21 research outputs found

    Altering adsorbed proteins or cellular gene expression in bone-metastatic cancer cells affects PTHrP and Gli2 without altering cell growth

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    AbstractThe contents of this data in brief are related to the article titled “Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II”. In this DIB we will present our supplemental data investigating Integrin expression, attachment of cells to various adhesion molecules, and changes in gene expression in multiple cancer cell lines. Since the interactions of Integrins with adsorbed matrix proteins are thought to affect the ability of cancer cells to interact with their underlying substrates, we examined the expression of Integrin β1, β3, and β5 in response to matrix rigidity. We found that only Iβ3 increased with increasing substrate modulus. While it was shown that fibronectin greatly affects the expression of tumor-produced factors associated with bone destruction (parathyroid hormone-related protein, PTHrP, and Gli2), poly-l-lysine, vitronectin and type I collagen were also analyzed as potential matrix proteins. Each of the proteins was independently adsorbed on both rigid and compliant polyurethane films which were subsequently used to culture cancer cells. Poly-l-lysine, vitronectin and type I collagen all had negligible effects on PTHrP or Gli2 expression, but fibronectin was shown to have a dose dependent effect. Finally, altering the expression of Iβ3 demonstrated that it is required for tumor cells to respond to the rigidity of the matrix, but does not affect other cell growth or viability. Together these data support the data presented in our manuscript to show that the rigidity of bone drives Integrinβ3/TGF-β crosstalk, leading to increased expression of Gli2 and PTHrP

    Matrix Rigidity Induces Osteolytic Gene Expression of Metastatic Breast Cancer Cells

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    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

    Expression and secretion of PTHrP by osteolytic, metastatic tumor cells increases with increasing substrate modulus.

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    <p>PTHrP mRNA normalized by 18S as measured by qPCR and secreted PTHrP as measured by IRMA for (A) MDA-MB-231 , (B) RWGT2, and (C) MCF-7 cells on substrates of increasing elastic modulus. *,# = p<0.05; **,## = p<0.01; ***,### = p<0.005 compared to 0.00045MPa value. Changes in MCF-7 cells were not significant. (D)–(F) Morphology of MDA-MB-231 cells cultured on substrates of varying elastic modulus 4 hours post-attachment. Nuclei were stained using DAPI (blue) and cells were visualized with both visible light and GFP (green). Similar effects of the modulus on cell morphology were observed for RWGT2 cells, while no effects were observed for MCF-7 cells.</p

    Expression of Gli2 and by MDA-MB-231 and RWGT2 cells increases with increasing substrate modulus.

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    <p>Expression of Gli2 mRNA normalized by 18S as measured by qPCR for (A) MDA-MB-231 and (B) RWGT2 cells on substrates of varying elastic modulus. Gli2 signaling increased 25-fold as the modulus increased from 3.3 to 1700 MPa (data not shown). *,# = p<0.05; **,## = p<0.01; ***,### = p<0.005 compared to 0.00045MPa value.</p

    TGF-ß mediates the response of MDA-MB-231 cells to substrate rigidity.

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    <p>(A) PTHrP gene expression increases significantly for MDA-MB-231 cells transfected with the plasmid control (MDA-pc), while MDA-MB-231 cells transfected to express a dominant negative form of the TGF-ß Type II receptor (MDA-TβRIIcyt ) and MCF-7 cells that do not express the TGF-ß Type II receptor show no increase in expression with rigidity. (B) TGF-ß signaling was measured in MDA-MB-231 cells transfected with the 3TP-Lux TGF-ß reporter construct. This showed an increase in TGF-ß signaling when cells are grown on rigid substrates. (C) TGF-ß1 mRNA expression by MDA-MB-231 cells increased on more rigid substrates. *,# = p<0.05; **,## = p<0.01; ***,### = p<0.005 compared to 0.00045MPa value.</p

    Mechanotransduction signals are regulated by values of the substrate modulus in the MPa range.

