Inhibition of STAT3 prevents bone metastatic progression of prostate cancer in vivo

Background: Prostate cancer (PC) metastasizes to the skeleton forming predominantly sclerotic lesions, and there is currently no cure for bone metastatic disease. The transcription factor signal transducer and activator of transcription 3 (STAT3) is implicated as a metastatic driver, but its potential as therapeutic target in bone metastasis has not been investigated. In this study, we evaluated for the first time a STAT3 inhibitor, Napabucasin, as a therapeutic option for bone metastatic PC.Methods: Effects of STAT3 inhibitors, Stattic and Napabucasin, on metastatic potential in PC cells were studied in vitro by assessment of migration capacity, self-renewal potential, and tumorsphere formation. For evaluation of the role of STAT3 in initial skeletal establishment of PC cells as well as in progressed castration-resistant PC (CRPC) in bone, human VCaP prostate cancer cells were inoculated in the tibia of mice which subsequently were treated with the STAT3 inhibitor Napabucasin. Bone specimens were analyzed using computed tomography (CT), immunohistochemistry, and quantitative polymerase chain reaction.Results: The small molecule STAT3 inhibitors Stattic and Napabucasin both effectively impaired metastatic potential of PC cells in vitro. Furthermore, treatment with Napabucasin prevented metastatic establishment in tibial bones in vivo and thereby also the tumor-induced sclerotic bone response seen in vehicle-treated VCaP xenografts. In addition, treatment with Napabucasin of established bone CRPC significantly decreased both tumor burden and tumor-induced trabecular bone volume compared with effects seen in vehicle-treated animals. Anti-mitotic effects were confirmed by decreased Ki67 staining in Napabucasin-treated xenografts compared with vehicle-treated xenografts. Alterations of gene expression in the femoral bone marrow (BM) niche toward the maintenance of hematopoietic stem cells and the myeloid lineage were demonstrated by quantitative real-time polymerase chain reaction and were further reflected by a substantial increase in the number of erythrocytes in BM of Napabucasin-treated mice. Furthermore, a unique pattern of STAT3 phosphorylation in osteoblasts/stromal cells surrounding the areas of tumor cells was demonstrated immunohistochemically in bone xenograft models using several different PC cell lines.Conclusion: Inhibition of STAT3 activity disrupts the bone metastatic niche and targets both the skeletal establishment of PC and advanced bone metastatic CRPC in mice, suggesting STAT3 as a candidate for molecular targeted therapies of skeletal metastatic disease.</div

High intratumoral dihydrotestosterone is associated with antiandrogen resistance in VCaP prostate cancer xenografts in castrated mice

Antiandrogen treatment resistance is a major clinical concern in castration-resistant prostate cancer (CRPC) treatment. Using xenografts of VCaP cells we showed that growth of antiandrogen resistant CRPC tumors were characterized by a higher intratumor dihydrotestosterone (DHT) concentration than that of treatment responsive tumors. Furthermore, the slow tumor growth after adrenalectomy was associated with a low intratumor DHT concentration. Reactivation of androgen signaling in enzalutamide-resistant tumors was further shown by the expression of several androgen-dependent genes. The data indicate that intratumor DHT concentration and expression of several androgen-dependent genes in CRPC lesions is an indication of enzalutamide treatment resistance and an indication of the need for further androgen blockade. The presence of an androgen synthesis, independent of CYP17A1 activity, has been shown to exist in prostate cancer cells, and thus, novel androgen synthesis inhibitors are needed for the treatment of enzalutamide-resistant CRPC tumors that do not respond to abiraterone.Peer reviewe

The regulatory role of osteoblasts in castration-resistant growth of prostate cancer

Bone metastasis of a predominantly osteoblastic (sclerotic) nature is the outcome for the vast majority of patients with castration-resistant prostate cancer (CRPC). Pathologically, osteoblastic tumors are characterized by excessive bone formation resulting in decreased quality of life due to severe pain, fractures, nerve compression, and a suppressed immune system. Despite the success of novel therapeutic approaches, castration-resistant tumors remain the primary unsolved obstacle for patient survival. Therefore, an improved understanding of the molecular mechanisms behind the osteoblastic growth of CRPC is important in the search for novel therapeutic strategies. The aim of this thesis was to investigate the specific role of osteoblasts in the growth of prostate cancer in bone. By establishing and characterizing a novel model of sclerotic CRPC, it was demonstrated that both osteoblasts and prostate cancer cells are potential mediators of bone formation. It was further demonstrated that osteoblasts promote the osteogenic and metastatic progression of CRPC cells and potentiate the cross talk between CRPC and bone cells. Moreover, it was shown that osteoblasts induce and alter steroidogenesis in the CRPC cells by increasing the expression of steroidogenic enzymes in a similar manner to what has previously been described in bone metastases from patients. Further studies reveled that Runt-related transcription factor 2 (Runx2) – which is under the control of osteoblasts – is a putative regulator of de novo steroid synthesis in osteogenic CRPC cells, and this mimics a mechanism of steroid synthesis previously only described in osteoblasts. Finally, a preclinical study with tasquinimod showed that this drug efficiently impaired the establishment of bone metastases in mice by interfering with the osteoblastic pre-metastatic niche and osteoblastic activity, thus emphasizing the role of osteoblasts in the early phases of the metastatic process. In summary, the studies performed in this thesis have characterized the role of osteoblasts in castration-resistant growth of prostate cancer in bone and suggest that osteoblasts could be an attractive target for the development of novel therapeutic approaches. A better understanding of the osteoblast–tumor cell interaction might facilitate the design of treatment strategies targeting the osteoblasts as a way to inhibit the metastatic process and thus bypass the castration resistance of CRPC bone metastases

