Targeting of cytotoxic luteinizing hormone-releasing hormone analogs to breast, ovarian, endometrial, and prostate cancers

Targeted chemotherapy is a modern approach aimed at increasing the efficacy of systemic chemotherapy and reducing its side effects. The peptide receptors expressed primarily on cancerous cells can serve as targets for a selective destruction of malignant tumors. Binding sites for LHRH (now known in genome and microarray databases as GNRH1), were found on 52% of human breast cancers, about 80% of human ovarian and endometrial cancers, and 86% of human prostatic carcinoma specimens. Because LHRH receptors are not expressed on most normal tissues, they represent a specific target for cancer chemotherapy with antineoplastic agents linked to an LHRH vector molecule. To test the efficacy of targeted chemotherapy based on LHRH analogs, we recently developed a cytotoxic analog of LHRH, designated AN-152, which consists of [D-Lys6]LHRH covalently linked to one of the most widely used chemotherapeutic agents, doxorubicin (DOX). In addition, we designed and synthesized a highly active derivative of DOX, 2-pyrrolino-DOX (AN-201), which is 500-1000 times more potent than DOX in vitro. AN-201 is active against tumors resistant to DOX, and noncardiotoxic. As in the case of DOX, AN-201 was coupled to carrier peptide [D-Lys6]LHRH to form a superactive targeted cytotoxic LHRH analog, AN-207. Both AN-152 and AN-207 can effectively inhibit the growth of LHRH receptor-positive human breast, ovarian, endometrial, and prostate cancers xenografted into nude mice. DOX-containing cytotoxic LHRH analog AN-152 is scheduled for clinical phase I/IIa trials in patients with advanced ovarian and breast cancers in 2005.     

Luteinizing hormone-releasing hormone analogs: their impact on the control of tumorigenesis

The development of the luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists and the principles of their clinical use were reviewed. In the 28 years that have elapsed since the elucidation of the structure of LH-RH, various applications in gynecology, reproductive medicine, and oncology have been established for LH-RH agonists and antagonists. These clinical applications are based on inhibition of the pituitary and the gonads. The advantage of the LH-RH antagonists is due to the fact that they inhibit the secretion of gonadotropins and sex steroids immediately after the first injection and thus achieve rapid therapeutic effects in contrast to the agonists, which require repeated administration. LH-RH antagonists should find applications in the treatment of benign gynecologic disorders and benign prostatic hypertrophy and in assisted reproduction programs. The primary treatment of advanced androgen-dependent prostate cancer is presently based on the use of depot preparations of LH-RH agonists, but antagonists like Cetrorelix already have been tried successfully. Antagonists of LH-RH might be more efficacious than agonists in treatment of patients with breast cancer as well as ovarian and endometrial cancer. Recently, practical cytotoxic analogs of LH-RH that can be targeted to LH-RH receptors on tumors have been synthesized and successfully tested in experimental cancer models. Targeted cytotoxic LH-RH analogs show a great promise for therapy of prostate, breast, and ovarian cancers.     

New approaches to the therapy of various tumors based on peptide analogues.

