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

The 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.


a b s t r a c t
The 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 Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/dib films which were subsequently used to culture cancer cells. Poly-llysine, 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.   1. Data

Gene expression changes in response to rigidity
The bone-metastatic breast cancer cell line MDA-MB-231, bone-metastatic lung cancer cell line RWGT2, and the bone-metastatic prostate cancer cell line PC3 were used to test the effects of matrix rigidity on gene expression of the bone destructive genes, PTHrP and Gli2. Integrin β3 was either over expressed by an Iβ3 construct or inhibited in the MDA-MB-231 cell line by genetic inhibition using a shRNA construct or pharmacological inhibition with LM609 or Cilengitide [1].
In addition to gene expression changes, Gli2 protein increases with respect to matrix rigidity (Fig. 1A). While Integrin gene expression changes correlate with rigidity [2], in the bone-metastatic MDA-MB-231 cells, Iβ1 and Iβ5 do not respond to matrix rigidity while Iβ3 increases with increasing substrate modulus (Fig. 1B D). Thus the cells are interacting with the matrix primarily through Iβ3.

Effects of adhesion Molecules on bone metastatic gene expression
Expression of Iβ3, Gli2, and PTHrP by MDA-MB-231 cells cultured on 2D compliant and rigid films and normalized to values measured for compliant films is shown in Fig. 2. With the exception of PTHrP expression on films treated with poly(L-lysine), no significant differences were observed between rigid and compliant films for poly(L-lysine), vitronectin, or type I collagen.

Molecular modulation of Iβ3 expression
OE β3 cells showed increased PTHrP expression compared to mock-transfected control cells (Fig. 4A). Additionally, when cultured on rigid and compliant PUR films, the OE β3 cells showed no statistical difference in PTHrP gene expression (Fig. 4B). Thus, the OE β3 and shβ3 cells were utilized as model cell systems with high and low Integrin expression, respectively (Figs. 4C and D).

Effects of matrix rigidity on TGF-β RII expression
Expression of TGF-β RII by MCF-7 (negative control), MDA-MB-231, and RWGT2 cells was measured by qPCR on rigid and compliant substrates. As anticipated, expression of TGF-β RII was significantly lower in MCF-7 cells compared to MDA-MB-231 and RWGT2 cells. There were no significant differences in expression as a function of matrix rigidity for any of the three cell types (Fig. 5A).

Effects of Fn concentration on physical interactions between Iβ3 and TGF-βRII
The effects of Fn concentration on the FRET signal in MDA-MB-231 cells was measured for Fn concentrations ranging from 0-50 μg/ml. The FRET signal was significantly higher on rigid substrates at all Fn concentrations (Fig. 5B).

Exogenous TGF-β stimulation of PTHrP and Gli2 is Iβ3-dependent
To investigate the role of TGF-β signaling in Iβ3 regulation of PTHrP and Gli2, RWGT2 or PC3 cells were treated with the Integrin inhibitory antibody LM609 and given exogenous TGF-β. PTHrP and Gli2 were analyzed by qPCR. TGF-β stimulates PTHrP and Gli2 mRNA expression in RWGT2 (black) or PC3 (white) cells, but is unable to stimulate expression when Iβ3 is inhibited with LM609 ( Fig. 6A and B) suggesting that both TGF-β and Iβ3 are required for regulating PTHrP and Gli2.

Effects of silencing Iβ3 on growth of and bone metastatic gene expression by MDA-MB-231 tumor cells
As shown in Fig. 7A, shβ3 cells exhibited a growth rate similar to that of the mock-transfected control. With the exception of Osteopontin (Opn), expression of bone metastatic genes by shβ3 cells was similar to that by mock-transfected control cells (Fig. 7B).

Western blot analysis
Cells were harvested 24 h after seeding on PUR substrates in a radioimmunoprecipitation buffer containing a cocktail of protease and phosphatase inhibitors (Pierce). Equal protein concentrations were prepared for loading with NuPAGE sample buffer (Life Technologies) and separated on a 10% SDS-PAGE gel (BioRad). Proteins were transferred to a PVDF membrane and blocked with 5% BSA in TBS containing 0.1% Tween-20 for 1 h at room temperature, followed by incubation with anti-Gli2 antibody (1:1000, SantaCruz) overnight at 4 1C. After washing, membranes were blotted with antigoat IgG (1:2000, SantaCruz), and bands were detected by enhanced chemiluminescence using an In-Vivo MS FX Pro (Bruker). Membranes were then stripped and reprobed using an antibody for β-actin (1:5000, Sigma) as a loading control. Analysis was performed using Image J software.

Quantitative real-time PCR
To measure changes in gene expression, mRNA reverse transcription was carried out using the qScript cDNA synthesis kit (Quanta, VWR) per manufacturer's instructions. Briefly, cells were harvested with trypsin after 24 h in culture and total RNA was extracted using the RNeasy Mini Kit (Qiagen). The qScript cDNA supermix was used to synthesize cDNA using 1 mg total RNA. The expression of PTHrP, Gli2, Iβ1, Iβ3, Iβ5, and TGF-β RII was measured in triplicate by quantitative qRT-PCR using validated TaqMan primers with the 7500 Real-Time PCR System (Applied Biosciences) using the following cycling conditions: 95 1C for 15 s and 60 1C for 1 min, preceded by an initial incubation period of 95 1C for 10 min. Quantification was performed using the absolute quantitative for human cells method using 18S as an internal control.

Adsorption of adhesion molecules
In addition to fibronection (Fn), compliant and rigid PUR films were incubated with a 4 mg/mL solution of vitronectin, type I collagen, or poly(L-lysine) in PBS overnight at 4 1C to mediate cell adhesion. MDA-MB-231 cells were cultured on the films for 48 h. Expression of Iβ3, Gli2, and PTHrP was measured by qPCR relative to the control 18S.