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Drug resistance is a major barrier for the development of effective and durable cancer therapies. Overcoming this challenge requires further defining the cellular and molecular mechanisms underlying drug resistance, both acquired and environment-mediated drug resistance (EMDR). Here, using neuroblastoma (NB), a childhood cancer with high incidence of recurrence due to resistance to chemotherapy, as a model we show that human bone marrow–mesenchymal stromal cells induce tumor expression of sphingosine-1-phosphate receptor-1 (S1PR1), leading to their resistance to chemotherapy. Targeting S1PR1 by shRNA markedly enhances etoposide-induced apoptosis in NB cells and abrogates EMDR, while overexpression of S1PR1 significantly protects NB cells from multidrug-induced apoptosis via activating JAK–STAT3 signaling. Elevated S1PR1 expression and STAT3 activation are also observed in human NB cells with acquired resistance to etoposide. We show in vitro and in human NB xenograft models that treatment with FTY720, an FDA-approved drug and antagonist of S1PR1, dramatically sensitizes drug-resistant cells to etoposide. In summary, we identify S1PR1 as a critical target for reducing both EMDR and acquired chemoresistance in NB. Mol Cancer Ther; 16(11); 2516–27. ©2017 AACR.

Supplemental Figure 1. Bone marrow stromal cells enhance NB resistance to etoposide; Supplemental Figure 2. Overexpression of S1PR1 in CHLA-171 cells activates STAT3 and renders cells resistant to etoposide via JAK2/STAT3 signaling; Supplemental Figure 3. Elevated S1PR1 expression and STAT3 activation in etoposide-resistant CHLA-171 NB cells; Supplemental Figure 4. Taxol and JAK inhibitor effects on NB drug resistance; Supplemental Figure 5. Stattic and W146 inhibit STAT3 phosphorylation in NB drug resistant cells; Supplemental Figure 6. Crosstalk between NB cells and BM-MSC cells upregulates Sphk1.

Supplemental Figure 1. Bone marrow stromal cells enhance NB resistance to etoposide; Supplemental Figure 2. Overexpression of S1PR1 in CHLA-171 cells activates STAT3 and renders cells resistant to etoposide via JAK2/STAT3 signaling; Supplemental Figure 3. Elevated S1PR1 expression and STAT3 activation in etoposide-resistant CHLA-171 NB cells; Supplemental Figure 4. Taxol and JAK inhibitor effects on NB drug resistance; Supplemental Figure 5. Stattic and W146 inhibit STAT3 phosphorylation in NB drug resistant cells; Supplemental Figure 6. Crosstalk between NB cells and BM-MSC cells upregulates Sphk1.

Suppl. Fig. S1 showing CTLA4 expression and CD86 cellular internalization by human BCL and human MM cells acquired by flow cytometry.

Suppl. Fig. S2 showing improved Th1 and Th2 maturation and increased CD3 T cell activation upon CTLA4 blockade in vivo.

CTL–associated antigen 4 (CTLA4) is a well-established immune checkpoint for antitumor immune responses. The protumorigenic function of CTLA4 is believed to be limited to T-cell inhibition by countering the activity of the T-cell costimulating receptor CD28. However, as we demonstrate here, there are two additional roles for CTLA4 in cancer, including via CTLA4 overexpression in diverse B-cell lymphomas and in melanoma-associated B cells. CTLA4-CD86 ligation recruited and activated the JAK family member Tyk2, resulting in STAT3 activation and expression of genes critical for cancer immunosuppression and tumor growth and survival. CTLA4 activation resulted in lymphoma cell proliferation and tumor growth, whereas silencing or antibody-blockade of CTLA4 in B-cell lymphoma tumor cells in the absence of T cells inhibits tumor growth. This inhibition was accompanied by reduction of Tyk2/STAT3 activity, tumor cell proliferation, and induction of tumor cell apoptosis. The CTLA4–Tyk2–STAT3 signal pathway was also active in tumor-associated nonmalignant B cells in mouse models of melanoma and lymphoma. Overall, our results show how CTLA4-induced immune suppression occurs primarily via an intrinsic STAT3 pathway and that CTLA4 is critical for B-cell lymphoma proliferation and survival. Cancer Res; 77(18); 5118–28. ©2017 AACR.

Suppl. Fig. S3 showing CTLA4 expression by tumor associated CD19 B cells

Figure S1. Increased TLR9 expression in induced glioma spheres. Figure S2. TLR9 and STAT3 form a feed-forward loop in GSCs. Figure S3. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits tumor growth. Figure S4. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits GSCs. Figure S5. Targeting Brain tumors systemically with CpG-siRNA reaches the tumor site.

Drug resistance is a major barrier for the development of effective and durable cancer therapies. Overcoming this challenge requires further defining the cellular and molecular mechanisms underlying drug resistance, both acquired and environment-mediated drug resistance (EMDR). Here, using neuroblastoma (NB), a childhood cancer with high incidence of recurrence due to resistance to chemotherapy, as a model we show that human bone marrow–mesenchymal stromal cells induce tumor expression of sphingosine-1-phosphate receptor-1 (S1PR1), leading to their resistance to chemotherapy. Targeting S1PR1 by shRNA markedly enhances etoposide-induced apoptosis in NB cells and abrogates EMDR, while overexpression of S1PR1 significantly protects NB cells from multidrug-induced apoptosis via activating JAK–STAT3 signaling. Elevated S1PR1 expression and STAT3 activation are also observed in human NB cells with acquired resistance to etoposide. We show in vitro and in human NB xenograft models that treatment with FTY720, an FDA-approved drug and antagonist of S1PR1, dramatically sensitizes drug-resistant cells to etoposide. In summary, we identify S1PR1 as a critical target for reducing both EMDR and acquired chemoresistance in NB. Mol Cancer Ther; 16(11); 2516–27. ©2017 AACR.