The discovery of hypothalamic hormones was briefly reviewed. The development of new hormonal methods for the therapy of various cancers based on analogues of hypothalmic hormones is then presented. My group isolated luteininzing hormone-releasing hormone (LH-RH), also known as Gn-RH, from pig hypothalmi, elucidated its amino acid sequence, and synthesized it in 1971. The interest in medical applications of LH-RH led to the synthesis of LH-RH analogues by various groups. LH-RH agonists substituted in positions 6 or 10 including Decapeptyl, Leuprolide and Zoladex are much more active than LH-RH and on continuous administration produce inhibition of pituitary and gonads. Chronic administration of LH-RH agonists is being utilized for the treatment of prostate and breast cancer. Octapeptide analogues of somatostatin have various applications in Oncology. In 1980 we developed a new endocrine therapy for advanced prostate cancer based on agonists of LH-RH, which is now preferred by 70-90% of prostate cancer patients for primary treatment. LH-RH antagonists such as Cetrorelix can be used for therapy of BPH. On the basis of the presence of specific receptors for hypothalamic peptides on human cancers, we developed targeted cytotoxic analogues of LH-RH, somatostatin, and bombesin/GRP linked to doxorubicin or 2-pyrrolinodoxorubicin. These analogues inhibit the growth of experimental human prostate, breast, ovarian and endometrial cancer, renal cell carcinoma, pancreatic, colorectal and gastric cancers, small cell lung carcinoma (SCLC) and non-SCLC, brain tumors, melanomas, and lymphomas. Cytotoxic LH-RH analogues are now in clinical trials. Recently we demonstrated that growth hormone-releasing hormone (GH-RH) also serves as an autocrine growth factor in many cancers. Antagonistic analogues of GH-RH synthesized in our laboratory inhibit the growth of diverse tumors. The discovery of LH-RH and somatostatin has led to clinical use of their analogues in the field of cancer treatment and GH-RH antagonists also show a great promise.     

Antitumor effects of analogs of LH-RH and somatostatin: Experimental and clinical studies

Many clinical approaches for the treatment of hormone-sensitive tumors are being developed based on analogs of LH-RH and somatostatin. Inhibition of the pituitary-gonadal axis forms the basis for oncological applications of LH-RH agonists like [ -Trp⁶]-LH-RH and new LH-RH antagonists free of edematogenic effects such as [Ac- -Nal(2)¹- -Phe(4Cl)²- -Pal(3)³, -Cit⁶, -Ala¹⁰]-LH-RH (SB-75). Agonists and antagonists of LH-RH have been used in patients with prostate cancer and might be also beneficial for the treatment of breast cancer and ovarian, endometrial and pancreatic carcinomas. Some of the effects of LH-RH analogs can be due to direct action since LH-RH receptors have been found in these cancers. The use of sustained delivery systems based on microcapsules of PLG, makes the treatment more efficacious. Octaeptide analogs of somatostatin such as -P s-Trp-NH₂ (RC-160) and related analogs were designed specifically for antitumor activity. These somatostatin analogs, by virtue of having a wide spectrum of activities appear to inhibit various tumors through multiple mechanisms. Direct antiproliferative actions of somatostatin analogs appear to be mediated by specific receptors located on tumor cells. High affinity binding sites for RC-160 and related analogs have been found in human pancreatic, prostate, breast and ovarian cancers and brain tumors such as meningiomas. In vivo administration of analog RC-160 inhibits the growth of Dunning R-3327 prostate cancers in rats, MXT mammary tumors in mice and BOP-induced ductal pancreatic cancers in hamsters. Combination of microcapsules of RC-160 with [ -Trp⁶]-LH-RH results in synergistic potentiation of the inhibition of these cancers. Somatostatin analog RC-160 and LH-RH antagonist SB-75 are the object of further experimental studies and clinical trials aimed at the exploration of their inhibitory effects on the processes of malignant growth.     

NPY receptors in human cancer: a review of current knowledge

Many peptide hormone receptors are over-expressed in human cancer, permitting an in vivo targeting of tumors for diagnostic and therapeutic purposes. NPY receptors are novel and promising candidates in this field. Using in vitro receptor autoradiography, Y1 and Y2 receptors have been found to be expressed in breast carcinomas, adrenal gland and related tumors, renal cell carcinomas, and ovarian cancers in both tumor cells and tumor-associated blood vessels. Pathophysiologically, tumoral NPY receptors may be activated by endogenous NPY released from intratumoral nerve fibers or tumor cells themselves, and mediate NPY effects on tumor cell proliferation and tumoral blood supply. Clinically, tumoral NPY receptors may be targeted with NPY analogs coupled with adequate radionuclides or cytotoxic agents for a scintigraphic tumor imaging and/or tumor therapy.     

