proteomic analysis of selected prognostic factors of breast cancer

Kylie RobertsKiron BhatiaPeter StantonRoger Lord

Department of Surgery,University of Tasmania,Hobart, Australia

Proteomic analysis of selected prognostic factorsof breast cancer

The incidence of breast cancer is on the rise but as yet there is no guaranteed bene-ficial treatment for many of the sufferers. The treatments specific for breast and otherhormone-sensitive cancers work well at times, however, the population of women thatthey will benefit is relatively small. Many are limited to surgical, chemotherapy, andradiotherapy options. Here, using two-dimensional electrophoresis (2-DE) in conjunc-tion with a silver stain and Western blotting approach, we attempt to locate selectedknown prognostic markers for breast cancer. With these results, we can exclude theseproteins from the future search for potential pharmaceutical targets, using the sametechniques. The proteins that were located include the estrogen receptor-a, b-casein,cytokeratin 7, calponin and bax. For each protein an estimated Mr and pI was gained.Each protein was found in multiple variants. By locating these proteins the number ofunknown proteins found on the 2-DE gel has been reduced, helping the future searchfor novel markers that are shown as being differentially expressed between healthyand cancerous tissue samples.

Keywords: Breast cancer / Immunoblotting / Prognostic factors / Two-dimensional gel electro-phoresis PRO 0633

1 Introduction

It has been reported that breast cancer was diagnosed inover 1 million people worldwide in 2000, ,99% of whomwere women [1]. Endeavours to find a cure have so farproven to be a long and incomplete task. However, treat-ments and diagnostic techniques are improving, screen-ing programs are in place, and awareness of the diseaseis increasing. Despite these advances, this disease wasdeemed responsible for the deaths of more than 350 000people in 2000 [1]. The treatment regime for breast canceris dependent upon several factors, of which one is theestrogen receptor-a (ER-a) status. For all breast cancersufferers the options of chemotherapy, radiation therapyand surgery exist, however, for 50–55% of women whosetumors are ER-a positive [2] there is a fourth option thatinvolves hormone-based adjuvant therapies.

The estrogen-based adjuvant therapies such as Tamoxi-fen and the third-generation aromatase inhibitor-basedtherapies which are still in clinical trials, will only provide

a substantial benefit for half of the women who are diag-nosed each year. This highlights the necessity for a treat-ment that is hormone independent, which in turn requiresthe recognition of new protein markers. To accomplishthis task, the techniques utilized in proteomics have beenemployed.

There are a large number of proteins that have been linkedto breast cancer either as a suspected cause of cancer oras a subsequent result of other cellular changes. Whencomparing the proteome of breast cancer cells to the pro-teome of healthy breast tissue cells, there are going to bea number of proteins that are differentially expressed. Thisis indicative of a protein marker of the disease and byexcluding the protein/s in certain locations (based on pIand Mr) on 2-DE gels, this will hasten the search for noveland more effective pharmaceutical targets.

This study aims to exclude the following known prognosticmarkers of breast cancer from the search for new markers,eliminating them from further complex and expensiveprotein purification and identification techniques. Theearliest known molecular prognostic factor for breast can-cer, the ER-a [3], was chosen because of its well-definedrole in estrogen-dependent tumors. To date the location ofthe ER-a has not been demonstrated using 2-DE of breastcancer tissue. It is expected that ER-a will have anincreased expression in many breast tumors and thereforeit is useful to exclude it from this study.

Correspondence: Dr. Roger Lord, Department of Surgery, Uni-versity of Tasmania, Private Bag 252–28, Hobart, 7001, AustraliaE-mail: [email protected]: 161-3-6226-4760

Abbreviations: ER-Æ, estrogen receptor-a; IHC, immunohisto-chemistry; K7, cytokeratin 7; OD, optical density; STAT 5, signaltransducers and activators of transcription

784 Proteomics 2004, 4, 784–792

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de

DOI 10.1002/pmic.200300633

Proteomics 2004, 4, 784–792 Selected prognostic factor of breast cancer 785

Cytokeratin 7 (K7) is known to be expressed in all breastcancers [4] although its presence in healthy breast tissueis limited. It is a structural protein of the Cytokeratin IIsub-family defined by the neutral-basic isoelectric pointsof its members. K7 is of significance in this study becauseit has been shown using immunohistochemistry to beexpressed in all breast cancer samples [4]. The locationof this protein will act as a positive control ensuringthe validity of the techniques being employed. No directlink between K7 and the prognosis of breast cancer hasbeen found although the differential expression betweenhealthy and cancerous tissue will be detected when usingPD-Quest Software (Bio-Rad), therefore knowing theposition of K7 on a proteomic map of breast cancer tissuewould be beneficial.

