Mouse Model Of Prostate Cancer Research Articles

Abstract 1568: A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer

Abstract Extracellular adenosine impairs immune function in tumors and limits the efficacy of anti-CTAL4 immune checkpoint blockade. We previously showed that adenosine 2A receptor (A2aR) blockade with AZD4635 moderately inhibited tumor growth, in part by improving antigen presentation and cytotoxic T cell (CTL) activity in mouse Pten-deficient prostate cancer. Here, we continue to investigate the antitumor activity of AZD4635 and its effects when combined with CTLA4 blockade (aCTLA4) in preclinical models of Pten-deficient prostate cancer. Prostate tumors from Pten conditional knockout (KO) mice treated with aCTLA4 for five days had increased expression levels of adenosinergic genes. Furthermore, gene expression, flow cytometric and IHC analyses showed increased T regulatory cell differentiation and accumulation that was offset with the addition of AZD4635. Moreover, expression levels of genes associated with Th 1 cell differentiation and T cell-mediated cytotoxicity were increased in AZD4635/aCTLA4 treated tumors. Adding AZD4635 to a four-week regimen of aCTLA4 therapy improved the antitumor response by two-fold in an early-stage intervention model of Pten-deficient prostate cancer. Androgen deprivation (AD) has the potential to promote T cell infiltration in prostate tumors and using the AZD4635/aCTLA4 treatment combination as neoadjuvant therapy to AD via surgical castration led to a greater antitumor response. The antitumor activity of this treatment combination was further examined and confirmed in a subcutaneous syngeneic genome-derived allograft model using tumor-bearing conditional Pten-deficient knockout mice grafted with Pten-deficient CRPC tumor fragments. In a Pten/Trp53 conditional double knockout (DKO) mouse model of advanced prostate cancer, the AZD4635/aCTLA4 treatment combination did not improve overall survival rates. However, when AZD4635/aCTLA4 was used as neoadjuvant therapy to AR inhibition with the anti-androgen apalutamide, it improved median survival time from 21 days in monotherapy treated mice to 34 days in AZD4635/aCTLA4 treated mice. These results provide preclinical evidence to support the rational combination of A2AR blockade with AZD4635 and aCTLA4 immune checkpoint inhibition for PTEN-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Alwin Schuller, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1568.

Abstract 1438: Acute immune responses to apalutamide in mouse Pten-deficient prostate cancer

Abstract The androgen receptor (AR) is a major therapeutic target for human prostate cancer and therapies targeting the AR pathway also impact local and systemic immunity. However, the influences over host immune function vary depending on the type of AR treatment. Androgen deprivation (AD) can improve immune cell infiltration by enhancing T cell trafficking, but it also attracts immunosuppressive myeloid cells and regulatory T cells. We previously showed that chronic dosing of apalutamide, an oral nonsteroidal AR antagonist, was effective in suppressing tumor growth in a preclinical mouse models of Pten-deficient prostate cancer and also changed the composition of the tumor immune microenvironment improving cytotoxic T cell activity. Moreover, we also showed that using AD as neoadjuvant therapy improved the antitumor activity and immune responses of combined JAK/PD-L1 blockade, whereas administered as concurrent or adjuvant therapy was ineffective. Our aim in this study is to determine the potential of using apalutamide in as combination therapy with immunotherapy and immune modulating agents. For this we have characterized the acute immune modulatory activity of apalutamide on mouse Pten-deficient and compared its activity against that of AD. Conditional prostate specific Pten/Trp53 double knockout mice were orchidectomized or treated with apalutamide (30 mg/kg/D) for five days. Tumor samples and lymphoid organs were collected and processed for immune profiling using a qRT-PCR focused panel, flow cytometry and/or immunohistochemistry. Prostates from orchidectomized mice tended to be smaller however there was no decrease in size for apalutamide treated mice. Notably, thymuses were significantly enlarged in both orchidectomized and apalutamide treated mice compared to control. Genes related to antigen presenting/dendritic cell (APC/DC) and activated DC were greater in apalutamide treated mice compared to vehicle treated or surgically castrated mice. This finding was consistent with an increased abundance of MHC-IIhi/CD80+ migratory DCs (CD11c+/XCR1+) in tumors of apalutamide treated mice. Tumor associated macrophage infiltration was also greater in apalutamide treated mice. Granulocyte infiltration was significantly greater after both castration and apalutamide therapy however the proportion of mature granulocytes was higher in castrated mice. T cell infiltration was comparable between orchidectomized and apalutamide treated mice, and regulatory T cell activity was increased in both groups compared to controls, but to a higher degree in orchidectomized mice. In summary, this provides critical data that will be essential when developing rational combinations with additional immunotherapies and immunomodulatory agents. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Kazuko Sakai, Nobutaka Shimizu, Eri Banno, Masahiro Nozawa, Kazuhiro Fujita, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Acute immune responses to apalutamide in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1438.

