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. Author manuscript; available in PMC: 2012 May 15.
Published in final edited form as: Cancer Res. 2011 Mar 28;71(10):3482–3493. doi: 10.1158/0008-5472.CAN-10-2665

Endoglin regulates cancer-stromal cell interactions in prostate tumors

Diana Romero 1,4, Christine O’Neill 1, Aleksandra Terzic 1, Liangru Contois 1, Kira Young 1,2, Barbara A Conley 1, Raymond C Bergan 3, Peter C Brooks 1,2, Calvin PH Vary 1,2
PMCID: PMC3096740  NIHMSID: NIHMS284275  PMID: 21444673

Abstract

Endoglin is an accessory receptor for transforming growth factor-β (TGF-β) that has been implicated in prostate cancer cell detachment, migration and invasiveness. However, the pathophysiological significance of endoglin to prostate tumorigenesis has yet to be fully established. In this study we addressed this question by investigation of endoglin-dependent prostate cancer progression in a TRAMP mouse model where endoglin was genetically deleted. In this model, endoglin was haploinsufficient such that its allelic deletion slightly increased the frequency of tumorigenesis, yet produced smaller, less vascularized, and less metastatic tumors than TRAMP control tumors. Most strikingly, TRAMP:eng+/− tumors lacked the pronounced infiltration of carcinoma-associated fibroblasts (CAFs) that characterize TRAMP prostate tumors. Studies in human primary prostate-derived stromal fibroblasts (PrSC) confirmed that suppressing endoglin expression decreased cell proliferation, the ability to recruit endothelial cells, and the ability to migrate in response to tumor cell-conditioned medium. We found increased levels of secreted insulin-like growth factor binding proteins (IGFBPs) in the conditioned media from endoglin-deficient PrSCs, and that endoglin-dependent regulation of IGFBP-4 secretion was crucial for stromal cell-conditioned media to stimulate prostate tumor cell growth. Together, our results firmly establish the pathophysiological involvement of endoglin in prostate cancer progression, and they show how endoglin acts to support the viability of tumor infiltrating CAFs in the tumor microenvironment to promote neovascularization and growth.

Keywords: endoglin, TRAMP, carcinoma-associated fibroblast, CAF, PrSC, IGFBP-4

Introduction

Prostate cancer is the second leading cause of male cancer death in the U.S., mainly because of metastatic disease (1). Endoglin expression is altered in prostate cancer (2) and high endoglin levels are associated with decreased survival in patients with tumor Gleason scores 6–7 (3). We have shown that endoglin, a TGFβ co-receptor, is involved in prostate cancer cell migration and invasion. Importantly, endoglin expression is lost in human metastatic prostate cancer cells (4). When restored, endoglin inhibits cell migration in vitro via modulation of both Smad-dependent and independent signaling mechanisms (5, 6). Endoglin expression in human prostate cancer cells also represses their tumorigenicity in SCID immunosuppressed mice (6), and metastasis in an orthotopic mouse model of prostate cancer (7). These studies however, did not address the mechanisms underlying endoglin function in terms of stromal cell support of tumor vascularization and growth.

Solid tumors are a heterogeneous population of malignant and non-malignant cell types. The latter include inflammatory cells, stem cells, fibroblasts, and endothelial cells (8). These cell populations constitute the tumor stroma, which provides key regulatory determinants for tumor progression and metastasis (9). We have previously described the effects of endoglin expression in prostate tumor cells in vitro (4–6), as well as in vivo (6, 7). However, the in vivo role of endoglin expression in other tumor cell types is unknown. To address this question, we developed a genetic model of prostate cancer that combined endoglin haploinsufficiency (eng+/−, (10)) with the TRAMP (transgenic adenocarcinoma mouse prostate) mouse, a well-characterized transgenic model for the study of prostate cancer (11). TRAMP mice express the SV40 virus large T antigen under the control of the prostate epithelium-specific probasin promoter, and develop prostate cancer from hyperplasia through more aggressive and metastatic stages (11, 12). In this model, the resulting level of endoglin in all eng+/− mouse-derived tissues is deficient as compared to eng+/+ tissues (10). Our results demonstrate that endoglin is required for the presence of carcinoma-associated fibroblasts (CAFs) in prostate tumors. Furthermore, data suggest that the prostate tumor CAFs impaired by endoglin deficiency in the TRAMP model are myogenic in origin, and that endoglin suppression impairs CAF-mediated endothelial cell recruitment and CAF migratory response to tumor-derived factors. Finally, data support the hypothesis that endoglin downregulation in affects CAF IGFBP-4 expression, supporting a novel mechanism of cancer-stromal cell crosstalk mediated through endoglin.