p130cas, also called BCAR 1 (breast cancer anti-estrogenresistance 1) is a recent addition to the list of proteins withlinks to breast cancer. It has been shown to have a role inaltering a tumor cell’s anti-estrogen resistance when thep130cas protein is overexpressed [5, 6]. p130cas has alsobeen linked to cell adhesion and other signalling cas-cades [7]. Its novelty and apparent role in critical cell func-tions have made this protein one of interest and has ledto its inclusion in this study.

b-Casein is a protein that is produced and secreted byhealthy myoepithelial breast cells during lactation dueto stimulation via lactogenic hormones such as prolactinand progesterone. Its presence in cancerous tissue ofthe breast has been shown in various studies, however,opinion is divided between those who believe it is signifi-cant [8, 9] and those who believe that there is no correla-tion [10–12]. Recent studies have suggested that insulin-like growth factor-1 (IGF-1) is involved in breast cancer.IGF-1 can stimulate the JAK 2 pathway stimulating STAT5 transcription [13], and STAT 5 has been identified as atranscription factor for b-casein [14], this indirectly sug-gests that IGF-1 can alter the b-casein levels. The inclu-sion of b-casein in this study was pursued because com-bined with the recent findings into IGF-1’s involvement inbreast cancer, the role of b-casein in non-lactating cells iscurrently unknown and the significance of its presence inbreast cancer is in dispute.

The same questionability over its role in breast cancer ledalso to the inclusion of the pro-apoptotic Bcl-2 familymember Bax. Bax is not currently seen as an accurateindependent indicator of prognosis. Some studies havereported that in combination with p53 and/or Bcl-2, Baxbecomes a more reliable prognostic marker [15–17].Others have suggested that Bax has no value when deter-mining prognosis [18, 19]. Calponin is a marker of myo-epithelial and smooth muscle cells [20]. Monsunjac, et al.[21] showed that, via immunohistochemistry (IHC), calpo-

nin is an accurate marker of malignancy in intraductalpapillary carcinoma. Malignancy is determined by theinability of a cell to differentiate and express a normalphenotype and genotype. A downregulation or lack ofcalponin may imply that an event has occurred resultingin malignancy. This has only been studied using onebreast tumor type and so it was seen as important toinclude. It should be noted, however, that if the previousdata are accurate then calponin will be absent from thecancerous samples and present in the healthy samples,the reverse of the other factors that are investigated inthis study. The aim of this study was to eliminate theseproteins from planned future studies to seek new targetsfor treatment using a 2-DE system.

2 Materials and methods

2.1 Sample collection and preparation

Ethics approval was obtained from the Royal HobartHospital’s Ethics Committee and completed via patientconsent from women undergoing either a lumpectomy ormastectomy from three hospitals in Hobart, Tasmania(Royal Hobart Hospital, St. Johns Hospital or HobartPrivate). Tumors were sent to the Pathology departmentof the corresponding hospital for routine examination,samples for this study were provided by the pathologistsand stored at 2707C until further processing occurred.Some of the specimens contained excessive amounts offatty tissue, which was dissected from around the tumorperimeter before storage at 2707C. 100 mg of breast can-cer tissue was homogenized using the sequential extrac-tion techniques outlined in the Bio-Rad Laboratorieshandbook [22]. The supernatant from each step of thesequential extraction was stored at 2207C until required.

2.2 Two-dimensional PAGE

2.2.1 First dimension

185 mL of sample was used to passively rehydrate each of2611 cm IPG strips, pI 3–10 (Bio-Rad Laboratories, Her-cules, CA, USA) with the same sample type, overnight.The strips were then placed into a Protean IEF cell (Bio-Rad) and focused for , 5 h according to the following pro-tocol: 250 V with linear climb for 20 min, 8000 V with linearclimb for 2.5 h and 8000 V with rapid climb until 20 000 Vhwas reached. Focused strips were then equilibrated twicewith 4 mL of equilibration buffer (36 g urea, 10 mL 20%SDS, 3.3 mL of 1.5 M Tris, 40 mL 50% glycerol) per IPGstrip. This was done for 10 min with 200 mg of DTT addedto the 4 mL of equilibration buffer which was decanted offand then for a further 10 min with 4 mL of equilibrationbuffer and 250 mg of iodoacetamide.


