Abstract 5999: TBX2 promotes prostate cancer bone-metastatic phenotype through exosomal microRNA-375-3p

Abstract

Abstract Introduction: Bone is the preferred site of metastasis in about 80% of advanced prostate cancer (PCa) patients. The molecular mechanisms that drive bone metastatic PCa are not well understood. Recent reports have increasingly shed light on the role of cancer-cell-derived extracellular vesicles (exosomes) in driving the metastatic cascade including priming the secondary site for metastatic manifestation. We have previously reported that TBX2 promotes multiple facets of the PCa bone metastatic cascade via a cell-intrinsic manner. In this study, we sought to determine if the bone metastasis promoted by TBX2 is driven by microRNAs (miRs) in an exosome-mediated paracrine manner. Methods: Two converse approaches were utilized for the genetic modulation of TBX2: a) TBX2 was blocked in the metastatic PC3 and C4-2B human PCa cells using a dominant negative (DN) approach, and b) TBX2 was over-expressed in the low-TBX2 expressing LNCaP human PCa cells. Exosomes isolated from TBX2-modulated human PCa cells were added to MC3T3 pre-osteoblastic cells or mouse calvaria bone cells to examine the differential effects of TBX2 modulation on osteoblast or osteoclast differentiation. Next, unbiased next generation sequencing (NGS) was performed to identify the differential miR expression in exosomes derived from the TBX2-blocked PC3 cells. Results: Our results demonstrate that compared with control exosomes, exosomes derived from TBX2-blocked PC3 cells slowed the proliferation and migration/wound healing when added to 22Rv1 cells. Further, compared with control exosomes, exosomes derived from TBX2-blocked C4-2B cells resulted in reduced osteoblast differentiation when added to: a) MC3T3 pre-osteoblastic cells, or b) mouse calvaria bone cells. Along similar lines, when compared with the control exosomes, exosomes derived from TBX2-blocked PC3 and C4-2B cells resulted in reduced osteoclast differentiation when added to bone-marrow derived cells. These results were confirmed by q RT-PCR analysis of the osteoblastic/osteolytic bone markers. In order to identify exosomal miRs downstream of TBX2 signaling, NGS analysis of exosomal miRs obtained from TBX2-blocked PC3 human PCa cells identified miR-375-3p as the top-most differentially-expressed (over-expressed miR) when compared with control exosomes. In silico analysis revealed that miR-375-3p could potentially bind and target the 3’UTR of RBPJ, an established driver of PCa bone metastasis. In agreement, q RT-PCR analysis revealed that rescue of miR-375-3p in TBX2 modulated human PCa cells rescued RBPJ expression. Conclusions: Our studies have identified the exosome-mediated TBX2/miR-375-3p/RBPJ signaling axis as a promoter of the bone-metastatic phenotype in human PCa. Citation Format: Girijesh Kumar Patel, Sayanika Dutta, Hamed Khedmatgozar, Manisha Tripathi, Srinivas Nandana. TBX2 promotes prostate cancer bone-metastatic phenotype through exosomal microRNA-375-3p [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5999.