Matrix Rigidity Induces Osteolytic Gene Expression of Metastatic Breast Cancer Cells

Abstract

Nearly 70% of breast cancer patients with advanced disease will develop bone metastases. Once established in bone, tumor cells produce factors that cause changes in normal bone remodeling, such as parathyroid hormone-related protein (PTHrP). While enhanced expression of PTHrP is known to stimulate osteoclasts to resorb bone, the environmental factors driving tumor cells to express PTHrP in the early stages of development of metastatic bone disease are unknown. In this study, we have shown that tumor cells known to metastasize to bone respond to 2D substrates with rigidities comparable to that of the bone microenvironment by increasing expression and production of PTHrP. The cellular response is regulated by Rho-dependent actomyosin contractility mediated by TGF-ß signaling. Inhibition of Rho-associated kinase (ROCK) using both pharmacological and genetic approaches decreased PTHrP expression. Furthermore, cells expressing a dominant negative form of the TGF-ß receptor did not respond to substrate rigidity, and inhibition of ROCK decreased PTHrP expression induced by exogenous TGF-ß. These observations suggest a role for the differential rigidity of the mineralized bone microenvironment in early stages of tumor-induced osteolysis, which is especially important in metastatic cancer since many cancers (such as those of the breast and lung) preferentially metastasize to bone.

Contribution of Bone Tissue Modulus to Breast Cancer Metastasis to Bone

“…1a, while mechanical stretch differs from cellular attachment to a rigid matrix, the mechanotransduction pathway is activated in both cases through the action of external forces on the cell. Thus, recent observations showing that Gli2 and PTHrP expression are upregulated on rigid matrices through both mechanically transduced and TGF-β signaling [4] are consistent with previous studies reporting increased PTHrP expression in response to mechanical stretch [31,32]. Interestingly, PTHrP and Gli2 expression were increased on rigid (compared to compliant) 2D substrates, but the effect was blocked when TGF-β or mechanically transduced signaling was inhibited.…”

“…The rigidity of the extracellular matrix is known to regulate tumor cell invasiveness at the primary site [51][52][53], and a recent in vitro study suggests that the rigid mineralized bone matrix contributes to the initiation of osteolytic gene expression by tumor cells when they become established in bone [46]. In this study, Gli2 and PTHrP expression increased when breast cancer cells were cultured on 2D substrates with elastic moduli exceeding that of the basement membrane (∼10,000 nN/μm2) [4,46]. Considering that all the substrates were coated with the same surface concentration of FN, differences in gene expression could not be attributed to differences in composition.…”

“…One of the major differences between bone and soft tissue is the rigidity of the mineralized extracellular matrix, which is approximately 1,000,000 times more rigid than normal breast tissue. The rigid bone matrix not only causes physical constraints on tumor growth, but it also alters both tumor cell gene expression and tumor cell response to growth factors [4]. While the rigid mineralized extracellular matrix is a major factor in the regulation of tumor cell gene expression, other characteristics of the bone microenvironment, such as stromal cells, may also mediate the behavior of the tumor cells when they become established in bone.…”

“…Overexpression of α v β 3 in breast cancer cells has been associated with increased levels of bone metastasis, tumor burden, and subsequent osteolysis [36,[42][43][44][45]. Considering the role of FN in tumor-induced bone disease, in vitro studies investigating the role of matrix rigidity on osteolytic gene expression have utilized FN as the matrix protein supporting binding of the tumor cells to the matrix [4,46]. Alternatively, tumor cells may bind to the lining cells directly in contact with the bone through the bone stromal ligand osteopontin [47], which is required for tumor cell colonization to bone [48].…”

“…Considering that all the substrates were coated with the same surface concentration of FN, differences in gene expression could not be attributed to differences in composition. Since ROCK [4] and α v β 3 integrin are required for the increased osteolytic gene expression on rigid matrices, in a later section we review the role of integrins and ROCK in regulating the ability of the cell to sense the rigidity of the matrix.…”