CTL–associated antigen 4 (CTLA4) is a well-established immune checkpoint for antitumor immune responses. The protumorigenic function of CTLA4 is believed to be limited to T-cell inhibition by countering the activity of the T-cell costimulating receptor CD28. However, as we demonstrate here, there are two additional roles for CTLA4 in cancer, including via CTLA4 overexpression in diverse B-cell lymphomas and in melanoma-associated B cells. CTLA4-CD86 ligation recruited and activated the JAK family member Tyk2, resulting in STAT3 activation and expression of genes critical for cancer immunosuppression and tumor growth and survival. CTLA4 activation resulted in lymphoma cell proliferation and tumor growth, whereas silencing or antibody-blockade of CTLA4 in B-cell lymphoma tumor cells in the absence of T cells inhibits tumor growth. This inhibition was accompanied by reduction of Tyk2/STAT3 activity, tumor cell proliferation, and induction of tumor cell apoptosis. The CTLA4–Tyk2–STAT3 signal pathway was also active in tumor-associated nonmalignant B cells in mouse models of melanoma and lymphoma. Overall, our results show how CTLA4-induced immune suppression occurs primarily via an intrinsic STAT3 pathway and that CTLA4 is critical for B-cell lymphoma proliferation and survival. Cancer Res; 77(18); 5118–28. ©2017 AACR.

COHCAP (City of Hope CpG Island Analysis Pipeline) is an algorithm to analyze single-nucleotide resolution DNA methylation data produced by either an Illumina methylation array or targeted bisulfite sequencing. The goal of the COHCAP algorithm is to identify CpG islands that show a consistent pattern of methylation among CpG sites. COHCAP is currently the only DNA methylation package that provides integration with gene expression data to identify a subset of CpG islands that are most likely to regulate downstream gene expression, and it can generate lists of differentially methylated CpG islands with ∼50% concordance with gene expression from both cell line data and heterogeneous patient data. For example, this article describes known breast cancer biomarkers (such as estrogen receptor) with a negative correlation between DNA methylation and gene expression. COHCAP also provides visualization for quality control metrics, regions of differential methylation and correlation between methylation and gene expression. This software is freely available at https://sourceforge.net/projects/cohcap/.

The participation of a specific subset of B cells and how they are regulated in cancer is unclear. Here, we demonstrate that the proportion of CD5(+) relative to interleukin-6 receptor α (IL-6Rα)-expressing B cells was greatly increased in tumors. CD5(+) B cells responded to IL-6 in the absence of IL-6Rα. IL-6 directly bound to CD5, leading to activation of the transcription factor STAT3 via gp130 and its downstream kinase JAK2. STAT3 upregulated CD5 expression, thereby forming a feed-forward loop in the B cells. In mouse tumor models, CD5(+) but not CD5(-) B cells promoted tumor growth. CD5(+) B cells also showed activation of STAT3 in multiple types of human tumor tissues. Thus, our findings demonstrate a critical role of CD5(+) B cells in promoting cancer.

Although obesity is known to be critical for cancer development, how obesity negatively impacts antitumor immune responses remains largely unknown. Here we show that increased fatty acid oxidation (FAO) driven by activated STAT3 in CD8(+) T effector cells is critical for obesity-associated breast tumor progression. Ablating T cell Stat3 or treatment with an FAO inhibitor in obese mice spontaneously developing breast tumor reduces FAO, increases glycolysis and CD8(+) T effector cell functions, leading to inhibition of breast tumor development. Moreover, PD-1 ligation in CD8(+) T cells activates STAT3 to increase FAO, inhibiting CD8(+) T effector cell glycolysis and functions. Finally, leptin enriched in mammary adipocytes/fat tissues downregulates CD8(+) T cell effector functions through activating STAT3-FAO and inhibiting glycolysis. We identify a critical role of increased oxidation of fatty acids driven by leptin and PD-1 through STAT3 in inhibiting CD8(+) T effector cell glycolysis and in promoting obesity-associated breast tumorigenesis.

Summary Although obesity is known to be critical for cancer development, how obesity negatively impacts antitumor immune responses remains largely unknown. Here, we show that increased fatty acid oxidation (FAO) driven by activated STAT3 in CD8+ T effector cells is critical for obesity-associated breast tumor progression. Ablating T cell Stat3 or treatment with an FAO inhibitor in obese mice spontaneously developing breast tumor reduces FAO, increases glycolysis and CD8+ T effector cell functions, leading to inhibition of breast tumor development. Moreover, PD-1 ligation in CD8+ T cells activates STAT3 to increase FAO, inhibiting CD8+ T effector cell glycolysis and functions. Finally, leptin enriched in mammary adipocytes and fat tissues downregulates CD8+ T cell effector functions through activating STAT3-FAO and inhibiting glycolysis. We identify a critical role of increased oxidation of fatty acids driven by leptin and PD-1 through STAT3 in inhibiting CD8+ T effector cell glycolysis and in promoting obesity-associated breast tumorigenesis.

The Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins, particularly STAT3, are among the most promising new targets for cancer therapy. In addition to interleukin-6 (IL-6) and its family members, multiple pathways, including G-protein-coupled receptors (GPCRs), Toll-like receptors (TLRs) and microRNAs were recently identified to regulate JAK-STAT signalling in cancer. Well known for its role in tumour cell proliferation, survival, invasion and immunosuppression, JAK-STAT3 signalling also promotes cancer through inflammation, obesity, stem cells and the pre-metastatic niche. In addition to its established role as a transcription factor in cancer, STAT3 regulates mitochondrion functions, as well as gene expression through epigenetic mechanisms. Newly identified regulators and functions of JAK-STAT3 in tumours are important targets for potential therapeutic strategies in the treatment of cancer.

Understanding supports for cancer stem–like cells in malignant glioma may suggest therapeutic strategies for their elimination. Here, we show that the Toll-like receptor TLR9 is elevated in glioma stem–like cells (GSC) in which it contributes to glioma growth. TLR9 overexpression is regulated by STAT3, which is required for GSC maintenance. Stimulation of TLR9 with a CpG ligand (CpG ODN) promoted GSC growth, whereas silencing TLR9 expression abrogated GSC development. CpG-ODN treatment induced Frizzled4-dependent activation of JAK2, thereby activating STAT3. Targeted delivery of siRNA into GSC was achieved via TLR9 using CpG–siRNA conjugates. Through local or systemic treatment, administration of CpG-Stat3 siRNA to silence STAT3 in vivo reduced GSC along with glioma growth. Our findings identify TLR9 as a functional marker for GSC and a target for the delivery of efficacious therapeutics for glioma treatment. Cancer Res; 74(18); 5218–28. ©2014 AACR.

