INTERACTIONS WITH MUSCLE CELLS BOOST FUSION, STEMNESS AND DRUG RESISTANCE OF PROSTATE CANCER CELLS

Summary.

Poorly understood interactions with non-malignant cells within the tumor microenvironment play an important role in cancer progression. Here, we explored interactions between prostate cancer and muscle cells that surround the prostate. We found that co-culturing of prostate cancer cells with skeletal or smooth muscle cells expands the subpopulations of cancer cells with features characteristic of cancer stem-like cells, including anchorage-independent growth, elevated CD133 expression and drug resistance. These changes in the properties of cancer cells depended on: (1) the muscle cell-induced increases in the concentrations of interleukins 4 and 13; (2) the cytokine-induced upregulation of the expression of syncytin 1 and annexin A5; and (3) cancer cell fusion. In human prostate cancer tissues, expression of syncytin 1 and annexin A5, proteins that we found to be required for the cell fusion, positively correlated with the cancer development suggesting that these proteins can be used as biomarkers to evaluate cancer progression and potential therapeutic targets.

INTRODUCTION

Interactions between cancer cells and non-malignant cells within the tumor microenvironment play a critical role in cancer progression from primary tumor to metastasis, which is responsible for most of cancer-related deaths (1,2). These interactions direct dramatic changes in the morphology and properties of the primary tumor cells, including the transdifferentiation of epithelial cell–originated prostate cancer cells, into more invasive mesenchymal cells (the epithelial-mesenchymal transition, EMT). Cells can drastically change their properties by cell-to-cell fusion. Cells fuse in fertilization, in placentogenesis, in skeletal muscle formation, and in osteoclastogenesis (3). The molecular mechanisms underlying these fusion processes are poorly understood. Several proteins, including annexin A5 (AnxA5) and syncytin 1 (Syn1), have been implicated in diverse fusion processes. AnxA5 is involved in myoblast fusion (4), osteoclast fusion (5) and in placental trophoblast fusion (6). Syn1, a captive retroviral envelope protein that acquires a functional conformation after interactions with the ubiquitous receptor ASCT2, has been implicated in placental trophoblast fusion (7) and osteoclast fusion (5,8). It has been suggested that cell–cell fusion, possibly involving Syn1, plays an important role in cancer (9–13). However, the very low efficiency of cancer cell fusion in many experimental models has hindered elucidation of the role of cell fusion in cancer initiation and progression. The specific role and place of cell fusion in the dynamic and complex network of interactions between tumor cells and non-malignant cells, the nature of fusion-triggering events, and proteins involved remains to be elucidated.

In this study, we focused on prostate cancer, the most frequently diagnosed cancer in American men in 2017. The prostate gland is surrounded by smooth muscle cells of the prostate stroma and, on the apical and anterior surfaces, blends with the skeletal myofibers of the rhabdosphincter. To explore the interactions between prostate cancer cells and muscle cells, we co-cultured PC3 cells (human prostate cancer cell line) and primary human prostate cancer cells (pPCa) with primary smooth or skeletal muscle cells. The co-culturing of cancer cells with different muscle cells caused the expansion of subpopulations of cancer cells with cancer stem-like cells (CSLC) characteristics, including anchorage-independent growth, CD133 expression and drug resistance. We found that these changes in the properties of cancer cells required co-culturing-induced increases in medium concentrations of IL-4 and IL-13, cytokines that had been linked to both prostate cancer progression (14,15) and fusion of muscle cells (16–18). In turn, IL-4 and IL-13 upregulated the expression of Syn1 and AnxA5 and induced fusion of cancer cells that was dependent on these proteins. Cancer cell fusion is required for the muscle cell-dependent changes in the properties of cancer cells. These data and our finding that levels of expression of Syn1 and AnxA5 are higher in human prostate cancer tissues than in normal and benign prostatic hyperplasia tissues suggest that in the tumor microenvironment, interactions with muscle cells increase the metastatic potential and drug resistance of the cancer cells by promoting cancer cell fusion. Thus, our work identifies a direct link between Syn1- and AnxA5-upregulation, cell fusion and prostate cancer progression.

Materials and Methods

Cells.

PC3 cells (ATCC, Manassas, VA), primary human myoblasts (hMYO) and primary prostate smooth muscle cells (pSMC), both obtained from Lonza, were maintained according to the manufacturer’s instructions. Primary murine myoblasts were isolated and grown as described in (4). Primary human prostate cancer cells (pPCa) developed from a human prostate adenocarcinoma sample (Gleason 8 (4+4), T2N0M0) were obtained from Celther, Polska (Poland) and cultured according to the manufacturer’s instructions.

Cancer cell - muscle cell co-cultures.

Cancer and muscle cells were co-cultured in a 1:4 ratio in the same medium either separately or in a direct contact. To measure RNA or protein expression levels in cancer cells or in muscle cells, to examine clonogenic potential under anchorage-independent conditions, and to examine drug resistance, we co-cultured the cells without direct physical contact using 0.4-μm cell culture inserts (Millipore, Cat# PICM03050). To examine cell fusion, cell surface CD133 expression, and CD133 promoter-driven GFP expression in cancer cells, we co-cultured cancer cells and muscle cells in direct contact. In all co-culturing experiments, including the controls with cancer cells without muscle cells, the cells were grown in the medium that was a 1:1 mix of the culture media conventionally used for cancer cells and for muscle cells. We observed no obvious changes in the properties of PC3 cells after growing them in the medium that we used to culture muscle cells.

Fusion between cancer cells.

To quantify fusion by content mixing and multinucleation assays, we labeled cancer cells with either CellTracker™ Green CMFDA dye or CellTrace™ Far Red dye. Labeled cancer cells were co-plated to unlabeled muscle cells that had been seeded 24 h earlier. At the end of the 72-h co-incubation of cancer cells and muscle cells, we labeled cell nuclei with Hoechst-33342 and imaged the cells on Zeiss LSM 510 laser confocal microscope. Fusion between cells labeled with different probes mixes their cytosols and generates double-labeled cells. Images of the cells in both fluorescent channels were carefully examined and all cells that showed cell-associated fluorescence in both channels were counted as double-labelled and “content-mixed”.

We quantified multinucleation (=generation of multinucleated cells) by normalizing the total number of nuclei in multinucleated cancer cells (defined as cells with ≥2 nuclei) to the total number of nuclei in the field. Note that in this study we are interested only in relative efficiencies of fusion under different conditions. Culturing of the cells for 72 h complicates unambiguous quantitative analysis of the number of fusion events based on the appearance of double-labeled cells, and reliable measurements of the fusion efficiency will likely require live imaging of the cells.

To examine the role of nuclear division without cytokinesis in generation of multinucleated cells, we suppressed DNA replication by application of DNA methylation inhibitor FdUrd to the co-cultured pPCa cells and hMYO for the last 24h of the 72h co-incubation at the time of the most robust development of multinucleation.

