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Annals of Clinical & Laboratory Science 34:251-262 (2004)
© 2004 Association of Clinical Scientists

Review

Morphoproteomic and Pharmacoproteomic Correlates in Hormone-Receptor-Negative Breast Carcinoma Cell Lines

Robert E. Brown1, Mingyue Lun2, Jeffrey W. Prichard1, Thomas M. Blasick2 and Ping L. Zhang1,2
1 Division of Laboratory Medicine, and 2 Weis Center for Research, Geisinger Medical Center, Danville, PA

Address correspondence to Robert E. Brown, M.D., Division of Laboratory Medicine, Geisinger Medical Center, Danville, PA 17822-0131, USA; tel 570 271 6332; fax 570 271 6105; e-mail: rebrown{at}geisinger.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The aim of this study was to elucidate protein circuitry in breast cancer based on the profiling of hormone-receptor-negative breast carcinomas using morphoproteomic and pharmacoproteomic techniques. Three human breast carcinoma cell lines (SKBR-3, MDA-175, MDA-231) were reacted by immunohisto-chemical (IHC) procedures to detect several categories of protein analytes. Immunoreactivities and cell compartmentalizations were scored from 0 to 3+ positivity using bright-field microscopy. An automated cellular imaging system (ACIS) was used to obtain a final combined score of staining intensity and positive cells in the IHC reactions, to enable comparisons with the visual scores and the rates of inhibition by pharmaceutical agents. FDA-approved inhibitors that target the protein circuitry were added to the cultures for 2–4 days. Proliferation assays were conducted, and in vitro inhibition rates were calculated as (control-treated)/control. Western blot analyses of whole cell lysates assessed the effects of the pharmaceutical agents on selected aspects of protein circuitry. Good to excellent correlation was observed between visual scores and ACIS scores (r values from 0.732 to 0.996 in 10 of 11 trials). Gleevec produced growth inhibition that correlated with the composite expressions of the platelet-derived growth factor (PDGF) family of ligands and receptors; captopril inhibited only MDA-175, consistent with its unique expression of plasmalemmal angiotensin-converting enzyme (ACE); and interferon (IFN)-{alpha} effected growth inhibition in accordance with the degree of conventional (c) protein kinase C (PKC)-{alpha} and phosphorylated (p)-PKC{alpha}/ßII expressions. Western blot analyses revealed correlative changes of several intracellular signals following incubation with these inhibitors. This study shows (a) a close association between the immunohistochemical expression of signal transduction markers and in vitro inhibition by pharmaceutical agents, and (b) correlations between the sites of action of the pharmaceutical agents and the downstream expression of proteins in hormone-receptor-negative breast cancer cell lines. Such morphoproteomic and pharmacoproteomic profiling of individual tumors may enable the pathologist and oncologist to design antitumor therapy that is customized for an individual patient.

(received 3 May 2004; accepted 24 May 2004)

Keywords: breast carcinoma, proteomics, cancer chemotherapy, immunohistochemistry, Western blotting


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Immunohistochemistry has become an invaluable tool for categorizing invasive carcinoma of the breast as hormone-receptor positive or negative and for helping to guide therapy with biological response modifiers (eg, tamoxifen and aromatase inhibitors) [1]. During the past several years, targeted therapies have been developed that can promote immune surveillance, interfere with signal transduction and anti-apoptotic pathways in breast carcinoma, or promote growth inhibition and apoptosis of tumor cells. Such agents include trastuzumab, a recombinant, DNA-derived, humanized, monoclonal antibody against the ectodomain of human epidermal growth factor receptor (HER)-2/neu (Herceptin® [2,3]); ZD1839, an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (Iressa, gefitinib [4]); STI571, an inhibitor of the platelet-derived growth factor receptor (PDGFR) family of signal transducers (Gleevec, imatinib mesylate [5,6]); statins and aminobisphosphonates, inhibitors of 3-hydroxy-3 methylglutaryl-coenzyme A (HMG CoA) reductase [7], and farnesyl diphosphate synthase [8–11] to interrupt prenylation of GTP-binding proteins that facilitate signal transduction; inhibitors of ACE, such as captopril [12,13], and inhibitors of angiotensin II type 1 receptor (AT1R) signaling, such as losartan [14,15], that could curb transactivation of cell signaling [16]; 8rapamycin and rapamycin analogs (eg, CCI-779 and RAD001) to interfere with the downstream signaling of the Akt pathway at the level of mTOR, thereby moderating the antiapoptotic and proliferative effects of this effector pathway [17–19]; retinoids such as 4-hydroxyphenylretinamide (4-HPR) to downregulate c-erb B–2 expression and to activate latent transforming growth factor (TGF)-ß1 and upregulate TGF-ßRII, leading to apoptosis [20–23]; and Velcade (bortezomib, PS-341) to inhibit NF-{kappa}B, reducing its potential growth promoting, tumor-igenic, and anti-apoptotic effects [24,25].

