The brain-penetrant cell-cycle inhibitor p28 sensitizes brain metastases to DNA-damaging agents.

Background: Brain metastases (BMs), the most common tumors of the central nervous system, are life-threatening with a dismal prognosis. The major challenges to developing effective treatments for BMs are the limited abilities of drugs to target tumors and to cross the blood-brain barrier (BBB). We aimed to investigate the efficacy of our therapeutic approach against BMs in mouse models that recapitulate the clinical manifestations of BMs. Methods: BMs mouse models were constructed by injecting human breast, lung cancer, and melanoma intracardially, which allowed the BBB to remain intact. We investigated the ability of the cell-penetrating peptide p28 to cross the BBB in an in vitro 3D model and in the BMs animal models. The therapeutic effects of p28 in combination with DNA-damaging agents (radiation and temozolomide) on BMs were also evaluated. Results: p28 crossed the intact BBB more efficiently than the standard chemotherapeutic agent, temozolomide. Upon crossing the BBB, p28 localized preferentially to tumor lesions and enhanced the efficacy of DNA-damaging agents by activating the p53-p21 axis. In the BMs animal models, radiation in combination with p28 significantly reduced the tumor burden of BMs. Conclusions: The cell-cycle inhibitor p28 can cross the BBB localize to tumor lesions in the brain and enhance the inhibitory effects of DNA-damaging agents on BMs, suggesting the potential therapeutic benefits of this molecule in BMs.

Cross-talk between cancer and Pseudomonas aeruginosa mediates tumor suppression.

Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in cancer, and therapies that incorporate bacteria have been tried in various types of cancer. We previously demonstrated that cupredoxin azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa, enters human cancer cells and induces apoptotic death1-4. However, the physiological interactions between P. aeruginosa and humans and their role in tumor homeostasis are largely unknown. Here, we show that P. aeruginosa upregulated azurin secretion in response to increasing numbers of and proximity to cancer cells. Conversely, cancer cells upregulated aldolase A secretion in response to increasing proximity to P. aeruginosa, which also correlated with enhanced P. aeruginosa adherence to cancer cells. Additionally, we show that cancer patients had detectable P. aeruginosa and azurin in their tumors and exhibited increased overall survival when they did, and that azurin administration reduced tumor growth in transgenic mice. Our results suggest host-bacterial symbiotic mutualism acting as a diverse adjunct to the host defense system via inter-kingdom communication mediated by the evolutionarily conserved proteins azurin and human aldolase A. This improved understanding of the symbiotic relationship of bacteria with humans indicates the potential contribution to tumor homeostasis.

Tumor-targeting cell-penetrating peptide, p28, for glioblastoma imaging and therapy.

Despite recent advances in cancer research, glioblastoma multiforme (GBM) remains a highly aggressive brain tumor as its treatment options are limited. The current standard treatment includes surgery followed by radiotherapy and adjuvant chemotherapy. However, surgery without image guidance is often challenging to achieve maximal safe resection as it is difficult to precisely discern the lesion to be removed from surrounding brain tissue. In addition, the efficacy of adjuvant chemotherapy is limited by poor penetration of therapeutics through the blood-brain barrier (BBB) into brain tissues, and the lack of tumor targeting. In this regard, we utilized a tumor-targeting cell-penetration peptide, p28, as a therapeutic agent to improve the efficacy of a current chemotherapeutic agent for GBM, and as a carrier for a fluorescence imaging agent for a clear identification of GBM. Here, we show that a near-infrared (NIR) imaging agent, ICG-p28 (a chemical conjugate of an FDA-approved NIR dye, indocyanine green ICG, and tumor-targeting p28 peptide) can preferentially localize tumors in multiple GBM animal models. Moreover, xenograft studies show that p28, as a therapeutic agent, can enhance the cytotoxic activity of temozolomide (TMZ), one of the few effective drugs for brain tumors. Collectively, our findings highlight the important role of the tumor-targeting peptide, which has great potential for intraoperative image-guided surgery and the development of new therapeutic strategies for GBM.

Nontoxic Tumor-Targeting Optical Agents for Intraoperative Breast Tumor Imaging.

