Rosalie Nolley

Tissue Slice Grafts : An in Vivo Model of Human Prostate Androgen Signaling

We developed a tissue slice graft (TSG) model by implanting thin, precision-cut tissue slices derived from fresh primary prostatic adenocarcinomas under the renal capsule of immunodeficient mice. This new in vivo model not only allows analysis of approximately all of the cell types present in prostate cancer within an intact tissue microenvironment, but also provides a more accurate assessment of the effects of interventions when tissues from the same specimen with similar cell composition and histology are used as control and experimental samples. The thinness of the slices ensures that sufficient samples can be obtained for large experiments as well as permits optimal exchange of nutrients, oxygen, and drugs between the grafted tissue and the host. Both benign and cancer tissues displayed characteristic histology and expression of cell-type specific markers for up to 3 months. Moreover, androgen-regulated protein expression diminished in TSGs after androgen ablation of the host and was restored after androgen repletion. Finally, many normal secretory epithelial cells and cancer cells in TSGs remained viable 2 months after androgen ablation, consistent with similar observations in postprostatectomy specimens following neoadjuvant androgen ablation. Among these were putative Nkx3.1(+) stem cells. Our novel TSG model has the appropriate characteristics to serve as a useful tool to model all stages of disease, including normal tissue, premalignant lesions, well-differentiated cancer, and poorly differentiated cancer.

The Significance of Monoamine Oxidase-A Expression in High Grade Prostate Cancer

PURPOSE Gleason grade 4/5 prostate cancer is a determinant for recurrence following radical prostatectomy. Monoamine oxidase-A is over expressed in grade 4/5 compared to grade 3 cancer. Monoamine oxidase-A is also expressed by normal basal cells and in vitro studies suggest that its function is to repress secretory differentiation. Therefore, monoamine oxidase-A in grade 4/5 cancer might reflect dedifferentiation to a basal cell-like phenotype. We investigated whether monoamine oxidase-A expression correlates with another basal cell protein, CD44, in high grade cancer and whether either is associated with an aggressive phenotype. MATERIALS AND METHODS A total of 133 grade 4/5 archival cancers from a cohort previously used to evaluate the prognostic significance of histomorphological variables were scored for monoamine oxidase-A and CD44 immunohistochemical labeling. Spearman rank correlations of the proteins, and histomorphological and clinical variables were determined. The univariate and multivariate value of each variable as a determinant of biochemical recurrence was assessed by logistic regression. RESULTS Monoamine oxidase-A expression correlated with CD44. Neither was prognostic for biochemical recurrence. However, monoamine oxidase-A expression positively correlated with preoperative serum prostate specific antigen and the percent of grade 4/5 cancer. CONCLUSIONS Concurrent expression of monoamine oxidase-A and CD44 suggests that grade 4/5 cancer may be basal cell-like in nature, despite the absence of other classic basal cell biomarkers such as cytokeratins 5 and 14, and p63. The correlation of monoamine oxidase-A expression with prostate specific antigen and the percent of grade 4/5 cancer suggests that monoamine oxidase-A may contribute to growth of high grade cancer and that antidepressant drugs that target monoamine oxidase-A may have applications in treating prostate cancer.

Elevated Serum MicroRNA Levels Associate with Absence of High-Grade Prostate Cancer in a Retrospective Cohort

To reduce treatment of indolent prostate cancer (PCa), biomarkers are needed to improve identification of patients with a low-risk of having aggressive disease. Over-treatment of these patients occurs because of uncertainty in the aggressiveness of the entire tumor based on the biopsies, which do not accurately sample multifocal tumors. Circulating microRNAs (miRNAs) are stable serum markers and differential miRNA levels occur in men with PCa. The goal of this study was to identify circulating miRNAs that were associated with aggressive or indolent PCa. We measured circulating miRNAs in 150 patients prior to surgery and compared the miRNA levels to the pathology of the entire radical prostatectomy specimen. For this study we used an exceptionally well-characterized cohort of patients who had benign prostatic hyperplasia (BPH), low-grade or high-grade PCa. Low-grade was defined as patients with 100% Gleason grade 3 tumor as determined by step-wise sectioning. High-grade PCa patients had 30-90% Gleason grade 4+5 in the tumor. BPH patients had at least two biopsies negative for PCa. Twenty one miRNAs were selected for analysis. The miRNAs were quantified by RT-qPCR and analyzed by logistic regression. High levels of 14 miRNAs were exclusively present in the serum from patients with low-grade PCa or BPH, compared to men with high-grade PCa who had consistently low levels. The expression levels of the 14 miRNAs were combined into a “miR Score” which had a negative predictive value (NPV) of 0.939 to predict absence of high-grade PCa among PCa and BPH patients. Biochemical recurrence (BCR) was known for the PCa patients and a combined “miR Risk Score” accurately classified a subset of patients with low risk of BCR (NPV 0.941). In summary, measurement of serum miRNAs may have pre-surgical utility in combination with clinical risk calculators to identify patients with low risk of harboring aggressive PCa.