Against this background, pathologists and clinical scientists are presented with the opportunity to play a role in customizing therapy for individual patients by detecting and correlating, as a molecular profile, those protein analytes in their tumors that might be potential targets for one or more of the aforementioned agents and reporting these pathways and options in a consultative format (consultative proteomics).

The purpose of this report is twofold: first, to begin to develop templates for consultative proteomics based on molecular profiling of hormone-receptor-negative breast carcinoma cell lines using immunohistochemistry (morpho-proteomics); and second, to test the validity of the morphoproteomic approach by observing the effects of various pharmaceutical agents that are known to inhibit the signaling pathways at specific points in the protein circuitry (pharmacoproteomics).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Human breast cancer cell lines.  Slides that each contained sections of 3 pelleted, formalin-fixed and paraffin-embedded human breast carcinoma cell lines (SKBR-3, MDA-175, MDA-231) were obtained as part of the standard Hercept® kit (DAKO Corporation, Carpinteria, CA). These cell lines are known to be hormone-receptor negative (ie, non-immunoreactive for estrogen or progesterone receptors [26]). Cell cultures of the 3 cell lines were obtained from American Type Culture Collection (ATCC) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM [Gibco-BRL, Gaithersburg, MD]), supplemented with 10% fetal bovine serum and cultured in 95% air/5% CO2. After reaching 80% confluence, the 3 cell types were placed into 96-well plates and allowed to attach to the wells for 3 days.

Immunohistochemistry.  The general immunohistochemical procedure has been previously described [27]. Positive controls using established immunoreactive tissues and negative controls utilizing the cell lines and case study materials were run concurrently and shown to react appropriately. A panel of antibodies was assembled to detect various protein antigens, as follows:

Mouse monoclonal anti-human platelet-derived growth factor (PDFG)-AB (clone 10106.3, IgG2b,k, R&D Systems) was used to detect the corresponding antigen, a cytokine that serves as a signaling ligand for PDGF receptor (R).

Rabbit polyclonal anti-human stem cell factor (SCF) antibody (catalog #sc-9132; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used to detect the corresponding antigen, the signaling ligand for c-kit.

Goat polyclonal anti-human interleukin (IL)-1{alpha} antibody (catalog # sc-1253; Santa Cruz Biotechnology) was used to detect the corresponding antigen, a polypeptide (cytokine) that plays a critical role in regulating the immune response.

Mouse monoclonal anti-human PDGFR-{alpha}antibody (clone 35264.11, IgG1k; R&D Systems) was used to detect the corresponding antigen, a protein that functions as a tyrosine kinase receptor and, when activated, as a signal transducer.

Mouse monoclonal anti-human PDGFR-ß (A-3) antibody (IgG2b, catalog #sc-6252; Santa Cruz Biotechnology) was used to detect the corresponding antigen, a tyrosine kinase receptor that functions as a signal transducer and a mitogen for mesenchymal and glia-derived cells.

Rabbit polyclonal anti-human c-kit (CD117) antibody (code #A4502; DAKO) was used to detect the corresponding antigen, a protein that functions as a transmembrane tyrosine kinase receptor.

Mouse monoclonal IgG1 anti-human conventional (c) protein kinase C (PKC)-{alpha} (H-7) and cPKC-ßII (F-7) antibodies (catalog# sc-8393 and sc-13149, respectively; Santa Cruz Biotechnology) were used to assess the corresponding antigens, both isozymes in the PKC superfamily of signaling molecules that play key roles in an array of physiological processes including growth and differentiation of tissue.

Rabbit polyclonal anti-human phosphorylated (p)-PKC{alpha}/ßII antibody (catalog # 9375L; Cell Signaling Technology, Inc., Beverly, MA) was used to detect the corresponding phosphorylated antigen with an epitope common to both isozymes in the PKC superfamily of signaling molecules.

Rabbit polyclonal anti-human cathepsin D (code #A0561; DAKO) was used to detect the corresponding antigen, a lysosomal enzyme involved in intracellular protein turnover.

Mouse monoclonal anti-human angiotensin-converting enzyme (ACE; clone CG2-1193-36-18; Accurate Chemical & Scientific Corp., Westbury, NY) was used to detect the corresponding antigen, an endopeptidase that generates angiotensin II from angiotensin I.

Rabbit polyclonal anti-human angiotensin II type 1 receptor (AT1R; catalog #sc-579; Santa Cruz Biotechnology) was used to detect the corresponding antigen, the protein receptor that mediates the effects of angiotensin II, which include the activation of several transduction pathways.

Scoring of immunoreactivity.  Immunoreactivities of the 3 cell lines and study cases were scored from 0 (negative) to 3+ positivity using bright-field microscopy. Instances in which the brown diaminobenzidine (DAB) chromogenic signal was faint (between negative and 1+) were assigned a "±" status (0.5 score). The final score for each protein analyte incorporated the range of signals and the relative percentages of positive cells within the individual cell line and reflected any heterogeneity of the tumor cells. In addition, an automated cellular imaging system (ACIS II, ChromaVision System, San Juan Capistrano, CA) was used for selected analytes along the signal transduction pathways to determine the percentage of cells that expressed the respective proteins and the intensity of staining.

