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Annals of Clinical & Laboratory Science 32:12-21 (2002)
© 2002 Association of Clinical Scientists

HER-2/neu-Positive Breast Carcinoma: Molecular Concomitants by Proteomic Analysis and their Therapeutic Implications

Robert E. Brown
Division of Laboratory Medicine, Geisinger Medical Center, Danville, Pennsylvania

Address correspondence to Robert E. Brown, M.D., Division of Laboratory Medicine, Geisinger Medical Center, Danville, PA 19822-0131, 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
 
Objective: To identify molecular events that occur concomitantly in HER-2/neu protein-receptor-positive breast carcinoma and to identify pathogenetic and growth-modulating sequences around its tyrosine-kinase-mediated cell proliferation and tumorigenesis that may be amenable to therapeutic interventions. Methods: Slides containing sections of 3 pelleted human breast carcinoma cell lines (DAKO HercepTest) and expressing HER-2/neu protein-receptor scored at 3+ (SKBR-3), 1+ (MDA-175), and 0 (MDA-231), respectively, were reacted in immunohistochemical procedures for the detection of the following antigens: HER-2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, cyclin D1, c-Jun, epidermal growth factor receptor (EGFR), transforming growth factor (TGF)–{alpha}, components of the JAK/STAT signal transduction pathway (gp130, interleukin [IL]–6, and IL-11), p21ras, farnesyl transferase (FT), and potential growth inhibitory/proapoptotic and antiapoptotic-related proteins (latency-associated peptide [LAP] of TGF-ß1, TGF-ß receptor [R] II, p53, and bcl-2 and cyclooxygenase [COX]-2). Immunoreactivities were graded using bright-field microscopy on a scale of 0 to 3+. Results: Commonalities noted among the 3 cell lines include absent (0) chromogenic signals for ER and PR, relatively high Ki-67 proliferation indices (54, 40, and 61%, respectively), and positive signals (1 to 3+) for IL-6, IL-11, TGF-{alpha}, EGFR, TGF-ß1 (LAP), TGF-ßRII,FT, p21ras, and p53. Strong intranuclear immunopositivities for cyclin D1 and c-Jun antigens were evident in the MDA-231 cell line but absent or rare (0 to ±) in SKBR-3. Conversely, gp130 antigen was readily detected in the SKBR-3 cell line but only weakly expressed (±) in MDA-231, whereas bcl-2 and COX-2 were expressed in the latter and not in SKBR-3 cells. Conclusions: These data suggest that signal transduction through farnesylated p21ras is part of the pathogenesis of tyrosine-kinase-mediated proliferation in HER-2/neu protein-receptor-positive breast carcinoma. Collaborations with the EGFR and JAK/STAT systems in these molecular events are also likely. Potential therapeutic agents include downregulators of c-erb-B1 (EGFR) and -B2 (HER-2) receptor expressions, inhibitors of tyrosine kinase and farnesylation, and activators of growth inhibitory/proapoptotic pathways.

(received 3 August 2001; accepted 21 September 2001)

Keywords: HER-2/neu, breast carcinoma, carcinogenesis, molecular pathways


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Amplification of the human epidermal growth factor receptor (HER)-2/neu oncogene occurs in approximately 15 to 30% of invasive breast carcinomas [1–3]. This results in the overexpression of the corresponding protein receptor, leading to proliferative responses in the tumor cells via a tyrosine-kinase-mediated signal [4–6]. Clinically such overexpression and/or amplification portends a worse prognosis for the patients in terms of both a shorter disease-free interval and overall survival [7]. The purpose of this report is twofold: first, to identify other molecular events occurring concomitantly in such tumors that provide additional insight into the pathogenesis of their growth; and second, to create a molecular profile of HER-2/neu protein-receptor-positive breast carcinoma with clearly defined pathways that present opportunities for therapeutic intervention.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Human breast cancer cell lines.  Slides, each containing sections of 3 pelleted, formalin-fixed and paraffin-embedded human breast carcinoma cell lines–SKBR-3, MDA-175, and MDA-231, respectively, were obtained as part of the standardized DAKO HercepTest® (DAKO Corporation, Carpinteria, CA).

Immunohistochemistry.  A panel of antibodies was assembled to detect the following antigens in the aforementioned cell lines: HER-2/neu protein receptor, Ki-67, cyclin D1, c-Jun, estrogen receptor (ER), progesterone receptor (PR), epidermal growth factor receptor (EGFR), transforming growth factor (TGF)- {alpha}, gp130, interleukin (IL)-6, IL-11, p21ras, farnesyl transferase (FT), the latency-associated peptide (LAP) of TGF-ß1, TGF-ß receptor (R) II, p53, bcl-2, and cyclooxygenase (COX)-2.

