Skeletal loading regulates breast cancer-associated osteolysis in a loading intensity-dependent fashion

Abstract

Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium (CM) and fluid flow-treated conditioned medium (FFCM) with 0.25 Pa and 1 Pa shear stress. Notably, CM and FFCM at 0.25 Pa induced the mesenchymal-to-epithelial transition (MET), but FFCM at 1 Pa induced the epithelial-to-mesenchymal transition (EMT). This suggested that the effects of fluid flow on conditioned media depend on flow intensity. Fluorescence resonance energy transfer (FRET)-based evaluation of Src activity and vinculin molecular force showed that osteopontin was involved in EMT and MET switching. A mouse model of tumor-induced osteolysis was tested using dynamic tibia loadings of 1, 2, and 5 N. The low 1 N loading suppressed tumor-induced osteolysis, but this beneficial effect was lost and reversed with loads at 2 and 5 N, respectively. Changing the loading intensities in vivo also led to changes in serum TGFβ levels and the composition of tumor-associated volatile organic compounds in the urine. Collectively, this study demonstrated the critical role of intensity-dependent mechanotransduction and osteopontin in tumor-osteocyte communication, indicating that a biophysical factor can tangibly alter the behaviors of tumor cells in the bone microenvironment.

References

[1] Huang C, Ogawa R. Mechanotransduction in bone repair and regeneration. FASEB J., 2010, 24:3625-3632

[2] Goldring SR. The osteocyte: key player in regulating bone turnover. RMD Open, 2015, 1 Suppl. 1

[3] Huang Q et al. Fluid shear stress and tumor metastasis. Am. J. Cancer Res., 2018, 8:763-777

[4] Liu S et al. Fluid shear stress induces epithelial-mesenchymal transition (EMT) in Hep-2 cells. Oncotarget, 2016, 7:32876-32892

[5] Tse JM et al. Mechanical compression drives cancer cells toward invasive phenotype. Proc. Natl Acad. Sci. USA, 2012, 109:911-916

[6] Chen YC, Sosnoski DM, Mastro AM. Breast cancer metastasis to the bone: mechanisms of bone loss. Breast Cancer Res., 2010, 12

[7] Weilbaecher KN, Guise TA, McCauley LK. Cancer to bone: a fatal attraction. Nat. Rev. Cancer, 2011, 11:411-425

[8] Bonecchi R, Locati M, Mantovani A. Chemokines and cancer: a fatal attraction. Cancer Cell, 2011, 19:434-435

[9] Chen A et al. Attraction and compaction of migratory breast cancer cells by bone matrix proteins through tumor-osteocyte interactions. Sci. Rep., 2018, 8

[10] Corsa C et al. The action of discoidin domain receptor 2 in basal tumor cells and stromal cancer associated fibroblasts is critical for breast cancer metastasis. Cell Rep., 2016, 15:2510-2523

[11] Vogel W, Gish GD, Alves F, Pawson T. The discoidin domain receptor tyrosine kinases are activated by collagen. Mol. Cell, 1997, 1:13-23

[12] Pichler K et al. Expression of matrix metalloproteinases in human growth plate chondrocytes is enhanced at high levels of mechanical loading: a possible explanation for overuse injuries in children. Bone Jt. J., 2013, 95:568-573

[13] Chang SH et al. Excessive mechanical loading promotes osteoarthritis through the gremlin-1-NF-kB pathway. Nature Commun., 2019, 10:1442

[14] Patel JB et al. Control of EVI-1 oncogene expression in metastatic breast cancer cells through microRNA miR-22. Oncogene, 2011, 30:1290-1301

[15] Kato Y et al. Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture. J. Bone Min. Res., 2001, 16:1622-1633

[16] Chiechi A et al. Role of TGF-β in breast cancer bone metastases. Adv. Biosci. Biotechnol., 2013, 4:15-30

[17] Ishizawar R, Parsons SJ. C-Src and cooperating partners in human cancer. Cancer Cell, 2004, 6:209-214

[18] McCarthy N. Signalling: SRC and survival. Nat. Rev. Cancer, 2012, 12:80-81

[19] Zhao H et al. The role of osteopontin in the progression of solid organ tumour. Cell Death Dis., 2018, 9

[20] Wan Q et al. Subcellular domain-dependent molecular hierarchy of Src and FAK in mechanotransduction and cytokine signaling. Sci. Rep., 2017, 7

[21] Li F et al. Vinculin force sensor detects tumor-osteocyte interactions. Sci. Rep., 2019, 9

