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Supplementary Figure S1. Expression and activation of YAP and other Hippo Pathway factors in CD4+ T cell subsets; Supplementary Figure S2. Characterization of the baseline immune profile of T cell-specific YAP-deficient mice; Supplementary Figure S3. Haematoxylin and eosin staining of lung, kidney, liver, small intestine and stomach sections from 21-day-old wild-type and YAPcKO mice; Supplementary Figure S4. The effect on iTreg generation under optimal TGFβ concentration; Supplementary Figure S5. Treg-specific YAP deficiency slows the growth of implanted MC38-colon tumors and boosts anti-tumor immunity; Supplementary Figure S6. Treg-specific YAP deficiency slows the growth of implanted EL4 thymomas and boosts anti-tumor immunity; Supplementary Figure S7. The effect of YAP inhibition monotherapy and combinational immunotherapy treatments on the immune constituents of the tumor microenvironment; Supplementary Figure S8. Correlation of gene expression in stimulated wild-type and YAP cKO derived Tregs; Supplementary Figure S9. Both Activin A and its receptor (ACVR1c) are upregulated during the course of iTreg differentiation; Supplementary Figure S10. Effect of AcVR1c deficiency on iTreg differentiation; Supplementary Figure S11. Supplemental Activin fails to rescue iTreg generation when SMAD2/3 levels are lacking; Supplementary Figure S12. Effects of GM-Vac and Anti-Activin treatment on B16 tumor progression in Wild Type and AcVR1c KO mice; Supplementary Figure S13. A model for YAP-mediated TGFβ/SMAD signaling enhancement

Regulatory T cells (Treg) are critical for maintaining self-tolerance and immune homeostasis, but their suppressive function can impede effective antitumor immune responses. FOXP3 is a transcription factor expressed in Tregs that is required for their function. However, the pathways and microenvironmental cues governing FOXP3 expression and Treg function are not completely understood. Herein, we report that YAP, a coactivator of the Hippo pathway, is highly expressed in Tregs and bolsters FOXP3 expression and Treg function in vitro and in vivo. This potentiation stemmed from YAP-dependent upregulation of activin signaling, which amplifies TGFβ/SMAD activation in Tregs. YAP deficiency resulted in dysfunctional Tregs unable to suppress antitumor immunity or promote tumor growth in mice. Chemical YAP antagonism and knockout or blockade of the YAP-regulated activin receptor similarly improved antitumor immunity. Thus, we identify YAP as an unexpected amplifier of a Treg-reinforcing pathway with significant potential as an anticancer immunotherapeutic target.Significance: Tregs suppress antitumor immunity, and pathways supporting their function can be novel immunotherapy targets. Here, the selective expression of YAP by Tregs, its importance for their function, and its unexpected enhancement of pro-Treg Activin/SMAD signaling are reported, as are validations of potential cancer-fighting antagonists of YAP and its regulatory targets. Cancer Discov; 8(8); 1026–43. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899

The increase of interleukin-4 (IL-4) level in tumor environment and the up-regulation of IL-4 receptor (IL-4R) on tumor cells have been long observed. However, their significance for tumor development has not been investigated. Here, we found that endogenous IL-4 promotes tumor growth because neutralizing IL-4 by 11B11 monoclonal antibody (mAb) significantly delayed the growth of MCA205 fibrosarcoma. We also observed that tumor cells with higher IL-4R expression have more chances to survive in immunocompetent mice. To investigate how endogenous IL-4 influences tumor growth, we established a pair of tumor cells with or without IL-4R expression from the common parental cells. IL-4R–competent tumors exhibit increased growth compared with its IL-4R–deficient counterparts when inoculated into syngeneic mice. This growth advantage was still kept in IL-4R knockout mice but was abrogated in mice given i.p. with IL-4 neutralizing mAb. In vitro analyses indicate that IL-4 neither affects the proliferation of tumor cells nor changes the expression of several immune-related molecules, such as MHC-I, Fas, and B7-H3. Nonetheless, IL-4 up-regulates antiapoptotic gene expression in tumor cells and reduces apoptosis of tumor cells in vivo, as evidenced by real-time PCR, immunoblotting, and TUNEL staining. These findings were helpful to understand the long clinical observation and revealed that endogenous IL-4, the product of host immune response, can be used by tumor cells to facilitate their growth. [Cancer Res 2008;68(21):8687–94]

Supplementary Figure 3 from Endogenous Interleukin-4 Promotes Tumor Development by Increasing Tumor Cell Resistance to Apoptosis

Supplementary Figure 1 from Endogenous Interleukin-4 Promotes Tumor Development by Increasing Tumor Cell Resistance to Apoptosis

Supplementary Figure 2 from Endogenous Interleukin-4 Promotes Tumor Development by Increasing Tumor Cell Resistance to Apoptosis

Regulatory T cells (Treg) are critical for maintaining self-tolerance and immune homeostasis, but their suppressive function can impede effective antitumor immune responses. FOXP3 is a transcription factor expressed in Tregs that is required for their function. However, the pathways and microenvironmental cues governing FOXP3 expression and Treg function are not completely understood. Herein, we report that YAP, a coactivator of the Hippo pathway, is highly expressed in Tregs and bolsters FOXP3 expression and Treg function in vitro and in vivo. This potentiation stemmed from YAP-dependent upregulation of activin signaling, which amplifies TGFβ/SMAD activation in Tregs. YAP deficiency resulted in dysfunctional Tregs unable to suppress antitumor immunity or promote tumor growth in mice. Chemical YAP antagonism and knockout or blockade of the YAP-regulated activin receptor similarly improved antitumor immunity. Thus, we identify YAP as an unexpected amplifier of a Treg-reinforcing pathway with significant potential as an anticancer immunotherapeutic target. Significance: Tregs suppress antitumor immunity, and pathways supporting their function can be novel immunotherapy targets. Here, the selective expression of YAP by Tregs, its importance for their function, and its unexpected enhancement of pro-Treg Activin/SMAD signaling are reported, as are validations of potential cancer-fighting antagonists of YAP and its regulatory targets. Cancer Discov; 8(8); 1026–43. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 899

