Patient-derived xenografts and organoids model therapy response in prostate cancer.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds.

miR-221-5p regulates proliferation and migration in human prostate cancer cells and reduces tumor growth in vivo.

BACKGROUND: Despite latest advances in prostate cancer (PCa) therapy, PCa remains the third-leading cause of cancer-related death in European men. Dysregulation of microRNAs (miRNAs), small non-coding RNA molecules with gene expression regulatory function, has been reported in all types of epithelial and haematological cancers. In particular, miR-221-5p alterations have been reported in PCa. METHODS: miRNA expression data was retrieved from a comprehensive publicly available dataset of 218 PCa patients (GSE21036) and miR-221-5p expression levels were analysed. The functional role of miR-221-5p was characterised in androgen- dependent and androgen- independent PCa cell line models (C4-2 and PC-3M-Pro4 cells) by miR-221-5p overexpression and knock-down experiments. The metastatic potential of highly aggressive PC-3M-Pro4 cells overexpressing miR-221-5p was determined by studying extravasation in a zebrafish model. Finally, the effect of miR-221-5p overexpression on the growth of PC-3M-Pro4luc2 cells in vivo was studied by orthotopic implantation in male Balb/cByJ nude mice and assessment of tumor growth. RESULTS: Analysis of microRNA expression dataset for human primary and metastatic PCa samples and control normal adjacent benign prostate revealed miR-221-5p to be significantly downregulated in PCa compared to normal prostate tissue and in metastasis compared to primary PCa. Our in vitro data suggest that miR-221-5p overexpression reduced PCa cell proliferation and colony formation. Furthermore, miR-221-5p overexpression dramatically reduced migration of PCa cells, which was associated with differential expression of selected EMT markers. The functional changes of miR-221-5p overexpression were reversible by the loss of miR-221-5p levels, indicating that the tumor suppressive effects were specific to miR-221-5p. Additionally, miR-221-5p overexpression significantly reduced PC-3M-Pro4 cell extravasation and metastasis formation in a zebrafish model and decreased tumor burden in an orthotopic mouse model of PCa. CONCLUSIONS: Together these data strongly support a tumor suppressive role of miR-221-5p in the context of PCa and its potential as therapeutic target.

Therapeutic Targeting of CD146/MCAM Reduces Bone Metastasis in Prostate Cancer.

Prostate Cancer is the most common cancer and the second leading cause of cancer-related death in males. When prostate cancer acquires castration resistance, incurable metastases, primarily in the bone, occur. The aim of this study is to test the applicability of targeting melanoma cell adhesion molecule (MCAM; CD146) with a mAb for the treatment of lytic prostate cancer bone metastasis. We evaluated the effect of targeting MCAM using in vivo preclinical bone metastasis models and an in vitro bone niche coculture system. We utilized FACS, cell proliferation assays, and gene expression profiling to study the phenotype and function of MCAM knockdown in vitro and in vivo. To demonstrate the impact of MCAM targeting and therapeutic applicability, we employed an anti-MCAM mAb in vivo. MCAM is elevated in prostate cancer metastases resistant to androgen ablation. Treatment with DHT showed MCAM upregulation upon castration. We investigated the function of MCAM in a direct coculture model of human prostate cancer cells with human osteoblasts and found that there is a reduced influence of human osteoblasts on human prostate cancer cells in which MCAM has been knocked down. Furthermore, we observed a strongly reduced formation of osteolytic lesions upon bone inoculation of MCAM-depleted human prostate cancer cells in animal model of prostate cancer bone metastasis. This phenotype is supported by RNA sequencing (RNA-seq) analysis. Importantly, in vivo administration of an anti-MCAM human mAb reduced the tumor growth and lytic lesions. These results highlight the functional role for MCAM in the development of lytic bone metastasis and suggest that MCAM is a potential therapeutic target in prostate cancer bone metastasis. IMPLICATIONS: This study highlights the functional application of an anti-MCAM mAb to target prostate cancer bone metastasis.

Bisphosphonates reduce biomaterial turnover in healing of critical-size rat femoral defects.

