Acquisition of estrogen independence and antiestrogen resistance in breast cancer : association with the invasive and metastatic phenotype

A significant percentage of human breast cancer (HBC) is dependent upon the ovarian hormone estrogen for its onset and progression. The presence or lack of estrogen receptors (ERs) in human breast cancer is an important determinant both of prognosis and of choice of treatment - a poorer prognosis being associated with ER–ve disease. Cell lines established from human breast cancer provide models for breast cancer in various stages of progression (Engel & Young 1978). When grown as tumors in athymic nude mice, these lines represent the major in vivo experimental model for HBC studies (Brünner et al 1987). The ease of both in vitro and in vivo maintenance, the human derivation of the tissue, and the similarities in plasma estrogen levels between ovariectomized nude mice and postmenopausal women (Seibert et al. 1983, Brünner et al. 1986), make the growth of human breast cancer cell lines in nude mice an attractive...

The orphan nuclear receptor LRH-1 promotes breast cancer motility and invasion

The orphan nuclear receptor liver receptor homologue-1 (LRH-1) has roles in the development, cholesterol and bile acid homeostasis, and steroidogenesis. It also enhances proliferation and cell cycle progression of cancer cells. In breast cancer, LRH-1 expression is associated with invasive breast cancer; positively correlates with ERα status and aromatase activity; and promotes oestrogen-dependent cell proliferation. However, the mechanism of action of LRH-1 in breast cancer epithelial cells is still not clear. By silencing or over-expressing LRH-1 in ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells, we have demonstrated that LRH-1 promotes motility and cell invasiveness. Similar effects were observed in the non-tumourigenic mammary epithelial cell line, MCF-10A. Remodelling of the actin cytoskeleton and E-cadherin cleavage was observed with LRH-1 over-expression, contributing to increased migratory and invasive properties. Additionally, in LRH-1 over-expressing cells, the truncation of the 120 kDa E-cadherin to the inactive 97 kDa form was observed. These post-translational modifications in E-cadherin may be associated with LRH-1-dependent changes to matrix metalloproteinase 9 expression. These findings suggest a new role of LRH-1 in promoting migration and invasion in breast cancer, independent of oestrogen sensitivity. Therefore, LRH-1 may represent a new target for breast cancer therapeutics.

lacZ transduced human breast cancer xenografts as an in vivo model for the study of invasion and metastasis

A number of human cancer cell lines have been described as being invasive and metastatic in immune incompetent animals. However, it is difficult to assess metastatic spread of a subcutaneously injected or inoculated cell line, since an exact detection of all microfoci of human tumour cells in the animals by usual histological procedures would require extensive sectioning of the whole animal. To overcome this problem, we transduced human breast cancer cells with a replication-defective Moloney murine leukaemia retroviral vector (M-MuLV) containing both neo(R) (neomycin resistance) and lacZ genes. The resulting cell lines were selected for antibiotic (G418) resistance, and cell-sorted for lacZ expression. lacZ continued to be expressed in cultured cells for at least 20 passages without further G418 selection. The lacE gene codes for β-D-galactosidase, and cells expressing this gene stain blue with the chromogenic substrate X-gal. The lacZ-expressing cells retained the pre-transduction ability to traverse Matrigel in vitro, to form subcutaneous tumours in nude mice, and to grow invasively with the formation of metastases. X-gal staining showed high specificity, staining the tumour cells but not the surrounding mouse tissue on either whole tissue blocks or histological sections. The staining procedure was highly sensitive, allowing detection of microfoci of human cancer cells, and quantitative estimation of the metastatic capacity of the cells. These results indicate that lacZ transduction of human tumour cells is a powerful means of studying human cancer cell invasion and metastases in vivo.

Assays for the study of human cancer cell invasion and metastasis

Two in vitro and two in vivo assays for the study of human cancer invasion and metastasis are described. The assays include in vitro invasiveness through an artificial basement membrane (Matrigel®), invasiveness and metastasis in nude mice of subcutaneously injected LacZ-transduced human tumor cells, in vitro adherence to basement membrane components, and LacZ-transduced human cancer cells injected intravenously into nude mice. In studies of the processes involved in human cancer cell invasion and metastasis, these four assays were found to be complementary, and thus provide a set of test systems for preclinical screening of agents which interfere with these processes.

