scatter factor expression and c-met in primary breast cancer

Surgical Oncology 1996; 5: 15-21

Hepatocyte growth factor/scatter factor expression and in primary breast cancer

c-met

J. NAGY, G. W. CURRY,* K. J. HILLAN,*t I. C. McKAY,:!: E. MALLON,* A. D. PURUSHOTHAM AND W. D. GEORGE University Department of Surgery, *Pathology and xlmmunotoqv. Western Infirmary, Dumbarton Road, Glasgow, G11 6NT, UK

Hepatocyte growth factor/scatter factor (HGF/SF) is a fibroblast-derived cytokine whose receptor is encoded by comet. Activation of comet promotes tumour cell proliferation, dissociation, invasiveness and angiogenesis. Aberrant expression of HGF/SF or comet may playa role in tumour progression. HGF/SF and comet were determined in 73 breast cancers (median follow up: 61 months) and 10 samples of tumour-free breast tissue. HGF/SF was detected at significantly higher concentrations in breast cancers (median 350, range 58-1604 ng per 100 mg total protein) when compared with normal breast tissue (median 108, range 66-213 ng per 100 mg total protein) (P<0.001). Comet was detected in all 10 samples of tumour-free breast tissue and in 26 breast cancers. HGF/SF concentrations correlated with disease relapse (P < 0.001) and reduced overall survival (P < 0.001). Tumours with detectable comet correlated significantly with disease-relapse (P = 0.012). Multivariate analysis demonstrated a significant interaction between HGF/SF and comet in relation to disease-relapse (P = 0.014). These results suggest a biological interaction involving HGF/SF and comet in promoting tumour progression in breast cancer. Surgical Oncology 1996: 5: 15-21. Keywords: breast cancer, comet, HGF/SF, metastasis, prognosis, proto-oncogene.

HGF/SF promotes mitogenesis, dissociation, motility and invasiveness in epithelial cells, and angiogenesis in endothelial cells [4, 13, 14]. These pleiotropic effects have been shown to be mediated through the c-met receptor [13,15, 16]. C-met was originally described as an activated oncogene in a chemically transformed human osteosarcoma cell line [17]. Overexpression of c-met has been reported in colorectal, gastric, thyroid, pancreatic and ovarian cancers [18-22]. In gastric and thyroid cancer such overexpression correlates with adverse prognostic factors. Cells co-expressing c-met and HGF/SF have been shown to induce tumours in mice, suggesting an autocrine transformation mechanism [23]. HGF/SF has been shown to accumulate in the stromal components of malignant tissue, and the expression of HGF/SF has been shown to be significantly higher in breast cancers when compared with normal breast tissue [24, 25]. The biological effects of HGF/SF, via its receptor c-met, suggest a possible role in carcinogenesis, tumour invasion and metastasis.

INTRODUCTION

Hepatocyte growth factor (HGF) was first identified as a mitogen for hepatocytes and scatter factor (SF) because of its ability to dissociate breast epithelial cells in culture [1, 2]. HGF and SF have since been shown to be identical. HGF/SF is a paracrine factor produced by cells of mesenchymal origin which acts on both epithelial and endothelial cell types [3-7J. The HGF/SF receptor, a 190 kDa heterodimeric protein, is encoded by the c-met protooncogene. The 50 kDa «-chaln. exposed at the extracellular surface of the plasma membrane, is disulphide linked to the transmembrane 145 kDa {3-chain that contains a tyrosine kinase domain [8-11]. It is expressed in a wide range of human epithelial cells and by vascular endothelium [12, 13]. In vitro and in vivo studies have shown that Correspondence: J. Nagy, University Department of Surgery, Western Infirmary, Dumbarton Road, Glasgow G11 6NT, UK. tCurrent address: Genentech lnc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA. © 1996 Blackwell Science Ltd

15

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J. Nagy et al.

Expression of HGF/SF and its c-met receptor have been reported in normal and malignant breast epithelium [24, 26, 27]. Furthermore, HGF/SF levels in breast cancer homogenates in vivo were found to correlate significantly with disease relapse and reduced survival [28]. The aims of this study were to determine HGF/SF levels and c-met in normal breast tissue and breast cancers and to determine possible correlations with c1inico-pathological variables, disease relapse and overall survival.