[22] Kuzu OF et al. The role of cholesterol in cancer. Cancer Res, 2016, 76:2063-2070

[23] Woollam M et al. Detection of volatile organic compounds (VOCs) in urine via gas chromatography-mass spectrometry QTOF to differentiate between localized and metastatic models of breast cancer. Sci. Rep., 2019, 21

[24] Grashoff C et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature, 2010, 466:263-266

[25] Ma Y et al. Mechanical regulation of breast cancer migration and apoptosis via direct and indirect osteocyte signaling. J. Cell. Biochem., 2018, 7:5665-5675

[26] Lynch ME et al. In vivo tibial compression decreases osteolysis and tumor formation in a human metastatic breast cancer model. J. Bone Min. Res., 2013, 28:2357-2367

[27] Menon S, Beningo K. Cancer cell invasion is enhanced by applied mechanical stimulation. PLoS ONE., 2011, 6

[28] Ishihara S, Inman DR, Li WJ, Ponik SM, Keely PJ. Mechano-signal transduction in mesenchymal stem cells induces prosaposin secretion to drive the proliferation of breast cancer cells. Cancer Res, 2017, 77:6179-6189

[29] Sottnik JL, Dai J, Zhang H, Campbell B, Keller ET. Tumor-induced pressure in the bone microenvironment causes osteocytes to promote the growth of prostate cancer bone metastasis. Cancer Res, 2015, 75:2151-2158

[30] Fritton SP, Weinbaum S. Fluid and solute transport in bone: flow induced mechanotransduction. Annu Rev. Fluid Mech., 2009, 41:347-374

[31] Danen EH, Yamada KM. Fibronectin, integrins, and growth control. J. Cell Physiol., 2001, 189:1-13

[32] Palmieria D et al. Human anti-nucleolin recombinant immunoagent for cancer therapy. Proc. Natl Acad. Sci. USA, 2015, 112:9418-9423

[33] Satelli A, Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol. Life Sci., 2011, 68:3033-3046

[34] Lakshmi et al. Transforming growth factor β suppresses peroxisome proliferator activated receptor γ expression via both SMAD binding and novel TGFβ inhibitory elements. Biochemical J., 2017, 474:1531-1546

[35] Jia R et al. Osteopontin facilitates tumor metastasis by regulating epithelial-mesenchymal plasticity. Cell Death Dis., 2016, 7

[36] He B, Mirza M, Weber G. An osteopontin splice variant induces anchorage independence in human breast cancer cell. Oncogene, 2006, 25:2192-2202

[37] Wai P, Kuo P. Osteopontin: regulation in tumor metastasis. Cancer Metastasis Rev., 2008, 27:103-118

[38] Wei R, Wong J, Kwok H. Osteopontin– a promising biomarker for cancer therapy. J. Cancer, 2017, 8:2173-2183

[39] Myoui A et al. C-SRC tyrosine kinase activity is associated with tumor colonization in bone and lung in an animal model of human breast cancer metastasis. Cancer Res., 2003, 63:5028-5033

[40] Courter DL, Lomas L, Scatena M, Giachelli CM. Src kinase activity is required for integrin aVb3 mediated activation of nuclear factor kB. J. Biol. Chem., 2005, 280:12145-12151

[41] Mon NN, Senga T, Ito S. Interleukin 1b activates focal adhesion kinase and Src to induce matrix metalloproteinase 9 production and invasion of MCF-7 breast cancer cells. Oncol. Lett., 2017, 13:955-960

[42] Kang KS, Robling AG. New insights into Wnt-Lrp5/6-b-catenin signaling in mechanotransduction. Frot Endocrinol., 2014, 5:246

[43] Liu D et al. Activation of extracellular signal regulated kinase (ERK1/2) by fluid shear is Ca2+ and ATP dependent in MC3T3-E1 osteoblasts. Bone, 2008, 42:644-652

[44] Liu S et al. Osteocyte-driven downregulation of snail restrains effects of Drd2 inhibitors on mammary tumor cells. Cancer Res., 2018, 78:3865-3876

[45] Dodge T et al. Mechanical loading, damping, and load-driven bone formation in mouse tibiae. Bone, 2012, 51:810-818

[46] Li J, Yang G, Siebert T, Shi MF, Yang L. A method of the direct measurement of the true stress-strain curve over a large strain range using multi-camera digital image correlation. Opt. Lasers Eng., 2018, 107:194-201

Funding

U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)(AR52144, R03 CA238555)