Robert D. Bruno

Reprogramming human cancer cells in the mouse mammary gland.

The tissue microenvironment directs stem/progenitor cell behavior. Cancer cells are also influenced by the microenvironment. It has been shown that, when placed into blastocysts, cancer cells respond to embryonic cues and differentiate according to the tissue type encountered during ontological development. Previously, we showed that the mouse mammary gland was capable of redirecting adult mouse testicular and neural stem/progenitor cells toward a mammary epithelial cell fate during gland regeneration. Here, we report that human embryonal carcinoma cells proliferate and produce differentiated mammary epithelial cell progeny when mixed with mouse mammary epithelial cells and inoculated into the epithelium-free mammary fat pads of athymic nude mice. Fluorescence in situ hybridization confirmed the presence of human cell progeny in the mammary outgrowths for human centromeric DNA, as well as immunochemistry for human-specific breast epithelial cytokeratins and human-specific milk proteins in impregnated transplant hosts. It was found that the number of human cells increased by 66- to 660-fold during mammary epithelial growth and expansion as determined by human cytokeratin expression. All features found in primary outgrowths were recapitulated in the secondary outgrowths from chimeric implants. These results show that human embryonal carcinoma-derived progeny interact with mouse mammary cells during mammary gland regeneration and are directed to differentiate into cells that exhibit diverse mammary epithelial cell phenotypes. This is the first demonstration that human cells are capable of recognizing the signals generated by the mouse mammary gland microenvironment present during gland regeneration in vivo.

Synthesis and biological evaluations of putative metabolically stable analogs of VN/124-1 (TOK-001): Head to head anti-tumor efficacy evaluation of VN/124-1 (TOK-001) and abiraterone in LAPC-4 human prostate cancer xenograft model

In a continuing study of our clinical candidate 5 VN/124-1 (TOK-001) and analogs as potential agents for prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using western blot analysis, we have generated a preliminary structure-activity relationship (SAR) of 5 and related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical trials. In our desire to optimize the potency of 5, compounds 6 (3ξ-fluoro-) and 9 (3β-sulfamate-) designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We showed, that on equimolar basis, compound 6 was ∼2-fold more efficacious versus LAPC-4 xenografts than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a potential treatment of prostate cancer.

17α-Hydroxylase/17,20 lyase inhibitor VN/124-1 inhibits growth of androgen-independent prostate cancer cells via induction of the endoplasmic reticulum stress response

Inhibitors of the enzyme 17α-hydroxylase/17,20 lyase are a new class of anti–prostate cancer agents currently undergoing preclinical and clinical development. We have previously reported the superior anticancer activity of our novel 17α-hydroxylase/17,20 lyase inhibitor, VN/124-1, against androgen-dependent cancer models. Here, we examined the effect of VN/124-1 on the growth of the androgen-independent cell lines PC-3 and DU-145 and found that the compound inhibits their growth in a dose-dependent manner in vitro (GI50, 7.82 μmol/L and 7.55 μmol/L, respectively). We explored the mechanism of action of VN/124-1 in PC-3 cells through microarray analysis and found that VN/124-1 up-regulated genes involved in stress response and protein metabolism, as well as down-regulated genes involved in cell cycle progression. Follow-up real-time PCR and Western blot analyses revealed that VN/124-1 induces the endoplasmic reticulum stress response resulting in down-regulation of cyclin D1 protein expression and cyclin E2 mRNA. Cell cycle analysis confirmed G1-G0 phase arrest. Measurements of intracellular calcium levels ([Ca2+]i) showed that 20 μmol/L VN/124-1 caused a release of Ca2+ from endoplasmic reticulum stores resulting in a sustained increase in [Ca2+]i. Finally, cotreatment of PC-3 cells with 5, 10, and 20 μmol/L VN/124-1 with 10 nmol/L thapsigargin revealed a synergistic relationship between the compounds in inhibiting PC-3 cell growth. Taken together, these findings show VN/124-1 is endowed with multiple anticancer properties that may contribute to its utility as a prostate cancer therapeutic. [Mol Cancer Ther 2008;7(9):2828–36]

The mouse mammary microenvironment redirects mesoderm-derived bone marrow cells to a mammary epithelial progenitor cell fate.

