Prostate cancer (PCa) is the most frequently diagnosed cancer in men, causing considerable morbidity and mortality. The P2X4 receptor (P2X4R) is the most ubiquitously expressed P2X receptor in mammals and is positively associated with tumorigenesis in many cancer types. However, its involvement in PCa progression is less understood. We hypothesized that P2X4R activity enhanced tumour formation by PCa cells. We showed that P2X4R was the most highly expressed, functional P2 receptor in these cells using quantitative reverse transcription PCR (RT-PCR) and a calcium influx assay. The effect of inhibiting P2X4R on PCa (PC3 and C4-2B4 cells) viability, proliferation, migration, invasion, and apoptosis were examined using the selective P2XR4 antagonists 5-BDBD and PSB-12062. The results demonstrated that inhibiting P2X4R impaired the growth and mobility of PCa cells but not apoptosis. In BALB/c immunocompromised nude mice inoculated with human PC3 cells subcutaneously, 5-BDBD showed anti-tumourigenic effects. Finally, a retrospective analysis of P2RX4 expression in clinical datasets (GDS1439, GDS1746, and GDS3289) suggested that P2X4R was positively associated with PCa malignancy. These studies suggest that P2X4R has a role in enhancing PCa tumour formation and is a clinically targetable candidate for which inhibitors are already available and have the potential to suppress disease progression.

Dear EditorBone is the most common site for metastasis in prostate cancer (PCa), and identifying clinically useful biomarkers to predict the risk of currently incurable bone metastases is a major priority in PCa research.Extracellular adenosine triphosphate (ATP) was shown to be a major biochemical constituent of the tumor microenvironment and regulates the tumor and host interactions by acting at P2 purinergic receptors. Among the P2 receptors, the ion channel P2X purinergic receptor 7 (P2X7R) has an unusually long C-terminus containing 200 amino acids, which forms the molecular basis for the unique bi-function of P2X7R promoting cancer cell proliferation or inducing membrane permeabilization and apoptosis, upon activation by a low or high concentration of ATP, respectively. A cysteine-rich region and a guanosine diphosphate or triphosphate (GDP/GTP)-binding site located in the C-terminus are pivotal for the structural rearrangement of the second transmembrane domain helix and the globular ballast underneath during the membrane pore permeabilization [1].Among the 10 human P2X7R splice variants (P2X7RA-J), the P2X7RA variant is the full-length "wild-type (WT)" receptor, while the truncated P2X7RB variant lacks the intracellular C-terminal tail. This naturally occurring P2X7RB isoform maintains ATP-stimulated channel activity but not pore permeabilization [2, 3]. P2X7RB is upregulated in epithelial cancer cells [3] and associated with the trophic activity of primary bone cancer [2,4,5]. These findings have highlighted the importance of defining the role of P2X7R C-terminal truncated variants in cancer progression, particularly considering that data on

Breast cancer is the most commonly diagnosed cancer and frequently metastasizes to bone. Previous evidence has suggested that cancer cells target the osteoblastic lineage cells to establish colonization of bone. It is well established that mechanical loading (e.g. load bearing exercise) elevates bone remodeling via stimulating osteoblastic activities. Whether mechanical loading facilitates the colonization of breast cancer cells in bone is not fully understood. To test this, we have firstly applied a murine tibial mechanical loading model and determined the effects of mechanical loading on bone remodeling in vivo. Six 10-week-old BALB/c nude female mice were subjected to a right tibial dynamic load (9N) onto a 2N pre-load using the ElectroForce 5500 Test Instrument, 40 load cycles with 10 sec intervals, 3 times a week for 1 week. Mice were then euthanized with additional 4 mice euthanized before mechanical loading as baseline controls. Quantitative micro-CT analysis suggested that bone mass related parameters significantly increased in the loaded tibias compared to contra-lateral non-loaded controls, while data in the baseline group showed no differences between left and right tibias. As previously published 2-week loading regimens, one week of loading was enough to induce a 24.3% increase in trabecular bone volume (P=0.001, paired t-test), a 9.0% increase in trabecular thickness (P= 0.01), and a 17.9% increase in cortical bone volume (P<0.0001). Using this model, we tested whether mechanical loading affects the arrival of breast cancer cells in bone. Human and mouse breast cancer cells (MDA-MB-231 and 4T1) were firstly labelled with vital fluorescent lipophilic dyes (Vybrant DiD) to allow tracking of individual tumor cells arriving in bone ex vivo. Cells were injected into the circulation (intracardiac) after mice (BALB/c nude and BALB/c) subjected to 1-week of mechanical loading. Mice were then loaded 3 times more to maintain bone mass before euthanized 7 days post-injection. DiD labelled cancer cells were examined and quantified in tibial bone marrow using multiphoton microscopy ex vivo. In both xenograft and syngeneic models, there were no significant differences found in the number of breast cancer cells arriving in bone marrow (MDA: P=0.32; 4T1: P=0.67) and their relative location to bone surfaces (MDA: P= 0.45; 4T1: P= 0.91) in loaded vs non-loaded bones. There was no significant difference in size of objectives (indicating size of micro tumors) detected in the MDA model (P= 0.21), comparing loaded to non-loaded control tibias but a significant 17.7% reduction in the 4T1 model in the loaded tibias (P=0.02). These pre-clinical data demonstrate that mechanical loading induced osteoblastic bone formation does not affect the initial colonization of breast cancer cells in bone, and suggest that load bearing exercise will not increase the risk of breast cancer bone metastasis in patients. Citation Format: Alexandria R. Sprules, Norain B. Latif, Janet E. Brown, Alison Gartland, Ning Wang. Mechanical loading induced osteogenic response does not interfere with the colonization of breast cancer in bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2904.

