (LTßR-Fc) or agonist (anti-LTßR) were administered post oncogene transfection. Initiated livers/tumours were investigated for changes in oncogene expression, tumour proliferation, progression, latency and pathology. Moreover, specific LTßR-mediated molecular events were investigated in human liver cancer cell lines and through transcriptional analyses of samples from patients with intrahepatic cholangiocarcinoma (ICC). RESULTS: AKT/ß-catenin-transfected livers displayed increased expression of LTß and LTßR, with antagonism of LTßR signalling reducing tumour progression and enhancing survival. Conversely, enforced LTßR-activation of AKT/ß-catenin-initiated tumours induced robust increases in proliferation and progression of hepatic tumour phenotypes in an AKT-dependent manner. LTßR-activation also rapidly accelerated ICC progression initiated by AKT/Notch, but not Notch alone. Moreover, LTßR-accelerated development coincides with increases of Notch, Hes1, c-MYC, pAKT and ß-catenin. We further demonstrate LTßR signalling in human liver cancer cell lines to be a regulator of Notch, pAKTser473 and ß-catenin. Transcriptome analysis of samples from patients with ICC links increased LTßR network expression with poor patient survival, increased Notch1 expression and Notch and AKT/PI3K signalling. CONCLUSIONS: Our findings link LTßR and oncogenic AKT signalling in the development of ICC.

Macrophage migration inhibitory factor induces epithelial to mesenchymal transition, enhances tumor aggressiveness and predicts clinical outcome in resected pancreatic ductal adenocarcinoma.

MIF is a proinflammatory cytokine and is implicated in cancer. A higher MIF level is found in many human cancer and cancer-prone inflammatory diseases, including chronic pancreatitis and pancreatic cancer. We tested the hypothesis that MIF contributes to pancreatic cancer aggressiveness and predicts disease outcome in resected cases. Consistent with our hypothesis we found that an elevated MIF mRNA expression in tumors was significantly associated with poor outcome in resected cases. Multivariate Cox-regression analysis further showed that MIF is independently associated with patients' survival (HR = 2.26, 95% CI = 1.17-4.37, p = 0.015). Mechanistic analyses revealed that MIF overexpression decreased E-cadherin and increased vimentin mRNA and protein levels in pancreatic cancer cell lines, consistent with the features of epithelial-to-mesenchymal transition (EMT). Furthermore, MIF-overexpression significantly increased ZEB1/2 and decreased miR-200b expression, while shRNA-mediated inhibition of MIF increased E-cadherin and miR-200b expression, and reduced the expression of ZEB1/2 in Panc1 cells. Re-expression of miR-200b in MIF overexpressing cells restored the epithelial characteristics, as indicated by an increase in E-cadherin and decrease in ZEB1/2 and vimentin expression. A reduced sensitivity to the chemotherapeutic drug, gemcitabine, occurred in MIF-overexpressing cells. Indicative of an increased malignant potential, MIF over-expressing cells showed significant increase in their invasion ability in vitro, and tumor growth and metastasis in an orthotopic xenograft mouse model. These results support a role of MIF in disease aggressiveness, indicating its potential usefulness as a candidate target for designing improved treatment in pancreatic cancer.

Expression of costimulatory TNFR2 induces resistance of CD4+FoxP3- conventional T cells to suppression by CD4+FoxP3+ regulatory T cells.

