TRAMP Tumors Research Articles

Abstract 2601: Lrig1 is a haplo-insufficient tumor suppressor and Lrig1-expressing cells can function as cells-of-origin of tumor development

Abstract LRIG1, leucine-rich repeats and immunoglobulin-like domains protein 1, is deleted or downregulated in many cancer types and has been reported to function as a tumor suppressor. LRIG1 is preferentially expressed in adult stem/progenitor cells and regulates their biological properties and quiescence by repressing the ERBB signaling. We recently reported LRIG1 to be overexpressed in human prostate cancer and to function as a feedback tumor suppressor. Human LRIG1 transgenically expressed in the mouse prostate inhibited the development and growth of both Hi-Myc and TRAMP tumors. The relationship between Lrig1-expressing adult stem/progenitor cells and cells-of-origin for cancer remains poorly understood. To address this, we deleted the Pten tumor suppressor gene in Lrig1-expressing cells by crossing Lrig1-CreERT2 mouse with Ptenfl/fl animals followed by Cre activation via tamoxifen administration. This knockout of Pten in Lrig1-expressing cells allowed us to assess the tumor suppressive function of Lrig1 as Lrig1-CreERT2 is a knock-in model in which one allele of Lrig1 is inactivated. We also generated a Lrig1-lineage tracing mouse model to label endogenous Lrig1-expressing cells by crossing Lrig1-CreERT2 mouse with CAG-tdTomato animals. Cre recombinase in these animals was activated by tamoxifen and epithelial tissues were harvested and analyzed for tomato signal by fluorescent microscopy. Lrig1-lineage tracing model showed that Lrig1-expressing cells in mouse prostate and oral epithelia are of CK5+ and CK8- phenotype suggesting their basal lineage. Targeted deletion of Pten in the Lrig1-expressing cellular compartments resulted in hyperplasic lesions in oral mucosa as early as 4 weeks and prevalent papilloma by 8 weeks. Pten deletion in the mouse prostate led to hyperplasia and high-grade prostate intraepithelial neoplasia (HGPIN) in a time-dependent manner. Consistent with these hyperplastic and tumor phenotypes, we observed elevated Ki-67 and pAkt staining indicating increased cell proliferation. Since Lrig1-CreERT2 is a knock-in model in which one allele of Lrig1 is inactivated, our data suggest that even one allelic loss of Lrig1 promotes more aggressive tumor phenotypes compared to those reported earlier with Pten deletion in CK5+ basal cells (CK5-CreERT2:Ptenfl/fl) in which similar lesions in oral mucosa and prostate tissues developed only after 20-24 weeks of Pten deletion. These results suggested that LRIG1 is a haplo-insufficient tumor suppressor as mono-allelic deletion of Lrig1 is sufficient to promote the tumorigenic process in mouse epithelial tissues induced by Pten loss. Our study also suggests that the Lrig-1 expressing cells can function as the cells-of-origin of cancer. We propose that Lrig1 may be used as a prognostic marker in cancers of epithelial origin and higher levels may portend a better prognosis. Citation Format: Moyi Wang, Qiuhui Li, Amanda Tracz, Jason Kirk, Anmbreen Jamroze, Rahul Kumar, Dean G. Tang. Lrig1 is a haplo-insufficient tumor suppressor and Lrig1-expressing cells can function as cells-of-origin of tumor development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2601.

Abstract 4861: Loss of YAP1 activates Wnt/beta-Catenin signaling in prostate cancer

Abstract Prostate cancer (PCa) is the most common non-cutaneous cancer among American men. In 2019, 174,650 new patients will be diagnosed from this disease. Most patients with localized PCa could be treated successfully. However, when PCa develops into castrate-resistant prostate cancer (CRPCa), the five-year survival rate drops below 30%. Neuroendocrine prostate cancer (NEPCa) is an aggressive subtype of PCa observed in 20%-30% CRPCa patients. Understanding how NEPCa progressed from prostate adenocarcinoma could reveal novel therapeutic targets. Previous studies indicate that activation of Wnt/beta-Catenin signaling promotes the progression to CRPCa with an increased NE differentiation. Additionally, animal studies suggest that Wnt/beta-Catenin signaling is active in NEPCa. Here we explored the mechanisms that promote Wnt/beta-Catenin signaling in NEPCa. Using TRAMP mice, which develop NEPCa after castration, we assessed the expression of Wnt-related genes and found that after androgen deprivation, the expression of Wnt7A is increased in prostate derived from both wild type and TRAMP mice but the expression of DKK1, a Wnt inhibitor, is decreased in wild type prostate but not in TRAMP prostate. The elevated expression of Wnt7a in combination with the lack of induction of Dkk1 in TRAMP tumors provides a mechanism to activate Wnt/beta-Catenin signaling in these tumors. Furthermore, we found the expression of YES-associated Protein (YAP1), a key transcriptional coactivator of Hippo pathway, is lost in NEPCa, in contrast to the elevated expression of YAP1 in high-grade prostate adenocarcinoma. YAP1 knockdown activated Wnt/beta-Catenin activity in PCa PC3 and 22Rv1 cells, indicating that the loss of YAP1 expression provides another mechanism to activate Wnt/beta-Catenin signaling, promoting NEPCa phenotype in PCa cells. Funding: This study is supported by: FWCC/Foundation Legacy Funds, DOD New Investigator Award, The Office of Research SEED awards, NIH R01 AND R03. Citation Format: Siyuan Cheng, Shu Yang, Nestor Prieto-Dominguez, Zachary M. Connelly, Yingli Shi, Xiuping Yu. Loss of YAP1 activates Wnt/beta-Catenin signaling in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4861.

