Infiltrating neutrophils promote renal cell carcinoma (RCC) proliferation via modulating androgen receptor (AR) → c-Myc signals

Cancer Letters 368 (2015) 71–78

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

Infiltrating neutrophils promote renal cell carcinoma (RCC) proliferation via modulating androgen receptor (AR) → c-Myc signals Wenbin Song a,b,1, Lei Li a,*,1, Dalin He a, Hongjun Xie a, Jiaqi Chen a, Chiuan-Ren Yeh b, Luke Sien-Shih Chang a, Shuyuan Yeh b, Chawnshang Chang b,c,** a

Sex Hormone Research Center, Department of Urology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China George Whipple Lab for Cancer Research, Departments of Urology and Pathology, University of Rochester Medical Center, Rochester, New York 14642, USA c Sex Hormone Research Center, China Medical University and Hospital, Taichung, 404, Taiwan b

A R T I C L E

I N F O

Article history: Received 30 April 2015 Received in revised form 21 July 2015 Accepted 22 July 2015 Keywords: Infiltrating neutrophils Renal cell carcinoma (RCC) Androgen receptor c-Myc signal

A B S T R A C T

Early studies found critical roles for neutrophils in renal cell carcinoma (RCC) progression. However, detailed mechanisms of how infiltrating neutrophils in the kidney tumor microenvironment impact RCC progression remain unclear. Here we found more neutrophils were infiltrated in human RCC lesions than those found in surrounding normal kidney tissues. Similarly, in vitro studies also revealed that RCC cells recruited more neutrophil HL-60N cells than normal kidney epithelial cells. Furthermore, in vitro and in vivo experiments also showed that the infiltrated neutrophils could promote RCC cell growth. Mechanism studies showed that co-culture of RCC cells with neutrophil HL-60N cells could selectively upregulate the androgen receptor (AR) signals, which might then alter the c-Myc signals. Interruption approaches using AR-siRNA to knock down AR in RCC cells blocked neutrophil-enhanced RCC cell proliferation. In vivo data using an orthotopically xenografted RCC mouse model also confirmed that infiltrated neutrophils could promote RCC proliferation via modulating the expressions of related cytokines. Together, these results conclude that infiltrated neutrophils may function through modulating the AR → c-Myc signals to promote RCC cell proliferation. Targeting this newly identified infiltrating neutrophil → AR → c-Myc signal pathway in the kidney tumor microenvironment may provide a new potential therapy to better suppress RCC progression. Published by Elsevier Ireland Ltd.

Introduction Renal cell carcinoma (RCC) accounts for approximately 3% [1] of adult malignancies and represents 90–95% of neoplasms arising from the kidney [2]. RCC is the most common type of kidney cancer with an estimated 65,150 new cases diagnosed in the United States in 2013 [3], and may represent the most lethal urologic malignancy with deaths of more than 100,000 per year worldwide. Surgical treatment remains the standard of care for localized, non-metastatic RCC. However, 1/3 of patients undergoing surgical resection for local disease will eventually develop metastatic disease. In the European Union, approximately 60,000 new cases were diagnosed in 2006 [4] and about 30,000 metastatic RCC patients a year are potential candidates for systemic therapy in the European Union alone [5]. Importantly, metastatic RCC is generally highly resistant to chemotherapy and radiotherapy [6], with a 5-year survival rate of less

* Corresponding author. E-mail address: [email protected] (L. Li). ** Corresponding author. E-mail address: [email protected] (C. Chang). 1 Contributed equally. http://dx.doi.org/10.1016/j.canlet.2015.07.027 0304-3835/Published by Elsevier Ireland Ltd.