Anti-HER-2/neu polyclonal antibody (code #K5205, HercepTest®) was used to confirm the cellular distribution and the immunoreactivity for HER-2/neu protein receptor expression as stated for the individual cell lines.

Mouse monoclonal anti-human Ki-67 antibody (clone MIB-1, DAKO) was used to detect the corresponding antigen, a non-histone nuclear protein associated with all active phases in the cell cycle (G1, S, G2, and M).

Mouse monoclonal anti-human cyclin D1 antibody (clone DCS-6, DAKO) was used to detect the corresponding antigen, a protein that positively regulate the cell cycle in the G1 to S phase.

Mouse monoclonal anti-c-Jun antibody with human immunoreactivity (clone 3, BD Transduction Laboratories, Becton, Dickinson and Co.) was used to assess the nuclear expression of the c-Jun antigen, a protein product of its corresponding proliferation-associated, immediate-early gene and an essential component of the activator protein transcription factor (AP-1).

Mouse monoclonal anti-human ER antibody (clone 1D5, DAKO) was used to assess the expression of the corresponding antigen, a largely intranuclear protein that mediates the action of estrogenic hormones.

Mouse monoclonal anti-human PR antibody (clone PgR636, DAKO) was used to assess the expression of the corresponding antigen, an intranuclear protein that functions as a transcription factor, mediating the effect of progestogenic hormones.

Mouse monoclonal anti-human EGFR antibody (clone 2-18C9, DAKO EGFR pharmDx) was used to detect the plasmalemmal expression of the corresponding antigen, a transmembrane protein with an extracellular domain that after interactions with EGF or TGF-{alpha} generates a tyrosine-kinase-mediated signal resulting in cell proliferation [8].

Mouse monoclonal anti-human TGF-{alpha} antibody (clone 9426.21, IgG1, R&D Systems, Inc. Minneapolis, MN) was used to detect the corresponding antigen, a protein (cytokine) with high affinity for EGFR.

Mouse monoclonal anti-human gp130 antibody (clone 28118.11, IgG1, R&D Systems) was used to detect the corresponding antigen, a transmembrane protein that is the signal-transducing subunit for IL-6 and IL-11 [9].

Mouse monoclonal anti-human IL-6 and IL-11 antibodies (clones 1936.14, IgG2b, and 22315.1, IgG2a,k, respectively, R & D Systems) were used to assess the expression of the corresponding antigens, proteins (cytokines) whose signals are mediated through gp130 and the JAK/STAT signal transduction pathway [9].

Mouse monoclonal anti-human p21ras antibody (clone NCC-RAS-001,DAKO) was used to detect the corresponding antigen, a protein encoded by the H-ras gene that functions as a guanine nucleotide-binding (G) protein involved in signal transduction.

Rabbit polyclonal anti-human FT{alpha} antibody (catalog #sc-487, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used to detect the corresponding antigen, a peptide mapping at the carboxy terminus of the 49 kD, {alpha} subunit common to farnesyl and geranylgeranyl transferases, that catalyze the prenylation and, thereby, activation of ras-related proteins.

Goat polyclonal antibody reactive with LAP of human TGF-ß1 (catalog #AB-246-NA, R&D Systems) was used in this study. This antibody against LAP has been shown to react with latent TGF-ß1 in immunohistochemical applications.

Rabbit polyclonal anti-human TGF-ßRII antibody (catalog #sc-220, Santa Cruz Biotechnology) was used to assess the expression of the corresponding antigen, a glycoprotein designed to mediate a signal from active TGF-ß.

Mouse monoclonal anti-human p53 antibody (clone BP53 12-1, BioGenex, San Ramon, CA) was used to assess the expression of the corresponding antigen, a primarily intranuclear protein that can exist in both wild-type and mutant forms, the latter reflecting a specific genetic change in malignant breast cancer.

Mouse monoclonal anti-human bcl-2 antibody (clone 124, DAKO) was used to investigate the expression of the corresponding antigen, a protein that plays a key role in the inhibition of apoptosis.

Rabbit polyclonal anti-human PGHS-2 (product #PG 27B, Oxford Biomedical Research, Inc., Oxford, MI) was used to assess the expression of the C-terminus of the COX-2 isoenzyme, a protein that is involved in the pathway leading to bcl-2 synthesis, thereby reducing apoptosis [10].