Loss of epithelial AR increase castration resistant stem-like prostate cancer cells and promotes cancer metastasis via TGF-β1/EMT pathway.

BackgroundPrevious study has reported that loss of epithelial androgen receptor (AR) may promote tumor progression and cause TRAMP mouse model die earlier. The detail mechanisms, however, remain unclear.MethodsImmunohistochemistry assay, Western blot and real-time PCR were used to detect the expression of epithelial and mesenchymal markers. RNA extraction, RT-PCR, quantitative RT-PCR, BrdU incorporation assays, flow cytometry and other experimental technics were also used in present work.ResultsDecreased expression of epithelial markers (Cytokeratin 8, NKX3.1 and E-cadherin) and increased expression of mesenchymal markers (α-SMA, Vimentin, and N-cadherin) in were found in AR knockout TRAMP tumors. Further investigation indicated that AR signal deprivation is associated with cell morphology transition, high cell mobility, high cell invasion rate and resistance to anoikis in TRAMP prostate tumor cells. Together, these findings implied knockout AR in TRAMP prostate tumor may lead to EMT, which may result in earlier metastasis, and then cause TRAMP mice die earlier. TGF-β1 is responsible for EMT in AR knockout TRAMP tumor cells.ConclusionsIn conclusion, ADT therapy induced hormone refractory prostate cancer may gain the ability of metastasis through cell’s EMT which is a phase of poor differentiation. Anti-EMT drugs should be developed to battle the tumor metastasis induced by ADT therapy.

HSP60 IS A NOVEL TARGET IN LETHAL PROSTATE CANCER

Currently, various therapeutic agents exist in the clinic to treat aggressive prostate cancer (PCa) by modulating androgen receptor (AR) activity, which is one of the main drivers of prostate cancer. However, resistance to these agents nearly always occurs and results in the production of a more aggressive form of cancer, typically referred to as castrate‐recurrent prostate cancer (CRPC). Patients within a subset of CRPC have tumors that undergo neuroendocrine (NE) transdifferentiation (NE‐CRPC). NE‐CRPC is poorly differentiated, readily metastasizes and is resistant to androgen deprivation therapy (ADT). Therefore, it is essential that alternative therapeutic targets that do not solely rely on AR modulation be better characterized for patients with this advanced disease.The mitochondrial unfolded protein response (mitoUPR) is responsible for maintaining mitochondrial quality control by preserving the integrity of the mitochondria in response to numerous stressors including hypoxia and chemotherapeutic agents. MitoUPR activation is a hallmark sign of mitochondrial dysfunction. Heat Shock Protein60 (Hsp60) is a mitochondrial chaperone and is a key player of the mitoUPR. Thus, we hypothesize that inhibition of Hsp60 renders aggressive NE‐like cells susceptible to stress, thereby alleviating tumorigenicity. We observed that protein expression of Hsp60 was upregulated in tumors derived from NE models such as the transgenic adenocarcinoma of mouse prostate tumor (TRAMP), DKO (pten−/−/rb1−/−) and TKO (rb1−/−/pten−/−/p53−/−) when compared to wild‐type controls. Transcript levels of HSPD1 positively correlated to Gleason Score and are upregulated in patients with NE‐like phenotypes from TCGA. Additionally, Hsp60 expression associated with severe mitochondrial dysfunction in TRAMP tumors. Specifically, mito‐nuclear imbalances and defects in oxidative phosphorylation activity were observed. Lastly, Inhibition of Hsp60 significantly increased sensitivity of NE cells to mitotoxic stressors such as hypoxia and the chemotherapeutic agent cisplatin.Together, our data indicate that inactivation of Hsp60 sensitizes NE cells to mitotoxic stress and has clinical and therapeutic relevance. Specifically, the use of cisplatin in small cell CRPC has been shown to have minimal effect, yet we determined that inhibition of Hsp60 sensitizes PCa cells to cisplatin. We envision that inactivation of Hsp60 will alleviate tumor burden by itself, but will also enhance sensitivity of chemo‐resistant PCa cells. Additionally, inhibition of Hsp60 may enhance therapeutic potential of anticancer agents in other types of cancers that overexpress Hsp60 including liver, lung and colon cancers. Thus, Hsp60 is a novel target in prostate cancers with NE‐like differentiation.Support or Funding InformationThis work was supported in part by the National Cancer Institute of the National Institutes of Health under Award no. R01CA160685This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LSD1 dual function in mediating epigenetic corruption of the vitamin D signaling in prostate cancer

