IL‐17A‐producing CD30+ Vδ1 T cells drive inflammation‐induced cancer progression

Although it has been suspected that inflammation is associated with increased tumor metastasis, the exact type of immune response required to initiate cancer progression and metastasis remains unknown. In this study, by using an in vivo tumor progression model in which low tumorigenic cancer cells acquire malignant metastatic phenotype after exposure to inflammation, we found that IL‐17A is a critical cue for escalating cancer cell malignancy. We further demonstrated that the length of exposure to an inflammatory microenvironment could be associated with acquiring greater tumorigenicity and that IL‐17A was critical for amplifying such local inflammation, as observed in the production of IL‐1β and neutrophil infiltration following the cross‐talk between cancer and host stromal cells. We further determined that γδT cells expressing Vδ1 semi‐invariant TCR initiate cancer‐promoting inflammation by producing IL‐17A in an MyD88/IL‐23‐dependent manner. Finally, we identified CD30 as a key molecule in the inflammatory function of Vδ1T cells and the blockade of this pathway targeted this cancer immune‐escalation process. Collectively, these results reveal the importance of IL‐17A‐producing CD30⁺ Vδ1T cells in triggering inflammation and orchestrating a microenvironment leading to cancer progression.     

Prognostic roles for IL‐2‐producing and CD69+ T cell subsets in colorectal cancer patients

Tumor infiltrating T cells are a predictor of patient outcome in patients with colorectal cancer (CRC). However, many T cell populations have been associated with both poor and positive patient prognoses, indicating a need to further understand the role of different T cell subsets in CRC. In this study, the T cell infiltrate from the tumor and nontumor bowel (NTB) was examined in 95 CRC patients using flow cytometry and associations with cancer stage and disease recurrence made. Our findings showed that IFN‐γ‐producing T cells were associated with positive patient outcomes, and CD69⁺ T cells were associated with disease recurrence. Inflammatory (IL‐17) and regulatory T cells were not associated with disease recurrence. Surprisingly, in a second cohort of 32 patients with long‐term clinical follow up data, tumor infiltrating IL‐2‐producing T cells correlated negatively with disease free survival (DFS) and a higher frequency of IL‐2‐producing T cells was found in the NTB of patients with poorly differentiated tumors. These results point toward the possibility of a negative impact of IL‐2 in tumor immune responses, which may influence future immunotherapy treatments in CRC patients.     

Overexpression of NLRP3, NLRC4 and AIM2 inflammasomes and their priming‐associated molecules (TLR2, TLR4, Dectin‐1, Dectin‐2 and NFκB) in Malassezia folliculitis

The activation of NLRP3, NLRC4 and AIM2 inflammasomes is pivotal for innate immunity against some pathogenic fungi, but their role in the pathogenesis of Malassezia folliculitis (MF) remains unclear. The objective of the study was to determine the expression of 4 canonical inflammasomes (NLRP1, NLRP3, NLRC4 and AIM2) and their priming‐associated molecules (TLR2, TLR4, Dectin‐1, Dectin‐2 and NFκB) in MF lesion. Expression of NLRP1, NLRP3, NLRC4, AIM2, caspase‐1, IL‐1β, TLR2, TLR4, Dectin‐1, Dectin‐2 and NFκB was detected by immunohistochemistry in skin lesion of 23 MF patients and normal skin of 12 healthy subjects. Furthermore, NLRP1, NLRP3, NLRC4, AIM2, caspase‐1 and IL‐1β mRNA was measured by quantitative real‐time PCR (qRT‐PCR) in 12 MF cases and 10 controls. Immunohistochemical analysis revealed that NLRP3, NLRC4, AIM2, Casp‐1, IL‐1β, TLR2, TLR4, Dectin‐1, Dectin‐2 and NFκB expression was up‐regulated in the epidermis and dermal inflammatory cells of MF lesion compared with control skin (P < .01‐.05), but NLRP1 expression was not different between both groups (P > .05). qRT‐PCR showed that levels of NLRP3, Casp‐1 and IL‐1β mRNA were significantly increased (P < .01‐.05), whereas those of NLRP1, NLRC4 and AIM2 mRNA were slightly augmented compared to control skin (P > .05). Our observation suggests that simultaneous activation of NLRP3, NLRC4 and AIM2 inflammasomes may play an important role in the pathogenesis of MF.     

