Mechanical suppression of breast cancer cell invasion and paracrine signaling to osteoclasts requires nucleo-cytoskeletal connectivity

Abstract

Exercise benefits the musculoskeletal system and reduces the effects of cancer. The effects of exercise are multifactorial, where metabolic changes and tissue adaptation influence outcomes. Mechanical signals, a principal component of exercise, are anabolic to the musculoskeletal system and restrict cancer progression. We examined the mechanisms through which cancer cells sense and respond to low-magnitude mechanical signals introduced in the form of vibration. Low-magnitude, high-frequency vibration was applied to human breast cancer cells in the form of low-intensity vibration (LIV). LIV decreased matrix invasion and impaired secretion of osteolytic factors PTHLH, IL-11, and RANKL. Furthermore, paracrine signals from mechanically stimulated cancer cells, reduced osteoclast differentiation and resorptive capacity. Disconnecting the nucleus by knockdown of SUN1 and SUN2 impaired LIV-mediated suppression of invasion and osteolytic factor secretion. LIV increased cell stiffness; an effect dependent on the LINC complex. These data show that mechanical vibration reduces the metastatic potential of human breast cancer cells, where the nucleus serves as a mechanosensory apparatus to alter cell structure and intercellular signaling.

References

[1] Sattelmair J et al. Dose response between physical activity and risk of coronary heart disease: a meta-analysis. Circulation, 2011, 124:789-795

[2] Warden SJ, Thompson WR. Become one with the force: optimising mechanotherapy through an understanding of mechanobiology. Br. J. Sports Med., 2017, 51:989-990

[3] Thune I, Furberg AS. Physical activity and cancer risk: dose-response and cancer, all sites and site-specific. Med. Sci. Sports Exerc., 2001, 33:S530-S550 discussion S609-510

[4] Moore SC et al. Association of leisure-time physical activity with risk of 26 types of cancer in 1.44 million adults. JAMA Intern. Med., 2016, 176:816-825

[5] Friedenreich CM, Orenstein MR. Physical activity and cancer prevention: etiologic evidence and biological mechanisms. J. Nutr., 2002, 132:3456S-3464S

[6] Li Y et al. Association between physical activity and all cancer mortality: Dose-response meta-analysis of cohort studies. Int J. Cancer, 2016, 138:818-832

[7] Pedersen L et al. Voluntary running suppresses tumor growth through epinephrine- and IL-6-dependent NK cell mobilization and redistribution. Cell Metab., 2016, 23:554-562

[8] Zhao M, Veeranki SP, Li S, Steffen LM, Xi B. Beneficial associations of low and large doses of leisure time physical activity with all-cause, cardiovascular disease and cancer mortality: a national cohort study of 88,140 US adults. Br. J. Sports Med, 2019, 53:1405-1411

[9] Frank LL et al. Effects of exercise on metabolic risk variables in overweight postmenopausal women: a randomized clinical trial. Obes. Res, 2005, 13:615-625

[10] Schmid D, Leitzmann MF. Association between physical activity and mortality among breast cancer and colorectal cancer survivors: a systematic review and meta-analysis. Ann. Oncol., 2014, 25:1293-1311

[11] Thompson WR, Yen SS, Rubin J. Vibration therapy: clinical applications in bone. Curr. Opin. Endocrinol., Diabetes, Obes., 2014, 21:447-453

[12] Rubin C, Turner AS, Bain S, Mallinckrodt C, McLeod K. Anabolism. Low mechanical signals strengthen long bones. Nature, 2001, 412:603-604

[13] Pongkitwitoon S, Uzer G, Rubin J, Judex S. Cytoskeletal configuration modulates mechanically induced changes in mesenchymal stem cell osteogenesis, morphology, and stiffness. Sci. Rep., 2016, 6

[14] Thompson WR et al. Mechanically activated Fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells. Stem Cells, 2013, 31:2528-2537

[15] Uzer G et al. Cell mechanosensitivity to extremely low-magnitude signals is enabled by a LINCed nucleus. Stem Cells, 2015, 33:2063-2076

[16] Patel VS et al. Incorporating refractory period in mechanical stimulation mitigates obesity-induced adipose tissue dysfunction in adult mice. Obesity (Silver Spring), 2017, 25:1745-1753

[17] Mogil RJ et al. Effect of low-magnitude, high-frequency mechanical stimulationon BMD among young childhood cancer survivors: a randomized clinical trial. JAMA Oncol., 2016, 2:908-914

