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Clinical Cancer Research Vol. 10, 7972-7977, December 1, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

c-erbB-2 Related Aggressiveness in Breast Cancer Is Hypoxia Inducible Factor-1{alpha} Dependent

Alexandra Giatromanolaki1, Michael I. Koukourakis2, Costantinos Simopoulos3, Alexandros Polychronidis3, Kevin C. Gatter4, Adrian L. Harris5 and Efthimios Sivridis1

Departments of 1 Pathology, 2 Radiotherapy/Oncology, and 3 Surgery, Medical School, Democritus University of Thrace, Alexandroupolis, Greece; and 4 Nuffield Department of Clinical Laboratory Sciences and 5 Cancer Research United Kingdom, Molecular Oncology Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Assessment of Antigen...
 RESULTS
 DISCUSSION
 REFERENCES
 
c-erbB-2–positive breast carcinomas are highly aggressive tumors. In vitro data on breast cell lines showed that c-erbB-2 enhanced translational efficiency of hypoxia inducible factor-1{alpha} (HIF1{alpha}) production (Laughner et al., Mol Cell Biol 2001;21:3995–4005). We investigated the clinical correlate of this observation to assess whether c-erbB-2 expression was related to HIF1{alpha} expression, angiogenesis, and prognosis. A series of 180 breast carcinomas of known c-erbB-2 status (90 c-erbB-2–positive and 90 c-erbB-2–negative carcinomas) were stained immunohistochemically for HIF1{alpha} and CD31 endothelial cell antigen. c-erbB-2 positivity was clearly related to HIF1{alpha} protein expression and high angiogenesis. However, prognosis was decreased only in cases with simultaneous c-erbB-2 and HIF1{alpha} expression. If activation of c-erbB-2 in humans results in overexpression of HIF1{alpha} independently of conditions of hypoxia, as occur in experimental studies, this interaction may represent a main pathway conferring clinical aggressiveness to c-erbB-2–positive breast tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Assessment of Antigen...
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia inducible factor-1 (HIF1) is a key transcription factor regulating the responses of a variety of genes related to angiogenesis, erythropoiesis, glycolysis, etc (1, 2, 3) . HIF1 is a heterodimer of two basic helix-loop-helix domain proteins, the HIF1{alpha} and HIF1ß (4) . Increased intracellular content of the HIF1{alpha} occurs after hypoxic stimulation, whereas HIF1ß is present constitutively and remains, by and large, unaffected by reduced oxygen tension. Hypoxic augmentation of HIF1{alpha} protein levels is a result of reduced rates of degradation by the ubiquitin-dependent proteasome pathway rather than increased mRNA transcription or translation (5) . Nevertheless, HIF1{alpha} stabilization or even overexpression may occur by mechanisms independent of hypoxic conditions. von Hippel-Lindau protein mutations (6) , for example, and the activation of HIFs by mitogen-activated protein kinases (7) or the insulin-like growth factor-2 pathway (8) are involved in the persistent HIF induction, despite the restoration of oxygenated conditions.

The c-erbB-2 gene (also known as neu or HER-2), the second member of the c-erbB/EGFR family of transmembrane proteins with tyrosine kinase activity (9) , is also thought to participate in the regulation of HIF1{alpha} expression. Experimental evidence suggests that neutralizing antibodies against the c-erbB-2 or the EGFR result in down-regulation of angiogenesis, probably through suppression of the VEGF gene (10) . Another mechanism for growth factor receptors to modulate angiogenesis is that mediated by HER-2 regulation of HIF1 expression, with increased translational efficiency (11) . However, this work is on cell lines, and clinical relevance of this interaction has not been investigated.

Investigating the expression of these proteins in human tumors is important for they may participate in intense activation of metabolic and pathogenic pathways related to invasion, metastasis and cancer cell survival. In this study, we examined the expression patterns of HIF1{alpha} protein and tumor angiogenicity with respect to c-erbB-2 protein membrane overexpression, providing evidence in support of a close coactivation of these pathways in breast carcinomas.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Assessment of Antigen...
 RESULTS
 DISCUSSION
 REFERENCES
 
A series of 180 infiltrating ductal carcinomas of breast, not otherwise specified, and of known c-erbB-2 status were retrieved from the files of the Department of Pathology, Medical School, Democritus University of Thrace. These were 90 sequential c-erbB-2–positive carcinomas of high reactivity and 90 sequential c-erbB-2–negative tumors. The immunohistochemical criteria for c-erbB-2–positive tumor selection are referred to below. The tissues were all from specimens obtained at operation and had been routinely fixed in 10% formol-saline; they were stained immunohistochemically for HIF1{alpha} and CD31 endothelial cell antigen. The results were interrelated with c-erbB-2 and correlated with prognosis. Survival data were available in 146 of 180 patients with a minimum follow up period of 12 months.