The ACIS II system consists of two major components: a microscope with electromechanical hardware and a computer with a frame grabber and image processor. The microscope system includes a stand and microscope mounted in a special shock-resistant frame with a video camera. The camera is a Sony progressive 3 CCD color video camera. The ACIS system is used to generate 2 indices: the percentage of positively stained tumor cells and the staining intensity score of the positive tumor cells in IHC sections (each equivalent to a 40x objective field). Multiplying these 2 indices obtained at 5 random areas of the tumor gives a final composite score with a mean ± SE for statistical comparisons. This approach has been previously used by one of us (PL Zhang) [28].

Inhibition studies.  Pharmaceutical agents used as potential inhibitors in the cell cultures included Gleevec (a gift from Novartis, Basel, Switzerland), captopril (Sigma, St. Louis, MO) and IFN-{alpha}(Sigma). Gleevec was dissolved in DMSO; captopril was dissolved in ethanol; and IFN-{alpha} was supplied in phosphate buffered saline. These stock solutions were diluted in culture medium and added to each of the cell lines to give final desired concentrations in µM, mM, and units (U)/ml, respectively. (Separate experiments were carried out to determine the impact of the vehicle alone on each of the 3 cell lines, and no effects were identified.) After 4 days of incubation, viable cells in each well were determined colorimetrically (CellTiter 96 AQueous ONE Solution Proliferation Assay, Promega, Madison, WI). Inhibition rates were calculated as the cell numbers in control groups minus those in treated groups, then divided by control [ie, (control-treated)/control].

Western blotting.  Control and pharmaceutical-agent-treated breast cancer cells were harvested and sonicated. Cell homogenates (30 µg total protein per lane) were separated on 6–12% SDS PAGE and transferred onto PVDF membranes. For immunostaining of phosphorylated (p)-Nuclear Factor (NF)-{kappa}B, rabbit anti-p-NF-{kappa}B antibody (Cell Signaling Technology, Beverly, MA; 1:1000 dilution) was used. The second antibody was horseradish-peroxidase-linked donkey anti-rabbit Ig whole antibody (Amersham Biosciences, Piscataway, NJ; 1:3000 dilution). Immunoreactive proteins were visualized by an enhanced chemiluminescence-Western blotting system (Amersham). Western blots with other antibodies were performed as described above. Primary antibodies included those against p-c-Jun N-terminal kinase (p-JNK; Affinity Bioreagents, Golden, CO); p-Akt (Cell Signaling), p-extracellular signal-regulated kinase (p-ERK1/2; Cell Signaling), I-{kappa}B (Santa Cruz Biotechnology, Santa Cruz, CA), and actin (Santa Cruz).

Data mining.  A computer-assisted search of the National Library of Medicine’s MEDLINE database was performed using the Ovid system, looking for information regarding these cell lines, their protein circuitry, and effects of pharmaceutical inhibitors.

Statistics.  ANOVA was used to determine the p value for group comparisons (p <0.05 statistically significant). Correlation coefficients were obtained by linear regression analysis. In vitro inhibitory rates were expressed as means ± SE. One-way ANOVA was used to compare the mean inhibitory rates among the 3 types of breast cancer cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
As shown in Table 1Go and Fig. 1Go, constitutive expressions of the protein analytes detected by immunohistochemistry in the 3 breast carcinoma cell lines revealed commonalities and relative differences. The latter included differences in cellular compartmentalization. The correlations between the visual scores and ACIS scores gave good to excellent r values, which ranged from 0.732 to 0.996 in 10 of 11 comparisons (Table 2Go).


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  		Table 1. Scoring and compartmentalization of protein analytes in 3 hormone-receptor-negative breast carcinoma cell lines.*
 


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  		Fig. 1. Composite digital images illustrating: cytoplasmic immunoreactivity for PDGFR-{alpha} expression (upper right) and subplasmalemmal/cytoplasmic expression of its signaling ligand, PDGF-AB (upper left) in MDA-175 cell line; cytoplasmic immunopositivity for c-kit (second row, right) and its signaling ligand, SCF (second row, left) in SKBR-3 cell line; cytoplasmic expression of cathepsin D (which converts angiotensinogen to angiotensin I) and plasmalemmal localization of ACE (third row, left and right respectively) in MDA-175 cell line; and strong immunohistochemical signal for cPKC-alpha (lower left ) and associated expression of activated, p-PKC-{alpha}/ßII (lower right) in MDA-231 cell line (DAB [brown]) chromogen; original magnification x600).

 

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  		Table 2. Correlation between visual scores and combined scores evaluated by the automated cellular imaging system (ACIS).
 
