Alterations of tumor microenvironment by nitric oxide impedes castration-resistant prostate growth

Himanshu Aroraa,b,1, Kush Panaraa, Manish Kuchakullaa, Shathiyah Kulandavelub, Kerry L. Burnsteinc, Andrew V. Schallyd,e,f,g,h,1, Joshua M. Hareb,i, and Ranjith Ramasamya,1

aDepartment of Urology, Miller School of Medicine, University of Miami, Miami, FL 33136; bThe Interdisciplinary Stem Cell Institute, Miller School of Medicine, University of Miami, Miami, FL 33136; cDepartment of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, FL 33136; dEndocrine, Polypeptide, and Cancer Institute Veterans Affairs Medical Center and South Florida Veterans Affairs Foundation for Research and Education, Miami, FL 33125; eDepartment of Medicine, Division of Hematology/Oncology, Miller School of Medicine, University of Miami, Miami, FL 33136; fDepartment of Medicine, Division of Endocrinology, Miller School of Medicine, University of Miami, Miami, FL 33136; gDepartment of Pathology, Miller School of Medicine, University of Miami, Miami, FL 33136; hSylvester Comprehensive Cancer Centre, Miller School of Medicine, University of Miami, Miami, FL 33136; and iDepartment of Medicine, Division of Cardiology, Miller School of Medicine, University of Miami, Miami, FL 33136

Contributed by Andrew V. Schally, August 24, 2018 (sent for review August 3, 2018; reviewed by K. C. Balaji, Vinata B. Lokeshwar, and Rama Soundararajan) Immune targeted therapy of nitric oxide (NO) synthases are being Others have investigated the tumoricidal implications of NO in considered as a potential frontline therapeutic to treat patients therapeutic resistance (21–23), cell survival, proliferation of tumors, diagnosed with locally advanced and metastatic prostate cancer. inhibition of tumor growth, and reduction in lung metastases (24) in However, the role of NO in castration-resistant prostate cancer many cancer types (25). Several NO donors, including S-nitrosothiols, (CRPC) is controversial because NO can increase in nitrosative stress organic nitrates, and Metal-NO complexes, have shown impacts on while simultaneously possessing antiinflammatory properties. Ac- cancer progression (26, 27). S-nitrosothiols such as S-nitroso-N- cordingly, we tested the hypothesis that increased NO will lead to acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) have tumor suppression of CRPC through tumor microenvironment. S- also shown promising effects as antineoplastic agents. However, these nitrosoglutathione (GSNO), an NO donor, decreased the tumor studies have only focused on certain aspects of NO such as its role in burden in murine model of CRPC by targeting tumors in a cell both progrowth and antigrowth effects (28), cellular localization, nonautonomous manner. GSNO inhibited both the abundance of endogenous expression of the androgen receptor (AR) (29), and AR antiinflammatory (M2) and expression of pERK, in- function inactivation by S-nitrosylation (30, 31). In the present study, dicating that tumor-associated macrophages activity is influenced we document the effect of NO on tumor suppression by targeting the by NO. Additionally, GSNO decreased IL-34, indicating suppression CRPC TME through TAMs. of tumor-associated differentiation. Cytokine pro- filing of CRPC tumor grafts exposed to GSNO revealed a significant Results decrease in expression of G-CSF and M-CSF compared with grafts Increased NO Levels Affect Testosterone and Luteinizing Hormone. not exposed to GSNO. We verified the durability of NO on CRPC We have previously shown that mice lacking the S-nitrosoglutathione tumor suppression by using secondary xenograft murine models. reductase (GSNOR) gene showed increased nitrosative stress and This study validates the significance of NO on inhibition of CRPC exhibited secondary hypogonadism (19). Therefore, in this study we tumors through tumor microenvironment (TME). These findings examined if increased NO levels are able to suppress testosterone (T) may facilitate the development of previously unidentified NO-based and luteinizing hormone (LH) in control C57BL/6J mice. For these therapy for CRPC. experiments, we administered GSNO (an NO donor) intraperito- neally (10 mg/kg) for 7 d and compared T and LH levels to mice nitric oxide | tumor microenvironment | CRPC | tumor-associated macrophages | immunotherapy Significance rostate cancer is the second most frequent cause of cancer- This study presents insights into the underexplored areas of related deaths in men. Men with prostate cancer that has P castration-resistant prostate cancer (CRPC) therapeutics—the recurred after local therapy usually respond to androgen depri- role of nitric oxide (NO) in CRPC reduction through its micro- vation therapy (ADT); however, despite this treatment, most environment. Results of this study provide important informa- patients eventually experience progression of the disease within 2 y, tion on the tumor reduction capabilities of increased NO levels a condition known as castration-resistant prostate cancer (CRPC) and its mechanistic aspect and demonstrates the potential long- (1). In trying to understand the causes of this androgen resistance term efficacy of NO on CRPC. An in-depth understanding of how that develops in CRPC, most research has focused directly on the NO affects the tumor microenvironment will allow development splice variants of the androgen receptor (ARVs) (2). However, the of chemotherapeutics based on NO for a CRPC cure. tumor microenvironment (TME) has been shown to play a major role in tumor progression, yet the response to therapy in other Author contributions: H.A., A.V.S., J.M.H., and R.R. designed research; H.A., K.P., and M.K. cancer types has been inadequately studied in CRPC (3–5). TME is performed research; H.A., K.P., S.K., K.L.B., A.V.S., and J.M.H. analyzed data; and H.A., comprised of a variety of cell types, including immune cells, fibro- S.K., A.V.S., J.M.H., and R.R. wrote the paper. blasts, , and tumor-associated macrophages (TAMs) (6). Reviewers: K.C.B., University of Florida; V.B.L., Augusta University; and R.S., The University TAMs are recruited to tumors from diverse signaling molecules of Texas MD Anderson Cancer Center. such as (CCL-2 and CCL-5) and cytokines (IL-34 and Conflict of interest: J.M.H. discloses a relationship with Vestion Inc. that includes equity, board membership, and consulting. J.M.H. is the Chief Scientific Officer, a compensated CSF-1) (7). While the exact mechanism is unknown, TAMs have consultant, and advisory board member for Longeveron and holds equity in Longeveron. been reported to play a key role in the progression of prostate J.M.H. is also the coinventor of intellectual property licensed to Longeveron. cancer through the secretion of cytokines, matrix metalloproteinases, Published under the PNAS license. and growth factors (8, 9). 1To whom correspondence may be addressed. Email: [email protected], andrew. A key molecule in the regulation of TME interactions is the [email protected], or [email protected]. ubiquitous nitric oxide (NO) (10–12). We have previously This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. established the importance of NO in the cardiovascular and 1073/pnas.1812704115/-/DCSupplemental. immune systems and in male secondary hypogonadism (13–20). Published online October 15, 2018.

