SLCO1B3 expression in PDX from lethal prostate cancer

Androgen receptor expression in lethal prostate cancer PDX models

Radium-223 dichloride (Ra-223) is a calcium-mimetic alpha-emitting radiopharmaceutical homing to bone, approved for the treatment of bone metastases in castration-resistant prostate cancer (CRPC), significantly prolonging overall survival in patients with advanced disease. This study integrates preclinical and clinical studies to investigate the effects of Ra-223 on tumor volume, bone structure, transcriptomics alterations, and to identify biomarkers of therapeutic response to Ra-223. Using patient-derived xenografts (MDA PCa PDX), which mimic the hallmark characteristics of clinical bone-forming metastases, mice with tumor-bearing and non-tumor-bearing bones were treated with Ra-223 or vehicle. Tumor burden was monitored via magnetic resonance imaging (MRI), bone parameters were assessed through microCT and bone histomorphometry, and transcriptomics changes were evaluated using RNA sequencing in both, tumor and stroma. Ra-223 treatment significantly reduced tumor volume and altered bone architecture, promoting bone remodeling and increasing bone density. Histomorphometric analysis revealed suppressed osteoclast activity in both tumor-bearing and non-tumor-bearing bones treated with Ra-223, with a more pronounced effect in non-tumor-bearing sites. Transcriptomic profiling identified differential expression of immune, metabolic, and cell-cycle pathways, with TP53 and MYC as central regulators. Additionally, we analyzed human serum samples from patients with CRPC bone metastases. We profiled novel candidate protein markers of Ra-223 response on serial serum samples collected at baseline (prior to initiation of Ra-223 treatment), on-treatment (OT), and at end of treatment (EoT) from 25 patients enrolled in a single-center, open-label clinical study (NCT02135484). Subjects received six intravenous doses of Ra-223 (50 kBq/kg) every four weeks, unless they experienced disease progression or toxicity, or until withdrawal. Our analysis highlighted IL18, SPARC, MET, TFF3 and TNFSF11 as survival-associated proteins significantly deregulated in patients receiving Ra-223 therapy. In particular, multivariable analyses showcased that elevated levels of TFF3 were independently linked to lower overall survival and possible resistance to Ra-223, suggesting its potential as a predictive biomarker of treatment efficacy. In summary, our findings suggest that Ra-223 has a more extensive impact on the tumor-bone microenvironment than previously understood. The increases in bone density and associated transcriptomics alterations pave the way for future investigations into combination therapies that enhance Ra-223’s benefits while addressing potential risks. Considering the clinical importance of TFF3, further validation in larger patient cohorts is essential to refine treatment strategies to improve outcomes for patients with CRPC bone metastases. Pablo Sanchis, Agustina Sabater, Juan Bizzotto, Gaston Pascual, Nicolas Anselmino, Jiabin Dong, Jun Yang, Ganiraju Manyam, Bradley Broom, Zhongcheng Shi, Shixia Huang, Rocio Seniuk, Eleonora Dondossola, Teocharis Panaretakis, John Araujo, Peter Shepherd, Patricia Troncoso, Nora Navone, Christopher J. Logothetis, Daniel E. Frigo, Elba Vazquez, Paul Corn, Geraldine Gueron, Estefania Labanca. Fracturing the tumor-bone alliance in metastatic prostate cancer with Ra-223 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3123.

Castration-resistant prostate cancer (CRPC) remains an incurable disease in need of improved treatments. CAMKK2 is an emerging therapeutic target whose oncogenic effects in prostate cancer have, to date, been largely attributed to its activation of AMP-activated protein kinase (AMPK). Here, we demonstrate that CAMKK2 promotes prostate cancer growth through an alternative downstream pathway involving CAMKI and CREB. Unbiased transcriptomics identify CREB-mediated transcription as a CAMKK2-regulated process, findings that we validate using diverse molecular, genetic, and pharmacological approaches in vitro and in vivo. CAMKK2 promotes CREB phosphorylation/activation through CAMKIα independently of AMPK, CAMKIV, or other CAMKI isoforms. Functionally, the CREB family members CREB1 and ATF1 exhibit close redundancy, necessitating co-targeting for optimal anti-tumor efficacy. An inhibitor of CREB1/ATF1 blocks CRPC with minimal side effects. Mechanistically, CAMKK2 and CREB increase CRPC growth through augmenting cholesterol metabolism. Together, these findings identify an oncogenic pathway that could be exploited for the treatment of CRPC.

