Endoglin regulates cancer-stromal cell interactions in ... · 1 Endoglin regulates cancer-stromal...

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Endoglin regulates cancer-stromal cell interactions in prostate tumors

Diana Romero, PhD1,4, Christine O’Neill1, Aleksandra Terzic, DVM, PhD1, Liangru Contois,

PhD1, Kira Young, BS 1,2, Barbara A. Conley, MS1, Raymond C. Bergan, MD 3, Peter C. Brooks,

PhD 1,2, and Calvin P.H. Vary, PhD 1,2

1Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME

04074, USA, 2The Graduate School of Biomedical Sciences, University of Maine, Orono, ME

04469, USA, and the 3Division of Hematology/Oncology, Department of Medicine,

Northwestern University Medical School, Chicago, IL 60611, USA. 4Current address: The

Institute of Reproductive and Developmental Biology, Imperial College London, London W12

0NN, UK.

Correspondence to:

Calvin P.H. Vary

Maine Medical Center Research Institute

81 Research Drive, Scarborough, ME 04074

Tel: (207) 396-8148; Fax: (207) 396-8179; email: [email protected]

Running title: Endoglin regulates tumor-stromal cell cross-talk

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

on May 31, 2020. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 28, 2011; DOI: 10.1158/0008-5472.CAN-10-2665

http://cancerres.aacrjournals.org/

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Abstract

Endoglin is an accessory receptor for transforming growth factor-ß (TGF-ß) 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.




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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 TGF� 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




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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.




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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).




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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-�-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




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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 x 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 �m, 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




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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 x 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 �m

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

ProteinPilotTM software (Life Technologies, Carlsbad, CA, USA). Detailed methods provided in

Supplemental Information.




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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).

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.




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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




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99% in TRAMP:eng+/- tumors.

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.

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




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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.

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-




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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)




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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).

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 TGF� 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




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knock down PrSC-derived medium, which is consistent with the reduced size of TRAMP:eng+/-

tumors.

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 (SM22�-positive cells), and less IGFBP-4 staining, which predominantly colocalized

with SM22� staining. In contrast, TRAMP:eng+/- tumor showed less SM22�-positive areas but

more prominent IGFBP-4 staining that was localized in the extracellular space adjacent to

SM22�-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.




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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




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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 TGF�-dependent myogenic differentiation (43) and cell

migration (44). The current data suggest that endoglin is primarily associated with

myofibroblast-related SM22�−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




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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




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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.




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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).

Conflict of interest

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

Acknowledgements

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.

Supplemental information

Supplementary information is available at Cancer Research website.




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27

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28

Legends to Figures

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

tumors.

A) Western blot for endoglin and �-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)).

Figure 2. Prostate tumorigenesis and tumor angiogenesis are altered in

TRAMP:eng+/+ versus TRAMP:eng+/- mice.

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




29

least eight fields/sample (n = 4). *, p < 0.05 (Student’s t-test).

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

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.

Figure 4. Endoglin is associated with tumor myofibroblasts.

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).

Figure 5. Endoglin knockdown reduces PrSC cell proliferation and affects PrSC-

dependent modulation of PC3-M cell proliferation.

PrSC were transfected with siRNAs directed against endoglin (siENG) or a control

scrambled sequence (siSC) for forty-eight hours. Endoglin expression was analyzed by




30

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 (25�g 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.

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

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




31

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.






















Published OnlineFirst March 28, 2011.Cancer Res Diana Romero, Christine F O'Neill, Aleksandra Terzic, et al. tumorsEndoglin regulates cancer-stromal cell interactions in prostate

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