Prostate Cancer Progression In Mice Research Articles

Dietary pro-oxidant therapy by a vitamin K precursor targets PI 3-kinase VPS34 function.

Men taking antioxidant vitamin E supplements have increased prostate cancer (PC) risk. However, whether pro-oxidants protect from PC remained unclear. In this work, we show that a pro-oxidant vitamin K precursor [menadione sodium bisulfite (MSB)] suppresses PC progression in mice, killing cells through an oxidative cell death: MSB antagonizes the essential class III phosphatidylinositol (PI) 3-kinase VPS34-the regulator of endosome identity and sorting-through oxidation of key cysteines, pointing to a redox checkpoint in sorting. Testing MSB in a myotubular myopathy model that is driven by loss of MTM1-the phosphatase antagonist of VPS34-we show that dietary MSB improved muscle histology and function and extended life span. These findings enhance our understanding of pro-oxidant selectivity and show how definition of the pathways they impinge on can give rise to unexpected therapeutic opportunities.

Abstract 1343: Mesenchymal cell populations associated with different stages of prostate cancer progression in mice and human

Abstract Mesenchymal cells in the prostate cancer (PCa) tumor microenvironment (TME) contribute to the biological and clinical history of PCa. Indeed, mesenchymal cells heavily interact with cancer cells, immune cells, and the other cellular and non-cellular components of the TME to favor or hinder carcinogenesis and tumor progression. Using a comprehensive array of genetically engineered mouse models (GEMMs) of prostate cancer, 8 mesenchymal populations with different transcriptional programs are preferentially enriched in specific GEMMs at different stages of PCa. Here, we determine the transferability of this mesenchymal cluster designation from mice PCa models to human PCa cases. To this end, we compared: a) Tmprss2-ERG (T-ERG) mouse and ERG+ human cases; b) Pb4-Cre+/-;Ptenf/f;LSL-MYCN+/+;Rb1f/f (PRN) mouse and PCa bone metastasis. We generated scRNA-seq data for > 8000 mesenchymal cells from ERG+ (n=6) and ERG- (n=3) PCa patients, and we retrieved data for bone metastasis mesenchymal cells (osteoblasts, osteoclasts, endothelial cells, pericytes; 1,872 total cells) from GSE143791. To transfer the stromal mouse clusters’ labels to human data, human gene symbols were converted to their mouse counterparts, then both datasets were restricted to overlapping genes. For the human PCa cases, label transfer was performed through ‘ingest’ using the scRNA-seq data from the mouse T-ERG model as reference for the human ERG+ cases and data from the remaining GEMMs as reference for the human ERG- cases. For bone metastases cases, mouse stromal data from all GEMMs were used to project the 8 stromal clusters to the mesenchymal cells in the bone metastases microenvironment. Not surprisingly, ERG+ human samples were enriched (> 60% of total stromal cells) in mouse stroma clusters predominantly present in T-ERG mouse model, characterized by the expression of Wnt regulators and AR. Common populations to all murine models, representing myofibroblasts and immunomodulatory fibroblasts (expressing Gpx3, C3, C7, Cfh), were also commonly present in patients, irrespectively to the ERG status. In the PCa bone metastases, mesenchymal clusters enriched in the PRN model were strongly represented in human bone metastases, comprising > 60% of total stromal cells. These cells were characterized by high expression of POSTN and MKI67, as well as bone-specific genes like BGN. Altogether, these findings suggest that our mesenchymal cluster designation developed using GEMMs can be meaningfully applied to human PCa, and that the different transcriptional programs we identified in distinct mesenchymal population are conserved across species. This lays the foundation for the utilization of defined genetically-engineered models in defining the interactions and cross-talks between different mesenchymal populations in relation to cancer and immune cells and other components of the TME in human prostate cancer. Citation Format: Mohamed Omar, Hubert Pakula, Filippo Pederzoli, Giuseppe N. Fanelli, Tania Panellinni, Ryan Carelli, Silvia Rodrigues, Caroline Fidalgo-Ribeiro, Pier V. Nuzzo, Lucie V. Emmenis, Mohammad Mohammad, Madhavi Jere, Caitlin Unkenholz, David Rickman, Christopher Barbieri, Brian Robinson, Luigi Marchionni, Massimo Loda. Mesenchymal cell populations associated with different stages of prostate cancer progression in mice and human [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 1343.