IFNγ plays a crucial role in immunity against a variety of transplanted tumors and methylcholanthrene-mediated tumorigenesis in mice. However, it is not clear whether and how endogenous IFNγ influences 7,12-dimethylbenz(a)anthracene (DMBA)–induced and 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced papilloma development. We found here that IFNγ expression was markedly up-regulated shortly after DMBA/TPA application to the skin. Surprisingly, neutralizing IFNγ activity in vivo did not increase but rather decreased tumor development. Furthermore, IFNγ receptor–deficient mice were also more resistant to papilloma development than their counterparts were. IFNγ acted mainly in the promotion stage of papilloma development by enhancing TPA-induced leukocyte infiltration and epidermal hyperproliferation. The up-regulation of tumor necrosis factor α, interleukin (IL)-6, and transforming growth factor β was largely dependent on host IFNγ responsiveness. Remarkably, up-regulation of both IL-17 expression in the skin and T helper 17 (Th17) cell number in draining lymph nodes after DMBA/TPA treatment was dependent on IFNγ signaling. Depletion of IL-17 not only decreased the DMBA/TPA–induced inflammation and keratinocyte proliferation but also delayed papilloma development. These results show that IFNγ, under certain conditions, may promote tumor development by enhancing a Th17-associated inflammatory reaction. [Cancer Res 2009;69(5):2010–7]

The increase of interleukin-4 (IL-4) level in tumor environment and the up-regulation of IL-4 receptor (IL-4R) on tumor cells have been long observed. However, their significance for tumor development has not been investigated. Here, we found that endogenous IL-4 promotes tumor growth because neutralizing IL-4 by 11B11 monoclonal antibody (mAb) significantly delayed the growth of MCA205 fibrosarcoma. We also observed that tumor cells with higher IL-4R expression have more chances to survive in immunocompetent mice. To investigate how endogenous IL-4 influences tumor growth, we established a pair of tumor cells with or without IL-4R expression from the common parental cells. IL-4R–competent tumors exhibit increased growth compared with its IL-4R–deficient counterparts when inoculated into syngeneic mice. This growth advantage was still kept in IL-4R knockout mice but was abrogated in mice given i.p. with IL-4 neutralizing mAb. In vitro analyses indicate that IL-4 neither affects the proliferation of tumor cells nor changes the expression of several immune-related molecules, such as MHC-I, Fas, and B7-H3. Nonetheless, IL-4 up-regulates antiapoptotic gene expression in tumor cells and reduces apoptosis of tumor cells in vivo, as evidenced by real-time PCR, immunoblotting, and TUNEL staining. These findings were helpful to understand the long clinical observation and revealed that endogenous IL-4, the product of host immune response, can be used by tumor cells to facilitate their growth. [Cancer Res 2008;68(21):8687–94]

Aristolochic acids (AA) are a family of structurally related nitrophenanthrene carboxylic acids, mainly consisting of aristolochic acid I (AAI) and aristolochic acid II (AAII), and are the active component of some herbal medicines. Use of dietary supplements and other botanical products containing AA have resulted in severe nephrotoxicity and consequent renal replacement therapy. Aristolochic acids are potent carcinogens as well, inducing urothelial cancers in humans and kidney tumors in rats. Previous studies have shown that the tumorigenicity of AA could result from AA-induced DNA adduct formation and mutation induction. To further understand the molecular mechanisms underlying AA-induced nephrotoxicity (e.g., carcinogenesis) and identify disease biomarkers, a quantitative proteomic analysis was conducted on kidneys from AA-treated rats. Animal treatment was conducted according to the approved protocol and followed the recommendations of the NCTR Institutional Animal Care and Use Committee. Six-week-old Big Blue rats were treated with AA as its sodium salt at concentrations of 0.1, 1.0, and 10.0 mg/kg body weight by gavage five times/week for 12 weeks or with 0.9% sodium chloride as the control using the same schedule. Six rats from each treatment group were sacrificed 1 day after the last treatment. The kidneys were isolated, frozen quickly in liquid nitrogen, and stored at −80 °C. Kidney tissues from the control and the highest AA dose treatment (10 mg/kg body weight) were selected for proteomic analysis. Proteins were extracted and digested into peptides. Trypsin-catalyzed 16O/18O stable isotope labeling was performed for each pair of control/treatment samples, which were then analyzed using two-dimensional liquid chromatography coupled online with tandem mass spectrometry (LC-MS/MS). Approximately 3000 proteins were quantified from each pair of samples, and more than 150 proteins were concordantly over expressed or under expressed across all the AA-treated samples. Pathway analysis indicated that significant changes include proteins involving xenobiotic metabolism, response to stimulus including DNA damage, cell proliferation and neoplasm, etc. Intriguingly, proteins indicating kidney pathological changes such as nephron degeneration were identified as well. Although further verification is needed, the quantitative proteomic study identified proteins related to potential toxicity and oncogenic pathways and proteins that could serve as urinary biomarkers of renal damage inflicted by aristolochic acid exposure. The views presented do not necessarily reflect those of the U. S. Food and Drug Administration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-438. doi:10.1158/1538-7445.AM2011-LB-438