11298–11303 | PNAS | October 30, 2018 | vol. 115 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1812704115 Downloaded by guest on September 24, 2021 administered with phosphate buffer saline (PBS). We found that T the overall tumor burden was decreased in mice treated with GSNO and LH levels were decreased 6× in the mice that received GSNO (high NO levels) (Fig. 2 B–E), significantly affecting the weight of (Fig. 1A). Further validation was obtained by oral administration of mice (Fig. 2C). We evaluated the NO levels in tumors isolated from GSNO(10mg/400mLbottlefor5wk)toC57BL/6Jmice,aswe mice treated with PBS or GSNO using Griess test and confirmed an found that T levels were undetectable in mice that received oral inverse association between tumor burden and NO levels (Fig. 2F). GSNO compared with untreated animals (Fig. 1B). To confirm if Tumors from mice that received GSNO treatment showed areas increased NO levels are capable of affecting the hypothalamic- with less necrosis (Fig. 2G and SI Appendix,Fig.S1). Furthermore, pituitary-gonadal axis to regulate LH and T levels, we checked GSNO-treated mice showed a reduced number of Ki-67– positive GnRHR expression in the brain (Fig. 1C), and our results con- cells, suggesting a reduced proliferation rate (Fig. 2H). firmed that increased NO levels were capable of reducing the expression of GnRHR in the brain. Together, these results in- NO Suppresses Tumor Burden in a Cell Nonautonomous Manner. Most dicated that increased NO levels affect receptors for GnRH, LH, chemotherapeutic drugs as well as ionizing radiation promote and T through the hypothalamic-pituitary-gonadal axis. autophagy in tumor cells (32–35). However, autophagy can have both cell autonomous and cell nonautonomous effects and influ- NO Reduces Tumor Burden in the Murine Model of CRPC. We hy- ence the outcome of therapy (35). Therefore, we evaluated whether pothesized that increased NO can lead to CRPC tumor suppression the effects of NO are tumor cell autonomous or nonautonomous. because of its ability to affect the hypothalamic-pituitary-gonadal We studied the cell proliferation rate after treating the 22Rv1 cells axis. After castration, 2.5 million 22RV1 cells were xenografted s.c. with varying concentrations of GSNO in vitro. Interestingly, we in each flank of SCID mice. GSNO (10 mg/kg per d i.p.) was ad- found that in vitro the proliferation of 22Rv1 cells was largely un- ministered to half of the mice (experimental group) and an equal affected (Fig. 3A), despite the efficacy of GSNO on tumor burden volume of PBS to the remaining animals. After 14 d of GSNO in vivo (Fig. 2). Because 22Rv1 cell growth is dependent on con- treatment, the grafts were harvested (Fig. 2A), and we found that stitutive signaling of the androgen receptor (AR), we examined the AR signaling markers involved in androgen-induced activation of endogenous AR, such as PSA and TMPRSS2 (29). Both PSA and TMPRSS2 levels were suppressed by escalating doses of GSNO (Fig. 3B). Taken together, changes in AR signaling without changes in cell proliferation in vitro suggest the effects of NO on 22Rv1 cells are likely cell nonautonomous.