Our previous study showed that TNFR2 is preferentially expressed by CD4(+)FoxP3(+) regulatory T cells (Tregs), and expression of this receptor identified maximally suppressive Tregs. TNFR2 is also expressed by a small fraction of CD4(+)FoxP3(-) conventional T cells (Tconvs) in normal mice, and its expression is upregulated by T cell activation. This raises questions about the role of TNFR2 signaling in the function of Tconv cells. In this study, by using FoxP3/gfp knock-in mice, we showed that TNFR2 signaling did not induce FoxP3(-) CD4 cells to become suppressive. Ki-67, a marker of proliferation, was concomitantly expressed with TNFR2 by CD4 cells, independent of forkhead box P3 expression, in normal mice and Lewis lung carcinoma-bearing mice. TNFR2 is associated with greater suppressive functions when expressed by Tregs and is associated with greater resistance to suppression when expressed by Tconv cells. In mice bearing 4T1 breast tumor or Lewis lung carcinoma, intratumoral Tconv cells expressing elevated levels of TNFR2 acquired the capacity to resist suppression by lymph node-derived Tregs. However, they remained susceptible to inhibition by more suppressive tumor-infiltrating Tregs, which expressed higher levels of TNFR2. Our data indicate that TNFR2 also costimulates Tconv cells. However, intratumoral Tregs expressing more TNFR2 are able to overcome the greater resistance to suppression of intratumoral Tconv cells, resulting in a dominant immunosuppressive tumor environment.

Co-expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood.

Previously, we found that co-expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3(+) cells present in CD25(high), CD25(low) and even CD25(-) subsets of CD4(+) cells expressed high levels of TNFR2. Consequently, TNFR2-expressing CD4(+)CD25(+) Treg included all of the FOXP3(+) cells present in the CD4(+)CD25(high) subset as well as a substantial proportion of the FOXP3(+) cells present in the CD4(+)CD25(low) subset. Flow cytometric analysis of PB identified five-fold more Treg, determined by FOXP3 expression, in the CD4(+)CD25(+)TNFR2(+) subset than in the CD4(+)CD25(high) subset. In addition, similar levels of FOXP3(+) cells were identified in both the CD4(+)CD25(+)TNFR2(+) and CD4(+)CD25(+)CD127(low/-) subsets. Furthermore, the CD4(+)CD25(+)TNFR2(+) subset expressed high levels of CTLA-4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4(+)CD25(+)TNFR2(+) cells were anergic and markedly inhibited the proliferation and cytokine production of co-cultured T-responder cells. In contrast, CD4(+)CD25(+)TNFR2(-) and CD4(+)CD25(-)TNFR2(+) T cells did not show inhibitory activity. As some non-Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.

Tumor-Infiltrating Myeloid Cells Co-Express TREM1 and TREM2 and Elevated TREM-1 Associates With Disease Progression in Renal Cell Carcinoma.

Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) contribute to cancer-related inflammation and tumor progression. While several myeloid molecules have been ascribed a regulatory function in these processes, the triggering receptors expressed on myeloid cells (TREMs) have emerged as potent modulators of the innate immune response. While various TREMs amplify inflammation, others dampen it and are emerging as important players in modulating tumor progression-for instance, soluble TREM-1 (sTREM-1), which is detected during inflammation, associates with disease progression, while TREM-2 expression is associated with tumor-promoting macrophages. We hypothesized that TREM-1 and TREM-2 might be co-expressed on tumor-infiltrating myeloid cells and that elevated sTREM-1 associates with disease outcomes, thus representing a possibility for mutual modulation in cancer. Using the 4T1 breast cancer model, we found TREM-1 and TREM-2 expression on MDSC and TAM and that sTREM-1 was elevated in tumor-bearing mice in multiple models and correlated with tumor volume. While TREM-1 engagement enhanced TNF, a TREM-2 ligand was detected on MDSC and TAM, suggesting that both TREM could be functional in the tumor setting. Similarly, we detected TREM-1 and Trem2 expression in myeloid cells in the RENCA model of renal cell carcinoma (RCC). We confirmed these findings in human disease by demonstrating the expression of TREM-1 on tumor-infiltrating myeloid cells from patients with RCC and finding that sTREM-1 was increased in patients with RCC. Finally, The Cancer Genome Atlas analysis shows that TREM1 expression in tumors correlates with poor outcomes in RCC. Taken together, our data suggest that manipulation of the TREM-1/TREM-2 balance in tumors may be a novel means to modulate tumor-infiltrating myeloid cell phenotype and function.

Nitric oxide, a mediator of inflammation, suppresses tumorigenesis.