The general immunohistochemical procedure has been previously described [11]. Positive controls using established immunoreactive tissues and a negative control using the 3 cell lines provided in the HercepTest® were shown to react appropriately.

Analysis of immunostains.  The scoring of the overexpression of HER-2/neu protein receptor was carried out with criteria outlined in the standardized procedure. All other immunoreactivities in the 3 cell lines were scored from 0 (negative) to 3+ positivity using bright-field microscopy.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Immunoreactivities for HER-2/neu protein-receptor expression in the 3 cell lines were confirmed at 3+ for SKBR-3, 1+ for MDA-175, and 0 for MDA-231. Circumferential plasmalemmal positivity characterized the expression in SKBR-3 cells (Fig. 1Go, panel A).



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  		Fig. 1. SKBR-3 human breast carcinoma cell line showing: A. strong (3+), plasmalemmal immunoreactivity for HER-2/neu (c-erbB-2) protein; B. moderate (2+), plasmalemmal immunoreactivity for epidermal growth factor receptor (EGFR[c-erbB-1]) protein; C. moderate (2+) cytoplasmic immunoreactivity for transforming growth factor (TGF)–{alpha}, the ligand for EGFR; D. strong (3+), cytoplasmic immunoreactivity for farnesyl transferase (FT)–{alpha} protein (DAB chromogen; original magnification x788).

 
Proteomic analysis by immunohistochemistry and scoring by bright-field microscopy revealed the following commonalities among these 3 breast carcinoma cell lines: relatively high proliferation indices with 54, 40, and 61% of nuclei showing Ki-67 antigen expression; absent (0) chromogenic signals for ER and P; and positive signals (1 to 3+) for IL-6, IL-11, EGFR (Fig. 1Go, panel B), TGF-{alpha}(Fig 1Go, panel C), TGF-ß1 (LAP), TGF-ßRII, FT (Fig. 1Go, panel D), p21ras, and p53. Strong intranuclear immunopositivities for cyclin D1 and c-Jun antigens, respectively, were evident in the MDA-231 cell line but absent or rare (0 to ±) in SKBR-3. Conversely, gp130 antigen was scored at 1+ in the SKBR-3 cell line but only weakly expressed (±) in MDA-231 cells. Finally, bcl-2 and COX-2 were detected in the cytoplasm of the MDA-231 cells but absent from the SKBR-3 cell line. Individual scores for each of these protein analytes according to cell line and cellular compartment are detailed in Table 1Go.


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  		Table 1. . Assessment of potential molecular concomitants in HER-2/neu protein-receptor-positive breast carcinoma using proteomic analysis by immunohistochemistry.*
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Proteomics, as defined in this study, is the detection, visual quantification, cellular compartmentalization, and functional grouping of proteins to create a molecular profile of tumor cells. Such analysis has identified molecular concomitants in the HER-2/neu protein-receptor-overexpressing and HER-2/neu gene-amplified human breast carcinoma cell line, SKBR-3[12]. The specific proteins fall into one of the following categories: those involved in the generation of an extracellular signal and its transduction to the nucleus, thereby triggering molecular events at the genomic level including cellular proliferation; and those that are potential growth inhibitors or proapoptotic. A third category includes those proteins that can create an environment that promotes tumorigenesis.

To expand on these, the cytokines IL-6 and IL-11 and their corresponding receptors complex with a shared signal-transducing subunit, gp130, to activate the JAK-STAT pathway [9]. The binding of activated STATs to DNA gamma activated sequence (GAS) elements leads to the induction of transcription and molecular expression of certain genes. In addition, IL-6 through gp130 signaling can activate other pathways that involve downstream effector molecules, including Ras [9]. Our demonstration of the co-expression of IL-6, IL-11, and gp130 antigens in the SKBR-3 cell line suggests that these molecular pathways may also be operative in HER-2/neu protein-receptor-positive breast carcinoma. Moreover, there is evidence that IL-6 can induce tyrosine phosphorylation of c-erbB-2 (HER-2/neu) and that the latter can form a complex with the gp130 subunit of the IL-6 receptor in an IL-6 dependent manner, leading in turn to signaling through the mitogen-activated protein (MAP) kinase pathway [13]. Similarly, the coexpression of EGFR (c-erbB-1) and its ligand, TGF-{alpha}, in the SKBR-3 cell line could initiate protracted signaling in an autocrine fashion, and tumoral proliferation through the MAP or extracellular signal-regulated kinase (ERK) pathway [14–18].

