Epigenetic Activation of WNT5A Drives Glioblastoma Stem Cell Differentiation and Invasive Growth.

Glioblastoma stem cells (GSCs) are implicated in tumor neovascularization, invasiveness, and therapeutic resistance. To illuminate mechanisms governing these hallmark features, we developed a de novo glioblastoma multiforme (GBM) model derived from immortalized human neural stem/progenitor cells (hNSCs) to enable precise system-level comparisons of pre-malignant and oncogene-induced malignant states of NSCs. Integrated transcriptomic and epigenomic analyses uncovered a PAX6/DLX5 transcriptional program driving WNT5A-mediated GSC differentiation into endothelial-like cells (GdECs). GdECs recruit existing endothelial cells to promote peritumoral satellite lesions, which serve as a niche supporting the growth of invasive glioma cells away from the primary tumor. Clinical data reveal higher WNT5A and GdECs expression in peritumoral and recurrent GBMs relative to matched intratumoral and primary GBMs, respectively, supporting WNT5A-mediated GSC differentiation and invasive growth in disease recurrence. Thus, the PAX6/DLX5-WNT5A axis governs the diffuse spread of glioma cells throughout the brain parenchyma, contributing to the lethality of GBM.

Telomerase reactivation following telomere dysfunction yields murine prostate tumors with bone metastases.

To determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-ß/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.

Prostate cancer-induced endothelial-cell-to-osteoblast transition drives immunosuppression in the bone-tumor microenvironment through Wnt pathway-induced M2 macrophage polarization.

Immune checkpoint therapy has limited efficacy for patients with bone-metastatic castration-resistant prostate cancer (bmCRPC). To improve immunotherapy for bmCRPC, we aimed to identify the mechanism of bmCRPC-induced changes in the immune microenvironment. Among bmCRPC patients, higher levels of a 32-gene M2-like macrophage signature in bone metastasis samples correlated with shorter overall survival. Immunohistochemistry showed that CD206-positive (CD206+) macrophages were enriched in bmCRPC bone biopsy specimens compared with primary tumors or lymph node metastases. In preclinical osteogenic prostate cancer (Pca) xenograft models, CD206+ macrophages were recruited to areas with tumor-induced bone. RNA sequencing (RNAseq) analysis showed higher expression of an M2-like gene signature, with activated canonical and noncanonical Wnt pathways, in tumor-associated macrophages isolated from osteogenic tumors (bone-TAMs) than in TAMs isolated from nonosteogenic tumors (ctrl-TAMs). Mechanistic studies showed that endothelial cells (ECs) that had undergone EC-to-osteoblast (EC-to-OSB) transition, the precursors of tumor-induced OSBs, produced paracrine factors, including Wnts, CXCL14, and lysyl oxidase, which induced M2 polarization and recruited M2-like TAMs to the bone-tumor microenvironment (bone-TME). Bone-TAMs suppressed CD8+ T cells' proliferation and cytolytic activity, and these effects were partially reversed by treating bone-TAMs with Wnt inhibitors. Genetic or pharmacological inhibition of Pca-induced EC-to-OSB transition reduced the levels of M2-like macrophages in osteogenic tumors. Our study demonstrates that Pca-induced EC-to-OSB transition drives immunosuppression in the bone-TME, suggesting that therapies that reduce Pca-induced bone formation may improve immunotherapeutic outcomes for bmCRPC.

Genetics and biology of prostate cancer.

Despite the high long-term survival in localized prostate cancer, metastatic prostate cancer remains largely incurable even after intensive multimodal therapy. The lethality of advanced disease is driven by the lack of therapeutic regimens capable of generating durable responses in the setting of extreme tumor heterogeneity on the genetic and cell biological levels. Here, we review available prostate cancer model systems, the prostate cancer genome atlas, cellular and functional heterogeneity in the tumor microenvironment, tumor-intrinsic and tumor-extrinsic mechanisms underlying therapeutic resistance, and technological advances focused on disease detection and management. These advances, along with an improved understanding of the adaptive responses to conventional cancer therapies, anti-androgen therapy, and immunotherapy, are catalyzing development of more effective therapeutic strategies for advanced disease. In particular, knowledge of the heterotypic interactions between and coevolution of cancer and host cells in the tumor microenvironment has illuminated novel therapeutic combinations with a strong potential for more durable therapeutic responses and eventual cures for advanced disease. Improved disease management will also benefit from artificial intelligence-based expert decision support systems for proper standard of care, prognostic determinant biomarkers to minimize overtreatment of localized disease, and new standards of care accelerated by next-generation adaptive clinical trials.