Abstract 2331: Metabolic evasion of mCRPC immunotherapy by ACOD1 production of itaconate

Abstract The use of immune checkpoint blockade (ICB) therapies in the form of αPD-1 & αCTLA-4 monoclonal antibodies (mAb) have made great strides in the battle against melanoma, lung, and breast cancers. However, these strategies are not universally effective. Roadblocks occur as certain cancers cannot be targeted by these therapies or may develop resistance to ICB. How resistance to ICB therapy arises represents a crucial barrier to more effective use of these therapies. Recent studies have demonstrated that the metabolic milieu of the tumor microenvironment (TME) plays a role in regulating the anti-tumor immune response. However, the specific mechanisms by which cancer cell secreted metabolites in the TME impact the responsiveness of cancer cells to ICB remain poorly understood. In order to better understand the factors that influence the effectiveness of ICB, we utilized a mouse model (Smad4-/-/Pten-/-/Trp53-/-) of male castration resistant prostate cancer (mCRPC) that is responsive to αCTLA-4 and αPD-1 ICB. Using serial culture and injection of residual tumor cells that survive ICB, we generated cell lines that had developed resistance to ICB. Unbiased metabolomic screening of these cell lines revealed a stark increase in the production of the anti-inflammatory metabolite itaconate in the resistant cell lines. This observation was supported by the increased abundance of the enzyme responsible for itaconate production, aconitate decarboxylase 1 (ACOD1) among the resistant lines. Reduction of this enzyme through shRNA compromises the viability of ICB resistant cells but not sensitive cells. Interestingly, conditioned media from the resistant cell lines restricts the in vitro activation of naïve CD8+ T cells. Furthermore, the reduction of ACOD1 among the resistant population results in increased activation of CD8+ T cells when treated with conditioned media from these resistant cells. These observations also extend to other, more established cell lines like the Lewis lung carcinoma (LLC). Taken together, these results indicate that cancerous epithelial cell production of itaconate, through the activity of ACOD1, may represent a mechanism that antagonizes T cell activation and thus promotes ICB evasion. Citation Format: James H. Schofield, Mark A. Hawk, Sharif Rahmy, Ryan D. Sheldon, Casey Leimbach, Seunghoon Sun, Xin Lu, Russell G. Jones, Zachary T. Schafer. Metabolic evasion of mCRPC immunotherapy by ACOD1 production of itaconate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2331.

Abstract 1780: Associations between gut microbiota and PD-L1 immunotherapy/JAK1/2 inhibition in mouse Pten-deficient prostate cancer

Abstract The gut microbiome is now known to influence host immune function, malignancies, and response to therapy. We previously showed that sequencing PD-L1/JAK1/2 blockade prior to androgen deprivation therapy (ADT) could promote immune activity to potentiate antitumor responses in mouse Pten-deficient prostate cancer. Here, we investigate the associations between the treatment pairings of PD-L1 antibody (aPD-L1, clone D265A, mouse/IgG1 kappa) blockade and the JAK1/2 inhibitor AZD1480 with ADT in mouse Pten-deficient prostate cancer and fecal gut microbiomes. We used 16S rRNA amplicon sequencing to survey fecal samples of tumor bearing conditional Pten-knockout (KO) mice treated for four weeks with aPD-L1 and AZD14870 alone and as combination therapy in intact mice, and as concurrent (Conc) therapy with ADT (via surgical castration), or sequenced as adjuvant (Adj) or neoadjuvant (NeoAdj) therapy. In this model, PD-L1 blockade was ineffective as monotherapy in all treatment settings and as combination therapy in intact and Conc combination settings and hyperprogression was observed in some mice treated with aPD-L1 monotherapy in intact and Adj settings. In the NeoAdj setting, aPDL1/AZD1480 therapy demonstrated superiority over monotherapy. Significant differences in microbial composition were observed between groups (P<0.001), drugs (P<0.001), castration status (P<0.001) and treatment responses (P<0.001). AZD1480 as monotherapy or in combination with aPD-L1 was associated with the greatest changes in microbial composition followed by surgical castration. In the neoadjuvant setting Allobaculum, Lactobacillus, Sutturella, Turcibacter and Prevotella were associated with responders whereas Hellicobacter, Oscillospora, and Ruminococcus gnavus were associated with hyperprogression. Functionally, enrichments in fatty acid biosynthesis/metabolism, tetracycline biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and glycolysis/gluconeogenesis were revealed in responders whereas lipopolysaccharide biosynthesis, glycosaminoglycan degradation, fructose and mannose metabolism, histidine metabolism, glyoxylate and dicarboxylate metabolism, and the citrate cycle (TCA) were enriched in low responders. This study provides insight into the complex interactions between gut microbiota and cancer-burdened hosts, and reveals associations between gut microbial composition and treatment responses to androgen deprivation, immune modulation via JAK1/2 inhibition and PD-L1 blockade in a mouse model of Pten-deficient prostate cancer. Citation Format: Marco A. De Velasco, Kazuko Sakai, Yurie Kura, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Associations between gut microbiota and PD-L1 immunotherapy/JAK1/2 inhibition in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1780.