An important intermediary step in this process is the activation of p21ras via farnesylation [17]; therefore, the immunoreactivity for farnesyl transferase (FT) antigen demonstrated in this study seems relevant. Furthermore, there appears to be collaboration between c-erbB-2 (HER-2/neu) and c-erbB-1 (EGFR) in promoting farnesylation, given the previous observation by Asslan and co-workers [19] that epidermal growth factor stimulates 3-hydroxy-3-methylglutaryl-coenzyme A reductase expression via the erbB-2 pathway in SKBR-3 cells (this enzyme represents an early but key step leading to the biosynthesis of farnesyl pyrophosphate, the substrate for farnesyl transferase) [20,21]. The prior demonstration of heterodimers of c-erB-1 and c-erbB-2 in the SKBR-3 cell line accords with such collaboration [22]. Finally, our finding of a relatively high proliferation (Ki-67) index at 54% [23] in the SKBR-3 cell line is consistent with the known ability of the activated ras/ERK pathway to promote DNA synthesis and cellular proliferations.

In short, proteomic analysis in this study has identified components of the JAK/STAT, EGFR, p21ras, and farnesylation pathways that provide opportunities for collaboration with overexpressed HER-2/neu protein-receptor in triggering cellular proliferation in this subset of human breast carcinomas.

As a potential counter to the relatively high cell cycle activity (Ki-67 antigen expression) in all three of these human breast carcinoma cell lines, there is the co-expression of TGF-ßRII and the latency-associated peptide of TGF-ß1. In general and following conversion from its latent to active form, TGF-ß1 can complex with TGF-ßRII to inhibit epithelial cell proliferation [10,24–27]. The exposure of the human breast carcinoma cell lines MDA-231 and SKBR-3 to TGF-ß1 effected growth inhibition in several studies [25,26]. In a related experiment, tamoxifen incubation with MDA-231 cells induced TGF-ß1 protein and was associated with a G1/G0 cell cycle blockade and with induction of apoptosis [28]. Furthermore, the demonstration by Arteaga and colleagues [29] of growth stimulation in MDA-231 cells following exposure to anti-transforming growth factor beta antibody provided evidence that cultured human breast cancer cells can utilize endogenously produced TGF-ß as an autocrine negative growth factor.

In addition to their potential roles in influencing events at the genomic level to promote or inhibit growth, respectively, IL-11 and the latency-associated peptide of TGF-ß1 could alter the extratumoral environment to promote tumorigenesis of human breast carcinoma. Specifically, IL-11 from MDA-231 cells has been implicated in the osteolytic bony metastasis of experimental animals by virtue of its ability to promote osteoclastogenesis [30,31]. In so doing, IL-11 could facilitate the growth and expansion within osseous tissues of those human breast carcinomas that produce it. The latency-associated peptide of TGF-ß1 might contribute to tumorigenesis by down-regulating host immune surveillance and, in particular, by interfering with natural killer cell and cytotoxic T-lymphocyte functions [26,32–34]. Ironically, it appears that both of these lymphocytic cell types possess an intrinsic ability to convert latent TGF-ß1 to its active form, leading to the down-regulation of immune surveillance [26,35].

By utilizing the molecular concomitants identified in this study and drawing on the observations of other investigators, one can envision a pathogenic sequence for the increased proliferative activity that is amenable to therapeutic intervention in HER-2/neu protein-receptor-positive breast carcinoma at multiple stages (Fig. 2Go). Specifically, because activation of p21ras via farnesylation is key to the downstream transduction of growth factor receptor signaling (vide supra), the use of farnesyl transferase or farnesyl diphosphate synthase inhibitors such as L739, 749, and aminobisphosphonates, respectively, may be worth considering [17,36–40].



