Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity

The immune system can act as an extrinsic suppressor of tumors. Therefore, tumor progression depends in part on mechanisms that downmodulate intrinsic immune surveillance. Identifying these inhibitory pathways may provide promising targets to enhance antitumor immunity. Here, we show that Stat3 is constitutively activated in diverse tumor-infiltrating immune cells, and ablating Stat3 in hematopoietic cells triggers an intrinsic immune-surveillance system that inhibits tumor growth and metastasis. We observed a markedly enhanced function of dendritic cells, T cells, natural killer (NK) cells and neutrophils in tumor-bearing mice with Stat3(-/-) hematopoietic cells, and showed that tumor regression requires immune cells. Targeting Stat3 with a small-molecule drug induces T cell- and NK cell-dependent growth inhibition of established tumors otherwise resistant to direct killing by the inhibitor. Our findings show that Stat3 signaling restrains natural tumor immune surveillance and that inhibiting hematopoietic Stat3 in tumor-bearing hosts elicits multicomponent therapeutic antitumor immunity.     

Stat3 activity in melanoma cells affects migration of immune effector cells and nitric oxide-mediated antitumor effects

Infiltration of immune effector cells in tumors is critical for antitumor immune responses. However, what regulates immune cell infiltration of tumors remains to be identified. Stat3 is constitutively activated with high frequency in diverse cancers, promoting tumor cell growth and survival. Blocking Stat3 signaling in tumors in vivo results in tumor growth inhibition that involves killing of nontransfected tumor cells and infiltration of immune effector cells, suggesting that Stat3 activity in tumor cells might affect immune cell recruitment. However, dying tumor cells can also attract immune cells. In this study, we show in isogenic murine melanomas that natural Stat3 activity is associated with tumor growth and reduction of T cell infiltration. Blocking Stat3 signaling in the melanoma cells containing high Stat3 activity results in expression of multiple chemoattractants, leading to increased migration of lymphocytes, NK cells, neutrophils, and macrophages. In addition, blocking Stat3 triggers tumor cells to produce soluble factors capable of activating macrophage production of NO in vitro and in vivo. TNF-alpha and IFN-beta, which are secreted by Stat3-inhibited tumor cells, are able to activate macrophage NO production, whereas neutralizing TNF-alpha in the tumor supernatant from Stat3-blocked tumor cells abrogates nitrite production. Moreover, interrupting Stat3 signaling in tumor cells leads to macrophage-mediated, nitrite-dependent cytostatic activity against nontransduced tumor cells. These results suggest that tumor Stat3 activity affects recruitment of diverse immune effectors and it can be manipulated to activate the effector phase of innate immune responses.     

Small interfering RNA targeted to IGF-IR delays tumor growth and induces proinflammatory cytokines in a mouse breast cancer model

Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2'-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2'-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines.     

A novel combination immunotherapy for cancer by IL-13Rα2-targeted DNA vaccine and immunotoxin in murine tumor models

Optimum efficacy of therapeutic cancer vaccines may require combinations that generate effective antitumor immune responses, as well as overcome immune evasion and tolerance mechanisms mediated by progressing tumor. Previous studies showed that IL-13Rα2, a unique tumor-associated Ag, is a promising target for cancer immunotherapy. A targeted cytotoxin composed of IL-13 and mutated Pseudomonas exotoxin induced specific killing of IL-13Rα2(+) tumor cells. When combined with IL-13Rα2 DNA cancer vaccine, surprisingly, it mediated synergistic antitumor effects on tumor growth and metastasis in established murine breast carcinoma and sarcoma tumor models. The mechanism of synergistic activity involved direct killing of tumor cells and cell-mediated immune responses, as well as elimination of myeloid-derived suppressor cells and, consequently, regulatory T cells. These novel results provide a strong rationale for combining immunotoxins with cancer vaccines for the treatment of patients with advanced cancer.     

