Ricardo M. Attar

Crystallographic structures of the ligand-binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone

The structures of the ligand-binding domains (LBD) of the wild-type androgen receptor (AR) and the T877A mutant corresponding to that in LNCaP cells, both bound to dihydrotestosterone, have been refined at 2.0 Å resolution. In contrast to the homodimer seen in the retinoid-X receptor and estrogen receptor LBD structures, the AR LBD is monomeric, possibly because of the extended C terminus of AR, which lies in a groove at the dimerization interface. Binding of the natural ligand dihydrotestosterone by the mutant LBD involves interactions with the same residues as in the wild-type receptor, with the exception of the side chain of threonine 877, which is an alanine residue in the mutant. This structural difference in the binding pocket can explain the ability of the mutant AR found in LNCaP cells (T877A) to accommodate progesterone and other ligands that the wild-type receptor cannot.

In vitro and in vivo antitumor effects of the dual insulin-like growth factor-I/insulin receptor inhibitor, BMS-554417.

The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 micromol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser473. At doses that inhibited proliferation, the compound also caused a G0-G1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo.

Constitutively Active Type I Insulin-Like Growth Factor Receptor Causes Transformation and Xenograft Growth of Immortalized Mammary Epithelial Cells and Is Accompanied by an Epithelial-to-Mesenchymal Transition Mediated by NF-κB and Snail

ABSTRACT Type I insulin-like growth factor receptor (IGF-IR) can transform mouse fibroblasts; however, little is known about the transforming potential of IGF-IR in human fibroblasts or epithelial cells. We found that overexpression of a constitutively activated IGF-IR (CD8-IGF-IR) was sufficient to cause transformation of immortalized human mammary epithelial cells and growth in immunocompromised mice. Furthermore, CD8-IGF-IR caused cells to undergo an epithelial-to-mesenchymal transition (EMT) which was associated with dramatically increased migration and invasion. The EMT was mediated by the induction of the transcriptional repressor Snail and downregulation of E-cadherin. NF-κB was highly active in CD8-IGF-IR-MCF10A cells, and both increased levels of Snail and the EMT were partially reversed by blocking NF-κB or IGF-IR activity. This study places IGF-IR among a small group of oncogenes that, when overexpressed alone, can confer in vivo tumorigenic growth of MCF10A cells and indicates the hierarchy in the mechanism of IGF-IR-induced EMT.

The Mechanisms of Differential Sensitivity to an Insulin-like Growth Factor-1 Receptor Inhibitor (BMS-536924) and Rationale for Combining with EGFR/HER2 Inhibitors

Overexpression and enhanced activity of insulin-like growth factor-I receptor (IGF-IR) in diverse tumor types make it an attractive target for cancer therapy. BMS-536924 is a potent small molecule inhibitor of IGF-IR, which shows antitumor activity in multiple tumor models, including sarcoma. To facilitate the development of IGF-IR inhibitors as cancer therapy, identification of biomarkers for selecting patients most likely to derive clinical benefit is needed. To do so, 28 sarcoma and neuroblastoma cell lines were screened for in vitro response to BMS-536924 to identify sensitive and resistant cell lines. Notably, Ewings sarcoma, rhabdomyosarcoma, and neuroblastoma are more responsive to BMS-536924, suggesting these specific subtypes may represent potential targeted patient subpopulations for the IGF-IR inhibitor. Gene expression and protein profiling were performed on these cell lines, and candidate biomarkers correlating with intrinsic and/or acquired resistance to BMS-536924 were identified. IGF-I, IGF-II, and IGF-IR were highly expressed in sensitive cell lines, whereas IGFBP-3 and IGFBP-6 were highly expressed in resistant lines. Overexpression of epidermal growth factor receptor (EGFR) and its ligands in resistant cell lines may represent one possible resistance mechanism by the adaptation of IGF-IR-independent growth using alternative signaling pathways. Based on cross-talk between IGF-IR and EGFR pathways, combination studies to target both pathways were performed, and enhanced inhibitory activities were observed. These results provide a strategy for testing combinations of IGF-IR inhibitors with other targeted therapies in clinical studies to achieve improved patient outcomes. Further exploration of mechanisms for intrinsic and acquired drug resistance by these preclinical studies may lead to more rationally designed drugs that target multiple pathways for enhanced antitumor efficacy.