CXCR2‐mediated tumor‐associated neutrophil recruitment is regulated by IFN‐β

The chemokine receptor CXCR2 and its ligands CXCL1, CXCL2 and CXCL5 play an important role in homing of tumor‐associated neutrophils (TANs) into developing tumors. TANs are known to support the development of blood vessels in growing solid tumors, hence contributing to tumor growth. Here, we show that the migration of neutrophils is influenced by endogenous interferon‐beta (IFN‐β) via regulation of such chemokines and their receptor. We could demonstrate that CXCL1 and CXCL2 gradients are formed in tumor‐bearing mice, i.e., low chemokine level in bone marrow (BM) and high level in the tumor. This supports migration of neutrophils into the tumor. Moreover, expression of CXCR2 was highest on neutrophils from BM and lowest in TANs. Importantly, although IFN‐β appears to have only a minor influence on the expression of CXCR2, it strongly regulates the CXCR2 ligands. In the absence of endogenous IFN‐β, they were expressed significantly higher in tumor‐infiltrating neutrophils. Treatment of such neutrophils from tumor‐bearing Ifnb1^?/? mice with recombinant IFN‐β downregulated CXCR2 ligand expression to wild‐type levels. This explains the reduced migration of neutrophils into tumors and the diminished tumor angiogenesis in IFN‐β‐sufficient mice. Our results add a novel functional aspect of the type I IFN system as effector molecules of natural cancer surveillance and open interesting possibilities for antineutrophil therapies against cancer.     

TNF‐α enhances TGF‐β‐induced endothelial‐to‐mesenchymal transition via TGF‐β signal augmentation

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer‐associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF‐β, which induces endothelial‐to‐mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor‐infiltrating inflammatory cells secrete various cytokines, including TNF‐α. However, the role of TNF‐α in TGF‐β‐induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF‐α on TGF‐β‐induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF‐β and TNF‐α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF‐β and TNF‐α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF‐β type I receptor, TGF‐β2, activin A, and integrin αv, suggesting that TNF‐α enhanced TGF‐β‐induced EndMT by augmenting TGF‐β family signals. Furthermore, oral squamous cell carcinoma‐derived cells underwent epithelial‐to‐mesenchymal transition (EMT) in response to humoral factors produced by TGF‐β and TNF‐α‐cultured ECs. This EndMT‐driven EMT was blocked by inhibiting the action of TGF‐βs. Collectively, our findings suggest that TNF‐α enhances TGF‐β‐dependent EndMT, which contributes to tumor progression.     

Repression of NF‐κB and activation of AP‐1 enhance apoptosis in prostate cancer cells

TNFα and TRAIL, 2 members of the tumor necrosis factor family, share many common signaling pathways to induce apoptosis. Although many cancer cells are sensitive to these proapoptotic agents, some develop resistance. Recently, we have demonstrated that upregulation of c‐Fos/AP‐1 is necessary, but insufficient for cancer cells to undergo TRAIL‐induced apoptosis. Here we present a prostate cancer model with differential sensitivity to TNFα and TRAIL. We show that inhibition of NF‐κB or activation of AP‐1 can only partially sensitize resistant prostate cancer cells to proapoptotic effects of TNFα or TRAIL. Inhibition of NF‐κB by silencing TRAF2, by silencing RIP or by ectopic expression of IκB partially sensitized resistant prostate cancer. Similarly, activation of c‐Fos/AP‐1 only partially sensitized resistant cancer cells to proapoptotic effects of TNFα or TRAIL. However, concomitant repression of NF‐κB and activation of c‐Fos/AP‐1 significantly enhanced the proapoptotic effects of TNFα and TRAIL in resistant prostate cancer cells. Therefore, multiple molecular pathways may need to be modified, to overcome cancers that are resistant to proapoptotic therapies. © 2008 Wiley‐Liss, Inc.     

Blockade of TGF‐β enhances tumor vaccine efficacy mediated by CD8+ T cells

Though TGF‐β inhibition enhances antitumor immunity mediated by CD8⁺ T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF‐β blockade, we tested the effect of anti‐TGF‐β combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti‐TGF‐β antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti‐TGF‐β antibodies compared to vaccinated mice without anti‐TGF‐β, suggesting that anti‐TGF‐β synergistically enhanced irradiated tumor vaccine efficacy. CD8⁺ T‐cell depletion completely abrogated the vaccine efficacy, and so protection required CD8⁺ T cells. Depletion of CD25⁺ T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti‐TGF‐β did not change the number of CD25⁺ T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d‐restricted NKT cells, which have been reported to induce TGF‐β production by MDSC through an IL‐13‐IL‐4R‐STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti‐TGF‐β enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF‐β levels found in patients with cancer and that the effect is not dependent on TGF‐β solely from CD4⁺CD25⁺ T regulatory cells or the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway.     