Immunogenic HER-2/neu peptides as tumor vaccines

During the last decade, a large number of tumor-associated antigens (TAA) have been identified, which can be recognized by T cells. This has led to renewed interest in the use of active immunization as a modality for the treatment of cancer. HER-2/neu is a 185-KDa receptor-like glycoprotein that is overexpressed by a variety of tumors including breast, ovarian, lung, prostate and colorectal carcinomata. Several immunogenic HER-2/neu peptides recognized by cytotoxic T lymphocytes (CTL) or helper T lymphocytes (TH) have been identified thus far. Patients with HER-2/neu over-expressing cancers exhibit increased frequencies of peripheral blood T cells recognizing immunogenic HER-2/neu peptides. Various protocols for generating T cell-mediated immune responses specific for HER-2/neu peptides have been examined in pre-clinical models or in clinical trials. Vaccination studies in animals utilizing HER-2/neu peptides have been successful in eliminating tumor growth. In humans, however, although immunological responses have been detected against the peptides used for vaccination, no clinical responses have been described. Because HER-2/neu is a self-antigen, functional immune responses against it may be limited through tolerance mechanisms. Therefore, it would be interesting to determine whether abrogation of tolerance to HER-2/neu using appropriate adjuvants and/or peptide analogs may lead to the development of immune responses to HER-2/neu epitopes that can be of relevance to cancer immunotherapy. Vaccine preparations containing mixtures of HER-2/neu peptides and peptide from other tumor-related antigens might also enhance efficacy of therapeutic vaccination. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004     

Absence of binding of targeted analogs of somatostatin carrying cytotoxic radicals or radionuclides to growth hormone secretagogue receptors on human myocardium

Various peripheral human tissues express receptors for growth hormone secretagogue (GHS), the highest density being in the myocardium. It was also reported that some octapeptide analogs of somatostatin (SRIH) can displace radiolabeled Tyr-Ala-hexarelin from GHS receptors on the human pituitary and heart. Thus, it is possible that radionuclide analogs of SRIH such as OctreoScan and recently developed cytotoxic SRIH analogs containing doxorubicin (DOX) intended for targeted tumor therapy, could bind to these GHS receptors, compromising the safety of compounds of this type. Therefore, we determined the binding of OctreoScan and two cytotoxic SRIH analogs consisting of octapeptide carrier RC-121 and DOX (AN-162) or 2-pyrrolino-DOX (AN-238) to human myocardium specimens. None of these compounds displayed specific binding to the human heart indicating that the clinical use of SRIH analogs linked to anthracyclines or radionuclides should not be associated with increased cardiac side effects.     

Antitumor effects of analogs of hypothalamic hormones in endocrine-dependent cancers

A new approach to the treatment of endocrine-dependent tumors based on analogs of hypothalamic hormones is in the early stages of development, but appears promising and significant. Administration of hypothalamic hormones can mimic hypophysectomy and gonadectomy, and is essentially devoid of side effects. A successful use of agonistic analogs of LH-RH for treatment of endocrine-dependent prostate cancer has been documented in several hundred patients. Experimental studies suggest that agonists and/or antagonists of LH-RH might be useful for treatment of breast cancer and pituitary tumors. Our work in animal models also indicates that analogs of somatostatin, alone or combined with LH-RH agonists, could be considered for therapy of chondrosarcomas, osteosarcomas, and pancreatic cancer. Experiments are in progress on the use of LH-RH analogs for treatment of ovarian cancer, neoplasms of the female genital tract, and for protection against gonadal damage during chemotherapy. These investigations should extend the concepts of endocrine treatment of cancers.     

