Faculty Bibliography

Signal transducer and activator of transcription-3 (STAT3) is critical for cancer progression by regulating tumor cell survival, proliferation, and angiogenesis. Herein, we investigated the regulation of STAT3 activation and the therapeutic effects of Icaritin, a prenyl flavonoid derivative from Epimedium Genus, in renal cell carcinoma (RCC). Icaritin showed significant anti-tumor activity in the human and mouse RCC cell lines, 786-O and Renca, respectively. Icaritin inhibited both constitutive and IL-6-induced phospho-STAT3 (STAT3(Y705)) and reduced the level of STAT3-regulated proteins Bcl-xL, Mcl-1, Survivin, and CyclinD1 in a dose-dependent manner. Icaritin also inhibited activation of Janus-activated kinase-2 (JAK2), while it showed minimal effects on the activation of other key signaling pathways, including AKT and MAPK. Expression of the constitutively active form of STAT3 blocked Icaritin-induced apoptosis, while siRNA directed against STAT3 potentiated apoptosis. Finally, Icaritin significantly blunted RCC tumor growth in vivo, reduced STAT3 activation, and inhibited Bcl-xL and Cyclin E, as well as VEGF expression in tumors, which was associated with reduced tumor angiogenesis. Overall, these results suggest that Icaritin strongly inhibits STAT3 activation and is a potentially effective therapeutic option for the treatment of renal cell carcinoma.

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.

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.