Abstract 2928: Evaluating Cks1-Skp2 interface as an effective therapeutic target for RB1-deficient metastatic castration-resistant prostate cancer (mCRPC)

Abstract Most prostate cancers are locally confined and can be cured by surgery, radiation therapy, and androgen deprivation therapy (ADT). Many prostate cancers initially respond to ADT but become resistant over time and ultimately relapse with a more aggressive, chemotherapy-resistant, and often metastatic disease, termed metastatic castration-resistant prostate cancer (mCRPC), with five-year survival rate <30%. Compared with regional and castration-sensitive prostate cancer, mCRPC harbor higher frequency of inactivating RB1 mutations. Unfortunately, no targeted therapy is available for RB1-deficient tumors, as it is not possible to restore this tumor suppressor in tumor cells. Therefore, we aim to identify downstream signaling targets of pRb which could serve as drug targets and thereby restore the critical functions of pRb. Our group previously identified the E3 ubiquitin ligase SCF-Skp2 as a direct repression target of pRb. Skp2 knockout completely blocks prostate tumorigenesis and cancer-related deaths in an Rb1 and Trp53 double deficient mouse model. As a substrate of Skp2, the CDK inhibitor p27 is upregulated upon Skp2 deletion, which slows cell cycle progression and thus cell proliferation. Our research findings indicate that Skp2 is a promising target for RB1 deficient mCRPC treatment. Skp2 post-translationally regulates a large number of proteins, while Cks1 serves as an essential adaptor protein only for Skp2-mediated p21/p27/p57 ubiquitination, and its Asn45 plays a critical role in binding with Skp2. A Cks1 N45R mutant has been reported to abolish the binding of Cks1 with Skp2. In order to specifically and effectively target Skp2's function in p27 degradation, we generated Cks1 N45R knockin mice and evaluated its anti-tumorigenesis effect. Here we show Cks1 N45R knockin completely inhibits tumorigenesis in the Rb1 and Trp53 double deficient prostate cancer mouse model, with p27 being elevated in the prostate to the comparable amount of that in Skp2 knockout. Surprisingly, Cks1 N45R leads to reduced Skp2 protein stability and consequently the total protein level of Skp2 decreases by more than 50%. We found that the CDK1/2 inhibitor Dinaciclib downregulates Skp2 protein levels by promoting Skp2's interaction and ubiquitination with the E3 ligase Cdh1. Our preliminary data suggests that increased p27 facilitates Cdh1-mediated Skp2 degradation by suppressing Cdk2/CyclinA-mediated Skp2 and Cdh1 phosphorylation. This study highlights the potential of developing small molecule inhibitors targeting the Cks1-Skp2 interface for RB1-mutant mCPRC, it also shows the regulation of Skp2 protein levels may be a viable strategy to phenocopy the antitumor effect of Skp2 knockout. Furthermore, we expect that the combination of Cdk1/2 inhibitors and Cks1-Skp2 interface inhibitors would increase the efficacy and selectivity of targeted therapy in mCRPC. Citation Format: Yingjiao Xue, Liang Zhu, Hongling Zhao, Edward L. Schwartz. Evaluating Cks1-Skp2 interface as an effective therapeutic target for RB1-deficient metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2928.

ATM/NEMO signaling modulates the expression of PD-L1 following docetaxel chemotherapy in prostate cancer

BackgroundThe efficacy of docetaxel-based chemotherapy is limited by the development of drug resistance. Recent studies demonstrated the efficacy of anti-programmed death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) immunotherapies in metastatic prostate...

Abstract LB136: Targeting GCN2 kinase-driven stress response inactivation by orally available small molecules to restore immune tumor microenvironment in prostate cancers

