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.

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

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.

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

IL-6/Jak2 signaling is viewed critical for persistent Stat3 activation in cancer. However, IL-6-induced Stat3 activity is transient in normal physiology. Here we identify a mechanism important for persistent Stat3 activation in tumor cells and the tumor microenvironment. We show that sphingosine-1-phosphate receptor 1 (S1PR1), a G-protein-coupled receptor for lysophospholipid sphingosine-1-phosphate (S1P), is elevated in Stat3-positive tumors. Stat3 is a transcription factor for the S1pr1 gene. Enhanced S1pr1 expression activates Stat3 and upregulates Il6 gene expression, thereby accelerating tumor growth and metastasis. Conversely, 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 demonstrate that Stat3-induced S1pr1 expression, as well as S1P/S1PR1 pathway, is important for persistent Stat3 activation in cancer cells and the tumor microenvironment and for malignant progression.

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.

SUMMARY 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. SIGNIFICANCE Recent studies suggest that two related cytokines, IL- 23 and IL- 12, play opposite roles in carcinogenesis. However, the underlying mechanisms regulating the balance between these cytokines in the tumor microenvironment have not been elucidated. Mechanisms by which IL-23 promotes tumor immune evasion also remain to be explored. Our results reveal that Stat3 signaling in the tumor microenvironment regulates the IL-12/IL-23 balance and further, that IL- 23 enhances the immunosuppressive activity of regulatory T cells within the tumor microenvironment, in part via IL-23 receptor dependent Stat3 activation. Because Stat3 is a point of convergence for signaling pathways commonly activated in cancer, our data reveal a mechanism by which oncogenic pathways regulate the immune microenvironment to promote tumor development.

SUMMARY Recent 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 pre-metastatic sites, leading to pre-metastatic niche formation. Targeting either S1PR1 or STAT3 in myeloid cells disrupts existing pre-metastatic 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.