[18] Mohammadi H, Sahai E. Mechanisms and impact of altered tumour mechanics. Nat. Cell Biol., 2018, 20:766-774

[19] Zhou ZL et al. Actin cytoskeleton stiffness grades metastatic potential of ovarian carcinoma Hey A8 cells via nanoindentation mapping. J. Biomech., 2017, 60:219-226

[20] Matsumoto A et al. Global loss of a nuclear lamina component, lamin A/C, and LINC complex components SUN1, SUN2, and nesprin-2 in breast cancer. Cancer Med., 2015, 4:1547-1557

[21] Lien SC et al. Mechanical regulation of cancer cell apoptosis and autophagy: roles of bone morphogenetic protein receptor, Smad1/5, and p38 MAPK. Biochim. Biophys. Acta, 2013, 1833:3124-3133

[22] Duffy MJ, Maguire TM, Hill A, McDermott E, O’Higgins N. Metalloproteinases: role in breast carcinogenesis, invasion and metastasis. Breast Cancer Res., 2000, 2:252-257

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

[24] Pagnotti GM et al. Low magnitude mechanical signals mitigate osteopenia without compromising longevity in an aged murine model of spontaneous granulosa cell ovarian cancer. Bone, 2012, 51:570-577

[25] Pagnotti GM et al. Low intensity vibration mitigates tumor progression and protects bone quantity and quality in a murine model of myeloma. Bone, 2016, 90:69-79

[26] Negishi-Koga T, Takayanagi H. Ca2⁺-NFATc1 signaling is an essential axis of osteoclast differentiation. Immunol. Rev., 2009, 231:241-256

[27] Guise, T. A. & Chirgwin, J. M. Transforming growth factor-beta in osteolytic breast cancer bone metastases. Clin. Orthop. Relat. Res. (415 Suppl), S32–S38 (2003).

[28] Sen B et al. Mechanical signal influence on mesenchymal stem cell fate is enhanced by incorporation of refractory periods into the loading regimen. J. Biomech., 2011, 44:593-599

[29] Sato K et al. RANKL synthesized by both stromal cells and cancer cells plays a crucial role in osteoclastic bone resorption induced by oral cancer. Am. J. Pathol., 2013, 182:1890-1899

[30] Cross SS et al. Expression of receptor activator of nuclear factor kappabeta ligand (RANKL) and tumour necrosis factor related, apoptosis inducing ligand (TRAIL) in breast cancer, and their relations with osteoprotegerin, oestrogen receptor, and clinicopathological variables. J. Clin. Pathol., 2006, 59:716-720

[31] Van Poznak C et al. Expression of osteoprotegerin (OPG), TNF related apoptosis inducing ligand (TRAIL), and receptor activator of nuclear factor kappaB ligand (RANKL) in human breast tumours. J. Clin. Pathol., 2006, 59:56-63

[32] Uzer G, Pongkitwitoon S, Ete Chan M, Judex S. Vibration induced osteogenic commitment of mesenchymal stem cells is enhanced by cytoskeletal remodeling but not fluid shear. J. Biomech., 2013, 46:2296-2302

[33] Uzer G et al. Gap junctional communication in osteocytes is amplified by low intensity vibrations in vitro. PLoS ONE, 2014, 9

[34] Uzer G et al. Separating fluid shear stress from acceleration during vibrations in vitro: identification of mechanical signals modulating the cellular response. Cell. Mol. Bioeng., 2012, 5:266-276

[35] Lei K et al. SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice. Proc. Natl Acad. Sci. USA, 2009, 106:10207-10212

[36] Chen CY et al. Accumulation of the inner nuclear envelope protein sun1 is pathogenic in progeric and dystrophic laminopathies. Cell, 2012, 149:565-577

[37] Chen Z-J et al. Dysregulated interactions between lamin A and SUN1 induce abnormalities in the nuclear envelope and endoplasmic reticulum in progeric laminopathies. J. Cell Sci., 2014, 127:1792-1804

[38] Lombardi ML et al. The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton. J. Biol. Chem., 2011, 286:26743-26753

[39] Brabek J, Mierke CT, Rosel D, Vesely P, Fabry B. The role of the tissue microenvironment in the regulation of cancer cell motility and invasion. Cell Commun. Signal., 2010, 8:22

[40] Provenzano PP, Keely PJ. Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling. J. Cell Sci., 2011, 124:1195-1205

[41] Katira P, Zaman MH, Bonnecaze RT. How changes in cell mechanical properties induce cancerous behavior. Phys. Rev. Lett., 2012, 108:028103

