New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Multi-drug loaded micelles delivering chemotherapy and targeted therapies directed against HSP90 and the PI3K/AKT/mTOR pathway in prostate cancer.

BACKGROUND:Advanced prostate cancers that are resistant to all current therapies create a need for new therapeutic strategies. One recent innovative approach to cancer therapy is the simultaneous use of multiple FDA-approved drugs to target multiple pathways. A challenge for this approach is caused by the different solubility requirements of each individual drug, resulting in the need for a drug vehicle that is non-toxic and capable of carrying multiple water-insoluble antitumor drugs. Micelles have recently been shown to be new candidate drug solubilizers for anti cancer therapy. METHODS:This study set out to examine the potential use of multi-drug loaded micelles for prostate cancer treatment in preclinical models including cell line and mouse models for prostate cancers with Pten deletions. Specifically antimitotic agent docetaxel, mTOR inhibitor rapamycin, and HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin were incorporated into the micelle system (DR17) and tested for antitumor efficacy. RESULTS:In vitro growth inhibition of prostate cancer cells was greater when all three drugs were used in combination compared to each individual drug, and packaging the drugs into micelles enhanced the cytotoxic effects. At the molecular level DR17 targeted simultaneously several molecular signaling axes important in prostate cancer including androgen receptor, mTOR, and PI3K/AKT. In a mouse genetic model of prostate cancer, DR17 treatment decreased prostate weight, which was achieved by both increasing caspase-dependent cell death and decreasing cell proliferation. Similar effects were also observed when DR17 was administered to nude mice bearing prostate cancer cells xenografts. CONCLUSION:These results suggest that combining these three cancer drugs in multi-drug loaded micelles may be a promising strategy for prostate cancer therapy

Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate

<div><p>The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene <i>Odc1</i>. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, <i>Nkx3</i>.<i>1</i>, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene <i>Notch1</i>. Other genes implicated in prostatic development including <i>Sox9</i>, <i>Wif1</i> and <i>Srd5a2</i> were unaffected by DFMO. Together these results indicate that <i>Odc1</i> and polyamines are required for androgens to exert their effect in mediating prostatic bud induction, and are required for the expression of a subset of prostatic developmental regulatory genes including <i>Notch1</i> and <i>Nkx3</i>.<i>1</i>.</p></div

Allowing prostatic buds to grow before polyamine depletion did not rescue <i>Nkx3</i>.<i>1</i> expression.

<p>We determined the time course of prostatic bud formation over 2 days of culture in testosterone, by culturing the UGS in the presence of testosterone for the time indicated, fixed the cultures, and stained them with E-Cadherin to examine prostatic bud formation. After 24 hours or 1 day in culture, prostatic buds were still not apparent (A). After 1.5 days in testosterone, several prostatic buds were observed (B, white arrowheads). The buds were longer and more numerous after 2 days in testosterone (C, white arrowheads). Culturing the UGS in testosterone for two days to allow buds to grow, followed by polyamine depletion did not rescue <i>Nkx3</i>.<i>1</i> expression (D). *, p<0.05.</p

Ornithine decarboxylase protein was present in the urogenital epithelium in the developing urogenital sinus.

<p>In the male UGS before budding at E15 (A) and E16 (B), ornithine decarboxylase protein (green) was present in the epithelium. As the buds emerged from the epithelium at E17 (E) and E18 (G), ornithine decarboxylase protein was still expressed in the epithelium and buds. In the female UGS, ornithine decarboxylase was present in the epithelium at E15 (B) and E16 (D), the period before prostatic buds are initiated in the male UGS. At E17 (F) and E18 (H), ornithine decarboxylase protein continued to be expressed in the urogenital epithelium. Ornithine decarboxylase staining is in green, and vimentin staining is in red. There was some co-localization of ornithine decarboxylase and vimentin (yellow) in the mesenchyme. Arrowheads denote prostatic buds. Abbreviations: E epithelium, M mesenchyme.</p

<i>Odc1</i> mRNA levels in the developing urogenital sinus and in separated UGS tissue compartments.

