Unnur P. Thorgeirsson

Role of collagenases in tumor cell invasion

SummaryCollagenases are a family of metalloproteinases which may play a role in facilitating tumor cell invasion of the extracellular matrix. Tumor cells traverse two types of extracellular matrix: basement membranes and interstitial stroma, at multiple stages of the metastatic process. The matrix is a dense meshwork of collagen, proteoglycans, elastin and glycoproteins. Normally the matrix does not contain open spaces large enough for cell movement. Therefore numerous investigators have postulated that collagenolytic proteases, secreted by tumor cells or associated host cells, breakdown the extracellular matrix during tumor cell invasion. A large number of animal and human tumors have been shown to contain collagenase at a higher level than corresponding benign tissues. Separate collagenolytic metalloproteinases have been identified which degrade specific types of collagen. A basement membrane collagenolytic protease was shown to be elevated in a series of metastatic murine tumor cells. Immunologic studies using antibodies specific for collagenase have demonstrated that in vivo, tumor cells can produce collagenase. Therefore identification of collagenase in cultured lines of tumor cells is not an artifact of in vitro cultivation. In some cases, tumor cells may induce host cells to produce collagenase. The best evidence to date that collagenases actually play a role in invasion is derived from experiments in which natural collagenase inhibitors block tumor cell invasion of extracellular matrix in vitro.

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.

Ras oncogene mediated induction of a 92kDa metalloproteinase; strong correlation with the malignant phenotype

We have previously demonstrated that activated ras oncogenes can induce the metastatic phenotype and type IV collagenolytic activity in NIH/3T3 cells (Thorgeirsson et al. Mol. Cell. Biol. 5:259-262, 1985). The present study demonstrates ras-mediated induction of a 92 kDa metalloproteinase, which degrades gelatin and type IV collagen. Association of the 92 kDa proteinase expression with the malignant phenotype was also observed in human tumor cell lines. Our data indicate that the 92 kDa gelatin-collagen IV degrading metalloproteinase is an important participant in the proteolytic process involving tumor cell invasion and metastasis.

Mammary carcinoma cells over‐expressing tissue inhibitor of metalloproteinases‐1show vascular endothelial growth factor expression

The tissue inhibitor of metalloproteinases‐1 (TIMP‐1) has at least 2 independent functions, i.e., regulation of matrix metalloproteinases and erythroid‐potentiating activity. We investigated the effects of TIMP‐1 over‐expression on tumor growth, using cloned lines derived from a TIMP‐1‐transfected rat breast carcinoma cell line. The in vitro growth rate of the TIMP‐1‐transfected clones was indistinguishable from that of the control. In contrast, the highest TIMP‐1‐producing clone (159.0 ng/ml), designated as T‐H, formed 4.6‐fold larger s.c. tumors than did the control after 14 days. Tumors derived from an intermediate TIMP‐1‐producing clone (45.4 ng/ml), designated as T‐M, were 1.9‐fold larger than the control. TIMP‐1 over‐expression was associated with increased vascular endothelial growth factor (VEGF) expression, vascularization and proliferative activity of the s.c. tumors. Similar to the rat breast carcinoma cells, transfection of TIMP‐1 cDNA into the human breast carcinoma cell line MCF‐7 resulted in up‐regulation of VEGF, with a linear relationship between TIMP‐1 and VEGF production in 9 cell clones examined. There was, however, no change in VEGF expression when the rat and human breast carcinoma cell lines were exposed to exogenous recombinant TIMP‐1. Our findings suggest that over‐expression of TIMP‐1 confers growth advantage on breast carcinoma cells in vivo and that up‐regulation of VEGF expression may play an important role in this TIMP‐1‐mediated, growth‐stimulating effect. Int. J. Cancer 75:81–87, 1998.Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

VEGF contributes to mammary tumor growth in transgenic mice through paracrine and autocrine mechanisms