To examine functional role of the AnxA5 in cancer cell fusion and multinucleation, we silenced AnxA5 gene expression in only PC3 cells or in both PC3 and hMYO cells with siRNA against AnxA5. PC3 cells transfected with either AnxA5 siRNA or non-targeting siRNA and 24h later overlaid with hMYO cells for 72h. To silence AnxA5 in both PC3 and hMYO cells, the cells were first overlaid and 24h later transfected with either AnxA5 siRNA or negative control siRNA and co-cultured for additional 72h.

Fusion between cancer and muscle cells.

We stained cancer cells and muscle cells with CellTracker™ Green CMFDA and CellTrace™ Far Red dyes, respectively, and co-cultured the cells for 72h. Then we labeled nuclei with Hoechst-33342 and took images on a Zeiss LSM 510 laser confocal microscope. To evaluate the percentage of cancer cells that were involved in fusion to muscle cells (identified as double-labeled), we normalized the number of cancer cell nuclei in the cells formed by fusion between cancer cells and muscle cells to the total number of cancer cell nuclei.

RNA transcription analysis.

Muscle cells were grown in collagen coated six-well plates. Cancer cells were grown on hydrophilic PTFE cell culture inserts with a pore size of 0.4 μm (Millipore, Cat. # PICM03050) placed in the same plate with muscle cells. At the end of the 72-h co-culturing RNA was extracted and analyzed.

CD133-Promoter Analysis.

To analyze CD133 gene expression, we transiently transfected PC3 cells with human CD133 promoter–driven GFP reporter construct obtained from Applied Biological Materials Inc. (Cat# MT-h68878) using Trans-IT transfection reagent (Mirus, Cat# MIR5400); 24h later, PC3 cells were overlaid with either hMYO or pSMC cells for 72h. The percentage of GFP-positive cells was determined from confocal microscopy images. The efficiency of the transfection was consistent from experiment to experiment at 80–85 % of the cells, as estimated using the GFP empty vector.

CD133 surface expression analysis.

Cells (1 × 10⁶) were plated in 100-mm dishes and cultured for 2 days before being stained with CD133 antibodies and analyzed by flow cytometry. To detect cell-surface CD133 expression in cancer cells co-cultured with muscle cells, PC3 cells stably expressing lentiviral GFP were co-cultured with muscle cells, and FACS analysis of the cell-surface CD133-PE was limited to the GFP-expressing PC3 cells.

To examine the effects of Syn1f expression in PC3 cells on cell surface CD133 expression, we transiently transfected PC3 cells with Syn1f; 24h later, we labeled PC3 cells overexpressing Syn1 with CellTrace™ Far Red dye and overlaid them with non-transfected PC3 cells labeled with CellTracker™ Green CMFDA dye. After a 72-h co-culturing, we stained the cells with CD133-PE antibody and analyzed CD133-PE expression in red-labeled, green-labeled and double-labeled (fused=yellow) PC3 cells.

Clonogenic potential under anchorage-independent conditions.

Cancer cells were grown on their own, co-cultured with different muscle cells, or treated with either IL-4 or IL-13 cytokines for 72 h. The cells were lifted and seeded as a single cell suspension onto an ultra-low-attachment surface (Corning) in growth factor-enriched medium (see Supplemental Materials for details). After 7–10 days of culturing, we counted the numbers of colonies (tumorspheres) operationally defined as dense, expanding, but not necessarily spherical, cell colonies that were stable after gentle media agitation, had size above 40μm and were present 7–10 day after plating.

Expression of Syn1 and Anx5 in tissue microarrays.

Paraffin embedded surgical resection specimens of prostate cancer, as well as normal and benign prostate tissues were obtained from the BioBank at Maine Medical Center Research Institute and were generated by the Histopathology Core at Maine Medical Center Research Institute. We have also used tissue microarrays generated by Cooperative Human Tissue Network (CHTN), Virginia University. The microarrays representing the stages of tumor progression in prostate adenocarcinoma were provided with tumor Gleason scores assigned by pathologists. Syn1 and AnxA5 were detected by immunostaining of paraffin-embedded prostate tissues with Syn1 antibody (Cat# NB-100–93579, Novus) and AnxA5 antibody (Cat# ab54775, Abcam). To quantify expression level of Syn1 and AnxA5, immunofluorescence staining was performed with 12 normal tissue specimens, 11 benign prostatic hyperplasia (BPH) specimens, 10 prostatic intraepithelial neoplasia (PIN) specimens, 11 cancer tissue specimens classified by pathologists as Gleason grade 5–6, 11 tissue specimens of Gleason grade 7, and 12 tissue specimens of Gleason grade 8–10.

Statistical Analysis.

We analyzed the data in the GraphPad Prism 6 graphing and analysis program using the Student’s paired t-test. A p-value < 0.05 was used to define statistically significant differences. *, < 0.05; **, < 0.001.

Supplemental Methods.

Cell culturing, reagents, plasmid, virus production and transfection, RNA isolation, qPCR, Western blot, flow cytometry, cell viability, gene silencing, cytokine expression and immunohistochemistry are described in detail in supplemental methods.

RESULTS

Co-culturing with muscle cells increases “stemness” and drug resistance of prostate cancer cells.

To study the dependence of cancer cells properties on the muscle cells in tumor microenvironment, we modeled the interphase between these cells using co-culturing experiments. We used highly malignant PC3 cells that have epithelial-like morphology and are widely employed to study EMT, drug resistance and CSLC (19,20), and primary prostate cancer cells (pPCa) developed from a human prostate adenocarcinoma. In contrast to PC3 cells, pPCa cells express PTEN, p53 and IL-4 (Fig. S1). Like PC3 cells, pPCa cells are deficient in androgen receptor expression.

We incubated PC3 cells for 72 h on transwell cell culture inserts placed into the cell culture wells with either primary human skeletal muscle cells (hMYO), primary prostate smooth muscle cells (pSMC), or primary murine myoblasts (mMYO). After the coculturing, we evaluated the clonogenic potential of the PC3 cells by plating them onto an ultra-low-attachment surface in a growth factor-enriched serum-free medium. The coculturing with the muscle cells increased a number of dense, 3-dimensionally expanding, but not necessarily spherical, larger than 40 μm colonies (“tumorspheres” (21)) of PC3 cells formed under these conditions (Fig. 1A,B). Similarly, coculturing with mMYO increased the number of pPCa-formed tumorspheres (Fig. S2).

Figure 1. PC3 and muscle cell co-cultures exhibit novel PC3 cell subpopulations characterized by elevated anchorage-independent growth, CD133 expression, and drug resistance.