Cytokine Signature Showed That NO Suppresses TAMs Affecting CRPC TME. The cell nonautonomous effects are related largely to the (35), TME, and subclone heterogeneity (36). Macrophages are known to increase specific circulating cytokines with progressive metastasis (37, 38). Therefore, to study the molecular events leading to NO-induced changes, we evaluated protein expression of 120 cytokines using the tumors from mice that received GSNO versus PBS (SI Appendix, Fig. S2). Among 120 cytokines assayed, there were 26 cytokines (CCL27, CD54, TIMP-1, ACRP30, G-CSF, AR, IL-17A, beta NGF, IL-2 R al- pha, CCL28, Axl, CCL7, CCL17, CXCL6, IL-1 F2, M-CSF, TGFBeta 3, CXCL13, BMP-6, CCL23, NT-3, CCL11, CCL1, IL-5, IL-6, and IGFBP4) that were suppressed more than 1.5 times (Fig. 3C). From previously published studies, we determined the role of these cytokines in cancer progression (SI Appendix,Table S2). Among the 26 affected cytokines, the macrophage colony stimulating factor (M-CSF) and granulocyte M-CSF (GM-CSF) play an essential role in the regulation of TAMs (39, 40). TAMs promote tumor progression and are resistant to various chemo- therapeutic agents (41, 42) in prostate cancer (43) by differentiating into either cytotoxic (M1) or tumor growth promoting (M2) states (44). Therefore, to study the implications of increased NO levels on

TAMs, we evaluated the markers of proinflammatory (M1) and MEDICAL SCIENCES antiinflammatory (M2) macrophages in tumors from mice treated with GSNO versus PBS. We found that following therapy with NO, expression of M2 macrophage markers (F4/80, CD206, Arginase) was suppressed and expression of M1 macrophage marker (iNOS) was increased (Fig. 3 D–G). This indicates that TAMs, a significant component of the antiinflammatory cell (M2) that infiltrates in prostate cancer (44), are suppressed by increased NO levels.

