Jennifer Santos

Preclinical trial of a new dual mTOR inhibitor, MLN0128, using renal cell carcinoma tumorgrafts

mTOR is a rational target in renal cell carcinoma (RCC) because of its role in disease progression. However, the effects of temsirolimus, the only first‐generation mTOR inhibitor approved by the FDA for first‐line treatment of metastatic RCC, on tumor reduction and progression‐free survival are minimal. Second‐generation mTOR inhibitors have not been evaluated on RCC. We compared the effects of temsirolimus and MLN0128, a potent second‐generation mTOR inhibitor, on RCC growth and metastasis using a realistic patient‐derived tissue slice graft (TSG) model. TSGs were derived from three fresh primary RCC specimens by subrenal implantation of precision‐cut tissue slices into immunodeficient mice that were randomized and treated with MLN0128, temsirolimus, or placebo. MLN0128 consistently suppressed primary RCC growth, monitored by magnetic resonance imaging (MRI), in three TSG cohorts for up to 2 months. Temsirolimus, in contrast, only transiently inhibited the growth of TSGs in one of two cohorts before resistance developed. In addition, MLN0128 reduced liver metastases, determined by human‐specific quantitative polymerase chain reaction, in two TSG cohorts, whereas temsirolimus failed to have any significant impact. Moreover, MLN0128 decreased levels of key components of the two mTOR subpathways including TORC1 targets 4EBP1, p‐S6K1, HIF1α and MTA1 and the TORC2 target c‐Myc, consistent with dual inhibition. Our results demonstrated that MLN0128 is superior to temsirolimus in inhibiting primary RCC growth as well as metastases, lending strong support for further clinical development of dual mTOR inhibitors for RCC treatment.

Establishment and serial passage of cell cultures derived from LuCaP xenografts

LuCaP serially transplantable xenografts derived from primary and metastatic human prostate cancer encompass the molecular and cellular heterogeneity of the disease and are an invaluable resource for in vivo preclinical studies. A limitation of this model, however, has been the inability to establish and passage cell cultures derived from the xenografts. Here, we describe a novel spheroid culture system that supports long‐term growth of LuCaP cells in vitro.

Induced Pluripotency of Human Prostatic Epithelial Cells

Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore, provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification.

Patient-derived tissue slice grafts accurately depict response of high-risk primary prostate cancer to androgen deprivation therapy

BackgroundEffective eradication of high-risk primary prostate cancer (HRPCa) could significantly decrease mortality from prostate cancer. However, the discovery of curative therapies for HRPCa is hampered by the lack of authentic preclinical models.MethodsWe improved upon tumorgraft models that have been shown to predict drug response in other cancer types by implanting thin, precision-cut slices of HRPCa under the renal capsule of immunodeficient mice. Tissue slice grafts (TSGs) from 6 cases of HRPCa were established in mice. Following androgen deprivation by castration, TSGs were recovered and the presence and phenotype of cancer cells were evaluated.ResultsHigh-grade cancer in TSGs generated from HRPCa displayed characteristic Gleason patterns and biomarker expression. Response to androgen deprivation therapy (ADT) was as in humans, with some cases exhibiting complete pathologic regression and others showing resistance to castration. As in humans, ADT decreased cell proliferation and prostate-specific antigen expression in TSGs. Adverse pathological features of parent HRPCa were associated with lack of regression of cancer in corresponding TSGs after ADT. Castration-resistant cancer cells remaining in TSGs showed upregulated expression of androgen receptor target genes, as occurs in castration-resistant prostate cancer (CRPC) in humans. Finally, a rare subset of castration-resistant cancer cells in TSGs underwent epithelial-mesenchymal transition, a process also observed in CRPC in humans.ConclusionsOur study demonstrates the feasibility of generating TSGs from multiple patients and of generating a relatively large number of TSGs from the same HRPCa specimen with similar cell composition and histology among control and experimental samples in an in vivo setting. The authentic response of TSGs to ADT, which has been extensively characterized in humans, suggests that TSGs can serve as a surrogate model for clinical trials to achieve rapid and less expensive screening of therapeutics for HRPCa and primary CRPC.

Radioprotection and cell cycle arrest of intestinal epithelial cells by darinaparsin, a tumor radiosensitizer.

PURPOSE It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. METHODS AND MATERIALS The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome. The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. RESULTS In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. CONCLUSIONS DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy.

