Beta-hCG/LH receptor (b-HCG/LH-R) expression is increased in invasive versus preinvasive breast cancer: implications for breast carcinogenesis?

Introduction The role of the b-HCG/LH/LH-R system in breast cancer is conflicting. Whereas some reports suggest a protective effect of b-HCG on breast epithelium, vitro studies implicate a role of b-HCG/LH-R in the development and growth of breast tumors.

In order to further investigate a possible involvement of b-HCG/LH-R in breast carcinogenesis, immunofluorescence analyses of b-HCG/LH-R expression was performed on 70 preinvasive and adjacent invasive breast cancer specimen using tissue microarrays (TMAs).

In 37 preinvasive samples available for further analysis, b-HCG/LH-R was found in 8/37 samples (21.6%; weak, intermediate and strong staining in 4/37 (10.8%), 2/37 (5.4%) and 2/37 (5.4%). In contrast, b-HCG/LH-R expression was observed in 19/27 (70.4%) adjacent invasive specimen with weak, moderate and strong immunostaining in 10/27 (37.0%), 6/27 (22.2%) and 3/27 (11.1%), respectively. This was statistically significant when compared to preinvasive components (P = 0.001, Chi Square Test).

Based on the observation that b-HCG/LH-R was found to be selectively upregulated in invasive tumor components, we suggest that under certain circumstances, sensitivity of ductal cells to hormones that target b-HCG/LH-R could favour mammary carcinogenesis.

HCG receptor, Breast cancer, DCIS, Invasive

Luteinizing hormone (LH) and beta-human chorionic gonadotropin (b-HCG) are structurally related glycoprotein hormones produced by the pituary gland and placenta, respectively (Licht et al. 2001, 2003). Binding to the G-protein coupled membrane receptor (b-HCG/LH-R) confers activation of adenylyl cyclase followed by an increase in cAMP levels thereby mediating hormonal effects (Licht et al. 2003; Minegishi et al. 1997). LH and b-HCG do regulate ovarian steroidogenesis (Minegishi et al. 1997), but have also been shown to exert various effects on non-gonadal tissues including endometrium (Shemesh et al. 2001), myometrium (Lei et al. 1992) and fallopian tubes (Lei et al. 1993a, b), such as an increase in uterine blood flow (Lei et al. 1992) or promotion of decidualization of human endometrium (Han et al. 1996). Recent evidence suggests that the action of LH and b-HCG might also contribute to malignant transformation of human cells by promoting pro-mitogenic effects. For example, b-HCG/LH-R levels have been found to be significantly elevated in endometrial carcinomas (Lin et al. 1994), and may be involved in the progression of some ovarian cancers (Lojun et al. 1997). In addition, expression of b-HCG/LH-R has been demonstrated in endometriosis and has been shown to be increased in patients with adenomyosis (Hudelist et al. 2008), (Lei et al. 1993a, b). However, reports on the biological consequences of elevated LH and/or b-HCG levels and expression of their receptor in breast tissue are conflicting. On one hand, in vitro observations suggest an anti-proliferative effect of b-HCG on breast cancer cell lines (Rao Ch et al. 2004) which could explain the decreased incidence of malignant breast tumors in early pregnancy. On the other hand, animal studies demonstrate that the development of the rudimentary ductal network can be boosted by creating LHbCTP transgenic mice resulting in extensive epithelial hyperplasia and subsequent development of malignant tumors in case of chronic exposure to elevated LH levels (Mann et al. 2003). Although there are some studies on the expression of the target receptor of LH and b-HCG in invasive breast tumors, data on the presence of b-HCG/LH-R in preinvasive lesions is lacking. In order to gain further information on the role of b-HCG/LH-R in breast carcinogenesis, we investigated the differential expression of b-HCG/LH-R in preinvasive and adjacent invasive breast cancer tissues.