Abstract In patients with metastatic castration-resistant prostate cancers (mCRPC) who have previously been treated with abiraterone or enzalutamide, the median overall survival is only 14 months. Unlike many other cancers, immunotherapies have had limited successes, due to the fact that there are very few T cells in the tumor microenvironment of prostate cancer (PC) patients. Identifying ways to boost immunotherapy responses could change the paradigm of mCRPC, a disease still difficult to treat. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the tumors, leads to depletion of nutrients such as functional/natural amino acids. This metabolically stressful milieu causes activation of nutrient stress pathways, autophagy, and repressed immune responses. A key meditator of this nutrient stress pathway is a cytoplasmic Ser/Thr protein kinase called General Control Nonderepressible 2 (GCN2), also called EIF2AK4. GCN2 switches on by a reduction of amino acids, and its activity results in T cell inactivation, T cell death, regulatory T cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs). We have developed and synthesized a series of novel small molecule immunotherapeutic agents that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit pharmacological responses in immune cells. GCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF-4 phosphorylation inhibition assays were performed and confirmed it's on-target efficacy and potency of lead GCN2 inhibitor HCI-1046. In these studies, our lead GCN2 kinase inhibitor HCI-1046 demonstrated potent activity with an IC50 of 36 nM in inhibiting GCN2. GCN2 expression has been detected in PC-3, DU-145, and LNCap cell lines, and HCI-1046 exhibited cellular efficacy with an IC50 of 0.9 to 8 μM range, reduced phosphorylation of GCN2, ATF-4 significantly and its downstream target eIF2α. HCI-1046 inhibited pGCN2 and pEIF2α in human prostate cancer clinical patient samples derived MDSC cultured in amino-acid starved conditions. HCI-1046, in a dose-dependent fashion, restored CD8+ T cell proliferation and function in clinical samples of PC patients. Our preliminary results support the hypothesis that inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in PC cellular models. In vivo PK studies of HCI-1046 in rodent species showed excellent PK properties; 55% oral bioavailability, low clearance, and >5 hours half-life. HCI-1046 is nominated as a pre-clinical agent. Additional 3D cellular efficacy studies, FACS for cellular apoptosis, cell migration, live PC cells including immuno-ELISA, ELISpot experimental results for MDSCs suppressive function of T cells, and restoration by HCI-1046 will be presented at the conference. PC mouse model efficacy studies will also be discussed. Citation Format: Kyle Medley, Zhaoliang Li, Dongqing Yan, Umang Swami, Neeraj Agarwal, Hariprasad Vankayalapati. Targeting GCN2 kinase-driven stress response inactivation by orally available small molecules to restore immune tumor microenvironment in prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB136.

Stress-Mediated Reprogramming of Prostate Cancer One-Carbon Cycle Drives Disease Progression.

One-carbon (1C) metabolism has a key role in metabolic programming with both mitochondrial (m1C) and cytoplasmic (c1C) components. Here we show that activating transcription factor 4 (ATF4) exclusively activates gene expression involved in m1C, but not the c1C cycle in prostate cancer cells. This includes activation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) expression, the central player in the m1C cycle. Consistent with the key role of m1C cycle in prostate cancer, MTHFD2 knockdown inhibited prostate cancer cell growth, prostatosphere formation, and growth of patient-derived xenograft organoids. In addition, therapeutic silencing of MTHFD2 by systemically administered nanoliposomal siRNA profoundly inhibited tumor growth in preclinical prostate cancer mouse models. Consistently, MTHFD2 expression is significantly increased in human prostate cancer, and a gene expression signature based on the m1C cycle has significant prognostic value. Furthermore, MTHFD2 expression is coordinately regulated by ATF4 and the oncoprotein c-MYC, which has been implicated in prostate cancer. These data suggest that the m1C cycle is essential for prostate cancer progression and may serve as a novel biomarker and therapeutic target. SIGNIFICANCE: These findings demonstrate that the mitochondrial, but not cytoplasmic, one-carbon cycle has a key role in prostate cancer cell growth and survival and may serve as a biomarker and/or therapeutic target.

The Combination of Radium-223 Dichloride and Bortezomib Effectively Reduces Tumor Burden and Lesion Area in the Syngeneic, Systemic 5TGM1 Multiple Myeloma Mouse Model

