Transforming growth factor ß-mediated suppression of antitumor T cells requires FoxP1 transcription factor expression.

Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the upregulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8⁺ T cells from proliferating and upregulating Granzyme-B and interferon-γ in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors and promoted protection against tumor rechallenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in preactivated CD8⁺ T cells in response to microenvironmental transforming growth factor-ß (TGF-ß), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-ß-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-ß signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation.

5α-Reductase, an enzyme regulating glucocorticoid action in the testis of Rhinella arenarum (Amphibia: Anura).

The reduction of A-ring of glucocorticoids to produce 5α-dihydro-derivatives by 5α-reductases has been considered as a pathway of irreversible inactivation. However, 5α-reduced metabolites of corticosterone and testosterone have significant biological activity. In this paper, we investigated whether toad testicular 5α-reductase (5α-Red) is able to transform corticosterone into 5α-dihydrocorticosterone. Furthermore, we studied the role of 5α-reduced metabolite of corticosterone as a glucocorticoid receptor (GR) agonist. The activity of 5α-Red was assayed in subcellular fractions with [(3)H]corticosterone or [(3)H]testosterone as substrate. The enzyme localizes in microsomes and its optimal pH is between 7 and 8. The activity is not inhibited by finasteride. These results support the conclusion that toad 5α-Red resembles mammalian type 1 isoenzyme. Kinetic studies indicate that neither K(m) nor V(max) for both corticosterone and testosterone were significantly different among reproductive periods. The K(m) value for testosterone was significantly higher than that for corticosterone, indicating that the C-21 steroid is the preferred substrate for the enzyme. Studies of the binding capacity of 5α-dihydrocorticosterone (5α-DHB) to the testicular GR show that 5α-DHB is able to displace the binding of [(3)H]dexamethasone to testicular cytosol with a similar potency than corticosterone. The inhibition constant (Ki) values for corticosterone and 5α-DHB were similar, 31.33±2.9 nM and 35.24±2.3 nM, respectively. In vitro experiments suggest that 5α-DHB is an agonist of toad testicular GR, decreasing the activity of the key enzyme for androgen synthesis, the cytochrome P450 17-hydroxylase, C17,20-lyase.

Follicle-Stimulating Hormone Receptor Is Expressed by Most Ovarian Cancer Subtypes and Is a Safe and Effective Immunotherapeutic Target.

PURPOSE: To define the safety and effectiveness of T cells redirected against follicle-stimulating hormone receptor (FSHR)-expressing ovarian cancer cells. EXPERIMENTAL DESIGN: FSHR expression was determined by Western blotting, immunohistochemistry, and qPCR in 77 human ovarian cancer specimens from 6 different histologic subtypes and 20 human healthy tissues. The effectiveness of human T cells targeted with full-length FSH in vivo was determined against a panel of patient-derived xenografts. Safety and effectiveness were confirmed in immunocompetent tumor-bearing mice, using constructs targeting murine FSHR and syngeneic T cells. RESULTS: FSHR is expressed in gynecologic malignancies of different histologic types but not in nonovarian healthy tissues. Accordingly, T cells expressing full-length FSHR-redirected chimeric receptors mediate significant therapeutic effects (including tumor rejection) against a panel of patient-derived tumors in vivo In immunocompetent mice growing syngeneic, orthotopic, and aggressive ovarian tumors, fully murine FSHR-targeted T cells also increased survival without any measurable toxicity. Notably, chimeric receptors enhanced the ability of endogenous tumor-reactive T cells to abrogate malignant progression upon adoptive transfer into naïve recipients subsequently challenged with the same tumor. Interestingly, FSHR-targeted T cells persisted as memory lymphocytes without noticeable PD-1-dependent exhaustion during end-stage disease, in the absence of tumor cell immunoediting. However, exosomes in advanced tumor ascites diverted the effector activity of this and other chimeric receptor-transduced T cells away from targeted tumor cells. CONCLUSIONS: T cells redirected against FSHR+ tumor cells with full-length FSH represent a promising therapeutic alternative against a broad range of ovarian malignancies, with negligible toxicity even in the presence of cognate targets in tumor-free ovaries. Clin Cancer Res; 23(2); 441-53. ©2016 AACR.

Tumor Cell-Independent Estrogen Signaling Drives Disease Progression through Mobilization of Myeloid-Derived Suppressor Cells.

The role of estrogens in antitumor immunity remains poorly understood. Here, we show that estrogen signaling accelerates the progression of different estrogen-insensitive tumor models by contributing to deregulated myelopoiesis by both driving the mobilization of myeloid-derived suppressor cells (MDSC) and enhancing their intrinsic immunosuppressive activity in vivo Differences in tumor growth are dependent on blunted antitumor immunity and, correspondingly, disappear in immunodeficient hosts and upon MDSC depletion. Mechanistically, estrogen receptor alpha activates the STAT3 pathway in human and mouse bone marrow myeloid precursors by enhancing JAK2 and SRC activity. Therefore, estrogen signaling is a crucial mechanism underlying pathologic myelopoiesis in cancer. Our work suggests that new antiestrogen drugs that have no agonistic effects may have benefits in a wide range of cancers, independently of the expression of estrogen receptors in tumor cells, and may synergize with immunotherapies to significantly extend survival. SIGNIFICANCE: Ablating estrogenic activity delays malignant progression independently of the tumor cell responsiveness, owing to a decrease in the mobilization and immunosuppressive activity of MDSCs, which boosts T-cell-dependent antitumor immunity. Our results provide a mechanistic rationale to block estrogen signaling with newer antagonists to boost the effectiveness of anticancer immunotherapies. Cancer Discov; 7(1); 72-85. ©2016 AACR.See related commentary by Welte et al., p. 17This article is highlighted in the In This Issue feature, p. 1.