Precise identification of the tumor margins during breast-conserving surgery (BCS) remains a challenge given the lack of visual discrepancy between malignant and surrounding normal tissues. Therefore, we developed a fluorescent imaging agent, ICG-p28, for intraoperative imaging guidance to better aid surgeons in achieving negative margins in BCS. Here, we determined the pharmacokinetics (PK), biodistribution, and preclinical toxicity of ICG-p28. The PK and biodistribution of ICG-p28 indicated rapid tissue uptake and localization at tumor lesions. There were no dose-related effect and no significant toxicity in any of the breast cancer and normal cell lines tested. Furthermore, ICG-p28 was evaluated in clinically relevant settings with transgenic mice that spontaneously developed invasive mammary tumors. Intraoperative imaging with ICG-p28 showed a significant reduction in the tumor recurrence rate. This simple, nontoxic, and cost-effective method can offer a new approach that enables surgeons to intraoperatively identify tumor margins and potentially improves overall outcomes by reducing recurrence rates.

Image-guided surgery with a new tumour-targeting probe improves the identification of positive margins.

BACKGROUND: Given the lack of visual discrepancy between malignant and surrounding normal tissue, current breast conserving surgery (BCS) is associated with a high re-excision rate. Due to the increasing cases of BCS, a novel method of complete tumour removal at the initial surgical resection is critically needed in the operating room to help optimize the surgical procedure and to confirm tumour-free edges. METHODS: We developed a unique near-infrared (NIR) fluorescence imaging probe, ICG-p28, composed of the clinically nontoxic tumour-targeting peptide p28 and the FDA-approved NIR dye indocyanine green (ICG). ICG-p28 was characterized in vitro and evaluated in multiple breast cancer animal models with appropriate control probes. Our experimental approach with multiple-validations and -blinded procedures was designed to determine whether ICG-p28 can accurately identify tumour margins in mimicked intraoperative settings. FINDINGS: The in vivo kinetics were analysed to optimize settings for potential clinical use. Xenograft tumours stably expressing iRFP as a tumour marker showed significant colocalization with ICG-p28, but not ICG alone. Image-guided surgery with ICG-p28 showed an over 6.6 × 103-fold reduction in residual normalized tumour DNA at the margin site relative to control approaches (i.e., surgery with ICG or palpation/visible inspection alone), resulting in an improved tumour recurrence rate (92% specificity) in multiple breast cancer animal models independent of the receptor expression status. ICG-p28 allowed accurate identification of tumour cells in the margin to increase the complete resection rate. INTERPRETATION: Our simple and cost-effective approach has translational potential and offers a new surgical procedure that enables surgeons to intraoperatively identify tumour margins in a real-time, 3D fashion and that notably improves overall outcomes by reducing re-excision rates. FUNDING: This work was supported by NIH/ National Institute of Biomedical Imaging and Bioengineering, R01EB023924.

Progesterone receptor antagonism inhibits progestogen-related carcinogenesis and suppresses tumor cell proliferation.

PURPOSE: Blockade of the progestogen-progesterone receptor (PR) axis is a novel but untested strategy for breast cancer prevention. We report preclinical data evaluating telapristone acetate (TPA), ulipristal acetate (UPA), and mifepristone. METHODS: Tumors were induced with medroxyprogesterone acetate (MPA) plus 7,12-dimethylbenz[a]anthracene (DMBA) in mice, and MPA or progesterone plus N-methyl-N-nitrosourea (MNU) in rats. Mammary gland histology, tumor incidence, latency, multiplicity, burden and histology were evaluated, along with immunohistochemical labeling of pHH3 (proliferation), CD34 (angiogenesis), and estrogen and progesterone receptors (ER and PR). A concentration gradient of TPA, UPA, and mifepristone was tested for growth inhibition of T47D spheroids. RESULTS: In mouse mammary glands, no tumors formed, but TPA opposed the pro-hyperplastic effects of MPA (p = 0.002). In rats, TPA decreased tumor incidence (p = 0.037 for MPA + TPA vs. MPA, and p = 0.032 for progesterone + TPA vs. progesterone) and tumor burden (p = 0.042 for progesterone + TPA vs. progesterone), with significant decreases in pHH3 and CD34 positive cells. TPA and UPA were superior to mifepristone in growth inhibition of T47D spheroids. CONCLUSION: TPA has consistent anti-tumorigenic effects in several models, which are accompanied by decreases in cell proliferation, angiogenesis, and hormone receptor expression.