High level of MT-MMP expression is associated with invasiveness of cervical cancer cells

MMP-2 (gelatinase A) has been associated with the invasive potential of many cancer cells both in vitro and in vivo. It is now becoming clear that the activation of this enzyme might be a key step in tumor invasion. This activation process has been shown to be a membrane-associated pathway inducible by various agents such as collagen type I, concanavalin A or TGF-β, but its physiological regulation is still largely unresolved. MT-MMP was recently discovered and described as a potential gelatinase-A activator. In the present study, we investigated the expression of MT-MMP (membrane-type metalloproteinase) in cervical cancer cells both in vitro and in vivo. Comparing several in vitro-transformed cervical cell lines, previously shown to display different invasive potentials, our results showed that the ability of cells to overexpress MT-MMP mRNA following ConA induction correlated with their ability to activate gelatinase A and with a highly invasive behavior. Moreover, using immunohistochemistry and in situ hybridization, we found a higher level of MT-MMP expression in invasive cervical carcinoma and lymphnode metastases compared to its expression in non-invasive CIN III lesions. Our in vivo observations also clearly demonstrated a cooperation between stromal and tumor cells for the production of MT-MMP. Taken together, our results clearly correlated high level MT-MMP expression with invasiveness, and thus suggested that MT-MMP might play a crucial role in cervical tumor invasion.

Upregulation of matrix metalloproteinases (MMPs) in breast cancer xenografts : a major induction of stromal MMP-13

In human breast cancer (HBC), as with many carcinoma systems, most matrix metalloproteinases (MMPs) are largely expressed by the stromal cells, whereas the tumour cells are relatively silent in MMP expression. To determine the tissue source of the most relevant MMPs, we xenografted HBC cell lines and HBC tissues into the mammary fat pad (MFP) or bone of immunocompromised mice and measured the expression of human and mouse MMP-2, -9, -11, -13, membrane-type-1 MMP (MT1-MMP), MT2-MMP and MT3-MMP by species-specific real-time quantitative RT-PCR. Our data confirm a stromal origin for most tumour-associated MMPs and indicate marked and consistent upregulation of stromal (mouse) MMP-13 and MT1-MMP in all xenografts studied, irrespective of implantation in the MFP or bone environments. In addition, we show increased expression of both human MMP-13 and human MT1-MMP by the MDA-MB-231 tumour cells grown in the MFP compared to in vitro production. MMP protein and activity data confirm the upregulation of MMP mRNA production and indicate an increase in the activated MMP-2 species as a result of tumour implantation. These data directly demonstrate tumour induction of MMP production by stromal cells in both the MFP and bone environments. These xenografts are a valuable means for examining in vivo production of MMPs and suggest that MMP-13 and MT1-MMP will be relevant targets for inhibiting breast cancer progression.

Neutrophil gelatinase-associated lipocalin (NGAL) an early-screening biomarker for ovarian cancer : NGAL is associated with epidermal growth factor-induced epithelio-mesenchymal transition

The expression of neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be upregulated in ovarian cancer cells. In this study, we report that the expression of immunoreactive NGAL (irNGAL) in ovarian tumors changes with disease grade and that this change is reflected in the concentration of NGAL in peripheral blood. A total of 59 ovarian tissues including normal, benign, borderline malignant and grades 1, 2 and 3 malignant were analyzed using immunohistochemistry. irNGAL was not present in normal ovaries and the NGAL expression was weak to moderate in benign tissues. Both borderline and grade 1 tumors displayed the highest amount of NGAL expression with moderate to strong staining, whereas in grade 2 and 3 tumors, the extent of staining was significantly less (p < 0.01) and staining intensity was weak to moderate. Staining in all cases was confined to the epithelium. NGAL expression was analyzed by ELISA in 62 serum specimens from normal and different grades of cancer patients. Compared to control samples, the NGAL concentration was 2 and 2.6-fold higher in the serum of patients with benign tumors and cancer patients with grade 1 tumors (p < 0.05) and that result was consistent with the expression of NGAL performed by Western blot. NGAL expression was evaluated by Western blot in an immortalized normal ovarian cell line (IOSE29) as well as ovarian cancer cell lines. Moderate to strong expression of NGAL was observed in epithelial ovarian cancer cell lines SKOV3 and OVCA433 while no expression of NGAL was evident in normal IOSE29 and mesenchyme-like OVHS1, PEO.36 and HEY cell lines. NGAL expression was downregulated in ovarian cancer cell lines undergoing epithelio-mesenchymal transition (EMT) induced by epidermal growth factor (EGF). Down-regulation of NGAL expression correlated with the upregulation of vimentin expression, enhanced cell dispersion and downregulation of E-cadherin expression, some of the hallmarks of EMT. EGF-induced EMT phenotypes were inhibited in the presence of AG1478, an inhibitor of EGF receptor tyrosine kinase activity. These data indicate that NGAL may be a good marker to monitor changes of benign to premalignant and malignant ovarian tumors and that the molecule may be involved in the progression of epithelial ovarian malignancies.

