Jiehui Deng

S1PR1-STAT3 Signaling Is Crucial for Myeloid Cell Colonization at Future Metastatic Sites

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.

Targeting Stat3 in the Myeloid Compartment Drastically Improves the In vivo Antitumor Functions of Adoptively Transferred T Cells

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-γ. 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.

S1PR1 is an effective target to block STAT3 signaling in activated B-cell like diffuse large B-cell lymphoma

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.

B Cells Promote Tumor Progression via STAT3 Regulated-Angiogenesis

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.

Icaritin Inhibits JAK/STAT3 Signaling and Growth of Renal Cell Carcinoma

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 (STAT3Y705) 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.

Prognostic Significance of B-Cells and pSTAT3 in Patients with Ovarian Cancer

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 (<50% PSCs). The Kaplan-Meier method with log-rank test was used to determine the association between clinicopathologic parameters and overall survival (OS). A multi-variate Cox proportional hazards regression analysis including nature of debulking (primary vs secondary), histology, tumor grade, receipt of prior chemotherapy, B-cell infiltration and pSTAT3 expression was performed. Results Median OS was 160.6 months in those patients with low B-cell expression vs 47.3 months in those with high B-cell expression (P = 0.0015). Similarly, median OS was improved in those patients with low pSTAT3 expression (160.6 vs 47.9 months, P = 0.02). In a multivariate model to predict survival, only the degree of B-cell infiltration and clinical stage were retained. pSTAT3 expression did not enter the final model, possibly be due to a high positive correlation with B-cell infiltration (r = 0.82, P<0.0001). Conclusions Increased B-cell infiltration and pSTAT3 expression in omental tissue are associated with poorer survival.

G-protein-coupled Receptor Agonist BV8/Prokineticin-2 and STAT3 Protein Form a Feed-forward Loop in Both Normal and Malignant Myeloid Cells

Background: Signaling pathways underlying BV8-mediated oncogenesis remain unknown. Results: BV8-STAT3 forms a feed-forward loop in both normal and malignant myeloid cells and promotes tumor growth. Conclusion: JAK2/STAT3 signaling plays critical roles in BV8-mediated myeloid cell-dependent oncogenesis. Significance: 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.

CD8+ T‐cell immunosurveillance constrains lymphoid premetastatic myeloid cell accumulation

Increasing evidence suggests that premetastatic niches, consisting mainly of myeloid cells, provide microenvironment critical for cancer cell recruitment and survival to facilitate metastasis. While CD8+ T cells exert immunosurveillance in primary human tumors, whether they can exert similar effects on myeloid cells in the premetastatic environment is unknown. Here, we show that CD8+ T cells are capable of constraining premetastatic myeloid cell accumulation by inducing myeloid cell apoptosis in C57BL/6 mice. Ag‐specific CD8+ T‐cell cytotoxicity against myeloid cells in premetastatic lymph nodes is compromised by Stat3. We demonstrate here that Stat3 ablation in myeloid cells leads to CD8+ T‐cell activation and increased levels of IFN‐γ and granzyme B in the premetastatic environment. Furthermore, Stat3 negatively regulates soluble Ag cross‐presentation by myeloid cells to CD8+ T cells in the premetastatic niche. Importantly, in tumor‐free lymph nodes of melanoma patients, infiltration of activated CD8+ T cells inversely correlates with STAT3 activity, which is associated with a decrease in number of myeloid cells. Our study suggested a novel role for CD8+ T cells in constraining myeloid cell activity through direct killing in the premetastatic environment, and the therapeutic potential by targeting Stat3 in myeloid cells to improve CD8+ T‐cell immunosurveillance against metastasis.

Use of VEGFR1 expression in benign pelvic lymph nodes to predict biochemical recurrence in patients with high-risk prostate cancer.

205 Background: High-risk prostate cancer (PCa) is a heterogeneous disease, and biomarkers that accurately predict clinical outcome within this subset are lacking. The pre-metastatic niche (PMN) represents one possible biomarker-this harbinger of future metastasis may be characterized by infiltration of VEGFR1+ cells [Kaplan et al Nature 139:820, 2005]. Given the predilection of PCa to spread to nodal sites, the association between VEGFR1 expression in benign pelvic lymph nodes and clinical outcome was examined. METHODS The City of Hope Prostate Cancer Registry (COH PCR) was used to identify 46 patients with high-risk PCa (baseline PSA > 20, pT3a-4 disease, or biopsy Gleason 8-10) who had undergone radical prostatectomy and pelvic lymph node dissection (PLND). The COH PCR prospectively collects clinical data associated with patients undergoing prostatectomy at the institution, and warehouses available clinical specimens. Benign tissue specimens derived from PLND were acquired for each patient, and were stained for VEGFR1 expression. VEGFR1+ cell clusters were counted within 8 distinct 40x fields, and the cluster count was averaged. RESULTS VEGFR1+ clustering in benign PLND specimens was a significant predictor of biochemical recurrence on multivariate Cox proportional hazards analysis, and outperformed other variables including established prognostic factors such as age, extracapsular spread, seminal vesicle invasion, and the aforementioned high-risk features. Patients with increased VEGFR1+ clustering pelvic lymph node tissue had a shorter interval to biochemical recurrence (HR 0.18, P<0.10). CONCLUSIONS These preliminary results indicate that increased VEGFR1+ cell clustering in benign nodal tissue may predict poorer clinical outcome in patients with high-risk PCa. VEGFR1+ cells (a purported constituent of the PMN) represents a targetable entity. A randomized, phase II study employing axitinib (VEGFR1 IC50=1 nM) as neoadjuvant therapy in patients with high-risk PCa is nearing initiation at City of Hope ( NCT01385059 ).

Chapter 26 – JAK/STAT Signaling in Myeloid Cells: Targets for Cancer Immunotherapy

Tumor-associated immune cells, in particular myeloid cells, have opposing roles during cancer development by facilitating antitumor immune responses and driving cancer-promoting inflammation. Defective antitumor immunity is prevalent in cancers, and it is now clear that overcoming the myeloid cell-mediated immunosuppressive microenvironment poses tremendous interest for future cancer therapies. JAK/STAT signaling has come to the forefront as a crucial pathway to induce immunosuppression and procancer inflammation. Specifically, STAT3 activation is critical for the phenotype of myeloid cells by regulating immunosuppressive and prometastatic factors, thereby providing myeloid cells with a multitude of tumor-promoting functions. Genetic ablation of STAT3 in the myeloid compartment induces potent innate and adaptive antitumor immunity along with an inhibition of tumor growth and metastasis. Recently, therapeutic targeting of JAK/STAT3 has shown great promise in blocking immunosuppression in preclinical models. One such example is the use of novel siRNA to selectively target STAT3 in myeloid cells, through conjugation to CpG oligonucleotides that agonize Toll receptor TLR9 on myeloid cells. Along with other novel therapeutic strategies to inhibit JAK/STAT signaling, it seems likely that future efforts to target this pathway will be made in single and combination approaches for effective anticancer immunotherapy.