Dysregulated inflammation in adipose tissue, marked by increased proinflammatory T-cell accumulation and reduced regulatory T cells (Tregs), contributes to obesity-associated insulin resistance. The molecular mechanisms underlying T-cell-mediated inflammation in adipose tissue remain largely unknown, however. Here we show a crucial role for signal transducer and activator of transcription 3 (Stat3) in T cells in skewing adaptive immunity in visceral adipose tissue (VAT), thereby contributing to diet-induced obesity (DIO) and insulin resistance. Stat3 activity is elevated in obese VAT and in VAT-resident T cells. Functional ablation of Stat3 in T cells reduces DIO, improves insulin sensitivity and glucose tolerance, and suppresses VAT inflammation. Importantly, Stat3 ablation reverses the high Th1/Treg ratio in VAT of DIO mice that is likely secondary to elevated IL-6 production, leading in turn to suppression of Tregs. In addition, Stat3 in T cells in DIO mice affects adipose tissue macrophage accumulation and M2 phenotype. Our study identifies Stat3 in VAT-resident T cells as an important mediator and direct target for regulating adipose tissue inflammation, DIO, and its associated metabolic dysfunctions.

SummaryRecent studies underscore the importance of myeloid cells in rendering distant organs hospitable for disseminating tumor cells to colonize. However, what enables myeloid cells to have an apparently superior capacity to colonize distant organs is unclear. Here, we show that S1PR1-STAT3 upregulation in tumor cells induces factors that activate S1PR1-STAT3 in various cells in premetastatic sites, leading to premetastatic niche formation. Targeting either S1PR1 or STAT3 in myeloid cells disrupts existing premetastatic niches. S1PR1-STAT3 pathway enables myeloid cells to intravasate, prime the distant organ microenvironment and mediate sustained proliferation and survival of their own and other stromal cells at future metastatic sites. Analyzing tumor-free lymph nodes from cancer patients shows elevated myeloid infiltrates, STAT3 activity, and increased survival signal.

Both STAT3 and NF-κB are persistently activated in diverse cancers and promote tumor cell proliferation, survival, angiogenesis, and metastasis through transcriptional activation of multiple common genes. Paradoxically, STAT3 also suppresses many NF-κB–inducible genes involved in innate and adaptive antitumor immunity in spite of elevated levels of NF-κB in tumors. In this study, we show that expression of many NF-κB downstream target genes in tumors depends on STAT3 DNA binding. When STAT3 is elevated in tumor cells and tumor-infiltrating immune cells, persistently activated NF-κB interacts with STAT3 and preferentially binds to genes with STAT3-binding site(s) in promoters. A large number of NF-κB downstream genes associated with oncogenesis and chronic inflammation contain STAT3 DNA-binding site(s). However, in contrast, many genes frequently associated with antitumor immunity lack STAT3 DNA-binding site(s) and can only be activated by NF-κB when STAT3 is inhibited in tumors. The introduction of STAT3 DNA-binding sequences by site-specific mutagenesis in an immunostimulatory gene promoter allows its transcriptional activation by NF-κB in tumor cells. Furthermore, STAT3 facilitates NF-κB binding to genes that are important for tumor growth while inhibiting its binding to Th-1 immunostimulatory genes in growing tumors, including in tumor-infiltrating immune cells. The results of this study provide insight into how some of the oncogenic/inflammatory and Th-1 immunostimulatory genes are differentially regulated in cancer. Cancer Res; 71(11); 3772–80. ©2011 AACR.

Interleukin-6 (IL-6)-Janus kinase (JAK) signaling is viewed as crucial for persistent signal transducer and activator of transcription-3 (STAT3) activation in cancer. However, IL-6-induced STAT3 activation is normally transient. Here we identify a key mechanism for persistent STAT3 activation in tumor cells and the tumor microenvironment. We show that expression of sphingosine-1-phosphate receptor-1 (S1PR1), a G protein-coupled receptor for the lysophospholipid sphingosine-1-phosphate (S1P), is elevated in STAT3-positive tumors. STAT3 is a transcription factor for the S1pr1 gene. Reciprocally, enhanced S1pr1 expression activates STAT3 and upregulates Il6 gene expression, thereby accelerating tumor growth and metastasis in a STAT3-dependent manner. Silencing S1pr1 in tumor cells or immune cells inhibits tumor STAT3 activity, tumor growth and metastasis. S1P-S1PR1-induced STAT3 activation is persistent, in contrast to transient STAT3 activation by IL-6. S1PR1 activates STAT3 in part by upregulating JAK2 tyrosine kinase activity. We show that STAT3-induced S1PR1 expression, as well as the S1P-S1PR1 pathway reciprocal regulation of STAT3 activity, is a major positive feedback loop for persistent STAT3 activation in cancer cells and the tumor microenvironment and for malignant progression.

Cancer stem cells (CSCs) are critical for cancer progression and chemoresistance. How lipid metabolism regulates CSCs and chemoresistance remains elusive. Here, we demonstrate that JAK/STAT3 regulates lipid metabolism, which promotes breast CSCs (BCSCs) and cancer chemoresistance. Inhibiting JAK/STAT3 blocks BCSC self-renewal and expression of diverse lipid metabolic genes, including carnitine palmitoyltransferase 1B (CPT1B), which encodes the critical enzyme for fatty acid β-oxidation (FAO). Moreover, mammary-adipocyte-derived leptin upregulates STAT3-induced CPT1B expression and FAO activity in BCSCs. Human breast-cancer-derived data suggest that the STAT3-CPT1B-FAO pathway promotes cancer cell stemness and chemoresistance. Blocking FAO and/or leptin re-sensitizes them to chemotherapy and inhibits BCSCs in mouse breast tumors in vivo. We identify a critical pathway for BCSC maintenance and breast cancer chemoresistance.

