Summary The anti-inflammatory actions of interleukin-10 (IL10) are thought to be mediated primarily by the STAT3 transcription factor, but pro-inflammatory cytokines such as interleukin-6 (IL6) also act through STAT3. We now report that IL10, but not IL6 signaling, induces formation of a complex between STAT3 and the inositol polyphosphate-5-phosphatase SHIP1 in macrophages. Both SHIP1 and STAT3 translocate to the nucleus in macrophages. Remarkably, sesquiterpenes of the Pelorol family, which we previously described as allosteric activators of SHIP1 phosphatase activity, could induce SHIP1/STAT3 complex formation in cells and mimic the anti-inflammatory action of IL10 in a mouse model of colitis. Using crystallography and docking studies we identified a drug-binding pocket in SHIP1. Our studies reveal new mechanisms of action for both STAT3 and SHIP1 and provide a rationale for use of allosteric SHIP1-activating compounds, which mimic the beneficial anti-inflammatory actions of IL10. Video Abstract

Interleukin-10 (IL10) is best studied for its inhibitory action on immune cells and ability to suppress an antitumour immune response. But IL10 also exerts direct effects on nonimmune cells such as prostate cancer epithelial cells. Elevated serum levels of IL10 observed in prostate and other cancer patients are associated with poor prognosis. After first-line androgen-deprivation therapy, prostate cancer patients are treated with androgen receptor antagonists such as enzalutamide to inhibit androgen-dependent prostate cancer cell growth. However, development of resistance inevitably occurs and this is associated with tumour differentiation to more aggressive forms such as a neuroendocrine phenotype characterized by expression of neuron specific enolase and synaptophysin. We found that treatment of prostate cancer cell lines in vitro with IL10 or enzalutamide induced markers of neuroendocrine differentiation and inhibited androgen receptor reporter activity. Both also upregulated the levels of PDL1, which could promote tumour survival in vivo through its interaction with the immune cell inhibitory receptor PD1 to suppress antitumour immunity. These findings suggest that IL10’s direct action on prostate cancer cells could contribute to prostate cancer progression independent of IL10’s suppression of host immune cells.

Macrophage cells form part of our first line defense against pathogens. Macrophages become activated by microbial products such as lipopolysaccharide (LPS) to produce inflammatory mediators, such as TNFα and other cytokines, which orchestrate the host defense against the pathogen. Once the pathogen has been eradicated, the activated macrophage must be appropriately deactivated or inflammatory diseases result. Interleukin-10 (IL10) is a key anti-inflammatory cytokine which deactivates the activated macrophage. The IL10 receptor (IL10R) signals through the Jak1/Tyk2 tyrosine kinases, STAT3 transcription factor and the SHIP1 inositol phosphatase. However, IL10 has also been described to induce the activation of the cyclic adenosine monophosphate (cAMP) regulated protein kinase A (PKA). We now report that IL10R signalling leads to STAT3/SHIP1 dependent expression of the EP4 receptor for prostaglandin E 2 (PGE 2). In macrophages, EP4 is a G αsprotein coupled receptor that stimulates adenylate cyclase (AC) production of cAMP, leading to downstream activation of protein kinase A (PKA) and phosphorylation of the CREB transcription factor. IL10 induction of phospho-CREB and inhibition of LPS-induced phosphorylation of p85 PI3K and p70 S6 kinase required the presence of EP4. These data suggest that IL10R activation of STAT3/SHIP1 enhances EP4 expression, and that it is EP4 which activates cAMP-dependent signalling. The coordination between IL10R and EP4 signalling also provides an explanation for why cAMP elevating agents synergize with IL10 to elicit anti-inflammatory responses.