B7-H3 Promotes Prostate Cancer Progression in Mice by Antagonizing Myeloid-Derived Suppressor Cell Apoptosis.

Background:B7-H3 is an important immunomodulatory molecule, and clinical studies have confirmed that its expression level is closely correlated with prostate cancer prognosis. However, the mechanism of its biological action is unclear.Methods:An engineered cell line overexpressing B7-H3 was constructed. Cell apoptosis, growth and proliferation assays in vitro and an animal model in vivo were performed to analyze the role and possible mechanism of B7-H3 in promoting prostate cancer progression.Results:Compared with the control cell line (Mock-RM-1), the B7-H3-overexpressing prostate cancer cell line (B7-H3-RM-1) showed no significant growth differences in vitro, whereas the in vivo tumorigenesis rate of B7-H3-RM-1 was significantly higher than that of Mock-RM-1. These results suggest that B7-H3indirectly, rather than directly, promotes prostate cancer progression. Further analysis revealed that significantly higher levels of myeloid-derived suppressor cells (MDSCs) accumulated in vivo in B7-H3-RM-1 tumor-bearing mice than in Mock-RM-1 mice. In vitro and in vivo experiments showed that B7-H3-RM-1 cells significantly antagonized MDSC apoptosis. To further confirm the role of MDSCs in B7-H3-mediated prostate cancer progression, model mice were pretreated with cyclophosphamide before inoculation to clear immune cells and achieve myelo suppression. The results showed no significant differences in tumor growth between the B7-H3-RM-1 group and the Mock-RM-1 group.Conclusions:We found, for the first time, that B7-H3 can antagonize MDSC apoptosis, leading to MDSC accumulation in the tumor microenvironment and thereby promoting prostate cancer progression.

Excess hepsin proteolytic activity limits oncogenic signaling and induces ER stress and autophagy in prostate cancer cells

The serine protease hepsin is frequently overexpressed in human prostate cancer (PCa) and is associated with matrix degradation and PCa progression in mice. Curiously, low expression of hepsin is associated with poor survival in different cancer types, and transgenic overexpression of hepsin leads to loss of viability in various cancer cell lines. Here, by comparing isogenic transfectants of the PCa cell line PC-3 providing inducible overexpression of wild-type hepsin (HPN) vs. the protease-deficient mutant HPNS353A, we were able to attribute hepsin-mediated tumor-adverse effects to its excess proteolytic activity. A stem-like expression signature of surface markers and adhesion molecules, Notch intracellular domain release, and increased pericellular protease activity were associated with low expression levels of wild-type hepsin, but were partially lost in response to overexpression. Instead, overexpression of wild-type hepsin, but not of HPNS353A, induced relocalization of the protein to the cytoplasm, and increased autophagic flux in vitro as well as LC3B punctae frequency in tumor xenografts. Confocal microscopy revealed colocalization of wild-type hepsin with both LC3B punctae as well as with the autophagy cargo receptor p62/SQSTM1. Overexpression of wild type, but not protease-deficient hepsin induced expression and nuclear presence of CHOP, indicating activation of the unfolded protein response and ER-associated protein degradation (ERAD). Whereas inhibitors of ER stress and secretory protein trafficking slightly increased viability, combined inhibition of the ubiquitin-proteasome degradation pathway (by bortezomib) with either ER stress (by salubrinal) or autophagy (by bafilomycin A1) revealed a significant decrease of viability during overexpression of wild-type hepsin in PC-3 cells. Our results demonstrate that a precise control of Hepsin proteolytic activity is critical for PCa cell fate and suggest, that the interference with ERAD could be a promising therapeutic option, leading to induction of proteotoxicity in hepsin-overexpressing tumors.