Fig. 1. Increased levels of NO affect levels of testosterone (T), FSH, and LH. NO Influences TAMs by Targeting Their Activity and Differentiation. (A) To validate the impact of increased NO levels on T, FSH, and LH, C57/BL6 Recent studies have suggested the importance of phospho- mice were treated with 10 mg/kg GSNO for 7 d and levels of T, FSH, and LH ERK1/2 levels with respect to combinations of lactate and hyp- were checked. (B) Additionally, C57/BL6 mice were kept on the oral dosage of oxia that eventually affect the fate of TAMs (45–48). Therefore, to GSNO at 10 mg per cage per week for 5 wk. Brains from i.p. GSNO-treated mice were harvested and analyzed for the presence of GnRHR (C), showing verify whether TAM activity is sensitive to increased levels of NO, that NO levels affect major pathways of production of hormones (T, LH, FSH) we checked the levels of pERK in tumors from mice which received by influencing hypothalamic-pituitary-gonadal axis regulation. (Scale bars: C, GSNO treatment and compared them with tumors from PBS- first three columns, 750 μm; C, Right,100μm.) treated mice using immunostaining and Western blot. We found

Arora et al. PNAS | October 30, 2018 | vol. 115 | no. 44 | 11299 Downloaded by guest on September 24, 2021 Fig. 2. Increased NO levels suppress tumor burden in CRPC mouse model (castrated SCID mice with s.c. xenograft of 22RV1). (A) Experiment with in vivo xenograft. (B) Xenograft tumors isolated from both flanks after 2 wk of treatment (PBS/GSNO) (each cup represents tumors from one mouse). (C) Animal weight. (D) Tumor volume. (E) Tumor weight. GSNO-treated CRPC mouse models have fewer necrotic areas and fewer proliferating cells in tumor grafts. (F) Relative comparison of nitrate levels between control and GSNO-treated mice. (G) H&E staining, showing the sections from tumor xenografts that received PBS (control) vs. treatment with GSNO (10 mg/kg per d i.p.). Grafts from PBS-treated mice have more areas of necrosis. (H) Ki-67 immunostaining of sections showing that proliferation is suppressed in GSNO-treated grafts. (Scale bars: 500 μm.)

that levels of pERK were suppressed upon GSNO treatment (Fig. 4 for a period of 4 wk (Fig. 5A). The results revealed that the overall A and B and SI Appendix,Fig.S5D). However, total levels of ERK tumor burden was significantly suppressed (Fig. 5B), with a re- protein remained similar in all of the conditions (Fig. 4A), In ad- duction in the number of Ki-67–positive cells in animals that re- dition, we determined the impact of increased levels of NO on other ceived cells from GSNO-treated mice (SI Appendix,Fig.S4). In factors which are markers of TAM activity, such as VEGF (an- addition, cells staining positive for M2 macrophages markers (F4/ giogenic marker), androgen receptor (AR), and androgen receptor 80, CD206) were decreased in animals that received cells from splice variant 7 (AR-V7) (a critical determinant of resistance de- GSNO-treated mice (Fig. 5 C and D). Taken together, the effect velopment in CRPCs) (49, 50), and found their levels to be con- of GSNO in secondary xenografts indicates the potential long- A sistently suppressed upon treatment of tumors with GSNO (Fig. 4 term therapeutic implications of increased NO levels on CRPC. and C and SI Appendix,Fig.S5A–C). Together, the results suggest a strong effect of NO levels on regulation of TAM activity. Discussion Another aspect of TAM regulation that we focused on is TAM Our study reveals an essential role for NO in CRPC tumor differentiation. Previous studies showed that tumor-derived factors suppression. We found that increased levels of NO, which are like IL-34 and M-CSF educate macrophages to become the alter- associated with lowering LH and T under physiological condi- natively activated M2 type to promote , tissue remod- tions, lead to inhibition of prostate tumor growth in a cell non- eling, and immune suppression (13, 24). For differentiation of autonomous manner. In addition, we established that increased TAM, the binding of cytokines (IL34, CSF) to the CSF1 receptor levels of NO down-regulate the translational activity of AR, (CSF1R) is critical (7). Enrichment analysis suggested the signifi- leading to a decreased expression of AR-V7 and pERK levels in cance of IL-34 and CSF1R in resistance development in CRPC tumors. In addition, we showed that NO levels have the potential patients (Fig. 4D and SI Appendix,Fig.S3A–E). Therefore, we to influence differentiation of TAM by affecting ligands like IL- explored the implications of increased NO levels on 22RV1 cells in 34 and the receptors to which they bind (CSF1R). NO levels the presence or absence of GW2580 (CSF1R inhibitor) at varying contribute to a decrease in the M2 macrophages (F4/80, CD206) concentrations(0,0.5,10,25,and50μM, respectively). Upon the suppression of IL-34–CSF1R interaction, expression of PSA and and the induction of M1 macrophages (iNOS). Finally, we TMRPSS2 was abrogated (Fig. 4E). Taken together, decreased studied whether the short half-life of NO affects its efficacy on levels of pERK, AR in tumors, and abrogated levels of PSA and tumor macrophages. The results show that tumor burden was TMRPSS2 upon CSF1R inhibition strongly supports the regulatory significantly suppressed in mice that received cells from mice role of NO on TAM activity and differentiation. treated with GSNO. In addition, the M2 macrophage marker was decreased in the tumor grafts that acquired cells from GSNO- Short Half-Life of NO Does Not Affect Its Long-Term Impact on TAMs. treated mice, thus supporting the potential long-term therapeu- We evaluated the efficacy of increased NO levels in the re- tic implications of increased NO levels on CRPC. duction of CRPC tumor considering the short half-life of NO. Several studies have demonstrated the tumoricidal effects of NO For this, the CRPC cells (5 million cells per mouse) that were in other cancer types. Schleiffer et al. (51) showed that NO likely isolated from animals in the two treatment groups (PBS and plays a role in neoplastic changes in a rat model of colon cancer. GSNO) were xenografted s.c. into castrated SCID mice (n = 4per This work revealed that preneoplastic changes were promoted in the group) (secondary xenograft). Tumors were then allowed to grow colon by decreasing the release of NO through the inhibition of