MATERIALS AND METHODS Patients Frozen tumour specimens from 73 patients with primary breast cancer were studied (median followup: 61 months). Local recurrence, systemic recurrence and overall survival were documented. The c1inico-pathological prognostic variables studied were: age, tumour size, grade, vascular invasion, axillary nodal and oestrogen receptor status. Ten samples of histologically normal breast tissue were also studied (nine samples of tumour-free breast tissue obtained at mastectomy and one sample of breast tissue from a reduction mammoplasty). Cell line The HT29 colonic carcinoma cell line was used as a positive and quality control for the detection of c-met protein [9]. Cells were grown at 3JOC in RPMI 1640 medium supplemented with 10% foetal calf serum in a 5% CO2-water saturated atmosphere until confluent. Protein extraction, electrophoresis and c-met immunostaining C-met protein was detected by Western blotting according to the method of Rygaard et al. [29]. Briefly, tissue samples and cells were homogenized in lysis buffer and centrifuged for 15 min at 12000 g. The protein concentration of the homogenate was determined using a bicinchoninic acid assay (Pierce, IL). Samples containing 100 pg total protein were reduced by boiling for 5 min in sample buffer containing mercaptoethanol. Samples were then electrophoresed in 7.5% SDS

polyacrylamide gels. Molecular weight markers in the 27-180 kDa range (Sigma, Dorset, UK) and HT 29 protein were co-electrophoresed. Separated proteins were then transferred onto nitrocellulose membranes (Millipore, Bedford, UK) by electroblotting (Bio-Rad, Hertfordshire, UK). The c-met protein was detected using the 19S murine monoclonal antibody which binds to the p-chain (donated by Dr G. F. Vande Woude, NCI, Frederick, USA) [30]. A secondary alkaline phosphatase conjugated rabbit anti-mouse antibody (Dako, Glostrup, Denmark) was added, and bound antibody was visualized using nitro-blue tetrazolium and 5-bromo-4-chloro3-indolyl phosphate. Controls included incubation with primary antibody pre-incubated with its competing protein pMet 50 and incubation without primary antibody [30]. The efficacy of the method was confirmed in each experiment by an HT 29 positive control. The blots were visually inspected by an independent observer in order to assess the presence or absence of c-met protein [12]. Assay for HGF/SF Tissue levels of immunoreactive HGF/SF were determined in tumour homogenates using a sandwich-type ELISA assay. Ninety-six-well microtitre plates were coated with the murine a-chain HGF-specific monoclonal antibody A 3.1.2 (Genentech Inc., San Francisco, CAl. Standard concentrations of human recombinant HGF/SF or homogenate were then added, followed by polyclonal sheep anti-HGF (Genentech Inc., San Francisco, CAl. Biontinylated donkey anti-sheep antibody (ICN Biomedicals, Thame, UK) was then added followed by avidin-horseradish peroxidase conjugate, 0.25% O-phenylenediamine and the reaction stopped by the addition of H2S04 , Absorbance was read at 492 nm by an automatic plate reader. HGF/SF concentrations were then calculated from a curve derived from HGF standards. The lower detection limit of this assay was 0.25 ng mr' (intra-ELISA variation: 1-5%, mean 2%). Tumour samples were analysed in duplicate or triplicate. The results for homogenates were converted into ng per 100 mg total protein. Statistical analysis The relationship between prognostic factors and disease-relapse or overall survival were tested by ©1996 Blackwell Science Ltd, Surgical Oncology, 5: 15-21

HGF/SF and c-met in primary breast cancer Table 1. Analysis of the relationships between HGF/SF, c-met and c1inico-pathological variables with disease relapse and overall survival

Variable HGF/SF c-met Lymph node status Tumour size (mm) Histological grade Oestrogen receptors Age Vascular invasion

Disease relapse

Overall survival

(P)

(P)

<0.001* 0.D12* 0.013* 0.027* 0.369 0.262 0.318 0.427

<0.001* 0.08 0.024* 0.115 0.205 0.283 0.588 0.472

Table 2. Clinico-pathological prognostic variables of 73 patients with breast cancers

Prognostic variables Age (years) <55 255 Tumour size (mm) <25 225 Lymph node status" Positive Negative Tumour gradet I

*Statistically significant.

II

III

Mann-Whitney or Kruskal-Wallis tests for categorical prognostic factors, or by Spearman's rank correlation for continuously variable prognostic factors (Table 1). The multivariate relationships between prognostic factors and disease relapse were tested by analysis of variance and covariance.

17

Oestrogen receptor statust Positive Negative Vascular invasion Present Absent

n 28 45 26 47 36 33 8 31 27 29 32 21 52

*Lymph node status unknown in 4 patients. Humours not graded (4 lobular, 2 mucoid, 1 medullary). :t:Oestrogen receptor status unknown in 12 patients.

