breast carcinoma–associated fibroblasts and their...

Breast Carcinoma–Associated Fibroblasts and Their Counterparts

Display Neoplastic-Specific Changes

Nahed M. Hawsawi,1Hazem Ghebeh,

2Siti-Faujiah Hendrayani,

1Asma Tulbah,

3Maha Al-Eid,

1

Taher Al-Tweigeri,4Dahish Ajarim,

4Ayodele Alaiya,

1Said Dermime,

2

and Abdelilah Aboussekhra1

1Department of Biological and Medical Research, 2Tumor Immunology/Stem Cell Therapy Program, 3Department of Pathology, and4Department of Oncology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia

Abstract

It has become clear that the initiation and progression of car-cinomas depend not only on alterations in epithelial cells, butalso on changes in their microenvironment. To identify thesechanges, we have undertaken cellular and molecular charac-terization of carcinoma-associated fibroblasts (CAF) and theirtumor counterpart fibroblasts (TCF) isolated from 12 breastcancer patients. Normal breast fibroblasts (NBF) from plasticsurgery were used as normal control. We present evidence thatboth CAFs and TCFs are myofibroblasts and show tumor-associated features. Indeed, the p53/p21 response pathway to;-rays was defective in 70% CAFs, whereas it was normal inall the TCF and NBF cells. In addition, the basal levels of the p53and p21 proteins were significantly low in 83% of CAFs andmodulated in the majority of TCFs compared with NBFs. Inter-estingly, both TCFs and CAFs expressed high levels of the cancermarker survivin and consequently exhibited high resistance tocisplatin and UV light. Moreover, most CAFs were positive forthe proliferation marker Ki-67 and exhibited high prolifera-tion rate compared with NBFs and TCFs. However, proliferatingcell nuclear antigen was highly expressed in both CAFs andTCFs. Using the two-dimensional gel electrophoresis technique,we have also shown that CAF, TCF, and NBF cells presentdifferent proteome profiles, with many proteins differentiallyexpressed between these cells. Taken together these resultsindicate that different genetic alterations can occur in breastCAFs and their corresponding adjacent counterparts, showingthe important role that stroma could play in breast carcino-genesis and treatment.[Cancer Res 2008;68(8):2717–25]

Introduction

Breast cancer is the most commonly occurring malignancy inwomen and is responsible for f500,000 deaths yearly worldwide(1). Carcinomas—the most frequent form of human cancer—resultmainly from the accumulation of somatic mutations in epithelialcells. Carcinoma cells, like normal epithelial cells, live in a complexmicroenvironment (stroma) that includes the extracellular matrixand cellular components, such as immune and inflammatory cells,blood vessel cells, and fibroblasts. All these cell types may critically

influence the multistep process of tumorigenesis (2–5). Fibroblasts,the predominant cells of the stroma, are responsible for the elabo-ration of most of the components of connective tissue (6, 7).In addition, stromal-epithelial interactions have a fundamental rolein the development of normal mammary gland. Therefore,modifications in the stromal fibroblasts can play a significant rolein overall cancer development (8). Nevertheless, a key questionremains: which comes first, the dysfunction of epithelial cells or thedysfunction of their microenvironment? In fact, several findingsfrom different laboratories have suggested that cancer cells them-selves can alter their adjacent stroma to form a permissive andsupportive environment for tumor progression (3). On the otherhand, there is compelling evidence that fibroblasts play major rolein the initiation and progression of carcinomas (3, 7–9). Theknowledge that stromal cells have the ability to stimulate onco-genenesis has been taken a step further by recent data showingthat stromal fibroblasts present in invasive human breastcarcinomas promote tumor growth and angiogenesis through ele-vated Sdf1 secretion (10). In another study, it has been shown thatextensive gene expression changes occur in all breast cell types,including fibroblasts (11). Furthermore, Kiaris et al. have shown anassociation between TP53 mutations in the stromal component ofepithelial tumors and carcinogenesis (12).Together, these findings indicate that stromal fibroblasts have an

active role in tumorigenesis and therefore should constitute acrucial target for cancer therapy, as well as for preventive strategies(13, 14). Thereby, a deep understanding of the epithelial-stromalbiochemical interactions and molecular signaling is mandatory.To this end, we undertook here molecular and cellular character-ization of breast carcinoma-associated fibroblasts (CAF) and theircorresponding adjacent fibroblasts. We have found that stromalfibroblasts, as well as their adjacent counterparts, show molecularand cellular modifications that are specific for tumor cells.