Role of fibroblast growth factor 8 in growth and progression of hormonal cancer

Hormonal cancers such as breast and prostate cancer arise from steroid hormone-regulated tissues. In addition to breast and prostate cancer hormonal regulation has also a role in endometrial, ovarian, testis and thyroid carcinomas. The effects of estrogens, androgens and progestagens on tumor growth are largely mediated by paracrine and autocrine target molecules which include growth factors and growth factor receptors. During cancer progression the hormonal growth regulation is often lost or overcome by an inappropriate activation of growth factor signaling cascades. One of the growth factors which have been associated with the regulation of growth and progression of hormonal cancer is fibroblast growth factor 8 (FGF8) which has also been recognized as an oncogene. FGF8 is widely expressed during embryonic development. It has been shown to mediate embryonic epithelial-mesenchymal transition and to have a crucial role in gastrulation and early organization and differentiation of midbrain/hindbrain, pharyngeal, cardiac, urogenital and limb structures. During adulthood FGF8 expression is much more restricted but in hormonal cancers it becomes frequently activated. High level of FGF8 expression in tumors is associated with a poor prognosis at least in prostate cancer. In experimental models FGF8 induces and facilitates prostate tumorigenesis and increases growth and angiogenesis of tumors. Several lines of evidence for autocrine and paracrine loops in the growth regulation of breast, prostate and ovarian cancer by FGF8 have been suggested.     

Development of hypoxia enhanced 111In-labeled Bombesin conjugates: design, synthesis, and in vitro evaluation in PC-3 human prostate cancer

The gastrin-releasing peptide receptor (BB2r) has shown great promise for tumor targeting due to the increase of the receptor expression in a variety of human cancers including prostate, breast, small-cell lung, and pancreatic cancer. From clinical investigations, prostate cancer has been shown to be among the most hypoxic of the cancers investigated. Many solid tumors contain regions of hypoxia due to poor organization and efficiency of the vasculature. However, hypoxia is typically not present in normal tissue. Nitroimidazoles, a thoroughly investigated class of hypoxia selective drugs, have been shown to be highly retained in hypoxic tissues. The purpose of this study is to determine if the incorporation of hypoxia trapping moieties into the structural paradigm of BB2r-targeted peptides will increase the retention time of the agents in prostate cancer tumors. The present work involves the design, syntheses, purification, and in vitro investigation of hypoxia enhanced (111)In-BB2r-targeted radioconjugates. A total of four BB2r-targeted conjugates (1-4) were synthesized and coupled with increasing numbers of 2-nitroimidazoles, a hypoxia trapping moiety. Conjugates were radiolabeled with (111)In and purified by HPLC prior to in vitro studies. Receptor saturation assays under both normoxic and hypoxic conditions showed that the BB2r receptor expression on the PC-3 human prostate cancer cell line was not significantly affected by oxygen levels. Competitive binding assays revealed that incorporation of 2-nitroimidazoles had a detrimental effect to BB2r binding when adequate spacer groups, between the hypoxia trapping agent and the pharmacophore, were not employed. All of the 2-nitroimidazole containing BB2r-targeted agents exhibited significantly higher longitudinal retention in PC-3 cells under hypoxic conditions compared to the analogous normoxic studies. Protein association analysis revealed a 3-fold increase in binding of a 2-nitroimidazole containing BB2r-targeted agent under hypoxic relative to normoxic conditions. The positive nature of these results indicate that further exploration into the potential of hypoxia selective trapping agents for BB2r-targeted agents, as well as other targeted compounds, is warranted.     

Effects of LHRH-analogues on mitogenic signal transduction in cancer cells

The expression of luteinizing hormone-releasing hormone (LHRH) and its receptors has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary, endometrium and prostate. These findings suggest the presence of an autocrine regulatory system based on LHRH. Recent studies in our laboratory have demonstrated that the function of LHRH produced by ovarian cancer cells is the inhibition of their proliferation. Dose-dependent antiproliferative effects of LHRH-agonists have been observed by several laboratories in cell lines derived from the above cancers. Interestingly, also LHRH-antagonists have marked antiproliferative activity in most of the ovarian, breast and endometrial cancer cell lines tested so far, indicating that the dichotomy of LHRH-agonists/LHRH-antagonists is not valid for the LHRH-system in cancer cells. In addition, our data suggest that the classical LHRH receptor signal transduction mechanisms known from the pituitary (phospholipase-C, protein kinase C, adenylyl cyclase) are not involved in the mediation of LHRH effects in cancer cells. Data obtained by several groups, including ours, rather suggest that LHRH analogs interfere with the signal transduction of growth-factor receptors and related oncogene products associated with tyrosine-kinase activity. The mechanism of action is probably an LHRH-induced activation of a phosphotyrosine phosphatase, counteracting the effects of receptor associated tyrosine kinase. In our hands, LHRH analogs virtually blocked the EGF-induced MAP-kinase activity of ovarian and endometrial cancer cells. The pharmacological exploitation of this mechanism might provide promising new therapies for these cancers.     