BackgroundLysine-specific demethylase 1A (LSD1) is a key regulator of the androgen (AR) and estrogen receptors (ER), and LSD1 levels correlate with tumor aggressiveness. Here, we demonstrate that LSD1 regulates vitamin D receptor (VDR) activity and is a mediator of 1,25(OH)2-D3 (vitamin D) action in prostate cancer (PCa).MethodsAthymic nude mice were xenografted with CWR22 cells and monitored weekly after testosterone pellet removal. Expression of LSD1 and VDR (IHC) were correlated with tumor growth using log-rank test. TRAMP tumors and prostates from wild-type (WT) mice were used to evaluate VDR and LSD1 expression via IHC and western blotting. The presence of VDR and LSD1 in the same transcriptional complex was evaluated via immunoprecipitation (IP) using nuclear cell lysate. The effect of LSD1 and 1,25(OH)2-D3 on cell viability was evaluated in C4-2 and BC1A cells via trypan blue exclusion. The role of LSD1 in VDR-mediated gene transcription was evaluated for Cdkn1a, E2f1, Cyp24a1, and S100g via qRT-PCR-TaqMan and via chromatin immunoprecipitation assay. Methylation of Cdkn1a TSS was measured via bisulfite sequencing, and methylation of a panel of cancer-related genes was quantified using methyl arrays. The Cancer Genome Atlas data were retrieved to identify genes whose status correlates with LSD1 and DNA methyltransferase 1 (DNMT1). Results were correlated with patients’ survival data from two separate cohorts of primary and metastatic PCa.ResultsLSD1 and VDR protein levels are elevated in PCa tumors and correlate with faster tumor growth in xenograft mouse models. Knockdown of LSD1 reduces PCa cell viability, and gene expression data suggest a dual coregulatory role of LSD1 for VDR, acting as a coactivator and corepressor in a locus-specific manner. LSD1 modulates VDR-dependent transcription by mediating the recruitment of VDR and DNMT1 at the TSS of VDR-targeted genes and modulates the epigenetic status of transcribed genes by altering H3K4me2 and H3K9Ac and DNA methylation. Lastly, LSD1 and DNMT1 belong to a genome-wide signature whose expression correlates with shorter progression-free survival and overall survival in primary and metastatic patients’ samples, respectively.ConclusionsResults demonstrate that LSD1 has a dual coregulatory role as corepressor and coactivator for VDR and defines a genomic signature whose targeting might have clinical relevance for PCa patients.

Development of high resolution 3D hyperpolarized carbon-13 MR molecular imaging techniques

The goal of this project was to develop and apply techniques for T2 mapping and 3D high resolution (1.5mm isotropic; 0.003cm3) 13C imaging of hyperpolarized (HP) probes [1-13C]lactate, [1-13C]pyruvate, [2-13C]pyruvate, and [13C,15N2]urea in vivo. A specialized 2D bSSFP sequence was implemented on a clinical 3T scanner and used to obtain the first high resolution T2 maps of these different hyperpolarized compounds in both rats and tumor-bearing mice. These maps were first used to optimize timings for highest SNR for single time-point 3D bSSFP acquisitions with a 1.5mm isotropic spatial resolution of normal rats. This 3D acquisition approach was extended to serial dynamic imaging with 2-fold compressed sensing acceleration without changing spatial resolution. The T2 mapping experiments yielded measurements of T2 values of >1s for all compounds within rat kidneys/vasculature and TRAMP tumors, except for [2-13C]pyruvate which was ~730ms and ~320ms, respectively. The high resolution 3D imaging enabled visualization the biodistribution of [1-13C]lactate, [1-13C]pyruvate, and [2-13C]pyruvate within different kidney compartments as well as in the vasculature. While the mouse anatomy is smaller, the resolution was also sufficient to image the distribution of all compounds within kidney, vasculature, and tumor. The development of the specialized 3D sequence with compressed sensing provided improved structural and functional assessments at a high (0.003cm3) spatial and 2s temporal resolution in vivo utilizing HP 13C substrates by exploiting their long T2 values. This 1.5mm isotropic resolution is comparable to 1H imaging and application of this approach could be extended to future studies of uptake, metabolism, and perfusion in cancer and other disease models and may ultimately be of value for clinical imaging.