SAT-326 INPP4B Suppresses Prostate Inflammation And Protects Mice Fed With High-fat Diet From The Development Of Prostate Intraepithelial Neoplasia

High fat diet leads to obesity, chronic inflammation, accelerated prostate cancer progression, and decreased survival. INPP4B, a dual specificity phosphatase, is a tumor suppressor which opposes Akt and PKC signaling pathways activated in response to obesity and prostate cancer progression. Previous research demonstrated that overexpression of PKC in mouse prostate leads to prostate intraepithelial neoplasia (PIN). Akt pathway stimulates prostate cancer progression in mice and is activated in all advanced prostate cancers in men. Using INPP4B knockout mouse model we tested whether INPP4B regulates prostate physiology in mice on high fat diet. The WT and Inpp4b-/- male mice were fed a low-fat diet (LFD) (11% fat) or high-fat diet (HFD) (58% fat) for 12 weeks. The mouse prostate was dissected into the anterior prostate (AP), dorsolateral prostate (DLP) and ventral prostate (VP). We observed that INPP4B and androgen receptor (AR) were expressed primarily in DLP and VP. INPP4B expression was decreased in the DLP of HFD fed mice. In the prostate of mice fed a LFD, we observed similar histological architecture in both WT and Inpp4b-/- mice. All the Inpp4b-/- mice fed a HFD developed PIN, whereas WT mice did not. Cells with polymorphic nuclei were abundant in PIN loci. The staining of α-SMA indicated that prostate epithelial cells invaded the stroma of HFD Inpp4b-/- mice. Consistent with the previous findings, HFD activated PKC, and the loss of INPP4B increased the levels of phospho-PKC βII and ζ in HFD mouse DLP. We previously reported that INPP4B loss changes AR transcriptional output in prostate cancer cells and normal mouse prostate. In HFD group, loss of INPP4B did not affect the level of AR in any of the three prostate lobes but led to an altered AR-dependent transcriptional activity. We show that the pro-inflammatory cytokines Il6, Tnfα and Il1β are expressed at low or undetectable levels in prostates of mice fed LFD. However, all three mediators were strikingly elevated in DLP of HFD Inpp4b-/- mice, where most of the PIN was also observed. Using an IL6 ELISA, we confirmed increases of IL6 protein levels in the prostates of the Inpp4b-/- HFD group. Consistent with elevated levels of inflammatory cytokines in prostates of Inpp4b-/- mice we detected an increase of macrophage specific biomarkers in the DLP of HFD groups, indicating HFD promotes macrophage infiltration in both WT and Inpp4b-/- mice. Importantly, no changes in PTEN were detected at both RNA and protein levels in Inpp4b-/- mice, indicating that increased inflammation and PIN development is due to the loss of INPP4B. Our data suggests that INPP4B suppresses oncogenic signaling pathways and opposes inflammation and neoplastic transformation in prostates of mice maintained on a high fat diet.

Cell surface GRP78 activation by anti‐GRP78 autoantibodies in relation to prostate tumour growth via tissue factor activation.