[42] Wirtz D, Konstantopoulos K, Searson PC. The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nat. Rev. Cancer, 2011, 11:512-522

[43] Thompson WR, Scott A, Loghmani MT, Ward SR, Warden SJ. Understanding mechanobiology: physical therapists as a force in mechanotherapy and musculoskeletal regenerative rehabilitation. Phys. Ther., 2016, 96:560-569

[44] Keller BV, Davis ML, Thompson WR, Dahners LE, Weinhold PS. Varying whole body vibration amplitude differentially affects tendon and ligament structural and material properties. J. Biomech., 2013, 46:1496-1500

[45] Thompson, W. R., Keller, B. V., Davis, M. L., Dahners, L. E. & Weinhold, P. S. Low-magnitude, high-frequency vibration fails to accelerate ligament healing but stimulates collagen synthesis in the achilles tendon. Orthop. J. Sports Med. 3, 2325967115585783 (2015).

[46] Sen B et al. mTORC2 regulates mechanically induced cytoskeletal reorganization and lineage selection in marrow-derived mesenchymal stem cells. J. Bone Miner. Res., 2014, 29:78-89

[47] Olcum M, Ozcivici E. Daily application of low magnitude mechanical stimulus inhibits the growth of MDA-MB-231 breast cancer cells in vitro. Cancer Cell Int., 2014, 14

[48] Chua HL, Serov Y, Brahmi Z. Regulation of FasL expression in natural killer cells. Hum. Immunol., 2004, 65:317-327

[49] Bachmeier BE, Nerlich AG, Lichtinghagen R, Sommerhoff CP. Matrix metalloproteinases (MMPs) in breast cancer cell lines of different tumorigenicity. Anticancer Res, 2001, 21:3821-3828

[50] Kohrmann A, Kammerer U, Kapp M, Dietl J, Anacker J. Expression of matrix metalloproteinases (MMPs) in primary human breast cancer and breast cancer cell lines: New findings and review of the literature. BMC Cancer, 2009, 9

[51] Brown SA, Guise TA. Cancer-associated bone disease. Curr. Osteoporos. Rep., 2007, 5:120-127

[52] Guise TA et al. Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. J. Clin. Invest, 1996, 98:1544-1549

[53] McCoy EM, Hong H, Pruitt HC, Feng X. IL-11 produced by breast cancer cells augments osteoclastogenesis by sustaining the pool of osteoclast progenitor cells. BMC Cancer, 2013, 13

[54] Shimo T et al. Pathogenic role of connective tissue growth factor (CTGF/CCN2) in osteolytic metastasis of breast cancer. J. Bone Miner. Res., 2006, 21:1045-1059

[55] Canon JR et al. Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis. Clin. Exp. Metastasis, 2008, 25:119-129

[56] Esposito M, Guise T, Kang Y. The biology of bone metastasis. Cold Spring Harb. Perspect. Med, 2018, 8:a031252

[57] Kulkarni RN, Voglewede PA, Liu D. Mechanical vibration inhibits osteoclast formation by reducing DC-STAMP receptor expression in osteoclast precursor cells. Bone, 2013, 57:493-498

[58] Sakamoto M et al. Vibration enhances osteoclastogenesis by inducing RANKL expression via NF-kappaB signaling in osteocytes. Bone, 2019, 123:56-66

[59] Mahoney L, Csima A. Clinical screening for breast cancer. N. Engl. J. Med, 1982, 306:546

[60] Janmey PA, Miller RT. Mechanisms of mechanical signaling in development and disease. J. Cell Sci., 2011, 124:9-18

[61] Cross SE et al. AFM-based analysis of human metastatic cancer cells. Nanotechnology, 2008, 19:384003

[62] Swaminathan V et al. Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines. Cancer Res., 2011, 71:5075-5080

[63] Maeda M et al. ARHGAP18, a GTPase-activating protein for RhoA, controls cell shape, spreading, and motility. Mol. Biol. Cell, 2011, 22:3840-3852

[64] Roduit C et al. Stiffness tomography by atomic force microscopy. Biophys. J., 2009, 97:674-677

[65] Thompson WR et al. Osteocyte specific responses to soluble and mechanical stimuli in a stem cell derived culture model. Sci. Rep., 2015, 5

[66] Thompson WR et al. LARG GEF and ARHGAP18 orchestrate RhoA activity to control mesenchymal stem cell lineage. Bone, 2018, 107:172-180

Funding

Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)(AR068332)

U.S. Department of Defense (United States Department of Defense)(BC150678)