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.

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.

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.

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.

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.

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.

Hemizygous disruption of Cdc25A inhibits cellular transformation and mammary tumorigenesis in mice.

CDC25A phosphatase activates multiple cyclin-dependent kinases (CDK) during cell cycle progression. Inactivation of CDC25A by ubiquitin-mediated degradation is a major mechanism of DNA damage-induced S-G(2) checkpoint. Although increased CDC25A expression has been reported in various human cancer tissues, it remains unclear whether CDC25A activation is a critical rate-limiting step of carcinogenesis. To assess the role for CDC25A in cell cycle control and carcinogenesis, we used a Cdc25A-null mouse strain we recently generated. Whereas Cdc25A(-/-) mice exhibit early embryonic lethality, Cdc25A(+/-) mice show no appreciable developmental defect. Cdc25A(+/-) mouse embryonic fibroblasts (MEF) exhibit normal kinetics of cell cycle progression at early passages, modestly enhanced G(2) checkpoint response to DNA damage, and shortened proliferative life span, compared with wild-type MEFs. Importantly, Cdc25A(+/-) MEFs are significantly resistant to malignant transformation induced by coexpression of H-ras(V12) and a dominant negative p53 mutant. The rate-limiting role for CDC25A in transformation is further supported by decreased transformation efficiency in MCF-10A human mammary epithelial cells stably expressing CDC25A small interfering RNA. Consistently, Cdc25A(+/-) mice show substantially prolonged latency in mammary tumorigenesis induced by MMTV-H-ras or MMTV-neu transgene, whereas MMTV-myc-induced tumorigenesis is not significantly affected by Cdc25A heterozygosity. Mammary tissues of Cdc25A(+/-);MMTV-neu mice before tumor development display less proliferative response to the oncogene with increased tyrosine phosphorylation of CDK1/2, but show no significant change in apoptosis. These results suggest that Cdc25A plays a rate-limiting role in transformation and tumor initiation mediated by ras activation.

Deregulated CDC25A expression promotes mammary tumorigenesis with genomic instability.

Checkpoint pathways help cells maintain genomic integrity, delaying cell cycle progression in response to various risks of fidelity, such as genotoxic stresses, compromised DNA replication, and impaired spindle control. Cancer cells frequently exhibit genomic instability, and recent studies showed that checkpoint pathways are likely to serve as a tumor-suppressive barrier in vivo. The cell cycle-promoting phosphatase CDC25A is an activator of cyclin-dependent kinases and one of the downstream targets for the CHK1-mediated checkpoint pathway. Whereas CDC25A overexpression is observed in various human cancer tissues, it has not been determined whether deregulated CDC25A expression triggers or promotes tumorigenesis in vivo. Here, we show that transgenic expression of CDC25A cooperates markedly with oncogenic ras or neu in murine mammary tumorigenesis. MMTV-CDC25A transgenic mice exhibit alveolar hyperplasia in the mammary tissue but do not develop spontaneous mammary tumors. The MMTV-CDC25A transgene markedly shortens latency of tumorigenesis in MMTV-ras mice. The MMTV-CDC25A transgene also accelerates tumor growth in MMTV-neu mice with apparent cell cycle miscoordination. CDC25A-overexpressing tumors, which invade more aggressively, exhibit various chromosomal aberrations on fragile regions, including the mouse counterpart of human 1p31-36, according to array-based comparative genomic hybridization and karyotyping. The chromosomal aberrations account for substantial changes in gene expression profile rendered by transgenic expression of CDC25A, including down-regulation of Trp73. These data indicate that deregulated control of cellular CDC25A levels leads to in vivo genomic instability, which cooperates with the neu-ras oncogenic pathway in mammary tumorigenesis.

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.

Short-term modulation of cell proliferation and apoptosis and preventive/therapeutic efficacy of various agents in a mammary cancer model.

PURPOSE: The methylnitrosourea (MNU)-induced mammary cancer model in rats is similar to estrogen receptor-positive breast cancer in women. In prevention studies using this model, tumor incidence and multiplicity were typically primary end points. The ability of various agents administered for a short period to modulate cell proliferation [proliferation index (PI)] and apoptosis [apoptotic index (AI)] in mammary cancers was compared with their efficacy in long-term prevention and therapy studies. EXPERIMENTAL DESIGN: Rats were injected with MNU to induce mammary cancers. For the prevention studies, agents were administered by gavage or in the diet beginning 5 days after MNU. For proliferation (PI) and apoptosis (AI) experiments, animals with a palpable mammary cancer were treated with the agents for only 4 to 7 days. PI was determined following 5-bromodeoxyuridine labeling whereas AI was determined using the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay. Therapeutic efficacy was evaluated by measuring cancer size over a 6-week period. RESULTS: Treatments with differing chemopreventive efficacy and mechanism(s) of action were examined: (a) hormonal treatments [tamoxifen, vorozole (an aromatase inhibitor), and ovariectomy]; (b) retinoid X receptor agonists (targretin, 9-cis retinoic acid, and UAB30); (c) inducers of drug-metabolizing enzymes (indole-3-carbinol, 5,6 benzoflavone, and diindoylmethane); (d) agents that alter signal transduction (R115777, a farnesyltransferase inhibitor); Iressa (an epidermal growth factor receptor inhibitor); sulindac and celecoxib (cyclooxygenase 1/2 and cyclooxygenase 2 inhibitors); and (e) diverse agents including meclizine, vitamin C, and sodium phenylbutyrate. Correlations between inhibition of PI, increase of AI, and chemopreventive efficacy were observed. Although most agents with moderate or low preventive efficacy suppressed PI, they minimally affected AI. CONCLUSIONS: The data confirmed that the short-term effects of various agents on cell proliferation and apoptosis in small mammary cancers can predict their preventive/therapeutic efficacy. Thus, these biomarkers can be used to help determine the efficacy of compounds in phase II clinical prevention trials.