RESULTS Patient data

Clinico-pathological prognostic variables from 73 patients with breast cancer are summarized in Table 2. The median follow-up was 61 months (range 7-89 months). Local recurrence was observed in three patients, local and systemic recurrence in nine patients and systemic recurrence in 16 patients. All patients with systemic recurrence died from metastatic disease. Positive nodal status correlated with disease relapse (P = 0.013) and reduced overall survival (P = 0.024, Table 1). Tumour size correlated with disease relapse (P = 0.027, Table 1). The remaining prognostic clinico-pathological variables did not correlate with either disease relapse or overall survival (Table 1). HGF/SF levels in breast cancer and tumour-free breast tissue

The concentration of HGF/SF was significantly higher in breast cancers (range 58-1604 ng per 100 mg total protein; mean 396 ng per 100 mg total protein) than in tumour-free breast tissue (range: 66-200 ng per 100 mg total protein; mean: 130 ng ©1996 Blackwell Science Ltd, Surgical Oncology, 5:

15~21

per 100 mg total protein, P < 0.001, Figure 1). The concentration of HGF/SF in breast cancers correlated with disease relapse (P < 0.001) and reduced overall survival (P <0.001). There were no significant correlations between HGF/SF concentrations and clinico-pathological prognostic variables (Table 3). C-met expression in breast cancer and tumourfree breast tissue Two protein bands of 145 kDa (corresponding to the f3 chain of the comet receptor, p145 Met) and 170 kDa (corresponding to the precursor of p145 Met) were detected (Figs 2 and 3). These protein bands were not detected in the absence of primary antibody or when primary antibody was pre-absorbed with pMet 50, confirming the specificity of the antibody. C-met protein was detected in all 10 samples of tumour-free breast tissue (Fig. 2). C-met protein was detected in 26 (36%) breast cancers, whereas in the remaining 47 (64%) breast cancers it was either absent or barely detectable (Fig. 3). Breast cancers demonstrating detectable c-met protein demonstrated a significant association with

J. Nagy et al.

18

2000

•

c

Q)

'0

a.

disease relapse (P = 0.012, Table 1). There was no significant correlation between c-met and clinicopathological prognostic variables (Table 3).

1500

Relationship between HGF/SF, c-met, prognostic variables and disease relapse

eli

2

01

E ,... ..... 0 0

I

1QOO

••

•••

01 C

U. (/)

u:: oI

t

500

oBo

0600

0

Breast Cancer

Tumour-free Breast tissue

Figure 1. Comparison between HGF/SF levels in tumourfree breast tissue and breast cancers.

A multivariate analysis of variance and covariance was performed to investigate the relationship between HGF/SF, c-met and clinico-pathological prognostic factors with respect to disease relapse. A significant statistical interaction was observed between HGF/SF and c-met in their relationship with disease relapse (P = 0.014). Furthermore, the observed correlation between c-met and disease relapse (Table 1) became insignificant in this analysis, the effect of c-met being attributed to its interaction with HGF/SF. This suggests the possiblity of a biological interaction between HGF/SF and c-met, resulting in disease progression.

DISCUSSION Table 3. Analysis of the relationship of HGF/SF and c-met with c1inico-pathological prognostic variables Variables Lymph node status Tumour size (mm) Histological grade Oestrogen receptors Age Vascular invasion

170

kD~

145

kD~

HGF/SF

c-met

(P)

(P)

0.99 0.56 0.44 0.87 0.54 0.53

0.14 0.89 0.76 0.86 0.52 0.057

t

In this study we have demonstrated that HGF/SF levels are significantly higher in breast cancer than in normal breast tissue. High levels of HGF/SF correlated with disease relapse and reduced survival. In addition, breast cancers with detectable c-met demonstrated a significant correlation with disease relapse. Multivariate analysis demonstrated that HGF/SF was an independent prognostic factor in this study. Furthermore, a statistically significant

t

t

Figure 2. Representative Western blot of c-rnet protein in tumour-free breast tissue. Lane 1: HT29 positive control. Lanes 2, 4 and 6: tumour-free breast tissue. Two c-met specific bands (p145 met and p170 met) were detected as labelled. Lanes 3 and 5 were blank. ©1996 Blackwell Science Ltd, Surgical Oncology, 5: 15-21

HGF/SF and c-met in primary breast cancer

19

170 kD~ 145

kD~

t

t

Figure 3. Representative Western blot of c-met prote in in breast cancers. Lane 1: HT29 positive contro l. Lanes 2-7:

breast cancers. Lanes 6 and 7: breast cancers w ith detectable c-met protein. Lanes 2, 3, 4 and 5: breast cancers with absent or barely detectable c-rnet protein. Two c-rnet specific bands (p145 met and p170 met) were detected as labelled .