Efficient delivery of siRNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We describe a novel siRNA-based approach – synthetically linking siRNA to an oligonucleotide TLR9 agonist – that targets and silences genes in TLR9+ myeloid cells and B cells, both of which are key components of the tumor microenvironment. Because Stat3 in tumor-associated immune cells suppresses antitumor immune responses and hinders TLR9-induced immune stimulation, we tested CpG-Stat3siRNA conjugates for anti-tumor effects. When injected locally at the tumor site or systemically through an intravenous route, the CpG-Stat3siRNA conjugates access tumor-associated dendritic cells, macrophages and B cells, inhibit Stat3 expression, leading to activation of tumor-associated immune cells, and ultimately potent anti-tumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.

The underlying molecular mechanisms that cause immune cells, mediators of our defense system, to promote tumor invasion and angiogenesis remain incompletely understood. Constitutively activated Stat3 in tumor cells has been shown to promote tumor invasion and angiogenesis. Therefore, we sought to determine whether Stat3 activation in tumor-associated inflammatory cells has a similar function. We found that Stat3 signaling mediates multidirectional crosstalk among tumor cells, myeloid cells in the tumor stroma, and ECs that contributes to tumor angiogenesis in mice. Myeloid-derived suppressor cells and macrophages isolated from mouse tumors displayed activated Stat3 and induced angiogenesis in an in vitro tube formation assay via Stat3 induction of angiogenic factors, including VEGF and bFGF. Stat3-regulated factors produced by both tumor cells and tumor-derived myeloid cells also induced constitutive activation of Stat3 in tumor endothelium, and inhibiting Stat3 in ECs substantially reduced in vitro tumor factor-induced endothelial migration and tube formation. In vivo assays demonstrated the requirement for Stat3 signaling in tumor-associated myeloid cells for tumor angiogenesis. Our results indicate that, by virtue of the ability of Stat3 in tumor cells and tumor-derived myeloid cells to upregulate expression of factors that activate Stat3 in ECs, Stat3 mediates multidirectional crosstalk among tumor cells, tumor-associated myeloid cells, and ECs that contributes to tumor angiogenesis.

Estrogens are female sex steroid hormones that control development, maintenance, and regulation of the female reproductive phenotype and behavior. They also stimulate the growth of normal and transformed epithelial cells of the female reproductive systems. The effect of estrogens is mediated through both estrogen receptors α and β (ERα and ERβ), which belong to the nuclear hormone receptor superfamily, a group of ligand-regulated, zinc finger-containing transcription factors (11, 40). The superfamily includes not only receptors for classical steroids such as estrogens, androgens, progesterones, and glucocorticoids, but also receptors for steroid analogues and nonsteroid ligands such as vitamin D, thyroid, and retinoic acids, as well as orphan receptors for which the ligand is unknown. Unlike the thyroid and vitamin D receptors, which reside in the nucleus in the absence of ligands, receptors for classical steroids such as ERα are targeted to the nucleus after binding with estrogens or selective estrogen receptor modulators such as tamoxifen. In contrast, the pure ERα antagonist ICI 182,780 directs the ERα to the cytoplasm (6). Of importance in this respect are three clusters of basic amino acids, similar to the nuclear localization signals found in simian virus 40 large T antigen, which were identified in the DNA binding domain and the hinge region of ERα (44). The nuclear localization signals are constitutively active and do not seem to explain the estrogen effect on the ERα nuclear localization (44). It is therefore possible that estrogen-induced targeting of ERα to the nucleus is mediated through other mechanisms. Studies in recent years have provided increasing evidence that nuclear localization is also controlled through nuclear export signals (17, 38, 42). A number of studies on exported proteins have shown that typical nuclear export signals are hydrophobic, leucine-rich sequences that signal the nuclear export complex containing exportin/Crm1 and RanGTP to transfer nuclear export signal-carrying proteins to the cytoplasm (8, 12, 29, 37). Besides ligands, nonsteroid stimuli such as kinase activators, phosphatase inhibitors, neurotransmitters, and growth factors were also shown to activate the ERα (35). They either activate the receptor ligand independently or enhance the ligand-induced activity. Consistent with the cross talk with kinase/phosphatase pathways, ERα has been found to be phosphorylated at different sites by various kinases, including the external signal-regulated kinase (4, 19), cyclin A-CDK2 (32), c-SRC (25), protein kinase A (5), and pp90RSK1 (16). Except for tyrosine-537, all known ERα phosphorylation sites are on serine residues (1). Single-site mutation or simultaneous mutation at multiple sites reduced the transcriptional activity of the receptor (13). With the exception of the serine-236 phosphorylation by protein kinase A (5), most studies indicated the general role of ERα phosphorylation to be the regulation of the transcriptional activity of the receptor by modulating the interaction between the ERα activation domains and transcriptional coactivators (9, 28, 39). In the present studies, we report that, in ERα-expressing endometrial cancer cells, 17β-estradiol activates the p38 mitogen-activated protein kinase (MAPK) pathway, which in turn mediates the phosphorylation of the ERα on threonine-311 (Thr311), promoting the receptor's nuclear localization and interaction with steroid receptor coactivators. Additional studies show that Thr311 phosphorylation in ERα is a critical determinant for its transcriptional and biological activities in endometrial cancer cells.

Nucleocytoplasmic shuttling of signaling molecules is crucial for regulating their activity. Regulation of signal transducer and activator of transcription 3 (STAT3) is critical for normal physiology while STAT3 dysregulation underlies many diseases such as cancer. However, the mechanism(s) underlying STAT3 nuclear egress remains unclear. Here, we show that exportin 7 is critical for STAT3 nuclear egress. Lysine acetylation at K685, frequently found in tumors and tumor cell lines, mediates STAT3 engagement with exportin 7. Blocking acetylation through drug administration or mutation of lysine K685 disrupted STAT3’s physical interaction with exportin 7, leading to STAT3 nuclear retention. Inhibition of STAT3 lysine acetylation significantly altered the functional localization of exportin 7 from the cell cytoplasm toward the nucleus which can be reversed by treating tumor with a lysine-acetylated peptide spanning STAT3 K685. Taken together, our results have identified exportin 7 as an essential karyopherin for STAT3 nucleocytoplasmic shuttling.