Advanced rat mammary cancers are growth hormone dependent.

Epidemiological studies suggest that the GH/IGF-I axis may promote human cancers. Animal models in which the GH/IGF-I axis can be controlled may be helpful in elucidating the role of these hormones during mammary cancer progression. Beginning at 3 or 5 wk of age, spontaneous dwarf rats (Gh(dr/dr)), which lack GH and have very low serum IGF-I, were treated with either rat or bovine GH twice daily. Other Gh(dr/dr) rats received vehicle, and wild-type Sprague Dawley rats (Gh(+/+), parent strain to SDR) received vehicle. One week later, all rats were exposed to a single injection of N-methyl-N-nitrosourea. Body weight gain and serum IGF-I levels were similar in Gh(+/+) and GH-treated Gh(dr/dr) rats. Furthermore, mammary tumor incidence, latency, and multiplicity were similar in Gh(+/+) and GH-treated Gh(dr/dr) rats. Vehicle-treated Gh(dr/dr) rats developed no tumors. Once advanced (> or =1 cm(3)) mammary cancers were established in GH-treated Gh(dr/dr) rats, GH treatments were halted and nearly all tumors regressed completely within 2 wk. Tumor regression was associated with loss of phospho-signal transducer and activator of transcription-3, but not alterations in IGF-I, IGF-I receptor, or GH receptor. These results demonstrate that Gh(dr/dr) rats, which are nearly refractory to mammary carcinogenesis, can be made vulnerable by restoring GH and IGF-I. Furthermore, advanced rat mammary cancers are dependent on GH and/or IGF-I for their survival. Therefore, therapeutics that target either GH or IGF-I may be effective at treating even advanced mammary cancers.

Anti-progestins suppress the growth of established tumors induced by 7,12-dimethylbenz(a)anthracene: comparison between RU486 and a new 21-substituted-19-nor-progestin.

In this report, we evaluate the effects of a 21-substituted-19-nor-progestin, CDB-4124, on 7,12,-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in rats in comparison with RU486. Sprague-Dawley female rats were treated with DMBA at 50 days of age in order to induce mammary tumors. When the tumors reached the size of 10-12 mm, the animals were treated for 28 days with the vehicle, RU486, progesterone, CDB-4124 at various doses, or CDB-4124 plus progesterone. Anti-progestins resulted in the regression in the size of the existing tumors, and in the suppressed development of new tumors and tumor multiplicity. Progesterone treatment, however, increased the size and multiplicity. Progesterone rendered an increased number of growing tumors as compared to the regression in the anti-progesterone treatment groups. The combination of CDB-4124 and high doses of progesterone opposed the efficacy of CDB-4124. The growth inhibitory effects of the anti-progestins were correlated with increased apoptosis and reduced cell proliferation. These results indicate that anti-progestins should be developed for the chemoprevention and treatment of hormone-responsive breast cancer.

Effects of the farnesyl transferase inhibitor R115777 (Zarnestra) on mammary carcinogenesis: prevention, therapy, and role of HaRas mutations.

The ability of the farnesyl transferase inhibitor R115777 to act as a cancer therapeutic/preventive agent and to modulate proliferation/apoptosis markers was determined in the methylnitrosourea-induced model of mammary carcinogenesis. Female Sprague-Dawley rats were given methylnitrosourea at 50 days of age. In the prevention study, R115777 (5, 16, or 50 mg/kg body weight/d), beginning 5 days after methylnitrosourea treatment, decreased the formation of mammary cancers by 6%, 42%, and 75%, respectively. Approximately 50% of the mammary cancers that developed had HaRas mutations. Only 1 of 15 tumors that grew out in the presence of R115777 (16 or 50 mg/kg body weight/d) had a HaRas mutation. In the therapeutic study, a surgical biopsy of a mammary cancer was done to determine HaRas status, and growth of the cancer was then followed during treatment of the rat with R115777. Virtually every cancer with a HaRas mutation underwent complete regression within 3 weeks, whereas tumors without a HaRas mutation had variable responses to the inhibitor. Both of these studies implied a high sensitivity of tumors with HaRas mutations to the effects of R115777. In order to understand the preferential susceptibility of tumors with HaRas mutations, rats with a palpable cancer were treated with R115777 for a period of 36 or 96 hours prior to sacrifice, and the proliferation and apoptosis levels in the cancers were determined. The proliferative index was significantly (>85%) decreased in all mammary cancers with HaRas mutations, whereas variable responses were observed in cancers without HaRas mutations. Apoptosis was also measured and a 5-fold increase was observed in HaRas mutant tumors, again with varying responses in the HaRas wild-type cancers. Thus, R115777 was active in the prevention and therapy of these chemically induced mammary cancers, but was strikingly more effective in cancers with HaRas mutations.