(A) Representative images of single-cell clones of PC3 cells (tumorspheres, marked by yellow arrows) developed in an anchorage-independent manner, in a serum-free medium enriched in growth factors by PC3 cells and PC3 cells that were co-cultured with either human skeletal muscle cells (hMYO), prostate smooth muscle cells (pSMC), or mouse myoblasts (mMYO). Scale bar, 90 μm (B) Numbers of tumorspheres formed by PC3 cells and PC3 cells that had been co-cultured with muscle cell of the indicated types. (C) Percentages of CD133 gene expressing cells among PC3 cells grown on their own (1), with hMYO (2), pSMC (3) or mMYO (4). To detect transcriptional activity of the CD133 gene in fluorescence microscopy as GFP expression, PC3 cells were transfected with pLenti lentiviral construct with GFP reporter driven by the human CD133 promoter. The transfection efficiency was consistent from experiment to experiment at 80–85 % of the cells, as estimated using the GFP empty vector. The cells used for all conditions within the same series of experiments were transfected in parallel 24h before the beginning of co-culturing experiments. Thus, both the percentage of the CD133 promoter–driven GFP reporter transfected prostate cancer cells and the distribution of the numbers of the reporter construct copies per cell within cell population are expected to be the same for all conditions. (D) Flow cytometry analysis of CD133 expression at the surface of PC3 cells or of PC3 cells co-cultured with either hMYO, pSMC, or mMYO. (E) qPCR analysis of RNA level expression of CD133 in PC3 cells. (F,G) PC3 cells and PC3 cells co-cultured with either hMYO cells or pSMC for 72 hours were treated with different concentrations of either doxorubicin (F) or cisplatin (G) for 24 h. The cell viability was determined from MTT assay. (H,I) Immunoblot of PC3 cells and PC3 cells co-cultured with hMYO (H) or pSMC (I) for different EMT markers: phosphorylated AKT (pAKT), AKT, MMP-9, Vimentin, E-Cadherin, Slug (I), and for Tubulin, as a loading control. (A-I) In all co-culturing experiments, cancer cells were grown on the inserts. All results are shown as means ± SEM (n ≥ 3).

Non-adherent anchorage-independent growth is a hallmark of the self-renewing drug-resistant prostate cancer cells, referred to as CSLC (22). A small population of these cells is hypothesized to play a key role in heterogeneity, growth, recurrence and metastasis of tumors (22). Analysis of the changes in CSLC population requires identification of these cells using stem cell markers. Since most of PC3 cells express CD44 (23), we identified CSLC as cells with elevated level of expression of CD133, a marker of CSLC from a variety of cancer cell lines and tumors, including prostate cancer (24). When we transiently transfected PC3 cells with a human CD133 promoter–driven GFP reporter lentiviral construct (25), ~25% of PC3 cells showed GFP expression and, thus, had active CD133 gene promoter (Fig. 1C). In agreement with earlier studies (26), fraction of PC3 cells expressing CD133 at their surface (CD133+ cells), detected by antibody staining was small (~ 1%, Fig. 1D, Fig. S3A). Thus, many cells showing CD133 gene promoter activity displayed no detectable CD133 at their surface. This discrepancy likely reflects the fact that cell surface expression of CD133 depends not only on transcriptional activity of the CD133 gene, but also on post-transcriptional regulation. Moreover, for the same cells, apparent protein expression varies widely depending on the antibody used (27). Both aforementioned assays and direct qPCR analysis of the endogenous CD133 transcript at the level of the cell population indicate that co-culturing of PC3 cells with different muscle cells upregulates CD133 expression (Fig. 1C-E). Coculturing of pPCa cells with hMYO or pSMC also upregulated CD133 expression at the RNA level (Fig. S4A). Promotion of anchorage-independent growth of cancer cells and upregulation of CD133 expression for cancer cells co-cultured with muscle cells points to an expansion of the CSLC population. CSLC have a higher resistance to chemotherapy treatments than main population of tumor cells (22). We examined whether interactions with muscle cells influence drug resistance of PC3 cells. We grew these cells on the inserts placed into the cell culture wells. hMYO or pSMC were grown on the bottom of the same wells. After 72h coculturing, we lifted PC3 cells from the inserts, and incubated them or PC3 cells grown on their own with either doxorubicin or cisplatin for 24h. We then analyzed the viability of PC3 cells by evaluating their metabolic activity using the MTT assay (Fig. 1F,G) and by counting live cells (Fig. S5A–C) and found that PC3-muscle cell co-culture moderately protected PC3 cells against the drugs. In similar experiments with pPCa cells, which, on their own, were more sensitive to doxorubicin treatment than PC3 cells, the effects of the co-culturing with muscle cells were even stronger (Fig. S5D–F).

Expansion of CSLC subpopulations, drug resistance, and elevated metastatic potential of cancer cells are often associated with EMT (28). We found that both PC3 and pPCa cells co-cultured with pSMC or hMYO had moderately higher levels of mesenchymal-cell-characteristic proteins, including elevated levels of AKT and its phosphorylated form (pAKT), MMP9, vimentin, and Slug, while expression of epithelial-cell-characteristic E-cadherin was decreased (29) (Fig. 1H,I; Fig. S5K,L). These changes in protein expression suggest that muscle cells promote EMT in prostate cancer cells. Since the data on CD133 expression, drug resistance and clonogenic potential suggest that only a fraction of cancer cells change their properties in the presence of the muscle cells, the analysis of the expression of different proteins in cell lysates likely underestimates the differences in the protein expression specific for the affected cells.

In summary, interactions with muscle cells expanded subpopulations of cancer cells demonstrating characteristic features of CSLC including anchorage-independent growth, elevated CD133 expression and drug resistance. Since these changes in the properties of cancer cells did not require direct contact between muscle and cancer cells, we focused our attention on the extracellular factors.

The effects of the muscle cells on cancer cells are mediated by IL-4 and IL-13 in the medium.

PC3 cells grown in the conditioned medium collected from PC3 cells co-cultured for 72h with different muscle cells demonstrated a moderate increase in resistance to doxorubicin (Fig. 2A) and in the clonogenic potential (Fig. S6A) compared with PC3 cells cultured in a fresh medium. Culturing pPCa cells in the conditioned media collected from either pPCa/pSMC or pPCa/hMYO cocultures also increased pPCa cells resistance to both doxorubicin and cisplatin (Fig. S5, G–J). Culturing PC3 cells in the conditioned media from different muscle cells grown on their own did not change the clonogenic potential, drug resistance and the numbers of CD133-gene expressing PC3 cells (Fig. S6, B,C,D). We also found no appreciable changes in the properties of PC3 cells grown on their own in the same medium for longer than 72h.

Figure 2. Interactions with muscle cells change the properties of the cancer cells by raising IL-4 and IL-13 contents in the medium.

(A) An increase in doxorubicin resistance observed for PC3 cells cultured in the conditioned medium collected from a 72-h co-culturing of PC3 and hMYO or mMYO or pSMC. We assessed changes in cell viability from counts of viable cells at the end of the 24 h treatment. IL-4 (B) and IL-13 (C) levels in the conditioned media collected from pPCa, pSMC, hMYO, PC3 cultures as well as from pPCa/hMYO, pPCa/pSMC, PC3/hMYO and PC3/pSMC co-cultures were evaluated using human IL-4 (B) or IL-13 (C) ELISA kits. (D) qPCR analysis of RNA level expression of IL-4 and IL-13 in PC3 cells and PC3 cells co-cultured with hMYO. (E) qPCR analysis of RNA level expression of IL-4 and IL-13 in hMYO and hMYO co-cultured with PC3 cells. (F) Numbers of tumorspheres formed by PC3 cells and by PC3 cells co-cultured with hMYO in the presence or in the absence of neutralizing antibodies to either IL-4 or IL-13. (G) Numbers of tumorspheres formed by PC3 cells, PC3 cells co-cultured with pSMC in the presence or in the absence of neutralizing antibodies to either IL-4 or IL-13. (H) Numbers of tumorspheres formed by PC3 cells and by PC3 cells co-cultured with mMYO in the presence or in the absence of neutralizing antibodies to either IL-4 or IL-13. (I) Flow cytometry analysis of CD133 expression at the surfaces of PC3 cells and of PC3 cells co-cultured with mMYO in the presence or in the absence of neutralizing antibodies to either IL-4 or IL-13. (B-I) In all co-culturing experiments, cancer cells were grown on the inserts. All results are shown as means ± SEM (n ≥ 3).