786 K. Roberts et al. Proteomics 2004, 4, 784–792

2.2.2 Second dimension

Focused IPG strips were set into place using 1% agarosesolution to the top of an 11 cm precast 4–15% Tris-HClgel (Bio-Rad). Tank buffer (30.28 g Tris, 144.14 g glycine,10 g SDS made up in 1 L Milli-Q water as stock and then100 mL of buffer was added to 900 mL of Milli-Q waterbefore use), was added and the sample was separatedfor 60 min at 200 V. One of these gels was then electro-blotted at 100 V for 30 min and the other placed in fixativesolution until staining was conducted.

2.3 Staining and imaging

The two gels were obtained (1 from each IPG strip) andwere subjected to one of two imaging protocols. Onewas silver-stained using Bio-Rad Silver Stain Plus kitreagents (Bio-Rad) according to the manufacturer’s in-structions. The other was Western-blotted. Briefly, pro-teins on the 2-D gels underwent electrotransfer at 100 Vfor 30 min to a Hybond membrane (Amersham PharmaciaBiotech, Uppsala, Sweden) and were labelled using oneof the primary antibodies outlined in Table 1. Gels were

then labelled with a secondary antibody (Table 1). Anti-body-labelled gels were then treated with ECL kit re-agents (Amersham) and then an autoradiograph wasobtained by exposure to Hyperfilm (Amersham).

2.4 Image analysis

The silver-stained gel and the autoradiograph werescanned using a Densitometer GS-800 (Bio-Rad) andthen analyzed using PD-Quest software (Bio-Rad) todetermine which of the proteins in the silver stain werethose that the antibodies bound to in the Western blot,this was done by overlaying the images from both theautoradiograph and from the silver stain to find the com-mon spots in each image. The approximate Mr and pIfor the proteins of interest were then established basedon the Mr standards (Bio-Rad Broad Range PrecisionStandards) and the pI standards (Bio-Rad IEF standards)that were incorporated into the experiment. Optical den-sity (OD) was obtained by using the Density tool providedin PD-Quest, using the silver-stained gel that was run intandem to the immunoblot for each prognostic marker.The intensity of the proteins, used for comparing cancer-

Table 1. Antibody list

Marker Host Clone Volumea)

(mL)Dilution Supplier

Primary antibodies

Bax Rabbit-anti-human A3533 20 1:500 Dako

Calponin Mouse-anti-human CALP 10 1:5000 Dako

Casein Mouse-anti-human F20.14 25 1:2000 Neomarkers

Estrogen receptor-a Mouse-anti-human 1D 5 50 1:800 Dako

Estrogen receptor-a Mouse-anti-human AER 611 50 1:50 Neomarkers

Keratin 7 Mouse-anti-human K72.7 10 1:5000 Neomarkers

p53 Mouse-anti-human DO-7 1

BP53–1250 1:1000 Neomarkers

p130cas Mouse-anti-human CAS-14 50 1:1000 Neomarkers

Progesteronereceptor

Mouse-anti-human hPRa2 1

hRPa350 1:1000 Neomarkers

Secondary antibodies

Anti-mouse Peroxidase conjugated goat-anti-mouse

10 1:1000 Zymed

Anti-rabbit(Bax only)

Peroxidase conjugated swine-anti-rabbit

10 1:1000 Dako

The list includes all antibodies, primary and secondary used to complete the immunoblotting proce-dure for each of the prognostic markers.a) Volume of antibody added to 10 mL of TBS.