The role of B cells in cancer and the underlying mechanisms remain to be further explored. Here, we show that tumor-associated B cells with activated STAT3 contribute to tumor development by promoting tumor angiogenesis. B cells with or without Stat3 have opposite effects on tumor growth and tumor angiogenesis in both B16 melanoma and Lewis Lung Cancer mouse models. Ex vivo angiogenesis assays show that B cell-mediated tumor angiogenesis is mainly dependent on the induction of pro-angiogenic gene expression, which requires Stat3 signaling in B cells. Furthermore, B cells with activated STAT3 are mainly found in or near tumor vasculature and correlate significantly with overall STAT3 activity in human tumors. Moreover, the density of B cells in human tumor tissues correlates significantly with expression levels of several STAT3-downstream pro-angiogenic genes, as well as the degree of tumor angiogenesis. Together, these findings define a novel role of B cells in promoting tumor progression through angiogenesis and identify STAT3 in B cells as potential therapeutic target for anti-angiogenesis therapy.

Signaling pathways underlying BV8-mediated oncogenesis remain unknown.BV8-STAT3 forms a feed-forward loop in both normal and malignant myeloid cells and promotes tumor growth.JAK2/STAT3 signaling plays critical roles in BV8-mediated myeloid cell-dependent oncogenesis.This study identifies a novel role of BV8-STAT3 signaling in mediating cross-talk between tumor microenvironment and tumor cells. An important role of BV8 in mobilization of myeloid cells and myeloid cell-dependent angiogenesis has been established. Recently, it has also been shown that granulocyte colony-stimulating factor (G-CSF)-induced BV8 expression is STAT3 dependent in CD11b(+)Gr1(+) myeloid cells. However, the BV8 downstream signaling pathway(s) intrinsic to myeloid cells crucial for angiogenesis, and potentially also for development of cancers of myeloid origin, remains largely unknown. Here we show that BV8 activates STAT3, which is critical for regulating genes important for both tumor cell proliferation/survival and tumor angiogenesis, in both normal and malignant myeloid cells. Further, BV8-induced STAT3 activation requires Janus-activated kinase 2 (JAK2) activity as shown by both genetic and pharmacologic inhibition. Knocking down BV8 in human myeloid leukemia cells inhibits STAT3 activity and expression of STAT3 downstream angiogenic and pro-proliferation/survival genes, leading to a decrease in tumor cell viability. BV8 shRNA expressing leukemia cells exhibit reduced STAT3 activity and tumor growth in vivo. Taken together, we have delineated a signaling pathway downstream of BV8 that plays critical roles in both the tumor microenvironment and malignant myeloid cells for angiogenesis and tumor cell proliferation/survival.

Background Several previous studies have identified a strong association between T-cell infiltration and clinical outcome in ovarian cancer. The role of B-cells remains controversial, however. Methods Forty-nine paraffin-embedded omental specimens derived from patients with high grade epithelial ovarian cancer were assessed. Immunohistochemical analyses were performed to characterize expression of CD19+ B-cells and pSTAT3 as high (>50% positively staining cells [PSCs]) or low (

STAT3 plays a crucial role in promoting progression of human cancers, including several types of B-cell lymphoma. However, as a transcription factor lacking its own enzymatic activity, STAT3 remains difficult to target with small-molecule drugs in the clinic. Here we demonstrate that persistent activated STAT3 colocalizes with elevated expression of S1PR1, a G-protein–coupled receptor for sphingosine-1-phosphate (S1P), in the tumor cells of the activated B cell–like subtype of diffuse large B-cell lymphoma patient specimens. Inhibition of S1PR1 expression by shRNA in the lymphoma cells validates that blocking S1PR1 affects expression of STAT3 downstream genes critically involved in tumor cell survival, proliferation, tumor invasion, and/or immunosuppression. Using S1PR1 shRNA, or FTY720, an antagonist of S1P that is in the clinic for other indications, we show that inhibiting S1PR1 expression down-regulates STAT3 activity and causes growth inhibition of the lymphoma tumor cells in vitro and in vivo. Our results suggest that targeting S1P/S1PR1 using a clinically relevant and available drug or other approaches is potentially an effective new therapeutic modality for treating the activated B cell–like subtype of diffuse large B-cell lymphoma, a subset of lymphoma that is less responsive to current available therapies.

The mechanisms underlying hypermethylation of tumor-suppressor gene promoters in cancer is not well understood. Here, we report that lysine acetylation of the oncogenic transcription factor STAT3 is elevated in tumors. We also show that genetically altering STAT3 at Lys685 reduces tumor growth, which is accompanied by demethylation and reactivation of several tumor-suppressor genes. Moreover, mutating STAT3 at Lys685 disrupts DNA methyltransferase 1–STAT3 interactions in cultured tumor cells and in tumors. These observations are confirmed by treatment with an acetylation inhibitor, resveratrol. Furthermore, reduction of acetylated STAT3 in triple-negative breast cancer cells leads to demethylation and activation of the estrogen receptor -α gene, sensitizing the tumor cells to antiestrogens. Our results also demonstrate a correlation between STAT3 acetylation and methylation of estrogen receptor -α in melanoma, which predicts melanoma progression. Taken together, these results suggest a role of STAT3 acetylation in regulating CpG island methylation, which may partially explain aberrant gene silencing in cancer. These findings also provide a rationale for targeting acetylated STAT3 for chemoprevention and cancer therapy.

Signal transducer and activator of transcription 3 (Stat3) has emerged as a critical regulator for tumor-associated inflammation. Activation of Stat3 negatively regulates the Th1-type immune response and promotes expansion of myeloid-derived suppressor cells (MDSCs) and regulatory T-cell functions in the tumor microenvironment. Mounting evidence suggests that Stat3 and related pathways may serve as a target for changing the tumor immunologic microenvironment to benefit cancer immunotherapies. Many recent studies support the use of certain tyrosine kinase inhibitors, through inhibition of Stat3, in decreasing immunosuppression in the tumor microenvironment. Other potential therapeutic avenues include the use of targeted delivery of Stat3 siRNA into immune cells. Here, we describe the role of Stat3 in regulating the immunologic properties of tumors as a background for Stat3-based therapeutic interventions.