than 10% [7]. Recently, targeted therapy provides a new therapeutic approach for advanced RCC patients. However, its effect is still limited for patients with selective pathological types [8,9]. Therefore, other treatment options, including immunotherapy, were considered [10]. Interestingly, recent reports also indicated that the growth of RCC cells could be greatly influenced by the immune system [11]. Several immune cells in the RCC tumor microenvironment, including T cells, natural killer (NK) cells, dendritic cells (DCs) and neutrophils, might be recruited into RCC to exert their functions on tumor proliferation and invasion [12]. Neutrophils are one of the key tumor-infiltrating myeloid cells in the tumor environment [13]. Similar to the myeloid macrophages, neutrophils also have a tumor-associated subpopulation (TAN) [14], and the relationship between TAN infiltration and prognosis in human cancers has been systematically discussed [15]. Epidemiological evidence suggested that neutrophil infiltration within human cancers was associated with a poor clinical outcome in the clear cell carcinomas and hepatocellular carcinoma [16]. Early studies suggested that most diseases with gender differences might link to their differential expression of sex hormone receptors [17]. Our recent results show Androgen receptor (AR) could promote RCC metastasis via modulating HIF2a-VEGF signaling [18], which also partially explains why RCC has a gender difference in

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tumor incidence (male-to-female ratio 1.6:1) [19], and may link AR signaling to RCC progression. Here we further studied the role of AR on infiltrating neutrophil-mediated RCC progression, and found that infiltrated neutrophils might function through modulating the AR → c-Myc signals to promote RCC cell proliferation.

Immuno-fluorescent (IF) staining IF staining was carried out as described previously [23,24]. Tumor sections were placed on slides and were incubated with the following primary antibodies: Rabbit monoclonal to Ki-67 (Cell Signaling D3B5 1:300) was used as well as anti-firefly (Santa Cruz Biotechnology) in 3% bovine serum albumin in phosphate-buffered saline overnight at 4 °C, followed by respective secondary antibodies. The stained slides were mounted and visualized by a fluorescent microscope.

Materials and methods Human samples and IHC staining To investigate the expression of CD66b+ neutrophils in human RCC tissues, we collected specimens of 37 malignant tissues and 11 adjacent normal kidney tissues from clear cell RCC patients who had undergone partial or radical nephrectomy for primary RCC at the Department of Urology, the First Affiliated Hospital of Medicine School, Xi’an Jiaotong University, between 2002 and 2012. All tumor tissues were diagnosed according to the 2009 edition of the TNM system and nuclear grading was performed according to WHO guidelines. Formalin-fixed, paraffin-embedded samples were cut to a thickness of 5 μm. Each tissue section was deparaffinized and rehydrated with graded ethanol. For antigen retrieval, the slides were boiled in EDTA (1 mM/pH 8.0) for 15 min in a microwave oven. Endogenous peroxidase activity was blocked with a 0.3% hydrogen peroxide solution for 10 min at room temperature. After rinsing with PBS, slides were incubated overnight at 4 °C with respective primary antibodies that include mouse antihuman CD66b monoclonal antibody (Thermo Fisher; dilution 1:50). After three washes in PBS, sections were incubated with biotinylated anti-mouse secondary antibody respectively for 30 min at room temperature. Immunostaining was performed using the Envision System with diaminobenzidine (Dako Cytomation, Glostrup, Denmark). Finally, the signal was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB), and all of the slides were counterstained with hematoxylin. Data were obtained by manually counting positively stained cells in five separate areas of intratumoral regions under 400× high-power magnification. Densities were determined by computing the mean number of positively stained cells per high power microscopic field (HPF) [20,21].

Cell culture and stable cell lines The human RCC cell lines SW839 (more expression of AR) and OSRC-2 (less expression of AR) were purchased from ATCC, and the normal human kidney proximal tubular cell line HKC-2/HKC-8 was kindly provided by Dr. Syed Khundmiri from the University of Louisville (Louisville, KY), and maintained in Dulbecco’s Minimum Essential Medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS). The AR knockdown of SW839 and OSRC-2 cells was performed by transfection using lentiviral transduction of siRNA AR.

Migration assay The CM from RCC cells and normal human kidney proximal tubular cell lines, HKC-2 and HKC-8, at 1 × 105 were plated into the lower chambers of the transwells with 5 μm pore polycarbonate membrane inserts. 1 × 105 HL-60N cells were plated onto the upper chambers. After 8 hrs, the cells which migrated into the lower chambers were collected and counted by the Bio-Rad TC10 automatic cell counter. Each data point was performed in triplicate and the experiments were independently repeated twice. The migration capability of RCC cells was determined using the same transwell assay. RCC cells were co-cultured with HL-60N for 7 days, then trypsinized and seeded with serum-free DMEM into the upper chambers at 5 × 104 cells/well, the bottom chambers contained DMEM with 20% FBS, and incubated for 24 hrs. The migrated RCC cells attached to the lower surface of the membrane were fixed by 4% paraformaldehyde and stained with 1% toluidine blue. Cell numbers were counted in five randomly chosen microscopic fields per membrane.