The pharmaceutical agents Gleevec, captopril, and IFN-{alpha} exhibited inhibitory effects on cell growth in the 3 cell lines that correspond to the morphoproteomic expression of their respective targets in the protein circuitry. Specifically, Gleevec’s inhibitory activity in MDA-175 and SKBR-3 cells coincided with the expression pattern of the tyrosine kinase targets among the platelet-derived growth factor family; captopril, with the expression of the angiotensin system of proteins and, in particular, cathepsin D and angiotensin-converting enzyme (ACE) in MDA-175 and IFN-{alpha} with the elements of the protein kinase C pathway and specifically c-PKC and p-PKC-{alpha}/ßII in MDA-231. Moreover, the correlation coefficients were good to excellent between these protein analyte expressions as determined by the ACIS combined score and the level of inhibition; dose-response relationships were documented. These are summarized in tabular and graphic form in Figs. 2Go–4Go.



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  		Fig. 2. The tabular data show good to excellent correlation coefficients (r) between members of platelet-derived growth factor family of signal transducers and ligands (except for c-kit) and the growth inhibitory rates produced by Gleevec. The chart of inhibitory rate vs increasing concentrations of Gleevec shows a dose-response effect against MDA-175 cell line and a slight inhibitory effect of Gleevec at 30 µM against the SKBR-3 cell line. *p<0.05 vs SKBR-3 and #p<0.05 vs MDA-175.

 


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  		Fig. 4 (in the left column). The tabular data show excellent correlation coefficients (r) between protein analytes of the protein kinase C alpha pathway (cPKC-{alpha} and phosphorylated [p]-PKC-{alpha}/ßII) and the growth inhibitory rate effected by IFN-{alpha}. The chart of inhibitory rates vs increasing concentrations of IFN-{alpha} shows a dose-response effect against the MDA-231 cell line with the strongest expression of both (Table 2Go). *p<0.05 vs SKBR-3 and #p< 0.05 vs MDA-175.

 
As shown in Fig. 5Go, Western blot analyses revealed a corresponding decrease in p-Akt and an increase in p-ERK1/2 in the MDA-175 and SKBR-3 cell lines following exposure to Gleevec; a decrease in p-Akt and an increase in p-ERK1/2 and p-JNK in MDA-175 following exposure to captopril, but with a decrease in both p-ERK1/2 and p-JNK in SKBR-3 and of p-JNK in MDA-231; and decreases in both p-NF-{kappa}B and I-{kappa}B in MDA-175 following exposure to captopril and Gleevec, respectively. Actin protein was expressed equally in each cell line without and after each inhibitor treatment.



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  		Fig. 5 (below). Western blots showing captopril (C), Gleevec (G), and interferon-alpha (I) lanes and depicting the effects of these agents on selected protein analytes in signal transduction pathways in the 3 breast carcinoma cell lines, compared to the non-exposed control (O) of each. Specifically, there are the following changes in band intensities: a corresponding decrease in phosphorylated (p)-Akt and an increase in p-ERK1/2 in the MDA-175 and SKBR-3 cell lines following exposure to 30 µM Gleevec for 48 hr; a decrease in p-Akt and increase in p-ERK1/2 and p-JNK in MDA-175 following exposure to 9 mM captopril for 48 hr, but with a decrease in both p-ERK1/2 and p-JNK in SKBR-3 and of p-JNK in MDA-231; and slight decreases in both p-NF-{kappa}B and its inhibitor, I-{kappa}B in MDA-175 following exposure to captopril and Gleevec, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Clinical proteomics has been defined as the characterization of cell circuitry and the impact of disease and therapy on cellular networks. Immunohistochemistry allows for an assessment of activation (eg, translocation and phosphorylation) and functional grouping of protein analytes in tumor cells, exposing circuitries that may be amenable to therapeutic intervention (morphoproteomics). Tissue sections of primary tumors examined by this approach provide for a concurrent contextual analysis of expression patterns of protein in companion stromal and endothelial cells that might be contributing in a paracrine fashion to signaling in tumor cells. (Although quantification of the overall immunoreactivity to determine the composite percentage of tumor cells expressing the protein analyte and the intensity of the signal can be performed by automated systems such as the ACIS used in this study, valuable information regarding the companion cell component and the cellular compartmentalization of the proteins depends upon the direct participation of the pathologist, at least for now.) Pharmacoproteomics assesses the impact of molecular inhibitors on tumor cells in vitro.

This study demonstrated correlations between the morphoproteomic and pharmacoproteomic analyses in hormone-receptor-negative breast carcinoma cell lines SKBR-3, MDA-175, and MDA-231. Gleevec (20 and 30 µM) demonstrated predominant inhibition in MDA-175 cells, corresponding to the significantly higher levels of expression of PDGFR-{alpha}, PDGFR-ß, and their signaling ligand, PDGF-AB, and showed a dose-response effect on the MDA-175 cells. Additionally, correlations were good to excellent between Gleevec’s inhibitory effects (20 and 30 µM) and PDGF-AB, PDGFR-{alpha}, and PDGFR-ß expressions estimated by the ACIS method in all 3 cell lines. Moreover, the results are consistent with the established inhibitory effects of Gleevec against c-kit and PDGFR-{alpha} and –ß tyrosine kinases [29,30].