BackgroundProstate cancer (PC) presentation is associated with the production of anti‐GRP78 autoantibodies that enhance tissue factor (TF) expression and procoagulant activity (PCA). We and others observed that GRP78 is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Normally, GRP78 is an endoplasmic reticulum resident chaperon that ensure the proper folding of newly synthesized polypeptides. We report that binding of these autoantibodies to cell surface GRP78 PC progression in mice, mediated by TF. Further, this observed increase in tumour growth can be reversed with the use of heparin or low molecular weight heparin molecules. We hypothesize that the disruption of the autoantibody/cell surface GRP78 complex represent a viable surrogate treatment of PC.MethodsWild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti‐GRP78 autoantibodies on tumor growth. Protein expression was determined using western blotting and qRT‐PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph's Hamilton.Results ‐Anti‐GRP78 autoantibodies correlate with PC stage: PC patients demonstrated high levels of anti‐GRP78 autoantibodies (60–100 μg/ml, pre‐prostatectomy stage) vs. healthy individuals (5–10 μg/ml). These titers were significantly reduced 24‐weeks post‐prostatectomy. ‐Anti‐GRP78 autoantibodies drive tumour progression: We show here that anti‐GRP78 autoantibodies activate the unfolded protein response, a pro‐survival cellular pathway in cancer cells. Furthermore, these autoantibodies were shown to accelerate tumor growth in a NOD/SCID mouse model. ‐Heparin and low molecular weight heparin abolished the effect of anti‐GRP78 autoantiboides on PC progression. In vitro: We show that heparin and low molecular weight heparin inhibit the binding of anti‐GRP78 autoantibodies. In vivo: a low molecular weight heparin was used as an intervention treatment for anti‐GRP78 autoantibodies and was shown to reverse the effect of anti‐GRP78 autoantibodies on increased tumour growth. ConclusionsWe have identified the function of an agent in patients' blood, anti‐GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.Support or Funding InformationThis work was supported in part by Prostate Cancer Canada (PCC) Discovery Grant (Discovery Grants # 2010‐590, and D2017‐1949), and McMaster Surgical Associate Grant 176725.

Cell surface GRP78 activation by anti-GRP78 autoantibodies confers prostate tumour growth via tissue factor activation

Introduction: Prostate cancer (PC) presentation is associated with the production of anti-GRP78 autoantibodies that enhance tissue factor (TF) expression and procoagulant activity (PCA). We and others observed that GRP78 is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Normally, GRP78 is an ER-resident chaperon that ensure the proper folding of newly synthesized polypeptides. We report that binding of these autoantibodies to cell surface GRP78 PC progression in mice, mediated by TF. Further, this observed increase in tumour growth can be reversed with the use of heparin or low molecular weight heparin molecules.

Cell surface GRP78 activation by anti-GRP78 autoantibodies in relation to prostate tumour growth via tissue factor activation.

31 Background: Prostate cancer (PC) is characterized by increased prothrombotic state due to enhanced tissue factor (TF) expression/procoagulant activity (PCA). We and others observed that GRP78, a molecular chaperone, is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Further, exposure of GRP78 on the surface of PC cells stimulates the production of anti-GRP78 autoantibodies in PC patients. We have reported that binding of these autoantibodies to cell surface GRP78 enhances TF PCA. We hypothesize that this autoantibody/cell surface GRP78 complex interaction increases PC progression and that disruption of this complex would represent a viable target for the treatment of advanced PC. Methods: Wild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti-GRP78 autoantibodies on tumor growth. Protein expression was determined using western blotting and qRT-PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph’s Hamilton. Results: Pre-prostatectomy patients demonstrated high levels of anti-GRP78 autoantibodies (60-100µg/ml) vs. healthy individuals (5-10µg/ml). These titers were significantly reduced 24-weeks post prostatectomy. We show here that anti-GRP78 autoantibodies upregulate TF and its PCA through a mechanism involving depletion of ER Ca2+ stores. This very effect on ER Ca2+ stores also caused activation of the unfolded protein response, a pro-survival cellular pathway. Furthermore, these autoantibodies were shown to accelerate tumor growth in a NOD/SCID mouse model. Finally, heparin and low molecular weight heparin were shown to interfere with the binding of these antibodies to cancer cells and prevent PC cell activation. Conclusions: We have identified the function of an agent in patients' blood, anti-GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.

Effect of anti-GRP78 autoantibodies on prostate cancer progression through tissue factor procoagulant activity.