Design of cyclic and d‐amino acids containing peptidomimetics for inhibition of protein‐protein interactions of HER2‐HER3

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2‐positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2‐mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18 ) exhibited antiproliferative activity in HER2‐overexpressing lung cancer cell lines with IC₅₀ values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d ‐amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18 , compound 32 , a cyclic, d ‐amino acid‐containing peptidomimetic, was found to have an IC₅₀ value in the nanomolar range in HER2‐overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d ‐amino acid analogs of 18 , compound 32 , binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.     

Somatostatin receptors in normal and tumoral tissue

Somatostatin receptors have been visualized with autoradiography and characterised biochemically in various somatostatin target tissues, such as brain, pituitary, pancreas and gastrointestinal tract, where they are likely to mediate the somatostatin actions. With the same methods, somatostatin receptors have been detected also in tumors originating from somatostatin target tissues: high receptor incidence is found in GH-producing pituitary adenomas as well as in some hormone-producing gastrointestinal tumors. These tumors are often highly responsive to somatostatin analogs in vivo. Among brain tumors, meningiomas usually contain a high density of receptors, suggesting a novel function for somatostatin in the human meninges. Among other human tumors tested, prostate, ovarian and endometrial carcinomas were free of receptors whereas 3 out of 39 mammary tumors contained somatostatin receptors.     

Immunohistological detection of BRAF25 in human prostate tumor and cancer specimens

BRAF25 is an alternatively spliced protein of BRAF35 (see associated paper). We have mapped the BRAF25 gene to chromosome sub-band 19p13.3, a region where loss of chromosomal heterozygosity has been reported in about 50% of ovarian cancers. Because of the high incidence of genetic links of prostate cancer to breast and ovarian cancers, we investigated the BRAF25 expression in the prostate specimens. Immunohistochemical analysis using antibodies specific for BRAF25 revealed a strong immunostaining in sections of the benign prostatic hyperplasia (BPH). The staining was concentrated on the nuclei of cells facing the lumen of prostatic glands, even though the sporadic nuclei of cells in stromas were also stained. However, the expression of BRAF25 was dramatically reduced in intermediate prostate cancer and absent in advanced prostate cancer. Preincubation of the antibody with the immunizing peptide abolished immunostaining in BPH specimens. Therefore, the expression of BRAF25 was gradually lost in prostate cancer.     

Potentiating effects of GHRH analogs on the response to chemotherapy

Growth hormone releasing hormone (GHRH) from hypothalamus nominatively stimulates growth hormone release from adenohypophysis. GHRH is also produced by cancers, acting as an autocrine/paracrine growth factor. This growth factor function is seen in lymphoma, melanoma, colorectal, liver, lung, breast, prostate, kidney, bladder cancers. Pituitary type GHRH receptors and their splice variants are also expressed in these malignancies. Synthetic antagonists of the GHRH receptor inhibit proliferation of cancers. Besides direct inhibitory effects on tumors, GHRH antagonists also enhance cytotoxic chemotherapy. GHRH antagonists potentiate docetaxel effects on growth of H460 non-small cell lung cancer (NSCLC) and MX-1 breast cancer plus suppressive action of doxorubicin on MX-1 and HCC1806 breast cancer. We investigated mechanisms of antagonists on tumor growth, inflammatory signaling, doxorubicin response, expression of drug resistance genes, and efflux pump function. Triple negative breast cancer cell xenografted into nude mice were treated with GHRH antagonist, doxorubicin, or their combination. The combination reduced tumor growth, inflammatory gene expression, drug-resistance gene expression, cancer stem-cell marker expression, and efflux-pump function. Thus, antagonists increased the efficacy of doxorubicin in HCC1806 and MX-1 tumors. Growth inhibition of H460 NSCLC by GHRH antagonists induced marked downregulation in expression of prosurvival proteins K-Ras, COX-2, and pAKT. In HT-29, HCT-116 and HCT-15 colorectal cancer lines, GHRH antagonist treatment caused cellular arrest in S-phase of cell cycle, potentiated inhibition of in vitro proliferation and in vivo growth produced by S-phase specific cytotoxic agents, 5-FU, irinotecan and cisplatin. This enhancement of cytotoxic therapy by GHRH antagonists should have clinical applications.     