The MDA-175 cell line in this study showed higher expression of markers in the angiotensin system, particularly plasmalemmal ACE, and was correspondingly inhibited by the ACE inhibitor captopril at both 3 and 9 mM when compared to SKBR-3 and MDA-231 cells. Similarly, there were excellent correlation coefficients between captopril’s inhibitory effects at 3 and 9 mM and ACE expressions measured by the ACIS method in all 3 cell lines. Inhibitors of the angiotensin system such as the ACE inhibitor captopril have been used extensively to treat hypertension or renal disorders in the clinic, but their role in inhibiting breast cancer is largely limited to the laboratory stage [31,32]. An additional possible molecular concomitant of the angiotensin system in the MDA-175 cell is the high expression of PDGF-AB when compared to that in the SKBR-3 and MDA-231 cell lines. This is consistent with the reported ability of the angiotensin system to stimulate the production of PDGF-AB [33]. Finally, a previously reported anecdotal observation by one of us (RE Brown) regarding a patient whose invasive carcinoma showed strong expression of ACE and who experienced a complete response to Herceptin while receiving captopril is intriguing [2].

IFN-{alpha} showed the greatest inhibitory activity against the MDA-231 cell line correlating with the level of expression of c-PKC-{alpha}, its translocation to the plasmalemmal aspect in some cells, and the companion expression of p-PKC-{alpha}/ßII. This is consistent with data from the literature indicating that IFN-{alpha} achieves growth inhibition in tumor cells coincidentally with the downregulation of PKC-{alpha}[34] and that it takes advantage of the PKC pathway to exert an antitumoral effect [35,36]. Moreover, there is clinical evidence of the efficacy of IFN-{alpha} in the treatment of Langerhans cell histiocytosis (LCH), a disease in which the lesional LCH cells show strong plasmalemmal immunoreactivity for PKC-{alpha} and similar immunopositivity for p-PKC-{alpha}/ßII [37]. Furthermore, our observation of cytoplasmic and, on occasion, plasmalemmal expression of PKC-{alpha} in MDA-231 (also referred to as MDA-MB-231) and SKBR-3, with a higher level of expression by both ACIS and visual scoring in the former, coincides with the finding of Lindemann and associates [38] on the respective protein levels of PKC-{alpha} in these 2 cell lines.

Additional morphoproteomic and pharmaco-proteomic correlates on one or more of these cell lines are available in the literature. These include the strong (3+) plasmalemmal expression of HER-2/neu in the SKBR-3 cell line and the inhibitory effect of Herceptin (trastuzumab) in vitro [39]; the moderate (2+) plasmalemmal expression of EGFR in SKBR-3 [26]; the additive effect of Iressa (ZD1839), an EGFR tyrosine kinase inhibitor, in association with trastuzumab in this cell line [40,41]; and the growth inhibitory effects of Iressa, both in vitro and in xenografts of MDA-MB-231 cells that strongly express EGFR [26,42].

Molecular concomitants of the inhibitory effects of captopril and Gleevec revealed by Western blot analyses in this study include a decrease in p-Akt in the MDA-175 cell line consistent with the reported role of the angiotensin system and platelet-derived growth factor receptor family in signaling the activation of the PI3-K/Akt pathway [30,43–46] and with the observation by McGary and coworkers [30] that Gleevec (STI571) inhibits both PDGFR-{alpha} and PDGFR-ß phosphorylation and the downstream phosphorylation target, Akt; an increase in p-ERK1/ 2 accumulation in both MDA-175 and, to a lesser extent, SKBR-3, consistent with the reported enhancing effect of STI571 (Gleevec) on hepatocyte growth factor-induced motility that includes ERK activation [47]; an increase in p-JNK in the plasmalemmal ACE-expressing MDA-175 cell line in response to captopril that accords with the recent report by Kohlstedt and colleagues [48,49] on the ability of ACE inhibitors via the phosphorylation of membrane ACE ser1270 to increase ACE-associated JNK activity; and, finally, reduced expression of p-NF-{kappa}B in MDA-175 breast cancer cells subsequent to captopril and Gleevec exposures is consistent with roles of the angiotensin system and the Akt pathway in promoting p-NF-{kappa}B formation through NF-{kappa}B-inducing kinase [50–52].