Hypoxia-inducible factor 1 (HIF-1) is a potent tumorigenic factor. Its α subunit (HIF-1α), which is tightly regulated in normal tissues, is elevated in tumors due to hypoxia and overactive growth signaling pathways. Although much is known about HIF-1α regulation in cancer cells, crucial molecular targets that affect HIF-1α levels modulated by both hypoxia and oncogenic signaling pathways remain to be identified. Additionally, whether and how the tumor microenvironment contributes to HIF-1α accumulation is unclear. This study shows a novel mechanism by which HIF-1α availability is regulated in both cancer cells and in myeloid cells in the tumor microenvironment. We show a requirement of signal transducer and activator of transcription 3 (Stat3) for HIF-1α RNA expression under both hypoxia and growth signaling conditions. Furthermore, tumor-derived myeloid cells express elevated levels of HIF-1α mRNA relative to their counterparts from normal tissues in a Stat3-dependent manner. Additionally, Stat3 activity in the nontransformed cells in the tumor milieu affects HIF-1α RNA expression of the entire growing tumor. Consistent with a role of Stat3 in regulating HIF-1α RNA transcription, elevated Stat3 activity increases HIF-1α promoter activity, and Stat3 protein binds to the HIF-1α promoter in both transformed cells and in growing tumors. Taken together, these findings show a novel mode by which HIF-1α is regulated not only in cancer cells but also in the tumor-associated inflammatory cells, suggesting Stat3 as an important molecular target for inhibiting the oncogenic potential of HIF-1 induced by both hypoxia and overactive growth signaling pathways prevalent in cancer. (Mol Cancer Res 2008;6(7):1099–105)

Interactions between tumor and immune cells either enhance or inhibit cancer progression. We show here that Stat3 signaling within the tumor microenvironment induces a pro-carcinogenic cytokine, IL-23, while inhibiting a central anti-carcinogenic cytokine, IL-12, thereby shifting the balance of tumor immunity towards carcinogenesis. Stat3 induces expression of IL-23, which is mainly produced by tumor-associated macrophages, via direct transcriptional activation of the IL-23/p19 gene. Furthermore, Stat3 inhibits NF-κB/c-Rel-dependent IL-12/p35 gene expression in tumor-associated dendritic cells. Tumor-associated regulatory T cells (Treg) express IL-23 receptor (IL-23R) which activates Stat3 in this cell type, leading to upregulation of the Treg-specific transcription factor, Foxp3, and the immunosuppressive cytokine, IL-10. These results demonstrate that Stat3 promotes IL-23-mediated pro-carcinogenic immune responses while inhibiting IL-12-dependent anti-tumor immunity.

Histone deacetylases (HDACs) are enzymes that regulate the functions of histones as well as nonhistones by catalyzing the removal of acetyl groups from lysine residues. HDACs regulate many biological processes, including the cell division cycle and tumorigenesis. Although recent studies have implicated HDAC8 in tumor cell proliferation, the molecular mechanisms linking HDAC8 to cell growth remain unknown. Here, we report that the human ortholog of the yeast ever-shorter telomeres 1B (EST1B) binds HDAC8. This interaction is regulated by protein kinase A-mediated HDAC8 phosphorylation and protects human EST1B (hEST1B) from ubiquitin-mediated degradation. Phosphorylated HDAC8 preferentially recruits Hsp70 to a complex that inhibits the CHIP (C-terminal heat shock protein interacting protein) E3 ligase-mediated degradation of hEST1B. Importantly, HDAC8 regulation of hEST1B protein stability modulates total telomerase enzymatic activity. Our findings reveal a novel mechanism by which HDAC8 contributes to tumorigenesis by regulating telomerase activity.