Radium-223 dichloride (radium-223, Xofigo®), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer with bone metastases. It is a calcium-mimetic that binds to hydroxyapatite in the bone and targets areas of increased bone turnover surrounding cancer lesions. It has been shown that osteoblasts incorporate Radium-223 into the bone matrix in vitro . Radium-223 inhibits disease progression by reducing tumor growth and tumor-induced pathological bone reaction in breast and prostate cancer mouse models. Multiple myeloma (MM) is a plasma-cell malignancy characterized by increased osteoclast and reduced or no osteoblast activity. Bortezomib (Velcade®), an approved treatment for MM, restores the impaired osteoblast activity in MM. Lenalidomide (Revlimid®) induces apoptosis in MM cells and disrupts the interaction between the myeloma cell and its microenvironment. Here, we report results including the effects of radium-223, bortezomib and their combination on mouse MM 5TGM1 cell proliferation in vitro . Furthermore, the anti-tumor effect of the combination of radium-223 with either bortezomib or lenalidomide on the bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model has been evaluated.Proliferation assays were performed with the mouse MM 5TGM1 cells using CellTiter-Glo® Luminescent Cell Viability Assay. In vivo experiments were performed in the syngeneic 5TGM1 mouse MM model. This model recapitulates many features of human MM including monoclonal paraprotein as well as pronounced osteolytic bone lesions. In the combination study radium-223 and bortezomib, female 7-week-old C57BL/KaLwRij mice (n=15/group) were inoculated with 5TGM1 cells via tail vein. The administration of radium-223 (300 kBq/kg, single i.v. injection) and/or bortezomib (1 mg/kg i.p., twice a week) or vehicle control was initiated 26 days later. In addition, the effect of the combination of radium-223 and lenalidomide was tested in a separate experiment using an early therapeutic setting in the syngeneic 5TGM1 MM model. The development of osteolytic lesions was monitored by radiography. Tumor cell apoptosis and necrosis were analyzed in histological samples of the hind limbs. TRACP-stained osteoclasts were counted at the tumor-bone interface. Hind limbs were also used for total activity measurement performed by a gamma-counter. The whole-body anti-tumor effect was determined measuring paraprotein (IgG2b) concentration in serum samples in the in vivo studies.Radium-223 inhibited proliferation of 5TGM1 cancer cells at 800 Bq/ml and bortezomib at 25 nM concentrations. Combination of radium-223 with bortezomib showed increased inhibition of cell proliferation in vitro . The 5TGM1 in vivo model demonstrated that both bortezomib and radium-223 decreased the tumor-induced osteolytic bone lesion area as monotherapy (p<0.05 and p<0.01, respectively), with the combination being more effective than either monotherapy alone (p<0.001). Bortezomib decreased the number of osteoclasts at the tumor-bone interface (p<0.05) and an additive decrease was observed with combination therapy with radium-223 (p<0.01) resulting in almost complete eradication of osteoclasts. The number of apoptotic cells was increased in the radium-223 group, and an increase of necrotic area was observed in the combination group of radium-223 and bortezomib. Tumor burden was lower in groups receiving either bortezomib or bortezomib and radium-223 combination as determined by serum paraprotein (IgG2b). Incorporation of radium-223 to bone was higher with combination therapy based on total activity measurements. All treatments were well tolerated in terms of fatal toxicities, cachexia and body weight loss.In conclusion, radium-223 therapy in combination with bortezomib decreased the osteolytic lesion area, tumor burden and almost completely eradicated tumor-associated osteoclasts in the 5TGM1 mouse model of myeloma bone disease. The incorporation of radium-223 to bone matrix around tumor lesions was improved, possibly via induction of osteoblast activity by bortezomib. These data provide a strong rationale for combination of radium-223 and bortezomib as possible effective therapy in MM. This is currently investigated in a Phase Ib/II trial in patients with early relapsed MM (NCT02928029). DisclosuresSuominen:Pharmatest Services Ltd.: Employment, Equity Ownership. Kähkönen:Pharmatest Services Ltd.: Employment. Mäki-Jouppila:Pharmatest Services Ltd.: Employment. Rissanen:Pharmatest Services Ltd.: Equity Ownership. Luostarinen:Pharmatest Services Ltd.: Employment. Fagerlund:Pharmatest Services Ltd.: Employment, Equity Ownership. Sjöholm:Aurexel Life Sciences Ltd.: Employment. Alhoniemi:Avoltus Oy: Employment. Kakonen:Aurexel Life Sciences Ltd.: Employment, Equity Ownership. Siemeister:Bayer AG: Employment, Equity Ownership, Patents & Royalties. Mumberg:Bayer AG: Employment, Equity Ownership, Patents & Royalties. Halleen:Pharmatest Services Ltd.: Employment, Equity Ownership. Ziegelbauer:Bayer AG: Employment, Equity Ownership, Patents & Royalties. Scholz:Bayer AG: Employment, Equity Ownership, Patents & Royalties.

PPARγ2 functions as a tumor suppressor in a translational mouse model of human prostate cancer.

Peroxisome proliferator-activated receptors γ (PPARγ) is a master regulator that controls energy metabolism and cell fate. PPARγ2, a PPARγ isoform, is highly expressed in the normal prostate but expressed at lower levels in prostate cancer tissues. In the present study, PC3 and LNCaP cells were used to examine the benefits of restoring PPARγ2 activity. PPARγ2 was overexpressed in PC3 and LNCaP cells, and cell proliferation and migration were detected. Hematoxylin and eosin (H&E) staining was used to detect pathological changes. The genes regulated by PPARγ2 overexpression were detected by microarray analysis. The restoration of PPARγ2 in PC3 and LNCaP cells inhibited cell proliferation and migration. PC3-PPARγ2 tissue recombinants showed necrosis in cancerous regions and leukocyte infiltration in the surrounding stroma by H&E staining. We found higher mixed lineage kinase domain-like (MLKL) and lower microtubule-associated protein 1 light chain 3 (LC3) expression in cancer tissues compared to controls by immunohistochemistry (IHC) staining. Microarray analysis showed that PPARγ2 gain of function in PC3 cells resulted in the reprogramming of lipid- and energy metabolism-associated signaling pathways. These data indicate that PPARγ2 exerts a crucial tumor-suppressive effect by triggering necrosis and an inflammatory reaction in human prostate cancer.