Identification of novel RARbeta2 transcript variants with short 5'-UTRs in normal and cancerous breast epithelial cells.

Functional significance of RARbeta2 as a putative tumor suppressor gene has been studied in breast cancer and other tumors. The long 5'-untranslated region (5'-UTR) of its transcript with multiple open-reading frames (uORFs) is considered as a regulatory unit for translation. Here, for the first time we identified RARbeta2 transcript variants with short 5'-UTRs in both normal and malignant breast epithelial cells. The 5'-RACE analysis of RARbeta2 mRNA in these cells demonstrated the existence of short RARbeta2 transcript variants that are identical to the sequence of known RARbeta2, but lack all the uORFs present in the full-length 5'-UTR. By RT-PCR analysis, we found that the expression of both transcripts with short and full-length 5'-UTR is mediated by retinoic acid, while cellular sensitivity is preferentially correlated to upregulation of short RARbeta2 transcript variants in response to retinoic acid. The transfection and in vitro translation assay indicated that the short 5'-UTR has no inhibitory effects on translation, while the presence of full-length 5'-UTR inhibited translation by 60%. In addition, no promoter activity was detectable in RARbeta2 full-length 5'-UTR region. Our data suggest that the RARbeta2 transcript variants with short 5'-UTR may serve as major transcripts for RARbeta2 protein translation as well as potential targets for retinoids in breast cancer prevention and therapy studies.

A novel RARbeta isoform directed by a distinct promoter P3 and mediated by retinoic acid in breast cancer cells.

Retinoids regulate gene transcription through activating retinoic acid receptors (RARs)/retinoic X receptors (RXRs). Of the three RAR receptors (alpha, beta, and gamma), RARbeta has been considered a tumor suppressor gene. Here, we identified a novel RARbeta isoform-RARbeta5 in breast epithelial cells, which could play a negative role in RARbeta signaling. Similar to RARbeta2, the first exon (59 bp) of RARbeta5 is RARbeta5 isoform specific, whereas the other exons are common to all of the RARbeta isoforms. The first exon of RARbeta5 does not contain any translation start codon, and therefore its protein translation begins at an internal methionine codon of RARbeta2, lacking the A, B, and part of C domain of RARbeta2. RARbeta5 protein was preferentially expressed in estrogen receptor-negative breast cancer cells and normal breast epithelial cells that are relatively resistant to retinoids, whereas estrogen receptor-positive cells that did not express detectable RARbeta5 protein were sensitive to retinoid treatment, suggesting that this isoform may affect the cellular response to retinoids. RARbeta5 isoform is unique among all of the RARs, because a corresponding isoform was not detectable for either RARalpha or RARgamma. RARbeta5 mRNA was variably expressed in normal and cancerous breast epithelial cells. Its transcription was under the control of a distinct promoter P3, which can be activated by all-trans-retinoic acid (atRA) and other RAR/RXR selective retinoids in MCF-7 and T47D breast cancer cells. We mapped the RARbeta5 promoter and found a region -302/-99 to be the target region of atRA. In conclusion, we identified and initially characterized RARbeta5 in normal, premalignant, and malignant breast epithelial cells. RARbeta5 may serve as a potential target of retinoids in prevention and therapy studies.

9-cis-retinoic acid but not 4-(hydroxyphenyl)retinamide inhibits prostate intraepithelial neoplasia in Noble rats.

In most previous studies, the incidence and multiplicity of chemically induced prostate tumors have been used as end points for assessing the efficacy of various chemopreventive agents. In this study, we used prostate intraepithelial neoplasia (PIN) in Noble rats as an intermediate end point to examine the chemopreventive efficacy of two retinoids, 9-cis-retinoic acid (9cRA) and 4-(hydroxyphenyl)retinamide, which in previous studies have shown promising inhibitory effects on various carcinogenesis models. We found that 80-100% of Noble rats treated for 36 weeks with testosterone + 17beta-estradiol developed multiple PIN lesions predominantly in the dorso-lateral prostate, which appears relevant to the place of origin of PIN and carcinoma in the human prostate. 9cRA at 50 or 100 mg/kg diet significantly decreased the multiplicity of PIN, whereas 4-(hydroxyphenyl) retinamide at 392 or 784 mg/kg diet, did not have an inhibitory effect on PIN. Thus, we provide for the first time evidence that the testosterone + 17beta-estradiol-induced PIN in Noble rats could be used as a potential intermediate end point in assessing the efficacy of retinoids and possibly of other agents on prostate carcinogenesis, and that 9cRA alone or in combination with other agents may have clinical promise in preventing the development of prostate cancer in men.

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.