Abstract 2117: Role of Neuropilin 2 in orchestrating the functions of osteoclasts in promoting prostate cancer bone metastasis

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Abstract Background: Bone metastasis is one of the major clinical concerns that causes skeletal-related malignancies and increased mortality. Bone is one of the preferred sites for metastatic prostate cancer. The metastatic prostate cancer cells interact with bone cells (osteoblasts and osteoclasts), resulting in an imbalance in the bone homeostasis leading to increased activation of osteoblasts over osteoclasts. Our preliminary data indicated a non-tyrosine kinase receptor Neuropilin 2 (NRP2) is expressed in osteoclasts (OC) induced by PCa cells and acts as a negative regulator of osteoclasts' function. We hypothesize that PCa-induced NRP2 expression in OC is necessary for low osteolytic activity and thus favors an osteoblastic lesion in PCa bone metastasis. Methods: Mouse OC precursors were isolated from bone marrow of C57BL/6 mice as well as transgenic CSF1R-cre; NRP2 Flox/Flox inducible mice where addition of 4-hydroxytamoxifen depletes NRP2 specifically from the myeloid cells. Differentiation of OCs was conducted under the conditions of RANKL and M-CSF and in conditioned medium (CM) collected from PCa cell line LNCaP C4-2B (promotes high osteoblastic and low osteoclastic activity) and PC3 (predominantly osteoclastic activity) to mimic the conditions in normal bone and PCa bone metastasis. NRP2 expression at protein and mRNA was evaluated. TRAP staining and pit formation were conducted to confirm the differentiation and function of OCs. Results and Discussion: We observed an increase in NRP2 expression in OCs induced by RANKL and M-CSF and in PC3 and LNCaP C4-2B CM simultaneously. TRAP staining and activity confirmed the differentiation of OCs under these conditions. Interestingly, depletion of NRP2 and treatment either in RANKL and M-CSF or LNCaP C4-2B CM exhibited a drastic increase in osteoclast differentiation and function. mRNA analysis revealed an increase in expression of osteoclastic genes following NRP2 depletion in RANKL and M-CSF and LNCaP C4-2B CM. However, NRP2-depleted OC precursors when treated with PC3 CM showed no change in osteoclastogenesis. These findings advocate a role of NRP2 in inhibiting osteoclastic activity in PCa bone metastasis and that osteolytic PCa evades NRP2 inhibition. Using in vitro and transgenic mice, we will elucidate the molecular mechanisms through which NRP2 regulates osteoclast differentiation and function in normal bone and in LNCaP C4-2B CM. We will also address how PC3 CM-induced OCs escapes the inhibition of NRP2. Together, this approach will elucidate the role of NRP2 axis on OCs in promoting PCa-induced bone metastasis and will aid in determining whether NRP2 axis can be a therapeutic target. Citation Format: Navatha Shree Polavaram, Arup Bag, Samikshan Dutta, Kaustubh Datta. Role of Neuropilin 2 in orchestrating the functions of osteoclasts in promoting prostate cancer bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2117.