Regulation of urokinase production by androgens in human prostate cancer cells: effect on tumor growth and metastases in vivo.

During the complex multistep process of tumor progression, prostate cancer is initiated as an androgen-sensitive, nonmetastatic cancer, followed by a gradual transition into a highly metastatic and androgen-insensitive variety that lacks the expression of functional androgen receptors (AR). Urokinase (uPA), a member of the serine protease family, has been implicated in the progression of various human malignancies, including prostate cancer. Although uPA production is regulated by various growth factors and cytokines, the role of sex steroids (androgens) in regulating uPA gene expression in prostate cancer is poorly understood. In the current study, we have examined the role of androgens in regulating uPA production and the invasive capacity of the androgen insensitive PC-3 cells transfected with the full-length human AR complementary DNA (PC-3T). Restoration of androgen responsiveness in PC-3T cells caused a marked decrease in cell doubling time. Treatment of PC-3T cells with dihydroxytestosterone (DHT) caused a dose-dependent decrease in uPA messenger RNA and protein production, resulting in their decreased ability to invade through the Matrigel. Nuclear runoff assays revealed that these effects were attributable to the ability of DHT to inhibit uPA gene transcription. AR antagonist flutamide (Flu) reversed the effect of DHT on proliferation and invasion of PC-3T cells. Both control (PC-3) and experimental (PC-3T) cells were injected into the right flank of male BALB/c nu/nu mice. Control animals developed palpable tumors and microscopic tumor metastases at lymph nodes, lungs, and liver at 6-week posttumor cell inoculation. In contrast to this, because of androgen sensitivity of PC-3T cells, palpable tumors were observed only at week 12, with occasional tumor metastases in lungs. Furthermore, inoculation of PC-3T cells into surgically castrated host animals resulted in the development of tumors at a much earlier time (week 10) and a high incidence of metastases, compared with regular animals receiving PC-3T cells. Collectively, these results demonstrate the ability of androgen to regulate uPA production, which may directly effect prostate cancer growth, invasion, and metastasis in vitro and in vivo.

Transcriptional regulation of urokinase (uPA) gene expression in breast cancer cells: role of DNA methylation.

Carcinoma of the breast is a leading hormone-dependent malignancy, resulting in a high rate of morbidity and mortality. During the complex multi-step process of tumor promotion, this common cancer is initiated as hormone-responsive (HR), non-metastatic cancer, followed by a gradual transition into a highly metastatic hormone-insensitive (HI) variety which lacks the functional estrogen receptor. This transition of cancer cells causes them to become refractory to hormonal treatment. Urokinase (uPA), a member of the serine protease family has been implicated in the progression of several malignancies including breast cancer. In the current study, we have examined the correlation between hormone sensitivity and uPA expression in HR normal mammary epithelial cells (HMEC) and in MCF-7 and T-47D breast cancer cell lines. Comparison was made with HI breast cancer cells MDA-231. uPA mRNA expression was seen only in the highly invasive, HI breast cancer cells MDA-231. Lack of uPA expression in HR normal (HMEC) and in minimally invasive, HR cells (MCF-7 and T-47D) was due to transcriptional suppression of uPA gene expression as determined by nuclear run-off assays. Since alteration of the DNA methylation status of CpG island in the 5' sequence of oncogenes and tumor suppressor genes has been demonstrated to change their expression, we examined DNA methylation as a potential molecular mechanism for regulating uPA gene transcription in these cancer cells. Southern blot analysis using methylation sensitive enzymes revealed that CpG island of uPA gene are methylated in HR, HMEC, MCF-7 and T-47D cells, whereas they are hypomethylated in HI and MDA-231 cells. Treatment of HR MCF-7 cells with cytosine DNA methyltransferase inhibitor 5' azacytidine caused a dose-dependent induction of uPA mRNA due to demethylation of the CpG island of the uPA gene which led to increased invasive ability of these HR cancer cells. Our results demonstrate that DNA methylation can regulate the transcription of the uPA gene to alter the invasive behaviour of these HR breast cancer cells.

Role of urokinase (uPA) and its receptor (uPAR) in invasion and metastasis of hormone-dependent malignancies.