Materials and Methods

Tissue collection. Breast cancer specimens were collected from primary

tumors of 12 patients who underwent surgery at King Faisal SpecialistHospital. Signed informed consent was obtained from all the patients.

After tumor resection, an anatomic pathologist grossly examined and

obtained a representative piece of the tumor tissue and an adjacent

‘‘histologically normal’’ breast tissue from the same affected breast. Forcomparison, we have also obtained normal tissue from healthy women after

plastic surgery.

Generation of primary fibroblast cells from breast cancer tissues.The obtained tissues were further processed to generate the primarycultures, as previously described (15). Fibroblast cells were cultured in

Medium 199 and Ham’s F12 mixed 1:1 and supplemented with 10% to 20%

FCS. All supplements were obtained from Sigma, except for antibiotics andantimycotics solutions, which were obtained from Life Technologies.

Note: Present address for N.M. Hawsawi: Department of Pathology, University ofNewcastle upon Tyne, Royal Victoria Infirmary, Queen Victoria Road, Newcastle uponTyne NE1 4LP, United Kingdom.

Requests for reprints: Abdelilah Aboussekhra, King Faisal Specialist Hospital andResearch Center, BMR, MBC 03-66, P.O. Box 3354, Riyadh 11211, Saudi Arabia.Phone: 966-1464-7272; Fax: 966-1-442-7858; E-mail: [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-08-0192

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Immunohistochemistry of frozen sections and cell cytospin. Freshtissues were snap frozen in liquid nitrogen and preserved at �80jC,sectioned, and adhered to slides as described (15). Cultured cells were

cytospined as described (15).

Single staining. Anti-CD90 (clone 5E10, 1:50; BD Biosciences), anti–a-smooth muscle actin (a-SMA) (clone 1A4, 1:50; R&D Systems), anti-

vimentin antibody (V9 clone, 1:3,000; Dako), anti–Ki-67 (Ki-67 clone, 1:50;

Dako), and anti–pan-cytokeratin antibody (AE1/AE3 clone, 1:500; Dako)

were used as primary antibodies. After washing, the following secondaryantibodies were used: Envision+ polymer (ready to use; Dako) was used for

Ki-67, CD-90, and a-SMA staining, whereas goat anti-mouse IgG1 (Southern

Biotech) was used for pan-cytokeratin and vimentin staining.

Double staining. Both anti-Ki-67 (Polyclonal, Vector Laboratories) andanti-vimentin antibodies were added together. Envision + polymer with

swine anti-rabbit IgG (Dako) was used as secondary antibodies. Color was

developed with 3,3¶-diaminobenzidine (Novocastra), Fuschin Red (DAKO),or AEC (Sigma), and instant hematoxylin (Shandon) was used for

counterstaining.

UV light and ;-ray treatments. For UV irradiation, the medium was

removed and the cell culture monolayers in dishes were covered with PBSand exposed to a germicidal UV lamp (254 nm) at fixed distance. The UV

dosimetry was done using a UV meter (Spectronics Corporation).

g-Radiation was done using Cobalt (Co) source at a dose rate of 0.60 Gy/min.

Cellular lysate preparation and immunobloting. This has been doneas previously described (16). Antibodies directed against p53 (DO-I), p21

(187), survivin (C-19), glyceraldehyde-3-phosphate dehydrogenase (GAPDH;

FL-335), proliferating cell nuclear antigen (PCNA; PC-10), and h-actin (C-11)were purchased from Santa Cruz.

Quantification of protein expression level. The expression levels of

the immunoblotted proteins were measured using the densitometer (BIO-

RAD GS-800 Calibrated Densitometer) as previously described (16).Cell death analysis by flow cytometry. Cells were challenged either

with UVC (30 J m�2) or with cisplatin, whereupon the monolayers were

incubated in DMEM with supplements. Detached and adherent cells were

then harvested after 72 h and processed as previously described (17).Cell proliferation analysis. Complete medium (100 AL) containing 2 to

4 � 103 cells was loaded in each well of the 96-well plate. The plate was

incubated for at least 30 min in a humidified (37jC) 5% CO2 incubator andthen was inserted into the real-time cell electronic sensing (RT-CES) system

(ACEA Biosciences, Inc.). Cell proliferation was monitored for 70 h.