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  		Fig. 2. Cartoon of breast carcinoma cell illustrating the findings in this study and those gleaned from the literature (*) on the HER-2/neu protein-overexpressing, and gene-amplified, SKBR-3 cell line. The molecular pathway created by functional grouping of these proteins provides for a pathogenetic sequence (red coloration) leading to growth of the tumor that involves: (1) heterodimerization of HER-2/neu (c-erbB-2) and transforming growth factor (TGF)-{alpha}-activated, epidermal growth factor receptor (EGFR[c-erbB-1]); (2) upregulation of 3-hydrox-3-methylglutaryl-coenzyme A (HMG-CoA) reductase; (3) farnesylation of p21(ras); and (4) downstream signaling through the extracellular signal-regulated kinase (ERK) culminating in nuclear DNA synthesis and cellular proliferation. Additional tumorigenic effects could include down-regulation (-) of TGF-ßRII by farnesylated p21(ras) and of natural killer (NK) cell activity by the latency-associated peptide (LAP) of TGF- ß1. Opportunities for therapeutic intervention into this pathogenetic sequence are depicted in blue coloration and include: (a) trastuzumab (anti-HER-2/neu antibody); (b) ZD 1839, an inhibitor of c-erbB-1 tyrosine kinase; (c) lovastatin-like agents to inhibit HMG-CoA; (d) aminobisphosphonates or farnesyl transferase inhibitors (eg, L739, 749) to interrupt farnesylation; (e) retinoids (such as 4-hydroxyphenyl retinamide, 4-HPR) to down-regulate c-erbB expression and to activate (+) latent TGF-b1, leading to apoptosis and enhanced NK cell activity.

 
In this regard, it is noteworthy that L739, 749 has been shown to cause tumor regressions of carcinomas in transgenic mice [41], and bisphosphonates have reduced the metastatic tumor burden of MDA-231 cells in bone in nude mice [42,43] and have been shown to have direct anti-tumor effects, causing apoptosis in human breast cancer cell lines [44]. Moreover, bisphosphonates are routinely used to treat the osteolytic lesions of metastatic breast carcinoma [45–47]. Alternatively, inhibitors of 3-hydroxy-3methylglutaryl-coenzyme A (HMG CoA) reductase such as lovastatin also might achieve the same effect by interfering in an earlier stage of farnesyl synthesis [48]. Correspondingly, the treatment of MDA-231 cells with mevinolin, an inhibitor of HMG CoA reductase, resulted in decreased DNA-synthesis and depressed their proliferation [49].

Attempting to interrupt any autocrine signaling initiated by TGF-{alpha} and p75 activations [17,50] of EGFR (c-erbB-1) and HER-2/neu (c-erbB-2), respectively, also would seem to be a logical approach. This might be achieved by the use of agents such as ZD 1839, an EGFR-tyrosine kinase inhibitor [51,52] and trastuzumab, a monoclonal antibody directed against the extracellular domain of the HER-2/neu receptor [7,53–55]. Both therapies are currently being used in clinical settings [52,55].

Finally, in order to promote apoptosis of the tumor cells and to improve host immune surveillance, one might consider using retinoids such as 4-hydroxyphenylretinamide (4-HPR) to convert the latency-associated peptide of TGF-ß1 to its active form (Fig 2Go) [56,57]. Retinoids also have been shown to down-regulate the expression of HER-2/neu protein receptor [58–60], possibly to reduce the incidence of second breast malignancies in premenopausal women [61], and to enhance natural killer cell functional activity [62].

Although seemingly paradoxic, the combined effect of c-erb B-1 (EGFR) and c-erb B-2 (HER-2/neu) would be to promote the action of retinoids through the up-regulation of retinoic acid receptor-alpha production [63]. Parenthetically, our finding of absent bcl-2 protein and COX-2 antigen expression in the SKBR-3 cell line suggests that intrinsic opposition to this apoptotic approach may be minimal [10]. To reiterate, these pathogenic, molecular pathways and opportunities for therapeutic intervention are illustrated in Fig. 2Go.

In summary, proteomic analysis by immunohistochemistry has identified molecular concomitants in HER-2/neu positive breast carcinoma that outline possible collaborations with the EGFR and JAK/STAT system and signal transduction through farnesylation of p21ras in promoting the tyrosine-kinase-mediated proliferations of such tumors.

Similar profiling of each individual patient’s tumor could identify such pathogenic pathways and thereby point to specific opportunities for customized therapeutic intervention. Reinforcement for such an approach can be found in the genomic studies of Perou and associates, who painted molecular portraits of human breast tumors using complementary DNA microarrays. Notably, they found that the tumors show great variation in their patterns of gene expression and that many different sets of genes show mainly independent patterns of variation [64]. Moreover, these investigators utilized immunohistochemical detection of proteins encoded by a particular gene in a cluster to identify the cell type involved and, thereby, to complement their work [64,65].


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The author is grateful to Glen Kauwell and Laurie Kneller of Geisinger Medical Center and Betsy Spaulding of DAKO Corp. for technical assistance in immunohistochemistry and to Tina Fahy for secretarial support. DAKO Corp. provided the HercepTest® control slides, and Dr. Weiping Jiang of R & D Systems provided the anti-TGF-{alpha} antibody used in this study.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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