Materials and Methods

Mouse strains

Endoglin-targeted mice were screened for the presence of a neo cassette in the truncated engineered endoglin allele, as previously described (10). TRAMP mice (The Jackson Laboratory, Bar Harbor, ME, USA) were screened for the presence of the SV40 large T antigen, as described on The Jackson Laboratory website (research.jax.org). Both TRAMP and endoglin heterozygous mice were maintained in the C57BL/6 background. Mice were bred, maintained, and experimentation was conducted according to the NIGH standards established in the Guidelines for the Care and Use of Experimental Animals.

Necropsy and analysis of mouse tissues

Mice were weighed and euthanized at 21 or 25-weeks of age. All mice were genotyped twice: after birth and following sacrifice. The internal organs were examined and dissected following established guidelines (13) and metastases determined as previously described (14). Harvested tumors and prostates were fixed in 4% paraformaldehyde for forty-eight hours and embedded in paraffin. H&E, Masson’s trichrome, and PECAM staining were performed as described (15). Antibodies used for immunohistochemistry were: anti-endoglin antibody MJ7/18 (Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA, USA). Anti-stromal-derived factor 1 (SDF-1); anti-smooth muscle actin (αSMA); anti-IGF-1; anti-IGF-IR (Abcam, Cambridge, MA, USA); anti-Ki67 (Dako, Glostrup, Denmark); and anti-IGFBP-4 were all obtained from R&D Systems (Minneapolis, MN, USA). TUNEL staining was performed with the In Situ Cell Death Detection kit from Roche (following manufacturer’s instructions, Basel, Switzerland).

For immunofluorescence analysis, anti-FSP-1 (S100A4 Ab-8 from NeoMarkers (Fremont, CA, USA, 1:50 dilution), anti-SM22α (Abcam; 1:200 dilution) and anti-IGFBP-4 (R&D Systems; 1:50 dilution) were used as previously described (16, 17).

The slides were examined with a Zeiss Axioskop microscope (Thornwood, NY, USA). Imaging was performed using the Scion Image software, and processed with Adobe Photoshop software as previously described (18). Human recombinant IGF-1, IGFBP-4 and IGFBP-6 proteins, and the neutralizing anti-IGFBP-4 were obtained from R&D Systems.

Protein analysis

The tumors were ground and homogenized in lysis buffer (150 mM NaCl, 300 mM sucrose, 1% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl pH 7.5) containing a cocktail of protease (Roche), and phosphatase (Calbiochem-EMD, Darmstadt, Germany) inhibitors. Immunoprecipitation and western blot analysis were performed with anti-endoglin (BD Transduction Laboratories, Palo Alto, CA, USA), and anti-β-actin (Sigma, St Louis, MO, USA) as previously described (16, 19).

Cell culture, gene silencing, and growth factor treatment

Human primary prostate stromal cells (PrSC, Clonetics, Lonza, Walkersville, MD, USA) were grown in stromal cell growth medium (SCGM, Clonetics, Lonza). PrSCs were used between passages 5 to 10. PC3-M-C and PC3-M-FL cells were grown as described in (6). Human primary umbilical vein endothelial cells (HUVEC, passage 3–6) were cultured as previously described (19). TRAMP-C2 cells were obtained from the American Type Culture Collection (Rockville, MD, USA), and maintained as described in the Supplemental information and (20).

siRNA for human endoglin interference was cloned in pSilencer 5.1 (Ambion, Austin, TX, USA). A pSilencer control (nonspecific) vector was purchased from the same company. The cells were transfected using Effectene (Qiagen, Valencia, CA, USA). RNA isolation and RT-PCR for endoglin and GAPDH were performed as previously described (6). Alternatively, constructs expressing 21-nucleotide endoglin-specific short hairpin RNAs (shRNA) targeting human endoglin (shENG(1), shENG(2), shENG(3)) or non-targeting control (shSC, Sigma, SHC002) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Constructs were packaged into lentivirus pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Transduction was performed by incubating PrSCs with lentivirus and stably transduced cells were subsequently used for studies without drug marker selection (see Supplemental information and Table s1). All cell lines were verified by morphology, mouse and human endoglin-specific PCR, certified mycoplasma-negative by PCR (Lonza), and primary cell cultures used within the indicated passage numbers.

Cell migration

Migration assays were performed as described (21). Briefly, 5 × 105 cells (HUVEC or PrSC) were suspended in migration buffer (stromal cell basal medium, SCBM, containing 1 mmol/L MgCl2, 0.2 mmol/L MnCl2, and 0.5% BSA), plated in the upper chamber of transwell migration chambers (8.0 μm, CoStar, Lowell, MA, USA), and allowed to invade through a polycarbonate membrane towards conditioned medium for 4h–8h at 37°C. Cells remaining on the topside were removed and cells that had migrated to the underside were stained with crystal violet. Cell migration was quantified in at least three independent experiments using triplicates, either by counting or by extraction of crystal violet and quantifying absorbance at 600 nm.