Treatment of osteoporotic patients with bisphosphonates (BPs) preserves bone mass and microarchitecture. The high prescription rate of the drugs brings about increases in the numbers of fractures and bone defects requiring surgical interventions in these patients. Currently, critical-size defects are filled with biomaterials and healing is supported with bone morphogenetic proteins (BMP). It is hypothesized that BPs interfere with biomaterial turnover during BMP-supported repair of defects filled with ß-tricalcium phosphate (ßTCP) ceramics. To test this hypothesis, retired breeder rats were ovariectomized ( OVX). After 8 weeks, treatment with alendronate (ALN) commenced. Five weeks later, 6 mm diaphyseal femoral defects were applied and stabilized with locking plates. ßTCP cylinders loaded with 1 µg and 10 µg BMP2, 10 µg L51P, an inhibitor of BMP antagonists and 1 µg BMP2/10 µg L51P were fitted into the defects. Femora were collected 16 weeks post-implantation. In groups receiving calcium phosphate implants loaded with 10 µg BMP2 and 1 µg BMP2/10 µg L51P, the volume of bone was increased and ßTCP was decreased compared to groups receiving implants with 1 µg BMP2 and 10 µg L51P. Treatment of animals with ALN caused a decrease in ßTCP turnover. The results corroborate the synergistic effects of BMP2 and L51P on bone augmentation. Administration of ALN caused a reduction in implant turnover, demonstrating the dependence of ßTCP removal on osteoclast activity, rather than on chemical solubility. Based on these data, it is suggested that in patients treated with BPs, healing of biomaterial-filled bone defects may be impaired because of the failure to remove the implant and its replacement by authentic bone.

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ALK1Fc Suppresses the Human Prostate Cancer Growth in in Vitro and in Vivo Preclinical Models.

Prostate cancer is the second most common cancer in men and lethality is normally associated with the consequences of metastasis rather than the primary tumor. Therefore, targeting the molecular pathways that underlie dissemination of primary tumor cells and the formation of metastases has a great clinical value. Bone morphogenetic proteins (BMPs) play a critical role in tumor progression and this study focuses on the role of BMP9- Activin receptor-Like Kinase 1 and 2 (ALK1 and ALK2) axis in prostate cancer. In order to study the effect of BMP9 in vitro and in vivo on cancer cells and tumor growth, we used a soluble chimeric protein consisting of the ALK1 extracellular domain (ECD) fused to human Fc (ALK1Fc) that prevents binding of BMP9 to its cell surface receptors and thereby blocks its ability to activate downstream signaling. ALK1Fc sequesters BMP9 and the closely related BMP10 while preserving the activation of ALK1 and ALK2 through other ligands. We show that ALK1Fc acts in vitro to decrease BMP9-mediated signaling and proliferation of prostate cancer cells with tumor initiating and metastatic potential. In line with these observations, we demonstrate that ALK1Fc also reduces tumor cell proliferation and tumor growth in vivo in an orthotopic transplantation model, as well as in the human patient derived xenograft BM18. Furthermore, we also provide evidence for crosstalk between BMP9 and NOTCH and find that ALK1Fc inhibits NOTCH signaling in human prostate cancer cells and blocks the induction of the NOTCH target Aldehyde dehydrogenase member ALDH1A1, which is a clinically relevant marker associated with poor survival and advanced-stage prostate cancer. Our study provides the first demonstration that ALK1Fc inhibits prostate cancer progression, identifying BMP9 as a putative therapeutic target and ALK1Fc as a potential therapy. Altogether, these findings support the validity of ongoing clinical development of drugs blocking ALK1 and ALK2 receptor activity.

Cancer cachexia associates with a systemic autophagy-inducing activity mimicked by cancer cell-derived IL-6 trans-signaling.

The majority of cancer patients with advanced disease experience weight loss, including loss of lean body mass. Severe weight loss is characteristic for cancer cachexia, a condition that significantly impairs functional status and survival. The underlying causes of cachexia are incompletely understood, and currently no therapeutic approach can completely reverse the condition. Autophagy coordinates lysosomal destruction of cytosolic constituents and is systemically induced by starvation. We hypothesized that starvation-mimicking signaling compounds secreted from tumor cells may cause a systemic acceleration of autophagy during cachexia. We found that IL-6 secreted by tumor cells accelerates autophagy in myotubes when complexed with soluble IL-6 receptor (trans-signaling). In lung cancer patients, were cachexia is prevalent, there was a significant correlation between elevated IL-6 expression in the tumor and poor prognosis of the patients. We found evidence for an autophagy-inducing bioactivity in serum from cancer patients and that this is clearly associated with weight loss. Importantly, the autophagy-inducing bioactivity was reduced by interference with IL-6 trans-signaling. Together, our findings suggest that IL-6 trans-signaling may be targeted in cancer cachexia.