Two-dimensional gel electrophoresis, scanning, and image analysis.Proteins (100 Ag) were loaded on each strip via rehydration using linear (pH4-7 ready IPG) strips (Bio-Rad). Two-dimensional gel electrophoresis (2-DE)

was done, and gels were stained with silver nitrate and scanned using a

laser densitometer. Data were analyzed using PDQUEST software (Bio-Rad)

as previously described (18, 19).Data preprocessing and statistical analysis. A difference of z2-fold

change was used as a threshold for marked quantitative difference between

pairs of samples. In addition to qualitative differences (on/off protein

spots), significantly differentially expressed protein spots were first selectedusing two different statistical methods (Student’s t test and partial least

square analysis) available in PDQuest 2-DE image analysis software. The

data from the match set were exported from PDQUEST in the form of datatable, with rows representing gels and columns representing spots. Datasets

were normalized before analysis (19, 20). The resulting data were subjected

to hierarchical clustering analysis using the J Express software.5

Results

Isolation of CAFs and their adjacent counterparts frominvasive human breast cancers. Fibroblast cells were extractedfrom 12 human invasive mammary ductal carcinomas obtainedfrom mastectomies. The tissues were dissociated, and various celltypes were separated to obtain CAFs. We also isolated from each of

the same 12 tissues a second fibroblast populations taken froma histologically noncancerous region of the breast at least 2 cmaway from the outer tumor margin (tumor counterpart fibro-blasts, TCF). We then verified the purity of the fibroblastic popu-lations by cell morphology under microscope and cytospin/immunostaining. Figure 1A shows the typical morphology offibroblasts. Furthermore, these cells strongly expressed thefibroblastic marker vimentin, whereas they were negative forcytokeratin (a marker for epithelial cells; Fig. 1A). This indicatesthat fibroblastic cells were well separated from the other type ofcells and, therefore, are considered to be highly homogeneous withminimal contamination.CAFs and TCFs express the myofibroblast A-SMA marker.

During the tumorigenesis process, stromal fibroblasts acquire someof the characteristics of smooth muscle cells that specificallyexpress a-SMA. Using anti–a-SMA antibody, we have found that allthe CAF and TCF cells examined were positive for this protein.However, a-SMA was almost undetectable in the fibroblastic cellseither in culture or in tissue from plastic surgery (normal breastfibroblasts 6, NBF6; Fig. 1B). The proportion of positive cells variedfrom 5% to 100% for CAFs, with an average of 42% and from 5 to90% for TCFs with an average of 53%. In recent studies, a linkbetween the expression of a-SMA and the cell surface glycoproteinCD90 (Thy1) was suggested (21). CD90 is expressed at differentlevels by fibroblasts from different organs (22). Figure 1B showsthat all CAF, TCF, and NBF cells and tissue were positive forCD-90 protein. The detection of this protein in normal NBF cellsindicates that most breast fibroblasts are also expressing thisprotein and that there is no direct link between the expression ofCD-90 and the transformation into myofibroblasts during breastcarcinogenesis.The p53/p21 DNA damage signaling pathway is defective in

most CAFs but not in their adjacent counterparts. Next, westudied the effect of UV light and g-rays on the up-regulation ofp53 and p21 proteins to determine their functional status and alsothe status of their upstream effectors. Ten CAFs/TCFs pairs andtwo NBFs were irradiated with UV light (5 J m�2) and g-rays (5 Gy).Irradiated cells were reincubated for different periods of time, andthen whole-cell extracts were prepared and used in immunoblotanalysis using specific p53 and p21 antibodies and GAPDH asinternal control. The used doses were previously shown to induceboth p53 and p21 proteins in breast fibroblast cells (23). Theincrease in the level of these proteins was considered as inductiononly when the protein level after the treatment became at leasttwice higher than in the control nonirradiated cells. Figure 2Ashows that p53 and p21 protein levels augmented in response toboth UV-light and g-rays in NBF6 cells. The maximum inductionsof p53 (f4.5-fold) and p21 (f3-fold) were reached 14 h post–UVirradiation. In response to g-rays, the maximum levels of inductionfor p53 (f3-fold) and p21 (4.5-fold) were reached 3 hours after thetreatment (Fig. 2A). Similar results were obtained with the NBF2cells (data not shown).Figure 2B shows also that, like in the NBF6 cells, p53 and p21