Despite our recent advances in characterizing the molecular basis of breast and prostate cancer and their early detection with the aid of new imaging and diagnostic techniques, these cancers continue to be the leading causes of cancer-related deaths. This limited success in achieving our ultimate goal of cancer control is due to our inability to block the production of various factors produced in the later stages of these cancers that cause this high rate of mortality. A key requirement in the complex process of tumor invasion is the ability of tumor cells to produce and recruit growth factors and proteolytic enzymes within the tumor cell environment to promote neovascularization, tumor growth and promote extracellular matrix (ECM) degradation to facilitate tumor metastasis. One such protease, urokinase (uPA), has been strongly implicated in the progression of several malignancies including breast and prostate cancer. Along with uPA, its cell surface receptor (uPAR) is also believed to be involved due to its ability to recruit uPA within the tumor cell environment. In recent years, novel in vivo models of breast and prostate cancer have been developed which have clearly demonstrated the significance of uPA and uPAR in the invasion and metastases of these hormone-dependent cancers. The availability of these in vivo models has now permitted us to evaluate the molecular, chemical and immunotherapeutic strategies targeted against the uPA/uPAR system. This review describes the mechanism of uPA actions in tumor progression and analyses the usefulness of these in vivo models to authenticate uPA/uPAR as a therapeutic target and evaluates the benefits of blocking uPA/uPAR interactions alone or in combination with currently available treatment modalities against this cancer. Based on these results, there is an urgent need to develop and optimize strategies which will ultimately allow us to control the progression of these malignancies and enhance our ability to effectively manage these patients.

Prevention of breast cancer growth, invasion, and metastasis by antiestrogen tamoxifen alone or in combination with urokinase inhibitor B-428.

Urokinase (urokinase plasminogen activator, uPA) and its cell surface receptor (uPA receptor, uPAR) play an important role in a variety of physiological and pathological processes requiring cell migration and tissue remodeling. Using our syngeneic model of uPAR overexpression by the rat breast cancer cell line Mat B-III, we have examined the ability of the nonsteroidal antiestrogen, tamoxifen (TAM), and of a selective synthetic inhibitor of uPA, 4-iodo benzo[b]thiophene-2-carboxamidine (B-428), to inhibit expression of uPA and uPAR as well as cell growth, invasion, and metastasis of wild-type Mat B-III cells and of cells overexpressing uPAR (Mat B-III-uPAR). Both TAM and B-428 inhibited uPAR gene transcription, mRNA expression, protein production and also decreased the proliferative and invasive capacity of Mat B-III and Mat B-III-uPAR. The effects of TAM and B-428 were more pronounced when these agents were tested in combination. Both control and experimental cells (1 x 10(6) cells) were inoculated orthotopically into the mammary fat pad of syngeneic female Fisher rats, and animals were infused i.p. with either TAM and B-428 alone or in combination for 2 weeks. Control animals receiving vehicle alone developed large tumors and macroscopic metastases to lungs, liver, and lymph nodes. In contrast to this, experimental animals receiving TAM and B-428 showed a significant decrease in primary tumor volume and metastases. Combination therapy had especially marked effects in blocking progression of the primary tumor in experimental animals inoculated with highly aggressive Mat B-III-uPAR cells. These results underscore the utility of anti-proteolytic agents (B-428) in addition to standard hormone therapy (TAM) in advanced breast cancer patients where the uPA/uPAR system plays a key role in tumor progression.

Overexpression of urokinase receptor in breast cancer cells results in increased tumor invasion, growth and metastasis.

We have examined the role of urokinase receptor (uPAR) in tumor invasion and metastasis by developing a homologous model of uPAR overexpression in a rat breast cancer cell line (Mat B III) using gene transfer technique. Control (pRc-CMV) and experimental plasmid (pRc-uPAR-S) were transfected into Mat B III cells by using Lipofectin reagent. Levels of uPAR production were accessed by Northern blotting, immunofluorescence, receptor binding and ELISA. At least 3 experimental clones (pRc-uPAR-S), expressing 3- to 5-fold higher levels of uPAR than control (pRc-CMV), were selected for further analysis. Experimental cells overexpressing uPAR showed a 4-to 5-fold higher invasive capacity compared with control cells in a Boyden chamber invasion assay. Both control and experimental cells (1 x 10(6) cells) were injected into the mammary fat pad of syngeneic female Fischer rats. Animals were sacrificed at timed intervals and evaluated for the development of tumor growth and metastasis. Animals receiving cells overexpressing uPAR had significantly larger tumor volume and weight throughout our study. Furthermore, due to increased uPAR expression, experimental animals developed large metastatic lesions in liver, spleen and lymph nodes. Our results therefore demonstrate the role of uPAR in tumor progression, due to its ability to localize uPA within the tumor cell milieu.