In situ expression of IFN-γ-inducible T cell α chemoattractant in breast cancer mounts an enhanced specific anti-tumor immunity which leads to tumor regression

Increased evidence indicates that chemokines are involved in tumor growth. ITAC, a key member of chemokines, possesses the ability to recruit T cells and enhance immune responses. Therefore, ITAC might contribute to antitumor immunity. In this study, we evaluated the relationship between the expression of ITAC and human breast cancer advancement. We further investigated whether forced expression of ITAC in tumor sites could mediate enhanced antitumor immunity in a murine breast cancer model. Results showed that ITAC expression level was down-regulated in 31 breast cancer specimens compared to normal mammary tissues, and associated negatively with the stages of breast cancer. Contrarily, forced expression of ITAC in murine 4T1 tumor cells resulted in tumor regression after initial growth upon injection into naïve Balb/c mice. More lymphocytes were recruited to the site of tumor inoculated by 4T1-ITAC and more than 80% of these T cells expressed the ITAC receptor, CXCR3. ITAC-recruited TILs exhibited 4T1-specific proliferation and cytotoxicity, and an increased IFN-γ but decreased IL-4 production. Importantly, forced expression of ITAC in 4T1 tumor nodules inhibited tumor growth. These findings demonstrated that the decreased expression of ITAC is associated with the advancement of breast cancer in patients. Forced expression of ITAC in tumor site not only induces increased T cell-recruitment and elicits a specific antitumor immunity, but also mediates regression of established 4T1 tumors, indicating the potential application of ITAC-expressing tumor cells in cancer immunotherapy and vaccine designing.     

IL-2 immunotoxin therapy modulates tumor-associated regulatory T cells and leads to lasting immune-mediated rejection of breast cancers in neu-transgenic mice

Studies in cancer patients have suggested that breast tumors recruit regulatory T cells (Tregs) into the tumor microenvironment. The extent to which local Tregs suppress antitumor immunity in breast cancer is unknown. We questioned whether inhibiting systemic Tregs with an IL-2 immunotoxin in a model of neu-mediated breast cancer, the neu-transgenic mouse, could impact disease progression and survival. As in human breast cancer, cancers that develop in these mice attract Tregs into the tumor microenvironment to levels of approximately 10-25% of the total CD4+ T cells. To examine the role of Tregs in blocking immune-mediated rejection of tumor, we depleted CD4+CD25+ T cells with an IL-2 immunotoxin. The treatment depleted Tregs without concomitant lymphopenia and markedly inhibited tumor growth. Depletion of Tregs resulted in a persistent antitumor response that was maintained over a month after the last treatment. The clinical response was immune-mediated because adoptive transfer of Tregs led to a complete abrogation of the therapeutic effects of immunotoxin treatment. Further, Treg down-modulation was accompanied by increased Ag-specific immunity against the neu protein, a self Ag. These results suggest that Tregs play a major role in preventing an effective endogenous immune response against breast cancer and that depletion of Tregs, without any additional immunotherapy, may mediate a significant antitumor response.     

CYP3A4 mediates growth of estrogen receptor-positive breast cancer cells in part by inducing nuclear translocation of phospho-Stat3 through biosynthesis of (±)-14,15-epoxyeicosatrienoic acid (EET)

CYP3A4 expression in breast cancer correlates with decreased overall survival, but the mechanisms are unknown. Cytochrome P450 gene profiling by RNAi silencing demonstrates that CYP3A or 2C8 gene expression is specifically required for growth of the breast cancer lines MCF7, T47D, and MDA-MB-231. CYP3A4 silencing blocks the cell cycle at the G(2)/M checkpoint and induces apoptosis in the MCF7 line, thereby inhibiting anchorage-dependent growth and survival. CYP3A4 was profiled for NADPH-dependent arachidonic acid (AA) metabolism and synthesized AA epoxygenase products (±)-8,9-, (±)-11,12-, and (±)-14,15-epoxyeicosatrienoic acid (EET) (total turnover of ～2 pmol/pmol CYP3A4/min) but not hydroxylase products (±)-15-, (±)-19-, or 20-hydroxyeicosatetraenoic acid. Furthermore, eicosanoid profiling revealed that MCF7 cells synthesize EETs in a CYP3A4-dependent manner. The (±)-14,15-EET regioisomer selectively rescues breast cancer cells from CYP3A4 silencing in a concentration-dependent fashion and promotes mitogenesis and anchorage-dependent cloning. Stat3 (Tyr-705) phosphorylation was inhibited by CYP3A4 silencing, providing a potential mechanism for CYP3A4 involvement in breast cancer cell growth. Silencing Stat3 blocks breast cancer cell growth and abrogates (±)-14,15-EET-induced proliferation, indicating a Stat3 requirement for (±)-14,15-EET-mediated cell growth. Although silencing of CYP3A4 reduces nuclear Tyr(P)-705-Stat3, (±)-14,15-EET restores this signaling process and promotes Tyr(P)-705-Stat3 translocation to the nucleus, suggesting that (±)-14,15-EET may be involved in an autocrine/paracrine pathway driving cell growth. These studies indicate that CYP3A4 is a highly active AA epoxygenase that promotes Stat3-mediated breast cancer cell growth in part through (±)-14,15-EET biosynthesis. Furthermore, these studies indicate an essential role for Stat3 as a mediator of epoxygenase activity in breast cancer.     