Background: Prostate cancer (PCa) preferentially metastasizes to bone, causing considerable mortality and morbidity. Exercise is a widely accepted intervention for improving bone health and can benefit overall health in patients with PCa. However, there are no direct studies defining the impact of various exercise regimens on the development of PCa bone metastasis. Hypothesis: Exercise regimens that induce osteogenic responses inhibit the growth of PCa cells in bone. Methods: Human PCa cells (PC3, 1x105 cells per injection) were intracardiacally injected into 8-week-old male BALB/c nude mice which were then subjected to right tibial dynamic mechanical loading (mimicking load bearing exercise, 9N peak force, 40 cycles with 10 second intervals, three times a week) or treadmill exercise (12 meters/minute, 30 minutes/day, 5 days/week) for 3 weeks. Another cohort of mice were firstly subjected to one week of mechanical loading before the inoculation of PC3 cells and then withdrawn from exercise regimen. The growth of skeletal tumours was tracked by bioluminescence for 3 weeks post-injection and bone structure was examined by micro-CT ex vivo. For mechanical loading experiments, comparison was made between loaded and contra-lateral non-loaded tibias, while treadmill exercise group was compared to sedentary controls in the treadmill study. Results: In the continuous mechanical loading group, tumours were detected in non-loaded tibias in all 12 mice (100%), whilst 8 out of 12 mice showed reduced skeletal tumours in the loaded tibias (67%), with 4 having no tumour in the loaded leg (33%). Tumour burden (Flux: photon/second) in the non-loaded tibias was 134% higher than that in the loaded tibias. Micro-CT analysis suggested that loaded tibias had significantly increased trabecular (BV/TV) and cortical bone volume and trabecular thickness (Tb.Th) (p<0.001). Surprisingly, treadmill exercised mice had higher tumour burden (~91%) in the hind limbs compared to the sedentary controls (P<0.05), with no statistically significant improvement in bone mass examined by micro-CT. In the loading withdrawal group, 6 out of 8 mice (75%) showed larger tumour size in the loading withdrawal tibias than in the non-loaded tibias, while only two mice (25%) had smaller or no tumour in the loading withdrawal tibias. Tumor burden were ~90% higher in the loading withdrawal tibias (p<0.05, t-test). This is associated with fast diminishing osteogenic response within a week after ceasing the loading stimulus, confirmed using micro-CT analysis by comparing percentage changes of BV/TV and Tb.Th among baseline, 1-week loading, and loading withdrawn groups. Conclusion: The continuity of high-intensity load bearing exercise is critical to prevent PCa growth in bone, possibly via maintaining the balance of bone remodelling towards osteoblastic bone formation. Mild aerobic exercise evaluated here was unable to significantly improve bone mass or achieve the benefits in inhibiting PCa bone metastases observed with high intensity loading. Citation Format: Ning Wang, Hector M. Arredondo, Alexandria R. Sprules, Colby L. Eaton. Continuous load bearing exercise inhibits the development of prostate cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3481.

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