258 Background: Prostate cancer (PC) is characterized by increased prothrombotic state due to enhanced tissue factor (TF) expression/procoagulant activity (PCA). We and others observed that GRP78, a molecular chaperone, is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Further, exposure of GRP78 on the surface of PC cells stimulates the production of anti-GRP78 autoantibodies in PC patients. We have reported that binding of these autoantibodies to cell surface GRP78 enhances TF PCA. We hypothesize that this autoantibody/cell surface GRP78 complex interaction increase PC progression and that disruption of this complex would represent a viable target for the treatment of advanced PC. Methods: Wild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti-GRP78 autoantibodies on tumour growth. Protein expression was determined using western blotting and qRT-PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph’s Hamilton. Results: Pre-prostatectomy patients demonstrated high levels of anti-GRP78 autoantibodies (60-100µg/ml) vs. healthy individuals (5-10µg/ml). These titres were significantly reduced 24-weeks post prostatectomy. We show here that anti-GRP78 autoantibodies upregulate TF and its PCA through a mechanism involving depletion of ER Ca2+ stores. This very effect on ER Ca2+ stores also caused activation of the unfolded protein response, a pro-survival cellular pathway. Furthermore, these autoantibodies were shown to accelerate tumour growth in a NOD/SCID mouse model. Finally, heparin and low molecular weight heparin were shown to interfere with the binding of these antibodies to cancer cells and prevent PC cell activation. Conclusions: We have identified the function of an agent in patients' blood, anti-GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.

Effect of Metformin, Rapamycin, and Their Combination on Growth and Progression of Prostate Tumors in HiMyc Mice.

In this study, we compared the effect of oral administration of metformin (MET) and rapamycin (RAPA) alone or in combination on prostate cancer development and progression in HiMyc mice. MET (250 mg/kg body weight in the drinking water), RAPA (2.24 mg/kg body weight microencapsulated in the diet), and the combination inhibited progression of prostatic intraepithelial neoplasia lesions to adenocarcinomas in the ventral prostate (VP). RAPA and the combination were more effective than MET at the doses used. Inhibition of prostate cancer progression in HiMyc mice by RAPA was associated with a significant reduction in mTORC1 signaling that was further potentiated by the combination of MET and RAPA. In contrast, treatment with MET alone enhanced AMPK activation, but had little or no effect on mTORC1 signaling pathways in the VP of HiMyc mice. Further analyses revealed a significant effect of all treatments on prostate tissue inflammation as assessed by analysis of the expression of cytokines, the presence of inflammatory cells and NFκB signaling. MET at the dose used appeared to reduce prostate cancer progression primarily by reducing tissue inflammation whereas RAPA and the combination appeared to inhibit prostate cancer progression in this mouse model via the combined effects on both mTORC1 signaling as well as on tissue inflammation. Overall, these data support the hypothesis that blocking mTORC1 signaling and/or tissue inflammation can effectively inhibit prostate cancer progression in a relevant mouse model of human prostate cancer. Furthermore, combinatorial approaches that target both pathways may be highly effective for prevention of prostate cancer progression in men.

Forced LIGHT expression in prostate tumors overcomes Treg mediated immunosuppression and synergizes with a prostate tumor therapeutic vaccine by recruiting effector T lymphocytes.

LIGHT, a ligand for lymphotoxin-β receptor (LTβR) and herpes virus entry mediator, is predominantly expressed on activated immune cells and LTβR signaling leads to the recruitment of lymphocytes. The interaction between LIGHT and LTβR has been previously shown to activate immune cells and result in tumor regression in a virally-induced tumor model, but the role of LIGHT in tumor immunosuppression or in a prostate cancer setting, where self antigens exist, has not been explored. We hypothesized that forced expression of LIGHT in prostate tumors would shift the pattern of immune cell infiltration toward an anti-tumoral milieu, would inhibit T regulatory cells (Tregs) and would induce prostate cancer tumor associated antigen (TAA) specific T cells that would eradicate tumors. Real Time PCR was used to evaluate expression of forced LIGHT and other immunoregulatory genes in prostate tumors samples. For in vivo studies, adenovirus encoding murine LIGHT was injected intratumorally into TRAMP-C2 prostate cancer cell tumor bearing mice. Chemokine and cytokine concentrations were determined by multiplex ELISA. Flow cytometry was used to phenotype tumor infiltrating lymphocytes and expression of LIGHT on the tumor cell surface. Tumor-specific lymphocytes were quantified via ELISpot assay. Treg induction and Treg suppression assays determined Treg functionality after LIGHT treatment. LIGHT in combination with a therapeutic vaccine, PSCA TriVax, reduced tumor burden. LIGHT expression peaked within 48 hr of infection, recruited effector T cells that recognized mouse prostate stem cell antigen (PSCA) into the tumor microenvironment, and inhibited infiltration of Tregs. Tregs isolated from tumor draining lymph nodes had impaired suppressive capability after LIGHT treatment. Forced LIGHT treatment combined with PSCA TriVax therapeutic vaccination delays prostate cancer progression in mice by recruiting effector T lymphocytes to the tumor and inhibiting Treg mediated immunosuppression. Prostate 75:280-291, 2015. © 2014 Wiley Periodicals, Inc.