<p><i>Odc1</i> transcripts were present in the male and female UGS throughout the period of prostatic bud induction from E15-E18, although they did not vary between males and females during this period (A). <i>Odc1</i> transcript abundance did not differ between the epithelium and mesenchyme at E16 (B), although there were significantly more <i>Odc1</i> transcripts in the mesenchyme compared to the epithelium at E18 (B). Abbreviations used: epi epithelium, mes mesenchyme. *, p<0.05.</p

Dose response of prostate cancer cells to individual drugs.

<p>(A-B) Cells were grown in either control regular media or media supplemented with DHT of increasing concentrations. Cell growth was assessed using Alamar Blue assay as described in Materials and Methods. Results are representative of 3 independent experiments. (C) Immunoblotting was performed from equal amount of total protein using the phospho-AKT, AKT, and β-actin antibodies as described in Materials and Methods. (D-I) Cytotoxic effects of increasing doses of docetaxel (D, G), rapamycin (E, H), and 17-AAG (F, I) in PTEN-P2 cells (D, E, F) and PTEN-CaP2 cells (G, H, I). Cells were exposed to the indicated concentration of drug-loaded micelle or empty micelle for 72 hours. Cell viability was assessed using Alamar Blue assay as described in Materials and Methods. * indicates statistically significant differences compared to micelle control (ANOVA, p < 0.05). Results are representative of 3 independent experiments.</p

Histology of DR17 and micelle control treated prostates.

<p>Pten het N = 9 (A, B, E, F, I, J) and Pten null N = 6 (C, D, G, H, K, L) mice were treated with empty micelles (Ctrl) or DR17 loaded micelles for 3 weeks. Animals were sacrifice two days after the last injection. Prostates were dissected and fixed in formalin. Prostate sections were used for H&E staining to assess overall morphology (A-D). Immunohistochemistry was performed to examine stromal smooth muscle actin (SMA, E-H) and epithelial cytokeratin (I-L) markers. Pictures were taken at 40X magnification. The black bar in each picture represents 30 microns.</p

Dose response of prostate cancer cells to multi-drug loaded micelles.

<p>Cytotoxic effects of DR17 in PTEN-P2 (A, C, E) and PTEN-CaP2 cells (B, D, F). Dose responses of PTEN-P2 (A, C) and PTEN-CaP2 (B, D) to DR17 free form or DR17 delivered by micelle (DR17). Cells were exposed to the indicated concentrations of free form DR17 or DMSO, or DR17 loaded micelle or empty micelle for 72 hours. Cell viability was assessed using Alamar Blue assay as described in Materials and Methods. E-F, Comparison of cytotoxic effects of three-drug combination DR17 to individual drug loaded micelles (C, D). For C and D, DR17 was added to the media to the final concentration of 105μM. Final concentrations of docetaxel, rapamycin, 17-AAG were calculated based on molarity ratio of each individual drug in the DR17 formula [docetaxel:rapamycin:17-AAG (1:1:8.5)], which are 12.38μM, 10.94μM, and 85.37μM, respectively. Cells were exposed to the indicated drug conditions for 72 hours. Cell viability was assessed using Alamar Blue assay as described in Materials and Methods. * indicates statistically significant differences compared to DR17 treatment (ANOVA, p < 0.05). Results are representative of 3 independent experiments.</p

Effects of DR17 on cell proliferation, apoptosis, and prostate basal cells.

<p>Pten het N = 9 (A, B, E, F, I, J) and Pten null N = 6 (C, D, G, H, K, L) mice were treated with empty micelles or DR17 loaded micelles for 3 weeks. Animals were sacrifice two days after the last injection. Prostates were dissected and fixed in formalin. Immunohistochemistry staining for proliferation marker Ki67 (A-D), apoptosis marker cleaved caspase 3 (E-H), and basal cell marker p63 (I-L) was performed on prostate tissue sections. Pictures were taken at 100X magnification. The black bar in each picture represents 10 microns. Quantification of Ki67 (M), cleaved caspase 3 (N), and p63 (O) labeling index was conducted as described in Materials and Methods. Differences from control were statistically significant as indicated by * (Student’s <i>t</i> test, p < 0.05).</p