Immunohistochemistry.
Sections were cut at 3 µm and stained immunohistochemically with the following techniques: (a) a standard streptavidin-biotin method for the detection of HIF1{alpha}, c-erbB-2, and estrogen receptor, progesterone receptor; refs. 12, 13, 14 ), and (b) the alkaline phosphatase/antialkaline phosphatase method for microvessel staining (15) . Details of the primary antibodies, the working dilutions, and the antigen retrieval methods used are given in Table 1Citation .


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  		Table 1 Details of the primary antibodies, dilutions, and antigen retrieval methods used

 
Known positive controls were included in each staining run. Omission of the primary antibody and substitution by nonspecific immunoglobulin at the same concentration were used as negative controls.

The Streptavidin-Biotin Method.
Sections were dewaxed, and endogenous peroxidase activity was quenched with methanol and 3% H2O2 for 15 minutes. Antigen retrieval was achieved by microwave treatment (three treatments for 4 minutes each). The primary antibody (1:20) was applied for 75 minutes at room temperature or overnight at 4°C (see Table 1Citation ). After washing with Tris buffered saline (TBS), sections were incubated with a secondary antirabbit antimouse antibody (Kwik biotinylated secondary; Shandon-Upshaw, Pittsburgh, PA) for 15 minutes and washed in TBS. The Kwik Streptavidin peroxidase reagent (Shandon-Upshaw) was applied for 15 minutes, and sections were again washed in TBS. The color was developed by a 15-minute incubation with 3,3'-diaminobenzidine solution, and the sections were weakly counterstained with hematoxylin.

The Alkaline Phosphatase/Antialkaline Phosphatase Method.
The JC70 monoclonal antibody (Dako, Glostrup, Denmark), which recognizes the CD31 pan endothelial antigen (platelet/endothelial cell adhesion molecule-1), and the alkaline phosphatase/antialkaline phosphatase procedure were used for blood vessel staining. Sections were dewaxed, rehydrated, and predigested with protease type XXIV (Sigma Chemical Co., St. Louis, MO) for 20 minutes at 37°C. The JC70 (1:20 dilution) was applied at room temperature for 30 minutes and washed in TBS. Rabbit antimouse antibody 1:50 (v/v) was applied for 30 minutes, followed by application of mouse alkaline phosphatase/antialkaline phosphatase complex 1:1 (v/v) for 30 minutes (Dako). After washing in TBS, the last two steps were repeated for 10 minutes each. The color was developed by incubation with New Fuchsin solution for 20 minutes.


   Assessment of Antigen Reactivity.
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Assessment of Antigen...
RESULTS
 DISCUSSION
 REFERENCES
 
HIF1{alpha}.
The HIF1{alpha} expression is purely cytoplasmic or mixed cytoplasmic and nuclear. Nuclear reactivity was scored as present (positive) or absent (negative). Cytoplasmic reactivity, when present (positive) was scored as weak and strong. The percentage of tumor cells showing nuclear or strong cytoplasmic HIF{alpha} reactivity was recorded semi-quantitatively at x200 magnification after examining the entire histologic section. Table 2Citation indicates the HIF grading system, as proposed in previous studies (12 , 16) . Cases lacking HIF1{alpha} reactivity (negative) were grouped in the same category with those having weak cytoplasmic reactivity.


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  		Table 2 HIF grading system based on the intensity and extent of cytoplasmic/nuclear cancer cell staining

 
Vascular Density.
Sections were first scanned at low power, and three areas of high vascular density ("hot spots") were chosen. Blood vessel counting was done at x200 magnification. The final vessel density was the mean score of the three vessel counts obtained. Only blood vessels with a clearly defined lumen or a well defined linear vessel shape, but not single endothelial cells, were taken into account for microvessel counting. The median vascular density (VD) was used to define groups of low and high VD.

c-erbB-2.
The percentage of tumor cells with a distinct membrane staining was recorded in all optical fields, and the mean value was calculated. Tissue samples with a mean value ≥20% were considered as positive for c-erbB-2 protein expression and were classed into three grades: tumors of low c-erbB-2 reactivity (20–35% positive cells); tumors of intermediate c-erbB-2 reactivity (36–50% positive cells); and tumors of high c-erbB-2 reactivity (>50% positive cells; ref. 17 ). For the purpose of this study, only tumors with a high c-erbB-2 reactivity were included in the positive group; these criteria define a subgroup of breast cancer patients having increased risk of recurrence and decreased overall survival (18) .

Estrogen receptor, Progesterone Receptor.
Positivity was indicated as a distinct brown nuclear staining of neoplastic cells. Estrogen receptor, progesterone receptor values above 10% were considered as positive (12) .