The differences in constitutive protein analyte expression in these cell lines uncovered by a morphoproteomic approach in this and a previous study [26] are sufficient to allow the design of protein circuitries unique to each. Furthermore, such heterogeneity among these phenotypically similar (hormone-receptor-negative) breast carcinoma cell lines coincides with the genomic studies of Perou and associates [53], who found that human breast tumors showed great variation in their patterns of gene expression and that many different sets of genes show mainly independent patterns of variation. Because of this heterogeneity, it appears that molecular profiling using morphoproteomics to define the protein circuitry and to uncover targets amenable to therapeutic intervention with specific inhibitors would be necessary to customize therapy for individual patients. This study has enabled us to move closer to that goal by defining distinctive differences in molecular protein circuitry that will contribute to the design of templates for the reporting of morphoproteomic analyses in tumors from breast cancer patients who have failed conventional chemotherapy (ie, consultative proteomics). Moreover, the pharmacoproteomic and molecular correlates increase our confidence in the predictive value of morphoproteomic analyses.

In summary, we have demonstrated morpho-proteomic and pharmacoproteomic correlates in 3 hormone-receptor-negative breast carcinoma cell lines and thereby have reinforced the utility of the morphoproteomic approach in the validation of therapeutic targets. Using these approaches, we have been able to uncover protein circuitries that are amenable to specific therapeutic intervention and that could contribute to the development of a template for customized therapy in breast cancer patients who have failed conventional therapy.



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  		Fig. 3. The tabular data show good to excellent correlation coefficients (r) between protein analytes of the angiotensin pathway, cathepsin D, and angiotensin-converting enzyme (ACE) and the growth inhibitory rates produced by captopril. The chart of inhibitory rate vs increasing concentrations of captopril illustrates a dose-response effect against the MDA-175 cell line, the only one with plasmalemmal expression of ACE. *p<0.05 vs SKBR-3 and #p<0.05 vs MDA-175.

 

