Breaking bad habits: Targeting MDSCs to alleviate immunosuppression in prostate cancer

ABSTRACT

The myeloid-derived suppressor cells (MDSCs) contribute to tumor immune evasion and still remain an elusive therapeutic target. Our study identified granulocytic MDSCs accumulating in prostate cancer patients during disease progression. We demonstrate the feasibility of using STAT3siRNA-based strategy for targeting MDSCs to alleviate arginase-dependent suppression of T cell activity.

KEYWORDS:

• Arginase
• immunotherapy
• MDSC
• myeloid-derived suppressor cells
• prostate cancer
• STAT3
• TLR9

Metastatic castration-resistant prostate cancers are known for being resistant to current standard therapies and even to the emerging immunotherapeutic regimens. Growing evidence suggests that advanced prostate cancers develop potently immunosuppressive microenvironment which partly relies on the heterogeneous population of MDSCs. The MDSCs play pivotal role in cancer progression and poor patients’ survival.¹ The MDSCs are immature myeloid cells which lack expression of multiple immune cell surface markers, including antigen-presenting molecules. Depending on the expression of myeloid lineage markers, human MDSCs can be divided into CD14⁺CD15^LOCD33^HI monocytic MDSCs (M-MDSCs) and CD14⁻CD15^HICD33^LO granulocytic MDSCs (G-MDSCs).¹ The M-MDSCs were previously found in prostate cancer patients and their levels correlated with levels of PSA (prostate-specific antigen) used as a marker of tumor burden.² Even though G-MDSCs were recently observed in other genitourinary tumors, the contribution of G-MDSCs to prostate cancer progression remained unknown.

Our recently published study focused on the phenotypic analysis of immune cell populations in blood derived from patients with localized or metastatic prostate cancers compared to healthy subjects.³ In contrast to earlier studies, our flow cytometric analysis revealed significant accumulation of granulocytic (CD15^HICD33^LO) but not monocytic (CD15^LOCD33^HI) cell populations in patients’ circulation with progression of the disease. Further studies of both myeloid cell populations confirmed that only CD15^HICD33^LO myeloid cells were functionally MDSCs as indicated by their potent inhibitory effect on T cell proliferation and activity. Due to complex identification which relies on markers common to other immune cell lineages, eliminating MDSCs using monoclonal antibodies is challenging. Thus, it seems more practical to target signaling molecules which control MDSC function in the specific tumor microenvironment. Our analysis of cytokine and growth factor (GF) profiles revealed elevated levels of factors related to recruitment and expansion of myeloid cells, such as G-CSF and IL-8 among others. Given the critical role of STAT3 transcription factor in the downstream signaling of G-CSF as well as other GFs present in the tumor microenvironment,³ we assessed levels of activated STAT3 (pSTAT3) in prostate cancer-associated G-MDSCs using flow cytometric and immunohistochemical analyses. We observed elevated pSTAT3 in circulating G-MDSCs and also in polymorphonuclear cells infiltrating prostate tissue biopsies from cancer patients. In addition, STAT3 activation clearly correlated with high levels of Arginase-1 (ARG1) expression in G-MDSCs compared to healthy granulocytes, and also with detectable ARG1 activity in blood samples from cancer patients. Highly elevated levels of ARG1 in G-MDSCs with weak induction of IDO and undetectable iNOS mRNA expression likely reflected different levels of STAT3 involvement in the regulation of these target genes. In G-MDSCs, STAT3 was shown to have potent and direct effect on ARG1, while it collaborates with noncanonical NF-κB signaling to promote IDO expression. In contrast, STAT3 activity is not required for iNOS expression which is driven by STAT1 and NF-κB signaling activated in monocytic MDSCs but not in G-MDSCs.¹ Altogether, these observations strengthened the evidence that G-MDSCs are commonly present in prostate cancer patients’ circulation and likely reach into the prostate tumor microenvironment.

TLR9 was recently found to be expressed by tumor-associated MDSCs in mice, with its highest levels observed in M-MDSCs.⁴ However, the pattern of TLR9 expression differs dramatically between mouse and human immune systems. While in mice TLR9 is ubiquitous in myeloid cells, it is expressed specifically by human plasmacytoid DCs in healthy subjects.⁵ Thus, the expression of TLR9 in prostate cancer patients’ MDSCs and specifically in G-MDSCs was a novel finding of our study. Whether downstream TLR9 signaling restricts or supports the immunosuppressive functions of G-MDSCs remains unclear. Treatments with synthetic TLR9 agonists, CpG oligodeoxynucleotides (ODN), can trigger MDSC differentiation and reduce their immunosuppressive potential in mice.⁴ However, other studies suggested that activation of TLRs in myeloid cells can promote expression of molecules critical for MDSC function, such as ARG1, S100A8, IL-10 and PGE2.¹ The functional dichotomy of TLR9 effects results from crosstalk with other signaling pathways operating under normal or tumor-induced inflammatory conditions. The outcome of TLR9 signaling seems to be defined by a multilayered negative feedback regulation through STAT3 regardless whether it is triggered by synthetic ligands or release of natural agonists (e.g. mitochondrial DNA) from dying cells.^6,7

We previously demonstrated that TLR9 agonists, CpG ODN, can be harnessed for delivery of therapeutic siRNA molecules into TLR9⁺ cells in both mouse and human systems.^8,9 Targeting STAT3 with simultanous TLR9 activation using CpG-STAT3siRNA was shown to eliminate tumorigenic effects of TLR9 signaling in mouse tumor models.⁷ Here, we demonstrated that CpG-STAT3siRNA allows for targeting of TLR9⁺ G-MDSCs to eliminate their immunosuppressive functions without myeloid cell depletion. Further studies should determine whether our approach only modulates G-MDSC activity or induces their differentiation into mature granulocytes or DCs/macrophages. In fact, we previously observed that CpG-STAT3siRNA induces activity of neutrophils against blood cancer xenotransplants in NSG mice.⁹ In a parallel study, we recently identified that prostate cancer stem cells in human tumors rely on TLR9 signaling through NF-κB and STAT3 signaling crucial for their self-renewal and tumor-propagating potential. We demonstrated that it is feasible to target tumorigenic signaling in cancer stem cells using CpG-siRNA approach.¹⁰ Taken together, our findings provide support for application of CpG-STAT3siRNA strategy to immunotherapy of advanced prostate cancers alone or in combination with immunotherapies, such as emerging T cell-based therapies (Fig. 1). Disruption of STAT3 signaling in the tumor microenvironment with concurrent TLR9 stimulation has potential to disrupt the central immunosuppressive circuit paving way to effective antitumor immune responses without toxicities associated with pharmacological agents.

Figure 1. TLR9-targeted STAT3 inhibition allows for 2-pronged therapeutic effect against prostate cancers. CpG-STAT3siRNA conjugates target STAT3 signaling in TLR9⁺ G-MDSCs, an immunosuppressive population of myeloid cells which accumulate during prostate cancer progression from localized to metastatic disease. STAT3 silencing reduces production of a potently immunosuppressive mediator, arginase-1 (ARG1), thereby restoring effector T cell proliferation and activity. As shown in a parallel study, CpG-siRNA strategy allows for delivery of therapeutic siRNAs to prostate cancer stem cells which express TLR9 and rely on NF-κB/STAT3 signaling for self-renewal and tumor-propagating potential. The combination of breaking immune suppression in the tumor microenvironment and decreasing cancer cell survival is likely to augment the overall therapeutic effect against prostate cancer.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.