Physiologically, STAT3 transcription factor is a critical negative regulator of tissue wound healing that induces pleiotrophic brake on inflammatory cytokines, chemokines, and angiogenic factors to prevent tissue injury during the chronic phase of wound healing (28
). It is not surprising that neoplastic epithelial cells have been selected for constitutively active STAT3 as a potent immune evasion mechanism. Previous work from our lab in the murine system showed that tumor cells could co-opt STAT3’s physiological function to prevent an anti-tumor host immune response (19
In this report, we demonstrated that STAT3 inhibition in human HNSCC cells could also induce upregulation and secretion of multiple cytokines and chemokines for potential antitumor response in the microenvironment. Both siRNA silencing and pharmacologic small molecule inhibition of STAT3 were used to substantially reduce STAT3 signaling in the human tumor cells. As demonstrated by this report, HNSCC differentially expressed multiple paracrine factors that can potentially inhibit LPS-induced DC maturation. STAT3 suppression in HNSCC cell lines dramatically reversed this immunosuppressive phenotype in vitro. We also showed that these STAT3 dependent tumor derived factors could stimulate migration of lymphocytes in vitro. The control tumor cells were treated with scrambled siRNA to ensure that variables that can be attributed to transfection or transduction of small RNA sequences did not confound the paracrine effects. Moreover, the two independent methods of STAT3 inhibition inducing comparable effects corroborate our hypothesis that these paracrine cross talk are STAT3-dependent.
We reasoned that focusing on a single cytokine or chemokine may not reveal the combinatorial effects of multiple factors that can direct cellular behaviors within the tumor microenvironment. To directly examine the various combination of STAT3 induced cytokines and chemokines, we tested the unbiased conditioned media from the STAT3 siRNA treated tumor cell line culture supernatants to examine the paracrine crosstalk between the human tumor cells and the isolated human immune cells. Although we tested for IP-10, IL-6, IL-8, and VEGF protein levels in the culture supernatants, this report does not address the possibility of the differential expression pattern of other proinflammatory factors that may shape the tumor microenvironment. Our preliminary experiments with VEGF to examine if a single STAT3 dependent factor can mediate some of the anti-tumor phenotypes of the tumor microenvironment suggest that STAT3 signaling induces transcriptional variations of multiple paracrine factors that work combinatorially to produce an anti-tumor response. We are currently investigating the presence of other important cytokines, such as IL12 and IL23, as well as combinatorial effects of multiple paracrine factors mediating DC activation and lymphocytic chemotaxis in vitro. It would be also very important to study the relationship between corresponding receptors of these proinflammatory mediators and STAT3 signaling suppression in the same tumor cells as well as in the immune cells.
Demonstrating IP10 upregulation consistently and significantly in all the cell lines treated with STAT3 siRNA was an interesting phenomena because it can potentially mediate antitumor activity through two independent mechanisms: by stimulation of leukocytic migration into tumor microenvironment and by inhibition of tumor angiogenesis. It is also well documented in mouse tumor models the synergistic antitumor effects of IP10 in conjunction with IL12 or with TNFα (30
). This “attraction-expansion” hypothesis is based on the idea that colocalization of proinflammatory and chemotactic mediators could enhance synergistically the anti-tumoral immune response (32
). Our results showed concomitant upregulation of various STAT3 dependent immunomodulatory factors that can act as the “recruiters” as well as “activators”.
The finding that blockade of STAT3 in HNSCC cancer cell lines could reverse the inhibitory effects of tumor cell supernatant on DC maturation suggested that VEGF may be a critical STAT3-dependent immunomodulator than can interfere with DC differentiation and function in cancer patients. Gabrilovich et al
. documented that there was a direct association between the elevated numbers of immature DCs in cancer patients and high levels of circulating VEGF (33
). In our in vitro studies, STAT3 dependent VEGF alone did not appear to directly mediate DC maturation. Interestingly, although we focused our initial studies on DC maturation, STAT3 dependent VEGF may induce other hematopoietic cells such as myeloid derived suppressor cells to shape the tumor microenvironment towards an immunosuppressive phenotype.
The role of IL-6 in tumorigenesis has been well documented, and a recent cohort study showed that serum IL-6 may serve as a biomarker for prognosis among head and neck cancer patients (34
). In our study, as well as in the murine models, we consistently noted increase level of IL-6 with STAT3 suppression. IL-6 is currently hypothesized as one cytokine that can mediate the STAT3 feed forward loop, which may be dependent on NF-kB signaling (22
). However, given the complex relationship between inflammation and tumorigenesis within the tumor microenvironment, the consequences of STAT3 dependent IL-6 variations may be difficult to predict. Since we did not observe any increased growth rate of the tumor cells tested in vitro
, this elevation of IL-6 is not correlated with STAT3-independent tumorigenesis. Despite IL-6’s possible use as a biomarker, however, it may not account for the full combinatorial effects of multiple cytokines and chemokines to direct the development of the tumor. Rather than focusing on a single cytokine, therefore, the focus of our study was to evaluate the role of STAT3 signaling in human tumor cells.
In terms of STAT3 signaling, our lab and others have shown that STAT3 signaling in the hematopoietic compartment also plays a significant role in the development of the murine tumor microenvironment (20
). Although our study was directed at the STAT3 activity from human carcinoma cells, murine studies have demonstrated that STAT3 signaling in the tumor microenvironment can also induce the expression of IL-23 on tumor infiltrating macrophages, while inhibiting NF-kB dependent IL-12/p39 gene expression (35
). STAT3 also appears to prolong NF-kB nuclear retention in both tumor cells as well as hematopoietic cells (36
). Moreover, STAT3 signaling regulates the level of Th17 cells in vivo
, and this proinflammatory environment has been shown to be pro-carcinogenic in the colon carcinoma models (12
). Cumulatively, STAT3 signaling affects multiple downstream pathways to promote a pro-carcinogenic “soil” that suppresses an anti-tumor response.
From a clinical standpoint, therefore, targeting STAT3 signaling in the tumor microenvironment appears to be attractive. The data presented in this report show that the paracrine effects of STAT3 activation in human carcinoma can regulate the human tumor microenvironment analogously to the murine system. Given that STAT3 signaling in tumor cells promotes proliferation and prevents apoptosis, our report provides more evidence that STAT3 targeting may be an avenue of translational research as immunomodulator for clinical trials. As a transcription factor, STAT3 may be difficult to target, but we were able to demonstrate that Stattic, a small molecule inhibitor available commercially, can inhibit STAT3 signaling in the human tumor cells. Interestingly, its initial report used several log-fold higher concentrations for induction of apoptosis (5
). Using doses from 1-10μM range, which did not induce apoptosis of the tumor cells in our studies, we showed that Stattic can induce an upregulation of RANTES, TNFα, IP-10, IL-8, IFNβ and IL-6 to a comparable level as seen with the STAT3 siRNA suppression. These results illustrate the feasibility of pharmacologic STAT3 blockade to induce an immunomodulatory effect.
Currently several clinical trials are underway that can block STAT3 signaling in various cancer patients (37
). Some are upstream regulators of STAT3 such as EGFR inhibitors and neutralizing antibodies, as well as Src inhibitors. Given the findings in our report, immunomodulatory effects of STAT3 signaling should be investigated as potential mechanisms of clinical efficacy among patients who are treated with biological agents that work upstream to STAT3 signaling.