Table 2. pI and Mr for all of the matched proteins obtained by overlaying the silver stain image withthe immunoblot image

Keratin Bax b-Casein p130cas Calponin ER

Mr pI Mr pI Mr pI Mr pI Mr pI Mr pI

70 6.7 16 – 22 – 140 7.9 50 6.9 63 7.356 6.6 16 7.9 22 – 140 – 50 7.0 80 7.956 6.7 16 – 22 7.1 280 – 35 –56 6.8 22 7.656 6.65 22 –56 6.7556 6.85

ous to tissue was based on a normalized value, designedto take into account staining variations, pipetting errorsand size and spot variations. Normalization is based onthe equation: raw spot quantity6scaling factor6pipet-ting error compensation factor (if any)/normalization fac-tor, where in this study scaling factor is 1 000 000 to gaina value in parts per million (ppm). There was no pipettingerror compensation factor and the normalization factorwas the total quantity (OD) of all valid spots.

3 Results and discussion

3.1 Cytokeratin 7

Cytokeratin 7 (K7) is one of the 20 members of the cyto-keratin family involved in the formation of the cytoskeletalintermediate filaments within every cell [23]. The cyto-keratin proteins belong to one family of proteins which isthen divided into 2 sub-families known as CK I and CK II;CK I contains the acidic cytokeratins whilst CKII containsthe rest, i.e. the neutral and the basic. Not every cell con-tains every form of cytokeratin, however, at least twowill be present, one from each sub-family. This is becausein order for the intermediate filaments of the cytoskeletonto be formed both an acidic and neutral/basic member ofthe cytokeratin family are required to form these inter-mediate filaments. The results for cytokeratin 7 (Fig. 1,Table 2) suggests that there are multiple forms of K7.It should be noted here that Figs. 1a and b demonstrateboth the silver-stained image (a) and the Western blotimage (b). Figures 2–6 are of the silver stain only, with thematching proteins, determined by image overlay from theWestern blots indicated. The western blots have beenomitted due to spacial considerations.

These multiple variants of K7 require further elucidation,however, it is thought that they are mostly likely the resultof post-translational modifications. Amino acid sequence

Figure 1. (a) 2-D separation using silver stain to visualizethe entire proteome. Numbers indicate the proteins witha corresponding protein on the immunoblot, as deter-mined by PD-Quest, for keratin 7 (see b). (b) Immunoblotof a 2-dimensionally separated sample using a monoclo-nal keratin 7 antibody.

data shows that there is a serine phosphorylation site inposition 13 [24], accounting possibly for the appearanceof one of the variants. The other visualized ‘spots’ couldsuggest that the protein is being actively synthesized,degraded or both. This is supported by the densitometryanalysis that was done using PD-Quest (Table 3). K7 is



Table 3. Densitometry results for prognostic markers investigated

Protein SSP OD Normalized intensity

Cancerous Normal

Bax 1 – 0.32 – –2 8202 0.29 8158.0 6 63.4 11439 6 15.83 9202 0.28 6256.6 6 54 42157.5 6 0.0

Calponin 1 – 0.24 – –2 5501 0.29 3876.8 6 33.4 –3 – 0.6 – –

b-Casein 1 – 0.37 –2 – 0.87 – –3 4301 0.79 5150.9 6 69.0 2784.0 6 121.34 5301 1.06 10230.8 6 86.0 6610.4 6 170.05 – 2.11 – –

ER 1 7503 0.06 5907.2 6 67.8 3071.3 6 75.92 6807 0.16 6337.9 6 61.2 –

Keratin 1 – 0.91 – –2 4501 1.63 11432.5 6 69 1776.8 6 44.33 4601 1.53 11556.7 6 65.9 2686.1 6 133.74 5601 1.77 11322.4 6 122.4 2626.8 6 118.75 5602 1.51 8628.3 6 78.7 907.1 6 12.66 4602 1.36 9862.5 6 64.5 –

P130cas 1 – 0.18 – –2 9801 0.43 11047.8 6 107.4 8165 6 83.73 9901 0.23 29539.9 6 114.4 5484.9 6 98.6

Data including raw optical density and normalized comparison of breast cancer samples and normalsamples with n = 27 and n = 10, respectivelySSP, spot number.

known as a positive control for breast cancer samplesand proteins 2–5 showed upregulation in all breast cancerspecimens looked at when compared to healthy breasttissue. Protein 6 was only present in cancerous tissueand protein 1 was too random in expression to draw anycorrelation between healthy and cancerous tissue.