Flow versus permeability weighting in estimating the forward volumetric transfer constant (Ktrans) obtained by DCE-MRI with contrast agents of differing molecular sizes

PurposeTo quantify the differential plasma flow- (Fp-) and permeability surface area product per unit mass of tissue- (PS-) weighting in forward volumetric transfer constant (Ktrans) estimates by using a low molecular (Gd-DTPA) versus high molecular (Gadomer) weight contrast agent in dynamic contrast enhanced (DCE) MRI. Materials and methodsDCE MRI was performed using a 7T animal scanner in 14 C57BL/6J mice syngeneic for TRAMP tumors, by administering Gd-DTPA (0.9kD) in eight mice and Gadomer (35kD) in the remainder. The acquisition time was 10min with a sampling rate of one image every 2s. Pharmacokinetic modeling was performed to obtain Ktrans by using Extended Tofts model (ETM). In addition, the adiabatic approximation to the tissue homogeneity (AATH) model was employed to obtain the relative contributions of Fp and PS. ResultsThe Ktrans values derived from DCE-MRI with Gd-DTPA showed significant correlations with both PS (r2=0.64, p=0.009) and Fp (r2=0.57, p=0.016), whereas those with Gadomer were found only significantly correlated with PS (r2=0.96, p=0.0003) but not with Fp (r2=0.34, p=0.111). A voxel-based analysis showed that Ktrans approximated PS (<30% difference) in 78.3% of perfused tumor volume for Gadomer, but only 37.3% for Gd-DTPA. ConclusionsThe differential contributions of Fp and PS in estimating Ktrans values vary with the molecular weight of the contrast agent used. The macromolecular contrast agent resulted in Ktrans values that were much less dependent on flow. These findings support the use of macromolecular contrast agents for estimating tumor vessel permeability with DCE-MRI.

Abstract 2407: Neuroendocrine and luminal progenitors drive cancer progression in prostate cancer

Abstract Neuroendocrine differentiation of prostate cancer (PCa) occurs frequently during the development of castration resistance and rarely in primary tumors. Using the transgenic mouse model of prostate cancer (TRAMP) in a B6/C57 background, we were able to detect, isolate and further characterize basal, luminal and neuroendocrine subsets of cancer cells. We performed allograft experiments in NSG mice to ensure cellular stem and progenitor properties as well as metastatic potential, and thus created a murine model of neuroendocrine prostate cancer. Whilst all three previously described cell populations were present in approximately three quarters of primary TRAMP tumors, some tumors lacked basal stem cells and showed a more aggressive phenotype. These tumors, mainly consisting of small cancer cells, expressed markers of neuroendocrine differentiation such as synaptophysin and chromogranin A as shown by immunohistochemistry and are further referred to as neuroendocrine carcinomas (NECs), as ruled by an experienced uropathologist of our institution. Adenocarcinoma-like tumors (ACs), in contrast, showed high expression of cytokeratins and retained glandular histology. Using fluorescence-activated cell sorting (FACS) against newly discovered NEC markers within the TRAMP model, we found a relative increase of neuroendocrine progenitors in prostate NECs compared to ACs (approximately 75 vs. 33%, respectively). In ACs, on the other hand, luminal progenitors were found to be the predominant drivers of cancer progression. To further evaluate this, we transplanted single-cell-suspensions into NSG mice without androgen supplementation and observed successful engraftment of both non-basal cell populations. Additionally, we were able to passage the resulted tumors for at least two generations and observed maintenance of histology and biological features for tumors of both luminal and neuroendocrine origin. Based on a RNA sequencing, we were able to define gene signatures for neuroendocrine and luminal progenitors, uncovering a number of novel potential therapeutic targets. Ultimately, we were able to evaluate the prognostic value of the signatures obtained from mice in the human disease by in-silico analyses of publically available gene expression profile databases. In conclusion, we created and characterized a murine model of neuroendocrine prostate cancer using flow cytometry and murine allografts. Further, we established gene expression signatures of luminal and neuroendocrine progenitors and translated them to the human disease. Our findings foster the understanding of neuroendocrine differentiation in prostate cancer and may help in developing new targeted approaches in this entity. Citation Format: Maximilian Marhold, Erwin Tomasich, Simon Udovica, Gerwin Heller, Corinna Altenberger, Andreas Spittler, Reinhard Horvat, Peter Horak, Michael Krainer. Neuroendocrine and luminal progenitors drive cancer progression in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2407.

Stromal hedgehog signaling maintains smooth muscle and hampers micro-invasive prostate cancer.