Retinoids induce cellular senescence in breast cancer cells by RAR-ß dependent and independent pathways: Potential clinical implications (Review).

Most studies on cellular senescence (CS) have been performed in vitro by employing cytotoxic agents, irradiation, chromatin and telomerase modulators or by activating certain oncogenes. All these approaches usually lead to DNA damage, gene instability and/or chromatin alterations that primarily affect p53-p21 signaling. Little is known on whether retinoids and rexinoids, which are cell differentiation agents, can also induce CS in vitro and in vivo, and which molecular mechanisms are involved in promoting the senescent phenotype. We reviewed the recent publications on CS induced by retinoids and rexinoids in ER+ and ER- breast cancer cell lines and in corresponding animal models of mammary carcinogenesis which simulate those of human breast cancer. The role of retinoic acid receptors ß2 and 5 (RARß2 and RARß5) and of receptor independent genes involved in mediating the senescence program of retinoids and rexinoids in ER+ and ER- breast cancer cells is discussed. Potential strategists for clinical implication of CS as biomarker of prognosis and of response to treatment with retinoids, rexinoids and with other cell differentiation and antitumor agents are outlined.

Methyl-substituted conformationally constrained rexinoid agonists for the retinoid X receptors demonstrate improved efficacy for cancer therapy and prevention.

(2E,4E,6Z,8Z)-8-(3',4'-Dihydro-1'(2H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,3,6-octatrienoinic acid, 9cUAB30, is a selective rexinoid for the retinoid X nuclear receptors (RXR). 9cUAB30 displays substantial chemopreventive capacity with little toxicity and is being translated to the clinic as a novel cancer prevention agent. To improve on the potency of 9cUAB30, we synthesized 4-methyl analogs of 9cUAB30, which introduced chirality at the 4-position of the tetralone ring. The syntheses and biological evaluations of the racemic homolog and enantiomers are reported. We demonstrate that the S-enantiomer is the most potent and least toxic even though these enantiomers bind in a similar conformation in the ligand binding domain of RXR.

MADD knock-down enhances doxorubicin and TRAIL induced apoptosis in breast cancer cells.

The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

Bexarotene induces cellular senescence in MMTV-Neu mouse model of mammary carcinogenesis.

Previous studies have shown that retinoids and rexinoids can prevent breast cancer in animal models and in women with increased risk of developing the disease. The cellular effects of these vitamin A analogues have been primarily associated with induction of differentiation and inhibition of proliferation. In this study, we tested the hypothesis that bexarotene (LGD1069, Targretin), a rexinoid, can not only inhibit cell proliferation but also induce cellular senescence in mammary epithelial cells, premalignant lesions, and tumors of the MMTV-Neu model of mammary carcinogenesis, which develops estrogen receptor-negative tumors. Mice with palpable mammary tumors were treated for 4 weeks with bexarotene at 80 or 40 mg/kg body weight, and senescent cells were determined by SA-ß-Gal assay. Bexarotene decreased in a dose-dependent manner the multiplicity of premalignant lesions and tumors, and this was associated with inhibition of cell proliferation and induction of cellular senescence and apoptosis. By double labeling of senescent cells, first by SA-ß-Gal and then by antibodies against genes related to cellular senescence, we found that p21, p16, and RARß, but not p53, were upregulated by bexarotene in mammary tumors and in breast cancer cell lines, suggesting involvement of multiple signaling pathways in mediating the senescence program of rexinoids. These findings indicate that, in addition to cell proliferation and apoptosis, cellular senescence could be used as a potential biomarker of response in breast cancer prevention and therapy studies with rexinoids and possibly with other antitumor agents.

A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt.

Amino acids 50-77 (p28) of azurin, a 128 aa cupredoxin isolated from Pseudomonas aeruginosa, is essentially responsible for azurin's preferential penetration of cancer cells. We now report that p28 also preferentially penetrates human umbilical vein endothelial cells (HUVEC), co-localized with caveolin-1 and VEGFR-2, and inhibits VEGF- and bFGF-induced migration, capillary tube formation and neoangiogenesis in multiple xenograft models. The antiangiogenic effect of p28 in HUVEC is associated with a dose-related non-competitive inhibition of VEGFR-2 kinase activity. However, unlike other antiangiogenic agents that inhibit the VEGFR-2 kinase, p28 decreased the downstream phosphorylation of FAK and Akt that normally precedes cellular repositioning of the cytoskeletal (F-actin), focal adhesion (FAK and paxillin), and cell to cell junction protein PECAM-1, inhibiting HUVEC motility and migration. The decrease in pFAK and pAkt levels suggests that p28 induces a pFAK-mediated loss of HUVEC motility and migration and a parallel Akt-associated reduction in cell matrix attachment and survival. This novel, direct antiangiogenic effect of p28 on endothelial cells may enhance the cell cycle inhibitory and apoptotic properties of this prototype peptide on tumor cell proliferation as it enters a Phase II clinical trial.