Diosgenin attenuates tumor growth and metastasis in transgenic prostate cancer mouse model by negatively regulating both NF-κB/STAT3 signaling cascades

Prostate cancer (PCa) is a common disease among men especially in the old age. The deregulated activation of oncogenic and pro-survival transcription factors has been linked with tumor progression in PCa patients. The consequence of diosgenin treatment on NF-κB/STAT3 activation in PCa cells as well as transgenic mouse model was determined. We also validated the hypothesis of targeting these transcription factors using in silico proteomics simulation model. Diosgenin abrogated NF-κB/STAT3 activation and this action was caused as a result of suppression of protein kinases and reporter gene activity that led to a substantial reduction in the expression of various tumorigenic gene products. In vivo, diosgenin (2% w/w) when mixed in diet and fed to mice abrogated tumor progression in transgenic mice. Diosgenin was also detected in serum and was well absorbed orally. Overall, our data highlights the promising efficacy of diosgenin in PCa therapy.

P0424 - Effects of cytoreductive primary tumor resection in an orthotopic mouse model of metastatic prostate cancer

P0424 - Effects of cytoreductive primary tumor resection in an orthotopic mouse model of metastatic prostate cancer

P0437 - Apalutamide and autophagy inhibition in xenograft mouse model of human prostate cancer

P0437 - Apalutamide and autophagy inhibition in xenograft mouse model of human prostate cancer

Nutritional AGEing and RAGEing as a Regulator of the Tumor Microenvironment

Abstract Objectives Advanced glycation end products (AGEs) are reactive metabolites formed endogenously by glyoxidative, oxidative and lipoxidative stresses. Foods associated with modern dietary habits are particularly AGE laden but despite increasing epidemiological evidence for oncogenic potential, cause and effect relationships are lacking. The objective was to provide detailed mechanistic insight and in vivo confirmation that AGEs found in the diet are oncogenic drivers of tumorigenesis. Methods We used the heat driven formation of glyoxidative, oxidative and lipoxidative stresses in experimental mouse chow to reproduce the wide spectrum of the AGEs found in vivo. Syngeneic xenograft and spontaneous prostate and breast cancer mouse models were then fed the AGE specific diets and the effects of chronic AGE consumption on tumor growth assessed. To gain mechanistic insight, human and mouse two compartment co-culture models using primary fibroblasts and matched tumor epithelial cells were then used to assess the effects of AGEs on extracellular crosstalk in the TME. Results A high impact finding from our research is that consumption of AGEs found in our diet promotes prostate tumor growth, aggression and metastasis by functioning as ligand to the transmembrane receptor for AGE (RAGE). Dietary-AGEs promoted neoplastic growth by functioning as ligand to RAGE expressed in the prostate tumor stroma not tumor epithelium. Dietary-AGE activation of stromal RAGE caused a regulatory program of ‘activated fibroblasts’ defined by the increased expression of cancer associated fibroblast markers, NFkB, MYC and pro-tumorigenic paracrine secretion. Fibroblast activation was accompanied by decreased expression of androgen receptor (AR) and the increased expression of neuroendocrine differentiation markers in tumor epithelial cells. AGE exposed primary fibroblasts isolated from patient tissue conferred tumor promoting abilities when cultured with patient matched tumor epithelial cells. Conclusions For the first time these data demonstrate a direct cause and effect relationship between dietary-AGEs and neoplastic growth. This may lay the foundation for strategic self-management strategies aimed at reducing AGE exposure in the diet to reduce cancer incidence and mortality. Funding Sources NIH/NCI; ACS.

In vivo flow cytometry reveals a circadian rhythm of circulating tumor cells

Circulating tumor cells (CTCs) is an established biomarker of cancer metastasis. The circulation dynamics of CTCs are important for understanding the mechanisms underlying tumor cell dissemination. Although studies have revealed that the circadian rhythm may disrupt the growth of tumors, it is generally unclear whether the circadian rhythm controls the release of CTCs. In clinical examinations, the current in vitro methods for detecting CTCs in blood samples are based on a fundamental assumption that CTC counts in the peripheral blood do not change significantly over time, which is being challenged by recent studies. Since it is not practical to draw blood from patients repeatedly, a feasible strategy to investigate the circadian rhythm of CTCs is to monitor them by in vivo detection methods. Fluorescence in vivo flow cytometry (IVFC) is a powerful optical technique that is able to detect fluorescent circulating cells directly in living animals in a noninvasive manner over a long period of time. In this study, we applied fluorescence IVFC to monitor CTCs noninvasively in an orthotopic mouse model of human prostate cancer. We observed that CTCs exhibited stochastic bursts over cancer progression. The probability of the bursting activity was higher at early stages than at late stages. We longitudinally monitored CTCs over a 24-h period, and our results revealed striking daily oscillations in CTC counts that peaked at the onset of the night (active phase for rodents), suggesting that the release of CTCs might be regulated by the circadian rhythm.