ABSTRACTIt is widely appreciated that reactive stroma or carcinoma-associated fibroblasts can influence epithelial tumor progression. In prostate cancer (PCa), the second most common male malignancy worldwide, the amount of reactive stroma is variable and has predictive value for tumor recurrence. By analyzing human PCa protein and RNA expression databases, we found smooth muscle cells (SMCs) are decreased in advanced tumors, whereas fibroblasts are maintained. In three mouse models of PCa, PB-MYC, ERG/PTEN and TRAMP, we found the composition of the stroma is distinct. SMCs are greatly depleted in advanced PB-MYC tumors and locally reduced in ERG/PTEN prostates, whereas in TRAMP tumors the SMC layers are increased. In addition, interductal fibroblast-like cells expand in PB-MYC and ERG/PTEN tumors, whereas in TRAMP PCa they expand little and stromal cells invade into intraductal adenomas. Fate mapping of SMCs showed that in PB-MYC tumors the cells are depleted, whereas they expand in TRAMP tumors and interestingly contribute to the stromal cells in intraductal adenomas. Hedgehog (HH) ligands secreted by epithelial cells are known to regulate prostate mesenchyme expansion differentially during development and regeneration. Any possible role of HH signaling in stromal cells during PCa progression is poorly understood. We found that HH signaling is high in SMCs and fibroblasts near tumor cells in all models, and epithelial Shh expression is decreased whereas Ihh and Dhh are increased. In human primary PCa, expression of IHH is the highest of the three HH genes, and elevated HH signaling correlates with high stromal gene expression. Moreover, increasing HH signaling in the stroma of PB-MYC PCa resulted in more intact SMC layers and decreased tumor progression (micro-invasive carcinoma). Thus, we propose HH signaling restrains tumor progression by maintaining the smooth muscle and preventing invasion by tumor cells. Our studies highlight the importance of understanding how HH signaling and stromal composition impact on PCa to optimize drug treatments.

The landscape of somatic chromosomal copy number aberrations in GEM models of prostate carcinoma.

Human prostate cancer is known to harbor recurrent genomic aberrations consisting of chromosomal losses, gains, rearrangements, and mutations that involve oncogenes and tumor suppressors. Genetically engineered mouse (GEM) models have been constructed to assess the causal role of these putative oncogenic events and provide molecular insight into disease pathogenesis. While GEM models generally initiate neoplasia by manipulating a single gene, expression profiles of GEM tumors typically comprise hundreds of transcript alterations. It is unclear whether these transcriptional changes represent the pleiotropic effects of single oncogenes, and/or cooperating genomic or epigenomic events. Therefore, it was determined whether structural chromosomal alterations occur in GEM models of prostate cancer and whether the changes are concordant with human carcinomas. Whole genome array-based comparative genomic hybridization (CGH) was used to identify somatic chromosomal copy number aberrations (SCNA) in the widely used TRAMP, Hi-Myc, Pten-null, and LADY GEM models. Interestingly, very few SCNAs were identified and the genomic architecture of Hi-Myc, Pten-null, and LADY tumors were essentially identical to the germline. TRAMP neuroendocrine carcinomas contained SCNAs, which comprised three recurrent aberrations including a single copy loss of chromosome 19 (encoding Pten). In contrast, cell lines derived from the TRAMP, Hi-Myc, and Pten-null tumors were notable for numerous SCNAs that included copy gains of chromosome 15 (encoding Myc) and losses of chromosome 11 (encoding p53). Chromosomal alterations are not a prerequisite for tumor formation in GEM prostate cancer models and cooperating events do not naturally occur by mechanisms that recapitulate changes in genomic integrity as observed in human prostate cancer.

Cell-based Hyper-interleukin 6 or Hyper-interleukin 11 secreting vaccines combined with low dose cyclophosphamide in an orthotopic murine prostate cancer model.

BackgroundCell based vaccines encoding Hyper-IL-6 (H6) and Hyper-IL-11 (H11) present high activity in murine melanoma and renal cancer model. We evaluated the efficacy of cellular vaccines modified with H6 or H11 combined with cyclophosphamide in orthotopic murine prostate cancer model.Material and methodsTRAMP cells were transduced with H6 and H11 cDNA (TRAMP-H6 and TRAMP-H11). An orthotopic TRAMP model based on the implantation of TRAMP cells into the dorsolateral lobe of the prostate of C57BL6/J mice was employed. The efficacy of TRAMP-H6 and TRAMP-H11 vaccines evaluated in the therapeutic setting was compared with the TRAMP cells modified with a mock transduced E1-deleted adenoviral vector (TRAMP-AdV) and non-modified irradiated TRAMP cells (TRAMP IRR) in relation to naive (non-immunized) mice. In the next experimental groups mice vaccinated with TRAMP-H6 and TRAMP-H11 received cyclophosphamide (CY). Detection of immune cells in the spleen in mice receiving vaccines combined with CY was evaluated.ResultsModification of TRAMP cells with H6 increased the efficacy of TRAMP-based whole-cell vaccine. The highest response rate was observed in mice receiving TRAMP-H6 alone and combined with CY. Vaccination with TRAMP-H6 alone and combined with CY and TRAMP H11 combined with CY extended median OS of mice bearing orthotopic TRAMP tumors in therapeutic setting. Low dose CY administered alone demonstrated some antitumor activity in employed model. TRAMP-H6 or TRAMP-H11 combined with CY strongly augmented generation of CD8+, CD4+ T lymphocytes and memory T cells. Immunization with TRAMP combined with or without CY suppressed generation of T regulatory cells.ConslusionsProstate cancer vaccines modified with H6 or H11 induce prostate tumour regression and increase mice survival by stimulating the immune system. Cyclophosphamide added to modified TRAMP vaccines demonstrated clinical benefit of treated mice and enhanced anti-tumour immune response.