Inflammation influences the development of cancer. The nitric oxide synthase (NOS2) is induced by inflammatory cytokines, e.g., tumor necrosis factor alpha and interleukin 1beta, and produces nitric oxide (NO*), a critical mediator of the inflammatory response. Because p53 governs NO* production by transcriptionally transrepressing NOS2, we used a genetic strategy to determine whether NO* and p53 cooperatively regulate tumorigenesis. Lymphomas developed more rapidly in p53-/-NOS2-/- or p53-/-NOS2+/- mice than in p53-/-NOS2+/+ mice that were cross-bred into a >95% C57BL6 background and maintained in a pathogen-free condition. Likewise, sarcomas and lymphomas developed faster in p53+/-NOS2-/- or p53+/-NOS2+/- than in p53+/-NOS2+/+ mice. When compared with the double knockout mice, p53-/-NOS2+/+ mice showed a higher apoptotic index and a decreased proliferation index with an increased expression of death receptor ligands, CD95-L and tumor necrosis factor-related apoptosis-inducing ligand, and the cell cycle checkpoint protein, p21(waf1), in the spleen and thymus before tumor development. Furthermore, mice deficient in both p53 and NOS2 produced a high level of anti-inflammatory interleukin 10 when compared with p53-deficient mice. These studies provide genetic and mechanistic evidence that NO* can suppress tumorigenesis.

Systemic IL-12 administration alters hepatic dendritic cell stimulation capabilities.

The liver is an immunologically unique organ containing tolerogenic dendritic cells (DC) that maintain an immunosuppressive microenvironment. Although systemic IL-12 administration can improve responses to tumors, the effects of IL-12-based treatments on DC, in particular hepatic DC, remain incompletely understood. In this study, we demonstrate systemic IL-12 administration induces a 2-3 fold increase in conventional, but not plasmacytoid, DC subsets in the liver. Following IL-12 administration, hepatic DC became more phenotypically and functionally mature, resembling the function of splenic DC, but differed as compared to their splenic counterparts in the production of IL-12 following co-stimulation with toll-like receptor (TLR) agonists. Hepatic DCs from IL-12 treated mice acquired enhanced T cell proliferative capabilities similar to levels observed using splenic DCs. Furthermore, IL-12 administration preferentially increased hepatic T cell activation and IFNγ expression in the RENCA mouse model of renal cell carcinoma. Collectively, the data shows systemic IL-12 administration enables hepatic DCs to overcome at least some aspects of the inherently suppressive milieu of the hepatic environment that could have important implications for the design of IL-12-based immunotherapeutic strategies targeting hepatic malignancies and infections.

Cutting edge: expression of TNFR2 defines a maximally suppressive subset of mouse CD4+CD25+FoxP3+ T regulatory cells: applicability to tumor-infiltrating T regulatory cells.

TNFR2 is predominantly expressed by a subset of human and mouse CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs). In this study, we characterized the phenotype and function of TNFR2(+) Tregs in peripheral lymphoid tissues of normal and tumor-bearing C57BL/6 mice. We found that TNFR2 was expressed on 30-40% of the Tregs of the peripheral activated/memory subset that were most highly suppressive. In contrast, TNFR2(-) Tregs exhibited the phenotype of naive cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4(+)CD25(-)TNFR2(+) cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2(+) Tregs was considerably more potent than that of reportedly highly suppressive CD103(+) Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2(+) Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity that may account for tumor-infiltrating lymphocyte-mediated immune evasion by tumors.

Stimulation through CD40 on mouse and human renal cell carcinomas triggers cytokine production, leukocyte recruitment, and antitumor responses that can be independent of host CD40 expression.