In eukaryotes, genomic DNA is wrapped tightly around core histones to form nucleosomes, the fundamental building blocks of chromatin. Nucleosomes, once regarded as inert structural particles, are now considered integral and dynamic components of the machineries responsible for gene regulation. Many different enzymes and protein complexes are known to bring about changes in the state of chromatin by numerous mechanisms, with resultant effects on gene expression. One class of complexes alters DNA packaging (remodels chromatin) in an ATP-dependent manner (4, 29). Another class of chromatin-altering factors acts by covalently modifying histone proteins (5). These modifications include acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation. The best-characterized posttranslational histone modification is acetylation, which is catalyzed by histone acetyltransferase (HAT) enzymes. Histone acetylation is a reversible process that is regulated by the opposing activities of HATs and histone deacetylases (HDACs). Generally, hyperacetylation of histones results in transcriptional activation whereas deacetylation correlates with transcriptional silencing. Consistent with this generalization, transcriptional activators are often associated with HAT activity whereas HDACs frequently form complexes with transcriptional repressors (24). Therefore, these two regulatory processes work in harmony to achieve appropriate levels of gene expression. Several oncogenes and tumor suppressors (pRb, BRCA-1, BRCA-2, PML-RAR, and a zinc finger protein mutated in leukemia) have been shown to be associated with HATs or HDACs (41). HDAC proteins are vital regulators of fundamental cellular events, including cell cycle progression, differentiation, and tumorigenesis (37, 45). A small-molecule inhibitor of HDAC, trichostatin A (TSA), arrests mammalian cells in both G1 and G2 (31, 44), while overexpression of HDAC1 in mouse cells reduces their growth rate by lengthening the duration of G2 and M (3). TSA induces terminal differentiation of mouse erythroleukemia cells and apoptosis of lymphoid and colorectal cancer cells. In addition, TSA treatment of cells expressing the PML zinc finger protein derepresses transcription and allows cells to differentiate normally (18). With this precedent, HDAC inhibitors are being actively explored as potential agents for the treatment of certain forms of cancer (22, 23, 27). The human HDACs are organized into three different classes based on their similarity to yeast HDAC proteins (37, 45). Class I enzymes are ubiquitously expressed and include HDAC1, -2, -3, and -8, which are homologous to the yeast RPD3 protein. Class II includes HDAC4, -5, -6, -7, -9, and -10, which are similar to yeast HDA1 and are expressed in a tissue-specific manner. The Sir2-like class III HDACs, including SIRT1 to -7, require NAD+ for enzymatic activity. The most recent addition to the human HDAC family, HDAC11, uniquely shares sequence homology with the catalytic regions of both class I and II HDAC enzymes (15). By far, the most frequently studied and best-characterized human HDACs are HDAC1 and HDAC2. Early studies elegantly demonstrated that HDAC1 and HDAC2 were associated with proteins that modulate their enzymatic activity and their recruitment to genomic regions. Three large multisubunit protein complexes, called Sin3, NuRD/Mi2, and CoREST, contain HDAC1 and HDAC2 (1, 17, 21, 25, 30, 38, 42, 46-48). In addition to complex formation, recent studies have revealed that the activity of class I HDACs is regulated by posttranslational modifications. For example, HDAC1 is a substrate for SUMO-1 (small ubiquitin-related modifier 1), and mutations of the target residues decrease transcriptional repression without affecting the ability of HDAC1 to associate with mSin3 (10). In addition, like those of many class II HDACs, the actions of HDAC1 and HDAC2 are regulated by phosphorylation. Phosphorylation of HDAC1 by protein kinase CK2 alters HDAC1's enzymatic activity and its capacity to form protein complexes (7, 13, 33). Similarly, phosphorylation of HDAC2 by protein kinase CK2 is essential for HDAC2's deacetylase activity and its association with mSin3, Mi2, Sp1, and Sp3 (36, 39). Our previous studies showed that, like HDAC1 and HDAC2, HDAC3 also is phosphorylated by protein kinase CK2 (39). Surprisingly, unlike other members of the class I HDAC family, HDAC8 is not phosphorylated by protein kinase CK2 (39). However, it is possible that kinases other than protein kinase CK2 phosphorylate HDAC8 and modulate its activity. A complete understanding of how phosphorylation regulates the actions of class I HDACs requires a thorough determination of whether HDAC8 is a phosphoprotein and, if so, what kinase is responsible and what the functional consequences are. HDAC8 cDNA was identified initially by three independent groups using sequence homology database searches with class I HDAC proteins (6, 20, 40). The HDAC8 gene encodes a 377-amino-acid protein with a predicted molecular mass of 45 kDa and is located on the X chromosome at position q21.2-q21.3 or q13 (6, 40). Protein sequence comparisons of HDAC8 reveal a 37% similarity to HDAC1. In Northern blot analyses, the size of HDAC8 mRNA is between 1.7 and 2.4 kb, and HDAC8 mRNA is expressed in multiple human organs, including the liver, heart, brain, lung, pancreas, placenta, prostate, and kidney. Consistent with the presence of a stretch of basic residues that could serve as a nuclear localization signal, HDAC8 is predominantly located in the nucleus. A recent report suggests that the inv (16) fusion protein specifically associates with HDAC8 (11). Although sequence analysis of HDAC8 revealed consensus phosphorylation sites for protein kinase A (PKA) and protein kinase CK2, our previous studies showed that HDAC8 was not phosphorylated by protein kinase CK2 in vitro (39). In the present study, we show that HDAC8 is phosphorylated instead by PKA both in vitro and in vivo. Most interestingly, phosphorylation of HDAC8 by PKA inhibits its deacetylase activity, which results in the hyperacetylation of histones H3 and H4. Thus, our findings uncover a novel mechanism of class I HDAC regulation.

Recent studies suggested that the protection of cell apoptosis by AKT involves phosphorylation and inhibition of FKHR and related FOXO forkhead transcription factors and that androgens provide an AKT-independent cell survival signal in prostate cancer cells. Here, we report receptor-dependent repression of FKHR function by androgens in prostate cancer cells. Transcriptional analysis demonstrated that activation of the androgen receptor caused an inhibition of both wild-type FKHR and a mutant in which all three known AKT sites were mutated to alanines, showing that the repression is AKT independent. In vivo and in vitro coprecipitation studies demonstrated that the repression is mediated through protein-protein interaction between FKHR and the androgen receptor. Mapping analysis localized the interacting domains to the carboxyl terminus between amino acids 350 and 655 of FKHR and to the amino-terminal A/B region and the ligand binding domain of the receptor. Further analysis demonstrated that the activated androgen receptor blocked FKHR's DNA binding activity and impaired its ability to induce Fas ligand expression and prostate cancer cell apoptosis and cell cycle arrest. These studies identify a new mechanism for androgen-mediated prostate cancer cell survival that appears to be independent of the activity of the receptor on androgen response element-mediated transcription and establish FKHR and related FOXO forkhead proteins as important nuclear targets for both AKT-dependent and -independent survival signals in prostate cancer cells.

Estrogens are mitogens that stimulate the growth of both normal and transformed epithelial cells of the female reproductive system. The effect of estrogens is mediated through the estrogen receptors, which are ligand-regulated transcription factors. Tamoxifen, a selective estrogen receptor modulator, functions as an estrogen receptor antagonist in breast but an agonist in uterus. In the current study, we show that coexpression of a constitutively active MEKK1, but not RAF or MEKK2, significantly increases the transcriptional activity of the receptor in endometrial and ovarian cancer cells. The expression of wild-type MEKK1 and an active Rac1, which functions upstream of MEKK1, also increased the activity of the receptor while coexpression of dominant negative MEKK1 blocked the Rac1 induction, indicating that endogenous MEKK1 is capable of activating the receptor. Additional experiments demonstrated that the MEKK1-induced activation was mediated through both Jun N-terminal kinases and p38/Hog1 and was independent of the known phosphorylation sites on the receptor. p38, but not Jun N-terminal kinases, efficiently phosphorylated the receptor in immunocomplex kinase assays, suggesting a differential involvement of the two kinases in the receptor activation. More importantly, the expression of the constitutively active MEKK1 increased the agonistic activity of 4-hydroxytamoxifen to a level comparable to that of 17beta-estradiol and fully blocked its antagonistic activity. These findings suggest that the uterine-specific agonistic activity of the tamoxifen compound may be determined by the status of kinases acting downstream of MEKK1.