   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank Laurie Kneller, HT (ASCP), and Glen Kauwell, HT (ASCP), for technical assistance and Kathy Fenstermacher and Sharon Coup for secretarial support. Novartis (Basel, Switzerland) kindly provided the Gleevec for this study.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Murray PA, Gomm J, Ricketts D, Powles T, Coombes RC. The effect of endocrine therapy on the levels of oestrogen and progesterone receptor and transforming growth factor-beta 1 in metastatic human breast cancer: an immunocytochemical study. Eur J Cancer 1994;30A: 1218–1222.[Medline]
  2. Brown RE, Bernath AM, Lewis GO. HER-2/neu protein-receptor-positive breast carcinoma: an immunologic perspective. Ann Clin Lab Sci 2000;30:249–258.[Abstract]
  3. Thor A. HER2 - a discussion of testing approaches in the USA. Ann Oncol 2001;12(Suppl 1):S101–107.[Abstract]
  4. Cohen MH, Williams GA, Sridhara R, Chen G, Pazdur R. FDA drug approval summary: gefitinib (ZD1839) (Iressa) tablets. Oncologist 2003;8:303–306.[Abstract/Free Full Text]
  5. Cohen MH, Dagher R, Griebel DJ, Ibrahim A, Martin A, Scher NS, Sokol GH, Williams GA, Pazdur R. U.S. Food and Drug Administration drug approval summaries: imatinib mesylate, mesna tablets, and zoledronic acid. Oncologist 2002;7:393–400.[Abstract/Free Full Text]
  6. Ross DM, Hughes TP. Cancer treatment with kinase inhibitors: what have we learnt from imatinib? Br J Cancer 2004;90:12–19.[Medline]
  7. Brower V. Of cancer and cholesterol: studies elucidate anticancer mechanisms of statins. J Natl Cancer Inst 2003;95:844–846.[Free Full Text]
  8. Luckman SP, Hughes DE, Coxon FP, Graham R, Russell G, Rogers MJ. Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J Bone Miner Res 1998;13:581–589.[Medline]
  9. Bergstrom JD, Bostedor RG, Masarachia PJ, Reszka AA, Rodan G. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch Biochem Biophys 2000; 373:231–241.[Medline]
  10. Lipton A. Bisphosphonates and metastatic breast carcinoma. Cancer 2003;97:848–853.[Medline]
  11. Fromigue O, Lagneaux L, Body JJ. Bisphosphonates induce breast cancer cell death in vitro. J Bone Miner Res 2000;15:2211–2221.[Medline]
  12. DiBianco R. Angiotensin converting enzyme inhibition. Unique and effective therapy for hypertension and congestive heart failure. Postgrad Med 1985;78:229–241.[Medline]
  13. Volpert OV, Ward WF, Lingen MW, Chesler L, Solt DB, Johnson MD, Molteni A, Polverini PJ, Bouck NP. Captopril inhibits angiogenesis and slows the growth of experimental tumors in rats. J Clin Invest 1996;98:671–679.[Medline]
  14. Fleming T, Borer J, Armstrong PW, Pfeffer M. Food and Drug Administration. Cardiovascular and Renal Drugs Advisory Committee. Food and Drug Administration: Cardiovascular and Renal Drugs Advisory Committee, 98th Meeting, January 6–7, 2003. Circulation 2003; 107:e9002–9003.[Free Full Text]
  15. Rivera E, Arrieta O, Guevara P, Duarte-Rojo A, Sotelo J. AT1 receptor is present in glioma cells; its blockage reduces the growth of rat glioma. Br J Cancer 2001;85:1396–1399.[Medline]
  16. Moriguchi Y, Matsubara H, Mori Y, Murasawa S, Masaki H, Maruyama K, Tsutsumi Y, Shibasaki Y, Tanaka Y, Nakajima T, Oda K, Iwasaka T. Angiotensin II-induced transactivation of epidermal growth factor receptor regulates fibronectin and transforming growth factor-beta synthesis via transcriptional and posttranscriptional mechanisms. Circ Res 1999;84:1073–1084.[Abstract/Free Full Text]
  17. Miller JL. Sirolimus approved with renal transplant indication. Am J Health Syst Pharm 1999;56:2177–2178.[Free Full Text]
  18. Hosoi H, Dilling MB, Shikata T, Liu LN, Shu L, Ashmun RA, Germain GS, Abraham RT, Houghton PJ. Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdo-myosarcoma cells. Cancer Res 1999;59:886–894.[Abstract/Free Full Text]
  19. Huang S, Houghton PJ. Targeting mTOR signaling for cancer therapy. Curr Opin Pharmacol 2003;3:371–377.[Medline]
  20. Grunt ThW, Dittrich E, Offterdinger M, Schneider SM, Dittrich Ch, Huber H. Effects of retinoic acid and fenretinide on the c-erbB-2 expression, growth and cisplatin sensitivity of breast cancer cells. Br J Cancer 1998;78:79–87.[Medline]
  21. Herbert BS, Sanders BG, Kline K. N-(4-hydroxyphenyl)-retinamide activation of transforming growth factor-beta and induction of apoptosis in human breast cancer cells. Nutr Cancer 1999;34:121–132.[Medline]
  22. Roberson KM, Penland SN, Padilla GM, Selvan RS, Kim CS, Fine RL, Robertson CN. Fenretinide: induction of apoptosis and endogenous transforming growth factor beta in PC-3 prostate cancer cells. Cell Growth Differ 1997;8:101–111.[Abstract]
  23. Jinno H, Steiner MG, Mehta RG, Osborne MP, Telang NT. Inhibition of aberrant proliferation and induction of apoptosis in HER-2/neu oncogene transformed human mammary epithelial cells by N-(4-hydroxyphenyl) retinamide. Carcinogenesis 1999;20:229–236.[Abstract/Free Full Text]
  24. Twombly R. First proteasome inhibitor approved for multiple myeloma. J Natl Cancer Inst 2003;95:845.[Free Full Text]
  25. Voorhees PM, Dees EC, O’Neil B, Orlowski RZ. The proteasome as a target for cancer therapy. Clin Cancer Res 2003;9:6316–6325.[Abstract/Free Full Text]
  26. Brown RE. HER-2/neu-positive breast carcinoma: molecular concomitants by proteomic analysis and their therapeutic implications. Ann Clin Lab Sci 2002;32:12–21.[Abstract/Free Full Text]
  27. Brown RE. Histogenesis of Reed-Sternberg and dendritic interdigitating cells in nodular sclerosing Hodgkin’s disease. Immunohistochemical evidence for monocytoid precursors. Ann Clin Lab Sci 1997;27:329–337.[Abstract]
  28. Sun W, Zhang PL, Herrera GA. p53 protein and Ki-67 overexpression in urothelial dysplasia of bladder. Appl Immunohistochem Mol Morphol 2002;10:327–331.[Medline]