CD40, a member of the TNFR superfamily, is expressed on a variety of host immune cells, as well as some tumors. In this study, we show that stimulation of CD40 expressed on both mouse and human renal carcinoma cells (RCCs) triggers biological effects in vitro and in vivo. Treatment of the CD40+ Renca mouse RCC tumor cells in vitro with an agonistic anti-CD40 Ab induced strong expression of the genes and proteins for GM-CSF and MCP-1, and induced potent chemotactic activity. Similarly, administration of alphaCD40 to both wild-type and CD40-/- mice bearing Renca tumors resulted in substantial amounts of TNF-alpha and MCP-1 in the serum, increased the number of total splenocytes and MHC class II+ CD11c+ leukocytes, and when combined with IFN-gamma, inhibited the progression of established Renca tumors in vivo in both wild-type and CD40-/- mice. Similarly, treatment of CD40+ A704 and ACHN human RCC lines with mouse anti-human CD40 Ab induced strong expression of genes and proteins for MCP-1, IL-8, and GM-CSF in vitro and in vivo. Finally, in SCID mice, the numbers of ACHN pulmonary metastases were dramatically reduced by treatment with species-specific human CD40 Ab. These results show that CD40 stimulation of CD40+ tumor cells can enhance immune responses and result in antitumor activity.

Multiprobe ribonuclease protection assay for simultaneous measurement of mRNA expression.

The multiprobe RNase protection assay enables investigators to monitor RNA expression of 8-12 genes with as little as 1 microg of total RNA. The commercial availability of numerous multi-gene template sets makes this assay practical for all basic research programs.

Synergistic engagement of an ineffective endogenous anti-tumor immune response and induction of IFN-gamma and Fas-ligand-dependent tumor eradication by combined administration of IL-18 and IL-2.

IFN-gamma is a critical component of the endogenous and many cytokine-induced antitumor immune responses. In this study we have shown that the combination of IL-18 and IL-2 (IL-18/IL-2) synergistically enhances IFN-gamma production both in vitro and in vivo, and synergizes in vivo to induce complete durable regression of well-established 3LL tumors in >80% of treated mice. We have observed a nascent, but ineffective, host immune response against 3LL that depends on endogenous IFN-gamma and IL-12 production and the Fas/Fas ligand (Fas-L) pathway. The combined administration of IL-18/IL-2 engages this endogenous response to induce tumor regression via a mechanism that is independent of NK and NKT cells or IL-12, but is critically dependent on CD8(+) T cells, IFN-gamma, and the Fas/Fas-L pathway. These studies demonstrate the importance of IFN-gamma as well as the Fas/Fas-L pathway in both endogenous and cytokine-driven antitumor immune responses engaged by IL-18/IL-2 and provide preclinical impetus for clinical investigation of this potent anti-tumor combination.

Activation of cutaneous protein kinase C alpha induces keratinocyte apoptosis and intraepidermal inflammation by independent signaling pathways.

Skin keratinocytes are major mediators of host immune responses. The skin is also a target for immunologically based inflammation in many pathological states. Activation of protein kinase C (PKC) can induce cutaneous inflammation, but the precise role of each of six cutaneous PKC isoforms (alpha, delta, epsilon, eta, zeta, mu) that regulate normal skin homeostasis or contribute to skin pathology has not been clarified. We generated transgenic mice that overexpress PKCalpha in the basal layer of the epidermis and the outer root sheath of hair follicles under the regulation of the bovine keratin 5 promoter. K5-PKCalpha transgenic mice exhibit severe intraepidermal neutrophilic inflammation and disruption of the epidermis and upper hair follicles when treated topically with 12-O-tetradecanoylphorbol-13-acetate (TPA). Both TPA and UVB cause apoptosis in transgenic skin, but only TPA evokes intraepidermal inflammation. TPA also induces apoptosis in cultured transgenic keratinocytes, and this is prevented by an AP-1 dominant-negative construct. However, inhibiting AP-1 in vivo does not abrogate intraepidermal inflammation. Transcripts for specific cytokines and chemokines are elevated in TPA-treated cultured transgenic keratinocytes, and conditioned culture medium from these cells promotes neutrophil migration in vitro. Chemokine expression and neutrophil migration are not diminished by inhibiting AP-1. Thus, PKCalpha activation induces keratinocyte apoptosis via an AP-1-dependent pathway and mediates chemokine induction and intraepidermal inflammation independently. This model system will be useful to define specific chemokines regulated by PKCalpha that promote intraepidermal neutrophilic inflammation, a condition that characterizes several human cutaneous diseases such as pustular psoriasis and acute generalized exanthematous pustulosis.