Prostate Cancer Growth In Mice Research Articles

The gut microbiome-prostate cancer crosstalk is modulated by dietary polyunsaturated long-chain fatty acids

The gut microbiota modulates response to hormonal treatments in prostate cancer (PCa) patients, but whether it influences PCa progression remains unknown. Here, we show a reduction in fecal microbiota alpha-diversity correlating with increase tumour burden in two distinct groups of hormonotherapy naïve PCa patients and three murine PCa models. Fecal microbiota transplantation (FMT) from patients with high PCa volume is sufficient to stimulate the growth of mouse PCa revealing the existence of a gut microbiome-cancer crosstalk. Analysis of gut microbial-related pathways in mice with aggressive PCa identifies three enzymes responsible for the metabolism of long-chain fatty acids (LCFA). Supplementation with LCFA omega-3 MAG-EPA is sufficient to reduce PCa growth in mice and cancer up-grading in pre-prostatectomy PCa patients correlating with a reduction of gut Ruminococcaceae in both and fecal butyrate levels in PCa patients. This suggests that the beneficial effect of omega-3 rich diet is mediated in part by modulating the crosstalk between gut microbes and their metabolites in men with PCa.

Abstract 5343: Depletion of gut microbiota with broad spectrum antibiotics drives Pten-null prostate cancer growth in mice

Abstract Gut microbes have the potential to be either promoters or inhibitors of cancer. Use of antibiotics during cancer therapy can lead to dysbiosis, which may affect treatment outcomes. Here, we examine the effects of broad-spectrum antibiotics on cancer progression in mice with Pten-null castration resistant prostate cancer (CRPC). After five or eight weeks, orchiectomized conditional Pten-null knockout (KO) mice aged 50 and 90 weeks were given two biweekly cycles of a broad-spectrum antibody cocktail (BSAbx), respectively. The effects of BSAbx on tumor burden were compared, and molecular and immunological responses were analyzed by quantitative IHC (qIHC) and qRt-PCR at week four after antibiotic treatment. BSAbx treatment resulted in tumor burdens of 1.2 and 2.4-fold higher than in control mice in mice aged 50 and 90 weeks, respectively. qIHC analysis of KI67 showed increased proliferation in stromal and cancer cells. Additionally, androgen receptor (AR) protein and gene expression levels of Ar and Ar target genes (Fkbp5, Tmprss2, and Timp4) were elevated in tumors of BSAbx-treated mice. Restoring gut microbiota by fecal microbiota transplantation (FMT) from KO mice CRPC after BSAbx restored AR transcriptional activity to basal levels and lessened tumor growth. FMT from non-castrated KO with castration-naïve prostate cancer or heathy wildtype mice further reduced AR Transcriptional activity. Moreover, altering the gut microbiota of CRPC mice with FMT from heathy mice resulted in a tendency for lower tumor burden, 21% reduction versus control mice. Immunologically, BSAbx treatment resulted in decreased CD11b+/GR1+ cells in tumors and were associated with decreased interferon-gamma (IFN-gamma), dendritic cell functions and T cell-mediated cytotoxicity gene signatures. Mice that received FMT form health mice showed enhanced T cell mediated immunity as demonstrated by higher IFN-gamma signatures and tumor abundances of CD8+ T cells expression IFN-gamma, TNF-alpha and the cytotoxic granule granzyme B. Our study shows that broad spectrum antibiotics caused dysbiosis and promoted prostate cancer growth in mice with Pten-null prostate cancer. The effects were partly mediated through the upregulation of AR signaling and the impairment of neutrophil and T cell-mediated immune responses in tumors. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Mitsuhisa Nishimoto, Yasunori Mori, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Depletion of gut microbiota with broad spectrum antibiotics drives Pten-null prostate cancer growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5343.

UBC9 deficiency enhances immunostimulatory macrophage activation and subsequent antitumor T cell response in prostate cancer.

The role of tumor-associated macrophages (TAMs), along with the regulatory mechanisms underlying distinct macrophage activation states, remains poorly understood in prostate cancer (PCa). Herein, we report that PCa growth in mice with macrophage-specific Ubc9 deficiency is substantially suppressed compared with that in wild-type littermates, an effect partially ascribed to the augmented CD8+ T cell response. Biochemical and molecular analyses revealed that signal transducer and activator of transcription 4 (STAT4) is a crucial UBC9-mediated SUMOylation target, with lysine residue 350 (K350) as the major modification site. Site-directed mutation of STAT4 (K350R) enhanced its nuclear translocation and stability, thereby facilitating the proinflammatory activation of macrophages. Importantly, administration of the UBC9 inhibitor 2-D08 promoted the antitumor effect of TAMs and increased the expression of PD-1 on CD8+ T cells, supporting a synergistic antitumor efficacy once it combined with the immune checkpoint blockade therapy. Together, our results demonstrate that ablation of UBC9 could reverse the immunosuppressive phenotype of TAMs by promoting STAT4-mediated macrophage activation and macrophage-CD8+ T cell crosstalk, which provides valuable insights to halt the pathogenic process of tumorigenesis.

PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation.

RationaleFructose-1,6-bisphosphatase (FBP1) is a tumor suppressor and a key enzyme negatively regulating Warburg effect in cancer. However, regulation of FBP1 protein expression and its exact role in prostate cancer (PCa) is largely unclear. Phosphatase and tensin homolog (PTEN) is one of the most frequently deleted tumor suppressor genes in human PCa. However, the role of PTEN loss in aberrant Warburg effect in cancer remains poorly understood.MethodsExpression of PTEN and FBP1 was analyzed in several PCa cell lines and prostate tumor tissues in mice. Western blot (WB) and RT-PCR approaches were used to examine how PTEN regulates FBP1 expression. Co-immunoprecipitation (co-IP) and in vivo ubiquitination assays were used to define the regulatory mechanisms. A PCa xenograft model was employed to determine the impact of PTEN regulation of FBP1 on PCa growth in vivo.ResultWe demonstrated that in a manner dependent of PI3K/AKT signal pathway PTEN regulated FBP1 expression in various PCa cell lines and tumors in mice. We confirmed that this regulation took place at the protein level and was mediated by SKP2 E3 ubiquitin ligase. Mechanistically, we showed that serine 271 phosphorylation of FBP1 by cyclin-dependent kinases (CDKs) was essential for SKP2-mediated degradation of FBP1 protein induced by PTEN loss. Most importantly, we further showed that loss of PTEN expression enhanced Warburg effect and PCa growth in mice in a manner dependent, at least partially on FBP1 protein degradation.ConclusionsOur results reveal a novel tumor-suppressive feature of PTEN in restraining FBP1 degradation and the Warburg effect. These results also suggest that prohibiting FBP1 protein degradation could be a viable therapeutic strategy for PTEN-deficient PCa.

A mitochondrial unfolded protein response inhibitor suppresses prostate cancer growth in mice via HSP60

Mitochondrial proteostasis, regulated by the mitochondrial unfolded protein response (UPRmt), is crucial for maintenance of cellular functions and survival. Elevated oxidative and proteotoxic stress in mitochondria must be attenuated by the activation of a ubiquitous UPRmt to promote prostate cancer (PCa) growth. Here we show that the 2 key components of the UPRmt, heat shock protein 60 (HSP60, a mitochondrial chaperonin) and caseinolytic protease P (ClpP, a mitochondrial protease), were required for the development of advanced PCa. HSP60 regulated ClpP expression via c-Myc and physically interacted with ClpP to restore mitochondrial functions that promote cancer cell survival. HSP60 maintained the ATP-producing functions of mitochondria, which activated the β-catenin pathway and led to the upregulation of c-Myc. We identified a UPRmt inhibitor that blocked HSP60’s interaction with ClpP and abrogated survival signaling without altering HSP60’s chaperonin function. Disruption of HSP60-ClpP interaction with the UPRmt inhibitor triggered metabolic stress and impeded PCa-promoting signaling. Treatment with the UPRmt inhibitor or genetic ablation of Hsp60 inhibited PCa growth and progression. Together, our findings demonstrate that the HSP60-ClpP–mediated UPRmt is essential for prostate tumorigenesis and the HSP60-ClpP interaction represents a therapeutic vulnerability in PCa.

Psychosocial Stress and Age Influence Depression and Anxiety-Related Behavior, Drive Tumor Inflammatory Cytokines and Accelerate Prostate Cancer Growth in Mice.

Prostate cancer (PCa) prevalence is higher in older men and poorer coping with psychosocial stressors effect prognosis. Yet, interactions between age, stress and PCa progression are underexplored. Therefore, we characterized the effects of age and isolation combined with restraint (2 h/day) for 14 days post-tumor inoculation on behavior, tumor growth and host defense in the immunocompetent, orthotopic RM-9 murine PCa model. All mice were tumor inoculated. Isolation/restraint increased sympathetic and hypothalamic-pituitary-adrenal cortical activation, based on elevated serum 3-methoxy-4-hydroxyphenylglycol/norepinephrine ratios and corticosterone levels, respectively. Elevated zero maze testing revealed age-related differences in naïve C57Bl/6 mice, and increased anxiety-like behavior in tumor-bearing mice. In open field testing, old stressed mice were less active throughout the 30-min test than young non-stressed and stressed, and old non-stressed mice, suggesting greater anxiety in old stressed mice. Old (18 month) mice demonstrated more depression-like behavior than young mice with tail suspension testing, without effects of isolation/restraint stress. Old mice developed larger tumors, despite similar tumor expression of tumor vascular endothelial growth factor or transforming growth factor-beta1 across age. Tumor chemokine/cytokine expression, commonly prognostic for poorer outcomes, were uniquely age- and stress-dependent, underscoring the need for PCa research in old animals. Macrophages predominated in RM-9 tumors. Macrophages, and CD4+ and CD4+FoxP3+ T-cell tumor infiltration were greater in young mice than in old mice. Stress increased macrophage infiltration in old mice. Conversely, stress reduced intratumoral CD4+ and CD4+FoxP3+ T-cell numbers in young mice. CD8+ T-cell infiltration was similar across treatment groups. Our findings support that age- and psychological stress interacts to affect PCa outcomes by interfering with neural-immune mechanisms and affecting behavioral responses.

Regular Voluntary Running Inhibits Androgen-Independent Prostate Cancer Growth in Mice

Benefits of regular physical exercise were demonstrated as preventive and coadjuvant nonpharmacological anticancer therapy. However, the role of exercise in modulating prostate cancer behavior has yet to be established. Prostate tumors were induced in C57BL/6 male mice (n = 28) by subcutaneous inoculation of a suspension of murine androgen-independent RM1 cells (1.5 × 105 cells/500 μL phosphate-buffered saline) in the dorsal region. Mice were randomly allocated into 2 study groups: sedentary tumor-induced (n = 14) and exercised tumor-induced (n = 14). Exercise consisted of voluntary running in wheeled cages. Mice (n = 7 per group) were sacrificed either 14 or 28days after cell inoculation to evaluate tumor weight and percentage of area occupied by immunohistochemistry stained cells for Ki-67 and TdT-mediated dUTP-biotin nick end labeling, used as surrogate markers of cell proliferation and apoptosis, respectively. Compared with sedentary tumor-induced mice, the tumors developed by exercised tumor-induced mice were significantly smaller at 14days (0.17 [0.12]g vs 0.48 [0.24]g, P < .05) and at 28days (0.92 [0.73]g vs 2.09 [1.31]g, P < .05), with smaller Ki-67 and greater TdT-mediated dUTP-biotin nick end-labeling stained areas (P < .05). These results suggest that regular voluntary running inhibits prostate cancer cell growth by reducing cell proliferation and enhancing apoptosis.

Tunable cytotoxic aptamer–drug conjugates for the treatment of prostate cancer

Therapies that can eliminate both local and metastatic prostate tumor lesions while sparing normal organ tissue are desperately needed. With the goal of developing an improved drug-targeting strategy, we turned to a new class of targeted anticancer therapeutics: aptamers conjugated to highly toxic chemotherapeutics. Cell selection for aptamers with prostate cancer specificity yielded the E3 aptamer, which internalizes into prostate cancer cells without targeting normal prostate cells. Chemical conjugation of E3 to the drugs monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) yields a potent cytotoxic agent that efficiently kills prostate cancer cells in vitro but does not affect normal prostate epithelial cells. Importantly, the E3 aptamer targets tumors in vivo and treatment with the MMAF-E3 conjugate significantly inhibits prostate cancer growth in mice, demonstrating the in vivo utility of aptamer-drug conjugates. Additionally, we report the use of antidotes to block E3 aptamer-drug conjugate cytotoxicity, providing a safety switch in the unexpected event of normal cell killing in vivo.

Bone marrow-derived mesenchymal stem cells induced by inflammatory cytokines produce angiogenetic factors and promote prostate cancer growth

BackgroundProstate is susceptible to infection and pro-inflammatory agents in a man’s whole life. Chronic inflammation might play important roles in the development and progression of prostate cancer. Mesenchymal stem cells (MSCs) are often recruited to the tumor microenvironment due to local inflammation. We have asked whether stimulation of MSCs by pro-inflammatory cytokines could promote prostate tumor growth. The current study investigated the possible involvement of MSCs stimulated by pro-inflammatory cytokines in promotion and angiogenesis of prostate cancer through relative pathway in vitro and in vivo.MethodsA syngeneic mouse model of C57 was established. The murine prostate cancer cells (RM-1) mixing with MSCs treated with tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) or vehicle were subcutaneously injected into C57 mice. Tumor volume of C57 mouse model was estimated and serum level of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) was test by Enzyme-linked Immunosorbent Assay (ELISA). A hen egg test-chorioallantoic membrane (HET-CAM) assay was applied to test the effect of conditioned media of stimulated MSCs in chorioallantoic membrane angiogenesis. Short interfering RNA (siRNA) knocked down either hypoxia-inducible factor-1alpha (HIF-1α) or nuclear factor-erythroid-2-related factor 2 (NRF2) were employed. mRNA of PDGF and VEGF in MSCs, as well as NRF2 and HIF-1α was test by Real time polymerase chain reaction (PCR) analyses. Protein expression levels of PDGF and VEGF from conditioned medium, NRF2, HIF-1α, as well as PDGF and VEGF in MSCs were detected by Western blot analysis.ResultsMSCs treated with TNF-α and IFN-γ promote tumor growth in C57 syngeneic mouse model, correlating with increased serum level of PDGF, VEGF. HET-CAM assay shows the angiogenic effect of conditioned medium of MSCs pre-treated with the pro-inflammatory cytokines. mRNA and protein levels of two pro-angiogenic factors (PDGF and VEGF) and key hypoxia regulators (HIF-1α and NRF2) in MSCs were induced after MSCs’ pretreatment. siRNA knockdown either HIF-1α or NRF2 results reduction of PDGF and VEGF expression.ConclusionsMSCs stimulated by pro-inflammatory cytokines increase the expression of PDGF and VEGF via the NRF2-HIF-1α pathway and accelerate prostate cancer growth in mice.

Mechanism of pro‐tumorigenic effect of BMP‐6: Neovascularization involving tumor‐associated macrophages and IL‐1α

INTRODUCTION. Overexpression of bone morphogenetic protein-6 (BMP-6) has been reported in human prostate cancer tissues. Previously we have demonstrated that BMP-6 enhances prostate cancer growth in mice and not in tissue culture. Herein, we have investigated the mechanism of BMP-6’s pro-tumorigenic effect in prostate cancer. METHODS. Tramp C2 murine and LNCaP human prostate cancer cell lines were co-cultured with RAW 264.7 and THP-1 cells, respectively. IL-1a knockout mice were used to confirm the role of BMP-6/IL-1a loop in vivo. Lastly, conditional macrophage null mice cd11b-DTR was used. RESULTS. The results demonstrated that BMP-6 induced the expression of IL-1a in macrophages via a cross-talk between NF-kB1 p50 and Smad1. When endothelial cells were treated with conditioned media harvested from macrophages incubated with BMP-6, tube formation was detected. In the presence of IL-1a neutralizing antibody, endothelial tube formation was blocked. In vivo, tumor growth and neovascularization decreased significantly when BMP-6 was expressed in IL-1a knockout and conditional macrophage-null mice. CONCLUSIONS. Prostate cancer-derived BMP-6 stimulates tumor-associated macrophages to produce IL-1a through a crosstalk between Smad1 and NF-kB1; IL-1a, in turn, promotes angiogenesis and prostate cancer growth.

Systemic delivery of therapeutic small interfering RNA using a pH-triggered amphiphilic poly-l-lysine nanocarrier to suppress prostate cancer growth in mice

Prostate cancer is associated with high mortality and new therapeutic strategies are necessary for improved patient outcome. The utilisation of potent, sequence-specific small interfering RNA (siRNA) to facilitate down-regulation of complementary mRNA sequences in vitro and in vivo has stimulated the development of siRNA-based cancer therapies. However, the lack of an effective siRNA delivery system significantly retards clinical application. Amphiphilic polycations with ‘stealth’ capacity have previously been synthesised by PEGylation of poly-l-lysine-cholic acid (PLL-CA). The benzoic imine linker between PEG and PLL-CA was designed to be stable at physiological pH but cleavable at lower pHs, consistent with the extracellular environment of tumours and the interior of endosomes/lysosomes. The selective hydrolysis of the PEG linker at these targeted sites should provide enhanced cellular uptake and endosomal escape while simultaneously ensuring prolonged blood circulation times. In this study, physicochemical profiling demonstrated nano-complex formation between the PLL derivatives and siRNA (200–280nm in diameter). At physiological pH only a slight cationic surface charge (<20mV) was detected, due to the masking effect of the PEG. In contrast, significantly higher positive charges (∼20 to 30mV and >40mV) were detected upon hydrolysis of the PEG linker at acidic pHs (pH=6.8 and 5.5, respectively). The PEGylated complexes were stable in serum without significant aggregation or decomplexation of siRNA for up to 48h. At the cellular level, PEG–PLLs were comparable with the commercial carrier INTERFRin™, in terms of cellular uptake, endosomal escape and in vitro reporter gene knockdown. In vivo, utilising a mouse model grafted with prostate carcinoma, significant tumour suppression was achieved using PEGylated complexes without marked toxicity or undesirable immunological response, this was accompanied by a simultaneous reduction in target mRNA levels. In summary, the advantages of these vectors include: the in vitro and in vivo silencing efficiency, and the low toxicity and immunogenicity.

Coevolution of Prostate Cancer and Bone Stroma in Three-Dimensional Coculture: Implications for Cancer Growth and Metastasis

Human bone stromal cells, after three-dimensional coculture with human prostate cancer (PCa) cells in vitro, underwent permanent cytogenetic and gene expression changes with reactive oxygen species serving as mediators. The evolved stromal cells are highly inductive of human PCa growth in mice, and expressed increased levels of extracellular matrix (versican and tenascin) and chemokine (BDFN, CCL5, CXCL5, and CXCL16) genes. These genes were validated in clinical tissue and/or serum specimens and could be the predictors for invasive and bone metastatic PCa. These results, combined with our previous observations, support the concept of permanent genetic and behavioral changes of PCa epithelial cells after being either cocultured with prostate or bone stromal cells as three-dimensional prostate organoids or grown as tumor xenografts in mice. These observations collectively suggest coevolution of cancer and stromal cells occurred under three-dimensional growth condition, which ultimately accelerates cancer growth and metastasis.

Prevention of human PC-346C prostate cancer growth in mice by a xenogeneic tissue vaccine.

Vaccination, as an approach to prostate cancer, has largely focused on immunotherapy utilizing specific molecules or allogeneic cells. Such methods are limited by the focused antigenic menu presented to the immune system and by immunotolerance to antigens recognized as "self". To examine if a xenogeneic tissue vaccine could stimulate protective immunity in a human prostate cancer cell line, a vaccine was produced by glutaraldehyde fixation of harvested PAIII prostate cancer cells tumors (GFT cell vaccine) from Lobund-Wistar rats. Immunocompetent Ncr-Foxn1<nu> mice were vaccinated with the GFT cell vaccine four times, 7 days apart. The control animals were either not vaccinated or vaccinated with media or glutaraldehyde-fixed PC346C human prostate cancer cells and adjuvant. About 8 days after the final boost, serum and spleens were harvested. The splenocytes were co-incubated with PC346C cells and then transplanted orthotopically into sygneneic immunodeficient nude mice. About 10 weeks later, the prostates were weighed and sampled for histolologic examination. The spleens were harvested from additional mice, and the splenocytes were cultured, either with or without pulsing by GFT cells, and the supernatants harvested 72 h later for cytokine analysis. Results showed that vaccination with GFT cells resulted in increased serum antibody to a PAIII cell lysate; reduced weight of the prostate/seminal vesicle complex and reduced incidence of prostate cancer in nude mice; increased splenocyte supernatant levels of TNF-alpha, IL-2, IFN-gamma and IL-12, cytokines associated with Th1 immunity; and increased splenocyte supernatant levels of IL-4 and IL-10, cytokines associated with Th2 immunity. In summary, the results suggest that use of a xenogeneic tissue vaccine can stimulate protective immunity against human prostate cancer cells.