It may also suggest that the antibody is binding with othercytokeratins in the CK II sub-family, possibly K8, normallyexpressed in the same tissue type, or K5/6, which havebeen associated with cancer formation [23]. The likeli-hood of this latter explanation is increased when consid-ering that whole tissue homogenate was used rather thana specific cell type. The protein that is seen at 70 kDacould also be the result of several options, either post-translational modification such as glycosylation or pro-tein-protein interactions. It is also feasible that K1 con-tamination may have occurred at some stage of tissueprocessing, K1 being the major keratin found in skin hasa Mr of 68 kDa, however, K1 is also highly insoluble [25]and consistent contamination in only the cancerous tis-sue specimens and not the healthy tissue is unlikely and,

therefore, may not be an appropriate speculation. Furtheranalysis is required to fully define the identity of the pro-tein/s that the K7 antibody has bound to.

3.2 ER-Æ

The ER-a is a well-documented prognostic factor ofbreast cancer, with current estimates of between 50 and55% of all breast tumors being ER-a positive and offeringbetter prognosis [2]. Some tumors develop an estrogendependency in which estrogen is essential for cell survi-val, effectively a pseudo-growth factor. If the tumor celldoes not receive the signals activated by estrogen, thecell cycle will be halted, stalling further progression andultimately leading to regression of the tumor. This is thebasis of adjuvant therapy treatment, to block the signal-ling. With a lot of data already known about this proteinthe need to rule it out of future investigations with regardsto novel markers is essential. The 2-DE analysis in thisstudy revealed the presence of two forms of ER-aisoforms (Fig. 2). The first is a 63 kDa molecule that



Figure 2. 2-D separation using silver stain, indicating theproteins which match an immunoblot using a monoclonalER antibody.

is thought to be the native form of ER-a, the second formis 80 kDa and has previously been only reported fromstudies on breast cancer cell lines [26].

The monoclonal antibody used to visualize ER-a wouldhave bound to both of the two isoforms of ER-a as the80 kDa form is caused by a repeat of both exon 6 and 7during transcription [26], this would result in only the joinsof the two exon 6’s and the two exon 7’s forming uniquesequences, neither of which would have been consideredin the development of the monoclonal antibody used inthis study, validating our observation of two ER-a iso-forms. Analysis using densitometry of the ER-a isoformsshowed that the 63 kDa form was upregulated in cancer-ous tissue samples. The 80 kDa form of ER-a was onlyseen in the cancerous samples and not in the healthysamples (Table 3). This clearly demonstrates the advan-tages of protein separation over in situ procedures suchas IHC where the differences in Mr variants are likely tonot have been detected.

3.3 Calponin

Calponin is an indicator of smooth muscle cells and itslack of appearance in myoepithelial cells of cancer tissueindicate malignancy [21]. Studies have revealed that thereare three variant forms of calponin, a basic (h1), a neutral(h2), and an acidic (h3) form all with Mr between 29 and36 kDa [27]. However, a recent prostate cancer studyrevealed an acidic 52 kDa form of calponin that was iden-tified by 2-DE protein separation techniques using thesame antibody [28]. Our study revealed that two of thethree proteins (Fig. 3), were approximately 50 kDa, how-ever, there was no evidence of an acidic nature with pIvalues calculated to be 6.9 and 7.0. The smaller Mr valueof these two 50 kDa proteins may suggest either degra-

Figure 3. 2-D separation using silver stain, indicating theproteins which match an immunoblot using a monoclonalcalponin antibody.

dation or synthesis was occurring hence the altered pIvalues. The 35 kDa protein that was located was too basicto establish an accurate pI value because the IEF markerswere only designed to incorporate the pI range of 4.6to 9.3. The theoretical pI of Calponin 1 is estimated to be,10.3 according to the SWISS-PROTentry for the protein[29], because of this and the estimated Mr being 35 kDa,also in accordance with theoretical data, it is assumedthat this protein is calponin h1, the basic isoform of calpo-nin.

Despite the antibody binding to three isoforms, analysisof the corresponding proteins in larger data sets usingdensitometry revealed that protein 2 was the only oneupregulated in all breast cancer samples. Both protein 1and 3 were expressed erratically and no correlation wasevident between its expression in cancerous and healthytissue (Table 3). This supports the evidence that the35 kDa form is a good indicator of malignancy. However,further investigation into the 50 kDa isoform that is up-regulated in the breast cancer samples is needed toascertain whether it too has a role in malignancy orwhether it would make an accurate potential prognosticmarker.