Mammary stem cells reside in protected tissue locales (niches), where their reproductive potency remains essentially unchanged through life. Disruption of the tissue leads to a reduced capacity of dispersed epithelial cells to recapitulate complete functional mammary structures. Previous studies demonstrate that during the reformation of mammary stem cell niches by dispersed epithelial cells in the mammary stroma, nonmammary cells of ectodermal germ origin may be sequestered and reprogrammed to perform mammary epithelial cell (MEC) functions, including those ascribed to mammary stem/progenitor cells. To test whether tissue cells from organs derived from different germ layers could respond to mammary epithelial-specific signals, we utilized fluorescence-activated cell sorting-purified Lin(-) and Lin(-)/cKit+adult male bone marrow cells to mix with MECs. Our evidence shows that the signals provided by the mammary microenvironment are capable of redirecting mesoderm-derived adult progenitor cells to produce functional MEC progeny.

Functional Characterization of Stem Cell Activity in the Mouse Mammary Gland

Any portion of the mouse mammary gland is capable of recapitulating a clonally derived complete and functional mammary tree upon transplantation into an epithelial divested mammary fat-pad of a recipient host. As such, it is an ideal model tissue for the study somatic stem cell function. This review will outline what is known regarding the function of stem/progenitor cells in the mouse mammary gland, including how progenitor populations can be functionally defined, the evidence for and potential role of selective DNA strand segregation, and the role of the niche in maintaining and controlling stem cell function.

Potent anti-prostate cancer agents derived from a novel androgen receptor down-regulating agent

The search for novel androgen receptor (AR) down-regulating agents by catalyst HipHop pharmacophore modeling led to the discovery of some lead molecules. Unexpectedly, the effect of these leads on human prostate cancer LNCaP cell viability did not correlate with the ability of the compounds to cause down-regulation of AR protein expression. Through rational synthetic optimization of the lead compound (BTB01434), we have discovered a series of novel substituted diaryl molecules as potent anti-prostate cancer agents. Some compounds (1-6) were shown to be extremely potent inhibitors of LNCaP cell viability with GI(50) values in the nanomolar range (1.45-83 nM). The most potent compound (4-methylphenyl)[(4-methylphenyl)sulfonyl]amine (5) with a GI(50) value of 1.45 nM is 27,000 times more potent than our lead compound BTB01434 (GI(50)=39.8 microM). In addition, some of the compounds exhibited modest anti-androgenic activities and one was also a potent inhibitor (GI(50)=850 nM) of PC-3 (AR-null) cell growth. A clear structure-activity relationship (SAR) has been established for activity against LNCaP cells, where potent molecules possess two substituted/unsubstituted aromatic rings connected through a sulfonamide linker. These novel compounds are strong candidates for development for the treatment of hormone-sensitive and importantly hormone-refractory prostate cancers in humans.

Embryonic Stem Cells Are Redirected to Non-Tumorigenic Epithelial Cell Fate by Interaction with the Mammary Microenvironment

Experiments were conducted to redirect mouse Embryonic Stem (ES) cells from a tumorigenic phenotype to a normal mammary epithelial phenotype in vivo. Mixing LacZ-labeled ES cells with normal mouse mammary epithelial cells at ratios of 1∶5 and 1∶50 in phosphate buffered saline and immediately inoculating them into epithelium-divested mammary fat pads of immune-compromised mice accomplished this. Our results indicate that tumorigenesis occurs only when normal mammary ductal growth is not achieved in the inoculated fat pads. When normal mammary gland growth occurs, we find ES cells (LacZ+) progeny interspersed with normal mammary cell progeny in the mammary epithelial structures. We demonstrate that these progeny, marked by LacZ expression, differentiate into multiple epithelial subtypes including steroid receptor positive luminal cells and myoepithelial cells indicating that the ES cells are capable of epithelial multipotency in this context but do not form teratomas. In addition, in secondary transplants, ES cell progeny proliferate, contribute apparently normal mammary progeny, maintain their multipotency and do not produce teratomas.

Notch-induced mammary tumorigenesis does not involve the lobule-limited epithelial progenitor.