Lactate Dehydrogenase-5.
To confirm the functionality of HIF1{alpha}, the expression of lactate dehydrogenase-5 (LDH5) was assessed in 30 cases with high and 30 cases with low HIF1{alpha} expression, as described previously (19 , 20) . LDH5 is transcriptionally regulated by HIF1{alpha} (21 , 22) . The sheep polycloncal ab9002 (Abcam, Cambridge, United Kingdom) raised against human LDH5 purified from human placenta was used for immunohistochemistry (23) . Ab9002 is an IgG fraction, the identity of which was confirmed by double diffusion against purified LDH5 and a known antihuman LDH5. Specificity has been done by Western blot against liver cell lysate. The cytoplasmic and/or nuclear expression of LDH5 was assessed in all fields, and the median percentage of positive cells was taken into account to define two groups of low and high LDH5 reactivity. Details on scoring have been reported previously (19) .

Statistical Analysis.
We did statistical analysis using the GraphPad Prism 2.01 package (GraphPad Software Inc., San Diego, CA). A Fisher’s exact test or unpaired two-tailed t test was used for testing relationships between noncontinuous categorical (contingency tables) and continuous categorical (comparison of the mean values from two sets of data) tumor variables, respectively. We plotted survival curves using the method of Kaplan and Meier, and the log-rank test was used to determine statistical differences between life tables. The end points were the overall survival from the day of surgery. A Cox proportional hazard model was used to assess the effect of tumor variables on overall survival. A P value of <0.05 was used for significance.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Assessment of Antigen...
 RESULTS
DISCUSSION
 REFERENCES
 
A high HIF1{alpha} reactivity, as defined in Table 2Citation , was detected in 89 of the 180 breast carcinomas studied; the HIF-1{alpha} expression was nuclear/cytoplasmic, or nuclear and cytoplasmic (Fig. 1)Citation . The functionality of HIF1{alpha} was confirmed by correlating the HIF1{alpha} with LDH5 expression in parallel tumor sections. Of 30 cases with HIF1{alpha} expression, strong LDH5 cancer cell reactivity was noted in 26 cases (86.6%). On the contrary only 9 of 30 (30%) cases with low HIF1{alpha} reactivity were reactive for LDH5 (P < 0.001; Fisher’s exact test).



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  		Fig. 1. Breast carcinoma showing expression of HIF1{alpha}. Note the intense nuclear and cytoplasmic reactivity in cancer cells.

 
The vascular density of the tumors ranged from 8 to 65 capillaries, with a median value of 30. Using this cutoff point, we considered 85 of 180 cases to bear high VD. By design, 90 of the 180 infiltrating ductal breast carcinomas were c-erbB-2 membrane-positive tumors of high reactivity (>50% of the tumor cells showing a strong membrane staining) and 90 ductal breast carcinomas were c-erbB-2 negative.

The HIF1{alpha} expression in breast carcinomas was associated with an increased incidence of multiple (>4) lymph node metastases but not with the size of the primary tumor or the histologic grade (Table 3)Citation . There was also a strong association of HIF1{alpha} with c-erbB-2 and increased vascular density (Table 4)Citation . An inverse association between HIF1{alpha} and c-erbB-2 with estrogen receptor was also noted.


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  		Table 3 Association of HIF1{alpha} and of c-erbB-2 with histological variables

 

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  		Table 4 Association of HIF1a and of c-erbB-2 with the molecular variables examined

 
Table 5Citation shows the results of univariate survival analysis. A high HIF1{alpha} or high c-erbB-2 reactivity was associated significantly with poorer prognosis (Fig. 2A and B)Citation . However, double stratification analysis, according to HIF1{alpha} and c-erbB-2 reactivity, showed that only the simultaneous overexpression of these factors was associated with poor prognosis (Fig. 2C)Citation .


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  		Table 5 Univariate and multivariate analysis of local relapse: histological and molecular variables

 


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  		Fig. 2. Kaplan-Meier curves of overall survival stratified for HIF1{alpha} expression (Fig. 2A)Citation , c-erbB-2 expression (Fig. 2B)Citation and for combined c-erbB-2 and HIF1{alpha} expression (Fig. 2C)Citation .