Synthetic essentiality of chromatin remodelling factor CHD1 in PTEN-deficient cancer.

Synthetic lethality and collateral lethality are two well-validated conceptual strategies for identifying therapeutic targets in cancers with tumour-suppressor gene deletions. Here, we explore an approach to identify potential synthetic-lethal interactions by screening mutually exclusive deletion patterns in cancer genomes. We sought to identify 'synthetic-essential' genes: those that are occasionally deleted in some cancers but are almost always retained in the context of a specific tumour-suppressor deficiency. We also posited that such synthetic-essential genes would be therapeutic targets in cancers that harbour specific tumour-suppressor deficiencies. In addition to known synthetic-lethal interactions, this approach uncovered the chromatin helicase DNA-binding factor CHD1 as a putative synthetic-essential gene in PTEN-deficient cancers. In PTEN-deficient prostate and breast cancers, CHD1 depletion profoundly and specifically suppressed cell proliferation, cell survival and tumorigenic potential. Mechanistically, functional PTEN stimulates the GSK3ß-mediated phosphorylation of CHD1 degron domains, which promotes CHD1 degradation via the ß-TrCP-mediated ubiquitination-proteasome pathway. Conversely, PTEN deficiency results in stabilization of CHD1, which in turn engages the trimethyl lysine-4 histone H3 modification to activate transcription of the pro-tumorigenic TNF-NF-κB gene network. This study identifies a novel PTEN pathway in cancer and provides a framework for the discovery of 'trackable' targets in cancers that harbour specific tumour-suppressor deficiencies.

Metal-Chelatable Porphyrinic Frameworks for Single-Cell Multiplexing with Mass Cytometry.

Single-cell multiplexing is key to exploration of the heterogeneous cell populations in biological systems. Although the state-of-the-art mass cytometry (CyTOF) possesses high resolution and multiple dimensions, the lack of suitable marker materials prohibits fully exploiting the available CyTOF detection channels. Here we report a new design strategy for CyTOF markers using functionalized mesoporous porphyrinic frameworks (MPFs) as scaffolds for chelating metals that have been unachievable by conventional approaches. We developed surface modification for stably dispersing the MPF nanoparticles (<40 nm) during the metalation and antibody conjugation processes. Our markers exhibit higher sensitivity and comparable specificity compared with a polymer-based commercial benchmark. Compatibility with commercial markers during co-staining was also confirmed. Furthermore, our markers show promising performance for immunophenotyping and potential implementation in CyTOF systems.

δ-Tocopherol inhibits the development of prostate adenocarcinoma in prostate specific Pten-/- mice.

The PTEN/PI3K/AKT axis plays a critical role in regulating cell growth, differentiation and survival. Activation of this signaling pathway is frequently found in human cancers. Our previous studies demonstrated that δ-tocopherol (δ-T) attenuates the activation of AKT by growth factor in prostate cancer cell lines, leading to inhibition of proliferation and induction of apoptosis. Herein, we investigated whether δ-T inhibits the development of prostate adenocarcinoma in prostate-specific Pten-/- (Ptenp-/-) mice in which the activation of AKT is the major driving force for tumorigenesis. By feeding Ptenp-/- mice with AIN93M or 0.2% δ-T supplemented diet starting at the age of 6 or 12 weeks, we found that δ-T treatment reduced prostate adenocarcinoma multiplicity at the age of 40 weeks by 53.3 and 42.7%, respectively. Immunohistochemical (IHC) analysis demonstrated that the phosphorylation of AKT (T308) was reduced in the prostate of the mice administered the δ-T diet. Consistently, proliferation was reduced and apoptosis was increased in prostate lesions of mice on the δ-T diet. Oxidative stress, as determined by IHC staining of 8-OH-dG, was not altered during prostate tumorigenesis, nor was it affected by administration of δ-T. In contrast, α-tocopherol (α-T) at 0.2% in the diet did not affect prostate adenocarcinoma multiplicity in the Ptenp-/- mice. This finding is consistent with data from our previous study that δ-T, but not α-T, inhibits the activation of AKT and the growth of prostate cancer cells. Together, these results demonstrate that δ-T inhibits the development of prostate adenocarcinoma in Ptenp-/- mice, mainly through inhibition of AKT activation.