Vascular endothelial growth factor (VEGF) has been identified as a vascular permeability factor, angiogenic cytokine, and a survival factor. To address its role in mammary carcinogenesis, we used transgenic mice with human VEGF165 targeted to mammary epithelial cells under the control of the mouse mammary tumor virus (MMTV) promoter. Metastatic mammary carcinomas were induced by mating the MMTV-VEGF mice with MMTV-polyoma virus middle T-antigen (MT) mice to generate VEGF/MT mice. Tumor latency was decreased in the VEGF/MT mice, which developed mammary carcinomas with increased vasodilatation at 4 weeks of age. There was increased incidence, multiplicity, and weight of the mammary tumors in 6- and 8-week-old VEGF/MT mice, compared to their MT-only littermates. Macro- and microscopic lung metastases were detected in the VEGF/MT mice but not the MT mice at 6 and 8 weeks of age. Enhanced tumor growth was attributed to increased microvascular density (MVD), as well as increased tumor cell proliferation and survival. Angiogenesis array analysis showed that 24 of 25 differentially expressed genes were upregulated in the VEGF/MT tumors. In vitro studies revealed increased proliferative activity and upregulation of Flk-1 in the VEGF/MT tumor cells, compared with the MT-only tumor cells. Moreover, there was decreased proliferative activity with downregulation of Flk-1 in tumor cells isolated from conditional knockout (VEGF−/−) MT-induced mammary carcinomas. The slow growing VEGF−/− tumor cells were accumulated in the G1/G0 phase of the cell cycle and this was associated with stimulation of p16ink4a and p21WAF1. Similarly, p16ink4a was stimulated in VEGFlox/lox/MT mammary tumor cells following Adeno-cre-mediated VEGF gene inactivation. Collectively, the data from these transgenic models indicate that VEGF contributes to mammary tumor growth through increased neovascularization, as well as autocrine stimulation of growth and inhibition of apoptosis.

Enhanced RNA expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) in human breast cancer

Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) is known to have at least 2 distinct types of activity, i.e., as a regulator of collagenolytic activity, and erythroid potentiating activity (EPA). In this study, we examined the expression of TIMP‐1 in human mammary carcinomas, non‐malignant breast tissues and benign breast tumors. A total of 53 samples were subjected to Northern‐blot analysis, including 23 of primary breast cancer, 26 of non‐malignant breast tissues, and 4 of benign tumors. Of the 53 samples, 10 were paired malignant and non‐malignant breast‐tissue samples from the same patient. TIMP‐1 RNA expression was significantly higher in the malignant tumor tissues than in the non‐malignant counterpart. Similar differences were observed in the level of TIMP‐1 protein expression in the paired breast samples examined. Moreover, breast‐cancer cell lines secreted larger amounts of TIMP‐1 in vitro than non‐neoplastic breast epithelial lines. The up‐regulation of TIMP‐1 expression in breast cancer may suggest that TIMP‐1 has an additional role to that of metalloproteinase inhibitor.

Metabolism of food-derived heterocyclic amines in nonhuman primates.

During the cooking of meats, several highly mutagenic heterocyclic amines (HCAs) are produced. Three HCAs, IQ, MeIQx, and PhIP have been under study for carcinogenicity in cynomolgus monkeys, and to date, IQ has been shown to be a potent hepatocarcinogen. Concomitantly, the metabolic processing of these HCAs has been examined. Metabolism studies show that the potent hepatocarcinogenicity of IQ is associated with the in vivo metabolic activation of IQ via N-hydroxylation and the formation of DNA adducts. In monkeys undergoing carcinogen bioassay with IQ, N-hydroxylation was confirmed by the presence of the N-hydroxy-N-glucuronide conjugate of IQ in urine. The N-hydroxylation of IQ appears to be carried out largely by hepatic CYP3A4 and/or CYP2C9/10, and not by CYP1A2, an isoform not expressed in liver of this species. Notably MeIQx is poorly activated in cynomolgus monkeys and lacks the potency of IQ to induce hepatocellular carcinoma after a 5-year dosing period. The poor activation of MeIQx appears to be due to the lack of constitutive expression of CYP1A2 and an inability of other cytochromes P450, such as CYP3A4 and CYP2C9/10, to N-hydroxylate the quinoxalines. MeIQx is detoxified in monkeys largely by conjugation with glucuronide at the N-1 position. Although the carcinogenicity of PhIP is not yet known, the metabolic data suggest that PhIP will be carcinogenic in this species. PhIP is metabolically activated in vivo in monkeys by N-hydroxylation, as discerned by the presence of the N-hydroxy-N-glucuronide conjugate in urine, bile, and plasma. PhIP also produces DNA adducts that are widely distributed in tissues. The results from these studies support the importance of N-hydroxylation in the carcinogenicity of HCAs in nonhuman primates and by analogy, the importance of this metabolic activation step in the possible carcinogenicity of dietary HCAs in humans.