Analysis of conditioned media

1.2 × 106 PrSCs were plated in 10 cm-diameter plates. Forty-eight hours later, they were rinsed three times in stromal cell basal medium (SCBM, Clonetics, Lonza), and 5 ml/plate of fresh SCBM were added. Forty-eight hours later, the conditioned media were filtered (0.2 μm pore), concentrated and stored at −20°C until further analysis.

For isotope-coded affinity tag (ICAT) tandem mass spectrometry, the conditioned media were concentrated by ultracentrifugation, labeled, and purified using the Cleavable ICAT Reagent Kit for Protein Labeling (Applied Biosystems, Foster City, CA, USA), and analyzed with a tandem quadrupole time-of-flight mass spectrometer (QSTAR, MDS-SCIEX, Toronto, Canada) as described in (19). Analysis of mass spectrometric data was conducted using ProteinPilot™ software (Life Technologies, Carlsbad, CA, USA). Detailed methods provided in Supplemental Information.

Results

TRAMP:eng+/− mice have more tumors than TRAMP:eng+/+ mice, which are smaller and less metastatic

To generate TRAMP:eng+/+ and TRAMP:eng+/− transgenic mice, we crossed endoglin heterozygous (eng+/−) males (10) with TRAMP females (12). We analyzed tumor formation in TRAMP:eng+/+ and TRAMP:eng+/− 21-week-old (n = 12), and 25-week-old males (n = 10), obtaining similar results.

Western blot analysis indicated that TRAMP:eng+/− tumors demonstrated lower levels of endoglin than TRAMP:eng+/+ tumors, although heterogeneity was observed as expected ((10), Figure 1A). Quantitative analysis indicated that endoglin protein expression in TRAMP:eng+/− tumors was approximately one-third of the levels detected in TRAMP:eng+/+ tumors (Figure 1B). The stromal cells and most of the cancer cells within TRAMP:eng+/+ derived tumors expressed endoglin, which was significantly reduced in TRAMP:eng+/− derived tumors (Figure 1C). Image analysis (Figure 1D) suggested that this reduction was consistent (30–40% of wild type) with the data shown in Figure 1B. Normal prostate tissue sections exhibited only diffuse background staining using anti-endoglin antibody. However, the stromal cells within TRAMP:eng+/+ tumors expressed endoglin, with significantly reduced endoglin expression in TRAMP:eng+/− tumors (Figure 1C).

Figure 1. Endoglin expression is reduced in TRAMP:eng+/+ and TRAMP:eng+/− tumors.

Figure 1

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A) Western blot for endoglin and β-actin (control) in tumors derived from TRAMP:eng+/+ and TRAMP:eng+/− 21-week-old mice (n = 3).

B) Quantitation of western blots by image densitometry using Scion image analysis software (average diffuse optical density ± standard deviation).

C) Immunohistochemistry for endoglin in tumors derived from normal prostate and tumors from TRAMP:eng+/+ and TRAMP:eng+/− 21-week-old mice. The slides were counterstained with hematoxylin. Bars: 300 μm.

D) Quantitation of TRAMP tumor endoglin staining using Scion image analysis software (average pixel density ± standard deviation, (16)).

The frequency of prostate tumorigenesis was slightly higher in TRAMP:eng+/− mice than TRAMP:eng+/+ mice (Figure 2A). Two-thirds of the TRAMP:eng+/− tumors were non-metastatic, whereas all the TRAMP:eng+/+ tumors were metastatic (Figure 2A). Metastases were observed in lung and lymph nodes with similar frequencies in TRAMP:eng+/+ and TRAMP:eng+/− mice: 50% of the metastases occurred in local lymph nodes and 50% in lungs.

Figure 2. Prostate tumorigenesis and tumor angiogenesis are altered in TRAMP:eng+/+ versus TRAMP:eng+/− mice.

Figure 2

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A) Frequency of prostate tumorigenesis and metastasis in TRAMP:eng+/+ and TRAMP:eng+/− 21-week-old mice (n = 12).

B) Tumor size in TRAMP:eng+/+ (n = 4) and TRAMP:eng+/− (n = 5) 21-week-old mice (average weigh ± SD).

C) Immunohistochemistry for PECAM-1 and endoglin (arrows) in TRAMP:eng+/+ and TRAMP:eng+/− tumors from 21-week-old mice, counterstained with hematoxylin. Bars: 300 μm.

D) The number of microvessels stained for PECAM-1 and endoglin, determined in at least eight fields/sample (n = 4). *, p < 0.05 (Student’s t-test).