Coordinate Loss of MAP3K7 and CHD1 Promotes Aggressive Prostate Cancer

Prostate cancer subtypes are poorly defined and functional validation of drivers of ETS rearrangement-negative prostate cancer has not been conducted. Here, we identified an ETS(-) subtype of aggressive prostate cancer (ERG(-)MAP3K7(del)CHD1(del)) and used a novel developmental model and a cell line xenograft model to show that cosuppression of MAP3K7 and CHD1 expression promotes aggressive disease. Analyses of publicly available prostate cancer datasets revealed that MAP3K7 and CHD1 were significantly codeleted in 10% to 20% of localized tumors and combined loss correlated with poor disease-free survival. To evaluate the functional impact of dual MAP3K7-CHD1 loss, we suppressed Map3k7 and/or Chd1 expression in mouse prostate epithelial progenitor/stem cells (PrP/SC) and performed tissue recombination experiments in vivo. Dual shMap3k7-shChd1 PrP/SC recombinants displayed massive glandular atypia with regions of prostatic intraepithelial neoplasia and carcinoma apparent. Combined Map3k7-Chd1 suppression greatly disrupted normal prostatic lineage differentiation; dual recombinants displayed significant androgen receptor loss, increased neuroendocrine differentiation, and increased neural differentiation. Clinical samples with dual MAP3K7-CHD1 loss also displayed neuroendocrine and neural characteristics. In addition, dual Map3k7-Chd1 suppression promoted E-cadherin loss and mucin production in recombinants. MAP3K7 and CHD1 protein loss also correlated with Gleason grade and E-cadherin loss in clinical samples. To further validate the phenotype observed in the PrP/SC model, we suppressed MAP3K7 and/or CHD1 expression in LNCaP prostate cancer cells. Dual shMAP3K7-shCHD1 LNCaP xenografts displayed increased tumor growth and decreased survival compared with shControl, shMAP3K7, and shCHD1 xenografts. Collectively, these data identify coordinate loss of MAP3K7 and CHD1 as a unique driver of aggressive prostate cancer development.

Inhibition of monoamine oxidase A promotes secretory differentiation in basal prostatic epithelial cells

Monoamine oxidase A (MAO-A) expression is associated with high-grade prostate cancer. Immunohistochemistry showed that MAO-A is also expressed in the basal epithelial cells of normal prostate glands. Using cultured primary prostatic epithelial cells as a model, we showed that MAO-A prevents basal epithelial cells from differentiating into secretory cells. Under differentiation-promoting conditions, clorgyline, an irreversible MAO-A inhibitor, induced secretory cell-like morphology and repressed expression of cytokeratin 14, a basal cell marker. More importantly, clorgyline induced mRNA and protein expression of androgen receptor (AR), a hallmark of secretory epithelial cells. In clorgyline-treated cells, androgen induced luciferase activity controlled by the promoter of prostate-specific antigen, an AR target gene, in a dose-dependent manner. This activity was blocked by the AR antagonist Casodex, showing that AR is functional. In turn, androgen decreased MAO-A expression in clorgyline-treated, secretory-like cells. Our results demonstrated that cultured basal epithelial cells have the potential to differentiate into secretory cells, and that inhibition of MAO-A is a key factor in promoting this process. Increased expression of MAO-A in high-grade prostate cancer may be an important contributor to its de-differentiated phenotype, raising the possibility that MAO-A inhibition may restore differentiation and reverse the aggressive behavior of high-grade cancer.

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.

Transcript Levels of Androgen Receptor Variant AR-V1 or AR-V7 Do Not Predict Recurrence in Patients with Prostate Cancer at Indeterminate Risk for Progression

PURPOSE AR-V7, a ligand independent splice variant of androgen receptor, may support the growth of castration resistant prostate cancer and have prognostic value. Another variant, AR-V1, interferes with AR-V7 activity. We investigated whether AR-V7 or V1 expression would predict biochemical recurrence in men at indeterminate (about 50%) risk for progression following radical prostatectomy. MATERIALS AND METHODS AR-V7 and V1 transcripts in a mixed grade cohort of 53 men in whom cancer contained 30% to 70% Gleason grade 4/5 and in a grade 3 only cohort of 52 were measured using a branched chain DNA assay. Spearman rank correlations of the transcripts, and histomorphological and clinical variables were determined. AR-V7 and V1 levels were assessed as determinants of recurrence in the mixed grade cohort by logistic regression and survival analysis. The impact of TMPRSS2-ERG gene fusion on prognosis was also evaluated. RESULTS Neither AR-V7 nor V1 levels in grade 3 or 4/5 cancer in the mixed grade cohort were associated with recurrence or time to recurrence. However, AR-V7 and V1 inversely correlated with serum prostate specific antigen and positively correlated with age. The AR-V1 level in grade 3 cancer in the grade 3 only cohort was higher than in grade 3 or grade 4/5 components of mixed grade cancer. TMPRSS2-ERG fusion was not associated with AR-V7, AR-V1 or recurrence but it was associated with the percent of grade 4/5 cancer. CONCLUSIONS The AR-V1 or V7 transcript level does not predict recurrence in patients with high grade prostate cancer at indeterminate risk for progression. Grade 3 cancer in mixed grade tumors may differ from 100% grade 3 cancer, at least in AR-V1 expression.