Short Chain Fatty Acid Acetate Protects against Ethanol-Induced Acute Gastric Mucosal Lesion in Mice.

Short chain fatty acids acetate and propionate have been demonstrated protective function in the intestinal mucosa. However, their impact on gastric mucosa has not yet been elucidated. The current study aimed to investigate the potential protective effects of acetate and propionate against ethanol-induced gastric mucosal lesion and the underlying mechanism in mice. ICR mice were orally treated with acetate and propionate, respectively, 30 min prior to the establishment of gastric mucosal injury model by challenge with absolute ethanol. The gastric samples were collected for the detection of oxidative, inflammatory and apoptotic related parameters. Acetate, but not propionate, attenuated the severity of gastric mucosal damage as evidenced by the gross changes of gastric mucosa, pathological aberrations. Acetate alleviated oxidative stress as shown by the increase in glutathione (GSH) content and superoxide dismutase (SOD) activities, and the decrease of malondialdehyde (MDA) level. The elevated concentrations of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and IL-6, and the activation of nuclear factor-kappaB (NF-κB) p65 by ethanol stimulation was also reduced by acetate. Moreover, the anti-inflammatory factors, IL-4, LXA4 and IL-10, were up-regulated in acetate treated group. With respect to gastric mucosal apoptosis, acetate suppressed caspase-3 activity and BAX expression in favor of cell survival. These favorable actions were maybe associated with up-regulation of the gastric MUC5AC, the key defense factor of gastric mucosal system. These findings accentuate the gastroprotective actions of acetate in ethanol-induced gastric injury which were mediated via concerted multi-prolonged actions, including suppression of gastric oxidation, inflammation and apoptosis and promotion of MUC5AC expression.

Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence.

Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on induction of the p19(Arf)-p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumour-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19(Arf)-p53 pathway nor DNA damage, but instead depend on Atf4, p27 and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19(Arf)-p53 response is impaired, whereas a Skp2-SCF complex inhibitor can trigger cellular senescence in p53/Pten-deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof-of-principle evidence that pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy.

Role of LRF/Pokemon in lineage fate decisions.

In the human genome, 43 different genes are found that encode proteins belonging to the family of the POK (poxvirus and zinc finger and Krüppel)/ZBTB (zinc finger and broad complex, tramtrack, and bric à brac) factors. Generally considered transcriptional repressors, several of these genes play fundamental roles in cell lineage fate decision in various tissues, programming specific tasks throughout the life of the organism. Here, we focus on functions of leukemia/lymphoma-related factor/POK erythroid myeloid ontogenic factor, which is probably one of the most exciting and yet enigmatic members of the POK/ZBTB family.

Targeting PRMT3 impairs methylation and oligomerization of HSP60 to boost anti-tumor immunity by activating cGAS/STING signaling.

Immune checkpoint blockade (ICB) has emerged as a promising therapeutic option for hepatocellular carcinoma (HCC), but resistance to ICB occurs and patient responses vary. Here, we uncover protein arginine methyltransferase 3 (PRMT3) as a driver for immunotherapy resistance in HCC. We show that PRMT3 expression is induced by ICB-activated T cells via an interferon-gamma (IFNγ)-STAT1 signaling pathway, and higher PRMT3 expression levels correlate with reduced numbers of tumor-infiltrating CD8+ T cells and poorer response to ICB. Genetic depletion or pharmacological inhibition of PRMT3 elicits an influx of T cells into tumors and reduces tumor size in HCC mouse models. Mechanistically, PRMT3 methylates HSP60 at R446 to induce HSP60 oligomerization and maintain mitochondrial homeostasis. Targeting PRMT3-dependent HSP60 methylation disrupts mitochondrial integrity and increases mitochondrial DNA (mtDNA) leakage, which results in cGAS/STING-mediated anti-tumor immunity. Lastly, blocking PRMT3 functions synergize with PD-1 blockade in HCC mouse models. Our study thus identifies PRMT3 as a potential biomarker and therapeutic target to overcome immunotherapy resistance in HCC.