Analysis of regulator of G-protein signalling 2 (RGS2) expression and function during prostate cancer progression

Prostate cancer (PC) represents the second highest cancer-related mortality among men and the call for biomarkers for early discrimination between aggressive and indolent forms is essential. Downregulation of Regulator of G-protein signaling 2 (RGS2) has been shown in PC, however the underlying mechanism has not been described. Aberrant RGS2 expression has also been reported for other carcinomas in association to both positive and negative prognosis. In this study, we assessed RGS2 expression during PC progression in terms of regulation and impact on tumour phenotype and evaluated its prognostic value. Our experimental data suggest that the RGS2 downregulation seen in early PC is caused by hypoxia. In line with the common indolent phenotype of a primary PC, knockdown of RGS2 induced epithelial features and impaired metastatic properties. However, increased STAT3, TWIST1 and decreased E-cadherin expression suggest priming for EMT. Additionally, improved tumour cell survival and increased BCL-2 expression linked decreased RGS2 levels to fundamental tumour advantages. In contrast, high RGS2 levels in advanced PC were correlated to poor patient survival and a positive metastatic status. This study describes novel roles for RGS2 during PC progression and suggests a prognostic potential discriminating between indolent and metastatic forms of PC

Osteoblasts promote castration-resistant prostate cancer by altering intratumoral steroidogenesis

The skeleton is the preferred site for prostate cancer (PC) metastasis leading to incurable castration-resistant disease. The increased expression of genes encoding steroidogenic enzymes found in bone metastatic tissue from patients suggests that up-regulated steroidogenesis might contribute to tumor growth at the metastatic site. Because of the overall sclerotic phenotype, we hypothesize that osteoblasts regulate the intratumoral steroidogenesis of castration resistant prostate cancer (CRPC) in bone. We here show that osteoblasts alter the steroidogenic transcription program in CRPC cells, closely mimicking the gene expression pattern described in CRPC. Osteoblast-stimulated LNCaP-19 cells displayed an increased expression of genes encoding for steroidogenic enzymes (CYP11A1, HSD3B1, and AKR1C3), estrogen signaling-related genes (CYP19A1, and ESR2), and genes for DHT-inactivating enzymes (UGT2B7, UGT2B15, and UGT2B17). The observed osteoblast-induced effect was exclusive to osteogenic CRPC cells (LNCaP-19) in contrast to osteolytic PC-3 and androgen-dependent LNCaP cells. The altered steroid enzymatic pattern was specific for the intratibial tumors and verified by immunohistochemistry in tissue specimens from LNCaP-19 xenograft tumors. Additionally, the overall steroidogenic effect was reflected by corresponding levels of progesterone and testosterone in serum from castrated mice with intratibial xenografts. A bi-directional interplay was demonstrated since both proliferation and Esr2 expression of osteoblasts were induced by CRPC cells in steroid-depleted conditions. Together, our results demonstrate that osteoblasts are important mediators of the intratumoral steroidogenesis of CRPC and for castration-resistant growth in bone. Targeting osteoblasts may therefore be important in the development of new therapeutic approaches

Inhibition of STAT3 prevents bone metastatic progression of prostate cancer in vivo

Background: Prostate cancer (PC) metastasizes to the skeleton forming predominantly sclerotic lesions, and there is currently no cure for bone metastatic disease. The transcription factor signal transducer and activator of transcription 3 (STAT3) is implicated as a metastatic driver, but its potential as therapeutic target in bone metastasis has not been investigated. In this study, we evaluated for the first time a STAT3 inhibitor, Napabucasin, as a therapeutic option for bone metastatic PC. Methods: Effects of STAT3 inhibitors, Stattic and Napabucasin, on metastatic potential in PC cells were studied in vitro by assessment of migration capacity, self-renewal potential, and tumorsphere formation. For evaluation of the role of STAT3 in initial skeletal establishment of PC cells as well as in progressed castration-resistant PC (CRPC) in bone, human VCaP prostate cancer cells were inoculated in the tibia of mice which subsequently were treated with the STAT3 inhibitor Napabucasin. Bone specimens were analyzed using computed tomography (CT), immunohistochemistry, and quantitative polymerase chain reaction. Results: The small molecule STAT3 inhibitors Stattic and Napabucasin both effectively impaired metastatic potential of PC cells in vitro. Furthermore, treatment with Napabucasin prevented metastatic establishment in tibial bones in vivo and thereby also the tumor-induced sclerotic bone response seen in vehicle-treated VCaP xenografts. In addition, treatment with Napabucasin of established bone CRPC significantly decreased both tumor burden and tumor-induced trabecular bone volume compared with effects seen in vehicle-treated animals. Anti-mitotic effects were confirmed by decreased Ki67 staining in Napabucasin-treated xenografts compared with vehicle-treated xenografts. Alterations of gene expression in the femoral bone marrow (BM) niche toward the maintenance of hematopoietic stem cells and the myeloid lineage were demonstrated by quantitative real-time polymerase chain reaction and were further reflected by a substantial increase in the number of erythrocytes in BM of Napabucasin-treated mice. Furthermore, a unique pattern of STAT3 phosphorylation in osteoblasts/stromal cells surrounding the areas of tumor cells was demonstrated immunohistochemically in bone xenograft models using several different PC cell lines. Conclusion: Inhibition of STAT3 activity disrupts the bone metastatic niche and targets both the skeletal establishment of PC and advanced bone metastatic CRPC in mice, suggesting STAT3 as a candidate for molecular targeted therapies of skeletal metastatic disease