The mouse mammary epithelial cell hierarchy contains both multipotent stem cell as well as lineage-limited duct and lobular progenitor cell functions. The latter—also termed parity-identified mammary epithelial cells (PI-MECs)—are marked by beta-galactosidase (β Gal) expression following pregnancy and involution in whey acidic protein promoter (WAP)-Cre/Rosa26-flox-stop-flox-lacZ (WC/R26) mice, and are the targets of tumorigenic transformation in mouse mammary tumor virus-erbB2 transgenic mice. In this study, we demonstrate that an epithelial population distinct from PI-MECs is transformed during WAP-Int3 tumorigenesis. As expected, WAP-Int3/WC/R26 triple-transgenic mice failed to undergo secretory alveolar development, failed to lactate and developed mammary tumors. Following pregnancy and involution, β Gal+ mammary epithelial cells were found in the normal mammary tissue, but the resulting mammary tumors were all β Gal−. WAP-Int3/WC/R26 mammary glands contained ample estrogen receptor alpha (ERα)+ MECs, but only rare (<1%) progesterone receptor (PR)+ and RANKL+ cells. In addition, dissociated MECs from WAP-Int3/WC/R26 glands failed to regenerate a mammary tree upon transplantation into a cleared fat-pad of a nu/nu recipient mouse. However, when mixed with normal MECs, PI-MECs from WAP-Int3/WC/R26 mice contributed progeny to the resulting functional outgrowth. The WAP-Int3/WC/R26-derived PI-MECs displayed all of the properties of fully functional lobular progenitors including giving rise to ERα+, PR+, smooth muscle actin+ and RANKL+ epithelial progeny. These results demonstrate that WAP-Int3 has no oncogenic effect upon PI-MECs and that the expansion of functional lobular progenitors is required for secretory alveolar development and lactation. Furthermore, lobular progenitor function is ultimately controlled by signals within its microenvironment.

Mammary Extracellular Matrix Directs Differentiation of Testicular and Embryonic Stem Cells to Form Functional Mammary Glands In Vivo

Previously, we demonstrated the ability of the normal mammary microenvironment (niche) to direct non-mammary cells including testicular and embryonic stem cells (ESCs) to adopt a mammary epithelial cell (MEC) fate. These studies relied upon the interaction of transplanted normal MECs with non-mammary cells within the mammary fat-pads of recipient mice that had their endogenous epithelium removed. Here, we tested whether acellular mammary extracellular matrix (mECM) preparations are sufficient to direct differentiation of testicular-derived cells and ESCs to form functional mammary epithelial trees in vivo. We found that mECMs isolated from adult mice and rats were sufficient to redirect testicular derived cells to produce normal mammary epithelial trees within epithelial divested mouse mammary fat-pads. Conversely, ECMs isolated from omental fat and lung did not redirect testicular cells to a MEC fate, indicating the necessity of tissue specific components of the mECM. mECM preparations also completely inhibited teratoma formation from ESC inoculations. Further, a phenotypically normal ductal outgrowth resulted from a single inoculation of ESCs and mECM. To the best of our knowledge, this is the first demonstration of a tissue specific ECM driving differentiation of cells to form a functional tissue in vivo.

Galeterone and VNPT55 disrupt Mnk‐eIF4E to inhibit prostate cancer cell migration and invasion

Metastatic castration‐resistant prostate cancer (mCRPC) accounts for a high percentage of prostate cancer mortality. The proprietary compound galeterone (gal) was designed to inhibit proliferation of androgen/androgen receptor (AR)‐dependent prostate cancer cell in vitro and in vivo and is currently in phase III clinical development. Additionally, clinical studies with gal revealed its superb efficacy in four different cohorts of patients with mCRPC, including those expressing splice variant AR‐V7. Preclinical studies with gal show that it also exhibits strong antiproliferative activities against AR‐negative prostate cancer cells and tumors through a mechanism involving phosphorylation of eIF2α, which forms an integral component of the eukaryotic mRNA translation complex. Thus, we hypothesized that gal and its new analog, VNPT55, could modulate oncogenic mRNA translation and prostate cancer cell migration and invasion. We report that gal and VNPT55 profoundly inhibit migration and invasion of prostate cancer cells, possibly by down‐regulating protein expression of several EMT markers (Snail, Slug, N‐cadherin, vimentin, and MMP‐2/‐9) via antagonizing the Mnk–eIF4E axis. In addition, gal/VNPT55 inhibited both NF‐κB and Twist1 transcriptional activities, down‐regulating Snail and BMI‐1 mRNA expression, respectively. Furthermore, profound up‐regulation of E‐cadherin mRNA and protein expression may explain the observed significant inhibition of prostate cancer cell migration and invasion. Moreover, expression of self‐renewal proteins, β‐catenin, CD44, and Nanog, was markedly depleted. Analysis of gal/VNPT55‐treated CWR22Rv1 xenograft tissue sections also revealed that observations in vitro were recapitulated in vivo. Our results suggest that gal/VNPT55 could become promising agents for the prevention and/or treatment of all stages of prostate cancer.