Prostate cancer (PCa) lethality largely stems from the progression to castration-resistant PCa (CRPC) bone metastasis. We previously reported that fibroblast growth factor receptor 1 (FGFR1) is overexpressed in a subset of human CRPC bone metastases, and that FGFR1 overexpression of both α and β isoforms drives bone metastases and reduces survival in experimental metastatic murine models with intracardiac PCa cells injection, posing its blockade as a promising therapeutic strategy for advanced PCa. An on-going clinical study at our department at MD Anderson is currently evaluating erdafitinib, an FGFR inhibitor, for treating men with CRPC bone metastases (ClinicalTrials.gov Identifier: NCT04754425). However, the dynamics behind FGFR-driven PCa progression remain elusive. As FGFR signaling has previously been linked to the development and proliferation of PCa stem-cells, we hypothesized that FGFR promotes PCa bone metastasis by modulating cell plasticity. In vitro assays confirmed that FGFR1α- and FGFRβ-expressing PCa cells exhibited increased colony formation and confluency, and a more stem-like phenotype, evidenced by altered morphology, smaller cell size, elevated aldehyde dehydrogenase (ALDH) levels, and enhanced tumorsphere formation (p<0.0001). Consistently, RNA-seq analysis of PCa cells and MDA PCa patient-derived xenografts (PDX) showed a significantly positive correlation between FGFR1 levels and stem-cell markers (p<0.05). Furthermore, PCa patients from TCGA-PRAD (497 primary PCa) and SU2C (81 metastatic CRPC) datasets with enrichment of FGFR-correlated stem-cell categories exhibited increased risk of progression (log-rank p<0.05). Among the stem-categories that correlate with FGFR expression, genes from Wnt signaling, a pathway involved in stem-cell traits regulation and implicated in PCa, emerged prominently (FERMT2, FZD7, NES, CORO1C, BMP4, MEF2C, GJA1, SFRP1, WNT2B, CDH2). Bioinformatics analysis also revealed significant correlation between TCF/LEF factors and FGFR1 in TCGA-PRAD patients. FGFR1-expressing PCa cells showed increased TCF reporter activity, indicating Wnt activation, and nuclear β-catenin immunostaining in FGFR1-expressing PDXs and human CRPC bone metastases further confirmed this activation. Overall, our findings suggest that FGFR1 drives PCa progression through Wnt signaling-induced stemness, conferring cancer cells the plasticity to adapt to the bone microenvironment. Future studies aim to further characterize the FGFR1-induced stemness and dedifferentiation mechanisms underlying PCa bone dissemination and evaluate FGFR-targeted therapies. Agustina Sabater, Pablo Sanchis, Nicolas Anselmino, Juan Bizzotto, Jun Yang, Jiabin Dong, Peter D. Shepherd, Paul G. Corn, Elba Vazquez, Christopher J. Logothetis, Nora Navone, Geraldine Gueron, Estefania Labanca. Bone voyage: FGFR1, stemness, and the Wnt trail to prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2967.

Bone metastases are the most common milestone in the lethal progression of prostate cancer and prominent in a substantial portion of renal malignancies. Interactions between cancer and bone host cells have emerged as drivers of both disease progression and therapeutic resistance. To best understand these central host-epithelial cell interactions, biologically relevant preclinical models are required. To achieve this goal, we here established and characterized tissue-engineered bone mimetic environments (BME) capable of supporting the growth of patient-derived xenograft (PDX) cells, ex vivo and in vivo. The BME consisted of a polycaprolactone (PCL) scaffold colonized by human mesenchymal stem cells (hMSCs) differentiated into osteoblasts. PDX-derived cells were isolated from bone metastatic prostate or renal tumors, engineered to express GFP or luciferase and seeded onto the BMEs. BMEs supported the growth and therapy response of PDX-derived cells, ex vivo. Additionally, BMEs survived after in vivo implantation and further sustained the growth of PDX-derived cells, their serial transplant, and their application to study the response to treatment. Taken together, this demonstrates the utility of BMEs in combination with patient-derived cells, both ex vivo and in vivo.Statement of significanceOur tissue-engineered BME supported the growth of patient-derived cells and proved useful to monitor the therapy response, both ex vivo and in vivo. This approach has the potential to enable co-clinical strategies to monitor bone metastatic tumor progression and therapy response, including identification and prioritization of new targets for patient treatment.