To identify coculture-dependent factors in the conditioned medium and to examine the mechanisms by which muscle cells influence the properties of cancer cells, we focused on two related cytokines IL-4/IL-13 that share receptors and have been suggested to promote progression of prostate cancer (14,15). While we did not detect IL-4 in the conditioned media of either pPCa, pSMC, or hMYO cells cultured on their own, the media collected after co-incubation of pPCa cells with either hMYO or pSMC contained significant amounts of IL-4 (Fig. 2B). In contrast, concentration of IL-13 in the medium of the co-cultured pPCa cells and hMYO was as low as in the medium from pPCa cultured on their own (Fig. 2C). The medium collected from pPCa/pSMC co-culturing had IL-13 concentration that was like that for pSMC cultured on their own and much higher than the concentration of IL-13 in the medium from pPCa cells cultured alone (Fig. 2C). Thus, co-culturing with pSMC exposed pPCa cells to higher IL-13 concentration than that characteristic for pPCa cells cultured on their own. The conditioned media from the co-cultures of PC3 cells with hMYO or pSMC also contained higher amounts of IL-13 than the medium collected from PC3 cells cultured on their own (Fig. 2C). The concentrations of IL-4 in the media from PC3 cells either cultured on their own, or co-cultured with hMYO or pSMC were too low for detection (Fig. 2B).

To individually evaluate the expression of the cytokines in cancer and muscle cells, we separately lysed cancer cells grown on cell culture inserts and muscle cells grown on the bottom of the wells and analyzed the level of IL-4 and IL-13 RNAs in lysates of the cells of each type. Expression of both cytokines was strongly increased in PC3 cells co-cultured with hMYO (Fig. 2D). In hMYO, co-culturing with PC3 cells boosted IL-13 expression but not IL-4 expression (Fig. 2E). The experiments, in which we co-cultured PC3 cells with mMYO and then analyzed the conditioned medium with a mouse ELISA test kit (Fig. S7), showed upregulation of the expression of both IL-4 and IL-13 in muscle cells. Thus, elevated amounts of IL-4 and IL-13 in the coculture media, dependent on the cytokine and on the cells, may be generated by either cancer or muscle cells or both.

Neutralizing antibodies to IL-4 and IL-13 suppressed the muscle-cell induced promotion of the anchorage-independent colony formation (Fig. 2 F,G,H) and blocked an increase in the number of cell surface CD133-positive PC3 cells (Fig. 2I, Fig. S3B). These data suggested that muscle cell-effects on cancer cells depend on the increased concentrations of IL-4 and IL-13 in the co-culturing medium.

We then examined whether application of recombinant IL-4 or IL-13 to cancer cells can substitute for the co-culturing with muscle cells. Indeed, PC3 cells cultured in the medium supplemented with IL-4 or IL-13 demonstrated more efficient anchorage-independent growth (Fig. 3A,B), elevated expression of CD133 gene (Fig. 3C), expanded subpopulations of PC3 cells expressing the CD133 gene (Fig. 3D,E), and CD133+ cells identified by cell surface labeling with the CD133 antibody (Fig. 3F, Fig. S3C). To test whether application of IL-4 or IL-13 increases drug resistance of cancer cells, we incubated PC3 cells with each cytokine for 72-h and found that the cytokine-treated cells better withstood the subsequent 24-h-long application of doxorubicin or cisplatin (Fig. 3G,H). Like PC3 cells co-cultured with muscle cells, PC3 cells treated with either IL-4 or IL-13 demonstrated moderately elevated levels of mesenchymal-cell-characteristic proteins AKT and pAKT, MMP9, Slug (for IL-4 treated cells) and vimentin and somewhat lowered expression of E-cadherin (Fig. 3 I,J).

Figure 3. Recombinant IL-4 and IL-13 expand subpopulations of PC3 cells characterized by anchorage-independent growth, CD133 expression, drug resistance and upregulation of EMT markers.

(A) Representative images of tumorspheres developed by PC3 cells incubated with 50 ng/ml of either IL-4 or IL-13 for 72 h. Scale bar, 90 μm. (B) Tumorsphere counts determined for PC3 cells treated or not treated with either IL-4 or IL-13. (C) qPCR analysis of RNA level expression of CD133 in PC3 cells incubated with either IL-4 or IL-13 or neither for 72 h. (D) Representative fluorescence images that illustrate the transcriptional activity of the CD133 gene in PC3 cells transfected with GFP reporter driven by the CD133 gene promoter and then incubated for 72 h with either IL-4, IL-13 or neither (Control). Scale bar, 25 μm. (E, F) Percentages of PC3 cells expressing CD133 gene as detected with GFP reporter (E) and expressing CD133 at the cell surface as detected by antibodies (F) for the cells treated with IL-4, IL-13 or neither for 72 h. (G, H) The effects of 72 h pre-incubation with either IL-4 or IL-13 on viability of PC3 cells subjected to doxorubicin (G) or cisplatin (H) were assessed from counts of viable cells. (I, J) Immunoblot of PC3 cells and PC3 cells treated with either IL-4 (I) or IL-13 (J) for 72h for EMT markers (pAKT, AKT, MMP-9, Vimentin, Slug (I), E-Cadherin) and for tubulin, as a loading control. All results are shown as means ± SEM (n ≥ 3).

In brief, our results suggest that increased concentrations of cytokines IL-4 and IL-13 play a key role in muscle cell-dependent promotion of the stemness and drug resistance of cancer cells. The effects of IL-4 and IL-13 can be modulated by other cytokines upregulated by the interactions between cancer and muscle cells.

Co-culturing with muscle cells promotes fusion of cancer cells.

Since cells can change their properties by fusion, we tested whether interactions with muscle cells resulted in fusion of prostate cancer cells. We co-plated PC3 cells pre-labeled with green cell tracker with PC3 cells pre-labeled with red cell tracker and with either pSMC or hMYO. After 72-h co-incubation, we observed some double-labeled PC3 cells (Fig. 4A) that were not observed when we co-cultured differently labeled PC3 cells in the absence of muscle cells (Fig. 4B,C). Exchange of cytosolic markers between the cells (hereafter referred to as content mixing) suggested that co-incubation of PC3 cells with either pSMC or hMYO promotes fusion between PC3 cells. The finding that co-incubation of PC3 and muscle cells also increased the numbers of multinucleated PC3 cells (defined as cells with two or more nuclei) (Fig. 4B,C) and the time-lapse microscopy (Supplementary Movie 1) further substantiated this interpretation. Similar muscle cell-induced promotion of content mixing and multinucleation was observed for pPCa cells (Fig. 4D,E, and Fig. S8A).