Immunostimulatory early phenotype of tumor-associated macrophages does not predict tumor growth outcome in the HLA-DR mouse model of prostate cancer (TUM4P.923)

Abstract Tumor-associated macrophages (TAM) were shown to support progression of many solid tumors. However, anti-tumor properties of TAM were also reported in several types of cancer. Here, we investigated the phenotype and functions of TAM in two transgenic mouse models of prostate cancer displaying striking differences in the tumor growth outcome. Mice expressing Prostate-Specific Antigen as a self-antigen in the prostate (PSA tg mice) rejected PSA-expressing TRAMP tumors with high frequency. However, introduction of HLA-DRB1*1501(DR2b) transgene aggravated the growth of TRAMP-PSA tumors in DR2bxPSA F1 mice. Despite the difference in the tumor growth outcome, tumors in both strains were equally and intensively infiltrated by TAM at the first week after tumor challenge. TAM exhibited mixed M1/M2 polarization characterized by the production of pro-inflammatory (TNFα, IL1β) and anti-inflammatory (IL10) cytokines. They also secreted T cell/NK cell-recruiting chemokines (CXCL10, CCL2, CCL3, CCL4, CCL5) and expressed high levels of MHC class II and co-stimulatory molecules. Functionally, TAM demonstrated pronounced immunostimulatory activity in a mixed leukocyte reaction and induced apoptosis of tumor cells. We conclude that during early stages of tumor progression, the phenotype and functional properties of TAM did not predict tumor growth outcome in our model. Furthermore, we demonstrate that at this stage TAM have a potential to activate T cell immunity and mediate anti-tumor effects.

S100A9 Interaction with TLR4 Promotes Tumor Growth

By breeding TRAMP mice with S100A9 knock-out (S100A9−/−) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b+ S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68+ macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9−/− and TLR4−/−, but not in RAGE−/− animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b+ cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.

Abstract B47: Identification of novel E2F3 transcriptional targets expands the role of the RB/E2F axis beyond cell cycle control

Abstract The RB/E2F axis is frequently disrupted in multiple tumors. Classical studies reported that the RB tumor suppressor exerts its transcriptional repressor function through an interaction with the E2F family of transcription factors (E2F1-3) to control the timely expression of genes required for DNA replication and cell cycle progression. The E2F3 locus encodes two E2F3 isoforms, a and b, which have unique N-terminal sequences. Multiple studies indicate that E2F3, rather than E2F1 or 2, is elevated in various cancers, including prostate. In prostate tumors elevated E2F3 expression is an independent prognostic factor of clinical outcome, but the direct transcriptional targets of E2F3a and E2F3b have not been characterized. We found that E2F3a and b expression was increased in prostate tumor derived cell lines when compared to non-transformed controls. An expression array analysis following knockdown (k/d) of total E2F3 identified several unexpected targets including Interleukin 6 receptor (IL-6R), a critical component of the IL6 signaling cascade and calpain 2, a protease that is essential in cellular migration and has been shown to be elevated during prostate tumorigenesis. Further studies showed that the two genes were regulated by different E2F3 isoforms- IL6R was a target of E2F3a, while calpain 2 was regulated by E2F3b. Expression of both genes was enhanced by RB ablation. E2F3a-dependent IL-6R regulation was apparent in PC3, LNCaP, CWR-R1, and 22Rv1 cells. Chromatin immunoprecipitation (ChIP) studies identified sequences in the IL-6R promoter that were bound by E2F3. Transient co-transfection studies using an E2F3a expression plasmid showed that E2F3a transactivated the IL-6R promoter in a dose dependent manner. The IL-6R initiated signaling cascade was perturbed following a k/d of E2F3a since the levels of ERK1/2 phosphorylation was reduced. An siRNA-mediated ablation of each isoform revealed that E2F3b, not E2Fa, was effective is reducing calpain 2 levels. ChIP studies indicated that E2F3 binds to the endogenous calpain 2 promoter, and in transient transfection studies E2F3b transactivated the calpain 2 promoter in a dose dependent manner. An analysis of the calpain 2 promoter identified an androgen receptor (AR) half site. Additional transfection studies showed that the AR can cooperate with E2F3b in transactivation of the calpain 2 promoter. Moreover, reduced expression of E2F3b impaired cellular migration in a wound assay. The expression of the E2F3 isoforms has not been previously analyzed in the TRAMP prostate tumor model. Western immunoblot studies of five TRAMP tumors and three age and strain matched prostates showed that TRAMP tumors had highly elevated expression of both E2F3 isoforms. These results indicate that increased expression of both E2F3 isoforms is a feature of human tumor-derived cell lines and the TRAMP mouse model of prostate tumorigenesis. Calpain 2, expression was readily detected in all of the TRAMP tumors, but not in the control tissue. Hence E2F3 overexpression and increased calpain levels are features of human and mouse tumors. This analysis broadens the role of E2F3 in prostate tumorigenesis beyond the regulation of cell cycle progression. E2F3a is a link between the E2F/RB and the IL-6 signaling cascade, while E2F3b regulates expression of a protease that is essential in cell adhesion and migration. Therefore E2F3 deregulation affects multiple signaling networks to promote tumorigenesis. Citation Format: Stephen J. Libertini, Maria Mudryj, Alan P. Lombard, Honglin Chen, Veronica Rodriguez, Carlos Perez-Stable, Bushra al-Bataina, Tilak Koilvaram, Michael George, Allen C. Gao. Identification of novel E2F3 transcriptional targets expands the role of the RB/E2F axis beyond cell cycle control [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 B47.

