Faculty Bibliography

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.