protein levels were up-regulated in response to both UV light andg-rays in the TCF142 cells. Comparable results were obtained in allthe other TCF cells (data not shown). On the other hand, 7 of 10CAFs (70%) exhibited a defect in the induction of p53 and itsdownstream effector p21 in response to g-rays. Indeed, p53 andp21 were up-regulated in CAF87 (Fig. 2B), CAF169, and CAF180(data not shown) in response to both UV light and g-rays. However,p53 and p21 were induced only after UV light in the seven other5 http://java.sun.com

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Cancer Res 2008; 68: (8). April 15, 2008 2718 www.aacrjournals.org



CAFs (64, 76, 84, 114, 118, 142, 153). By contrast, following g-rays,these cells showed neither p53 nor p21 induction (Fig. 2B and datanot shown).These results indicate that the p53/p21 response pathway to

g-rays is defective in most CAFs but normal in all their corres-ponding counterparts.Basal expression of p21 and p53 proteins in CAFs and TCFs.

Next, the basal expression levels of p21 and p53 proteins wereassessed in 12 pairs CAFs/TCFs and two NBFs. Interestingly,in 10 of 12 cases (83%), the level of p53 was significantly lowerin CAFs than in the normal NBFs, and eight CAFs (66%) showedlow p53 level compared with their corresponding TCFs. How-ever, the level was similarly low in both CAF/TCF169 and 84.On the other hand, CAF180 exhibited higher p53 level than in itscorresponding TCF180 and the NBF cells (Fig. 2C and D). However,only four TCFs (69, 84, 118, and 169) showed significantly low p53levels. On the other hand, p53 level was higher in five TCFscompared with the NBF cells (Fig. 2C and D). These results indicatethat in most cases the level of p53 is lower in CAFs compared withtheir adjacent TCFs and the normal controls, and that the p53expression is also modulated in the majority of the TCFs.For p21, 10 of 12 CAFs (83%) exhibited low p21 level than in the

NBF cells and 5 of 12 CAFs (41%) showed lower expression than intheir corresponding TCFs. However, p21 level was similarly low in

four cases of CAFs and TCFs and was higher in the CAF142 andCAF180 than in their corresponding TCFs (Fig. 2C and D).Interestingly, the level of p21 was significantly lower in 6 of 12(50%) TCFs and higher in TCF153 than in the control NBF cells(Fig. 2C and D). Therefore, p21 expression is low in most CAFs andalso in a significant proportion of TCFs.Interestingly, a strong correlation exists between the expression

levels of p53 and p21 proteins in these cells (Fig. 2C), whichparallels the well known control of p21 expression by p53.Survivin is highly expressed in both CAFs and TCFs. To see

whether these fibroblasts display cancer-associated features, wesought to explore the expression level of survivin, the mostimportant cancer-associated protein (24, 25). To this end, whole-cell extracts were prepared from 12 pairs CAFs/TCFs and threeNBFs were used as normal control. Specific anti-survivin and anti–h-actin (used as internal control) were used for immunoblottinganalysis. As expected, the level of survivin was very low toundetectable in the normal NBF fibroblasts (Fig. 3A). However,survivin levels were higher in 100% CAFs and TCFs compared withthe level detected in the three NBF cells. The increase in survivinexpression was 2-fold in the CAF/TCF69 and reached 17-fold inthe CAF/TCF114 (Fig. 3B). Interestingly, in 9 of 11 cases, the levelsof survivin were comparable in CAFs and their correspondingadjacent fibroblasts (Fig. 3A and B). This shows that like for cancer

Figure 1. Breast CAFs and their adjacent counterparts are myofibroblasts. A, in vitro cultured fibroblasts were first visualized under a microscope and then the cytospinattached cells were immunostained with the indicated antibodies. Hematoxylin was used for counterstaining. Photomicrographs are at magnification of 260�. B,immunohistochemistry of frozen sections and cell cytospin were done using the indicated antibodies and cells. The circles indicate a region rich in fibroblastic cells.