Figure 4. Interactions with muscle cells promote content mixing and multinucleation of cancer cells.

(A). Fluorescence microscopy images of PC3 cells labeled with either green cell tracker or red cell tracker and co-cultured with either pSMC or hMYO for 72 h. Yellow and white arrows mark, respectively the co-labeled and multinucleated cells (white arrows). Bar, 25 μm. (B, C) PC3 cells labeled with either green cell tracker or red cell tracker and cultured by themselves, or co-cultured with hMYO (B) or pSMC (C) for 72h. Content mixing (bars 1 and 2) and multinucleation (bars 3 and 4) are quantified as the ratio of the number of nuclei either in double-labeled cells (fusion) or in multinucleated cells (multinucleation) to the total number of nuclei in the field of view. (D, E) Multinucleation (D) and content mixing (E) were quantified for pPCa cells cultured by themselves or co-cultured with hMYO or co-cultured with hMYO in the presence of FdUrd for 72 h. (F). Fluorescence microscopy images of co-cultured pPCa labeled with green cell-tracker and either pSMC or hMYO labeled with red cell-tracker. Arrows mark cells generated by pPCa-muscle cell fusion and labeled with both cell trackers. Bar, 25 μm. (G) Quantification of fusion between pPCa and pSMC and between pPCa and hMYO. (H) Quantification of fusion between PC3 cells and either pSMC (1), hMYO (2) or mMYO (3). (A-H). Cancer and muscle cells were co-cultured in a direct contact. All results are shown as means ± SEM (n ≥ 3).

Multinucleation can develop not only from cell fusion but also from nuclear division without cytokinesis. The latter mechanism is expected to be inhibited by blocking DNA replication with fluorodeoxyuridine (FdUrd). While, as expected, FdUrd application to pPCa/hMYO cocultures suppressed cell proliferation (Fig. S9), this reagent did not suppress the formation of multinucleated pPCa (Fig. 4D), indicating that formation of these cells involves cell fusion. Unexpectedly, we found FdUrd to increase the numbers of double-labeled cells generated by co-incubation of pPCa cells with hMYO (Fig. 4E). We speculate that suppressing cell division lowers the numbers of contacts within cell-division-generated clusters of same-colored cells and, thus, increases the frequency of green cell/red cell contacts which, upon fusion yield double labeled cells.

In addition to homotypic fusion between cancer cells, co-culturing with human muscle cells also induced heterotypic fusion between cancer cells and muscle cells, observed as the appearance of double-labeled cells after a 72-h co-incubation of green cell tracker-labeled pPCa cells with redcell tracker-labeled pSMC or hMYO (Fig. 4F,G). Heterotypic fusion was also observed for PC3 cells and either hMYO or pSMC (Fig. 4H, Fig. S8B). Interestingly, in contrast to hMYO, murine myoblasts (mMYO) did not fuse with PC3 cells (Fig. 4H).

To summarize, co-incubation of cancer cells with skeletal or smooth muscle cells promoted fusion of cancer cells, and heterotypic fusion between cancer cells and human muscle cells.

Muscle cell-promoted fusion of cancer cells depends on Syn1 and AnxA5.

We found that co-culturing of cancer and muscle cells boosts the expression levels of Syn1 and AnxA5, proteins associated with several fusion processes (4,6,7,30). The coculturing with hMYO increased levels of Syn1 and AnxA5 gene expression (Fig. 5A) in the PC3 lysates. Western blot analysis has shown a corresponding increase in the expression of these proteins, compared with PC3 cells cultured on their own (Fig. 5B,C). pPCa cells co-cultured with either pSMC or hMYO also showed elevated protein expression except for AnxA5 in pPCa cocultured with hMYO (Fig. S10).

Figure 5. Muscle cell-promoted fusion of cancer cells depends on Syn1 and AnxA5.

(A) qPCR analysis of Syn1 and AnxA5 expression in PC3 cells grown on the inserts on their own or co-cultured with hMYO (A) grown on the bottom of the same wells. (B,C) Western blot analysis of expression of Syn1 and AnxA5 in PC3 lysates with tubulin as a loading control for PC3 and for PC3 co-cultured with either hMYO (B) or pSMC (C). (D) Western blot analysis of AnxA5 expression in PC3 or PC3/hMYO cocultures transfected either with AnxA5 siRNA or non-targeting (NT) siRNA, and tubulin as a loading control. (E, F) The effects of siRNA suppression of AnxA5 expression in PC3 cells and in PC3 cells cocultured for 72 h in direct contact with hMYO (PC3/hMYO) on content mixing (E) and on multinucleation of PC3 cells (F). 1 – PC3 cells transfected with NT siRNA; 2 – PC3/hMYO coculture transfected with NT siRNA. 3 – PC3 transfected with AnxA5 siRNA and cocultured with hMYO. 4 - PC3/hMYO coculture transfected with AnxA5 siRNA. (G) Western blots for PC3 cells cultured on their own or with mMYO. (H) Multinucleation for PC3 cells (1), PC3 cells co-cultured for 72 h with mMYO (2), and PC3 cells co-cultured with AnxA5^−/− mMYO (3). (I) Multinucleation for PC3 cells (1), PC3 cells co-cultured for 72h with mMYO (2), and PC3 co-cultured for 72 h with mMYO in the presence of 50 μM Syn1 peptide (4) or 50 μM scrambled Syn1 peptide (3). (J) At the top, Western blots of PC3 cells transduced with either non-targeting control shRNA (PC3-NT) or with Syn1 shRNAs (PC3-Syn1 shRNA) to evaluate levels of expression of Syn1 with tubulin as a loading control. Below, multinucleation of PC3-NT cells (1), for PC3-NT cells co-cultured for 72 h with hMYO (2), and for PC3-Syn1 shRNA co-cultured with hMYO (3). (K) Quantification of heterotypic fusion between hMYO and either PC3 cells or PC3-Syn1 shRNA after 72h of co-culturing. (A-D) Cancer cells were grown on the inserts. (E-I) In all fusion experiments, cancer and muscle cells were co-cultured in a direct contact. All results are shown as means ± SEM (n ≥ 3).

Both AnxA5 and Syn1 are already implicated in cancer biology (30,31), and we focused on involvement of these proteins in the muscle-cell-induced fusion and multinucleation of cancer cells. Anx A5 deficiency is known to suppress myoblast fusion (4). We found the siRNA suppression of human AnxA5 in only PC3 cells or in both PC3 cells and hMYO to inhibit content mixing and multinucleation of PC3 cells observed after PC3/hMYO coculturing (Fig. 5D,E,F). To further explore the effects of AnxA5, we verified that mMYO isolated from wild-type mice, promote expression of Syn1 and AnxA5 and multinucleation in PC3 cells (Fig. 5G,H). In contrast, mMYO isolated from AnxA5-deficient mice did not promote multinucleation of PC3 cells (Fig. 5H). These data indicated that muscle-cell induced fusion between PC3 cells depends on AnxA5 produced both in cancer cells and in muscle cells.