Tumor necrosis factor receptor modulator spermatogenesis‐associated protein 2 is a novel predictor of outcome in ovarian cancer

Inflammation plays a crucial role in the pathogenesis of cancer with tumor necrosis factor‐α (TNF‐α) as a key mediator. Recently, spermatogenesis‐associated protein 2 (SPATA2) was identified as a TNF receptor modulator which is required for TNF‐induced inflammation and apoptosis. The available data on TNF‐α in ovarian cancer (OC) are inconsistent, and SPATA2 is completely uncharacterized in tumorigenesis. We analyzed expression of SPATA2 and TNFA by quantitative real‐time polymerase chain reaction in tissues of 171 patients with low‐grade serous (LGSOC), high‐grade serous (HGSOC), endometrioid and clear cell OC compared with 28 non‐malignant control tissues. We stimulated OC cells (OVCAR3) with pro‐inflammatory (TNF‐α, interleukin [IL]‐1β) and mitogenic stimuli (IL‐6, lysophosphatidic acid) to establish a direct effect between inflammatory signaling and SPATA2. Pro‐inflammatory, but not mitogenic stimuli, potently induced SPATA2 expression in OC cells. Expression of TNFA and SPATA2 was higher in OC compared with control tissues (P = 0.010 and P = 0.001, respectively) and correlated with each other (P = 0.034, r_s = 0.198). When compared with grade 1 cancers, SPATA2 was expressed higher in grade 2 and 3 tumors (P = 0.011) as well as in HGSOC compared with LGSOC (P = 0.024). Multivariate survival analyses revealed that OC with high SPATA2 expression were associated with reduced progression‐free survival (P = 0.048) and overall survival (P < 0.001). In conclusion, SPATA2 expression is regulated by TNF‐α and IL‐1β and is found to independently affect clinical outcome in OC patients. These data implicate a role of SPATA2 in tumorigenesis which warrants further investigation in gynecological malignancies.     

Tumor‐associated macrophage‐derived inflammatory cytokine enhances malignant potential of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an asbestos‐related aggressive malignant neoplasm. Due to the difficulty of achieving curative surgical resection in most patients with MPM, a combination chemotherapy of cisplatin and pemetrexed has been the only approved regimen proven to improve the prognosis of MPM. However, the median overall survival time is at most 12 mo even with this regimen. There has been therefore a pressing need to develop a novel chemotherapeutic strategy to bring about a better outcome for MPM. We found that expression of interleukin‐1 receptor (IL‐1R) was upregulated in MPM cells compared with normal mesothelial cells. We also investigated the biological significance of the interaction between pro‐inflammatory cytokine IL‐1β and the IL‐1R in MPM cells. Stimulation by IL‐1β promoted MPM cells to form spheroids along with upregulating a cancer stem cell marker CD26. We also identified tumor‐associated macrophages (TAMs) as the major source of IL‐1β in the MPM microenvironment. Both high mobility group box 1 derived from MPM cells and the asbestos‐activated inflammasome in TAMs induced the production of IL‐1β, which resulted in enhancement of the malignant potential of MPM. We further performed immunohistochemical analysis using clinical MPM samples obtained from patients who were treated with the combination of platinum plus pemetrexed, and found that the overexpression of IL‐1R tended to correlate with poor overall survival. In conclusion, the interaction between MPM cells and TAMs through a IL‐1β/IL‐1R signal could be a promising candidate as the target for novel treatment of MPM (Hyogo College of Medicine clinical trial registration number: 2973).     

Liver specific overexpression of platelet‐derived growth factor‐B accelerates liver cancer development in chemically induced liver carcinogenesis

