Effect of nitric oxide inhibition in Bacillus Calmette-Guerin bladder cancer treatment

Highlights

• INOS expression is associated with bad prognosis in patients with bladder cancer.

• We found an association between iNOS and the immunosuppressive protein S100A9.

• INOS inhibition using l-NAME increases CD8⁺ and NK cells and reduces Treg and MDSC.

• INOS inhibition using l-NAME reduces the expression of S100A9 and TGF-β.

• l-NAME improves BCG tumor growth inhibition and the antitumor immune response.

Abstract

Background

Bacillus Calmette-Guerin (BCG) is the standard treatment for patients with high-risk non-muscle invasive bladder cancer (BC). Despite its success, about 30–50% of patients are refractory. It was reported that inducible nitric oxide synthase (iNOS) tumor expression is presented in 50% of human BC, associated with bad prognosis and BCG failure.

Objective

to evaluate in human bladder tumors the association between iNOS expression and the tumor microenvironment focusing on the immunosuppressive protein S100A9. Also, investigate in a preclinical murine MB49-BC model the tumor immunoresponse induced by BCG in combination with the nitric oxide production inhibitor l-NAME.

Results

In human bladder tumors, we detected a positive association between iNOS and S100A9 tumor expression, suggesting a relationship between both immunomodulatory proteins. We also found a positive correlation between iNOS tumor expression and the presence of S100A9⁺ tumor-infiltrating cells, suggesting an immunosuppressive tumor microenvironment induced by the nitric oxide production.

Using the subcutaneous murine BC model, we show that similarly to the human pathology, MB49 tumors constitutively expressed iNOS and S100A9 protein. MB49 tumor-bearing mice presented an immunosuppressive systemic profile characterized by fewer cytotoxic cells (CD8⁺ and NK) and higher suppressor cells (Treg and myeloid-derived suppressor cells -MDSC-) compared to normal mice. BCG treatment reduced tumor growth, increasing local CD8⁺-infiltrating cells and induced a systemic increase in CD8⁺ and a reduction in Treg. BCG combined with l-NAME, significantly reduced tumor growth compared to BCG alone, diminishing iNOS and S100A9 tumor expression and increasing CD8⁺-infiltrating cells in tumor microenvironment. This local response was accompanied by the systemic increase in CD8⁺ and NK cells, and the reduction in Treg and MDSC, even more than BCG alone. Similar results were obtained using the orthotopic BC model, where an increase in specific cytotoxicity against MB49 tumor cells was detected.

Conclusion

The present study provides preclinical information where NO inhibition in iNOS-expressing bladder tumors could contribute to improve BCG antitumor immune response. The association between iNOS and S100A9 in human BC supports the hypothesis that iNOS expression is a negative prognostic factor and a promising therapeutic target.

Introduction

Bladder cancer (BC) is a common malignancy of the urological tract and ranks fourth in malignant cancer frequency in men in developed countries [1]. Immunotherapy with Bacillus Calmette-Guerin (BCG) has been used as gold standard therapy for in situ and non-muscle invasive (NMI) high histological grade BC treatment, to prevent recurrence and tumor progression [2]. However, it is still unknown why there are about 30–50% of patients that either fail to respond initially or relapse within the first years after treatment [3]. BCG successful depends on host immune system activation, which may exert an effective Th1-cytotoxic response, orchestrating the activity of CD8⁺, NK cells, macrophages and granulocyte cells [4]. A reduction in CD8⁺ and NK cytotoxic cells, with an increase in negative regulatory Treg and myeloid derived suppressor cells (MDSC) have been associated with low tumor immunogenicity [5,6].

Tumor nitric oxide (NO) production by inducible nitric oxide synthases (iNOS) have been suggested as responsible for BCG therapy failure [7]. Furthermore, patients whose bladder tumors express iNOS, were related to bad prognosis, high invasion status and lower recurrence free time [8,9]. Using a murine preclinical BC model developed in our laboratory, we proved that NO inhibition using the NOS inhibitor l-NAME is a good therapeutic strategy for tumors that constitutively express iNOS. l-NAME reduces tumor growth, invasion, development of metastases, angiogenic process, and enhances tumor growth inhibition induced by BCG [10,11]. In cancer, high iNOS activity depletes l-arginine, necessary for an effective immune response activation [12]. In tumor microenvironment, iNOS leads to the suppression of T cell expansion, inhibiting IL-2 production by T cells and recruiting immunosuppressive cells such as MDSC [12,13]. NO also reduces the affinity of T cell receptor to MHC-antigen complex, inhibiting cytotoxic activity [14]. NO inhibitors demonstrated to reduce breast tumor growth, associated with the restore of the immune response, by reducing MDSC and expanding T cells [13,15]. MDSC is a heterogeneous group that include immature granulocytes, macrophages, dendritic cells and myeloid progenitors. The increase in MDSC was associated with tumor progression in patients with BC [5,16]. iNOS expression is a key event in acute inflammatory processes and this induction is physiologically reversed by transforming growth factor beta (TGF-β) [[17], [18], [19]]. However, TGF-β pathway is up-regulated in BC, associated with cancer-specific death [20]. In a chronic inflammatory environment as occurs in BC, both, iNOS and TGF-β are overexpress, generating an immunosuppressing tumor microenvironment, at least in part, by inducing S100A9 expression [21]. S100A9 is a member of S100 low molecular weight Ca²⁺-binding proteins. The majority of S100A9 forms heterodimers with S100A8 (other member of S100 family) and could interact with three cell surface receptors in a wide range of different cells [22]. These receptors are Advanced glycation end product (RAGE), Toll-like receptor 4 (TLR4) and Extracellular matrix metalloproteinase inducer (CD147) [22]. In tumor cells, the interaction between S100A9 and these receptors activate the transcription factor NF-κB, inducing angiogenesis, tumor migration and proliferation [22,23]. Alternatively, in immature myeloid cells, the binding of S100A9 to TLR4 induce MDSC differentiation [24]. S100A9 protein could be expressed by myeloid, as well as by tumor cells [24,25]. Up-regulation of S100A9 in tumor cells also induces MDSC accumulation and Treg differentiation, which leads to the inability of host immune system to attack the tumor [24,26,27]. We previously reported that exist a positive correlation between iNOS and S100A9 tumor expression, suggesting an association among these two proteins [25]. Even more, we observed an increased number in S100A9⁺ tumor-infiltrating cells in invasive bladder tumors compared to NMI [25], suggesting that these immunosuppressive cells could be related to bad prognosis and the lack of BCG response.

Using the NMI MB49 murine preclinical BC model that express iNOS and produces NO, we observed that these tumors also express S100A9 and present high number of S100A9⁺ tumor-infiltrating cells as happens in tumors from patients with BC. Treatment with l-NAME reduced S100A9 expression either in vitro and in vivo [25].

In the present study, we evaluated in human bladder tumor samples the association between iNOS tumor expression and S100A9 in tumor cells or in tumor-infiltrating cells. In addition, to understand if NO production could induce S100A9 and an immunosuppressive profile, we have evaluated the immune response induced in the preclinical MB49 high-grade NMI BC model that naturally expresses iNOS. We also studied the BCG immune response in these iNOS-expressing tumors and the modulation by the NOS inhibitor, l-NAME. Our results, either in BC patients or in our murine BC model, showed that iNOS tumor expression is associated with the expression of S100A9 in tumor cells and also correlates with the presence of S100A9⁺ tumor-infiltrating cells. The NOS inhibition with l-NAME can induce a cytotoxic immune response and improved the BCG immunotherapy.