  29. Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, Lydon NB. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000;295:139–145.[Abstract/Free Full Text]
  30. McGary EC, Weber K, Mills L, Doucet M, Lewis V, Lev DC, Fidler IJ, Bar-Eli M. Inhibition of platelet-derived growth factor-mediated proliferation of osteosarcoma cells by the novel tyrosine kinase inhibitor STI571. Clin Cancer Res 2002;8:3584–3591.[Abstract/Free Full Text]
  31. Small W Jr, Molteni A, Kim YT, Taylor JM, Chen Z, Ward WF. Captopril modulates hormone receptor concentration and inhibits proliferation of human mammary ductal carcinoma cells in culture. Breast Cancer Res Treat 1997;44:217–224.[Medline]
  32. Small W Jr, Molteni A, Kim YT, Taylor JM, Ts’ao CH, Ward WF. Mechanism of captopril toxicity to a human mammary ductal carcinoma cell line in the presence of copper. Breast Cancer Res Treat 1999;55:223–229.[Medline]
  33. Wong J, Rauhoft C, Dilley RJ, Agrotis A, Jennings GL, Bobik A. Angiotensin-converting enzyme inhibition abolishes medial smooth muscle PDGF-AB biosynthesis and attenuates cell proliferation in injured carotid arteries: relationships to neointima formation. Circulation 1997; 96:1631–1640.[Abstract/Free Full Text]
  34. Rosewicz S, Weder M, Kaiser A, Riecken EO. Antiprolif-erative effects of interferon alpha on human pancreatic carcinoma cell lines are associated with differential regulation of protein kinase C isoenzymes. Gut 1996;39:255–261.[Abstract/Free Full Text]
  35. Tiefenbrun N, Kimchi A. The involvement of protein kinase C in mediating growth suppressive signals of interferons in hematopoietic cells. Oncogene 1991;6: 1001–1007.[Medline]
  36. Powell CB, Manning K, Collins JL. Interferon-alpha (IFN-alpha) induces a cytolytic mechanism in ovarian carcinoma cells through a protein kinase C-dependent pathway. Gynecol Oncol 1993;50:208–214.[Medline]
  37. Brown RE. Interferon-alpha therapy, protein kinase C-alpha and Langerhans cell histiocytosis. Med Pediatr Oncol 2003;41:63–64.[Medline]
  38. Lindemann RK, Braig M, Ballschmieter P, Guise TA, Nordheim A, Dittmer J. Protein kinase Calpha regulates Ets1 transcriptional activity in invasive breast cancer cells. Int J Oncol. 2003;22:799–805.[Medline]
  39. Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL. Herceptin-induced inhibition of phospha-tidyinositol-3 kinase and Akt is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res. 2002;62:4132–4141.[Abstract/Free Full Text]
  40. Moulder SL, Yakes FM, Muthuswamy SK, Bianco R, Simpson JF, Arteaga CL.Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res 2001;61:8887–8895.[Abstract/Free Full Text]
  41. Normanno N, Campiglio M, De LA, Somenzi G, Maiello M, Ciardiello F, Gianni L, Salomon DS, Menard S. Cooperative inhibitory effect of ZD1839 (Iressa) in combination with trastuzumab (Herceptin) on human breast cancer cell growth. Ann Oncol 2002;13:65–72.[Abstract/Free Full Text]
  42. Anderson NG, Ahmad T, Chan K, Dobson R, Bundred NJ. ZD1839 (Iressa), a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, potently inhibits the growth of EGFR-positive cancer cell lines with or without erbB2 overexpression. Int J Cancer 2001;94: 774–782.[Medline]
  43. Takahashi T, Taniguchi T, Konishi H, Kikkawa U, Ishikawa Y, Yokoyama M. Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells. Am J Physiol 1999;276:H1927–1934.[Medline]
  44. Chen EY, Mazure NM, Cooper JA, Giaccia AJ. Hypoxia activates a platelet-derived growth factor receptor/ phosphatidylinositol 3-kinase/Akt pathway that results in glycogen synthase kinase-3 inactivation. Cancer Res 2001; 61:2429–2433.[Abstract/Free Full Text]
  45. Lokker NA, Sullivan CM, Hollenbach SJ, Israel MA, Giese NA. Platelet-derived growth factor (PDGF) auto-crine signaling regulates survival and mitogenic pathways in glioblastoma cells: evidence that the novel PDGF-C and PDGF-D ligands may play a role in the development of brain tumors. Cancer Res 2002;62:3729–3735.[Abstract/Free Full Text]
  46. Park CS, Schneider IC, Haugh JM. Kinetic analysis of platelet-derived growth factor receptor/phosphoinositide 3-kinase/Akt signaling in fibroblasts. J Biol Chem 2003;278:37064–37072.[Abstract/Free Full Text]
  47. Frasca F, Vigneri P, Vella V, Vigneri R, Wang JY. Tyrosine kinase inhibitor STI571 enhances thyroid cancer cell motile response to hepatocyte growth factor. Oncogene 2001;20:3845–3856.[Medline]
  48. Kohlstedt K, Brandes RP, Muller-Esterl W, Busse R, Fleming I. Angiotensin-converting enzyme is involved in outside-in signaling in endothelial cells. Circ Res 2004; 94:60–67.[Abstract/Free Full Text]
  49. Ryan MJ, Sigmund CD. ACE, ACE inhibitors, and other JNK. Circ Res 2004;94:1–3.[Free Full Text]
  50. Costanzo A, Moretti F, Burgio VL, Bravi C, Guido F, Levrero M, Puri PL. Endothelial activation by angiotensin II through NF-kappaB and p38 pathways: Involvement of NF-kappaB-inducible kinase (NIK), free oxygen radicals, and selective inhibition by aspirin. J Cell Physiol 2003;195:402–410.[Medline]
  51. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB. NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999;401:82–85.[Medline]
  52. Burow ME, Weldon CB, Melnik LI, Duong BN, Collins-Burow BM, Beckman BS, McLachlan JA. PI3-K/AKT regulation of NF-kappaB signaling events in suppression of TNF-induced apoptosis. Biochem Biophys Res Commun 2000;271:342–345.[Medline]
  53. Perou CM, Jeffrey SS, van de Rijn M, Rees CA, Eisen MB, Ross DT, Pergamenschikov A, Williams CF, Zhu SX, Lee JCF, Lashkari D, Shalon D, Brown PO, Botstein D. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci USA 1999;96:9212–9217.[Abstract/Free Full Text]



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