The Thoc1 ribonucleoprotein and prostate cancer progression.

The majority of newly diagnosed prostate cancers will remain indolent, but distinguishing between aggressive and indolent disease is imprecise. This has led to the important clinical problem of overtreatment. THOC1 encodes a nuclear ribonucleoprotein whose expression is higher in some cancers than in normal tissue. The hypothesis that THOC1 may be a functionally relevant biomarker that can improve the identification of aggressive prostate cancer has not been tested. THOC1 protein immunostaining was evaluated in a retrospective collection of more than 700 human prostate cancer specimens and the results associated with clinical variables and outcome. Thoc1 was conditionally deleted in an autochthonous mouse model (n = 22 or 23 per genotype) to test whether it is required for prostate cancer progression. All statistical tests were two-sided. THOC1 protein immunostaining increases with higher Gleason score and more advanced Tumor/Node/Metastasis stage. Time to biochemical recurrence is statistically significantly shorter for cancers with high THOC1 protein (log-rank P = .002, and it remains statistically significantly associated with biochemical recurrence after adjusting for Gleason score, clinical stage, and prostate-specific antigen levels (hazard ratio = 1.61, 95% confidence interval = 1.03 to 2.51, P = .04). Thoc1 deletion prevents prostate cancer progression in mice, but has little effect on normal tissue. Prostate cancer cells deprived of Thoc1 show gene expression defects that compromise cell growth. Thoc1 is required to support the unique gene expression requirements of aggressive prostate cancer in mice. In humans, high THOC1 protein immunostaining associates with prostate cancer aggressiveness and recurrence. Thus, THOC1 protein is a functionally relevant molecular marker that may improve the identification of aggressive prostate cancers, potentially reducing overtreatment.

Abstract 673: A Monoclonal Antibody, MORAb-075, Targeting Claudin-3 & Claudin-4 Inhibits human ovarian, pancreatic and prostate cancer progression in mice

Abstract Claudin (CLDN) is a family of integral membrane proteins. They are crucial for tight junction formation and functions. Studies have found CLDN3 and CLDN4 proteins are overexpressed in majority of primary ovarian, prostate, and pancreatic cancers. We have generated a rat-human chimeric monoclonal antibody, MORAb-075, which binds to CLDN3 and CLDN4 with high affinity in vitro. In order to evaluate its therapeutic potential in vivo, we have established a number of human cancer models where the tumors are grown orthotopically in nude mice. These tumors were confirmed positive for human CLDN3 or/and CLDN4 expressions via immunohistochemistry staining. The models include a CLDN3/4-expressing prostate cancer PC-3; a CLDN4-expressing pancreatic cancer, BxPc-3; a CLDN3-expressing ovarian cancer, IGROV-1. In these orthotopic tumor models, MORAb-075 demonstrated significant antitumor activities. When the mAb was administered intravenously at 25 mg/kg, twice weekly for 4 weeks, the tumor growth inhibition (TGI) of PC-3 tumors reached 50%; TGI of BxPC-3 tumors was nearly 60% and TGI of IGROV-1 tumors was greater than 75%. Our findings demonstrate the potency of MORAb-075, as a single agent, in inhibiting solid tumor growth in these models. These results suggest that mAb therapy targeting CLDN3 & CLDN4 is a valuable option for therapeutic application in cancer therapy that may be improved in combination with standard of care chemotherapies for CLDN3 or CLDN4 positive cancers. Citation Format: Jian-Min Lin, allis soto, Christopher maddage, earl Albone, yuhong zhou, luigi grasso. A Monoclonal Antibody, MORAb-075, Targeting Claudin-3 & Claudin-4 Inhibits human ovarian, pancreatic and prostate cancer progression in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2014-673