For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.

Abstract Previous studies have demonstrated the non-canonical anti-tumor effect of heme-oxygenase 1 (HO-1) in prostate cancer (PCa). Although HO-1 is crucial for free heme degradation, its nuclear expression unveils non-canonical functions beyond its enzymatic function. Understanding the specifics of its non-canonical role remains a critical unmet need. In this study, we identified nuclear interactors of HO-1 and assessed their association with PCa.PCa cells were treated with hemin (80 µM, 24 h), a specific pharmacological inducer of HO-1. Nuclear HO-1 immunoprecipitation and LC-ESI MS/MS analysis identified 11 differential nuclear associated-HO-1 proteins between control and hemin-treated PCa cells (ILF3, ILF2, BCLAF1, SAFB, DDX17, SLC25A5, CASP14, PRDX1, BRIX1, CCDC175, and GPATCH1). Next, we performed an Ingenuity Pathway Analysis (QIAGEN) showing that ILF3 appears as a master regulator of this signature. To assess the clinical relevance of these factors in PCa, we analyzed overall survival (OS), progression-free survival (PFS), relapse free survival (RFS) in multiple PCa datasets (GSE34312, GSE35988, GSE3933, GSE46602, GSE6956, GSE70768, TCGA-PRAD, GSE70770, GSE16560, GSE24136; n=1064). We performed univariable and multivariable analyses for these factors and identified the ones that significantly and independently affected the OS, RFS, PFS. Next, a risk score model was built based on the expression of 9 genes (∑ni=1(Coefi×Expri)) for the GSE70770 dataset using these factors identifying a subpopulation of PCa patients with high-risk of RFS (HR: 7.39 High vs Low score, p<0.0001); ascertaining the critical role of this signature in PCa. To elucidate the association of these factors with the aggressive phenotype of PCa, we analyzed RNA-seq expression data from the MDA-PCa-PDXs series (PCa Patient Derived Xenografts Program; MD Anderson Cancer Center), capturing PCa heterogeneity. Unsupervised clustering analysis revealed that samples with high expression of HO-1 nuclear interactors corresponded to neuroendocrine tumors, negative for androgen receptor staining. Principal Component Analysis identified ILF3 as the most relevant HO-1 interactor driving PDXs’ samples variance, correlating with a significant decrease in relapse-free survival of PCa patients. Further, we used ChIP-Atlas to assess the epigenomic landscapes for these nuclear HO-1 interactors. Results indicated that 40% of HO-1 nuclear interactors were potential transcription regulators. Strikingly, KEGG pathways analyses revealed that their regulomes were significantly associated with neurodegenerative disorders, highlighting their relevance in neural processes. In conclusion, our findings suggest that HO-1 and its nuclear interactors may play a pivotal role in neuroendocrine PCa, shedding light on potential therapeutic targets for this aggressive form of the disease. Citation Format: Seniuk A. Rocio, Agustina Sabater, Pablo Sanchis, Juan Bizzotto, Gastón Pascual, Estefania Labanca, Nicolas Anselmino, Nora Navone, Elba Vazquez, Pia Valacco, Javier Cotignola, Ayelén Toro, Geraldine Gueron. Decoding the non-canonical functions of HO-1 in prostate cancer: A nuclear perspective and its association with a neuroendocrine signature [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 5648.