Breast CAFs and TCFs Exhibit Tumor-like Features




cells, CAFs and their adjacent counterpart fibroblasts also expresshigh levels of the cancer marker survivin.Response of CAFs and TCFs to cisplatin, UV light, and

;-rays. Because survivin is expressed in CAF/TCF cells andbecause it is a potent inhibitor of apoptosis, we sought toinvestigate the response of these fibroblasts to cisplatin, UV light,and g-rays. To this end, 10 CAFs and their corresponding TCFs aswell as two NBF cells were treated with cisplatin (30 Ag/mL), UVlight (30 Jm�2), or g-rays (30 Gy) and then reincubated for72 hours. Subsequently, cell death was assessed by flow cytometry(Fig. 4A). Figure 4A and B show that the normal control cells NBFare sensitive to cisplatin, which triggered cell death in f40% ofthese cells. However, 9 of 10 CAFs (90%) and 8 of 10 TCFs (80%)showed very high resistance to cisplatin. The proportions ofcell death induced in these fibroblasts were between 0% and 5%(Fig. 4B). The TCF76 cells were more sensitive with 38% killed withthe cisplatin treatment (Fig. 4B). On the other hand, the CAF/TCF114 pair exhibited very high sensitivity, with more that 70% celldeath (Fig. 4B).In response to UV light, f35% of the normal NBF cells

underwent cell death (Fig. 4A and C). However, 9 of 10 CAF cells(90%) exhibited high resistance to the killing effect of UV light

(Fig. 4C). Importantly, in 7 of 10 pairs, the CAF cells were sig-nificantly more resistant than their counterparts TCF cells. Indeed,CAFs 180, 84, 118, and 148 showed very high resistance to UV lightwith 1% to 2% cell death (Fig. 4C). On the other hand, only 50% ofthe TCFs were significantly resistant to UV light compared with thecontrol NBF cells, whereas the other five TCFs were only slightlysensitive (Fig. 4C). This shows that in most cases the TCF cellsshowed an intermediate response between the normal fibroblastsand the CAFs.Concerning the response to g-rays, all the cells including the

normal NBFs exhibited very high resistance (0–5% cell death), withthe exception of the CAF/TCF87 pair, wherein the proportion ofcell death reached 18% (Fig. 4D). Nevertheless, CAF and TCF cells,which showed similar responses, were slightly more sensitive thanthe NBF cells to the killing effect of g-rays.Proliferation rate and expression of PCNA and Ki-67 in CAFs

and TCFs. It was very obvious that CAFs grow faster than theircorresponding TCFs and the control normal NBF cells duringin vitro culturing. Thereby, we used the real-time cell electronicsensing system to study cell proliferation and show the growingdifference. We have found that the CAF114 and CAF118 grow fourto five times faster than their corresponding counterparts TCFs

Figure 2. p53 and p21 expression and response to UV light and g-rays in CAFs and TCFs. A and B, confluent cells were either mock treated or challenged withUV light (5 J m�2) or g-rays (5 Gy) and then reincubated for different periods of time, as indicated. After cell lysate preparation, 30 Ag of proteins were used forimmunoblot analysis using the indicated antibodies. C, whole-cell lysates were prepared from the indicated cells and used for immunoblot analysis using the indicatedantibodies. D, histograms showing p53 and p21 expression levels. The values were determined by densitometry and normalized against GAPDH. N1 and N6correspond to NBF1 and NBF6. Error bars represent SDs of at least three different experiments.

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(Fig. 5A). Similar results were obtained with other CAF/TCF pairs(data not shown), showing that the CAF cells acquired fasterproliferation rates compared with their adjacent counterpartfibroblasts. To further elucidate this, the level of the proliferationmarker Ki-67 was assessed in different CAF/TCF pairs. Figure 5Bshows that the Ki-67 protein was expressed at very low levels in theTCF fibroblasts. By contrast, most of the CAF cells were positive forthe proliferation antigen, although to different extents (Fig. 5B).Similar results were found in breast cancer tissues, where Ki-67 wasfound expressed in six of seven CAFs, but was undetectable in theircorresponding TCFs (data not shown). Figure 5C shows theexpression of Ki-67 in epithelial cells and their correspondingstromal fibroblasts from the same tissue. The expression of Ki-67 inboth cell types shows that the increase in the proliferation rate isnot restricted to breast carcinomas, but it is also present in theirstromal fibroblasts.Next, we assessed the level of the other proliferation marker