Endothelial-to-osteoblast transition in normal mouse bone development.

Metastatic prostate cancer (PCa) in bone induces bone-forming lesions. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone EC-to-osteoblast (OSB) transition. Here, we investigated whether EC-to-OSB transition also occurs during normal bone formation. We developed an EC and OSB dual-color reporter mouse (DRM) model that marks EC-OSB hybrid cells with red and green fluorescent proteins. We observed EC-to-OSB transition (RFP and GFP co-expression) in both endochondral and intramembranous bone formation during embryonic development and in adults. Co-expression was confirmed in cells isolated from DRM. Bone marrow- and lung-derived ECs underwent transition to OSBs and mineralization in osteogenic medium. RNA-sequencing revealed GATA family transcription factors were upregulated in EC-OSB hybrid cells and knockdown of GATA3 inhibited BMP4-induced mineralization. Our findings support that EC-to-OSB transition occurs during normal bone development and suggest a new paradigm regarding the endothelial origin of OSBs.

PRMT3-mediated arginine methylation of IGF2BP1 promotes oxaliplatin resistance in liver cancer.

Although oxaliplatin-based chemotherapy has been effective in the treatment of hepatocellular carcinoma (HCC), primary or acquired resistance to oxaliplatin remains a major challenge in the clinic. Through functional screening using CRISPR/Cas9 activation library, transcriptomic profiling of clinical samples, and functional validation in vitro and in vivo, we identify PRMT3 as a key driver of oxaliplatin resistance. Mechanistically, PRMT3-mediated oxaliplatin-resistance is in part dependent on the methylation of IGF2BP1 at R452, which is critical for the function of IGF2BP1 in stabilizing the mRNA of HEG1, an effector of PRMT3-IGF2BP1 axis. Also, PRMT3 overexpression may serve as a biomarker for oxaliplatin resistance in HCC patients. Collectively, our study defines the PRTM3-IGF2BP1-HEG1 axis as important regulators and therapeutic targets in oxaliplatin-resistance and suggests the potential to use PRMT3 expression level in pretreatment biopsy as a biomarker for oxaliplatin-resistance in HCC patients.

Cyclosporine A-resistant CAR-T cells mediate antitumour immunity in the presence of allogeneic cells.

Chimeric antigen receptor (CAR)-T therapy requires autologous T lymphocytes from cancer patients, a process that is both costly and complex. Universal CAR-T cell treatment from allogeneic sources can overcome this limitation but is impeded by graft-versus-host disease (GvHD) and host versus-graft rejection (HvGR). Here, we introduce a mutated calcineurin subunit A (CNA) and a CD19-specific CAR into the T cell receptor α constant (TRAC) locus to generate cells that are resistant to the widely used immunosuppressant, cyclosporine A (CsA). These immunosuppressant-resistant universal (IRU) CAR-T cells display improved effector function in vitro and anti-tumour efficacy in a leukemia xenograft mouse model in the presence of CsA, compared with CAR-T cells carrying wild-type CNA. Moreover, IRU CAR-T cells retain effector function in vitro and in vivo in the presence of both allogeneic T cells and CsA. Lastly, CsA withdrawal restores HvGR, acting as a safety switch that can eliminate IRU CAR-T cells. These findings demonstrate the efficacy of CsA-resistant CAR-T cells as a universal, 'off-the-shelf' treatment option.

Prostate cancer-induced endothelial-to-osteoblast transition generates an immunosuppressive bone tumor microenvironment.