Abstract B32: Patient-derived tissue slice grafts of high-risk primary prostate cancer: An authentic preclinical model for synthetic lethality-based therapy

Background: Effective eradication of high-risk primary prostate cancer (HRPCa) could significantly decrease mortality from prostate cancer. Drugs exploiting synthetic lethality have gained attention as novel anticancer agents. However, the discovery of such therapies for HRPCa is hampered by the lack of authentic preclinical models. Methods: We improved upon tumorgraft models that have been shown to predict drug response in other cancer types by implanting thin, precision-cut slices of HRPCa under the renal capsule of immunodeficient mice. Tissue slice grafts (TSGs) from 6 cases of HRPCa were established in mice. Following androgen deprivation by castration, TSGs were recovered and the presence and phenotype of cancer cells were evaluated. Results: High-grade cancer in TSGs generated from HRPCa displayed characteristic Gleason patterns and biomarker expression. Response to androgen deprivation therapy (ADT) was as in humans, with some cases exhibiting complete pathologic regression and others showing resistance to castration. As in humans, ADT decreased cell proliferation and prostate-specific antigen expression in TSGs. Adverse pathological features of parent HRPCa were associated with lack of regression of cancer in corresponding TSGs after ADT. Castration-resistant cancer cells remaining in TSGs showed upregulated expression of androgen receptor target genes, as occurs in castration-resistant prostate cancer (CRPC) in humans. Finally, a rare subset of castration-resistant cancer cells in TSGs underwent epithelial-mesenchymal transition, a process also observed in CRPC in humans. Conclusions: Our study demonstrates the feasibility of generating TSGs from multiple patients and of generating a relatively large number of TSGs from the same HRPCa specimen with similar cell composition and histology among control and experimental samples in an in vivo setting. The authentic response of TSGs to ADT, which has been extensively characterized in humans, suggests that TSGs can serve as a surrogate model for clinical trials to achieve rapid and less expensive screening of synthetic lethality based therapeutics for HRPCa and primary CRPC. Citation Format: Hongjuan Zhao, Alan Thong, Rosie Nolley, Stephen Reese, Jennifer Santos, Alexandre Ingels, Donna Peehl. Patient-derived tissue slice grafts of high-risk primary prostate cancer: An authentic preclinical model for synthetic lethality-based therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B32.

Abstract A3: A tumorgraft model of castration-resistant primary prostate cancer

Abstract Androgen receptor (AR) signaling plays a central role in all stages of prostate cancer (PCa). Androgen deprivation therapy (ADT) is used as adjuvant therapy for high-risk PCa as well as the first-line therapy for metastatic PCa. However, tumor remissions are temporary and patients almost inevitably progress to become castration resistant prostate cancer (CRPC). Most CRPC still expresses AR and depends on AR for growth, therefore, there is an urgent need to understand the regulation of AR expression and to find novel ways of inhibiting this pathway in CRPC. We developed a novel realistic model for primary CRPC by establishing tumorgrafts with thin, precision-cut tissue slices derived from high-grade primary PCa under the renal capsule of immunodeficient mice supplemented with subcutaneous testosterone pellets. One month after the implantation, the testosterones pellets were removed and the mice were castrated. Tumorgrafts from control and castrated mice were harvested one month after castration and fixed in formalin. Immunohistochemistry was performed to examine the expression of genes of interest in the grafts. Tumorgrafts derived from high-grade primary PCa resemble the original tumors in expression of all genes examined. Specifically, they are positive for AR, prostate specific antigen (PSA), cytokeratin 18, and AMCAR, but negative for p63. After castration, cancer cells remained in grafts derived from two of four tumors, whereas cancer cells in grafts derived from the other two tumors were almost completely eliminated, suggesting that some tumors respond to androgen deprivation therapy (ADT) better than the others. In the castration-resistant tumorgrafts (CRTs), PSA expression was diminished in cancer cells, but AR and cytokeratin 18 expression remained, similar to responses of human prostate to ADT. In addition, cancer cells in CRTs were less proliferative than in control grafts as determined by Ki67 staining consistent with the observation that ADT inhibit cell proliferation in human prostate. More importantly, CRTs expressed AR target genes that have been reported to be upregulated under androgen ablation in other experimental models. For example, the number of cyclin A-expressing cells was significantly higher in CRTs than in control grafts. Our results demonstrated that our novel CRT model has the appropriate characteristics to serve as a useful tool to model primary CRPC. Our results demonstrated that our novel CRT model has the appropriate characteristics to serve as a useful tool to model primary CRPC and may be used as a surrogate model for clinical trials to achieve rapid and less expressive screening of therapeutic agents for neoadjuvant or adjuvant therapy of PCa. Citation Format: Hongjuan Zhao, Alan Thong, Rosie Nolley, Stephen Reese, Jennifer Santos, Donna Peehl. A tumorgraft model of castrationresistant primary prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A3.