PCNA and showed that PCNA levels were higher in both CAFs andTCFs compared with the control NBF1 and NBF6 cells (Fig. 5D).When comparing the PCNA level in each CAF/TCF pair, we havefound that in most cases PCNA expression is only slightly higher inCAF than in TCF cells (Fig. 5D). Interestingly, the PCNA levels werefound to be considerably higher in CAF87 and CAF142 comparedwith their corresponding TCF cells (Fig. 5D). On the other hand,the CAF180 exhibited significantly lower level of PCNA than theTCF180 (Fig. 5D).Protein expression patterns of CAFs, TCFs, and NBF. Whole-

cell lysates were prepared from one normal (NBF6) and two CAF/TCF pairs (114 and 76) and were analyzed by 2-DE. Representative2-DE maps are shown in Fig. 6A . An average total number of 969spots were resolved, and minimum of 84% of the spots werematched between all the gels. Interestingly, marked quantitativeand qualitative changes were observed in the protein expressionpattern between NBF, TCF, and CAF samples. Using the correlationanalyses between pairs of samples, we observed high degree ofsimilarities between the two CAFs (r = 0.71) and the two TCFs

(r = 0.81). On the other hand, an average correlation coefficient of0.61 was observed between pairs of TCF and CAF cells. Similarcorrelation was observed between NBFs and TCFs (0.61). However,we observed higher degree of heterogeneity in the proteinexpression pattern between the normal control NBF and CAFs,with a correlation of only 0.49.Hierarchical cluster analysis of differentially expressed

proteins in the NBF, TCF, and CAF samples. The differences inprotein expression patterns between the matched spots in thedifferent gels were studied. A total of 483 spots were matched toall the five samples. In an effort to reduce the dataset, multivariatedata analysis of qualitative and quantitative differences werecarried out to generate sets of variables for more accurate use inthe clustering analysis. Using Student’s t test, a total of 95 poly-peptides were significantly (P < 0.05 with 98% confidence interval)differentially expressed between CAFs and TCFs. A similar analysisusing PLS resulted in 25 protein spots that differed significantly.Subsequently, we used these 95 and 25 separate datasets for

possible classification of the samples into their respective groupsusing the hierarchical cluster analysis, and all the samples werecorrectly classified (data not shown). A total of 15 protein spots fallin the intersection of the above two datasets and were used in thecluster analysis of all the samples (Fig. 6B). The same dataset wassubjected to correspondence analysis (Fig. 6B), and all sampleswere distinctly separated. These analysis showed differentialprotein expression pattern between the normal cells (NBF), TCFs,and CAFs, with the NBF and TCFs sharing more similarities in theirprotein expression patterns (Fig. 6B).The differential expressions of some of these protein spots are

shown in Fig. 6C . This figure shows the expression levels of sixdifferent proteins. Panels 1, 2, 3, and 4 show that these proteinscompletely disappeared in the CAF cells, whereas they were presentin the corresponding TCF cells and also in the normal NBFcells (Fig. 6C). On the other hand, panels 5 and 6 showed theoverexpression of proteins that were undetectable in the NBF cellsand appeared in the TCF cells (Fig. 6C). Together these figures

Figure 3. Survivin is highly expressed in both CAFs andtheir corresponding TCFs. Whole-cell extracts wereprepared from confluent cells and used for immunoblottinganalysis using the indicated antibodies. A, immunoblots.B, histogram showing survivin expression levels. Thevalues were determined by densitometry and normalizedagainst h-actin. A and C correspond respectively toCAF and TCF, whereas N1, N2, and N3 correspondrespectively to NBF1, NBF2, and NBF3. Error barsrepresent SDs of at least three different experiments.





show that simultaneous analysis of multiple polypeptides by 2-DEin combination with hierarchical cluster analysis enabled cleardiscrimination between CAFs, TCFs, and the NBFs.