Human prostate cancer ZIP1/zinc/citrate genetic/metabolic relationship in the TRAMP prostate cancer animal model

Prostate cancer is the second leading cause of cancer deaths among men. The availability of animal models that represent the events and factors that exist in the natural history and biology of human prostate cancer is essential in dealing with prostate cancer. In recent decades and presently, emphasis has been directed at the development and employment of prostate cancer induced in transgenic mice. However, the important consistent hallmark characteristic and event of decrease in zinc and citrate and downregulation of ZIP1 zinc transporter in prostate malignancy has not been studied or identified in any animal model. We investigated the status of these parameters in TRAMP tumors as compared with human prostate cancer. The results show that citrate levels are markedly decreased in the developing and advancing stages of malignancy in TRAMP. Zinc levels are also decreased and ZIP1 transporter is lost in TRAMP tumors. In vitro studies show that zinc treatment of TRAMP C2 cells exhibits cytotoxic effects. Collectively, these results mimic the ZIP1, zinc, and citrate status and relationship that exist in human prostate cancer. This is the first report that establishes the existence of the human prostate zinc/citrate hallmark characteristic and relationship in an animal model. It now appears that the TRAMP model will be suitable for studies relating to the implications and role of zinc- and citrate-related metabolism in the development and progression of human prostate cancer.

SU‐E‐J‐130: Hyperpolarized 13C Biomarkers of Response to Prostate Cancer Radiation Therapy

Purpose: Prior 13C MR imaging studies of hyperpolarized (HP) [1‐13C]pyruvate and 13C urea in the TRAMP murine model of prostate cancer have demonstrated the ability to monitor changes in metabolism and perfusion. Radiation therapy represents a common treatment of prostate cancer that could clinically benefit from an early assessment of therapeutic efficacy. The goal of this work was to investigate serial changes in perfusion (HP urea) and HP pyruvate metabolism in TRAMP tumors following exposure to increasing doses of radiation therapy in order to better understand the potential clinical value of HP 13C MR for predicting prostate cancer radiation therapy. Methods: Three TRAMP tumors (mean tumor size = 4.3 ± 1 cc) were exposed to varying doses of radiation by placing a single radioactive seed source using a Nucletron microSelectron‐HDR applicator on the surface of the tumor for ∼10 min. dwell periods. This resulted in doses of 14 Gy (close to the seed) to 5 Gy (deeper within the tumor). MR imaging studies were acquired serially at baseline, 1 day, 4 days and 8 days following therapy using a 14T, 600WB micro‐imaging spectrometer. Results: There are visually clear dose dependent changes in the lactate/pyruvate ratio over time. HP urea and total HP carbon significantly decreased in tumor regions receiving both high (p &lt; 0.01; and 0.05, respectively) and intermediate doses (p&lt; 0.01, for both) by 1 day after treatment. Whereas HP urea initially increased in the low dose regions and then decreased. For all three doses, the lactate/pyruvate ratios significantly decreased (p &lt; 0.01 for all doses) by 8 days following treatment. Conclusions: Significant, dose‐dependent decreases in perfusion and pyruvate‐to‐lactate flux were observed after radiation therapy. Ongoing serial radiation studies of TRAMP tumors are investigating the relationship between perfusion and metabolic changes and therapeutic efficacy.