Circulating microRNAs: Understanding the Limits for Quantitative Measurement by Real‐Time PCR

Ever since the discovery of small, non-coding RNAs in circulation, microRNAs have gained significant attention as biomarkers or, in some instances, regulators of disease and/or associated complications. Until now, most articles published use real-time quantitative PCR (qPCR) for assessment of

High fiber diet prevents prostate cancer progression in mice

High fiber diet prevents prostate cancer progression in mice

Deregulation of FoxO3a accelerates prostate cancer progression in TRAMP mice

Forkhead box, class "O" (FoxO) transcription factors are involved in multiple signaling pathways and possess tumor suppressor functions. Loss of PTEN and activation of PI3K/Akt is frequently observed in prostate cancer, which may potentially inactivate FoxO activity. We therefore investigated the role of FoxO transcription factors in prostate cancer progression, in particular FoxO3a, in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, which mimics progressive forms of human disease. Prostate cancer progression in TRAMP mice was followed from 8 to 28 weeks. Expression patterns of Akt, FoxO1a, FoxO3a, FoxO4, and their phosphorylated form, DNA binding activity and downstream signaling molecules during different stages of disease progression were examined by immunoblotting, immunoprecipitation, enzyme-linked immunoabsorbant assay (ELISA), and immunohistochemistry. Inhibition of FoxO3a activity was attained by using FoxO3a peptide treatment to TRAMP mice. In TRAMP mice, FoxO3a activity is negatively regulated by Akt/PKB through post-translational modification. Progressive increase in Akt activation during prostate cancer progression led to increase phosphorylation of FoxO3a and binding with 14-3-3, which potentially affected its transcriptional activity in age-specific manner. Furthermore, blocking FoxO3a activity resulted in accelerated prostate cancer progression in these mice, which was associated with the loss of cell cycle control and increased proliferation and survival markers. Restoration of FoxO3a activity represents an attractive therapeutic target in the chemoprevention and possibly in inhibition of progression of prostate cancer.

Abstract 5423: Diet induced obesity, inflammation and prostate cancer progression in Hi-Myc mice

Abstract Obesity occurs when energy intake exceeds expenditure and is associated with increased risk and/or mortality rates for various types of cancers. In contrast, calorie restriction (CR) has been shown to act as a potent inhibitor of tumorigenesis in many tissues. We have recently reported that diet induced obesity (DIO) enhanced prostate cancer progression in the ventral prostate of the Hi-Myc mice (Blando et al, CAPR, 2011). Significant increases in inflammatory gene expression were observed as well as accumulation of inflammatory cells. In the current study, we have further investigated changes in inflammatory genes as well as cellular changes associated with DIO effects on prostate cancer progression in this mouse model. Initial studies were conducted to look at some of the cell populations that reside in the ventral prostate (VP) of Hi-Myc mice on the 30% CR, overweight control (AIN76A) and DIO diets (60% Kcal fat). Staining for perilipin (a lipid associated protein that marks adipocytes) revealed that adipocytes were restricted to the periphery of the VP of Hi-Myc mice on the 30% CR diet. In contrast, adipocytes could be seen infiltrating the VP of Hi-Myc mice on the overweight control diet. Notably, in mice on the DIO diet, many of the glands in the VP were completely surrounded by adipocytes. Additionally, we performed double staining for either perilipin and CD3 (T-lymphocytes) or perilipin and RM0029-11H3 (macrophages). Few if any inflammatory cells were seen in VP of Hi-Myc mice on the 30% CR diet. In contrast, adipocytes and inflammatory cells (both T-cells and macrophages) were seen in close proximity to adipocytes that had infiltrated into the VP in both the overweight control and DIO diet groups. This was particularly evident in Hi-Myc mice on the DIO diet. In addition, we have also stained sections of VP from Hi-Myc mice on the different diets for both the IL-23 receptor (IL-23R) and Cox-2. IL-23R expression was dramatically upregulated in the epithelial cells of the VP from Hi-Myc mice on the DIO compared to the other diet groups. These data suggest that IL-23 may play a role not only in driving further inflammatory cell activation (e.g., TH17+/ CD4+ T-cells) but also have direct effects on prostate epithelial cells that facilitate prostate cancer progression. Cox-2 expression was also dramatically upregulated in VP of Hi-Myc on the DIO diet. This finding is consistent with the high level of NFkB activation seen in prostate tissues for the same mice and diet group. Thus, Cox-2 and subsequent generation of arachidonic metabolites may also play a role in the effects of DIO on prostate cancer progression in Hi-Myc mice. Collectively, changes in the levels of IL-23R and COX-2 and the elevated presence of inflammatory cells together with changes in the adipose tissue, suggest that the tumor associated microenvironment may play a key role in DIO effects on prostate cancer progression in Hi-Myc mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5423. doi:1538-7445.AM2012-5423