11300 | www.pnas.org/cgi/doi/10.1073/pnas.1812704115 Arora et al. Downloaded by guest on September 24, 2021 Fig. 3. NO targets CRPC in cell nonautonomous manner. (A) Cell proliferation assay (MTT) using GSNO concentrations of 0, 5, 10, 25, 50, and 100 μMon 22RV1 cells showed no specific inhibition, indicating effects of NO on 22Rv1 cells are likely cell nonautonomous. (B) Relative expression of PSA and TMRPSS2 upon varying concentrations of GSNO ranging from 10, 25, 50, and 100 μM at RNA levels. (C) Twenty-six cytokines (CCL27, CD54, TIMP-1, ACRP30, G-CSF, AR, IL-17A, beta NGF, IL-2 R alpha, CCL28, Axl, CCL7, CCL17, CXCL6, IL-1 F2, M-CSF, TGFBeta 3, CXCL13, BMP-6, CCL23, NT-3, CCL11, CCL1, IL-5, IL-6, and IGFBP4) that were found to be suppressed more than 1.5-fold in tumors isolated from mice that received GSNO treatment compared with control mice that received PBS. (D) Impact of NO on TAM M1 macrophage (iNOS) induced and M2 (CD206 and Arginase-1) at RNA levels. (E–G) Impact of GSNO on M2 (F4/80 and CD206) and M1 macrophage (iNOS) at protein levels. (Scale bars: E, 750 μm; F, 100 μm; G,75μm.)

iNOS (51). A separate study (24) demonstrated the role of NO reduction in lung metastases (24). The tumoricidal effects of in renal cancer cells and the ability of NO to inhibit tumor NO are understudied in prostate cancer. Usually, tumor growth growth, as authors induced NO production by transfection of in therapeutics of prostate cancer is controlled through steroid tumor cells with iNOS. Their data indicate that the high levels management by blocking the AR or by decreasing circulating of NO are associated with inhibition of tumor growth and a androgens. However, this method has been limited due to the MEDICAL SCIENCES

Fig. 4. Impact of NO on TAM activity and differentiation. To validate the impact of increased levels of NO on TAM activity, the expression of pERK, ERK, AR- V7, and VEGF was checked in tumor grafts isolated from animals treated with PBS or GSNO. (A) Western blot confirmed reduced levels of AR-V7 as well as pERK upon increased NO. Immunostaining confirmed a reduced number of cells staining positive for pERK (B) and VEGF (C) upon increased NO levels. (Scale bars: B, 500 μm; C, 100 μm.) (D) To validate the impact of increased levels of NO on TAM differentiation, we evaluated expression of IL-34 in 22RV1 cells treated with 10, 25, 50, and 100 μM GSNO. (E) To establish the significance of suppression of IL-34–CSF1R interaction, CSF1R was blocked by GW2580 at 0.5, 10, 25, and 50 μM, followed by treating 22RV1 cells with GSNO. Inhibiting CSF1R abrogated GSNO suppressive impacts on PSA and TMPRSS2 levels.