TRAMP:eng+/− tumors were smaller than TRAMP:eng+/+ tumors (Figure 2B). Quantification of the percentage of cells positive for the proliferation marker Ki67 and TUNEL staining indicated that proliferation and apoptotic rates were similar in the tumor cells of TRAMP:eng+/+ and TRAMP:eng+/− mice (data not shown), suggesting that the tumor microenvironment promoted more sustained growth of TRAMP:eng+/+ tumors over time.

TRAMP:eng+/+ are more vascularized than TRAMP:eng+/− mice

Endoglin is a marker of tumor neoangiogenesis (reviewed in (22)). To investigate differences in tumor vascularization, the endothelial cell marker PECAM-1, as well as endoglin (Figure 2C), were used to quantify the microvascular density (Figure 2D). The number of PECAM-1 positive vessels was five-fold higher in TRAMP:eng+/+ tumors versus TRAMP:eng+/− tumors, whereas endoglin positive vessels were 25–30% higher in TRAMP:eng+/+ tumors versus TRAMP:eng+/− tumors, suggesting that TRAMP:eng+/+ tumors benefit from higher amounts of metabolites and oxygen.

Endoglin is associated with CAF investment of TRAMP:eng+/+ tumors

Hematoxylin and eosin (H&E), and Masson’s trichrome staining revealed that TRAMP:eng+/+ and TRAMP:eng+/− tumors were poorly differentiated adenocarcinomas, with a predominant solid mass of epithelial-derived cells and very rare gland formation, as defined in (13). We also observed that TRAMP:eng+/+ tumors contained areas enriched in fibroblast-like cells. In contrast, all the TRAMP:eng+/− tumors analyzed were non-fibrotic indicating the absence of stromal fibroblasts (Figure 3A and 3B). Image analysis confirmed that the average area occupied by epithelial-like cells was approximately 75% in TRAMP:eng+/+ tumors versus 99% in TRAMP:eng+/− tumors.

Figure 3. TRAMP:eng+/− tumors lack carcinoma-associated fibroblasts.

Figure 3

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A) H&E and Masson’s trichrome staining in TRAMP:eng+/+ and TRAMP:eng+/− tumors revealed fibroblast-enriched areas in TRAMP:eng+/+ tumors (arrow). Bars: 300 μm.

B) Frequency of fibrotic and non-fibrotic tumors in TRAMP:eng+/+ and TRAMP:eng+/− 21-week-old mice. At least three sections per tumor were analyzed (n = 4).

C) Immunohistochemistry for stromal markers SMA and SDF-1 in TRAMP:eng+/+ and TRAMP:eng+/− tumors counterstained with hematoxylin. Bars: 300 μm.

D) Immunofluorescence for SM22α and FSP-1 in TRAMP:eng+/+ and TRAMP:eng+/−. The nuclei were stained with DAPI. Bars: SM22α, 300 μm; FSP-1, 200 μm.

Carcinoma-associated fibroblasts (CAFs) are a major and heterogeneous constituent of the tumor stroma (23). CAFs are characterized by the expression of smooth muscle actin (αSMA) and the stromal-derived factor 1 (SDF-1) (8), which were both detected in the TRAMP:eng+/+ but not TRAMP:eng+/− CAFs (Figure 3C).

One of the cellular components of CAFs is the SM22α-positive myofibroblast (24), which plays an important role in tumor behavior (25). SM22α was restricted to the TRAMP:eng+/+ stromal fibroblast, yet was largely absent from TRAMP:eng+/− tumors (Figure 3D). Immunofluorescence staining for fibroblast-specific protein 1 (FSP-1) was more pronounced in TRAMP:eng+/+ tumors confirming the identity of prostate-associated fibroblasts (17). However, double immunofluorescence analysis using anti-endoglin, and either anti-SM22α or anti-FSP-1 antibodies revealed that endoglin expression was associated with SM22α-positive cells but not FSP-1-positive cells (Figure 4A and 4B). These results indicate that TRAMP:eng+/+ tumors are largely comprised of endoglin-expressing myofibroblast-derived CAFs.

Figure 4. Endoglin is associated with tumor myofibroblasts.

Figure 4

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A) Double immunofluorescence for endoglin, SM22α, and FSP-1 in TRAMP:eng+/+ tumors. The nuclei were stained with DAPI. Arrows: endoglin and SM22α double-positive cells. Bars: 200 μm.

B) The number of endoglin-SM22α and -FSP-1 double-positive cells were counted in at least five fields/sample (average ± SD) (18).