3.4 �-Casein

b-Casein is one of three members of the casein family(a, b, and k). It is a protein that is produced by myoepithe-lial cells and secreted during lactation into milk. The roleof b-casein is to transport calcium. This occurs withinmicelles that are formed by the k-casein component ofthe milk. b-Casein along with the other casein compo-nents are considered to be extremely important in thetransfer of nutrients from mother to child. b-Casein is tran-scribed under the control of STAT 5 (signal transducers



Figure 4. 2-D separation using silver stain, indicating theproteins which match an immunoblot using a monoclonalb-casein antibody.

and activators of transcription) which is regulated by bothlactogenic hormones such as prolactin [30] and by growthhormones [13] via the activation of the STAT 5 pathway.The action of b-casein on the cell in the absence of lacta-tion is not yet defined and its impact on the fate of the cellthat produces it is also unknown.

2-DE analysis revealed b-casein in 22 forms in non-lactat-ing breast tumor tissue (Fig. 4). Seventeen of these arethought to be degraded forms of b-casein which arefound in the region between 15 and 20 kDa and pI valuesof 3 and 4.5, the intensity of antibody binding and thesmaller quantity are the basis for this assumption. Therewere also 5 other proteins at 22 kDa that were moreintensely stained and in larger spots suggesting a largerconcentration of the protein. It has been demonstratedthat b-casein in the phosphorylated forms travels throughthe gel quicker than the unphosphorylated form explain-ing the smaller Mr value than what was expected [31].There are very few sequence homologies between thethree forms of casein (a, b, and k). Despite the epitope ofthe antibody not being established and 1-DE experiments(data not shown) confirming Mr of target epitope was incorrect vicinity, it was assumed all proteins are b-casein.It is suggested that these 5 forms of b-casein seen arevarying phosphorylation states of b-casein as the Mr issimilar for all, but the pI differ substantially. This explana-tion is supported by data that b-casein has 6 phosphoryl-ation states [32]. Densitometry analysis suggests that iso-form 4 may have a role as a prognostic indicator in breastcancer. Erratic expression of proteins 1, 2, and 5 was alsofound whilst there was little difference between cancer-ous and healthy for protein 3 (Table 3). This may explainwhy there are conflicting ideas surrounding whetherb-casein is a potential prognostic marker.

3.5 Bax

Bax is a pro-apoptotic protein and a product of the largeBcl-2 gene family. On its own there is doubt as to whetherBax is directly associated to breast cancer occurrenceand therefore speculation as to whether it can be usedaccurately as a prognostic factor in breast cancer. How-ever, reports do suggest that in combination with otherproteins such as Bcl-2 or p53, an assessment of apatient’s prognosis can be made [15, 16]. One of thesestudies also showed that when concentration in Bax wascompared to the apoptotic index (AI), a percentage ofthe number of cells that stained with antideoxygenin(a marker of apoptosis) versus the total number of cellsobserved, the results showed a positive correlation, con-firming Bax’s actions as a pro-apoptotic factor [15]. Theresults obtained for Bax suggest a slightly smaller formof Bax (Fig. 5). This discrepancy is attributed to the pro-tein being located at the very bottom of the gel, whichmay inadvertently affect the separation of the protein.Future studies specific to Bax would benefit from a morehighly concentrated gel to slow down the migration. Thiswas not done in this study in order to preserve uniformitybetween the different protein experiments.

Bax undergoes translocation to the mitochondria underthe stress of a cytotoxic event [33] and this translocationmay reflect on the possibility of a conformational changeof some form providing an explanation for two forms ofBax within the cell. The occurrence of three forms, how-ever, may be due to the likelihood of further phosphoryla-tion or alternate post-translational changes. Degradationor synthesis is also quite possible as many tumour cellsstem from a deregulation of the proteins involved in apop-totic regulation [34]. Although it was not possible to gain acorrelation for protein 1 between cancerous and healthytissue, proteins 2 and 3 both had a substantially increasedconcentration in healthy tissue compared to canceroustissue when analyzed using PD-Quest (Table 3). This

Figure 5. Image from a 2-D separation using silver stain,indicating the proteins which match an immunoblot usinga polyclonal Bax antibody.



result was expected as Bax is a promoter of apoptosis, aprocess inhibited in cancer, suggesting an alteration notof Bax in breast cancer but of one of the factors that in-fluences its transcription.