Early in vitro passages of breast cancer cells are differentially susceptible to retinoids and differentially express RARß isoforms.

The effect of retinoids on breast cancer has been predominantly studied in vitro, on established cell lines, which in biology differ significantly from primary tumor cells. Little is known on whether early in vitro passages of breast cancer cells (EPBCCs) are differentially sensitive to retinoids and differentially express retinoid acid receptors (RARs) and retinoid X receptors (RXRs). We have previously identified a novel RARß isoform (RARß5) and hypothesized that it may serve as a potential target of retinoids in EPBCCs. Breast cancer cells isolated from primary tumors were cultured in vitro for 6-12 passages (EPBCCs) and their epithelial origin was confirmed by a cocktail of antibodies against cytokeratins. EPBCCs were treated for 4 days with 1.0 µM of all-trans retinoic acid (atRA), 9-cis retinoic acid (9cRA) or 4-hydroxy-phenylretinamide (4-HPR) and their viability determined by MTT assay. Among nine EPBCCs consistently grown in vitro, three were resistant to the above retinoids, five were susceptible to atRA, four to 4-HPR and two to 9cRA, suggesting that patients with breast carcinomas may differentially respond to various retinoids. All EPBBCs differentially expressed RARα, RARγ, RXRα, RXRß proteins and RARß5 and RARß2 mRNAs. However, only one EPBCC (BCA-2) expressed RARß5 at mRNA and protein level and it was resistant to retinoids, both in vitro and in a xenograft tumor assay. RARß5 suppression by siRNA in BCA-2 cells increased their susceptibility to atRA. No correlation was found between sensitivity of EPBCCs to the above retinoids and RARß5 and RARß2 mRNA expression. atRA reduced RARß expression in most EPBCCs suggesting that this retinoid receptor is most probably the prime target of retinoids in breast cancer. These data may have clinical implication in selecting patients with breast cancer that would benefit the most from clinical trials with retinoids.

Cdk2-null mice are resistant to ErbB-2-induced mammary tumorigenesis.

The concept of targeting G(1) cyclin-dependent kinases (CDKs) in breast cancer treatments is supported by the fact that the genetic ablation of Cdk4 had minimal impacts on normal cell proliferation in majority of cell types, resulting in near-normal mouse development, whereas such loss of Cdk4 completely abrogated ErbB-2/neu-induced mammary tumorigenesis in mice. In most human breast cancer tissues, another G(1)-regulatory CDK, CDK2, is also hyperactivated by various mechanisms and is believed to be an important therapeutic target. In this report, we provide genetic evidence that CDK2 is essential for proliferation and oncogenesis of murine mammary epithelial cells. We observed that 87% of Cdk2-null mice were protected from ErbB-2-induced mammary tumorigenesis. Mouse embryonic fibroblasts isolated from Cdk2-null mouse showed resistance to various oncogene-induced transformation. Previously, we have reported that hemizygous loss of Cdc25A, the major activator of CDK2, can also protect mice from ErbB-2-induced mammary tumorigenesis [Cancer Res (2007) 67(14): 6605-11]. Thus, we propose that CDC25A-CDK2 pathway is critical for the oncogenic action of ErbB-2 in mammary epithelial cells, in a manner similar to Cyclin D1/CDK4 pathway.

CDB-4124, a progesterone receptor modulator, inhibits mammary carcinogenesis by suppressing cell proliferation and inducing apoptosis.