To examine the contributions of Syn1, we used a Syn1-derived synthetic peptide that blocks fusogenic restructuring of Syn1 and Syn1-mediated cell fusion (32). We found that this peptide, but not its scrambled version, lowered the numbers of multinucleated PC3 cells observed in PC3/mMYO co-cultures (Fig. 5I), suggesting that PC3 fusion depends on Syn1. Since murine cells do not have Syn1 gene, Syn1 must be expressed in PC3 cells. The importance of Syn1 in muscle cell-induced fusion between cancer cells was further substantiated by the experiments with PC3 cells stably expressing either non-targeting shRNA-GFP or Syn1 shRNA-GFP (PC3-NT and PC3-Syn1 shRNA cells, respectively). As expected, in the latter case, Syn1 expression was strongly downregulated (Fig. 5J). We co-cultured PC3-Syn1 shRNA or PC3-NT shRNA cells (bars 3 and 2) with hMYO cells for 72 h and found that suppression of Syn1 expression in PC3 cells inhibited both generation of multinucleated PC3 cells (Fig. 5J) and heterotypic fusion between PC3 cells and hMYO (Fig. 5K). Murine cells do not carry receptors for Syn1 and do not support Syn1-mediated fusion (33), thus providing additional support for the conclusion that both homotypic fusion between cancer cells and heterotypic fusion of these cells with muscle cells involve Syn1 and explaining the lack of fusion between mMYO and PC3 cells (Fig. 4H).

Together, our data suggest that Syn1 and AnxA5 upregulated in cancer and muscle cell co-cultures are required for cancer cell fusion.

An increase in the numbers of CD133-expressing and drug resistant cancer cells depends on cell fusion.

Application of recombinant IL-4 and/or IL-13 promoted fusion of PC3 cells (Fig. 6A,B) and pPCA cells (Fig. 6C,D, Fig. S11) and expression of Syn1 and AnxA5 in PC3 cells (Fig. 6E,F). Moreover, neutralizing antibodies to IL-4 and IL-13 inhibited fusion of cancer cells (Fig. 6G).

Figure 6. IL-4 and IL-13 dependence of muscle cell induced fusion of cancer cells.

(A) Fluorescence microscopy images of PC3 cells grown in the presence of either recombinant IL-4 (50 ng/ml), recombinant IL-13 (50 ng/ml) or neither for 72 h. Arrows mark examples of the co-labeled fused cells. Bar, 25 μm. (B, C) Quantification of content mixing between PC3 cells (B) and between pPCa cells (C) grown in the presence of IL-4 or IL-13 or neither. (D) Multinucleation for pPCa cells grown with and without IL-4 and IL-13. (E, F) Western blot analysis of expression of tubulin (as a loading control) and Syn1 and AnxA5 for PC3 cells and PC3 cells grown in the presence of 50 ng/ml IL-4 (E) or IL-13 (F). (G) Percentage of multinucleated cells for PC3 cells (1), PC3 cells co-cultured in a direct contact with mMYO and non-specific IgG (2), and PC3 co-cultured with mMYO in the presence of neutralizing antibody against IL-4 (3) or IL-13 (4). All results are shown as means ± SEM (n ≥ 3).

We hypothesized that muscle cell-induced increases in stemness and drug resistance of cancer cells depend on fusion between these cells. PC3 cells that were neither incubated with muscle cells nor exposed to IL-4/IL-13 were transfected to express exogenous Syn1 with a fusion-enhancing deletion in the cytoplasmic domain (Syn1f) (34). Syn1 overexpression in the transfected PC3 cells was confirmed by Western blots and qPCR (Fig. 7A). The transfected PC3 cells labeled with red cell tracker were co-plated, 24 h after transfection, with non-transfected PC3 cells labeled with green cell tracker. As expected, after a 48-h incubation of the cells, we observed many double-labeled (“yellow”) and multinucleated PC3 cells (Fig. 7B). We found that PC3 cell fusion induced by exogenous expression of Syn1f increased the clonogenic potential of cancer cells under anchorage-independent conditions (Fig. 7C). Syn1f-expressing PC3 cells demonstrated dramatically increased fractions of cells expressing the CD133 gene (Fig. 7D) and CD133 protein at the cell surface (Fig. 7E). Using content mixing assay, we separated our PC3 cells into 3 subpopulations: yellow cells that contain only fused cells, red cells which are a mixture of fused and unfused Syn1f transfected cells and green cells that are neither transfected nor fused. Fraction of CD133+ cells positively correlated with the presence of fused cells (Fig. 7E) increasing from 1% for green cells to 20% for yellow cells. In addition, a higher percentage of CD133-positive cells observed for yellow cells than for red cells indicated that the promotion in the CD133 expression depends on Syn1f-mediated fusion rather than just on expression of Syn1f. These findings suggested that a major increase in the stemness of PC3 cells depends on Syn1-mediated fusion. We also found that PC3 cell fusion induced by exogenous expression of Syn1 raised the AKT, pAKT, MMP-9 and vimentin content and lowered E-cadherin content, both characteristic of the EMT (Fig. 7F).

Figure 7. Muscle cells-induced expansion of subpopulations of prostate cancer cells characterized by anchorage-independent growth, CD133 expression, and drug resistance depends on cell fusion.

(A) Western blot and qPCR analysis of Syn1 expression in PC3 cells transfected to express Syn1f (PC3-Syn1). (B) Fluorescence microscopy images of PC3 cells labeled with green cell tracker and PC3 cells expressing Syn1f and labeled with red cell tracker labelling. Arrows mark the colabeled cells. Bar, 25 μm. Content mixing between PC3 cells and between PC3-Syn1 cells was quantified by flow cytometry. (C). Representative images (scale bar 90 μm) and quantification of tumorspheres developed by PC3 cells and by PC3-Syn1 cells. (D) Quantification of CD133 gene expression detected as GFP expression driven by the human CD133 promoter for PC3 cells and for PC3-Syn1 cells. (E) Flow cytometry analysis of cell surface CD133 expression for the co-cultured PC3 cells labeled with green cell tracker and the PC3-Syn1 cells labeled with red cell tracker. Results averaged over 3 independent experiments are presented as percentages of CD133+ cells among green PC3 cells, red PC3-Syn-1 cells and yellow cells generated by PC3/PC3-Syn1 fusion cells (F) Western blot analysis of expression of pAKT, AKT, MMP-9, Vimentin, E-Cadherin, and tubulin, as a loading control, in PC3 cells and PC3-Syn1 cells. (G) PC3 cells stably expressing lentiviral GFP (PC3-GFP cells), and PC3-GFP cells co-cultured with hMYO in the presence of either Syn1 peptide or scrambled Syn1 peptide. After co-culturing the cells were treated or not treated with doxorubicin. The dependence of the co-culturing-induced increase in doxorubicin resistance of PC3 cells on cell fusion. Numbers of viable PC3 cells after a 24 h application of the drug were normalized to the numbers in corresponding controls not treated with the drug. PC3 cells (1); PC3 cells grown in the presence of either Syn1 peptide (3) or scrambled Syn1 peptide (2); PC3 cells co-cultured in a direct contact with hMYO (4), PC3 cells co-cultured with hMYO in the presence of either Syn1 peptide (6) or scrambled Syn1 peptide (5). All results are shown as means ± SEM (n ≥ 3). (H) Fluorescent images of PC3 cells stably expressing lentiviral GFP (PC3-GFP cells), and PC3-GFP cells co-cultured in a direct contact with hMYO in the presence of either Syn1 peptide or scrambled Syn1 peptide. Cell were either treated with doxorumicin or not treated with doxorubicin. (I) Representative fluorescent images of prostate tissue microarray samples including BPH, PIN, Gleason grade 5–6, 7 and 8–10 stained with Syncytin 1 and Annexin A5. (J) Quantification of Syncytin 1 and Annexin A5 in BPH, PIN, Gleason grade 5–6, 7 and 8–10 prostate tissue samples. Means ± SEM (n ≥ 10). (K) Suggested pathway by which muscle cells in the microenvironment of prostate cancer cells promote its progression.