Immunization with murine breast cancer cells treated with antisense oligodeoxynucleotides to type I insulin-like growth factor receptor induced an antitumoral effect mediated by a CD8+ response involving Fas/Fas ligand cytotoxic pathway

We have demonstrated that in vivo administration of phosphorothioate antisense oligodeoxynucleotides (AS[S]ODNs) to type I insulin-like growth factor receptor (IGF-IR) mRNA resulted in inhibition of C4HD breast cancer growth in BALB/c mice. The present study focused on whether in vivo administration of C4HD tumor cells pretreated with IGF-IR AS[S]ODN and irradiated could provide protection against C4HD wild-type tumor challenge and also on elucidating the mechanism mediating this effect. Our results showed that mice immunized with IGF-IR AS[S]ODN-treated C4HD cells experienced a growth inhibition of 53.4%, 61.6%, and 60.2% when compared with PBS-treated mice, wild-type C4HD cell-injected mice, or phosphorothioate sense oligodeoxynucleotide-treated C4HD cell-injected mice, respectively. The protective effect was C4HD-specific, because no cross-protection was observed against other syngeneic mammary tumor lines. The lack of protection against tumor formation in nude mice indicated that T cells were involved in the antitumoral response. Furthermore, cytotoxicity and splenocyte proliferation assays demonstrated that a cellular CD8(+)-dependent immune response, acting through the Fas/Fas ligand death pathway, could be mediating the antitumor effect induced by immunization with AS[S]ODN-treated cells. Immunization also induced splenocytes to produce Ag-dependent IFN-gamma, indicating the presence of a type 1 response. We demonstrated for the first time that IGF-IR AS[S]ODN treatment of breast cancer cells induced expression of CD86 and heat shock protein 70 molecules, both involved in the induction of the immunogenic phenotype. Immunization with these tumor immunogens imparted protection against parental tumor growth through activation of a specific immune response.     

Stat1 is a suppressor of ErbB2/Neu-mediated cellular transformation and mouse mammary gland tumor formation

The anti-tumor function of Stat1 as a regulator of innate immunity and tumor immune surveillance has been long studied and is well understood; however, less clear is its tumor-site specific role. Although Stat1 phosphorylated at tyrosine (Y) 701 and serine (S) 727 is essential for interferon (IFN) signalling, its function in signalling induced in breast cancer cells is not understood. Herein, we show that Stat1 Y701 phosphorylation is increased in human breast tumor cells with elevated levels of ErbB2/HER-2 and in cells transfected with ErbB2/Neu. However, pharmacological inhibition of ErbB2/HER-2 results in the inhibition of Stat1 Y701 phosphorylation indicating an atypical role of phosphorylated Stat1 in the inhibition of ErbB2/HER-2 signalling. Consistent with this notion, we found that Stat1 suppresses tumor development by an activated form of ErbB2/Neu in mouse embryonic fibroblasts in xenograft tumor assays; however, this anti-tumor function of Stat1 does not rely on Y701 and S727 phosphorylation. Experiments with transgenic mice demonstrated that Stat1 acts to suppress Neu-mediated breast tumorigenesis through immune regulatory and tumor-site specific mechanisms. Our data reveal a previous uncharacterized anti-tumor activity of Stat1 in ErbB2/Neu-mediated cell transformation and breast oncogenesis with possible implications in the diagnosis and treatment of ErbB2-positive breast cancers.     

Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells

Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8(+)CXCR6(+) activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into "inflamed" peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy.     

CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis

Background

Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/Principal Findings

In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/Significance

The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.     