Castration-Resistant Prostate Cancer: Locking Up the Molecular Escape Routes

The understanding of the key role that androgens play on the normal and pathological physiology of the prostate guided the development of different therapies for the treatment of locally advanced or metastatic prostate cancer (PCa). These so-called androgen deprivation therapies include surgical or chemical castration, achieved by the administration of gonadotropin-releasing hormone analogs; inhibition of steroidogenic enzymes; and finally, blocking of the binding of androgens to their receptor (AR) by the use of antiandrogens. Despite an excellent initial response, in approximately 2 to 3 years, most of these patients will succumb to the castration resistant form of the disease. Remarkably, even in the presence of castration levels of circulating androgens, these tumors are still dependent on a functional AR, and several molecular mechanisms have been proposed to explain this phenomenon. These include: (1) gene amplification and increased expression of the AR mRNA and protein, (2) selection of mutations in the AR that confer broader ligand specificity, (3) changes in the ratios or expression between the AR and its coregulators, (4) increased expression of steroidogenic enzymes, and (5) up-regulation of cross-talk signal transduction pathways that can activate the AR in a ligand-independent manner. We will summarize how these molecular hypotheses are being tested in the clinic by the latest therapeutic modalities.

The current state of preclinical prostate cancer animal models.

Prostate cancer continues to be a major cause of morbidity and mortality in men around the world. The field of prostate cancer research continues to be hindered by the lack of relevant preclinical models to study tumorigenesis and to further development of effective prevention and therapeutic strategies. The Prostate Cancer Foundation held a Prostate Cancer Models Working Group (PCMWG) Summit on August 6th and 7th, 2007 to address these issues. The PCMWG reviewed the state of prostate cancer preclinical models and identified the current limitations of cell line, xenograft and genetically engineered mouse models that have hampered the transition of scientific findings from these models to human clinical trials. In addition the PCMWG identified administrative issues that inhibit the exchange of models and impede greater interactions between academic centers and these centers with industry. The PCMWG identified potential solutions for discovery bottlenecks that include: (1) insufficient number of models with insufficient molecular and biologic diversity to reflect human cancer, (2) a lack of understanding of the molecular events that define tumorigenesis, (3) a lack of tools for studying tumor–host interactions, (4) difficulty in accessing model systems across institutions, and (5) addressing why preclinical studies appear not to be predictive of human clinical trials. It should be possible to apply the knowledge gained molecular and epigenetic studies to develop new cell lines and models that mimic progressive and fatal prostate cancer and ultimately improve interventions. Prostate 68: 629–639, 2008.

The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens

BackgroundA crucial event in Prostate Cancer progression is the conversion from a hormone-sensitive to a hormone-refractory disease state. Correlating with this transition, androgen receptor (AR) amplification and mutations are often observed in patients failing hormonal ablation therapies. β-Catenin, an essential component of the canonical Wnt signaling pathway, was shown to be a coactivator of the AR signaling in the presence of androgens. However, it is not yet clear what effect the increased levels of the AR could have on the Wnt signaling pathway in these hormone-refractory prostate cells.ResultsTransient transfections of several human prostate cancer cell lines with the AR and multiple components of the Wnt signaling pathway demonstrate that the AR overexpression can potentiate the transcriptional activities of Wnt/β-Catenin signaling. In addition, the simultaneous activation of the Wnt signaling pathway and overexpression of the AR promote prostate cancer cell growth and transformation at castration levels of androgens. Interestingly, the presence of physiological levels of androgen or other AR agonists inhibits these effects. These observations are consistent with the nuclear co-localization of the AR and β-Catenin shown by immunohistochemistry in human prostate cancer samples. Furthermore, chromatin immunoprecipitation assays showed that Wnt3A can recruit the AR to the promoter regions of Myc and Cyclin D1, which are well-characterized downstream targets of the Wnt signalling pathway. The same assays demonstrated that the AR and β-Catenin can be recruited to the promoter and enhancer regions of a known AR target gene PSA upon Wnt signaling. These results suggest that the AR is promoting Wnt signaling at the chromatin level.ConclusionOur findings suggest that the AR signaling through the Wnt/β-Catenin pathway should be added to the well established functional interactions between both pathways. Moreover, our data show that via this interaction the AR could promote prostate cell malignancy in a ligand-independent manner.

Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors.

In developing inhibitors of the LIM kinases, the initial lead molecules combined potent target inhibition with potent cytotoxic activity. However, as subsequent compounds were evaluated, the cytotoxic activity separated from inhibition of LIM kinases. A rapid determination of the cytotoxic mechanism and its molecular target was enabled by integrating data from two robust core technologies. High-content assays and gene expression profiling both indicated an effect on microtubule stability. Although the cytotoxic compounds are still kinase inhibitors, and their structures did not predict tubulin as an obvious target, these results provided the impetus to test their effects on microtubule polymerization directly. Unexpectedly, we confirmed tubulin itself as a molecular target of the cytotoxic kinase inhibitor compounds. This general approach to mechanism of action questions could be extended to larger data sets of quantified phenotypic and gene expression data. [Mol Cancer Ther 2008;7(11):3490–8]

HER receptor signaling confers resistance to the insulin-like growth factor-I receptor inhibitor, BMS-536924

We have reported previously the activity of the insulin-like growth factor-I (IGF-IR)/insulin receptor (InsR) inhibitor, BMS-554417, in breast and ovarian cancer cell lines. Further studies indicated treatment of OV202 ovarian cancer cells with BMS-554417 increased phosphorylation of HER-2. In addition, treatment with the pan-HER inhibitor, BMS-599626, resulted in increased phosphorylation of IGF-IR, suggesting a reciprocal cross-talk mechanism. In a panel of five ovarian cancer cell lines, simultaneous treatment with the IGF-IR/InsR inhibitor, BMS-536924 and BMS-599626, resulted in a synergistic antiproliferative effect. Furthermore, combination therapy decreased AKT and extracellular signal-regulated kinase activation and increased biochemical and nuclear morphologic changes consistent with apoptosis compared with either agent alone. In response to treatment with BMS-536924, increased expression and activation of various members of the HER family of receptors were seen in all five ovarian cancer cell lines, suggesting that inhibition of IGF-IR/InsR results in adaptive up-regulation of the HER pathway. Using MCF-7 breast cancer cell variants that overexpressed HER-1 or HER-2, we then tested the hypothesis that HER receptor expression is sufficient to confer resistance to IGF-IR-targeted therapy. In the presence of activating ligands epidermal growth factor or heregulin, respectively, MCF-7 cells expressing HER-1 or HER-2 were resistant to BMS-536924 as determined in a proliferation and clonogenic assay. These data suggested that simultaneous treatment with inhibitors of the IGF-I and HER family of receptors may be an effective strategy for clinical investigations of IGF-IR inhibitors in breast and ovarian cancer and that targeting HER-1 and HER-2 may overcome clinical resistance to IGF-IR inhibitors. [Mol Cancer Ther 2008;7(9):2589–98]

Drug safety is a barrier to the discovery and development of new androgen receptor antagonists

Androgen receptor (AR) antagonists are part of the standard of care for prostate cancer. Despite the almost inevitable development of resistance in prostate tumors to AR antagonists, no new AR antagonists have been approved for over a decade. Treatment failure is due in part to mutations that increase activity of AR in response to lower ligand concentrations as well as to mutations that result in AR response to a broader range of ligands. The failure to discover new AR antagonists has occurred in the face of continued research; to enable progress, a clear understanding of the reasons for failure is required.