To test involvement of Syn1-mediated fusion in muscle cell-induced increase in the drug resistance of cancer cells, we blocked fusion with the Syn1 peptide. Addition of the Syn1 peptide, but not its scrambled version, inhibited increase in the doxorubicin-resistance observed after GFP-expressing PC3 cells were co-cultured with hMYO (Fig. 7G,H). Interestingly, Syn1 peptide inhibited drug resistance not only for the PC3 cells co-cultured with hMYO but also for the PC3 cells that were cultured on their own, suggesting a possible role of Syn1-mediated fusion in generation of the drug resistant cells that are normally present in the PC3 cell population.

In summary, our findings support the importance of cell fusion in the muscle cell-induced increases in the numbers of CD133+ and drug-resistant prostate cancer cells.

Elevated expression of Syncytin 1 and Annexin A5 in prostate cancer tissues.

Since our findings implicated Syn1 and AnxA5 in prostate cancer progression, we examined the expression of these proteins in the human prostate tissue microarrays by staining with antibodies to Syn1 and AnxA5 (Fig. 7I and J). We analyzed microarrays with normal prostate, benign prostatic hyperplasia (BPH) and malignant prostate tissue samples characterized by different Gleason score, a well-established histopathology grading system used to evaluate the aggressiveness of prostate cancer: the higher the score the more abnormal is the tissue. We found that while in normal tissue and in BPH the expression levels of either of these proteins were indistinguishable, in moderately differentiated carcinoma (Gleason score 5–6), Syn1 expression and AnxA5 expression were 60% and 180%, respectively, higher than in the normal tissues. Syn1 expression in prostatic intraepithelial neoplasia (PIN), a prostate abnormality preceding the development of prostate adenocarcinoma, was also higher than in non-cancerous and benign tissues.

The observation that Syn1 and AnxA5 expression is increased in cancer tissues compared with non-malignant tissues is consistent with our in vitro analysis supporting the important role of these proteins in cancer progression.

DISCUSSION

Interactions between tumor cells and between tumor cells and non-malignant cells in the tumor microenvironment play important roles in cancer progression (1). For example, signaling from prostatic smooth muscle cells to the prostatic epithelium influences dedifferentiation of epithelial cells and the progression of human prostatic adenocarcinomas (35). In the present study, to model the heterotypic cell-cell interactions at the interphase between the prostate gland and adjacent smooth muscle and striated muscle cells, we devised novel co-culture systems for cancer cells to grow in the presence of skeletal and smooth muscle cells. We found coculturing to promote the cancer cell stemness, defined here as increases in the fractions of CD133-expressing cells, anchorage-independent growth, drug resistance and EMT-characteristic changes in the protein expression, all known hallmarks of cancer progression. The mechanisms by which muscle cells induce these changes depend on the co-culture induced robust increases in IL-4 and IL-13 concentrations in the medium. In turn, elevated levels of IL-4 and IL-13 upregulated the expression of Syn1 and AnxA5 and promoted fusion of cancer cells. Finally, cell fusion increased stemness of cancer cells. Therefore, our findings suggest that progression of prostate cancer depends on IL-4/IL-13 mediated interactions between tumor cells and adjacent muscle cells. Finding that co-incubation with muscle cells or recombinant IL-4/IL-13 similarly changes the properties of two distinct types cancer cells (PC3 and pPCa cells) in the presence of different muscle cells suggests that these changes are not unique to a specific combination of cancer cells and muscle cells. Both of our cell lines are AR-negative and although most primary prostate tumors contain AR-negative cells (36) it will be important to extend our work to AR-positive prostate cancer cells.

IL-4 and IL-13 mediate muscle-induced changes in prostate cancer cells.

Progression of prostate cancer is influenced by cytokines in the tumor microenvironment and, specifically, by IL-4 and IL-13, (14,15,37). IL-4 promotes anti-apoptotic pathways that contribute to drug resistance of cancer cells and cancer stem cells (38) and that activate the pAKT pathway in cancer cells (39). IL-4 and IL-13 have been also implicated in myoblast fusion and macrophage fusion (16–18). Here we found that interactions between cancer cells and skeletal or smooth muscle cells exposed cancer cells to increased concentrations of IL-4 and IL-13. Several lines of evidence suggest that IL-4 and IL-13 play a pivotal role in muscle-cells-induced fusion and elevated stemness of the cancer cells. First, we found an increased concentration of these cytokines in the medium after the co-culturing. Second, muscle-cell-induced fusion of cancer cells was suppressed by neutralizing antibodies to IL-4 and IL-13. Third, application of recombinant IL-4 and IL-13 to cancer cells promoted fusion and stemness, similarly to the effects of the co-culturing. The concentrations of recombinant IL-4 and IL-13 that induce these effects were ~1,000-fold higher than their concentrations in the medium of the co-cultured cells. This large difference may reflect the contributions of other factors present in the medium, a redued biological activity of the recombinant cytokine, and a higher local concentration of cytokines at the surface of the contacting cells.

The mechanisms by which the co-culturing of cancer cells with muscle cells upregulates expression and secretion of IL-4 and IL-13 in these cells are yet to be clarified. Our finding that blocking cancer cell fusion suppressed IL-4/IL-13 dependent increase in stemness, indicates that this increase depends on the cytokine-induced cell fusion. More research is needed to elucidate the roles of these cytokines and other secreted factors in the effects of heterotypic cell interactions in the microenvironment of prostate cancer on its progression.

Formation of multinucleated prostate cancer cells depends on cell fusion, Syn1 and AnxA5.

The slow development of cancer cell fusion (2–3 days) is consistent with characteristic times of cell–cell fusion in the generation of myotubes, osteoclasts, and syncytiotrophoblasts (3). Development of the fusion competency of cancer cells is accompanied by upregulation of the expression of Syn1 and Anx5 implicated in some fusion processes (4,30). We found both proteins to be involved in fusion of cancer cells. Heterotypic cancer-muscle cell fusion also depended on Syn1.