interaction between HGF/SF and c-met was demonstrated in relation to disease relapse. Factors that regulate cell dissociation, motility, invasiveness and angiogenesis are fundamental to understanding processes that underlie tumour invasion and metastasis. The pleiotropic effects of comet activation by HGF/SF suggest a link with tumour progression . Furthermore, HGF/SF has been uniquely shown to promote mammary epithelial cell growth and invasion in the process of branching morphogenesis in vitro [31, 32). The results of this study confirm those of Yamashita et el., and provide strong evidence for a link between HGF/SF and tumour progression [28). Although Yamashita et al. demonstrated a significant correlation between high HGF/SF levels and tumours > 5 cm in diameter, HGF/SF levels were independent of other prognostic variables in both studies. This suggests that HGF/SF levels in breast cancer may serve as an independent marker of prognosis: however, the pathological process underlying this requ ires further investigation. The expression of c-met in normal breast tissue and breast cancer has been the subject of controversy . The initial studies conducted by Oi Renzo et al. and Prat et al. which reported conflicting results. were based on relatively small samples [12, 33). In a study of 50 breast cancers, Tsarfaty et al . reported reduced c-met immunostaining in breast cancer epithelial cells when compared with adjacent tumour-free breast epithe lium [26]. Similarly, in this study c-met was detected in all samples of tumour-free breast tissue but not in the majority of breast cancers. However, a proportion of breast cancers demonstrated detectable c-met © 1996 Blackwell Science Ltd, Surgical Oncology, 5: 15-21

protein and this group demonstrated a significant correlation with disease relapse. In vitro studies suggest that c-met may be expressed at low levels in well -differentiated breast cancer cell lines and at higher levels in normal and poorly-differentiated cell lines [27). The results of this study did not demonstrate a relationship between c-met and tumour grade. Further studies measuring the levels of c-met expression are therefore necessary. Although Tsarfaty et al. reported reduced c-met immunostaining in malignant breast epithelial cells, we determined c-met in breast cancer homogenates. We are therefore unable to comment on the contribution of various cell types to the expression of c-met in breast cancer. C-met is expressed by both epithelial and endothelial cells. Therefore, the detectable c-met in the breast cancers observed in this study may be a function of angiogenesis (endothelial cell expression) rather than tumour cell expression [13). The express ion of c-met in breast cancer is unique when compared to other epithelial malignancies such as gastric, colorectal, pancreatic, ovarian and thyroid cancers where overexpression is observed. It has been suggested that the reduced c-met expression observed in breast cancer may be related to loss of heterozygosity (LOH) of this proto-oncogene [26). However, the frequency of LOH of c-met in breast cancer as reported by Bieche et al. has not been confirmed by other studies [34-36). The mechanisms that regulate c-met expression in vivo are ill-understood. In vitro studies suggest that inflammatory cytok ines such as interleukin (lL)10:, IL-6, tumour necrosis factor-s and transforming growth factor-s (TGF-fJ) may up-regulate c-met expression in breast cancer [37).

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J. Nagy et al.

The cytokine TGF-{J has recently been identified as a potent inhibitor of HGF/SF expression

in vitro

and may therefore playa central role in the regulation of these factors [38). It is interesting to note that reduced TGF-{J levels in breast cancer correlate

with

positive

nodal

status

and

disease

relapse [39). Whether TGF-{J is linked to HGF/SF and

c-met expression in breast cancer remains

unknown. Multivariate analysis demonstrated a statistical

c-met In vitro studies

interaction between HGF/SF expression and in relation to suggest that

disease relapse. HGF/SF

is

expressed

mesenchymal cells, whereas

mainly

by

c-met is expressed in

epithelial and endothelial cells [3,4,13,33,40,41).

In situ hybridization studies in mice support these observations [7). Furthermore, breast cancer cells

in vitro do not express HGF/SF [3, 24, 41, 42). These findings strongly suggest that HGF/SF and

c-met may interact in a paracrine or stromalepithelial fashion. However, the co-expression of HGF/SF and has been

c-met in human breast cancer cells

reported and

therefore an autocrine

mechanism of interaction cannot be excluded [43). In conclusion, HGF/SF expression is significantly increased in breast cancers and this increased expression correlates with disease relapse and reduced

survival.

Furthermore,

between HGF/SF and

an

interaction

c-met is observed in relation

to disease relapse.

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