The relationship of HIF2 and the progression of PTEN-deficient mouse prostate tumors.

30 Background: The hypoxia-inducible factor (HIF) transcription factor has already cemented its oncogenic role in the development of renal cell carcinoma (RCC). However, its role in the tumorigenesis of other solid tumors remains unspecificed. Our studies focus on a novel link between HIF and prostate carcinogenesis. Methods: Using both in vitro cell culture studies as well as in vivo studies (orthotopic xenograft and genetically engineered mouse models) we investigate the role of HIF in prostate cancer cell proliferation, invasion, and progression. Results: Both HIF1 and HIF2 appear to be necessary for the proliferation and invasion of prostate cancer cell lines in vitro. Preliminary analysis of a PTEN deficient mouse model of prostate cancer suggests that expression of a stabilized form of HIF2 promotes the development of a larger prostate tumor burden and a more aggressive histology (high grade prostate intraepithelial neoplasia [PIN] at earlier stages). Moreover, PTEN-deficient prostate tumors producing HIF2 are more proliferative and vascular and express increased levels of genes associated with epithelial to mesenchymal transition (EMT). Conclusions: There has been much interest in the role of angiogenesis and hypoxia in prostate cancer progression. Our preliminary data suggest that HIF2 is able to promote PTEN-deficient prostate cancer progression in mice by increasing proliferation, angiogenesis, and EMT. No significant financial relationships to disclose.

SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Abrogation of De novo Lipogenesis by Stearoyl-CoA Desaturase 1 Inhibition Interferes with Oncogenic Signaling and Blocks Prostate Cancer Progression in Mice

Increased de novo fatty acid (FA) synthesis is one hallmark of tumor cells, including prostate cancer. We present here our most recent results showing that lipid composition in human prostate cancer is characterized by an increased ratio of monounsaturated FA to saturated FA, compared with normal prostate, and evidence the overexpression of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1) in human prostate cancer. As a new therapeutic strategy, we show that pharmacologic inhibition of SCD1 activity impairs lipid synthesis and results in decreased proliferation of both androgen-sensitive and androgen-resistant prostate cancer cells, abrogates the growth of prostate tumor xenografts in nude mice, and confers therapeutic benefit on animal survival. We show that these changes in lipid synthesis are translated into the inhibition of the AKT pathway and that the decrease in concentration of phosphatidylinositol-3,4,5-trisphosphate might at least partially mediate this effect. Inhibition of SCD1 also promotes the activation of AMP-activated kinase and glycogen synthase kinase 3alpha/beta, the latter on being consistent with a decrease in beta-catenin activity and mRNA levels of various beta-catenin growth-promoting transcriptional targets. Furthermore, we show that SCD1 activity is required for cell transformation by Ras oncogene. Together, our data support for the first time the concept of targeting the lipogenic enzyme SCD1 as a new promising therapeutic approach to block oncogenesis and prostate cancer progression.