Multinucleated cells labeled with only one of our probes can be also generated by endoreplication, i.e., nuclear divisions without accompanying cytokinesis. We present several lines of evidence (content mixing, videomicroscopy and multinucleation in the presence of DNA replication inhibitor) that coculturing-induced multinucleated cells are formed by fusion.

Fusion between cancer cells and non-malignant cells such as macrophages, endothelial cells, and epithelial cells and very rare events of fusion between non-malignant cells have been suggested to promote the formation and progression of tumors (12,13,40). Fusion of cancer cells with leukocytes generates cells exhibiting increased metastatic behavior and the number of these fusion hybrids in peripheral blood of human cancer patients correlates with disease stage (41). On the other hand, it has been suggested that fusion between cancer cells could suppress their invasive phenotype and that Syn1 expression could be used as a positive prognostic marker (42). Our work documents fusion of cancer cells in the context of their interactions with smooth and skeletal muscle cells, implicates Syn1 and AnxA5, and establishes the central place of cancer cell fusion in the pathway by which these cells acquire CSLC characteristics and become drug-resistant in vitro. Increased expression of Syn1 and AnxA5 in prostate cancer tissues compared with non-malignant tissues substantiates the hypothesis that Syn1 and AnxA5 play important role in cancer progression in vivo. These observations also suggest that changes in the expression of these proteins could be used to evaluate a progression potential of the prostate cancer cells.

Muscle cell-induced progression of prostate cancer cells.

We propose that interactions with smooth and skeletal muscle cells bring about the hallmarks of prostate cancer progression by the following mechanism (Fig. 7K). First, interactions between muscle and cancer cells upregulate the expression and secretion of IL-4 and IL-13. Then, IL-4 and IL-13 upregulate expression of the fusion-related proteins including AnxA5 and Syn1. While AnxA5 is upregulated in many tumors and cultured cancer cells and has been suggested to be involved in the tumor progression (31), the specific contributions of this protein remain unclear. Our data are the first evidence that AnxA5 is upregulated in prostate cancer in vivo and is involved at the cell-fusion stage of the progression. Alternatively, AnxA5 expression levels may influence pre-fusion processes, including cytokine expression. Similarly, while Syn1 was reported to be expressed in some cancer cell lines (9), and RNA encoding envelope protein of another endogenous retrovirus HERV-E was found in prostate carcinoma tissues and cell lines (43), our study is the first report showing upregulation of Syn1 expression in prostate cancer tissue. Moreover, we report that Syn1 contributes to cancer progression by fusing cancer cells, as evidenced by the finding that a peptide that specifically inhibits Syn1-mediated fusion also inhibits the cancer cell progression. An earlier finding that IL-4 promotes ASCT2 expression in some breast cancer cells (44) suggests that, in addition to Syn1 upregulation, IL-4/IL-13 can further promote cancer cell fusion by upregulating expression of Syn1 receptor.

Importantly, the progression of cancer cells was triggered by fusion between cancer cells rather than by fusion between cancer and muscle cells. Indeed, mMYO, which did not fuse with cancer cells, promoted muscle cell-induced progression of prostate cancer cells towards their more dedifferentiated and malignant state. Whereas here we focused on the effects of muscle cells on cancer cells, interactions with cancer cells clearly influenced the muscle cells. For instance, we observed hMYO and mMYO co-cultured with cancer cells to form some multinucleated myotubes that we have not observed for either of these skeletal muscle cells grown in the same medium on their own. 3-days of coculturing with cancer cells also resulted in a notable loss of muscle cells that may resemble the loss of smooth muscle and their replacement with fibroblasts and myofibroblasts in cancerous stroma surrounding developing tumor (45). The specific effects of cancer cells on the smooth and skeletal muscle cells in their environment still wait to be explored. It will be also interesting to compare the effects of mononucleated satellite cells, precursors to skeletal muscle cells used in our study, with the effects of fully differentiated multinucleated myotubes.

In earlier studies, large multinucleated cells have been noted in several cancer cell lines and in different human cancer tissues (46,47). Moreover, the presence of multinucleated cells in human cancer tissues has been reported to correlate with aggressiveness of the tumor and poor prognosis (46,47). Chemotherapeutic treatments increase the numbers of large multinucleated cells in cancer tissues (48). Since multinucleated cells undergo asymmetric division (producing mononuclear cells) and self-renewal in vitro and in vivo, these cells can function as cancer stem cells (49). Muscle-cell-induced and IL-4/IL-13–mediated fusion of cancer cells increases cell content of mesenchymal-cell-characteristic proteins including AKT, and, specifically, pAKT, MMP9, vimentin, Slug, and downregulates epithelial-cell-characteristic E-cadherin. These changes in protein expression in cancer cells may directly contribute to their increased aggressiveness. For instance, the EMT marker Slug plays a major role in prostate cancer progression through regulation of proliferation and promotes pAKT activity via suppression of PTEN (50). Increased Slug expression could initiate EMT in the prostate cancer–muscle microenvironment.

Importantly, we found that, within 3 days of the co-culturing, the interactions with muscle cells notably changed the properties of only a fraction of cancer cells: most of the cells in the population did not express CD133 and remained mononucleated and sensitive to the drugs. In the physiological context, the muscle- cell-induced changes in cancer cells can be expected to be slower and less frequent than in our co-cultures to be consistent with, fortunately, relatively inefficient progression of the primary prostate tumors.

Conclusions.

The prostate gland is surrounded by smooth muscle cells and contacts the skeletal myofibers of the rhabdosphincter. Our study identifies a novel pathway in which interactions between prostate cancer cells and muscle cells increase local concentrations of IL-4 and IL-13. These cytokines upregulate expression of Syn1 and AnxA5, and promote fusion between cancer cells. In turn, this fusion expands populations of CD133-expressing and drug-resistant cells, an expansion which may represent significant progression of the cancer. This novel pathway of microenvironment-driven progression of prostate cancer identified in in vitro experiments and substantiated by the in vivo findings showing upregulated expression of Syn1 and AnxA5 in cancerous tissues may present new therapeutic targets and may have analogies in other tumors.

Implications:

The discovered effects of muscle cells on prostate cancer cells reveal a novel and specific pathway by which muscle cells in the microenvironment of prostate cancer cells promote cell fusion and cancer progression.

Abbreviations:

Anx

annexin

CSLC

cancer stem-like cells

EMT

epithelial–mesenchymal transition

hMYO

primary human muscle cells

IL-4

interleukin 4

IL-13

interleukin 13

mMYO

primary murine myoblasts

PC3-NT shRNA

PC3 cells stably expressing non-targeting shRNA-GFP

PC3-Syn1 shRNA

PC3 cells stably expressing Syn1 shRNA-GFP

pPCa

primary prostate cancer cells

pSMC

primary prostate smooth muscle cells

Syn1

syncytin 1

Syn1pep

Syn1-derived synthetic peptide