CDB-4124 (Proellex or telapristone acetate) is a modulator of progesterone receptor (PR) signaling, which is currently employed in preclinical studies for prevention and treatment of breast cancer and has been used in clinical studies for treatment of uterine fibroids and endometriosis. Here we provide evidence for its action on steroid hormone-signaling, cell cycle-regulated genes and in vivo on mammary carcinogenesis. When CDB-4124 is given to rats at 200 mg/kg for 24 months, it prevents the development of spontaneous mammary hyperplastic and premalignant lesions. Also, CDB-4124 given as subcutaneous pellets at two different doses suppressed, dose dependently, N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis. The high dose (30 mg, over 84 days) increased tumor latency from 66 ± 24 days to 87 ± 20 days (P < 0.02), decreased incidence from 85% to 35% (P < 0.001), and reduced multiplicity from 3.0 to 1.1 tumors/animal (P < 0.001). Tumor burden decreased from 2.6 g/animal to 0.26 g/animal (P < 0.01). CDB-4124 inhibited cell proliferation and induced apoptosis in MNU-induced mammary tumors, which correlated with a decreased proportion of PR(+) tumor cells and with decreased serum progesterone. CDB-4124 did not affect serum estradiol. In a mechanistic study employing T47D cells we found that CDB-4124 suppressed G(1)/G(0)-S transition by inhibiting CDK2 and CDK4 expressions, which correlated with inhibition of estrogen receptor (ER) expression. Taken together, these data indicate that CDB-4124 can suppress the development of precancerous lesions and carcinogen-induced ER(+) mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer.

p27(Kip1) deficiency promotes prostate carcinogenesis but does not affect the efficacy of retinoids in suppressing the neoplastic process.

BACKGROUND: p27 is a cell cycle suppressor gene, whose protein is a negative regulator of cyclin/cdk complexes. p27 is also a potential target of retinoids in cancer prevention studies. In benign prostate hyperplasia (BPH), and in most carcinomas, p27(Kip1) is down-regulated, suggesting its potential resistance to retinoids. To test this hypothesis, we examined the efficacy of 9-cis retinoic acid (9cRA) to suppress prostate cell proliferation (PECP) and carcinogenesis in p27(Kip1) deficient mice. METHODS: p27(Kip1) deficient (-/-), heterozygous (+/-) and homozygous (+/+) mice were treated for 7 days with testosterone, 9cRA, or with both, and cell proliferation in dorsolateral prostate (DLP) was determined by BrdU labeling. Prostate carcinogenesis was induced by N-Methyl-N-Nitrosourea (MNU) and hormone stimulation. RESULTS: PECP in DLP of two-month-old mice of all genotypes was similar but significantly increased in old p27-/- mice only. Testosterone treatment increased PECP in all three p27 genotypes with the highest values in p27-/- mice. p27(Kip1) deficiency did not affect the response of PEC to 9cRA and to 9cRA+testosterone. The decrease of p27(Kip1) in p27+/- and p27-/- mice progressively increased the incidence and frequency of PIN and tumors. 9cRA suppressed PIN in all three p27 genotypes and this was associated with decreased PECP and increased cellular senescence. CONCLUSIONS: This data indicates that p27(Kip1) deficiency promotes prostate cell proliferation and carcinogenesis but does not affect 9cRA's potential to suppress prostate carcinogenesis, suggesting that patients with PIN and carcinomas lacking or having a low level of p27(Kip1) expression may also benefit from clinical trials with retinoids.

A peptide fragment of azurin induces a p53-mediated cell cycle arrest in human breast cancer cells.

We report that amino acids 50 to 77 of azurin (p28) preferentially enter the human breast cancer cell lines MCF-7, ZR-75-1, and T47D through a caveolin-mediated pathway. Although p28 enters p53 wild-type MCF-7 and the isogenic p53 dominant-negative MDD2 breast cancer cell lines, p28 only induces a G(2)-M-phase cell cycle arrest and apoptosis in MCF-7 cells. p28 exerts its antiproliferative activity by reducing proteasomal degradation of p53 through formation of a p28:p53 complex within a hydrophobic DNA-binding domain (amino acids 80-276), increasing p53 levels and DNA-binding activity. Subsequent elevation of the cyclin-dependent kinase inhibitors p21 and p27 reduces cyclin-dependent kinase 2 and cyclin A levels in a time-dependent manner in MCF-7 cells but not in MDD2 cells. These results suggest that p28 and similar peptides that significantly reduce proteasomal degradation of p53 by a MDM2-independent pathway(s) may provide a unique series of cytostatic and cytotoxic (apoptotic) chemotherapeutic agents.

