(513b) Invasive Behavior in Osteolytic Metastatic Cancers Is Dictated by Mechanical Cues

Many cancers, such as those of the lung and breast, are known to preferentially metastasize to bone. To invade the mineralized bone matrix, cancer cells change their gene expression. Specifically, PTHrP levels increase in patients with osteolytic metastases. Currently, the initiator of PTHrP expression in osteolytic metastases is unclear. It is, however, established that cells respond to mechanical cues; and that cancer cells exhibit a more invasive phenotype in response to increased substrate rigidity in soft tissue. Despite this, the effect of rigid tissues such as bone on cell behavior has not been investigated. We hypothesize that the extreme rigidity of the bone matrix is the initiator of PTHrP expression in bone metastases. To address this, we examined changes in genes of known importance in bone metastases in response to substrate rigidity in the osteolytic tumor cell lines MDA231 and RWGT2, as well as the non-osteolytic MCF7 line. We found that PTHrP expression increased 6-fold in MDA231 cells and 5-fold in RWGT2 cells in response to bone-like substrates as compared to soft tissue-like substrates. Substrate rigidity did not induce a change in PTHrP expression for MCF7 cells. These qPCR results were verified by radioactive immunoassays for PTHrP. Additionally, Gli2 expression, a known regulator of PTHrP, was increased in response to rigid substrates while expression of osteopontin (OPN), a gene important in primary metastases, was increased on soft substrates in MDA231 and RWGT2 but not MCF7. Interleukin-11 (IL11) expression was unaffected by substrate rigidity in all cell lines. It has previously been shown that substrate rigidity influences cell behavior through mechanotransduction. Inhibiting cell contractility in mammary epithelial cells prevents invasive phenotype development in response to substrate rigidity. Additionally, inhibition of rho-associated kinase (ROCK), which is important in mechanotransduction, prevents bone metastases in breast cancer. We used two inhibitors of mechanotransduction, Y27632 (ROCK-1) and blebbistatin (actinomyosin contractility) to investigate mechanotransduction effects on gene expression in MDA231 and RWGT2. Both inhibitors blocked the upregulation of PTHrP and increased expression of OPN on rigid substrates in both cell lines. Mechanotransduction inhibition did not affect IL11 expression in either cell line. Taken together these data suggest that the rigidity of bone is one of the initiators of PTHrP expression in cancer-induced bone disease.