Endoglin expression is necessary for the viability of cultured prostate stromal cells

We attempted to establish primary cultures of CAFs derived from TRAMP:eng+/+ and TRAMP:eng+/− tumors. However, whereas we were able to propagate TRAMP:eng+/+ CAFs in culture, the TRAMP:eng+/− derived CAFs were not viable under a variety of culture conditions (data not shown). To overcome this limitation, we used human primary prostate stromal cells (PrSC). Consistent with TRAMP immunohistochemistry, human PrSCs robustly expressed endoglin, as detected by RT-PCR (Figure 5A, left panel). Endoglin expression was transiently knocked down in PrSCs with a specific interfering RNA construct, siENG. The efficiency of endoglin RNA silencing was approximately 60%, as detected both by RT-PCR and immunoprecipitation (Figure 5A, right panel). This reduction of endoglin protein level approximated the difference seen in tumors (Figure 1A), and was sufficient to significantly impair PrSC cell growth in vitro (Figure 5B, left panel), suggesting that endoglin expression promotes prostate tumor CAF proliferation.

Figure 5. Endoglin knockdown reduces PrSC cell proliferation and affects PrSC-dependent modulation of PC3-M cell proliferation.

Figure 5

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PrSC were transfected with siRNAs directed against endoglin (siENG) or a control scrambled sequence (siSC) for forty-eight hours. Endoglin expression was analyzed by RT-PCR and immunoprecipitation (A). PC3-M-C and endoglin-expressing PC3-M-FL cells (6): negative and positive control, respectively. HC, immunoglobulin heavy chain.

B) Left panel: PrSC proliferation following siENG transfection: Two independent experiments using triplicates were performed. r: ratio of siENG- versus siSC-treated cells. Middle panel: PrSC siENG- or siSC siRNA-derived conditioned stromal cell basal medium (SCBM) was used to treat new cultures of PrSC. r: number of cells divided by the number of siSC cells in basal media. Right panel: PC3-M-C or PC3-M-FL cells were prepared in SCBM or PrSC-conditioned medium. The number of cells/well was determined forty-eight hours after as described above. r: number of cells divided by the number of cells in basal media.

C) PrSCs were transduced with shRNA constructs targeting human endoglin (left panel insert). Endoglin western blot of PrSC. (left panel) HUVECs were tested for ability to migrate towards basal PrSC shSC- or shENG(1–3)-medium (25μg protein). CM and BM, conditioned and basal medium, respectively.

D) TRAMP-C2 cells were used to prepare conditioned medium as described above. Following shRNA transduction, PrSC were used for migration assays as above.

* p < 0.05, and ** p < 0.005 (Student’s t-test). See Supplemental Information for detailed methods.

Because growth factor secretion is a recognized CAF function (23), we analyzed the effect of the conditioned medium from PrSCs in their proliferation. PrSC growth was stimulated when they were cultivated in their own conditioned medium. Moreover, PrSC-conditioned medium partially rescued the inhibitory effect of endoglin knock down in PrSCs. The conditioned medium from endoglin knock down in PrSCs failed to stimulate PrSC cell growth, or to rescue the inhibitory effect of decreased endoglin levels (Figure 5B, middle panel). These results suggest that endoglin affects prostate stromal cell viability via secretion of soluble factors.

Stromal fibroblasts stimulate the proliferation of prostate cancer cells through an endoglin-dependent mechanism

CAFs contribute to tumor development in part because they stimulate tumor cell proliferation (8). To further investigate the link between endoglin expression in PrSCs and prostate cancer cell proliferation, we used PC3-M cells that did not express endoglin (4, 6) (PC3-M-C, control), or that stably overexpressed endoglin (PC3-M-FL, full-length) (6). PC3-M-C and PC3-M-FL cells were grown in the presence of basal medium, or in the presence of conditioned medium from PrSCs transfected with an interfering RNA against endoglin or non-targeting control. Control PrSC-conditioned medium strongly stimulated the proliferation of both PC3-M-C and PC3-M-FL cells. The conditioned medium from endoglin knock down in PrSCs had a lower stimulatory effect in PC3-M-C cells, and no effect in PC3-M-FL cells (Figure 5B, right panel). Taken together, these results are consistent with the view that endoglin expression in stromal cells is necessary to stimulate cancer cell proliferation via a mechanism that involves secreted factors.

Endoglin deficiency in PrSCs impairs endothelial cell migration and tumor cell recruitment

To further suppress endoglin expression in PrSCs, three separate shRNA constructs were delivered using lentivirus (26). PrSC shENG(1–3) shRNAs resulted in either partial or complete suppression of endoglin protein levels, respectively (Figure 5C, inset). Conditioned medium collected from shENG1, shENG2, and shENG3 all reduced the ability of HUVEC migration, reflecting the degree of endoglin suppression. TRAMP-C2-conditioned medium was also tested for its ability to recruit PrSCs. Endoglin-deficient PrSCs were significantly impaired in their capacity to migrate in response to tumor cell-conditioned medium (Figure 5D). TRAMP-C2 endoglin knockdown did not affect cell recruitment (Data not shown), suggesting that endoglin is required for CAF-dependent recruitment of endothelial cells and their response to tumor cell factors.

