Prostate cancer (PCa) is the most common cancer among men in Western developed countries. While the majority of PCa diagnosed by PSA screening are indolent, advanced and metastatic disease has a significant mortality and morbidity. Bone metastases are extremely common in PCa and identification of bone metastasis associated genes may provide insights into PCa progression and assist in finding new drug targets. However, the genetic study of bone metastases is very limited due to the difficulty of sampling. We performed genome-wide analysis of six fresh frozen PCa bone metastases. We found several alterations commonly present in advanced PCa, including gains at: 1q32.1, broad gains of 8q (MYC, NCOA2), 9q33.2-34.3, 11q13.1-14.1 (CCND1), 12q24.23-24.31, 16p13.3, 16p12.1-11.2 and Xq12-13.1 (AR) as well as losses at: 5q11.1-22.1, 5q14.3-23.1, 6q14.1-22, 8p23.2-p21, 13q13.2-31.1 (RB1), 17p13.1-12 (TP53) and 18q11.1-22.3. Two cases also showed PTEN loss and one sample had deletion indicative of TMPRSS2-ERG fusion. For downstream analysis we concentrated on CCND1 oncogene at 11q13 and FBXL4 at 6q16 as potential drivers of these genomic changes. Using fluorescence in situ hybridisation we found common CCND1 gain and FBXL4 loss in PCa bone metastases (54.5%, 12/22 and 47.8%, 11/23, respectively), much less frequent in primary tumours (7%, 10/142 and 13.8%, 20/145, respectively) and absent in BPH cases (0/55). The expression levels of cyclin D1 protein, coded by CCND1 correlated with CCND1 copy number gain (p < 0.0001) and were higher in metastatic tumours than in primary PCa (p = 0.015), confirming cyclin D1 involvement in advanced PCa. Presence of FBXL4 loss in early stage primary PCa strongly correlated with current PCa prognostic markers and with worse patient survival. Therefore, we propose that FBXL4 may be a tumour suppressor gene in prostate, whose loss in early PCa could be indicative of more aggressive disease. Using in vitro experiments we demonstrated that FBXL4 regulates cells motility and invasion. We confirmed that ERLEC1, an ER lectin involved in ER stress response pathway is a degradation target of FBXL4. As activation of ER stress response pathway is linked to enhanced cell migration and invasion, loss of FBXL4 could be one of the mechanisms by which cancer cells increase their efficiency to respond to stress and to escalate their metastatic potential through stabilisation of ERLEC1. Further studies of FBXL4 - ERLEC1 axis are necessary to establish how they contribute to PCa progression. This knowledge can potentially help to develop novel targeted therapies for aggressive disease harbouring FBXL4 abnormalities.

Bisphosphonates (BPs) have been used clinically as anti-resorptive/cancer agents with confounded clinical outcomes and uncertain/conflicting biological understanding. This study was designed to evaluate the impact of clinically used anti-resorption drug alendronate (ALN) on cancer-bone metastasis and bone biology using novel 3D cancer-bone interaction model systems. To test the effects of ALN on the cancer-bone metastasis/interactions and bone biology we have utilized a novel 3D Co-cultures of live mouse neonatal calvarial bone organs with oral squamous cancer cells in a roller tube model systems (Curtin et al, 2012) in the absence and presence of ALN. These model systems under bone resorption and formation conditions were evaluated by chemical, biochemical, and histological analyses of the used media and calvarial bones. At the end of 8 days, the calvarial bones co-cultured with oral cancer cell lines in the absence and presence of ALN were processed for histological observations, TRAP and ALP enzyme activities, and neutral red staining. These studies were complemented by the effects of ALN on oral cancer cells under 2D classic cell culture conditions. In 3D-bone organ cultures under resorption conditions, oral cancer cells induce differentiation of osteoclasts and bone resorption and inclusion of ALN inhibited cancer-induced bone resorption. However, in both bone resorption and formation models the oral cancer cells colonized the bone and while treatment with ALN inhibits bone resorption, no effect on bone colonization was evident. Contrary to those under 2D cell culture conditions exposure to ALN of confluent and non-confluent oral cancer cells in the absence of live bone impacted oral cancer cells significantly in a dose dependent manner. Our studies using live bone organ cultures with oral cancer cells under specific dissociated bone remodeling stages, viz., resorption or formation only, revealed major and significant biological events which led to the conclusions that: (a) In the absence of bone in 2D cultures oral cancers are sensitive to ALN treatment whereas in the 3D live bone microenvironment tumors are resistant to ALN drug therapy, and (b) oral cancer-bone metastasis is independent of bone remodeling stage.