Discussion

In the present report, we provide strong evidence that CAFs, aswell as their adjacent counterparts, show several tumor-specificfeatures. Indeed, both are active myofibroblasts that express highlevels of PCNA and survivin, they are highly resistant to the killingeffect of cisplatin, most of them express low levels of the tumorsuppressor p53 and p21 proteins, and their protein expressionprofiles are different from that of NBF cells. Interestingly, mostCAFs but not TCF cells were defective in the g-ray–dependentinduction of p53 and p21 proteins. Furthermore, like tumor cells,CAFs expressed high levels of Ki-67 and exhibited high proliferationrates. These results provide the first clear indication that TCFs,although histologically normal and are not in contact with cancercells, bear genetic changes that distinct them from NBFs and alsofrom their neighbor CAF cells.We have shown here that 70% of CAF cells are defective in the

g-ray–dependent up-regulation of p53 and its downstream effectorp21, which are normally induced after UV light (Fig. 2B). This maysuggest a defect in the p53-upstream effectors that govern the g-ray

signaling pathway, such as Atm and Chk2, which play importantroles in breast cancer (26–28). Interestingly, it has been recentlyshown that the presence of an association between the existence ofp53 mutations and loss of heterozygosity/allelic imbalance at theATM locus in the stromal compartment of breast cancer and tumorgrade (29). Furthermore, Kiaris et al. have shown an associationbetween p53 mutations in the stromal component of epithelialtumors and carcinogenesis (12). Together, these findings show theimportance of the ATM/p53 tumor suppressor pathway in thestromal compartment as well. The fact that this pathway is normalin the adjacent counterpart fibroblasts suggests that the defect inthe g-ray–dependent induction of p53 that has been observedin CAFs resulted from a selective pressure from the tumor cells.In addition, the levels of p53 and p21 decreased in 80% CAFscompared with NBF cells. However, only 33% and 50% of TCF cellsshowed lower levels of p53 and p21 proteins, respectively. Thesefindings present the first indication that p53 and p21 expressionlevels decrease in CAFs and their adjacent counterparts, whichcould have a link with the development/progression of carcinomas.Indeed, in a mouse model of prostate cancer, it has been recentlyshown that initiating tumorigenesis in the epithelium by pRBinactivation led to the loss of p53 expression in stromal tumorfibroblasts and increased mesenchymal cell proliferation andtumor progression. Subsequently, some epithelium regions have

Figure 4. Response of CAFs and TCFs to the killing effect of cisplatin, UV light, and g-rays. Subconfluent cells were either mock-treated or challenged withcisplatin (30 Ag/mL), UV light (30 J m�2), or g-rays (30 Gy) and then reincubated for 72 h. Cell death was measured using flow cytometry. A, FACScan analysis.B-D, histograms showing the proportions of induced cell death. N2 and N6 correspond to NBF2 and NBF6.

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also lost p53 expression (30). Furthermore, it has been recentlyshown that p53 suppresses the production of the chemokine Sdf1in cultured fibroblasts (31). Because the secretion of this proteinby stromal fibroblasts contributes to tumor promotion (10), adecrease in p53 level in CAFs will lead to an increase in theproduction and secretion of Sdf1 and, therefore, enhances tumorformation. This explains the decrease in p53 levels in most CAFsexamined in this study, which further prove the nonautonomouseffect of p53 tumor suppressor in carcinogenesis. A decrease in thelevel of the universal cyclin-dependent kinase inhibitor p21 mayalso have a tremendous effect on tumorigenesis because it is amodulator of both cell cycle and apoptosis and also controls theexpression of various cancer-related genes (32–34).Furthermore, we have shown that the level of the survivin

protein is similarly higher in CAFs and TCFs compared with thenormal NBF cells (Fig. 3). The increase in survivin expression isknown to occur in most cancer cells and tissues as a consequenceof activation of oncogenes or loss of tumor suppressor genes