28The anti-inflammatory actions of interleukin-10 (IL10) are thought to be mediated primarily by the STAT3 29 transcription factor, but pro-inflammatory cytokines such as interleukin-6 (IL6) also act through STAT3. 30We now report that IL10, but not IL6 signaling, induces formation of a complex between STAT3 and the 31 inositol polyphosphate-5-phosphatase SHIP1 in macrophages. Both SHIP1 and STAT3 translocate to the 32 nucleus in macrophages. Remarkably, sesquiterpenes of the Pelorol family we previously described as 33 allosteric activators of SHIP1 phosphatase activity, could induce SHIP1/STAT3 complex formation in cells, 34 and mimic the anti-inflammatory action of IL10 in a mouse model of colitis. Using crystallography and 35 docking studies we identified a drug-binding pocket in SHIP1. Our studies reveal new mechanisms of 36 action for both STAT3 and SHIP1, and provide a rationale for use of allosteric SHIP1-activating compounds 37 which mimic the beneficial anti-inflammatory actions of IL10. 38 39 1999) much like an IL10 -/mouse (Kuhn et al., 1993, Zigmond et al., 2014. However, STAT3 becomes 63 tyrosine phosphorylated and activated by many stimuli including the pro-inflammatory cytokine 64 interleukin-6 (IL6) (Garbers et al., 2015), so STAT3 activation must differ downstream of IL10 and IL6 65 signaling in order to mediate their opposing actions. 66The SHIP1 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase is a cytoplasmic protein expressed 67 predominantly in hematopoietic cells (Hibbs et al., 2018, Fernandes, 2013 #1400, Huber et al., 1999 68 Krystal, 2000, Pauls and Marshall, 2017). In response to extracellular signals, SHIP1 can be recruited to 69 the cell membrane and one of its actions can be to turn off phosphoinositide 3-kinase (PI3K) signaling 70 (Brown et al., 2010) by dephosphorylating the PI3K product PIP3 into PI(3,4)P2 (Fernandes et al., 2013, 71 Huber et al., 1999, Krystal, 2000, Pauls and Marshall, 2017. We have shown that SHIP1 phosphatase 72 activity is allosterically activated by its product PI(3,4)P2 and that small molecules of the pelorol family 73 (ZPR-MN100 and ZPR-151) also allosterically enhance SHIP1 phosphatase activity (Meimetis et al., 2012, 74 Ong et al., 2007. These data suggest that stimulating SHIP1 phosphatase activity with small molecule 75 SHIP1 activators could be used to treat inflammatory diseases caused by inappropriately sustained PI3K 76 production of PI(3,4)P2. 77However, in addition to its enzymatic function in hydrolyzing PIP3, SHIP1 can also act as a docking protein 78 for assembly of signaling complexes (Pauls and Marshall, 2017). We previously showed that IL10R 79 signaling requires SHIP1 to inhibit TNFα translation (Chan et al., 2012) but whether SHIP1 and STAT3 80 worked independently or together was not determined. We now report that a SHIP1 protein containing 81 point mutations, which inactivates its phosphatase activity, could still mediate the anti-inflammatory 82 action of IL10, and that SHIP1 and STAT3 associate with each other in respo...

Augmenting adaptive immunity is a critical goal for developing next-generation cancer therapies. T and B cells infiltrating the tumor dramatically influence cancer progression through complex interactions with the local microenvironment. Cancer cells evade and limit these immune responses by hijacking normal immunologic pathways. Current experimental models using conventional primary cells, cell lines, or animals have limitations for studying cancer-immune interactions directly relevant to human biology and clinical translation. Therefore, engineering methods to emulate such interplay at local and systemic levels are crucial to expedite the development of better therapies and diagnostic tools. In this review, we discuss the challenges, recent advances, and future directions toward engineering the tumor-immune microenvironment (TME), including key elements of adaptive immunity. We first offer an overview of the recent research that has advanced our understanding of the role of the adaptive immune system in the tumor microenvironment. Next, we discuss recent developments in 3D in-vitro models and engineering approaches that have been used to study the interaction of cancer and stromal cells with B and T lymphocytes. We summarize recent advancement in 3D bioengineering and discuss the need for 3D tumor models that better incorporate elements of the complex interplay of adaptive immunity and the tumor microenvironment. Finally, we provide a perspective on current challenges and future directions for modeling cancer-immune interactions aimed at identifying new biological targets for diagnostics and therapeutics.

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