Immune checkpoint therapy has limited efficacy for patients with bone metastatic castrate-resistant prostate cancer (bmCRPC). In this study, we revealed a novel mechanism that may account for the relative resistance of bmCRPC to immune checkpoint therapy. We found that prostate cancer (PCa)-induced bone via endothelial-to-osteoblast (EC-to-OSB) transition causes an ingress of M2-like macrophages, leading to an immunosuppressive bone tumor microenvironment (bone-TME). Analysis of a bmCRPC RNA-seq dataset revealed shorter overall survival in patients with an M2-high versus M2-low signature. Immunohistochemical (IHC) analysis showed CD206 + M2-like macrophages were enriched in bmCRPC specimens compared with primary tumors or lymph node metastasis. In osteogenic PCa xenografts, CD206 + macrophages were enriched adjacent to tumor-induced bone. FACS analysis showed an increase in CD206 + cells in osteogenic tumors compared to non-osteogenic tumors. Genetic or pharmacological inhibition of the EC-to-OSB transition reduced aberrant bone and M2-like macrophages in osteogenic tumors. RNAseq analysis of tumor-associated macrophages from osteogenic (bone-TAMs) versus non-osteogenic (ctrl-TAMs) tumors showed high expression of an M2-like gene signature, canonical and non-canonical Wnt pathways, and a decrease in an M1-like gene signature. Isolated bone-TAMs suppressed T-cell proliferation while ctrl-TAMs did not. Mechanistically, EC-OSB hybrid cells produced paracrine factors, including Wnts, CXCL14 and LOX, which induced M2 polarization and recruited M2-like TAMs to bone-TME. Our study thus links the unique EC-to-OSB transition as an "upstream" event that drives "downstream" immunosuppression in the bone-TME. These studies suggest that therapeutic strategies that inhibit PCa-induced EC-to-OSB transition may reverse immunosuppression to promote immunotherapeutic outcomes in bmCRPC. Significance: The insight that prostate cancer-induced bone generates an immunosuppressive bone tumor microenvironment offers a strategy to improve responses to immunotherapy approaches in patients with bone metastatic castrate-resistant prostate cancer.

Novel roles of Skp2 E3 ligase in cellular senescence, cancer progression, and metastasis.

S-phase kinase-associated protein 2 (Skp2) belongs to the F-box protein family. It is a component of the SCF E3 ubiquitin ligase complex. Skp2 has been shown to regulate cellular proliferation by targeting several cell cycle-regulated proteins for ubiquitination and degradation, including cyclin-dependent kinase inhibitor p27. Skp2 has also been demonstrated to display an oncogenic function since its overexpression has been observed in many human cancers. This review discusses the recent discoveries on the novel roles of Skp2 in regulating cellular senescence, cancer progression, and metastasis, as well as the therapeutic potential of targeting Skp2 for human cancer treatment.

Dynamic Immune Landscape and VZV-Specific T Cell Responses in Patients With Herpes Zoster and Postherpetic Neuralgia.

Objectives: Varicella-zoster virus (VZV) can induce herpes zoster (HZ) and postherpetic neuralgia (PHN). Immune cells play an important role in regulating HZ and PHN pathogenesis, but the dynamic immune profiles and molecular mechanisms remain unclear. This study aimed to screen dynamic immune signatures during HZ progression and elucidate the mechanism of VZV-specific T cells in PHN. Methods: We used cytometry by time-of-flight (CyTOF) to analyze peripheral blood mononuclear cells (PBMC) samples from 45 patients with HZ and eight age-sex-matched healthy controls, eight PHN samples and seven non-PHN samples. Correlations between the immune subsets and clinical pain-related scores were performed. Further, the characteristics of VZV-specific T cells between PHN and non-PHN patients were evaluated by VZV peptide pools stimulation. The expression level of cytokines, including granzyme B, interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α was performed via cytometric bead array. Finally, we analyzed the alteration of Ca2+ signals in dorsal root ganglion (DRG)-derived cells after TNF-α stimulation. Results: We investigated the dynamic characteristics of the immune landscape of peripheral blood samples of patients with HZ and PHN, and depicted two major dynamic signatures in NK, CD4+ and CD8+ T subsets in patients with HZ, which closely correlated with clinical pain-related scores. The frequency of PD-1+CD4+ T cells, VZV-specific PD-1+CD4+ T cells, and the amount of TNF-α produced by VZV-specific T cells were higher in patients with PHN than without PHN. Furthermore, we showed that TNF-α could induce calcium influx in DRG-derived cells in a dose-dependent manner. Conclusions: Our results profiled the dynamic signatures of immune cells in patients with HZ and highlighted the important role of VZV-specific T cells in the pathogenesis of PHN.

Prostate tumor-induced stromal reprogramming generates Tenascin C that promotes prostate cancer metastasis through YAP/TAZ inhibition.