Patient and tumor characteristics Paraffin-embedded tissue samples) deriving from 70 postmenopausal patients with DCIS and adjacent invasive breast tumors were included in the present analysis. Tumor tissue had been obtained during curative surgical treatment of the primary tumor. None of the patients had received any prior chemo- or endocrine therapy for breast cancer. In all patients, the malignant breast tumor consisted of both, DCIS components (“coexistent DCIS”) and adjacent invasive components (IBC). DCIS specimen were classified as low grade, intermediate grade and high grade based on the criteria outlined by Holland and colleagues (Bocker et al. 1997) which considers architectural features and nuclear grading (Figs. 1, 2). Tissue microarrays (TMA) Paraffin-embedded tumor specimens were obtained from archived breast biopsies of the Department of Pathology, University of Muenster, Germany. In order to identify the location of coexistent DCIS and IBC, sections from each tumor block were stained with haematoxylin and eosin and representative regions were circled on the slides and the remaining tissue block. Following this, four or more 0.6 mm cylindrical cores were punched out of the marked areas on the tissue block and arrayed into a new recipient paraffin block (20 × 35 mm) by using a manual tissue arrayer (Beecher Instruments, Silver Spring, MD, US). Tumor biopsies were arrayed in quadruplicate to ensure representative analysis. The tissue set finally used for the analysis consisted of two TMA blocks containing 116–306 preinvasive and/or invasive tissue cores. Before immunohistochemical detection of proteins of interest, the presence of coexistent DCIS and adjacent IBC was confirmed by re-evaluating haematoxylin–eosin sections of TMAs (Figs. 3, 4). Beta-HCG-receptor (b-HCG/LH-R) immunofluorescence After deparaffinization and rehydration of paraffin sections, antigen retrieval was performed by boiling the slides twice in the microwave oven for 5 min, 850 W in 10 mM Na–Citrate-buffer (pH 6) followed by cooling and rinsing the sections in washing buffer. All washing steps were done three times for 5 min in a buffer containing 100 mM Tris–Cl, (pH 7.5); 150 mM NaCl, 0,1% Tween 20. After blocking of sections in TSA blocking buffer for 30 min at RT slides were incubated overnight with a sheep anti-human LH/CG receptor antibody (dilution 1:100, Acris Antibodies GmbH, Im Himmelreich, Germany) at 4°C. For immunofluorescence detection, FITC-conjugated anti sheep antibodies (dilution 1:500, molecular probes) were used. Finally, nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI 1:2,000, Sigma) and the sections were embedded with fluoromount G (Soubio). Control cells were incubated with replacement of the specific primary antibody by unspecific sheep isotype control (1:100, Ab37385, Abcam, Cambridge, UK; Fig. 5). Immunofluorescence quantification A semiquantitative scoring system (immunoreactive score; IRS) according to Remmele et al. (Remmele and Stegner 1987) was used to allow for a reproducible evaluation of protein expression levels in ductal and stromal components of immunofluorescence-stained tissue sections. Tissue sections were scored independently by two experienced pathologists. The IRS was calculated according to the following formula: IRS = staining intensity (0–3) × percentage of positive cells or nuclei (“0” = <10%; “1” = 10–25%; “2” = 26–50%; “3” = 51–75%; “4” = 76–100%). Possible scores ranged from 0 to 12. Zero to two points was considered to be negative (0). 3 to 5 points was defined as weak staining (+), 6–8 points was considered to be intermediate (++) and 9–12 points was considered to be strong staining (+++). Statistical analysis A Chi Square test was used to identify differences in b-HCG/LH-R protein expression levels in preinvasive and invasive tissue components of breast cancer specimens. For all analyses, P-value <0.05 was considered statistically significant. Win-SAS V 8 (SAS Institute GmbH, Heidelberg, Germany) statistical software system was used for all calculations.