Pain related behaviors and disease related outcomes in an immunocompetent mouse model of prostate cancer induced bone pain

The purpose of this study is to characterize a syngeneic mouse model of prostate cancer induced bone pain. We stably transfected a prostate cancer cell line (RM-1) with green fluorescent protein (GFP) or red fluorescent protein (RFP) and luciferase reporters in order to visualize tumor growth longitudinally in vivo and to assess the relationship between sensory neurons and tumor cells within the bone microenvironment. Following intra-femoral injection of the RM-1 prostate cancer cell line or Hank's buffered saline into male C57Bl6 mice, we observed a progressive increase in spontaneous guarding of the inoculated limb between 12-21 days post inoculation. Daily running wheel performance was assessed as a measure of functional impairment and movement evoked pain. We observed a progressive reduction in the distance traveled and percentage of time at an optimal velocity between 12-21 days post inoculation. We utilized histological, radiographic and μCT analysis to examine tumor induced bone remodeling and observed osteolytic lesions within the distal femur as well as extra-periosteal aberrant cortical bone formation in the tumor bearing femur similar to clinical findings in patients with mixed bone metastatic prostate cancer. Tumor bearing mice displayed significant increases in markers of central sensitization (pERK, pDyn), astrocyte activation (GFAP) but not microglial activation (IBA1) in the ipsilateral spinal cord compared to sham operated mice by three weeks post inoculation. We observed reorganization of blood vessels (endomucin) and alterations in nerve fibers (PGP 9.5) in the intramedullary space of the tumor bearing femur. This immunocompetent mouse model will be useful when combined with transgenic Cre/Flpo driver mice to examine tumor, immune cell and sensory neuron interactions in the bone microenvironment and their role in pain and disease progression associated with bone cancer. Supported by DOD grants W81XWH-17-1-0542 to CMP and W81XWH-17-1-0541 o YS.

Histone deacetylase inhibition can revert induced resistance to immune checkpoint blockade in a mouse model of prostate cancer.

Abstract Castration resistant prostate cancer (CRPC) is one of the leading causes of death in men in the United States, with a 5-year survival rate of 28%. Immune checkpoint blockade (PD-1 &amp; CTLA4 inhibition, ICB) is a novel therapeutic strategy based on restoring the tumoricidal activity of cytotoxic T-lymphocytes. ICB therapy has produced spectacular results in the treatment of melanoma but shows almost no effect in CRPC. We have developed a model system of advanced prostate cancer (Pten−/−/Tp53−/−/Smad4−/−) consisting of a parental (ICB sensitive) cell line and an ICB resistant subline (PPS-ICBR). We analyzed the transcriptome and found significant changes associated with ICB resistance. Subsequently we reversed some of those changes with Vorinostat, a histone deacetylase inhibitor. Mice injected subcutaneously with the PPS-ICBR cell line showed no response to Vorinostat or ICB therapy, but when combined 40% of the mice were completely cured, and another 40% showed significant reductions in tumor growth. We dissociated the tumors and used mass cytometry to determine the changes in the tumor immune microenvironment. ICB + Vorinostat therapy causes a significant increase in CD4+ &amp; CD8+ T-cells, and a significant decrease in M2 macrophages. Concurrent with this decrease in M2 macrophages we observed a significant increase of TNFα+/iNOS+ monocyte derived dendritic cells. Our goal is to study the underlying mechanisms that can transdifferentiate an immuno-suppressive tumor immune microenvironment towards a tumor-suppressive immune microenvironment and improve clinical outcomes.

Evaluation of the PSMA-Binding Ligand 212Pb-NG001 in Multicellular Tumour Spheroid and Mouse Models of Prostate Cancer.