MTT assay We plated 5000 RCC cells into each well of 24 well-plates, then divided these into 2 groups: RCC cells cultured with regular media; RCC cells co-cultured with HL-60N cells. On 0, 2, 4, 6 days, cells were collected and MTT assay was performed: 100 μl of 5 mg/ml MTT was added to each well. We included one set of wells with MTT but no cells (control), then incubated for 3 hours at 37 °C in a culture hood, removed media and added 150 μl DMSO, covered the plates with foil, agitated the cells on an orbital shaker for 15 min, and then read the absorbance at 570 nm.

Cell transformation with soft agar colony formation assay The colony formation assay was performed as described previously [22]. Briefly, 1000 cells were plated into 6-well plates. After 10 days, cultures were examined with an inverted phase microscope at 100× and 200× for final colony counts.

Quantitative PCR Total RNA was extracted from each cell line using Trizol (Invitrogen, Grand Island, NY). Reverse transcription was performed using the iScript reverse transcription kit (Bio-Rad). Quantitative real-time PCR (qRT-PCR) was conducted using a Bio-Rad CFX96 system with SYBR green to determine the level of mRNA expression of a gene of interest. Expression levels were normalized to the expression of GAPDH RNA. Western Blot assay Western Blot analyses of the expressions of AR and their related proteins were determined by Western blot according to the previous study [25]. All analyses were performed in triplicate. In vivo metastasis studies Male 6–8 weeks old nude mice were purchased from NCI. SW839 cells were engineered to express luciferase reporter gene (PCDNA3.0-luciferase) by stable transfection and the positive stable clones were selected and expanded in culture. 6 mice were injected with 1 × 106 SW839-Luc cells (mixed with Matrigel, 1:1 v/v) and the other 6 mice were co-injected with SW839-Luc cells (9 × 105) and HL-60N cells (1 × 105) into renal capsules. The tumor sizes were measured using a Fluorescent Imager (IVIS Spectrum, Caliper Life Sciences, Hopkinton, MA) at 3 different time points (2, 4, and 6 wks after injection). After monitoring with the Imager, mice were sacrificed and the tumors were further examined by H&E staining. Immunohistochemistry The tumor samples from in situ kidney tumors were fixed in 4% neutral buffered para-formaldehyde and embedded in paraffin. IHC staining was conducted using the VECTASTAIN® ABC System (Victor Laboratories, Burlingame, CA). The primary antibodies of the Rabbit polyclonal antibody against AR (N-20, 1:500 diluted) and rabbit anti-cMyc monoclonal antibody (ab32072, 1:50 diluted) were used for immunohistochemical staining, and the slides were analyzed using an E800 microscope (Nikon, Melville, NY) and a SPOT camera (Diagnostic Instruments, Arnold, MD). The protein expressions were scored as negative (score = 1), weak (score = 2), moderate (score = 3), or strong (score = 4) using a system that has been validated previously [26]. The positive staining signals were quantitated by Image J software. Statistical analysis Data are expressed as mean ± SEM from at least 3 independent experiments. Statistical analyses involved paired t-test with SPSS 17.0 (SPSS Inc., Chicago, IL). In the in vivo studies, measurements of tumors size among the two groups were analyzed through one-way ANOVA coupled with the Newman–Keuls test. P < 0.05 was considered statistically significant.

Table 1 Summary of pathological and clinical data. Characteristics

Informative cases

Median age (range), years Sex Male Female Stage T1 T2 T3 Grade G1 G2 G3 Metastasis M0 M1

56.4 (29–71)

M0, tumor without metastasis; M1, tumor with metastasis.