The novel RARbeta isoform (beta5) is a potential target of retinoids in breast cancer.

Retinoic acid receptor beta 2 (RARbeta2) isoform has been considered a putative tumor suppressor because it is expressed in normal cells but is lacking in most tumors, including breast cancer. Recently, we identified a novel RARbeta isoform (beta5) in breast cancer cells, which may sereve as a potential target of retinoids in cancer prevention and therapy studies. In this review are summarized the data on the expression of RARbeta5 and of the previously identified RARbeta4 and RARbeta' isoforms in various breast cancer cell lines. We found that RARbeta5 may serve as a potential biomarker of resistance of breast cancer cells to retinoids and thus may have clinical implication in selecting patients that may benefit the most from clinical trials with retinoids.

Noncationic peptides obtained from azurin preferentially enter cancer cells.

Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, alpha-helical peptides are unique. Unlike the cationic cell-penetrating peptides, alpha-helical antennapedia-like, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively.

Effects of gefitinib (Iressa) on mammary cancers: preventive studies with varied dosages, combinations with vorozole or targretin, and biomarker changes.

The ability of the epidermal growth factor receptor inhibitor gefitinib (Iressa) to prevent/treat methylnitrosourea (MNU)-induced mammary cancers and to modulate biomarkers in female Sprague-Dawley rats was examined. Rats were given a single dose of MNU (75 mg/kg body weight) at 50 days of age. In the prevention studies, continual treatment with Iressa at 10, 3, or 1 mg/kg body weight per day beginning 5 days after MNU reduced tumor multiplicity by 93%, 43%, and 20%, respectively. Treatment of rats bearing small palpable cancers with Iressa (10 mg/kg body weight per day) resulted in the complete regression of 70% of the tumors. Short-term treatment of tumor-bearing rats with Iressa caused decreases in cell proliferation and phosphorylated epidermal growth factor receptor and increases in apoptosis. To examine treatment regimens that might decrease the skin toxicity associated with Iressa, both intermittent treatments and combinations of lower doses of Iressa with other effective agents were evaluated. Treatment with Iressa (10 mg/kg body weight per day) continually or intermittently (either "3 weeks on/3 weeks off" or "4 days on/3 days off") reduced cancer multiplicity by 91%, 24%, and 68%, respectively. However, all regimens reduced tumor weights >85%. Finally, combining suboptimal doses of Iressa with suboptimal doses of vorozole (an aromatase inhibitor) or targretin (a retinoid X receptor agonist) yielded greater chemopreventive efficacy than any of these agents given alone.

Disruption of growth hormone signaling retards prostate carcinogenesis in the Probasin/TAg rat.

We asked whether down-regulation of GH signaling could block carcinogenesis in the Probasin/TAg rat, a model of aggressive prostate cancer. The Spontaneous Dwarf rat, which lacks GH due to a mutation (dr) in its GH gene, was crossed with the Probasin/TAg rat, which develops prostate carcinomas at 100% incidence by 15 wk of age. Progeny were heterozygous for the TAg oncogene and homozygous for either the wild-type GH gene (TAg/Gh(+/+)) or the dr mutation (TAg/Gh(dr/dr)). Prostate tumor incidence and burden were significantly reduced, and tumor latency was delayed in TAg/Gh(dr/dr) rats relative to TAg/Gh(+/+) controls. At 25 wk of age, loss of GH resulted in a 20 and 80% decrease in the area of microinvasive carcinoma in the dorsal and lateral lobes, respectively. By 52 wk of age, invasive prostate adenocarcinomas were observed in all TAg/Gh(+/+) rats, whereas the majority of TAg/Gh(dr/dr) did not develop invasive tumors. Suppression of carcinogenesis could not be attributed to alterations in prostate expression of TAg or androgen receptor or changes in serum testosterone levels. As carcinogenesis progressed in TAg/Gh(+/+) rats, prostate GHR mRNA and protein expression increased significantly, but prostate IGF-I receptor mRNA and protein levels dropped. Furthermore, serum IGF-I and prostate IGF-I levels did not change significantly over the course of carcinogenesis. These findings suggest that GH plays a dominant role in progression from latent to malignant prostate cancer driven by the powerful probasin/TAg fusion gene in rats and suggest that GH antagonists may be effective at treating human prostate cancer.