Biologic differences between peripheral and transition zone prostate cancer

Prostate cancer arises in the transition zone (TZ) in approximately 20–25% of cases. Modern biopsy and surveillance protocols, and advances in prostate cancer imaging, have renewed interest in TZ prostate cancers. We compared TZ and PZ prostate cancer to determine if cancer location is independently associated with better outcomes.

Optimization and comprehensive characterization of a faithful tissue culture model of the benign and malignant human prostate

Few preclinical models accurately depict normal human prostate tissue or primary prostate cancer (PCa). In vitro systems typically lack complex cellular interactions among structured prostatic epithelia and a stromal microenvironment, and genetic and molecular fidelity are concerns in both in vitro and in vivo models. ‘Tissue slice cultures’ (TSCs) provide realistic preclinical models of diverse tissues and organs, but have not been fully developed or widely utilized for prostate studies. Problems encountered include degeneration of differentiated secretory cells, basal cell hyperplasia, and poor survival of PCa. Here, we optimized, characterized, and applied a TSC model of primary human PCa and benign prostate tissue that overcomes many deficiencies of current in vitro models. Tissue cores from fresh prostatectomy specimens were precision-cut at 300 μm and incubated in a rotary culture apparatus. The ability of varied culture conditions to faithfully maintain benign and cancer cell and tissue structure and function over time was evaluated by immunohistological and biochemical assays. After optimization of the culture system, molecular and cellular responses to androgen ablation and to piperlongumine (PL), purported to specifically reduce androgen signaling in PCa, were investigated. Optimized culture conditions successfully maintained the structural and functional fidelity of both benign and PCa TSCs for 5 days. TSCs exhibited androgen dependence, appropriately undergoing ductal degeneration, reduced proliferation, and decreased prostate-specific antigen expression upon androgen ablation. Further, TSCs revealed cancer-specific reduction of androgen receptor and increased apoptosis upon treatment with PL, validating data from cell lines. We demonstrate a TSC model that authentically recapitulates the structural, cellular, and genetic characteristics of the benign and malignant human prostate, androgen dependence of the native tissue, and cancer-specific response to a potentially new therapeutic for PCa. The work described herein provides a basis for advancing the experimental utility of the TSC model.

Darinaparsin: Solid Tumor Hypoxic Cytotoxin and Radiosensitizer

Purpose: Hypoxia is an important characteristic of the solid tumor microenvironment and constitutes a barrier for effective radiotherapy. Here, we studied the effects of darinaparsin (an arsenic cytotoxin) on survival and radiosensitivity of tumor cells in vitro under normoxia and hypoxia and in vivo using xenograft models, compared to effects on normal tissues. Experimental Design: The cytotoxicity and radiosensitization of darinaparsin were first tested in vitro in a variety of solid tumor cell lines under both normoxia and hypoxia and compared with arsenic trioxide (ATO, an arsenical with reported cytotoxic and radiosensitizing activities on tumor cells). The effects were then tested in mouse models of xenograft tumors derived from tumor cell lines and clinical tumor specimens. The potential mechanisms of darinaparsin effects, including reactive oxygen species (ROS) generation, cellular damage, and changes in global gene expression, were also investigated. Results: In comparison with ATO, darinaparsin had significantly higher in vitro cytotoxic and radiosensitizing activities against solid tumor cells under both normoxia and hypoxia. In vivo experiments confirmed these activities at doses that had no systemic toxicities. Importantly, darinaparsin did not radiosensitize normal bone marrow and actually radioprotected normal intestinal crypts. The darinaparsin-mediated antitumor effects under hypoxia were not dependent on ROS generation and oxidative damage, but were associated with inhibition of oncogene (RAS and MYC)-dependent gene expression. Conclusion: Darinaparsin has significant and preferential cytotoxic and radiosensitizing effects on solid tumors as compared with normal cells. Darinaparsin may therefore increase the therapeutic index of radiation therapy and has near term translational potential. Clin Cancer Res; 18(12); 3366–76. ©2012 AACR.