(24, 25). This suggests that CAFs, as well as their correspondingTCFs, acquired tumor-like changes that are most likely necessaryfor tumor growth. Because the survivin level is higher in both CAFsand their counterparts than in the normal controls, it is possiblethat survivin up-regulation occurs early and has a promoting roleduring cancer development. Therefore, the increase in the survivinlevel could constitute an important cancer predisposing factor,especially with its dual role in inhibiting apoptosis and activatingcell proliferation. Indeed, Temme et al. have reported that over-expression of survivin increased human fibroblast proliferation (35).Interestingly, like for cancer cells, the increase in the level of this

antiapoptosis protein was accompanied with an increase in theresistance of these cells to the killing effect of UV light andcisplatin. Whereas CAFs and TCFs were similarly highly resistant tocisplatin, most CAFs showed higher resistance to UV light thantheir counterparts TCFs. To further elucidate the tumor-likephenotype of these stromal fibroblasts, we have shown that CAFcells and tissues express high levels of the proliferation marker

Figure 5. Cell proliferation and the expression of Ki-67 and PCNA in NBF, TCFs, and CAFs. A, 2,000 cells were seeded on 96-well plates and were incubated for theindicated periods of time. Cell proliferation rate was determined using the real-time cell electronic sensing system. B, in vitro cultured fibroblast cells were cytospinattached to slides and stained by Ki-67 antibody. Hematoxylin was used for counterstaining. Representative immunohistochemical staining of Ki-67 (brown , nuclear) ofthe indicated cell cultures is shown. Photomicrographs are at �320. C, immunohistochemistry using anti–Ki-67 antibody on breast cancer tissue. The arrows show thestained epithelial and fibroblast cells. D, 30 Ag of cell lysates were immunoblotted using the indicated antibodies. A and C correspond to CAF and TCF, respectively.PCNA corresponding levels are indicated below the corresponding bands. Representative of two experiments.





Ki-67 and showed enhanced proliferation than their adjacentcounterparts TCFs. However, PCNA was found to be highlyexpressed in both CAFs and TCFs. This explains the higher abilityof these cells in proliferating and may be their predisposition toreceive signals from cancer cells.Finally, using 2-DE and hierarchical cluster analysis, we classified

CAFs, TCFs, and NBF cells into three distinct groups. This showsthat CAFs and TCFs are different from each other and also differentfrom NBFs. These results also indicated that TCFs are closer to theNBF than the CAFs. Indeed, the analysis of individual spots showedvarious proteins in the TCFs that exhibited a level of intermediateexpression between the NBF and the CAFs (Fig. 6C). These resultsprovide the first clear indication that the proteome of the TCFs isnot normal but also different from that of the CAFs, which cor-roborates our findings regarding the basal and induced level ofp53/p21, the response to UV light, and cell proliferation.These findings support the importance of analysis of stromal and

tumor-adjacent tissue proteome in the discovery of potentialbiomarkers related to breast carcinomas that may have prognostic

and/or predictive values. Indeed, the presence of significant asso-ciations between loss of heterozygosity/allelic imbalance in thebreast cancer stroma and tumor grade and regional lymph nodemetastasis has been recently shown. In fact there were morecorrelation between clinicopathologic features and loss of hetero-zygosity/allelic imbalance in the stroma than in the epithelium(29). Therefore, the identification of some of the differentiallyexpressed proteins in the stroma may shed light on the initiationand progression of breast tumor and further highlights theirpotential usefulness as marker of malignancy.

Acknowledgments

Received 1/16/2008; revised 1/28/2008; accepted 1/28/2008.Grant support: King Abdulaziz City for Science and Technology. This work was

performed under the RAC proposal 2031091.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. K. Abu-Khabar and L. Al-Haj for their help with the RT-CES systemand Dr. K. Al-Hussein and P.S. Manogaran for their help with the flow cytometry.

Figure 6. Protein expression patterns of CAF, TCF, and NBF cells. Whole-cell lysates were prepared and subjected to 2-DE using readymade IPG strips (pH 4–7) inthe first and 12.5% homogeneous SDS PAGE in the second dimension. A, two-dimensional maps showing representative examples of 2-DE gels. B, left, clusteranalysis of the indicated samples using expression dataset from 15 polypeptides derived from Boolean analyses of t test and PLS; right, correspondence analysis plotof the same dataset. C, gel segments showing six protein spots that are differentially expressed between the different indicated samples.

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2008;68:2717-2725. Cancer Res Nahed M. Hawsawi, Hazem Ghebeh, Siti-Faujiah Hendrayani, et al. Counterparts Display Neoplastic-Specific Changes

Associated Fibroblasts and Their−Breast Carcinoma

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