Publications
Nitric oxide S-nitrosylates CSF1R to augment the action of CSF1R inhibition against castration resistant prostate cancer
During progression of prostate cancer, sustained oxidative overload in cancer cells potentiates the overall tumor microenvironment (TME). Targeting the TME using colony-stimulating factor 1 receptor (CSF1R) inhibition is a promising therapy for castration-resistant prostate cancer (CRPC). However, the therapeutic response to sustained CSF1R blockade therapy (CSF1Ri) is limited as a monotherapy. We postulated that one of the causative agents for reduced efficacy of CSF1Ri and increased oxidation in CRPC is endothelial nitric oxide syntheses (eNOS). Results showed that in high grade PCa human specimens, eNOS is positively correlated with CSF1-CSF1R signaling and remains in an un-coupled state. The uncoupling disables eNOS to generate sufficient Nitric oxide (NO) that are required for inducing effective S-nitrosylation of CSF1R molecule at specific cysteine sites (Cys 224, Cys 278 and Cys 830). Importantly, we found that S-nitrosylation of CSF1R molecule at Cys 224, Cys 278 and Cys 830 sites is necessary for effective inhibition of tumor promoting cytokines (which are downstream of CSF1-CSF1R signaling) by CSF1R blockade. In this context, we studied if exogenous NO treatment could rescue the side effects of eNOS uncoupling. Results showed that exogenous NO treatment (using S-nitrosoglutathione (GSNO)) is effective in not only inducing S-Nitrosylation of CSF1R molecule, but it helps in rescuing the excess oxidation in tumor regions, reducing overall tumor burden, suppresses the tumor promoting cytokines which are ineffectively suppressed by CSF1R blockade. Together these results postulated that NO therapy could act as an effective combinatorial partner with CSF1R blockade against CRPC. In this context, results demonstrated that exogenous NO treatment successfully augment the anti-tumor ability of CSF1Ri in murine models of CRPC. Importantly, the overall tumor reduction was most effective in NO-CSF1Ri therapy compared to NO or CSF1Ri mono therapies. Moreover, Immunophenotyping of tumor grafts showed that the NO-CSF1Ri combination significantly decreased intratumoral percentage of anti-inflammatory macrophages, myeloid derived progenitor cells and increased the percentage of pro-inflammatory macrophages, cytotoxic T lymphocytes, and effector T cells respectively. Together, our study suggests that the NO-CSF1Ri combination has the potential to act as a therapeutic agent that restore control over TME and improve the outcomes of PCa patients.
Alterations of tumor microenvironment by nitric oxide impedes castration-resistant prostate cancer growth
Immune targeted therapy of nitric oxide (NO) synthases are being considered as a potential frontline therapeutic to treat patients diagnosed with locally advanced and metastatic prostate cancer. However, the role of NO in castration-resistant prostate cancer (CRPC) is controversial because NO can increase in nitrosative stress while simultaneously possessing antiinflammatory properties. Accordingly, we tested the hypothesis that increased NO will lead to tumor suppression of CRPC through tumor microenvironment. S-nitrosoglutathione (GSNO), an NO donor, decreased the tumor burden in murine model of CRPC by targeting tumors in a cell nonautonomous manner. GSNO inhibited both the abundance of antiinflammatory (M2) macrophages and expression of pERK, indicating that tumor-associated macrophages activity is influenced by NO. Additionally, GSNO decreased IL-34, indicating suppression of tumor-associated macrophage differentiation. Cytokine profiling of CRPC tumor grafts exposed to GSNO revealed a significant decrease in expression of G-CSF and M-CSF compared with grafts not exposed to GSNO. We verified the durability of NO on CRPC tumor suppression by using secondary xenograft murine models. This study validates the significance of NO on inhibition of CRPC tumors through tumor microenvironment (TME). These findings may facilitate the development of previously unidentified NO-based therapy for CRPC.
Leptin secreted from testicular microenvironment modulates hedgehog signaling to augment the endogenous function of Leydig cells
factors responsible for TD remain largely unknown. Leydig stem cells (LSCs) differentiate into adult Leydig cells (ALC) and produce testosterone in the testes under the pulsatile control of luteinizing hormone (LH) from the pituitary gland. However, recent studies have suggested that the testicular microenvironment (TME), which is comprised of Sertoli and peritubular myoid cells (PMC), plays an instrumental role in LSC differentiation and testosterone production under the regulation of the desert hedgehog signaling pathway (DHH). It was hypothesized that the TME releases paracrine factors to modulate LSC differentiation. For this purpose, cells (Sertoli, PMCs, LSCs, and ALCs) were extracted from men undergoing testis biopsies for sperm retrieval and were evaluated for the paracrine factors in the presence or absence of the TME (Sertoli and PMC). The results demonstrated that TME secretes leptin, which induces LSC differentiation and increases testosterone production. Leptin's effects on LSC differentiation and testosterone production, however, are inversely concentration-dependent: positive at low doses and negative at higher doses. Mechanistically, leptin binds to the leptin receptor on LSCs and induces DHH signaling to modulate LSC differentiation. Leptin-DHH regulation functions unidirectionally insofar as DHH gain or loss of function has no effect on leptin levels. Taken together, these findings identify leptin as a key paracrine factor released by cells within the TME that modulates LSC differentiation and testosterone release from mature Leydig cells, a finding with important clinical implications for TD.
Subcutaneous Leydig Stem Cell Autograft: A Promising Strategy to Increase Serum Testosterone
Exogenous testosterone therapy can be used to treat testosterone deficiency; however, it has several adverse effects including infertility due to negative feedback on the hypothalamic-pituitary-gonadal (HPG) axis. Leydig stem cell (LSC) transplantation could provide a new strategy for treating testosterone deficiency, but clinical translatability of injecting stem cells inside the testis is not feasible. Here, we explore the feasibility of subcutaneously autografting LSCs in combination with Sertoli and myoid cells to increase testosterone. We also studied whether the grafted LSCs can be regulated by the HPG axis and the molecular mechanism behind this regulation. LSCs were isolated from the testes of 12-week-old C57BL/6 mice, and subcutaneously autografted in combination with Sertoli cells and myoid cells. We found that LSCs alone were incapable of self-renewal and differentiation. However, in combination with Sertoli cells and myoid cells, LSCs underwent self-renewal as well as differentiation into mature Leydig cells. As a result, the recipient mice that received the LSC autograft showed testosterone production with preserved luteinizing hormone. We found that testosterone production from the autograft was regulated by hedgehog (HH) signaling. Gain of function and loss of function study confirmed that Desert HH (DHH) agonist increased and DHH antagonist decreased testosterone production from autograft. This study is the first to demonstrate that LSCs, when autografted subcutaneously in combination with Sertoli cells and myoid cells, can increase testosterone production. Therefore, LSC autograft may provide a new treatment for testosterone deficiency while simultaneously preserving the HPG axis.