21 16 24 9 4 19 15 3 33 4

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Fig. 1. The infiltration of neutrophils in RCC tissue and cancer cells. A. The representative (upper panels) and quantitative (lower panels) IHC staining of neutrophil marker CD66b+ in RCC (scale bar: 20 μm). The clinico-pathological parameters of these tumors are listed in Table 1. B. The expression of N2 type markers in HL-60N cells. C. The quatitative data and cartoon show the recruitment of neutrophils by RCC cells. 1 × 105 HL-60N cells were plated onto the upper chamber with 5 μm pore polycarbonate membrane to determine the HL-60N migration rate toward conditioned media from RCC or non-malignant kidney cells. After 8 hrs, the HL60-N cells that migrated into the lower chamber were collected and counted by the Bio-Rad TC10 automatic cell counter. Compared to the normal kidney epithelial cell lines (HKC-2 and HKC-8), RCC cells (SW839 and OSRC-2) recruited more HL-60N cells (*P < 0.05).

Results RCC tissues have a better capacity than surrounding normal kidney tissues to recruit neutrophils To examine the potential impacts of neutrophils on RCC progression, IHC staining with neutrophil marker CD66b+ was performed to compare neutrophil infiltration in RCC and normal kidney tissues. Our results showed that more CD66b+ neutrophils were recruited to the RCC lesions than to normal kidney tissues (Fig. 1A). We then applied the in vitro migration assay to confirm the clinical findings by using a transwell Boyden chamber migration system. By treating with 1.25% DMSO for 5 days before migration assay, we observed that HL-60 cells were successfully differentiated to neutrophil-like HL-60N cells. We also found that neutrophil markers CD11b and MPO, and N2 type marker hARG-1, were expressed in N2 type neutrophils (HL-60N) (Fig. 1B). Importantly, the migration assay revealed that RCC SW839 and OSRC-2 cells recruited more HL-60N cells than non-malignant kidney tube epithelial HKC-2/ HKC-8 cells. (Fig. 1C). Together, results from Fig. 1A–C suggest that RCC cells have a better capacity to recruit neutrophils than their surrounding normal kidney tubular epithelial cells.

Infiltrated neutrophils enhanced the RCC cell proliferation via upregulating of AR signaling in RCC cells To further study the consequences of infiltrated neutrophils on RCC progression, we validated the AR expression in various RCC cell lines and found that AR was strongly expressed in SW839 and weakly expressed in OSRC-2 cells (Fig. 2A). To examine the role of neutrophils on RCC growth, SW839 cells were co-cultured with or without neutrophils for 7 days (Fig. 2B). Then the RCC cells were re-seeded in the 24-well plate (5 × 103/well) for proliferation evaluation by MTT assay. As shown in Fig. 2C (left), the results revealed that RCC cells co-cultured with neutrophils had better proliferation than RCC cells cultured alone. We then applied the 2nd different cell growth assay using colonyformation to confirm above finding. The results revealed that RCC SW839 cells with co-culture of infiltrated HL-60N cells also showed better proliferation capacity (Fig. 2C, middle, *P < 0.05). The 3rd cell growth assay with Ki67 staining also showed that RCC cells co-cultured with neutrophil cells have higher proliferation capacity than RCC cells alone (Fig. 2E). Importantly, to prove the above finding is not cell specific, we then replaced RCC SW839 cells with OSRC-2 cells, and similar results were observed (Fig. 2D, *P < 0.05).

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Fig. 2. Infiltrated neutrophils promoted RCC proliferation. A. AR expression in different RCC cell lines by Western blot. B. Illustration shows the RCC proliferation following co-culture with HL-60N cells. C. SW839 cell proliferation capacities following co-cultures with neutrophil cells were determined by MTT (left panel) and colony formation (middle panel). The quantitative proliferation data of SW839 are shown in the right panel (*P < 0.05). D. OSRC cell proliferation capacities following co-cultures with neutrophil cells were determined by MTT (left panel) and colony formation (middle panel).The quantitative proliferation data of OSRC are shown in the right panel (*P < 0.05). E. The Ki67 expression of SW839 plus/minus co-culture with neutrophils was detected using immunofluorescence.