Endoglin-dependent modulation of IGFBP-4 secretion by PrSCs is involved in the regulation of tumor cell growth

To identify peptides secreted by PrSCs, we performed isotope-coded affinity tag (ICAT) mass spectrometry (27) to compare the conditioned media from control and endoglin knock down-PrSCs. Among the proteins overexpressed by endoglin knock down in PrSCs were: (i) tissue inhibitors of metalloproteinases 1 and 2 (TIMP1, TIMP2), (ii) sulfhydryl oxidase 1, (iii) SPARC, and (iv) two members from the insulin-like growth factor binding protein (IGFBP) family: IGFBP-4 and IGFBP-6 (Table 1). These proteins are implicated in the induction of cell growth arrest, as well as in cell invasiveness (15, 28–31).

Table 1.

Summary of proteins identified and quantified by ICAT MSMS in PrSC conditioned media: siENG/siSC PrSCs.

Accession no. Name 1 H vs. L 3 N Function
P16035 tissue inhibitor of metalloproteinases 2 (TIMP-2) 3.026 18 ECM degradation
P09382 galectin-1 1.947 6 cell-ECM interaction
P00338 L-lactate dehydrogenase A chain 1.794 6 metabolism
P07355 annexin A2 1.750 8 signal transduction
P01033 tissue inhibitor of metalloproteinases 1 (TIMP-1) 1.633 1 ECM degradation
P14618 pyruvate kinase isozymes M1/M2 1.615 21 metabolism
O00391 sulfhydryl oxidase 1 1.559 5 induced in quiescent fibroblasts
O76061 stanniocalcin-2 1.554 11 calcium homeostasis
P27797 calreticulin 1.448 14 calcium homeostasis
P22692 2insulin-like growth factor-binding protein 4 1.439 3 IGFBP family
P12109 collagen alpha-1(VI) chain 1.347 12 ECM
P24592 2insulin-like growth factor-binding protein 6 1.357 4 IGFBP family
P60174 triosephosphate isomerase 1.289 16 metabolism
Q99497 DJ-1 1.247 18 chaperone
P23142 fibulin-1 1.220 7 ECM
P08123 collagen alpha-2(I) chain 1.190 8 ECM
P29400 collagen alpha-5(IV) chain 1.184 14 ECM
P09486 SPARC 1.126 24 inhibition of cancer cell proliferation
Q12841 follistatin-related protein 1 0.849 27 actin binding
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1

H/L: heavy isotope (siENG)- versus light isotope (siSC)-tagged peptide, average of confirmed samples.

3

N: number of peptides identified and quantified >95% confidence

Mass spectrometric sequencing of the putative IGFBP-4 and IGFBP-6 peptides confirmed their identities and corroborated the quantitative data indicating their upregulation in endoglin-deficient PrSCs (Supplemental Figures s1–s7). IGFBPs play important roles neoplastic processes and prostate cancer (32, 33) and TGFβ signaling regulates tumor-stromal interactions via IGF-1 (34). Therefore, we quantified the cell growth of PC3-M-C cells in response to recombinant IGF-1, IGFBP-4, and IGFBP-6 treatment. IGF-1 and IGFBP-6 stimulated PC3-M-C proliferation (Figure 6A). IGFBP-4 alone did not affect cell proliferation; however in combination with IGF-1, it inhibited IGF-1-dependent stimulation of cell proliferation (Figure 6A). When these treatments were performed in PrSC-conditioned medium, the growth stimulation effect of IGF-1 and IGFBP-6 was enhanced, and, surprisingly, IGFBP-4 alone inhibited cell proliferation. These effects were likely due to the presence of PrSC-derived IGF-1 in the medium (35). It is reasonable to postulate that IGFBP-4 inhibits PC3-M proliferation through an IGF-dependent mechanism because PC3 cells express IGF signaling components (36). A similar response was detected in PC3-M-FL cells (data not shown). The use of a blocking antibody for IGFBP-4 partially prevented its inhibition of PC3-M-C cell proliferation when the treatment was performed in control PrSC-conditioned medium (Figure 6B). When added in the presence of endoglin knock down PrSC-conditioned medium, the neutralizing antibody had the same partial blocking effect on IGFBP-4-dependent inhibition of PC3-M-C cell growth (Figure 6B). This experimental approach confirmed the presence of functional IGFBP-4 in endoglin knock down PrSC-derived medium, which is consistent with the reduced size of TRAMP:eng+/− tumors.