Metastatic prostate cancer (PCa) in bone induces bone-forming lesions that enhance PCa progression. How tumor-induced bone formation enhances PCa progression is not known. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition by tumor-secreted bone morphogenetic protein 4 (BMP4). Here, we show that EC-to-OSB transition leads to changes in the tumor microenvironment that increases the metastatic potential of PCa cells. We found that conditioned medium (CM) from EC-OSB hybrid cells increases the migration, invasion, and survival of PC3-mm2 and C4-2B4 PCa cells. Quantitative mass spectrometry (Isobaric Tags for Relative and Absolute Quantitation) identified Tenascin C (TNC) as one of the major proteins secreted from EC-OSB hybrid cells. TNC expression in tumor-induced OSBs was confirmed by immunohistochemistry of MDA PCa-118b xenograft and human bone metastasis specimens. Mechanistically, BMP4 increases TNC expression in EC-OSB cells through the Smad1-Notch/Hey1 pathway. How TNC promotes PCa metastasis was next interrogated by in vitro and in vivo studies. In vitro studies showed that a TNC-neutralizing antibody inhibits EC-OSB-CM-mediated PCa cell migration and survival. TNC knockdown decreased, while the addition of recombinant TNC or TNC overexpression increased migration and anchorage-independent growth of PC3 or C4-2b cells. When injected orthotopically, PC3-mm2-shTNC clones decreased metastasis to bone, while C4-2b-TNC-overexpressing cells increased metastasis to lymph nodes. TNC enhances PCa cell migration through α5ß1 integrin-mediated YAP/TAZ inhibition. These studies elucidate that tumor-induced stromal reprogramming generates TNC that enhances PCa metastasis and suggest that TNC may be a target for PCa therapy.

A simple quantitative PCR assay to determine TRAMP transgene zygosity.

BACKGROUND: The TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model remains one of the most widely used transgenic mouse models of prostate cancer. This is due to its ability to recapitulate with ~100% penetrance multiple aspects of the human disease such as prostatic intraepithelial neoplasia lesions, invasive carcinoma, progression to castration-resistant prostate cancer including aggressive neuroendocrine prostate cancer and metastasis. Despite its popularity, the use of TRAMP mice is limited/slowed by the inability to distinguish the zygosity of the TRAMP transgene. This is especially true for breeding strategies implementing multiple crosses and alleles and when the rapid generation of large animal cohorts with the desired genotype is needed. METHODS: We developed a quantitative PCR (qPCR) approach to determine the relative TRAMP transgene copy number of mice. RESULTS: This method was validated by three independent laboratories across two institutions, which successfully identified the genotype of the mice 98.2% of the time (165/168) in the first attempt. The genotypes of the uncertain mice were correctly identified in the repeated experiments. CONCLUSIONS: We develop the first straightforward, qPCR approach to reliably determine the TRAMP transgene zygosity. The development of this qPCR-based genotyping method enables researchers to streamline breeding strategies when creating complex genetic mouse models involving TRAMP mice; thus, ultimately reducing the required animal numbers, cost, and investigator time.

Multiple pathways coordinating reprogramming of endothelial cells into osteoblasts by BMP4.

Cell type transition occurs during normal development and under pathological conditions. In prostate cancer bone metastasis, prostate cancer-secreted BMP4 induces endothelial cell-to-osteoblast (EC-to-OSB) transition. Such tumor-induced stromal reprogramming supports prostate cancer progression. We delineate signaling pathways mediating EC-to-OSB transition using EC lines 2H11 and SVR. We found that BMP4-activated pSmad1-Notch-Hey1 pathway inhibits EC migration and tube formation. BMP4-activated GSK3ß-ßcatenin-Slug pathway stimulates Osx expression. In addition, pSmad1-regulated Dlx2 converges with the Smad1 and ß-catenin pathways to stimulate osteocalcin expression. By co-expressing Osx, Dlx2, Slug and Hey1, we were able to achieve EC-to-OSB transition, leading to bone matrix mineralization in the absence of BMP4. In human prostate cancer bone metastasis specimens and MDA-PCa-118b and C4-2b-BMP4 osteogenic xenografts, immunohistochemical analysis showed that ß-catenin and pSmad1 are detected in activated osteoblasts rimming the tumor-induced bone. Our results elucidated the pathways and key molecules coordinating prostate cancer-induced stromal programming and provide potential targets for therapeutic intervention.