Successful adoptive immunotherapy of murine poorly immunogenic tumor with specific effector cells generated from gene-modified tumor-primed lymph node cells

We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-gamma, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-gamma and B7-1 (MCA102/B7-1/IFN-gamma) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-gamma and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-gamma tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-gamma tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers.     

IL-4-induced Stat6 activities affect apoptosis and gene expression in breast cancer cells

IL-4-induced Stat6 signaling is active in a variety of cell types, including immune cells and cancer cells, and plays an important role in the regulation of gene expression. Using EMSA gel shift assay and an antibody to Stat6, we phenotyped two breast cancer cell lines, ZR-75-1 being active Stat6(high) phenotype and BT-20 being defective Stat6(null) phenotype, respectively. Breast cancer cells carrying Stat6(null) phenotype exhibited increased spontaneous apoptosis compared with those carrying Stat6(high) phenotype. Expression microarray analyses demonstrated that IL-4 upregulated CCL26, SOCS1, CISH, EGLN3, and SIDT1, and downregulated DUSP1, FOS, and FOSB, respectively, in these breast cancer cells. Among those genes, CCL26 and SOCS1 were known genes regulated by IL-4/Stat6 pathway, but CISH, EGLN3, SIDT1, DUSP1, FOS, and FOSB were novel genes demonstrated to be IL-4 responsive for the first time. IL-4 also upregulated 38 genes unique to Stat6(null) BT-20 cells and 23 genes unique to Stat6(high) ZR-75-1 cells, respectively. Furthermore, Stat6(high) and Stat6(null) cells showed very different profiles of constitutively expressed genes relevant to apoptosis and metastasis among others, which serve as a valuable expression database and warrant for detailed studies of IL-4/Stat6 pathway in breast cancer.     

Replicative senescence of CD8 T cells: potential effects on cancer immune surveillance and immunotherapy

The process of replicative senescence, which stringently limits the proliferative potential of normal T cells, constitutes a potential problem for cancer immunotherapy. The ability of CD8 T cells to recognize and destroy tumor cells has been well-established, but the requirement for massive, prolonged proliferative T-cell expansion and maintenance of functional integrity poses a significant obstacle to the success of cancer immunotherapy. Cancer immune surveillance may also be compromised by the long-term exposure of T cells to tumor antigens, particularly those of latent viruses, which could drive certain T cells to replicative senescence. This review summarizes the major characteristics of T-cell replicative senescence and raises the possibility that this process has the potential to affect both cancer development and treatment. Experimental strategies aimed at preventing T-cell replicative senescence are discussed in the context of cancer immunotherapy and vaccines.This article forms part of the Symposium in Writing Tumor escape from the immune response, published in Vol. 53.     

Cancer-associated adipocytes promote the invasion and metastasis in breast cancer through LIF/CXCLs positive feedback loop

Cancer-associated adipocytes (CAAs), which are adipocytes transformed by cancer cells, are of great importance in promoting the progression of breast cancer. However, the underlying mechanisms involved in the crosstalk between cancer cells and adipocytes are still unknown. Here we report that CAAs and breast cancer cells communicate with each other by secreting the cytokines leukemia inhibitory factor (LIF) and C-X-C subfamily chemokines (CXCLs), respectively. LIF is a pro-inflammatory cytokine secreted by CAAs, which promotes migration and invasion of breast cancer cells via the Stat3 signaling pathway. The activation of Stat3 induced the secretion of glutamic acid-leucine-arginine (ELR) motif CXCLs (CXCL1, CXCL2, CXCL3 and CXCL8) in tumor cells. Interestingly, CXCLs in turn activated the ERK1/2/NF-κB/Stat3 signaling cascade to promote the expression of LIF in CAAs. In clinical breast cancer pathology samples, the up-regulation of LIF in paracancerous adipose tissue was positively correlated with the activation of Stat3 in breast cancer. Furthermore, we verified that adipocytes enhanced lung metastasis of breast cancer cells, and the combination of EC330 (targeting LIF) and SB225002 (targeting C-X-C motility chemokine receptor 2 (CXCR2)) significantly reduced lung metastasis of breast cancer cells in vivo. Our findings reveal that the interaction of adipocytes with breast cancer cells depends on a positive feedback loop between the cytokines LIF and CXCLs, which promotes breast cancer invasion and metastasis.