3.6 p130cas

p130cas (BCAR 1) is a protein that is being revealed as anintegral part of many signalling pathways. 2-DE analysisin this study showed that there are three forms ofp130cas, each of a different pI (Fig. 6). Analysis of the liter-ature revealed that there have been two constant Mr ofp130cas found, 116 kDa [6, 35] and 135 kDa [36]. In thisstudy, estimates based on the standard curves of thestandard molecular weight markers and the distance trav-elled through the gel for the relevant proteins have beenthe basis of Mr determination, it is possible to postulatetherefore that the presence of two forms of p130cas at theestimated 140 kDa and varying pI (Table 2), is actually oneof each of the 116 kDa and 135 kDa Mr variants. Anotherexplanation for the difference in pI may be the possibil-ity of phosphorylation as p130cas is known to have sev-eral phosphorylation sites including a BH3 region usedin the activation of various proteins such as the srconcoprotein [37].

Figure 6. Image from a 2-D separation using silver stain,indicating the proteins which match an immunoblot usinga monoclonal p130cas antibody.

A basic 280 kDa protein was also seen in this study. Thismay be caused by binding of p130 cas to one of its bindingpartners, of which more are being found constantly. Itmay also be caused by homodimerization of the p130cas

protein. The 250 kDa protein was the highest Mr markerthat was incorporated in the standards used for this study,so the Mr of this protein has been extrapolated and maybe the cause of a slight deviation from the expected Mr

if p130cas was to dimerize. Whilst densitometry analysisrevealed that there is no correlation between cancerous

and healthy tissue for protein 1, protein 2 and 3 indicatedincreases of 35% and 539% respectively, supporting thetheory that p130cas may play a major role in breast cancer.

4 Concluding remarks

Identified for the first time are the locations of five prog-nostic markers of breast cancer within breast tumourtissue in 2-DE maps. The ER-a subtypes being presentintroduce the possibility that IHC detection of ER-a maynot be enough for accurate diagnosis and the ratios ofone to the other may be an even better way to establishprognosis of a patient. The multiple forms of K7 suggestthat there is most likely active synthesis of protein occur-ring, with the possibility of degradation. To our knowl-edge, this is the first time that a proteomic map has beengenerated to demonstrate the possible catabolism, syn-thesis, post-translational modifications, and predictedlength of the K7 protein. The 70 kDa protein indicates thepossibility of the formation of a very strong bond betweenK7 and one of its smaller associated proteins. The pres-ence of 5 full length b-caseins despite there being 6 phos-phorylation states, suggesting that either the phosphoryl-ation of site 6 or the unphosphorylated state of b-caseinmay result in a conformational change, and altering theantibodies epitope, hence no binding is seen for a sixthstate of b-casein. Is this the key for b-casein transloca-tion?

All of these points raise more questions than can beanswered through the purpose of this study, however, itdoes support use of proteomics in disease examination,as a way to find answers and new questions. Ultimately,the long-term goal of future studies is to establish a mark-er, or markers, that can be used to detect cancer at anearlier stage than currently possible and which might atthe same time offer new prognostic information.

The use of some diagnostic and prognostic markers aredependent upon the stage which the tumour is currentlyin, as most protein markers are expressed intermittentlyin the cell’s cycle. A limitation of this study concernsthe staging of the samples used. A large number of thesamples obtained were found through pathology testingto be in stage 3. This increases the chances that anymarkers found may only be late stage proteins thereforelimiting their usefulness. The early-stage tumors that werecollected will undoubtedly prove very important in deter-mining which of the differentially expressed proteins willbe more closely looked at first. The discovery of a proteinthat is expressed at multiple if not all stages would beinstrumental in changing the way that a particular canceris approached. Until a marker of such versatility is found,knowing all that is possible about the markers that we



have is crucial and this study has shown some new find-ings about these known prognostic markers as well asraised many questions.

This study was supported by grants from the Royal HobartHospital Research Foundation, Cancer Council Tasmania,and the University of Tasmania.

Received May 23, 2003Revised July 31, 2003Accepted September 1, 2003

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proteomic analysis of selected prognostic factors of breast cancer

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