Extrapolation of Heterocyclic Amine Carcinogenesis Data from Rodents and Nonhuman Primates to Humans

Twenty different heterocyclic amines have been isolated and identified from cooked foods especially beef, fish, pork and fowl. Other HCAs have also been isolated but their structure remains to be elucidated and new HCAs are likely to be identified in the future. The HCAs are highly mutagenic and all ten HCAs that have been tested for carcinogenic activity, produce tumors in mice and rats. For humans the average daily intake of HCAs is in quantities of 10-20 mg/person/day. The HCAs are procarcinogens and are activated by the cytochrome P450 system especially CYP 1A2. Rodents, monkeys and humans have the capacity to activate HCAs. Studies using hepatic microsomes demonstrated that humans have a greater capacity to activate the majority of HCAs tested than rodents or cynomolgus monkeys. Three HCAs are currently under evaluation in nonhuman primates for carcinogenic activity and one, IQ, is highly carcinogenic inducing primary hepatocellular carcinomas in the majority of cynomolgus monkeys treated. Epidemiological studies, although not definitive, are supportive of an association of HCAs intake to the etiology of human cancer. Risk assessments from animal data show a risk of HCAs to humans in the range of 10(-3) to 10(-4) which is an order of magnitude greater than compounds currently regulated by the U.S. Food and Drug Administration or the Environmental Protection Agency. Taken together evidence from mutagenicity data, activation by various species including humans, carcinogenicity in animals, human consumption data, epidemiological studies and risk assessment, supports the conclusion that HCAs are probable human carcinogens.

Mammary gland carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Sprague-Dawley rats on high- and low-fat diets

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic amine derived from cooked meat. Mammary gland tumors were induced in female Sprague-Dawley rats given 10 doses of PhIP (75 mg/kg po) once per day from 43 days of age and then placed on a defined high-fat (23.5% corn oil) or low-fat (5% corn oil) diet for 25 weeks. Mammary tumor incidence was 49% (44 of 90 rats) and 31% (27 of 88 rats) in the high- and low-fat groups, respectively. No tumors were found in vehicle control rats on the high-or the low-fat diet (n = 44 and 43, respectively). The higher tumor incidence in the high-fat group was due to an increase specifically in carcinomas (classified as tubulopapillary carcinomas) rather than benign tumors (tubular adenomas and fibroadenomas). The incidence of carcinomas was 45% and 24% in PhIP-treated rats on the high- and low-fat diets, respectively. In addition, the percentage of carcinomas showing stromal invasion was highest in the high-fat diet group (22% vs. 8%, high- vs. low-fat diet). Proliferating cell nuclear antigen immunostaining (PCNA) index revealed six times more proliferation in carcinomas from rats on the high-fat diet than in rats on the low-fat diet. Adenomas from rats on different diets had similar PCNA indexes. The tumor apoptotic index, quantitated by immunohistochemical detection (terminal deoxynucleotidyl transferase nick end labeling), was twice as high in carcinomas from rats on the high-fat diet as in carcinomas from rats on the low-fat diet but was similar between the two groups of adenomas. The PCNA-to-apoptosis ratio was 43 and 17 in carcinomas from rats on the high- and low-fat diets, respectively, indicating that the growth rate of carcinomas was greater in rats on the high-fat diet. The results from this study show that the high-fat diet increases the incidence, invasiveness, and growth of PhIP-induced mammary gland carcinomas.

Altered Localization of Retinoid X Receptor α Coincides with Loss of Retinoid Responsiveness in Human Breast Cancer MDA-MB-231 Cells

ABSTRACT To understand the mechanism of retinoid resistance, we studied the subcellular localization and function of retinoid receptors in human breast cancer cell lines. Retinoid X receptor α (RXRα) localized throughout the nucleoplasm in retinoid-sensitive normal human mammary epithelial cells and in retinoid-responsive breast cancer cell line (MCF-7), whereas it was found in the splicing factor compartment (SFC) of the retinoid-resistant MDA-MB-231 breast cancer cell line and in human breast carcinoma tissue. In MDA-MB-231 cells, RXRα was not associated with active transcription site in the presence of ligand. Similarly, ligand-dependent RXR homo- or heterodimer-mediated transactivation on RXR response element or RARE showed minimal response to ligand in MDA-MB-231 cells. Infecting MDA-MB-231 cells with adenoviral RXRα induced nucleoplasmic overexpression of RXRα and resulted in apoptosis upon treatment with an RXR ligand. This suggests that nucleoplasmic RXRα restores retinoid sensitivity. Epitope-tagged RXRα and a C-terminus deletion mutant failed to localize to the SFC. Moreover, RXRα localization to the SFC was inhibited with RXRα C-terminus peptide. This peptide also induced ligand-dependent transactivation on RXRE. Therefore, the RXRα C terminus may play a role in the intranuclear localization of RXRα. Our results provide evidence that altered localization of RXRα to the SFC may be an important factor for the loss of retinoid responsiveness in MDA-MB-231 breast cancer cells.