Figure 6. IGF-1 signaling and PrSC-dependent modulates PC3-M cell proliferation.

Figure 6

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A) PC3-M-C and PC3-M-FL cells were prepared in SCBM or PrSC-conditioned medium, with or without 50 ng/ml IGF-1, 50 ng/ml IGFBP-4, and 50 ng/ml IGFBP-6. 19,000 cells/well were plated in 24-well plates. Forty-eight hours after, the number of cells/well was determined. Two independent experiments using triplicates were performed. r: number of cells divided by the number of untreated cells in basal media. * p < 0.05, and ** p < 0.005 (Student’s t-test). Asterisk-tagged bar statistics are referenced to lane 1.

B) PC3-M-C and PC3-M-FL cells were trypsinized and resuspended in SCBM or PrSC-conditioned medium, with or without 50 ng/ml IGFBP-4, and 100 ng/ml anti-IGFBP-4 antibody. Proliferation assay was performed as described for panel (A).

C) Immunohistochemistry for IGFBP-4, IGF-1 and IGF-IR in tumors derived from TRAMP:eng+/+ and TRAMP:eng+/− 21-week-old mice, counterstained with hematoxylin. Bars: 300 μm.

D) Immunofluorescence for SM22α and IGFBP-4 in tumors derived from TRAMP:eng+/+ and TRAMP:eng+/− 21-week-old mice. Arrows: IGFBP-4 staining. Bars: 300 μm.

TRAMP:eng-derived tumor sections were stained for these IGF signaling components. In TRAMP:eng+/+ tumors, IGFBP-4 was detected in both fibroblast-like and epithelial-derived cancer cells. The epithelial staining appeared to be peripheral, suggesting that most of the IGFBP-4 detected was associated with the stromal compartment (Figure 6C, arrow). TRAMP:eng+/− tumors showed minimal staining for IGFBP-4 (Figure 6C), due to the lack of CAFs. IGF-1 and IGF-IR receptor were detected mainly in the non-stromal compartment (Figure 6C).

Immunofluorescence analysis of TRAMP:eng+/+ tumor showed more myofibroblast incursion (SM22α-positive cells), and less IGFBP-4 staining, which predominantly colocalized with SM22α staining. In contrast, TRAMP:eng+/− tumor showed less SM22α-positive areas but more prominent IGFBP-4 staining that was localized in the extracellular space adjacent to SM22α-positive cells (Figure 6D). Thus, the expression pattern of IGFBP-4 in these tumors is consistent with endoglin-dependent modulation of IGFBP-4 availability and affects stromal investment in prostate tumors.

Discussion

The role of endoglin in tumorigenesis in vivo has been principally studied using tumor cell xenografts. Such studies indicate that endoglin expression represses migration and invasiveness of prostate cancer cells (4, 5), and that it attenuates their tumorigenicity (6). However, more accurate animal models are needed to elucidate the behavior of particular tumor types in their microenvironment. The present work is the first to study the effect of endoglin haploinsufficiency in an autologous model of cancer. This bigenic model is based on the TRAMP mouse, which develops in situ and invasive carcinoma of the prostate (11), and ultimately late stage metastatic cancer (37).

Endoglin expression inhibits prostate cancer cell migration in vitro (4, 5) but, surprisingly, the frequency of metastasis in our in vivo model was higher in TRAMP:eng+/+ mice than TRAMP:eng+/− mice. The increased vascularization of TRAMP:eng+/+ tumors is likely the reason for this difference, as the intravasation of tumor cells into the blood stream is the first step in the establishment of distant site metastatic lesions (9).

Histologic and immunohistochemical examination of TRAMP:eng+/+ versus TRAMP:eng+/− tumors showed that endoglin was required for the presence of CAFs in the tumor. This phenotype is much more profound than expected from endothelial cell haploinsufficiency (50% reduction in endoglin level) or the asymptomatic reduction of endoglin systemically. Interestingly, studies of the effect of endoglin haploinsufficiency on xenografted Lewis lung carcinoma 3LL cell-derived tumors showed no such CAF phenotype (38). Moreover, endoglin expression in endothelial cells of eng+/+ versus eng+/− mice cause relatively small effects (compared to the tumor CAF phenotype) in the context of skin carcinogenesis (39). These observations suggest that the endoglin-dependent CAF phenotype is specific to the prostate tumor stroma.

