Faculty Bibliography

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 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-kappaB 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-kappaB-inducible genes involved in innate and adaptive antitumor immunity in spite of elevated levels of NF-kappaB in tumors. In this study, we show that expression of many NF-kappaB 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-kappaB interacts with STAT3 and preferentially binds to genes with STAT3-binding site(s) in promoters. A large number of NF-kappaB 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-kappaB 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-kappaB in tumor cells. Furthermore, STAT3 facilitates NF-kappaB 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.

Adoptive cell therapy with engineered T cells to improve natural immune response and antitumor functions has shown promise for treating cancer. However, the requirement for extensive ex vivo manipulation of T cells and the immunosuppressive effects of the tumor microenvironment limit this therapeutic modality. In the present study, we investigated the possibility to circumvent these limitations by engineering Stat3 -deficient CD8(+) T cells or by targeting Stat3 in the tumor microenvironment. We show that ablating Stat3in CD8(+) T cells prior to their transfer allows their efficient tumor infiltration and robust proliferation, resulting in increased tumor antigen-specific T-cell activity and tumor growth inhibition. For potential clinical translation, we combined adoptive T-cell therapy with a Food and Drug Administration-approved tyrosine kinase inhibitor, sunitinib, in renal cell carcinoma and melanoma tumor models. Sunitinib inhibited Stat3 in dendritic cells and T cells and reduced conversion of transferred FoxP3(-) T cells to tumor-associated regulatory T cells while increasing transferred CD8(+) T-cell infiltration and activation at the tumor site, leading to inhibition of primary tumor growth. These data show that adoptively transferred T cells can be expanded and activated in vivo either by engineering Stat3-silenced T cells or by targeting Stat3 systemically with small-molecule inhibitors.

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.

Improving effector T-cell functions is highly desirable for preventive or therapeutic interventions of diverse diseases. Signal transducer and activator of transcription 3 (Stat3) in the myeloid compartment constrains Th1-type immunity, dampening natural and induced antitumor immune responses. We have recently developed an in vivo small interfering RNA (siRNA) delivery platform by conjugating a Toll-like receptor 9 agonist with siRNA that efficiently targets myeloid and B cells. Here, we show that either CpG triggering combined with the genetic Stat3 ablation in myeloid/B cell compartments or administration of the CpG-Stat3siRNA drastically augments effector functions of adoptively transferred CD8+ T cells. Specifically, we show that both approaches are capable of increasing dendritic cell and CD8(+) T-cell engagement in tumor-draining lymph nodes. Furthermore, both approaches can significantly activate the transferred CD8(+) T cells in vivo, upregulating effector molecules such as perforin, granzyme B, and IFN-gamma. Intravital multiphoton microscopy reveals that Stat3 silencing combined with CpG triggering greatly increases killing activity and tumor infiltration of transferred T cells. These results suggest the use of CpG-Stat3siRNA, and possibly other Stat3 inhibitors, as a potent adjuvant to improve T-cell therapies.

Efficient delivery of small interfering (si)RNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We show that siRNA synthetically linked to a CpG oligonucleotide agonist of toll-like receptor (TLR)9 targets and silences genes in TLR9(+) myeloid cells and B cells, both of which are key components of the tumor microenvironment. When a CpG-conjugated siRNA that targets the immune suppressor gene Stat3 is injected in mice either locally at the tumor site or intravenously, it enters tumor-associated dendritic cells, macrophages and B cells. Silencing of Stat3 leads to activation of tumor-associated immune cells and ultimately to potent antitumor 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.

Hypoxia-inducible factor 1 (HIF-1) is a potent tumorigenic factor. Its alpha subunit (HIF-1alpha), 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-1alpha regulation in cancer cells, crucial molecular targets that affect HIF-1alpha levels modulated by both hypoxia and oncogenic signaling pathways remain to be identified. Additionally, whether and how the tumor microenvironment contributes to HIF-1alpha accumulation is unclear. This study shows a novel mechanism by which HIF-1alpha 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-1alpha RNA expression under both hypoxia and growth signaling conditions. Furthermore, tumor-derived myeloid cells express elevated levels of HIF-1alpha 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-1alpha RNA expression of the entire growing tumor. Consistent with a role of Stat3 in regulating HIF-1alpha RNA transcription, elevated Stat3 activity increases HIF-1alpha promoter activity, and Stat3 protein binds to the HIF-1alpha promoter in both transformed cells and in growing tumors. Taken together, these findings show a novel mode by which HIF-1alpha 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.