One of the most recently recognized signaling pathways that regulate tumor cell proliferation and survival involves signal transducers and activators of transcription (STAT) proteins. The STAT family of proteins has seven known members: Stat1, 2, 3, 4, 5A, 5B, and 6. Interestingly, it has become evident that dif ferent STAT proteins can serve either tumor suppressing or oncogenic roles. Stat1, for example, functions as a tumor suppressor in several capacities ( 1 – 5 ) , whereas Stat3 and to a lesser extent, Stat5, play a critical role in malignant progression at multiple levels ( 6 – 9 ) . Although it has been demonstrated that interferon-induced Stat1 activity can cause growth arrest and can induce apoptosis, it is not clear whether a lack of Stat1 in human cancer directly contributes to cancer cell proliferation and survival. If so, what keeps Stat1 from functioning in this manner in tumor cells also remains largely unknown. The article by Xi et al. ( 10 ) in this issue of the Journal presents evidence that Stat1 expression in squamous cell carcinoma of the head and neck (SCCHN) is lower than that of normal tissues from individuals without cancer. Restoring Stat1 expression in the tumor cells leads to growth inhibition in vitro and in xenograft tumors, which is accompanied by an increase in p21 expression. Their work further demonstrates that the low expression of Stat1 in SCCHN tumors is associated with Stat1 promoter methylation and that treatment with a methylation inhibitor increases STAT1 and p21 expression and sensitizes SCCHN tumor cells to cisplatin. These fi ndings are novel in several respects. Many independent studies, which involve mainly mouse tumor models or cell lines, have suggested that Stat1 can function as a tumor suppressor ( 1 – 5 ) . How does Stat1 function as a tumor suppressor? Stat1’s ability to mediate host immune defenses against tumors has been elegantly demonstrated in mice ( 2 ) . Increasing Stat1 activity by interferon treatment also leads to cell growth inhibition, which can be explained by Stat1’s capacity to induce p21waf and caspase expression ( 1 , 5 ) . Moreover, Stat1defi cient cells have defects both in S-phase and G 2 – M checkpoints in response to DNA damage ( 11 ) . Also, Stat1and p53-null mice show more frequent and rapid tumor development than wild-type mice. The basal expression level of the p53 inhibitor Mdm2 is higher in Stat1 − / − cells than in wild-type cells, suggesting that Stat1 is a negative regulator of Mdm2 expression ( 12 ) . The fi ndings by Xi et al. provide direct evidence supporting the

Objectives: High-risk prostate cancer (PC) represents a heterogeneous disease, and metastatic potential varies within this subset of patients. The occurrence of metastasis involves both tumor cells and tumor-associated stromal cells. In the present study, murine models were used to examine the association between Stat3 activation and tumor-associated stromal cells. The latter may confer metastatic potential through the formation of pre-metastatic niches. These parameters were subsequently assessed in the benign lymph nodes of patients with high-risk PC. Methods: To induce pre-metastatic conditions, we injected tumor-conditioned media from murine tumor cell lines over-expressing the Stat3 activator, S1PR1, into C57BL/6 mice. Subsequently, mice were challenged with parental tumor cells via tail vein injection. For assessing a potential role of Stat3 in formation of the pre-metastatic niche, we analyzed mouse lung tissue by immunofluorescent staining and Western blot. We also increased and decreased myeloid cell Stat3 activity by retrovirus transduction and genetic ablation, respectively. For clinical correlation, paraffin-embedded lymph node tissue derived from 50 patients with high-risk PC was acquired. High-risk PC was defined by standard criteria as follows: (1) baseline prostate specific antigen (PSA) ≫ 20, (2) clinical stage T3a disease, or (3) Gleason score 8-10. Immunohistochemistry (IHC) staining was performed to assess pStat3 levels and CD68+ myeloid cell clustering. Results: In the murine models examined, increasing Stat3 activity in tumor cells by its newly identified activator, S1PR1, produced factors that activated Stat3 in lung tissue. This led to a dramatic increase in myeloid clustering and metastasis. Up-regulating Stat3 activity within myeloid cells enhanced the interaction between myeloid cells and endothelial cells in vivo, increased angiogenesis, and promoted subsequent formation of metastasis. In contrast, ablation of Stat3 in myeloid cells prevented the formation of pre-metastatic niches and metastasis. IHC staining of benign lymph node tissue in patients with high-risk PC yielded a spectrum of pStat3 levels. Higher levels of pStat3 were qualitatively associated with infiltration of CD68+ macrophages, appearing in distinct clusters. Conclusion: The current study indicates a critical role of Stat3 in promoting metastatic potential, possibly through mediating myeloid cell infiltration and formation of pre-metastatic niches. In the context of high-risk PC, differential levels of pStat3 and a potential association with myeloid cell clustering was observed. While correlation between these parameters and clinical outcome is ongoing, these preliminary findings suggest a putative role for Stat3 as a therapeutic target to prevent and/or reduce pre-metastatic niche formation and metastasis. 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 405. doi:10.1158/1538-7445.AM2011-405

NF-kappaB (RelA) is constitutively active in many cancers, where it upregulates antiapoptotic and other oncogenic genes. While proinflammatory stimulus-induced NF-kappaB activation involves IKK-dependent nuclear translocation, mechanisms for maintaining constitutive NF-kappaB activity in tumors have not been elucidated. We show here that maintenance of NF-kappaB activity in tumors requires Stat3, which is also frequently constitutively activated in cancer. Stat3 prolongs NF-kappaB nuclear retention through acetyltransferase p300-mediated RelA acetylation, thereby interfering with NF-kappaB nuclear export. Stat3-mediated maintenance of NF-kappaB activity occurs in both cancer cells and tumor-associated hematopoietic cells. Both murine and human cancers display highly acetylated RelA, which is associated with Stat3 activity. This Stat3/NF-kappaB interaction is thus central to both the transformed and nontransformed elements in tumors.