Arora et al. PNAS | October 30, 2018 | vol. 115 | no. 44 | 11301 Downloaded by guest on September 24, 2021 Fig. 5. Long-term implications of NO on CRPC. (A) To validate the efficacy of NO on CRPC, we used secondary xenograft models in which castrated SCID mice were xenografted s.c. with 22RV1 cells followed by GSNO treatment in half of them for 4 wk. Cells from the two groups (control and experimental) of mice were harvested and reinjected into another set of castrated SCID mice, which were maintained for 4 wk but not treated (secondary xenograft). (B) The latter group showed a significant decrease in tumor burden. Mice that received cells from GSNO-treated animals showed a lower percentage of cells staining positive for M2 macrophages like F4/80 (C) and CD206 (D). (Scale bars: 100 μm.)

progression of the disease to a castration-resistant prostate cancer profound in noncastrate mice. However, this assumption still needs in which steroid manipulation becomes ineffective (52). Previous to be explored; (iii) the dose-dependent effect of NO on CRPC is studies have demonstrated that even modest increases in AR and not validated; and (iv) we have used 22Rv1 cells as a model cell line ARV7 expression may contribute to the development of resistance to investigate the implications of NO; however, use of cells from and progression of disease (14702632) (24909511). Therefore, human xenograft models of CRPC which were beyond the scope of increased NO capability to decrease AR expression (SI Appendix, the present study, but are ongoing in our laboratory, may further Fig. S5) may promote the susceptibility of PCa tumor cells to confirm and extend the findings discovered in this current study. current therapies. Recently, it has been reported that elevated NO In conclusion, this study shows the regulatory role of NO in the levels are able to inhibit tumor growth in androgen-dependent as TME of CRPC. To better understand the role of NO in the ther- well as CRPC cell lines such as LNCAP (30). However, the in vivo apeutics of CRPC, a further in-depth evaluation of other NO do- regulatory mechanisms behind the impact of increased NO levels norsandtheireffectsontheTMEofCRPCaswellasalong-term on CRPC tumors have largely been unknown. Our study reveals that follow-up to determine how the loss of function of NO could revert the TME could be the target of NO for suppression of tumori- the TAMs and affect CRPC is currently being addressed. genesis in CRPC cell lines like 22RV1. There are several strengths and limitations in our study. The study Materials and Methods reports four major findings: (i) This investigation demonstrates that Animal experiments were carried out in compliance with the Institutional NO levels increased by GSNO are capable of arresting tumor bur- ii Animal Care and Use Committee of University of Miami. Molecular analyses den in highly tumorigenic 22RV1 CRPC xenografts; ( )thework were performed using standard procedures. A more detailed description and shows that NO deffects on CRPC are cell nonautonomous effects additional data are provided in SI Appendix, Materials and Methods. and are targeting the TME; (iii) increased NO affects both activity and differentiation of TAMs in CRPC; and (iv) the study validates ACKNOWLEDGMENTS. We thank all the mentors (Dr. Dipen J. Parekh), the efficacy and durability of NO using a secondary xenograft collaborators (Dr. Alan Pollack, Dr. Chad Ritch), and interns (Aysswarya mouse model. The limitations of our study are as follows: (i)The Manoharan, Khushi Shah) for their insights, suggestions, and support during exact mechanisms by which NO-affected TAM differentiation re- this study. Additionally, we thank the American Urological Association ii Research Scholar Award and Stanley Glaser Award (for R.R.) and the Sexual main unexplored; ( ) increased NO levels are capable of affecting Medicine Society of North America (SMSNA) (for H.A.). J.M.H. is supported by hypogonadism and lead to CRPC tumor suppression, therefore NIH Grants 1R01 HL137355, 1R01 HL107110, 1R01 HL134558, 5R01 CA136387, leading to the strong possibility that the tumors inhibition could be and 5UM1 HL113460, and the Soffer Family Foundation.

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