TU‐A‐BRC‐02: Hyperpolarized 13C MRSI for Assessing Radiation Response of Prostate Cancer in Transgenic Mice

Purpose: To assess the tumor response to radiation in transgenic mouse model of prostatic adenocarcinoma (TRAMP) by hyperpolarized MR spectroscopic imaging (MRSI) of [1–13C]pyruvate.Methods: The TRAMP mouse model used in this study develops an autochthonous tumor in the prostate at the onset of puberty. The progression of tumors was volumetrically monitored by T2‐weighted MRI at 7 T. Four mice with tumor volumes of 400–1500 mm3 underwent hyperpolarized 13C MRSI at 3 T. Two of them received fractionated X‐ray radiation (200 kV, 20 mA, 1.21 cGy/s, 1 mm Cu HVL) doses of 10 and 20 Gy to the tumors, followed by the 13C MRSI at the fourth day of postirradiation. Dynamic 13C MRSI was performed following an intravenous bolus injection of 300 μL of 80 mM hyperpolarized [1–13C]pyruvate to obtain the transient fate of 13C‐labeled metabolites in a 3D volume encompassing the tumor with a nominal spatial resolution of 2.7×2.7×3 mm3. The average time‐resolved signal intensities of pyruvate and its downstream products lactate and alanine were measured in the tumors from reconstructed metabolic images. Metabolic response was characterized in terms of lactate/pyruvate ratio and apparent rate constant of pyruvate‐to‐lactate conversion, kpl, by fitting data to a two‐site exchange model. Results: Based on MRI volumetry, the TRAMP tumors exponentially grew with doubling time of 7±3 d and shrank in response to radiation by 50% in volume within 4±1 d. The untreated tumors that grew to larger volumes over a similar postirradiation period exhibited increased lactate/pyruvate ratio and kpl compared to the treated tumors. Alanine was in noise levels and hence not considered for analysis. Conclusions: Preliminary data indicate the feasibility of utilizing hyperpolarized 13C MRSI to assess therapeutic response of prostate cancer to radiation in TRAMP mice. Further investigations are underway to obtain additional data.This work is partly supported by DOD PCRP (W81XWH‐10‐10498) research award.

Role of vitamin D receptor in the antiproliferative effects of calcitriol in tumor-derived endothelial cells and tumor angiogenesis in vivo.

Calcitriol (1,25-dihydroxycholecalciferol), the major active form of vitamin D, is antiproliferative in tumor cells and tumor-derived endothelial cells (TDEC). These actions of calcitriol are mediated at least in part by vitamin D receptor (VDR), which is expressed in many tissues including endothelial cells. To investigate the role of VDR in calcitriol effects on tumor vasculature, we established TRAMP-2 tumors subcutaneously into either VDR wild-type (WT) or knockout (KO) mice. Within 30 days post-inoculation, tumors in KO mice were larger than those in WT (P < 0.001). TDEC from WT expressed VDR and were able to transactivate a reporter gene whereas TDEC from KO mice were not. Treatment with calcitriol resulted in growth inhibition in TDEC expressing VDR. However, TDEC from KO mice were relatively resistant, suggesting that calcitriol-mediated growth inhibition on TDEC is VDR-dependent. Further analysis of the TRAMP-C2 tumor sections revealed that the vessels in KO mice were enlarged and had less pericyte coverage compared with WT (P < 0.001). Contrast-enhanced magnetic resonance imaging showed an increase in vascular volume of TRAMP tumors grown in VDR KO mice compared with WT mice (P < 0.001) and FITC-dextran permeability assay suggested a higher extent of vascular leakage in tumors from KO mice. Using ELISA and Western blot analysis, there was an increase of hypoxia-inducible factor-1alpha, vascular endothelial growth factor, angiopoietin 1, and platelet-derived growth factor-BB levels observed in tumors from KO mice. These results indicate that calcitriol-mediated antiproliferative effects on TDEC are VDR-dependent and loss of VDR can lead to abnormal tumor angiogenesis.

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy

Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.

The mechanism of exogenous B7.1-enhanced IL-12-mediated complete regression of tumors by a single electroporation delivery

Electroporation-based mono-gene therapy has received great interest in recent years but coadministration of different therapeutic genes for treatment of tumors has not been well explored. We hypothesize that electroporation is capable of delivering multiple genes that induce an additive or synergistic antitumor effect. To test this hypothesis, we used mice that were bearing SCCVII or TRAMP tumors. Established tumors with a diameter of 4-5 mm were injected with control plasmid DNA or plasmid DNA encoding B7.1, IL-12 or both via electroporation. Tumor regression, CTL activity and the level of B7.1, IL-12 and Stat1 expression were determined in both wild-type mice and in mice with a knock-out of the Stat1 gene. Remarkably, a single coadministration of the plasmids that encoded IL-12 and B7.1 eradicated tumors in 80% of mice. The therapeutic effect was associated with high levels of endogenous B7.1 expression, activity of cytotoxic lymphocytes, and activation of Stat1. Both exogenous B7.1 and IL-12 were required for inducing a high level of Stat1 activation in tumors, which occurred through a mechanism that was independent of the host Stat1. Both stimulators were also required for inducing the strong cytotoxic lymphocyte activity and for increasing the level and extending the duration of endogenous B7.1 expression. We therefore propose a 2-signal stimulation model to explain the synergistic effect of the coadministration of IL-12 and B7.1 on the regression of the tumors.