Targeting breast and prostate cancers through their hormone receptors

A targeted treatment that effectively destroys human breast, prostate, ovarian, and testicular cancer cells that express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors has been developed. The treatment consists of a conjugate of a membrane-disrupting lytic peptide (Hecate, Phor14, or Phor21) and a 15-amino acid segment of the beta chain of CG. Because these conjugates act primarily by destroying cell membranes, their effects are independent of cell proliferation. The conjugates are relatively small molecules, are rapidly metabolized, and are not antigenic. In a series of independent experiments conducted in three different laboratories, the validity of the concept has been established, and it has been shown that the LH/CG receptor capacity of the cancer cells is directly related to the sensitivity of the lytic peptide conjugates. Sensitivity to the drugs can be increased by pretreating prostate or breast cancer cells with FSH or estradiol to up-regulate LH/CG receptors. A series of 23 in vivo experiments involving a total of 1630 nude mice bearing xenografts of human prostate or breast cancer cells showed convincingly that all three lytic peptide-betaCG compounds were highly effective in destroying tumors and reducing tumor burden. Hecate-betaCG was less effective in mice bearing ovarian epithelial cancer cell xenografts, but was highly effective in treating granulosa cell tumors in transgenic mice. In addition, Hecate-betaCG and Phor14-betaCG were highly effective in targeting and destroying prostate and breast cancer cell metastases in the presence or absence of the primary tumors. Although effective in vitro, neither Hecate nor Phor14 alone were effective in reducing primary tumor volume or burden in nude mice bearing prostate or breast cancer xenografts.     

In vitro and in vivo evaluation of 64Cu-labeled DOTA-linker-bombesin(7-14) analogues containing different amino acid linker moieties

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a variety of tumor types and has been targeted with radiolabeled peptides for detection and therapy of these cancers. Analogues of the 14 amino acid bombesin (BN) peptide have been radiolabeled with both gamma- and positron-emitting radionuclides for detection of GRPR-expressing tumors. We have previously evaluated BN analogues radiolabeled with the positron-emitter, copper-64 (64Cu), that contained various aliphatic linkers placed between the BN peptide and the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. These studies showed that the analogues could be used for positron-emission tomographic (PET) imaging of GRPR-positive tumors in mice but clinical translation would be hindered by significant uptake in background tissues. Therefore, the purpose of this study was to determine if the use of amino acid linkers placed between the DOTA chelate and the BN peptide would reduce nontarget tissue uptake, while maintaining good prostate tumor uptake. The linkers studied utilized three amino acid combinations of glycine (G), serine (S), or glutamic acid (E). In vitro assays in PC-3 cells showed that the glutamic acid-containing linkers had poor binding and internalization, while the other analogues had IC50 values <100 nM and good internalization. In vivo, these same analogues demonstrated tumor-specific uptake and good imaging characteristics that were comparable to, or better than the previously reported 64Cu-labeled DOTA-BN analogues. Overall, this study shows that BN analogues containing amino acid linkers can be used for the PET imaging of GRPR-expressing prostate cancer and that these linkers lead to lower background tissue uptake.     

Self-renewing Pten-/- TP53-/- protospheres produce metastatic adenocarcinoma cell lines with multipotent progenitor activity

Prostate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem/progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease.