Prostate Cancer Bone Metastases Research Articles

Myelodysplastic Syndrome Related to Successful Treatment With177Lu-PSMA for Metastatic Castration-Refractory Prostate Cancer.

177Lu-vipivotide tetraxetan (177Lu-PSMA-617/LuPSMA) received recent EMA approval for metastatic castration-resistant prostate cancer, with promising data for earlier stages. Secondary myeloid neoplasm following exposure to DNA-damaging therapy (therapy-related myelodysplastic syndrome [MDS]) is a rare severe complication of 177Lu-oxodotreotide. We present a 77-year-old man, with synchronous liver, bone, and lymph node metastatic prostate cancer, having developed a low-risk MDS with SF3B1 mutation, 1 month after the sixth administration of LuPSMA. Although on partial metabolic and biological response with PSA nadir at 7 months after therapy, life quality was significantly altered by MDS.

Immunogenicity of radiotherapy on bone metastases from prostate adenocarcinoma: What is the future for the combination with radiotherapy/immunotherapy?

Bone metastatic prostate cancers (PCa) are resistant to usual immunotherapies such as checkpoint inhibitors. The main hypothesis related to this immunoresistance is the lack of antigens to stimulate anti-tumor immunity. External radiation is a potential inducer antigens presentation and thus to immunotherapy proprieties. The aim of this review is to describe the tumor microenvironment specificities, especially in bone metastasis and the immune modifications after radiation therapy on a metastatic castration-resistant PCa population. PCa microenvironment is immunosuppressive because of many tumor factors. The complex interplay between PCa cells and bone microenvironment leads to a 'vicious circle' promoting bone metastasis. Furthermore, the immune and bone systems, are connected through an osteoclastogenic cytokine: the Receptor Activator Nuclear Factor Kappa B ligand. Adapted doses of ionizing radiation play a dual role on the tumor. Indeed, radiotherapy leads to immunogenicity by inducing damage associated with molecular patterns. However, it also induces an immunosuppressive effect by increasing the number of immunosuppressive cells. Interestingly, the abscopal effect could be used to optimize immunotherapy potential, especially on bone metastasis. Radiotherapy and immunotherapy combination is a promising strategy, however further studies are necessary to determine the more efficient types of radiation and to control the abscopal effect.

TempEasy 3D Hydrogel Coculture System Provides Mechanistic Insights into Prostate Cancer Bone Metastasis.

Patients diagnosed with advanced prostate cancer (PCa) often experience incurable bone metastases; however, a lack of relevant experimental models has hampered the study of disease mechanisms and the development of therapeutic strategies. In this study, we employed the recently established Temperature-based Easy-separable (TempEasy) 3D cell coculture system to investigate PCa bone metastasis. Through coculturing PCa and bone cells for 7 days, our results showed a reduction in PCa cell proliferation, an increase in neovascularization, and an enhanced metastasis potential when cocultured with bone cells. Additionally, we observed increased cell proliferation, higher stemness, and decreased bone matrix protein expression in bone cells when cocultured with PCa cells. Furthermore, we demonstrated that the stiffness of the extracellular matrix had a negligible impact on molecular responses in both primary (PCa cells) and distant malignant (bone cells) sites. The TempEasy 3D hydrogel coculture system is an easy-to-use and versatile coculture system that provides valuable insights into the mechanisms of cell-cell communication and interaction in cancer metastasis.

Abstract 161: Aged mice decelerate bone metastasis of prostate cancer by decreasing infiltration of M2 macrophages

Abstract Bone metastasis is a major contributor to the mortality of patients with prostate cancer and results in the development of an immunosuppressive microenvironment in the bone marrow. While advanced age increases the risk of bone metastasis, effects of age-related changes on immune microenvironmental homeostasis are not yet clearly understood. To investigate interactions between cancer cells and the immune microenvironment in elderly individuals, we utilized murine-derived specific bone metastatic prostate cancer cells (RM1-BM), which we had previously isolated from bone metastatic mice and transfected with a luciferase-based reporter gene. We then injected the cells into the left ventricle of both 3‒6 months and 22‒24 months, male, wild-type mice to induce bone metastasis, using age-matched mice injected with PBS as a control. We employed in vivo imaging to compare the metastatic levels in both groups of mice every other day. On day 14, we extracted bone marrow cells for CyTOF analysis and femurs and tibias for Hematoxylin and Eosin staining. Interestingly, our preliminary results demonstrated that healthy aged mice exhibited a lower susceptibility to develop bone metastasis when compared to their younger counterparts. A further investigation of the immune subtypes revealed an increase in the percentage of macrophages in CD45+ immune cells in young mice that developed bone metastasis as opposed to age-matched controls. However, the percentage decreased in aged mice, going from 12.20% to 9.92% (p = 0.001). Lastly, macrophages were isolated from the bone marrow of 3‒6 months and 22‒24 months old mice and were co-cultured with ovalbumin-transfected RM1-BM cells (RM1-BM-ova) in vitro for 24 hours. The flow cytometry results indicated that both groups consist mainly of CD206+ (M2) macrophages. Nonetheless, those obtained from aged mice exhibited reduced ovalbumin presentation capability while maintaining a steady level of type I major histocompatibility complex (MHC-I). These findings suggested that increased infiltration of pro-inflammatory M2 macrophages within the tumor microenvironment may contribute to the bone metastasis of prostate cancer. Therefore, inhibiting M2 macrophages recruitment to the bone marrow microenvironment may prove a useful approach for treating patients with prostate cancer bone metastasis. This study was supported by the NSFC projects (81972420, 81972766, 82173336), and grants from the Shenzhen Science and Technology Innovation Commission (JCYJ20190809161811237, JCYJ20210324104214040). Citation Format: Xiao Zhang, Wei Yu, Xin Huang, Yi Lu, Jian Zhang. Aged mice decelerate bone metastasis of prostate cancer by decreasing infiltration of M2 macrophages [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 161.

Comparison of the relative diagnostic performance of 68Ga-DOTA-IBA and 18F-NaF for the detection of bone metastasis.

We aimed to compare the relative diagnostic efficacy of 68Ga-Labeled DOTA-ibandronic acid (68Ga-DOTA-IBA) to that of18F-NaF PET/CT as a mean of detecting bone metastases in patients with a range of cancer types. This study retrospectively enrolled patients with bone metastases associated with various underlying malignancies. All patients underwent both 68Ga-DOTA-IBA and 18F-NaF PET/CT scans. Histopathology and follow-up CT or MRI imaging results were used as reference criteria, with a minimum follow-up period of 3 months. The maximum Standardized Uptake Value (SUVmax) and number of bone metastases were recorded. The Target-Background Ratio (TBR) was calculated along with the detection rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of 68Ga-DOTA-IBA and 18F-NaF PET/CT imaging for overall and partial primary solid tumor bone metastases. Pearson chi-square test, McNemar test, and Kappa test was conducted to assess the correlation and consistency of diagnostic efficiency between the two imaging agents. Receiver Operating Characteristic curve (ROC curve) was performed to compare diagnostic performance and the area under the curve of the two imaging agents, determining optimal critical values for SUVmax and TBR in diagnosing bone metastasis. Differences in SUVmax and TBR values between the two imaging agents for detecting bone metastases were analyzed using the Wilcoxon signed rank test. The difference was statistically significant when P < 0.05. A total of 24 patients (13 women and 11 men) were included in this study, with a mean age of 52 (interquartile range, 49-64 years). The detection rate, sensitivity, specificity, PPV, NPV, accuracy, and AUC of 68Ga-DOTA-IBA and 18F-NaF PET/CT for bone metastases were 81%, 90%, 62%, 95%, 43%, 88%, 0.763, and 89%, 99%, 59%, 95%, 89%, 95%, 0.789, respectively. There was no significant difference between the two imaging methods (P < 0.01), and there was a significant correlation (X2=168.43, P < 0.001) and a strong consistency (Kappa=0.774,P < 0.001) between the diagnostic results of the two imaging agents. The SUVmax values of lesions measured by 68Ga-DOTA-IBA and 18F-NaF imaging in 22 patients with bone metastasis were 5.1 ± 5.4 and 19.6 ± 15.1, respectively, with statistically significant differences (P<0.05). The TBR values of the two imaging methods were 5.0 ± 5.0 and 6.7 ± 6.4, respectively, with statistically significant differences (P<0.05). The AUC of the SUVmax of 68Ga-DOTA-IBA and 18F-NaF curves were 0.824 and 0.862, respectively, with no statistically significant difference (P=0.490). No significant difference was found in the AUC of the TBR of 68Ga-DOTA-IBA and 18F-NaF (0.832 vs 0.890; P=0.248). Subgroup analysis showed significant correlation between the two imaging agents in the diagnosis of bone metastases in lung cancer and breast cancer, with consistent diagnostic results. However, in the diagnosis of bone metastases in prostate cancer, there was a significant difference (P<0.001) and lack of consistency (P=0.109). The diagnostic efficacy of 68Ga-DOTA-IBA for bone metastasis lesions is comparable to that of 18F-NaF. This finding holds significant clinical importance in terms of diagnosis of bone metastasis and selecting treatment plans for patients with malignant tumors.

Abstract 103: Prostate cancer bone metastasis: Therapeutic targeting of tumor associated macrophages

Abstract Introduction: Castration-resistant bone metastatic prostate cancer is a morbid and deadly disease with an exceedingly low response rates to available immunotherapies. Understanding the mechanisms behind immune evasion and tumor progression is critical to improving therapy. Historically, bone metastases have been challenging to model in the laboratory. Our foundational work was built upon fresh patient samples taken directly from the operating room for analysis across multiple platforms: IHC, single cell expression profiling, and spatial transcriptomics. We compared tumors from patients with bone metastatic disease to tumors from patients with disease localized to the prostate. Our work (Cancer Cell, 2021 and Nature Communications, 2023) has consistently pointed towards dysregulated myeloid populations within the tumor microenvironment, particularly TREM2+ tumor associated macrophages (TAMs), as key drivers of disease progression. Methods: Our patient samples and murine pre-clinical models of prostate cancer highlighted several specifically upregulated molecules and pathways in TAMs. Here we focus on TAMs in vitro and in vivo using a new and genetically tractable myeloid model to interrogate the mechanistic role of key pathways, and how they may contribute to T-cell immune suppression. The ER-Hoxb8 system of conditional myeloid differentiation permitted the generation of isogenic cell lines to interrogate the roles of key molecules TREM2, MSR1, APOE, and MERTK through overexpression or CRISPR/Cas9 knockout. Results: Using a system of co-culture in vitro, we quantified the role of these proteins in suppressing T-cell proliferation and cytokine production, as well as their role in TAM differentiation and function. In vivo, these lines were co-injected subcutaneously with syngeneic RM1-BM3 tumor cells to quantify their effect on tumor growth and T-cell suppression in vivo. In addition to localized subcutaneous tumors, bone metastatic disease was established by intracardiac injection. Furthermore, established tumors were treated with TAM-directed small molecule therapy (e.g., MERTK inhibition) as well as immune checkpoint inhibition (anti-PD1) alone, and in combination. Conclusions: TAMs are immune suppressive myeloid cells that are not found in normal tissues. They are specifically recruited to tumors and highly enriched in bone metastatic disease. Transcriptomic analyses highlighted signaling pathways potentially amenable to productive therapeutic targeting. The ER-Hoxb8 system was highly effective in generating a genetically tractable model of TAM for study in vitro and in vivo. Our work supports the hypothesis that the specific targeting of TAMs, by focusing on critical signaling pathways, can be effective in the treatment of pre-clinical models of prostate cancer. This new approach merits further study as a potential strategy for the treatment of patients with bone metastases. Citation Format: Hanyu Zhang, Shenglin Mei, Nathan E. Jeffries, Douglas M. Dahl, Chin-Lee Wu, Philip J. Saylor, David B. Sykes. Prostate cancer bone metastasis: Therapeutic targeting of tumor associated macrophages [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 103.

Abstract 5387: Host-derived CCL2 drives prostate cancer bone metastasis via CCND2/STAT3 pathway

Abstract Overexpression of tumor-derived CCL2 has been shown to partially promote prostate cancer (PCa) bone metastasis, but the function of host-derived CCL2 is not fully understood. We first developed a mouse model using intracardiac injection and obtained highly metastatic PCa cell subclones through in vivo selection. The highly metastatic tumor cells were injected intracardially into CCL2 knockout (CCL2 KO) mice and wild-type (WT) controls, and the tumor cell growth and metastasis were monitored weekly by bioluminescence imaging. The osteolytic lesions were evaluated by radiography, micro-computed tomography (microCT) and immunohistochemistry (IHC) staining. The cells in the bone microenvironment were analyzed by flow cytometry. Also, RNA sequencing (RNA-seq) analysis was performed on the cells either from CCL2 KO mice or WT controls. We found that a loss of host-derived CCL2 significantly retarded tumor growth in bone and prolonged mouse survival. In addition, bone density analysis revealed a decrease in osteolytic lesion in CCL2 KO mice, compared to WT controls. Systemically, CCL2 deficiency reduced the proportion of granulocyte-myeloid-derived suppressor cell (G-MDSC) populations with immunosuppressive function and decreased the PCa conditioned medium (CM)-induced osteoclast formation. The enrichment analysis revealed that the dysregulated genes in bone metastatic cells from CCL2 KO mice were significantly enriched in several biological processes, including vitamin metabolism, monoatomic anion homeostasis, immune response, and bone growth. Additionally, we also confirmed that the CCND2/STAT3 pathway is apparently suppressed in the cells from CCL2 KO mice. This study demonstrated that targeting the host-derived CCL2 might be a novel potential anti-metastatic approach. Supported by the NSFC projects (81972766, 81972420, 82173336), as well as grants from the Science and Technology Project of Shenzhen (JCYJ20190809161811237, JCYJ20210324104214040). Key words Host-derived CCL2; Prostate cancer; Bone metastasis; osteoclast; CCND2 Citation Format: Jie Meng, Ming Chang, Siyuan Qin, Xin Huang, Weiping Liang, Haibo Tong, Jian Zhang. Host-derived CCL2 drives prostate cancer bone metastasis via CCND2/STAT3 pathway [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 5387.

Abstract 2815: Immunotherapy development landscape for bone metastasis - need of predictive preclinical efficacy evaluation for de-risking clinical development

Abstract Most cancer deaths are due to metastases, and bone metastases are challenge especially in breast and prostate cancer, being developed in 70-90% of advanced-stage patients. Bone metastases remain incurable causing high mortality, skeletal-related effects and decreased quality of life. Currently used immunotherapies are ineffective in bone metastases. This creates a need to improve understanding immune microenvironment in bone metastases, namely interactions between tumor, immune and bone cells according to the osteoimmuno-oncology (OIO) concept, and ultimately to develop immunotherapies that primarily target bone metastases. We used 1stOncology database, a cancer drug development resource to identify novel immunotherapies in development for breast and prostate cancer bone metastasis. Twenty-four immunotherapies that included evaluation of effects on bone metastasis were identified. Efficacy of only three of the identified therapies had been evaluated in preclinical bone metastasis models, one reason being that such models have not been commonly available. The use of clinically relevant and predictive preclinical bone metastasis models would significantly de-risk decision making when moving to clinical development in bone metastasizing cancers. To support predictive preclinical evaluation of immunotherapies for bone metastatic cancers, we describe a preclinical bone metastasis technology platform for evaluating efficacy of immunotherapies. The platform utilizes tumors growing in bone microenvironment, mimicking growth of bone metastases in patients, and generates tumor-induced bone effects in a cancer-type specific manner. Syngeneic and/or humanized mouse models with tumor and immune cells of the same species are needed for development of immunotherapies, allowing interactions of tumor and immune cells in the bone metastatic microenvironment to support mode-of-action studies. The platform is clinically predictive, as demonstrated with observed effects that align with clinical findings. As an example, anti-PD-1 antibody had no effects on breast and prostate cancer bone metastases, predicting recent clinical findings that demonstrate lack of efficacy in clinical prostate cancer trials. We conclude that tumor microenvironment in bone metastases has unique characteristics and is understudied as a potential primary cause of lack of efficacy of many immunotherapies, especially in breast and prostate cancer. It is prime time to focus on bone metastases by increasing understanding of the immune landscape in the bone tumor microenvironment according to the OIO concept. The biologically relevant and clinically predictive preclinical bone metastasis technology platform should be routinely used to study the mechanism-of-action and efficacy of therapies before entering clinical development in bone metastasizing cancers. Citation Format: Tiina E. Kähkönen, Jussi M. Halleen, Gary MacRitchie, Ronnie M. Andersson, Jenni Bernoulli. Immunotherapy development landscape for bone metastasis - need of predictive preclinical efficacy evaluation for de-risking clinical development [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 2815.

Abstract 1501: Engineering tissue models to elucidate prostate cancer bone metastasis dynamics

Abstract Prostate cancer (PCa) ranks as the second most frequently diagnosed cancer in men and the fifth leading cause of male cancer-related deaths globally Advanced PCa exhibits a propensity for bone metastasis which significantly contributes to morbidity. It is challenging to understand its mechanisms and develop effective treatments due to complicated tumor microenvironments. The current study addresses this gap by focusing on the construction of in vitro and in vivo models to mimic tissue-specific microenvironments resembling primary prostate tumor sites and metastatic bone tissue, shedding light on the complex processes of cancer cell dissemination, osteomimicry, and bone metastasis. To recapitulate the complex interplay between primary tumor sites and bone microenvironments, the study employs a combination of tunable extracellular matrix mechanics, bone-derived growth factors, and bone marrow-derived stromal cells. These engineered niches emulate the contexts of both primary tumor and bone tissue. LNCAP-C4-2B prostate cancer cells are introduced into these niches to analyze various aspects, including cell proliferation, osteoblastic differentiation, dissemination, and in vivo tumor formation. The study introduces a 3D organoid culture that mimics the pre-metastatic niche, providing a platform for studying PCa cell proliferation and dissemination. A negative relationship is observed between 3D matrix stiffness and cell proliferation, while a positive link is identified with cell dissemination. Bone matrix-derived soluble factors (BMSF) are identified as key modulators of LNCAP-C4-2B cell behavior, suppressing LNCAP-C4-2B proliferation but enhancing osteomimicry in vitro, Notably, bone marrow-derived stromal cells, primed with bone matrix-derived growth factors, amplify tumor cell proliferation and chemotaxis in vitro, contributing to increased tumor formation in vivo. In vivo experiments demonstrate BMSFs eliciting local inflammatory responses and inhibiting in vivo tumor formation. Moreover, osteogenic bone marrow stromal cells are shown to play a promotive role in LNCAP-C4-2B proliferation and migration, emphasizing the influence of the bone microenvironment on the prostate-to-bone progression of PCa cells. In conclusion, this study highlights the potential of the engineered tissue models for dissecting tumor progression and screening therapeutic interventions. By leveraging tissue-engineered soft and hard tissues, our approach offers a versatile toolbox for comprehensively investigating PCa progression and bone metastasis. These insights extend to potential therapeutic screening applications, providing a valuable resource for advancing our understanding of cancer pathogenesis and treatment strategies. Citation Format: Bo Han, Zhi Yang, Shuqing Zhao, Gangning Liang, Ba Xuan Hoang, Josephine Fang. Engineering tissue models to elucidate prostate cancer bone metastasis dynamics [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 1501.

Abstract 1798: Glyoxalase 1 (GLO1) as an oncometabolic enabler of prostate cancer progression: CRISPR/Cas9-based GLO1-deletion from human DU145-Luc2 cells blocks EMT and bone metastasis in SCID mice

Abstract Glyoxalase 1 (encoded by GLO1) is a glutathione-dependent enzyme detoxifying the glycolytic byproduct methylglyoxal (MG), an oncometabolite involved in metabolic reprogramming. Recently, we have demonstrated that GLO1 is overexpressed in human prostate cancer cells and patient tumors. In order to inform the ongoing debate on the role of GLO1 as an oncometabolic enabler of tumor glucose metabolism and malignant progression, we performed CRISPR/Cas9-based GLO1-deletion in human DU145-Luc2 malignant prostate cancer cells. NanoString nCounterTM ('PanCancer-Progression-Panel') comparative gene expression profiling (GLO1_KO versus GLO1_wt) revealed a significant downregulation of EMT-related pathways in GLO1_KO cells; concordantly, phenotypical screening indicated a pronounced attenuation of matrigel invasiveness observable in GLO1_KO cells. Likewise, inclusion of MG or a small molecule GLO1 inhibitor (TLSC-702) blocked invasiveness of DU145 GLO1_wt cells. Downregulation of EMT-related genes (including MMP3, SPP1, CXCL8) was accompanied by increased expression of TXNIP (thioredoxin-interacting protein), a master regulator of cellular energy metabolism and redox homeostasis. In a bioluminescent SCID mouse bone metastasis model (intracardial injection of DU145-Luc2 GLO1_wt and GLO1_KO cells), GLO1 expression was necessary to cause mandibular bone metastases (as evidenced by the complete absence of bone metastases after GLO1 deletion). Given the availability of drug-like small molecule inhibitors of GLO1 enzymatic activity these data suggest that GLO1 represents a novel molecular target for the pharmacological suppression of prostate cancer bone metastasis. Citation Format: Jana Jandova, Anne E. Cress, Georg T. Wondrak. Glyoxalase 1 (GLO1) as an oncometabolic enabler of prostate cancer progression: CRISPR/Cas9-based GLO1-deletion from human DU145-Luc2 cells blocks EMT and bone metastasis in SCID 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 1798.

Abstract 2032: Osteoblasts alter their protein expression profile in the presence of prostate cancer cells to facilitate their invasion

Abstract Bone metastasis is the leading cause of death in prostate cancer patients, for which there is currently no effective treatment. Since the bone microenvironment plays an important role in this process, attention has been directed to the interactions between cancer cells and the bone microenvironment, including osteoblasts. Here, we investigated the mechanism of interactions between prostate cancer cells (DU-145 and PC-3) and osteoblast cell line hFOB.1.19. We are investigating the broader effects of four different nucleoside reverse transcriptase inhibitor (NRTI) analogs 5-fluoro-1-((1R,4R)-4-hydroxycyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (KBMD-E), 3-benzoyl-5-fluoro-1-((1S,4S)-4-((tetrahydro-2H-pyran-2-yl)oxy)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (KBMD-G), 1-((1R,4R)-4-hydroxycyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (KBMD-H) and 1-(5-hydroxymethyl)-2,5-dihydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (KBMD-S) along with aPKC specific inhibitors ICA-1S and ζ-Stat to develop as potential anti-cancer drugs. Our previous publications suggested that aPKC-ι and ζ are involved in prostate cancer progression and metastasis. Current data suggested that aPKC specific inhibitors along with KBMD compounds induced apoptosis and pyroptosis of DU-145 and PC-3 while having lesser effects on hFOB.1.19 cell line. Our data demonstrate that osteoblasts assist prostate cells to establish in the bone microenvironment by producing metastatic stimulating chemokines while upregulating responsible transcription factors such as c-Jun. In addition, our preliminary data suggested that PKC-ι and ζ are crucial for driving critical steps of the metastatic cascades of prostate cancer cells. Preliminary data suggested that all 6 compounds upregulated interleukin (IL)-18 and IL-1β while downregulating IL-8, IL-6 and CXCL-1. Upregulation of Caspase 8 with cleaved gasdermin D in prostate cells indicates an increase in pyroptosis. Our preliminary results suggest that all 6 compounds can be used to disrupt the main steps of prostate cancer bone metastasis which can be targeted to develop customized, tailored therapies for bone metastatic prostate cancer which merit further research. Citation Format: Aaron J. Todman, Wishrawana S. Ratnayake, Luke Lajmi, Sloan Breedy, Abiral H. Shourav, Kirpal Bisht, Mildred Acevedo-Duncan. Osteoblasts alter their protein expression profile in the presence of prostate cancer cells to facilitate their invasion [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 2032.

Abstract 3064: Unmasking the deadly tango: PKA and lipid genes in prostate cancer's bone invasion

Abstract About 88% of metastatic prostate cancer (PCa) cases involve bone metastasis. When PCa cells spread to the bone, a metabolic adaptation to the new environment takes place, partly influenced by factors secreted by the homing organ. Thus, we hypothesize that metabolic reprogramming, both in the tumor and bone niche drive PCa bone metastasis. In this work, we carried out an integrative in vitro, in vivo, and bioinformatics approach to identify key regulators governing the metabolic behavior of PCa cells. We performed an indirect co-culture between PCa (PC3) and bone progenitor (MC3T3 or Raw264.7) cells. PCa cells displayed a strong transcriptional (RNA-seq) activation of lipid metabolism, including PPAR-signaling, fat digestion, and PI3K-Akt pathway. Accordingly, treatment with the conditioned media (CM) of the co-culture promoted lipid accumulation in PC3 cells (Bodipy 493/503 staining). To assess the clinical relevance of the in vitro model, we performed an unsupervised clustering analysis using transcriptomics data from human PCa and bone metastatic samples (GSE74685). Results showed that the metabolic genes deregulated in PC3 by the co-culture, could accurately cluster samples in primary tumor or bone metastasis. We next performed a multivariable Cox regression analysis and built a prognostic model, identifying a novel signature of 5 lipid-associated genes, PPARA, VDR, SLC16A1, PAPSS2 and GPX1, associated with a 23-fold higher risk of death in PCa patients (SU2C-PCF dataset). The expression of this signature was validated in a PDX pre-clinical model (MDA PCa PDX series) when comparing MDA-PCa-183 growing intrafemorally vs. subcutaneously. In order to identify the central hubs regulating this transcriptional phenotype, we performed an Ingenuity Pathway Analysis (QIAGEN), showcasing that the lipid-related signature deregulated by the bone niche is indeed modulated by the Protein Kinase A (PKA). Accordingly, the expression of these signature was downregulated by PKA inhibition (H89; PKA inhibitor). Moreover, secretome analyses (ESI MS/MS) revealed that bone secreted type I collagen (Col1a1) and fibronectin (Fn1) regulate PKA activity in PCa cells. Further, we investigated whether PKA could modulate critical mechanisms for bone progression, focusing on osteopontin (SPP1), a secreted protein involved in the adhesion and invasion of PCa cells, whose regulatory mechanism in tumor cells is yet to be elucidated. Strikingly, we observed not only that the activation of PKA using Forskolin in PCa cells activated SPP1 expression, but also that PKA inhibition led to a significant impairment of the SPP1 induction triggered by bone soluble factors secreted during the PC3/MC3T3 co-culture. Altogether, we showcase PKA as a central hub of PCa energetic metabolism during bone progression and outline a lipid-associated gene signature that can be targeted to halt metastatic PCa. Citation Format: Pablo Sanchis, Nicolas Anselmino, Estefania Labanca, Agustina Sabater, Juan Bizzotto, Gaston Pascual, Rocio Seniuk, Sofia Lage-Vickers, Antonina Mitrofanova, Pia Valacco, Ayelen Toro, Javier Cotignola, Nora Navone, Elba Vazquez, Geraldine Gueron. Unmasking the deadly tango: PKA and lipid genes in prostate cancer's bone invasion [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 3064.

Abstract 2752: ICA1 promotes prostate cancer progression and metastasis via AR down-regulation

Abstract Cancer metastasis, one of the main characteristics in malignancy, contributes to high mortality in cancer patients. Prostate cancer (PCa) is the most common cancer among men and the second leading cause of cancer-related death in Europe and the United States. In China, its incidence has been dramatically increased over past years. However, the mechanism of PCa progression and metastasis remains unclear. The androgen receptor (AR) has been demonstrated to play an important role in PCa progression and metastasis. We have previously established mouse models of PCa bone metastasis and PCa lung metastasis. We have obtained cell clones with bone-specific or lung-specific metastatic capability. By transcriptomic analysis of highly metastatic cell clones and their parental cells, we found a gene group including islet cell autoantigen 1 (ICA1) that are highly expressed in both metastatic cell clones. We further compared these results with RNA array data in clinical samples. Data comparison and correlation analysis showed that ICA1 was negatively correlated with AR expression. Survival analysis of PCa and lung cancer patients showed higher expression of ICA1 but with shorter overall survival. Down-regulation of ICA1 expression in highly metastatic tumor cells significantly inhibited cell growth, colony formation, migration, and invasion. ICA1 overexpression in parental RM1 cells apparently increased cell growth, migration, and metastasis. Interestingly, down-regulation of ICA1 also significantly activated AMPK/mTOR signaling pathway. These findings are anticipated to shed light on the novel pathogenic mechanism underlying PCa and propose a compelling theoretical basis for the application of ICA1 in PCa. This work supported by the NSFC projects (81972420, 81972766, 82173336), as well as grants from the Science and Technology Project of Shenzhen (JCYJ20210324104214040, JCYJ20190809161811237).Keywords: prostate cancer, metastasis, ICA1, androgen receptor Citation Format: Fuhao Wang, Yu He, Xin Huang, Ming Chang, Xiao Zhang, Xiaoyi Li, Xinyu Ye, Yi Lu. ICA1 promotes prostate cancer progression and metastasis via AR down-regulation [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 2752.

Abstract 5899: Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy

Abstract Recent research into prostate cancer treatment has unveiled a promising contender known as Up284. In laboratory experiments, this compound has exhibited a remarkable preference for targeting prostate cancer cells as opposed to normal cells. Specifically, in vitro cell viability assays using MTT have shown that prostate cancer cells display high sensitivity to Up284 at low nanomolar IC50 values, while primary human prostate epithelial cells and mouse keratinocytes exhibit minimal sensitivity. This establishes a notable therapeutic window between cancerous and non-cancerous cells, with Up284 significantly inhibiting colony formation in PC3 cells. Additional functional assays have confirmed Up284's potential, including the 4UbFL Reporter Gene assay, which has revealed robust, dose-dependent proteasome inhibition. This effect is supported by the accumulation of polyubiquitinated proteins in various prostate cancer cell lines following Up284 treatment, suggesting a distinct mechanism of toxicity compared to bortezomib. Furthermore, Up284 has been found to induce apoptosis in PC3 cells, surpassing the efficacy of bortezomib. Competitive binding assays have shown that Up284 exhibits a stronger binding affinity to the proteasomal receptor RPN13 than the prototype inhibitor RA190B. This is further supported by a Cellular Thermo Stabilization Assay (CETSA), which indicates that Up284 stabilizes RPN13 in cells. A preliminary in vivo study utilizing the bone metastatic prostate cancer genetically engineered mouse model(BMPC GEMM) model for metastatic prostate adenocarcinoma has demonstrated that Up284 leads to significant tumor regression and increased levels of poly-ubiquitinated proteins in residual tumor tissues. Moreover, when Up284 is conjugated with an E3 ligase ligand, it successfully degrades RPN13, resulting in the death of PC3 cells. Up284 has also shown favorable pharmacokinetics/pharmacodynamics (PK/PD) profiles and safety in animal models. It is well-tolerated in mice and holds promise for oral administration, demonstrating a proteasome-dependent reporter protein stabilization effect superior to ixazomib. Importantly, Up284 has induced tumor regression and improved survival rates in various cancer models, including ovarian and breast cancers. In conclusion, Up284 emerges as a lead candidate for targeted prostate cancer therapy due to its high specificity, substantial therapeutic index, and potent anti-cancer activity both in vitro and in vivo. Further development and clinical investigation of Up284 are warranted to fully explore its therapeutic potential. Citation Format: Balasubramanyam Karanam, Ravi Anchoori, Yung Chang. Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy [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 5899.

Abstract 5967: Blockade of Wnt5A/ROR1 signaling as cancer stem cell targeting therapy for metastatic prostate cancer

Abstract BACKGROUND– Wnt5A and its non-canonical Wnt receptor, ROR1, have emerged as a promising new signaling pathway target for lethal, metastatic prostate cancer. Wnt5A has emerged as a significant marker of poor prognosis in circulating tumor cells (CTCs) of metastatic castration resistant prostate cancer (mCRPC) patients. ROR1 is expressed in lethal types of mCRPC, especially neuroendocrine prostate cancer (NEPC). A therapeutic ROR1 antibody, Zilovertamab, has been clinically tested in chronic lymphocytic leukemia (CLL) and metastatic breast cancer and shown to be safe. We sought to investigate Zilovertamab-based therapies for metastatic prostate cancer in pre-clinical model systems. HYPOTHESIS – Wnt5A/ROR1 signaling may mediate a cancer stem cell program which makes cells resistant to therapies which target the cell cycle and proliferation. Blocking ROR1 may reveal vulnerabilities which sensitize cancer cells to chemotherapies like docetaxel. METHODS– ROR1 expression was determined using RNASeq, qRT-PCR, FACS, IHC and Western blotting. ROR1 signaling was blocked using the anti-ROR1 therapeutic antibody, Zilovertamab, or CRISPRCas9 ROR1 knock out. Cell growth was measured using an Incucyte real time imaging system. Stem cell activity was assessed in 3D organoids. Cell cycle analysis was performed in live cells in 2D cultures and 3D organoids using the Fucci2BL bicistronic Fluorescent, Ubiquitination-based Cell Cycle Indicator reporter system. PDX PCSD13 tumor growth was measured via calipers and IVIS. RNASeq was performed on tumors. RESULTS– We showed that ROR1 was expressed at high levels in mCRPC cell lines: PC3, DU145, and in the bone metastatic prostate cancer PDX: PCSD13. CRISPR/Cas9 Knock out of ROR1 in PC3 and DU145 cells showed increased sensitivity to docetaxel inhibition of proliferation in vitro in 2D real time Incucyte proliferation assays and in 3D organoids. Organoid formation was significantly reduced in ROR1 KO cells. Cells expressing the FUCCI live cell cycle tracker showed docetaxel led to G2 arrest and ROR1 signaling inhibition increased efficacy of docetaxel induced cell cycle arrest. Treatment of PCSD13 PDX in vivo with Zilovertamab plus docetaxel synergistically increased tumor growth inhibition in vivo and modulated cancer stem cell and cell cycle expression profiles. CONCLUSIONS– Cancer stem cells represent the fundamental precursors from which all diverse tumor subpopulations evolve. Thus, therapeutic targeting of these tumor initiating stem/progenitor cells may prevent the evolutionary diversification of a tumor and overcome a critical clinical barrier to cancer treatment. We showed synergistic response in our PDX and PCs cell line models to Zilovertamab plus docetaxel. We are now conducting a phase 1b clinical trial with zilovertamab plus docetaxel in patients with metastatic CRPC (CirmD, NCT05156905, PI R Mckay). Citation Format: Christina A.M. Jamieson, Jamillah Murtadha, Christopher S. Oh, Michelle T. Muldong, Niloofar Etemadfard, Evodie Koutouan, Young E. Yoon, Sanghee Lee, Oh Kwon, Jong J. Oh, Min S. Kim, Karl Willert, Nicholas A. Cacalano, Catriona H.M. Jamieson, Terry Gaasterland, Rana R. Mckay, Christopher J. Kane, Anna A. Kulidjian, Charles E. Prussak. Blockade of Wnt5A/ROR1 signaling as cancer stem cell targeting therapy for metastatic prostate cancer [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 5967.

Abstract 5538: Efferocytosis-induced HIF-1α enhances tumor vascularization and promotes osteoblastic bone remodeling in metastatic prostate cancer

Abstract Metastatic prostate cancer (PCA) exhibits a predilection for bone metastasis, initiating a complex interplay with bone cells, leading to dysregulated remodeling and mixed-pathological bone alterations. Human PCA primarily yields osteoblastic bone lesions, linked to bone pain and an adverse prognosis. In this study, we explored the activation of HIF-1α in myeloid phagocytic cells by apoptotic cancer cells in the context of bone metastatic PCA. Our previous findings demonstrated HIF-1α stabilization in bone macrophages engulfing apoptotic PCA cells in vitro. This study extends our understanding to an in vivo setting using mouse prostate cancer cells designed to express inducible caspase 9, triggering apoptosis (and subsequent efferocytosis) when exposed to the dimerizer molecule AP20187 (AP). Our assessment of PCA tumor growth in bone utilized a syngeneic mouse model, introducing a loss-of-function Hif1a mutation into myeloid lineage cells using LysM-Cre mice crossed with Hif1a-floxed mice (Hif1amut (LysM-Cre+/- Hif1aflox)), compared with the LysM-Cre-/- Hif1aflox (WT). In the first model, cancer cells were inoculated into the tibiae of these mice and apoptosis was induced with AP on days 3 and 5 post-inoculation (AP model). Results were compared to a model where apoptosis was not induced after inoculation (No-AP). In the AP model, fractional bone volume (BV/TV) in the medullae was significantly higher in tumor-inoculated tibiae than in non-inoculated (No-tumor) contralateral bone, highlighting the predominance of osteoblastic lesions. Conversely, in the No-AP model, increased BV/TV significance was achieved in WT mice (tumor vs. No-tumor), but not in mutant mice. Trabecular thickness (Tb.Th) was increased in both models in the presence of tumor, regardless of the Hif1a phenotype. Importantly, significant differences in BV/TV and Tb.Th between WT and Hif1amut tumor tibiae were exclusively noted in the context of induced apoptosis. Histological differences between tumors in the AP model were observed by immunofluorescence (IF) staining, which revealed a reduced fractional F4/80+ area in Hif1amut vs. WT tumors, with no differences in TRAP+ (osteoclasts) lining bone surfaces. RNAseq analysis of HIF1amut vs. WT macrophages efferocytosing apoptotic cancer cells showed downregulated processes related to vasculature development and blood vessel morphogenesis (p&amp;lt; 10-5). In correlation, IF analysis with endomucin (EMCN) demonstrated a reduction in stained area (EMCN+) relative to tumor area in Hif1amut tibiae vs. WT, suggesting a role of efferocytosis-induced HIF-1α in tumor vascularization. This study unveils intricacies of metastatic tumor growth in bone and proposes a connection between efferocytosis-induced HIF-1α in myeloid phagocytic cells and the promotion of tumor vascularization, alongside the enhancement of osteoblastic bone remodeling. Citation Format: Gabriel G. Kleer, Maria Molina Sanchez, Veronica Mendoza-Reinoso, Amy J. Koh, John Rubin, Laurie K. McCauley, Hernan Roca. Efferocytosis-induced HIF-1α enhances tumor vascularization and promotes osteoblastic bone remodeling in metastatic prostate cancer [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 5538.

The regulatory process and practical significance of non-coding RNA in the dissemination of prostate cancer to the skeletal system.

Prostate cancer is a major contributor to male cancer-related mortality globally. It has a particular affinity for the skeletal system with metastasis to bones seriously impacting prognosis. The identification of prostate cancer biomarkers can significantly enhance diagnosis and patient monitoring. Research has found that cancer and metastases exhibit abnormal expression of numerous non-coding RNA. Some of these RNA facilitate prostate cancer bone metastasis by activating downstream signaling pathways, while others inhibit this process. Elucidating the functional processes of non-coding RNA in prostate cancer bone metastasis will likely lead to innovative treatment strategies for this malignant condition. In this review, the mechanistic role of the various RNA in prostate cancer is examined. Our goal is to provide a new avenue of approach to the diagnosis and treatment of bone metastasis in this cancer.

In vivo genome-wide CRISPR screening identifies CITED2 as a driver of prostate cancer bone metastasis.

Most cancer deaths are due to metastatic dissemination to distant organs. Bone is the most frequently affected organ in metastatic prostate cancer and a major cause of prostate cancer deaths. Yet, our partial understanding of the molecular factors that drive bone metastasis has been a limiting factor for developing preventative and therapeutic strategies to improve patient survival and well-being. Although recent studies have uncovered molecular alterations that occur in prostate cancer metastasis, their functional relevance for bone metastasis is not well understood. Using genome-wide CRISPR activation and inhibition screens we have identified multiple drivers and suppressors of prostate cancer metastasis. Through functional validation, including an innovative organ-on-a-chip invasion platform for studying bone tropism, our study identifies the transcriptional modulator CITED2 as a novel driver of prostate cancer bone metastasis and uncovers multiple new potential molecular targets for bone metastatic disease.

Research Progression in the Mechanism of Bone Metastasis and Bone-Targeted Drugs in Prostate Cancer

Prostate cancer is a particularly slow growing cancer, the early stage of the disease is not easy to detect, the some major clinical manifestations include low back pain, urgent and frequent urination, urinary pain, and other urethral symptoms. These symptoms are often experienced after surgical resection or drug castration treatment. Early-stage, prostate cancer is curable, and with disease progression many clinical symptoms become worse with high probability of metastasis. Bone is the most common site of advanced metastasis of prostate cancer. Bone metastasis is a continuous and complex pathological process regulated by tumor cells and bone microenvironment, in which epithelial-mesenchymal transformation, homing and dormancy, reactivation, and proliferation of tumor cells are closely related to its occurrence and development. Several cytokines such as Receptor activator of NF-κB ligand (RANK-L) is overexpressed in bone microenvironment and prostate cancer. RANKL, chemokine family, and integrins are involved in bone metastasis of prostate cancer through complex interaction mechanisms. A variety of bone-targeting drugs such as bisphosphonates, RANKL inhibitors (denosumab) and radiotherapy drugs (radium-223, strontium-89, samarium-153), tyrosine kinase inhibitors, integrin-targeted drugs, etc. are approved for the prevention and treatment of skeletal related events caused by bone metastasis in prostate cancer patients. In this review, the biological mechanism of bone metastasis in prostate cancer and the research progress of bone-targeting drugs are reviewed.

Pretreatment level of serum sialic acid predicts both qualitative and quantitative bone metastases of prostate cancer.

Recently, serum sialic acid (SA) has emerged as a distinct prognostic marker for prostate cancer (PCa) and bone metastases, warranting differential treatment and prognosis for low-volume (LVD) and high-volume disease (HVD). In clinical settings, evaluating bone metastases can prove advantageous. We aimed to establish the correlation between SA and both bone metastasis and HVD in newly diagnosed PCa patients. We conducted a retrospective analysis of 1202 patients who received a new diagnosis of PCa between November 2014 and February 2021. We compared pretreatment SA levels across multiple groups and investigated the associations between SA levels and the clinical parameters of patients. Additionally, we compared the differences between HVD and LVD. We utilized several statistical methods, including the non-parametric Mann-Whitney U test, Spearman correlation, receiver operating characteristic (ROC) curve analysis, and logistic regression. The results indicate that SA may serve as a predictor of bone metastasis in patients with HVD. ROC curve analysis revealed a cut-off value of 56.15 mg/dL with an area under the curve of 0.767 (95% CI: 0.703-0.832, P < 0.001) for bone metastasis versus without bone metastasis and a cut-off value of 65.80 mg/dL with an area under the curve of 0.766 (95% CI: 0.644-0.888, P = 0.003) for HVD versus LVD. Notably, PCa patients with bone metastases exhibited significantly higher SA levels than those without bone metastases, and HVD patients had higher SA levels than LVD patients. In comparison to the non-metastatic and LVD cohorts, the cohort with HVD exhibited higher levels of alkaline phosphatase (AKP) (median, 122.00 U/L), fibrinogen (FIB) (median, 3.63 g/L), and prostate-specific antigen (PSA) (median, 215.70 ng/mL), as well as higher Gleason scores (> 7). Multivariate logistic regression analysis demonstrated that an SA level of > 56.15 mg/dL was independently associated with the presence of bone metastases in PCa patients (OR = 2.966, P = 0.018), while an SA level of > 65.80 mg/dL was independently associated with HVD (OR = 1.194, P = 0.048). The pretreatment serum SA level is positively correlated with the presence of bone metastases.