Together, results from Fig. 2A–D demonstrated that infiltrated neutrophils could enhance the RCC cell proliferation. Mechanism(s) of how infiltrated neutrophils promote RCC cell proliferation To further dissect the molecular mechanism(s) of neutrophilmediated RCC cell proliferation, we focused on AR, as recent studies [18,27] indicated that AR plays positive roles in RCC progression. As shown in Fig. 3A, SW839 cells co-cultured with neutrophil HL60N cells had higher AR expression than those SW839 cells cultured alone. Similar results were also obtained with OSRC-2 cells. To validate the essential role of AR on infiltrated neutrophilenhanced RCC cell proliferation, we then applied the interruption approaches to knock down AR expression in RCC cells. The MTT assay results revealed that knockdown of AR blocked the neutrophilenhanced RCC proliferation (Fig. 3B). We also applied the 2nd different cell growth assay using colony-formation assay to confirm

the above finding and results revealed that knockdown of AR in RCC SW839 cells can decrease RCC cell proliferation, but with coculture with HL-60N cells showed better proliferation capacity (Fig. 3C). Together, results from Fig. 3A–C indicated that infiltrated neutrophils may function through modulating AR signals to impact the RCC cell proliferation.

Mechanism(s) of how infiltrated neutrophil-increased AR expression promotes RCC cell proliferation We applied Q-PCR-based focus-array to search for the key genes related to RCC cell growth. Among many genes, we found that IGF1, TGFβ-1 and c-Myc in SW839 cells and OSRC-2 cells could be further modulated by co-culture with neutrophils (Fig. 4A). Western-Blot analyses also confirmed up-regulation of c-Myc protein expressions in RCC cells after co-culture with HL-60N cells,

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Fig. 3. Mechanism dissection shows how infiltrated neutrophils promote RCC cell proliferation. A. WB detected AR expression in RCC SW839 and OSRC-2 cells with/ without co-culture with neutrophil HL-60N. B. The growth abilities were assayed by MTT following knocking-down AR RCC cells plus/minus HL-60N cell. C. The representative and quatitative data of RCC growth were determined by colony formation assay.

and knock-down of AR impaired c-Myc up-regulation in RCC cells co-cultured with HL-60N (Fig. 4B). Together, results from Fig. 4A and B suggest that infiltrated neutrophils may function through modulating the AR-cMyc signals to promote RCC cell proliferation. Infiltrated neutrophils enhanced RCC proliferation in the in vivo mouse model To further confirm the findings in vitro, we then applied a renal capsule xenografted mouse model of of SW839 (cells transduced with pCDNA3-luciferase vector) and HL-60N cells at 9:1 ratio, along with subcutaneous xenografts for monitoring tumor growth visually and via IVIS imaging [28]. Using IVIS image assay, we noticed the bigger tumors in the mice co-implanted with SW839 cells than in those co-implanted with HL-60N cells in 6 weeks (Fig. 5A). Histology analyses of RCC tumors formed in nude mice were performed by HE staining (Fig. 5B). IHC staining indicated higher expressions of AR and c-Myc in SW839 plus HL-60N xenografted mice than SW839 cells alone mice. These data were consistent with the above findings from the in vitro study (Fig. 5C). Together, results from in vivo mouse model studies (Fig. 5A–C) confirmed the above in vitro studies and demonstrated that infiltrated neutrophils could enhance RCC cell proliferation via modulating AR → c-Myc signals.

Discussion Previous studies showed that RCC was not generally considered as a sex hormone-dependent tumor; however, it has been reported that there is a huge difference in RCC incidence between males and females. An early study observed that testosterone treatment increased the incidence of carcinogen Fe-NTA induced RCC initiation in the rat model, indicating that sex hormone signaling might contribute to the RCC carcinogenesis [29]. Our recent study further revealed that targeting AR could inhibit RCC cell migration and invasion by modulating HIF-2α/VEGF signals [18], suggesting that RCC initiation and progression was highly linked to the AR signaling. Besides tumor cells, the tumor microenvironment is composed of a wide spectrum of immune cell types, which are significantly involved in cancer progression and prognosis. Tumor-infiltrating neutrophils (TIN) are known to make up a significant part of these immune cells within the tumor microenvironment in different types of human cancers [30–32]. Despite their origin in the peripheral blood, TINs have been shown to exhibit impaired bactericidal and enhanced angiogenesis activities [33]. The presence of intratumoral neutrophils has been reported to be associated with poor prognosis in primary breast cancer [34] and RCC [35]. Consistently, depletion of neutrophils led to an inhibited tumor growth [36], limited metastasis numbers [37] and reduced endothelial cell recruitment to the tumors [38]. Our results confirm the rather anti-tumor roles of