A genetic basis of hepatocellular carcinoma (HCC) has been well‐established and major signaling pathways, such as p53, Wnt‐signaling, transforming growth factor‐β (TGF‐β) and Ras pathways, have been identified to be essential to HCC development. Lately, the family of platelet‐derived growth factors (PDGFs) has shifted to the center of interest. We have reported on spontaneously developing liver fibrosis in PDGF‐B transgenic mice. Since HCC rarely occurs in healthy liver, but dramatically increases at the cirrhosis stage of which liver fibrosis is a preliminary stage, we investigated liver cancer development in chemically induced liver carcinogenesis in these mice. HCC induction was performed by treatment of the mice with diethylnitrosamine and phenobarbital. At an age of 6 months, the tumor development of these animals was analyzed. Not only the development of dysplastic lesions in PDGF‐B transgenic mice was significantly increased but also their malignant transformation to HCC. Furthermore, we were able to establish a key role of PDGF‐B signaling at diverse stages of liver cancer development. Here, we show that development of liver fibrosis is likely through upregulation of TGF‐β receptors by PDGF‐B. Additionally, overexpression of PDGF‐B also leads to an increased expression of β‐catenin as well as vascular endothelial growth factor and platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31), all factors with established roles in carcinogenesis. We were able to extend the understanding of key genetic regulators in HCC development by PDGF‐B and decode essential downstream signals.     

Immune checkpoint blockade combined with IL‐6 and TGF‐β inhibition improves the therapeutic outcome of mRNA‐based immunotherapy

Improved understanding of cancer immunology has provided insight into the phenomenon of frequent tumor recurrence after initially successful immunotherapy. A delicate balance exists between the capacity of the immune system to control tumor growth and various resistance mechanisms that arise to avoid or even counteract the host's immune system. These resistance mechanisms include but are not limited to (i) adaptive expression of inhibitory checkpoint molecules in response to the proinflammatory environment and (ii) amplification of cancer stem cells, a small fraction of tumor cells possessing the capacity for self‐renewal and mediating treatment resistance and formation of metastases after long periods of clinical remission. Several individual therapeutic agents have so far been developed to revert T‐cell exhaustion or disrupt the cross‐talk between cancer stem cells and the tumor‐promoting microenvironment. Here, we demonstrate that a three‐arm combination therapy—consisting of an mRNA‐based vaccine to induce antigen‐specific T‐cell responses, monoclonal antibodies blocking inhibitory checkpoint molecules (PD‐1, TIM‐3, LAG‐3), and antibodies targeting IL‐6 and TGF‐β—improves the therapeutic outcome in subcutaneous TC‐1 tumors and significantly prolongs survival of treated mice. Our findings point to a need for a rational development of multidimensional anticancer therapies, aiming at the induction of tumor‐specific immunity and simultaneously targeting multiple resistance mechanisms.     

Modulation of TGF‐β activity by latent TGF‐β‐binding protein 1 in human malignant glioma cells

High biological activity of the transforming growth factor (TGF)‐β‐Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF‐β has become a novel target for the experimental treatment of these tumors. TGF‐β is processed by furin‐like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency‐associated peptide (LAP). Latent TGF‐β‐binding protein 1 (LTBP‐1) covalently binds to this small latent TGF‐β complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP‐1 protein in vivo increase with the grade of malignancy in gliomas. LTBP‐1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP‐1 into the medium is decreased by the inhibition of FLP activity. Gene‐transfer mediated overexpression of LTBP‐1 in glioma cell lines results in an increase inTGF‐β activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF‐β activity is enhanced. Conversely, LTBP‐1 gene silencing reduces TGF‐β activity and Smad2 phosphorylation without affecting TGF‐β protein levels. Collectively, we identify LTBP‐1 as an important modulator of TGF‐β activation in glioma cells, which may contribute to the malignant phenotype of these tumors. © 2009 UICC     

C/EBPβ promotes angiogenesis through secretion of IL‐6, which is inhibited by genistein,in EGFRvIII‐positive glioblastoma

To study the mechanisms underlying the IL‐6‐promoted angiogenic microenvironment in EGFRvIII‐positive glioblastoma, VEGF expression in EGFRvIII‐positive/negative tumors was determined by optical molecular imaging. Next, the HUVEC tube formation assay, Western blot, qPCR, RNA silencing, chromatin immunoprecipitation, luciferase reporter and ELISA assays were performed to examine the role of IL‐6 and C/EBPβ in the formation of the angiogenic microenvironment in EGFRvIII‐positive tumors. Finally, in vitro and in vivo genistein treatment experiments were conducted to challenge the interaction between the IL‐6 promoter and C/EBPβ. Optical imaging revealed greater VEGF expression in EGFRvIII‐positive tumor‐bearing mice, suggesting an angiogenic microenvironment. In vitro experiments demonstrated that C/EBPβ‐mediated regulation of IL‐6 was indispensable for maintenance of this angiogenic microenvironment. In contrast, genistein‐mediated upregulation of CHOP impeded C/EBPβ interaction with the IL‐6 promoter, thus disturbing the angiogenic microenvironment. This more malignant microenvironment in EGFRvIII glioblastoma is generated, at least in part, by greater VEGF, IL‐6 and C/EBPβ expression. Interaction of C/EBPβ with the IL‐6 promoter maintains this angiogenic microenvironment, while disturbance of this dynamically balanced interaction inhibits EGFRvIII tumor proliferation by reducing both VEGF and IL‐6 expression.     