OBJECTIVE: To determine the effect of clinically used zoledronate (ZOL) and docetaxel on breast cancer cells and bone biology under both bone remodeling stages and the rate of tumor dissemination and state of dormancy. METHODS: The effect of clinically used zoledronate (ZOL) was examined on MDA-MB-231 and MDA-BO cells in a roller tube system under bone resorption and formation conditions. Three groups; calvaria alone, calvarial co-cultured with tumor cells, and calvaria with tumor cells treated with four repeat doses of 2 µM of ZOL were cultured for 8, 14 and 20 days. The formation groups were supplemented with 150 µg/ml ascorbic acid. Cell counts were performed on trypsinized calvaria harvested at 2, 8 and 14 days. Media was changed every 2 days and the changed media was re-seeded in a 24-well for 20 days. To test the impact of chemotherapy agents on cancer-bone metastasis the effect of 10 µM of docetaxel was tested on breast cancer cells under formation and resorption conditions using the above design. Tumor burden was assessed at 8 days. RESULTS: Tumor burden: no statistically significant difference between ZOL treated and untreated groups under resorption and formation conditions in both cell lines. Exposure to docetaxel revealed that ~30% of the cells were affected by chemotherapy in formation model, while ~70% was affected in resorption model in both cell types. Dissemination model: the dissemination rate for MDA and BO cells under formation condition is significantly less than for resorption conditions. Fluorescent microscopy: MDA and BO tumor-calvaria were treated with Ki 67 antibody showed that under bone resorption conditions the cancer-bone cells colony were predominantly in proliferation stage while under formation conditions cancer cells were in dormancy. Confocal microscopy: observation confirmed the relation of the mode of cancer cell attachment to bone endosteal cell layer with the dormancy and cell proliferation states. CONCLUSIONS: Both cancer cell lines showed resistance to ZOL under formation and resorption conditions. Drug resistance to docetaxel was more evident under formation condition, where cells are dormant and not proliferating. The dissemination rate is significantly higher in resorption condition, suggesting that cells in formation are dormant with lower dissemination rate. / 2019-07-23T00:00:00Z

FGFR4 and β-Klotho in Metastatic Prostate Cancer by Derek LaMar Shenefelt Fibroblast growth factors and fibroblast growth factor receptors have been associated with the aggressiveness and progression of Prostate Cancer (PCa). Also, β-Klotho is a known co-receptor with FGFR4 for FGF19 in the liver however, the role of this co-receptor pair remains unclear in the setting of PCa. I demonstrated that FGFR4 and KLB mRNA and protein are highly expressed in PCa cells when compared to bone marrow stromal cells, a common site of metastasis. I also provide support for the association of FGFR4 and KLB in PCa, suggesting a functional co-receptor pair capable of altering cellular signaling. FGFR4-KLb may also provide some level of protection to PCa cells from chemotherapeutics. This analysis of FGFR4 and KLB expression and signaling in PCa has provided novel insights into phenotypic alterations during PCa progression while also providing new avenues of study to further explore the role and importance of this exciting co-receptor complex.

Pain is the most feared symptom of cancer and can impact patients' lives more than the cancer itself. Despite improvements in cancer prevention and detection, pain is often the first sign of cancer, with an estimated 70-75% of advanced stage cancer patients presenting with skeletal metastases. Cancer metastasis to the bone is associated with persistent pain that increases in intensity over time. Current treatments follow the World Health Organization (WHO) analgesic ladder for cancer pain management suggesting non-steroidal anti-inflammatory drugs (NSAIDs) for mild to moderate pain and opioids for moderate to severe pain. However, estimates indicate as many as 50-80% of cancer patients worldwide receive inadequate pain management. Moreover, opioid doses required for these patients are associated with adverse side effects further diminishing quality of life. Development of improved non-opioid therapies is dependent on increased understanding of mechanisms driving cancer pain and its relief. The objective of this dissertation was to characterize a rat model of cancer-induced bone pain, to develop approaches to measure both ongoing and breakthrough pain and to investigate the contribution of underlying inflammatory mechanisms to pain, bone destruction and bone remodeling. Using female Fischer F344/NhSD rats, histocompatible MAT B III mammary adenocarcinoma cells were sealed into the intramedullary space of the right rear tibia for a time course of 13 days. Ongoing pain was characterized based on the WHO 3-step ladder for pain management utilizing novel behavioral and neurochemical assays. Morphine and peripheral nerve block were sufficient to control ongoing pain, whereas NSAID treatment failed to provide pain relief. Cancer-bearing rats selectively displayed movement-induced breakthrough pain to a background of morphine-controlled ongoing pain. Furthermore, we determined that breakthrough pain is initiated, but not maintained, by peripheral afferent input from the tumor-bearing tibia using lidocaine administration prior to or following movement. For the final part of this study, we investigated the role of transient receptor potential vanilloid 1 (TRPV1) and interleukin-6 (IL-6) blockade, as these have been shown to be important mediators in animal models CIBP. Acute blockade of TRPV1 channels by AMG9810 selectively reversed inflammatory-induced pain, but failed to control evoked or ongoing CIBP. Acute blockade of interleukin-6 signaling by TB-2-081, an IL-6 receptor antagonist, successfully reversed evoke pain responses, but like AMG9810, failed to control ongoing pain. Sustained administration of TB-2-081 reversed cancer-induced tactile hypersensitivity and tumor-induced bone remodeling of the tibia. Further in vitro analysis revealed TB-2-081 functions by inhibiting the Jak/STAT cascade on both tumor cells and osteoblasts, suggesting that blockade of IL-6 signaling can effectively modulate the bone microenvironment to reduce tumor burden and pain. Combined, our data introduce a rat model of breast cancer bone metastasis, in which the underlying mechanisms of ongoing and breakthrough CIBP can be effectively studied. From this, novel therapeutic agents can be developed and investigated to help improve quality of life in patients suffering from this disease.