Notch3 promotes prostate cancer-induced bone lesion development via MMP-3

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Prostate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. Elevated Notch activity is associated with high-grade disease and metastasis. To address how Notch affects prostate cancer bone lesions, we manipulated Notch expression in mouse tibia xenografts and monitored tumor growth, lesion phenotype, and the bone microenvironment. Prostate cancer cell lines that induce mixed osteoblastic lesions in bone expressed 5–6 times more Notch3, than tumor cells that produce osteolytic lesions. Expression of active Notch3 (NICD3) in osteolytic tumors reduced osteolytic lesion area and enhanced osteoblastogenesis, while loss of Notch3 in osteoblastic tumors enhanced osteolytic lesion area and decreased osteoblastogensis. This was accompanied by a respective decrease and increase in the number of active osteoclasts and osteoblasts at the tumor–bone interface, without any effect on tumor proliferation. Conditioned medium from NICD3-expressing cells enhanced osteoblast differentiation and proliferation in vitro, while simultaneously inhibiting osteoclastogenesis. MMP-3 was specifically elevated and secreted by NICD3-expressing tumors, and inhibition of MMP-3 rescued the NICD3-induced osteoblastic phenotypes. Clinical osteoblastic bone metastasis samples had higher levels of Notch3 and MMP-3 compared with patient matched visceral metastases or osteolytic metastasis samples. We identified a Notch3–MMP-3 axis in human prostate cancer bone metastases that contributes to osteoblastic lesion formation by blocking osteoclast differentiation, while also contributing to osteoblastogenesis. These studies define a new role for Notch3 in manipulating the tumor microenvironment in bone metastases.

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Funding for this project was provided by a DOD Postdoctoral Fellowship W81XWH-16-1-0136 (SSG), DOD W81XWH-14-1-0479 (SBF, LT, CKM), University of Arizona Cancer Center (UACC) NIH/NCI P30CA023074 (KS, CKM), the Van Andel Research Institute (VARI) (GH, XL), and the University of Arizona (CKM). The TMA obtained from the Prostate Cancer Biorepository Network (PCBN) was supported by funds from the DOD Prostate Cancer Research Program: W81XWH-14-2-0182, W81XWH-14-2-0183, W81XWH-14-2-0185, W81XWH-14-2-0186, and W81XWH-15-2-0062. We wish to thank the following: Dr Denise Roe, Director of Biostatistics Shared Resource at UACC for biostatistics support; Dr Chunyan Liu at Ventana Medical Services, Oro Valley, AZ for her pathology services; Dr Colm Morrissey in the Department of Urology, University of Washington, Seattle WA, for his assistance with providing pathological assessment of bone metastasis lesions in the PCBN TMA. Alexandra VanderArk, Veronique Schulz, and Ghada Y.T Mohsen at Van Andel Research Institute for their technical expertise; Zachary Madaj for statistical analysis expertise; Lisa Turner, Kristin Feenstra, and Bree Berghuis of the VARI Pathology and Biorepository Core for their pathology and Aperio expertise; Su Yanli and Staff of the VARI Vivarium and Transgenics core for technical assistance with animal experiments; David Nadziejka for technical editing of the paper; Jeanie Wedberg and Michelle Minard at the Van Andel Institute and David Alvarado at University of Arizona for their administrative support.

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