Rho GDP‐dissociation inhibitor α is a potential prognostic biomarker and controls telomere regulation in colorectal cancer

Rho GDP‐dissociation inhibitor α (RhoGDIα) is an essential regulator for Rho GTPases. Although RhoGDIα may serve as an oncogene in colorectal cancer (CRC), the underlying mechanism is still unclear. We investigated the function, mechanism, and clinical significance of RhoGDIα in CRC progression. We founded that downregulation of RhoGDIα repressed CRC cell proliferation, motility, and invasion. Overexpression of RhoGDIα increased DNA damage response signals at telomeres, and led to telomere shortening in CRC cells, also being validated in 26 pairs of CRC tissues. Mechanistic studies revealed that RhoGDIα could promote telomeric repeat factor 1 (TRF1) expression through the phosphatidylinositol 3‐kinase–protein kinase B signal pathway. Moreover, RhoGDIα protein levels were strongly correlated with TRF1 in CRC tissues. A cohort of 297 CRC samples validated the positive relationship between RhoGDIα and TRF1, and revealed that RhoGDIα and TRF1 levels were negatively associated with CRC patients' survival. Taken together, our results suggest that RhoGDIα regulate TRF1 and telomere length and may be novel prognostic biomarkers in colorectal cancer.     

Glycogen synthase kinase‐3β is a pivotal mediator of cancer invasion and resistance to therapy

Tumor cell invasion and resistance to therapy are the most intractable biological characteristics of cancer and, therefore, the most challenging for current cancer research and treatment paradigms. Refractory cancers, including pancreatic cancer and glioblastoma, show an inextricable association between the highly invasive behavior of tumor cells and their resistance to chemotherapy, radiotherapy and targeted therapies. These aggressive properties of cancer share distinct cellular pathways that are connected to each other by several molecular hubs. There is increasing evidence to show that glycogen synthase kinase (GSK)‐3β is aberrantly activated in various cancer types and this has emerged as a potential therapeutic target. In many but not all cancer types, aberrant GSK3β sustains the survival, immortalization, proliferation and invasion of tumor cells, while also rendering them insensitive or resistant to chemotherapeutic agents and radiation. Here we review studies that describe associations between therapeutic stimuli/resistance and the induction of pro‐invasive phenotypes in various cancer types. Such cancers are largely responsive to treatment that targets GSK3β. This review focuses on the role of GSK3β as a molecular hub that connects pathways responsible for tumor invasion and resistance to therapy, thus highlighting its potential as a major cancer therapeutic target. We also discuss the putative involvement of GSK3β in determining tumor cell stemness that underpins both tumor invasion and therapy resistance, leading to intractable and refractory cancer with dismal patient outcomes.     

PD‐L1 expression enhancement by infiltrating macrophage‐derived tumor necrosis factor‐α leads to poor pancreatic cancer prognosis

Immunotherapy using anti‐PD‐1/PD‐L1 antibodies for several types of cancer has received considerable attention in recent decades. However, the molecular mechanism underlying PD‐L1 expression in pancreatic ductal adenocarcinoma (PDAC) cells has not been clearly elucidated. We investigated the clinical significance and regulatory mechanism of PD‐L1 expression in PDAC cells. Among the various cytokines tested, tumor necrosis factor (TNF)‐α upregulated PD‐L1 expression in PDAC cells through NF‐κB signaling. The induction of PD‐L1 expression was also caused by co‐culture with activated macrophages, and the upregulation was inhibited by neutralization with anti‐TNF‐α antibody after co‐culture with activated macrophages. PD‐L1 expression in PDAC cells was positively correlated with macrophage infiltration in tumor stroma of human PDAC tissues. In addition, survival analysis revealed that high PD‐L1 expression was significantly associated with poor prognosis in 235 PDAC patients and especially in patients harboring high CD8‐positive T‐cell infiltration. These findings indicate that tumor‐infiltrating macrophage‐derived TNF‐α could be a potential therapeutic target for PDAC.