The origin of CAFs is unclear. Candidate CAF precursors include activated quiescent local fibroblasts (8), and circulating bone marrow mesenchymal stem cells (40). Moreover, recent work suggests the intriguing possibility that CAFs result from endothelial cells undergoing endothelial-mesenchymal transition (41). Our studies suggest that endoglin is required for continuous tumor CAF investment. CAFs are also compared to myofibroblasts, defined as activated fibroblasts involved in processes such as wound healing (23). Endoglin is a marker of myofibroblasts (42), and its expression is increased in these cell type during atherosclerosis-related and vascular TGFβ-dependent myogenic differentiation (43) and cell migration (44). The current data suggest that endoglin is primarily associated with myofibroblast-related SM22α–positive fibroblasts. Based on our previous studies (45), we propose that endoglin expression is required for the viability or the lineage specification of the myofibroblast-related CAF precursors.

To study the role of endoglin in CAF function, we isolated CAFs from TRAMP:eng+/+ and TRAMP:eng+/− tumors. However, we were not able to establish cell cultures of TRAMP:eng+/− derived CAFs. PrSC human primary prostate stromal cells were utilized as an alternative. Two studies showed that co-injection of PrSCs together with prostate cancer cells in mice enhances tumor incidence and growth (35, 46). We demonstrated that endoglin is expressed in PrSCs and found that PrSC cell growth is impaired in conditions of reduced endoglin expression. In addition, reduction of endoglin expression in human prostate stromal cells reduced their ability to recruit endothelial cells and their capacity to migrate in response to tumor secreted factors. These results suggest that endoglin is required for multiple aspects of CAF function including viability, endothelial cell recruitment and tumor-induced migration.

CAFs recruit several cell types to the tumor area via growth factor secretion (8, 23). Therefore, decreased tumor angiogenesis in TRAMP:eng+/− mice may be directly related to the absence of CAFs needed to recruit endothelial cell precursors. However, the signals that CAFs use to communicate with adjacent tissue are poorly understood.

Quantitative isotope peptide tagging methods suggested that endoglin regulated PrSC secretion of several potentially important secreted proteins involved in cell recruitment. For example, endoglin knockdown resulted in increased TIMP1 and TIMP2 detected in PrSC-conditioned medium (Table 1). Previous studies implicate tumor-stromal interactions in the regulation of TIMP expression and its role in prostate cancer progression (30), consistent with the view that reduced endoglin expression raised TIMP levels, impairing CAF invasion of the tumor.

Mass spectrometry data suggested that the IGF signaling system is an important mediator of endoglin-dependent cancer-stromal cell interactions. This hypothesis is supported by studies showing that IGF-1 stimulates cancer cell proliferation (33) and promotes cell growth in several cancer cell lines including PC3, the precursors of PC-3-M cells (47). PrSCs secrete IGF-1, promoting the proliferation of human prostate cancer cells (35). IGFBP-4 and IGFBP-6 are modifiers of IGF pathway signaling. IGFBP-4 antagonizes the growth stimulatory effect of IGF-1 (31), and inhibits the proliferation and tumorigenicity of human prostate cancer cells (48). Additionally, inhibition of IGFBP-6 expression promotes colon cancer cell proliferation (49). Here we provide evidence suggesting that PrSCs secrete IGFBP-4 and -6 in response to decreased endoglin expression, which may repress tumor growth. In our experimental model, IGFBP-4 inhibits IGF-1-dependent stimulation of prostate cancer cell growth. Our interpretation is that PrSCs secrete IGF-1 and several modulators of its activity. Under wild type conditions of endoglin expression (eng+/+), the balance is switched toward the stimulation of prostate cancer cell proliferation. Therefore, we suggest endoglin expression is necessary for PrSC/IGF-dependent modulation of tumor growth, potentially by regulation of TGFβ signaling in CAFs (34). ICAT studies did not reveal endoglin-dependent contributions from other secreted factors including Wnt family members. Future studies are needed to elucidate the mechanisms underlying endoglin-dependent modulation of IGFBP secretion.

The present study supports the view that endoglin plays a critical role in prostate cancer stromal cell function in the microenvironment. Experiments in the TRAMP:eng mouse model, combined with conditional transgenic approaches (16) will help elucidate the effect of systemic endoglin levels on stromal investment at several stages of tumorigenesis.

Supplementary Material

1

Acknowledgments

Funding

This work was supported by the Maine Cancer Foundation and the National Institutes of Health National Center for Research Resources P20-RR-15555 (CPHV, PCB); NIH Grants HL083151 (CPHV), CA91645 (PCB), CA122985 and Prostate SPORE CA90386 (RCB).

The authors would like to thank Kathleen Carrier (Maine Medical Center Research Institute, Scarborough, ME, USA) for her excellent technical assistance, Dr. Michael Jones (Department of Pathology, Maine Medical Center) for analysis of TRAMP tumor pathology, and Norma Albrecht for critical review.

Footnotes

Conflict of interest

No authors have any financial interests relating to work described in this manuscript.

Supplemental information

Supplementary information is available at Cancer Research website.

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