Prostate cancer (PCa) bone metastasis (BM) plays a crucial role in disease outcome and poses a significant clinical obstacle due to its heterogeneity. In this sense, Osteopontin (SPP1/OPN) is elevated in a subset of patients with advanced disease. However understanding its regulation is an unmet need. In this work we investigated the factors controlling SPP1/OPN expression in a subgroup of patients with BM. We performed a comprehensive transcriptomics analysis (DEG, Ingenuity Pathway Analysis (IPA)) of publicly available patients datasets (GSE74685, SU2C-PCF and Westbrooke et al), comparing PCa on different metastatic sites and/or treatment status. Using an indirect co-culture (24h) to mimic the dialogue between PCa cells (PC3/C42B) and osteoblast precursors (MC3T3) in vitro, we integrated transcriptomics (RT-qPCR and RNAseq) and secretomic (ESI-MS/MS of conditioned media (CM)) data to dissect key players involved in the bi-directional crosstalk between PCa and bone cells. Protein Kinase A (PKA) pathway implication was evaluated by its induction (forskolin 1uM) or suppression (H89 10uM). Clinically relevant patient derived xenograft (PDX) models growing intrafemorally (i.f.) were used for in vivo validation. Across different datasets, we consistently found a sub-population of patients with BM characterized by high levels of SPP1 expression, associated with an active PKA pathway. By functionalizing this response experimentally, we demonstrated that specific bone secreted factors, particularly Col1a1 and Fn1, significantly induce SPP1 expression in PCa cells via PKA activation. Further, experimental and clinical settings suggest that the androgen receptor (AR) is implicated on this bone-induced PKA/SPP1 axis. Notably, longitudinal analysis of patients samples revealed an increased SPP1 expression alongside signs of PKA pathway activation, in a subpopulation of BM upon AR signaling inhibition with enzalutamide. Herein, we uncovered PKA as a novel upstream regulator of SPP1/OPN expression in PCa triggered by the bone microenvironment that could possibly be implicated in treatment resistance. These results highlight the potential of SPP1/OPN as a marker of tumors with active PKA to improve disease management.

Detail of manually curated full or partial copy number/ transcript loss

Radium 223 (Ra-223) is an α-emitting bone-homing radiopharmaceutical that targets tumor-induced osteoblasts and is used to reduce bone pain and prolong overall survival in men with bone-metastatic, castrate-resistant prostate cancer. However, increased fracture risk in skeletal sites with no bone metastasis has been observed in patients treated with Ra-223. Both luciferase- or green fluorescence protein (GFP)-labeled osteoblast reporter mice were used to monitor the effect of Ra-223 on resident osteoblasts and normal bone structure. Upon Ra-223 treatment, 70% of resident osteoblasts were reduced within 2 days, and the osteoblast reduction lasted for at least 18 weeks without detectable recovery, as measured by in vivo bioluminescent imaging. In GFP-labeled osteoblast reporter mice, Ra-223 mainly reduced osteoblasts localized in the trabecular bone areas; the osteoblasts in the growth plates were less affected. Micro-computed tomography analyses showed that Ra-223 significantly reduced bone mineral density and bone microstructure in the trabecular area of femurs but not in the cortical bone. Tumor-induced bone was generated by inoculating osteogenic TRAMP-BMP4 prostate cancer cells into the mouse femurs; Ra-223 treatment significantly reduced tumor-induced osteoblasts. Our study shows that Ra-223 affects bone structures that are not involved in bone metastasis. Strategies that improve bone health may reduce fracture risk in patients receiving Ra-223.

BackgroundThere are relatively few widely used models of prostate cancer compared to other common malignancies. This impedes translational prostate cancer research because the range of models does not reflect the diversity of disease seen in clinical practice. In response to this challenge, research laboratories around the world have been developing new patient-derived models of prostate cancer, including xenografts, organoids, and tumor explants.MethodsIn May 2023, we held a workshop at the Monash University Prato Campus for researchers with expertise in establishing and using a variety of patient-derived models of prostate cancer. This review summarizes our collective ideas on how patient-derived models are currently being used, the common challenges, and future opportunities for maximizing their usefulness in prostate cancer research.ResultsAn increasing number of patient-derived models for prostate cancer are being developed. Despite their individual limitations and varying success rates, these models are valuable resources for exploring new concepts in prostate cancer biology and for preclinical testing of potential treatments. Here we focus on the need for larger collections of models that represent the changing treatment landscape of prostate cancer, robust readouts for preclinical testing, improved in vitro culture conditions, and integration of the tumor microenvironment. Additional priorities include ensuring model reproducibility, standardization, and replication, and streamlining the exchange of models and data sets among research groups.ConclusionsThere are several opportunities to maximize the impact of patient-derived models on prostate cancer research. We must develop large, diverse and accessible cohorts of models and more sophisticated methods for emulating the intricacy of patient tumors. In this way, we can use the samples that are generously donated by patients to advance the outcomes of patients in the future.

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.

Percentage of reads matching with mouse DNA

Fusion genes expression levels. CNV details on TMPRSS2-ERG fusion and AR enhancer region. Putative PCa drivers in models lacking alterations in four major genes

Fusions identified in MDA PCa PDX cohort

T200.1 panel: genes implicated in the pathogenesis of solid cancers