IL15 Agonists Overcome the Immunosuppressive Effects of MEK Inhibitors.

Many signal transduction inhibitors are being developed for cancer therapy target pathways that are also important for the proper function of antitumor lymphocytes, possibly weakening their therapeutic effects. Here we show that most inhibitors targeting multiple signaling pathways have especially strong negative effects on T-cell activation at their active doses on cancer cells. In particular, we found that recently approved MEK inhibitors displayed potent suppressive effects on T cells in vitro However, these effects could be attenuated by certain cytokines that can be administered to cancer patients. Among them, clinically available IL15 superagonists, which can activate PI3K selectively in T lymphocytes, synergized with MEK inhibitors in vivo to elicit potent and durable antitumor responses, including by a vaccine-like effect that generated resistance to tumor rechallenge. Our work identifies a clinically actionable approach to overcome the T-cell-suppressive effects of MEK inhibitors and illustrates how to reconcile the deficiencies of signal transduction inhibitors, which impede desired immunologic effects in vivo Cancer Res; 76(9); 2561-72. ©2016 AACR.

Satb1 Overexpression Drives Tumor-Promoting Activities in Cancer-Associated Dendritic Cells.

Special AT-rich sequence-binding protein 1 (Satb1) governs genome-wide transcriptional programs. Using a conditional knockout mouse, we find that Satb1 is required for normal differentiation of conventional dendritic cells (DCs). Furthermore, Satb1 governs the differentiation of inflammatory DCs by regulating major histocompatibility complex class II (MHC II) expression through Notch1 signaling. Mechanistically, Satb1 binds to the Notch1 promoter, activating Notch expression and driving RBPJ occupancy of the H2-Ab1 promoter, which activates MHC II transcription. However, tumor-driven, unremitting expression of Satb1 in activated Zbtb46(+) inflammatory DCs that infiltrate ovarian tumors results in an immunosuppressive phenotype characterized by increased secretion of tumor-promoting Galectin-1 and IL-6. In vivo silencing of Satb1 in tumor-associated DCs reverses their tumorigenic activity and boosts protective immunity. Therefore, dynamic fluctuations in Satb1 expression govern the generation and immunostimulatory activity of steady-state and inflammatory DCs, but continuous Satb1 overexpression in differentiated DCs converts them into tolerogenic/pro-inflammatory cells that contribute to malignant progression.

Microbially driven TLR5-dependent signaling governs distal malignant progression through tumor-promoting inflammation.

The dominant TLR5(R392X) polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

Initiation of metastatic breast carcinoma by targeting of the ductal epithelium with adenovirus-cre: a novel transgenic mouse model of breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of αß and γδ T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Pathological mobilization and activities of dendritic cells in tumor-bearing hosts: challenges and opportunities for immunotherapy of cancer.

A common characteristic of solid tumors is the pathological recruitment of immunosuppressive myeloid cells, which in certain tumors includes dendritic cells (DCs). DCs are of particular interest in the field of cancer immunotherapy because they induce potent and highly specific anti-tumor immune responses, particularly in the early phase of tumorigenesis. However, as tumors progress, these cells can be transformed into regulatory cells that contribute to an immunosuppressive microenvironment favoring tumor growth. Therefore, controlling DC phenotype has the potential to elicit effective anti-tumor responses while simultaneously weakening the tumor's ability to protect itself from immune attack. This review focuses on the dual nature of DCs in the tumor microenvironment, the regulation of DC phenotype, and the prospect of modifying DCs in situ as a novel immunotherapeutic approach.

Reprogramming tumor-associated dendritic cells in vivo using miRNA mimetics triggers protective immunity against ovarian cancer.

Modulating the activity of miRNAs provides opportunities for novel cancer interventions. However, low bioavailability and poor cellular uptake are major challenges for delivering miRNA mimetics specifically to tumor cells. Here, we took advantage of the spontaneous enhanced endocytic activity of ovarian cancer-associated dendritic cells (DC) to selectively supplement the immunostimulatory miRNA miR-155. In vivo processing of nanoparticles carrying oligonucleotide duplexes mimicking the bulged structure of endogenous pre-miRNA (but not siRNA-like oligonucleotides) dramatically augmented miR-155 activity without saturating the RNA-induced silencing complex. Endogenous processing of synthetic miR-155 favored Ago2 and, to a lesser extent, Ago4 loading, resulting in genome-wide transcriptional changes that included silencing of multiple immunosuppressive mediators. Correspondingly, tumor-infiltrating DCs were transformed from immunosuppressive to highly immunostimulatory cells capable of triggering potent antitumor responses that abrogated the progression of established ovarian cancers. Our results show both the feasibility and therapeutic potential of supplementing/replenishing miRNAs in vivo using nonviral approaches to boost protective immunity against lethal tumors. Thus, we provide a platform, an optimized design, and a mechanistic rationale for the clinical testing of nonviral miRNA mimetics.