Regulation of breast cancer cells by hormones and growth factors : effects on proliferation and basement membrane invasiveness

The current understanding of the regulation of breast cancer cell proliferation and invasiveness by hormones and growth factors is reviewed. It has been shown that polypeptide growth factors are involved in hormone-independent breast cancer, and are sometimes oestrogen-regulated in hormone-responsive models. Basement-membrane invasiveness, relating to the metastatic potential of these cells, is also stimulated by oestrogen in hormone-dependent models, elevated in hormone-independent models, and is growth factor sensitive. Further understanding of the differential effects of growth factors on breast cancer cell proliferation and invasiveness should facilitate better therapeutic exploitation of regulation at this level.

Hormones and breast cancer in vitro

Breast cancer is characterized by hormonal regulation. The current article reviews the role of estrogen and polypeptide growth factors in control of proliferation and basement membrane invasion of breast cancer cells in vitro. The role of antiestrogens to regulate proliferation, invasion, and growth factor secretion is further highlighted. Finally, the use of in vitro cultures of breast cancer cells to model steps in the malignant progression of the disease is emphasized. The availability of hormone dependent and independent breast cancer cell lines should allow screening for better antiestrogens, antimetastatic drugs, and antagonists of local action of growth factors.

Interleukin-6 is a potent inducer of S100P, which is up-regulated in androgen-refractory and metastatic prostate cancer

Elevated circulating interleukin-6 (IL6) and up-regulated S100P in prostate cancer (PCa) specimens correlate independently with progression to androgen-independent and metastatic PCa. The cause of up-regulated S100P levels in advanced PCa remains to be determined. We investigated the possibility that IL6 is an inducer of S100P. Determination of mRNA and protein levels by real-time PCR and Western blotting revealed that IL6 is a more potent inducer of S100P than the synthetic androgen, R1881, in the LNCaP/C4-2B model of PCa progression. IL6 did not require androgen to induce S100P in these cells, which express a functional androgen receptor (AR). Like R1881, IL6 was unable to induce S100P in PC3 cells that lack a functional AR. IL6 did not strongly induce the AR-dependent genes PSA and KLK2 and, contrary to R1881, down-regulated Cyr61/CCN1, a potential marker that is down-regulated in PCa. Epidermal growth factor (EGF), which like IL6 is a non-androgen activator of the AR, did not induce S100P. The data identifies a unique gene-induction profile for IL6 and suggests that IL6 may require a functional AR for S100P induction. A link between elevated IL6 and up-regulated S100P in androgen-refractory and metastatic PCa is postulated.

Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.

Laminin adhesion-selected primary human colon-cancer cells are more tumorigenic than the parenteral and nonadherent cells

Laminin has been shown to promote the malignant phenotype and the level of the 32/67 Kd laminin receptor has been found to correlate with Dukes' staging of colon cancer. A biopsy of a Dukes' stage B2 human colon carcinoma formed a tumor in a nude mouse after coinjection with Matrigel. The parental tumor and the murine tumor appeared identical at the histological level. A cell line LCC-C1 was established from the murine tumor. The cell line appeared moderately differentiated although it did not produce mucin in vitro; however, the xenograft in vivo did produce low levels of mucin. Laminin adherent and non-adherent cell lines were selected. The parental and the laminin-selected cell subclones adhered equally well to plastic and to fibronectin and showed similar growth rates on plastic. When injected subcutaneously into nude mice, the laminin-adherent cells formed relatively undifferentiated tumors that were twice as large as the parental cell tumors whereas the laminin non adherent cells formed very small, but highly differentiated tumors. These data demonstrate that subpopulations of tumor cells which differ in their tumorigenic properties can be selected based on their adhesion to laminin and thus provide models for studying the mechanisms of tumor growth.

Expression of β1 integrin in laminin-adhesion-selected human colon cancer cell lines of varying tumorigenicity

Laminin has been shown to promote the malignant phenotype and the expression of certain laminin receptors has been correlated with the malignant character of the tumors. Here new cell lines were isolated from a human colon cancer cell line (LCC-C1) based on their adhesiveness to laminin. The laminin-adherent subclone formed large tumors in nude mice, whereas the laminin-nonadherent subclone failed to form sizable tumors. Only the laminin-adherent subclone adhered to laminin and invaded through Matrigel-coated filters. The adhesive and invasive ability of the cells was almost completely blocked by low concentrations (1.0 μg/ml) of anti-β1 integrin antibody. The amounts of total cellular β1 integrin protein were similar in the two subclones when compared by Western blot, and the mRNA levels also did not differ. The localization of β1 integrin laminin receptor varied in the two subclones; the laminin-adherent subclone showed a linear distribution along the cell-cell junctions, while the laminin-nonadherent subclone did not stain between the cells. Using laminin-Sepharose affinity chromatography, more β1 integrin was obtained from the laminin-adherent subclone. These findings suggest that alterations in the affinity of β1 integrin for laminin and in its membrane distribution might be involved in the increased tumorigenicity observed in colon cancer cells.