Beta-HCG/LH-R protein expression in DCIS and adjacent IBC Immunoreactivity for b-HCG/LH-R was detected in the membrane of pre-invasive and invasive epithelial cells. Out of 70 DCIS and IBC samples, 37 (52.8%) and 27 (38.6%) were available for further analysis due to loss of tissue cores during the staining procedure (Table 1). Eight out of 37 DCIS cases (21.6%) expressed b-HCG/LH-R; a weak, moderate and strong immunoreaction for b-HCG/LH-R was detected in 4/37 (10.8%), 2/37 (5.4%) and 2/37 (5.4%). Twenty-nine out of 37 DCIS samples (78.4%) lacked b-HCG/LH-R expression. In contrast, b-HCG/LH-R expression was observed in 19/27 (70.4%) adjacent invasive samples with weak, moderate and strong immunostaining in 10/27 (37.0%), 6/27 (22.2%) and 3/27 (11.1%), respectively. Eight out of 27 IBC tissues (29.6%) did not exhibit immunoreactivity for b-HCG/LH-R. Table 1. | b-HCG/LH-R | DCIS (n = 37) | IBC (n = 27) | P-value | |---|---|---|---| | Weak | 4/37 (10.8%) | 10/27 (37.0%) | | | Moderate | 2/37 (5.4%) | 6/27 (22.2%) | | | Strong | 2/37 (5.4%) | 3/27 (11.1%) | | | None | 29/37 (78.4%) | 8/27 (29.6%) | | | 0.001 | When the DCIS components were compared to corresponding IBC areas of the same individuals, b-HCG/LH-R was expressed in significantly higher amounts in IBC samples compared to preinvasive components (P = 0.001, Chi Square Test).

The b-HCG/LH/LH-R system has been shown to possess both, stimulatory and growth-inhibiting effects on ductal epithelium in vitro. For example, Rao Ch et al. (2004) demonstrated that HCG decreased proliferation and invasive properties of MCF-7 breast cancer cells via inhibition of NF-kappaB and AP-1 activation. Similarly, other groups (Lojun et al. 1997) showed decreased growth of b-HCG/LH-R expressing MCF-7 cells via treatment with highly purified b-HCG. In contrast, Popnikolov et al. (Popnikolov et al. 2001) was able to stimulate growth of breast epithelial cells implanted into athymic mice by administration of b-HCG. Within this, Kuorelahti and colleagues (Kuorelahti et al. 2007) found that promotion of lobuloalveolar development in transgenic mice expressing hCG was accompanied by abnormal expression of Wnt genes suggesting that b-HCG-induced deregulated Wnt signalling might contribute to breast carcinogenesis. Within this, Rulli et al. (Rulli et al. 2002) linked b-HCG with marked lobuloalveolar development followed by mammary tumorigenesi possibly via ovary-derived factors. Although b-HCG/LH-R expression has been investigated in malignant and non-malignant mammary tissues from breast cancer patients demonstrating varying degrees of expression levels in both, cancerous and non-cancerous epithelium, information on preinvasive tumor components is lacking so far. The present analysis demonstrates significantly higher b-HCG/LH-R expression levels in invasive tumors compared to adjacent in situ compounds suggesting upregulation of b-HCG/LH-R expression in invasive breast cancer. Whether this observation is a cause or a consequence of transformation of preinvasive to invasive tumoral tissue is yet not fully understood. High serum LH levels have been shown to up-regulate b-HCG/LH-R expression in tumor-bearing mice instead of expected down-regulation of receptor expression prompting authors to suggest that chronic receptor activation acts as a tumor-promotor similar to other G-protein-linked receptors (Dufau 1998). Recent evidence supports this hypothesis by demonstrating that a common b-HCG/LH-R gene variant increases receptor activity consequently shortening disease-free survival in breast cancer patients (Piersma et al. 2006). We suggest that under certain circumstances, b-HCG/LH-R function favours breast carcinogenesis. Factors influencing these pro-mitogenic receptor properties remain to be further investigated since they might be of therapeutic value in treating b-HCG/LH-R expressing breast cancer. Acknowledgments The authors wish to thank Barbara Weidinger and Martha Weinhaeusl for technical assistance with the immunohistochemical procedures.

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