 
In multivariate analysis, vascular density was the only variable examined that had an independent prognostic significance. HIF1{alpha} and c-erbB-2 did not show an independent significance, probably because of their close correlation. However, when the HIF-1{alpha}/c-erbB-2 status was taken as a single variable in a multivariate model, the combined variable was an independent prognostic factor (Table 5)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Assessment of Antigen...
 RESULTS
 DISCUSSION
REFERENCES
 
There are multiple mechanisms by which c-erbB-2 confers an unfavorable prognosis to breast cancer patients, including disruption of tumor cell adhesion, increasing cell mobility (24 , 25) , as well as enhancing proliferation, protecting from apoptosis, and conferring hormone-independent activation of the estrogen receptor (18) . Recent experimental evidence indicates that the HIF1{alpha} protein and the downstream molecular cascade were activated in MCF-7 breast cancer cells, following c-erbB-2 stimulation by its ligands (11) . This effect was the net result of increased HIF1{alpha} synthesis; the half-life of HIF1{alpha} protein remained unaffected. Furthermore, the increased HIF1{alpha} transcription rate was dependent on the activity of phosphatidylinositol 3-kinase, AKT (protein kinase B), and its effector FKBP-rapamycin–associated protein (FRAP; ref. 11 ). Zhong et al. (26) showed in human prostate cancer cells that basal-, growth factor-, and mitogen-induced transcription of HIF1{alpha} was blocked by LY294002 and rapamycin, inhibitors of phosphatidylinositol 3-kinase and FRAP, respectively. In this context, it is most interesting that treatment of two breast cancer patients with Herceptin, a monoclonal antibody blocking the membrane expression c-erbB-2, resulted in disappearance of the HIF1{alpha} immunohistochemical staining (27) .

It seems, therefore, that HIF1{alpha} overexpression in c-erbB-2–positive human breast carcinomas may result from tyrosine kinase receptor activation, independently of intratumoral hypoxic conditions. This finding suggests that the unfavorable prognosis of breast carcinomas with c-erbB-2 amplification and overexpression may not be attributed simply to activated proliferation and migration pathways (24 , 25) , but other pathways, mediated through the activation of a down-stream HIF1{alpha} transcription of proteins related to angiogenesis, glycolysis, and inhibition of apoptosis, may be of equal importance (28) . It is notable that in our study, both HIF1{alpha} and c-erbB-2 overexpression were associated with an intensified intratumoral angiogenesis.

The direct association of c-erbB-2 expression with HIF1{alpha} reactivity has been raised in earlier clinicopathologic studies in breast and lung carcinomas (29 , 30) . A more recent study by Bos et al. (31) confirmed this association in breast carcinomas. c-erbB-2 is a marker of decreased overall survival (18) and an indicator of resistance of breast carcinomas to chemotherapy, antiestrogen treatment, and radiotherapy (32 , 33) . HIF1{alpha} is another marker linked with poor survival in breast cancer patients (31 , 34) . In our study, both c-erbB-2 and HIF1{alpha} were associated with unfavorable prognosis. Combined analysis, however, revealed that the poor survival was only for tumors exhibiting c-erbB-2 and HIF1{alpha} reactivity simultaneously. Although these findings should be confirmed in other series of patients, current evidence suggests that c-erbB-2–mediated tumor aggressiveness in breast cancer should be attributed partly to HIF1{alpha} activation. The coactivation of angiogenesis and migration pathways in the HIF1{alpha}(+)/c-erbB-2(+) group of patients may be the reason for the ominous prognosis of this group. High angiogenesis in absence of cancer cell migration ability (i.e., c-erbB-2 negativity) or, high migration ability in lack of intense angiogenic potential may result in reduced metastatic or impaired installation ability of cancer cells in distant organs, respectively. Indeed, in the present study, cases with highVD/c-erbB-2(+) phenotype had a significantly poorer survival than highVD/c-erbB-2(–) and lowVD/c-erbB-2(+) ones (data not shown). The intense anaerobic metabolism and intratumoral acidity, predicted by the HIF1{alpha} and LDH5 up-regulation, or the resistance of HIF1{alpha}+ cancer cells to apoptotic stimuli may also account for the poorer survival in the HIF1{alpha}(+)/c-erbB-2(+) group (35 , 36) .

As several inhibitors of the HIF1{alpha} are in the preclinical and clinical stage of development (37) , the coactivation and cooperation of c-erbB-2 and HIF1{alpha} in the development of a clinically aggressive tumor phenotype underlines the eventual therapeutic benefit from a double blocking of this pathway.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Alexandra Giatromanolaki, P.O. Box 12, Alexandroupolis 68100, Greece. Phone: 0030-25510-75118; Fax: 0030-25510-30349; E-mail: targ{at}her.forthnet.g

Received 6/ 1/04; revised 8/16/04; accepted 9/ 2/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Assessment of Antigen...
 RESULTS
 DISCUSSION
 REFERENCES
 

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C. Spangenberg, E. U. Lausch, T. M. Trost, D. Prawitt, A. May, R. Keppler, S. A. Fees, D. Reutzel, C. Bell, S. Schmitt, et al.
ERBB2-Mediated Transcriptional Up-regulation of the {alpha}5{beta}1 Integrin Fibronectin Receptor Promotes Tumor Cell Survival Under Adverse Conditions.
Cancer Res., April 1, 2006; 66(7): 3715 - 3725.
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