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    <p>(A) Western blot analysis of myosin light chain phosphorylation shows higher pMLC expression by MDA-MB-231 cells seeded on more rigid (1700 MPa) relative to softer (3.3 MPa) PUR substrates. (B) Pharmacological inhibition of actomyosin contractility (blebbistatin) and ROCK (Y-27632) decreases PTHrP gene expression and protein secretion in MDA-MB-231 cells. Similar patterns were observed for RWGT2 cells (data not shown). The optimal doses of blebbistatin and Y-27632 were identified to be 50 uM and 20 uM, respectively through dose-response experiments (data not shown). (C) PTHrP is over-expressed and does not increase with rigidity in MDA-MB-231 cells genetically modified to express a constitutively active form of ROCK (MDA-4 cells). Similarly, PTHrP expression does not increase with rigidity in MDA-MB-231 cells genetically modified to express a dominant negative form of ROCK (MDA-KD4 cells). MDA cells transfected with a plasmid control (MDA-pc) show significant increases in PTHrP expression with rigidity. *,# = p<0.05; **,## = p<0.01; ***,### = p<0.005.</p

    Schematic of materials synthesis and characterization.

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    <p>(A) Synthesis of PUR networks from an LDI quasi-prepolymer (red) and a poly(ε-caprolactone-co-glycolide) triol with molecular weight M<sub>n</sub> = 3<i>w</i>, where <i>w</i> is the equivalent weight (g eq<sup>−1</sup>). (B) Experimental values of the elastic modulus <i>E</i> of PUR networks as a function of equivalent weight <i>w</i>. (C) Physical characteristics of polymer networks as calculated from rubber elasticity theory. E<sub>m</sub> and G<sub>m</sub> represents measured elastic and shear moduli, is the fraction of polymer in the swollen mass, M<sub>c</sub> is the calculated molecular weight between cross-links and E<sub>c</sub> and G<sub>c</sub> are the calculated elastic and shear moduli. (D) Schemes for providing a uniform surface concentration of fibronectin (Fn) for polyurethane networks and polyacrylamide gels.</p

    Inhibition of mechanotransduction and ROCK suppresses PTHrP expression induced by exogenous TGF-ß.

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    <p>Treatment with blebbistatin inhibits expression of (A) Gli2 and (B) PTHrP by MDA-MB-231 cells cultured on TCPS. Treatment with 5 ng/ml exogenous TGF-ß increases PTHrP and Gli2 expression, and treatment with both TGF-ß and blebbistatin reduces expression to levels observed for blebbistatin alone. Similarly, treatment of MDA-MB-231 cells with the ROCK inhibitor Y-27632 inhibits (C) Gli2 and (D) PTHrP expression. In the presence of both exogenous TGF-ß and Y-27632, both PTHrP and Gli2 expression are significantly reduced, but PTHrP expression is only partially inhibited relative to treatment with Y-27632 alone. Expression of (E) Gli2 and (F) PTHrP in MDA-MB-231 cells genetically modified to express a dominant negative form of ROCK (MDA-MB-231 KD4) is significantly lower than that measured for plasmid control MDA-MB-231 cells. Treatment with exogenous TGF-ß does not significantly increase Gli2 or PTHrP expression in KD4 cells. *,# = p<0.05; **,## = p<0.01; ***,### = p<0.005.</p

    Cabozantinib Inhibits Growth of Androgen-Sensitive and Castration-Resistant Prostate Cancer and Affects Bone Remodeling

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    <div><p>Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. <i>In vivo</i>, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease. </p> </div

    Expression of MET, P-MET, and VEGFR2 in primary and metastatic patient samples.

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    <p>IHC and analyses were performed as described in the Methods section. Graphical profiles illustrating distributions of staining intensity were constructed by calculating simple averages across all non-missing sections in each staining category. In each site, the mean staining index is marked by a filled orange circle and orange bars represent 95% CIs. Representative examples of staining are shown for each protein. A. MET is strongly expressed in both primary and metastatic PCa, though it is significantly increased in BM and decreased in soft tissue metastases vs. primary PCa. B. P-MET levels are higher in BM, LN and other soft tissue metastases, while no alteration was detected in liver metastases when compared to primary PCa. C. VEGFR2 expression is significantly increased across PCa metastatic lesions as compared to primary PCa. Images were taken at 400 x magnification.</p
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