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Fig. 4. Mechanism how infiltrated neutrophil-increased AR expression promotes RCC cell proliferation. A. Screening gene profile changes in neutrophil-co-cultured with RCC cells by Q-PCR. B. Western blot was used to detect AR and c-Myc expressions in RCC after co-culture with HL-60N cells. GAPDH was used as an internal control. C. Luciferase assay to detect c-Myc activity when the RCC cells are co-cultured with neutrophils.

neutrophils as we found a significantly higher ratio of neutrophils in dissociated tumor cell suspension from advanced G2-G3 RCC patients. In our study, we have shown that RCC patients have higher proportions of neutrophil infiltration in tumor samples than in adjacent normal tissue. A higher percentage of neutrophils was found in the tumor tissue in the high-grade RCC patients than in the lowgrade RCC patients. These findings may suggest the possibility of cancer-induced systemic as well as local immunosuppression and further promote cancer progression via modulation of neutrophils, which both seem to be the early event in the course of the disease. Neutrophils are composed of N1 and N2 sub-types, which are functionally different. Previous studies suggested that the activity of N1 neutrophils might enhance the expression of immuneactivating cytokines and chemokines including TNF-β, IL12 and INF-γ with a higher capacity to directly kill tumor cells. In contrast, the N2 neutrophils play roles in carcinogenesis, angiogenesis and immunosuppression [39,40]. We found that the expression of N2 markers (hARG-1) is markedly increased in neutrophils after

treating with 1.25% DMSO for 5 days, indicating the transformation of neutrophils from the N1 to N2 subtype. Interestingly, we further found that infiltrated N2 neutrophils could promote RCC proliferation via up-regulation of AR expression, which is in agreement with our previous data [18] that AR could have a positive role in RCC progression from the view of tumor environment inflammation cells. Early studies reported that activation of AR signaling could suppress the growth of adrenocortical and thyroid epithelial cells, which is likewise associated with a downregulation of c-Myc transcription [41]. We dissected the mechanisms of N2 neutrophil mediated up-regulation AR in RCC, and through the present coculture system, we found that AR suppresses human RCC cell proliferation via inhibition of c-Myc transcription. In summary, our results (see the outline in Fig. 5D) showed that infiltrating HL-60N could promote RCC proliferation via modulation of the AR → c-Myc signaling. This newly identified signal pathway may provide us with a new potential therapeutic approach to better battle RCC progression via targeting these newly identified signals.

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Fig. 5. Neutrophils could promote RCC proliferation in an orthotopically implanted RCC model. A. The representative foci IVIS image (left panel), and quantitative tumor weights of SW839 cells (9 × 105) and HL-60N cells (1 × 105) were orthotopically and subcutaneously co-implanted into the renal capsule of nude mice (n = 6 for each group). B. Histology analyses of RCC tumors formed in nude mice by HE staining. C. IHC results indicated the expression of AR and c-Myc in HL-60N co-implanted RCC tumors or tumor alone. The quantitative data confirmed HL-60N co-implanted RCC tumors have more expression of AR and c-Myc than cells alone. D. Summary of mechanisms and regulatory pathways of neutrophil-promoted RCC progression. RCC cells can better recruit neutrophils into tumor microenvironments. The infiltrated neutrophils further promote RCC proliferation via increasing AR and c-Myc expression.

Acknowledgements This work was supported by NIH grants (CA155477 and CA156700) and The National High Technology Research and Development Program of China (863 Program, NO. SS2014AA020607). George Whipple Professorship Endowment and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104-TDU-B-212-113002). Conflict of interest None. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.07.027.

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