Radioligand therapy targeting the prostate-specific membrane antigen (PSMA) is rapidly evolving as a promising treatment for metastatic castration-resistant prostate cancer. The PSMA-targeting ligand p-SCN-Bn-TCMC-PSMA (NG001) labelled with 212Pb efficiently targets PSMA-positive cells in vitro and in vivo. The aim of this preclinical study was to evaluate the therapeutic potential of 212Pb-NG001 in multicellular tumour spheroid and mouse models of prostate cancer. The cytotoxic effect of 212Pb-NG001 was tested in human prostate C4-2 spheroids. Biodistribution at various time points and therapeutic effects of different activities of the radioligand were investigated in male athymic nude mice bearing C4-2 tumours, while long-term toxicity was studied in immunocompetent BALB/c mice. The radioligand induced a selective cytotoxic effect in spheroids at activity concentrations of 3–10 kBq/mL. In mice, the radioligand accumulated rapidly in tumours and was retained over 24 h, while it rapidly cleared from nontargeted tissues. Treatment with 0.25, 0.30 or 0.40 MBq of 212Pb-NG001 significantly inhibited tumour growth and improved median survival with therapeutic indexes of 1.5, 2.3 and 2.7, respectively. In BALB/c mice, no signs of long-term radiation toxicity were observed at activities of 0.05 and 0.33 MBq. The obtained results warrant clinical studies to evaluate the biodistribution, therapeutic efficacy and toxicity of 212Pb-NG001.

Rutaecarpine administration inhibits cancer cell growth in allogenic TRAMP-C1 prostate cancer mice correlating with immune balance in vivo

BackgroundRutaecarpine (Rut) is a plant alkaloid abundant in Euodia ruticarpa which is a Chinese herbal medicine used for treating various cancers. However, the Rut administration effect on prostate cancer in vivo remains unclear. AimIn the present study we established an allogenic TRAMP-C1 prostate cancer mouse model to evaluate the Rut administration effect and mechanism in vivo. MethodsTo unravel the Rut administration effect on prostate cancer in vivo, C57BL/6J male mice (8 weeks old) were randomly grouped (n = 9), subcutaneously loaded with TRAMP-C1 prostate cancer cells and immediately given daily by gavage with Rut dissolved in soybean oil at 7 mg (low dose), 35 mg (medium dose), and 70 mg/kg b.w./day (high dose) for successive 39 days. ResultsRut administration significantly and dose-dependently reduced both tumor volume and solid prostate cancer weight in allogenic TRAMP-C1 male mice. Rut administration markedly increased (TNF-α+IFN-γ) (Th1-)/IL-10 (Th2-) cytokine secretion ratios by splenocytes and TNF-α (M1-)/IL-10 (M2-) cytokine secretion ratios by macrophages as compared to those of dietary control group, suggesting that Rut administration in vivo regulates the immune balance toward Th1- and M1-polarized characteristics. Decreased CD19+, CD4+ and CD8+ lymphocytes in the peripheral blood of allogenic TRAMP-C1 mice were significantly elevated by Rut administration. Tumor weights positively correlated with TNF-α secretions by splenocytes, suggesting that there is a tumor cachexia in the tumor-bearing mice. Tumor weights negatively correlated with IgG (Th1-antibody) levels in the sera, suggesting that Th1-polarized immune balance may inhibit prostate cancer cell growth. ConclusionsOur results evidenced that Rut administration suppresses prostate cancer cell growth in mice subcutaneously loaded with TRAMP-C1 cells and correlated the anti-cancer effects with Th1-polarized immune balance in vivo.

NF-κB Blockade with Oral Administration of Dimethylaminoparthenolide (DMAPT), Delays Prostate Cancer Resistance to Androgen Receptor (AR) Inhibition and Inhibits AR Variants.

NF-κB activation has been linked to prostate cancer progression and is commonly observed in castrate-resistant disease. It has been suggested that NF-κB-driven resistance to androgen-deprivation therapy (ADT) in prostate cancer cells may be mediated by aberrant androgen receptor (AR) activation and AR splice variant production. Preventing resistance to ADT may therefore be achieved by using NF-κB inhibitors. However, low oral bioavailability and high toxicity of NF-κB inhibitors is a major challenge for clinical translation. Dimethylaminoparthenolide (DMAPT) is an oral NF-κB inhibitor in clinical development and has already shown favorable pharmacokinetic and pharmacodyanamic data in patients with heme malignancies, including decrease of NF-κB in circulating leuchemic blasts. Here, we report that activation of NF-κB/p65 by castration in mouse and human prostate cancer models resulted in a significant increase in AR variant-7 (AR-V7) expression and modest upregulation of AR. In vivo castration of VCaP-CR tumors resulted in significant upregulation of phosphorylated-p65 and AR-V7, which was attenuated by combination with DMAPT and DMAPT increased the efficacy of AR inhibition. We further demonstrate that the effects of DMAPT-sensitizing prostate cancer cells to castration were dependent on the ability of DMAPT to inhibit phosphorylated-p65 function. IMPLICATIONS: Our study shows that DMAPT, an oral NF-κB inhibitor in clinical development, inhibits phosphorylated-p65 upregulation of AR-V7 and delays prostate cancer castration resistance. This provides rationale for the development of DMAPT as a novel therapeutic strategy to increase durable response in patients receiving AR-targeted therapy.