Abstract
BackgroundMany human cancer cells express filamin A (FLNA), an actin-binding structural protein that interacts with a diverse set of cell signaling proteins, but little is known about the biological importance of FLNA in tumor development. FLNA is also expressed in endothelial cells, which may be important for tumor angiogenesis. In this study, we defined the impact of targeting Flna in cancer and endothelial cells on the development of tumors in vivo and on the proliferation of fibroblasts in vitro.MethodsFirst, we used a Cre-adenovirus to simultaneously activate the expression of oncogenic K-RAS and inactivate the expression of Flna in the lung and in fibroblasts. Second, we subcutaneously injected mouse fibrosarcoma cells into mice lacking Flna in endothelial cells.ResultsKnockout of Flna significantly reduced K-RAS–induced lung tumor formation and the proliferation of oncogenic K-RAS–expressing fibroblasts, and attenuated the activation of the downstream signaling molecules ERK and AKT. Genetic deletion of endothelial FLNA in mice did not impact cardiovascular development; however, knockout of Flna in endothelial cells reduced subcutaneous fibrosarcoma growth and vascularity within tumors.ConclusionsWe conclude that FLNA is important for lung tumor growth and that endothelial Flna impacts local tumor growth. The data shed new light on the biological importance of FLNA and suggest that targeting this protein might be useful in cancer therapeutics.
Highlights
Many human cancer cells express filamin A (FLNA), an actin-binding structural protein that interacts with a diverse set of cell signaling proteins, but little is known about the biological importance of FLNA in tumor development
The genotype of offspring was confirmed by genomic PCR for floxed” conditional Flna knockout mice (Flnafl) (Figure 1B) and Kras2LSL (Figure 1C)
To visualize the vascular network within fibrosarcomas inoculated into VE-CadCre/+Flnao/fl and VE-CadCre/+Flnao/+ mice, whole-mounted tumor tissues were stained with antibodies detecting endothelial cells (PECAM) and pericytes (NG2) (Figure 5D)
Summary
Mice All mice included in this study had a C57Bl/6 genetic background. Male heterozygous mice containing a floxed stop codon (LSL) before the constitutively active Kras promoter (Kras2LSL) [11,12] were bred with female mice homozygous for a conditional “floxed” allele of Flna (Flnafl/fl) [10]. Isolation of cardiac and pulmonary endothelial cells and RT-PCR Whole mouse hearts or lungs were placed in ice-cold PBS, minced into 1 mm pieces and digested using 100 μg/ml Collagenase type III (Sigma) in Hanks’ balanced salt solution (HBSS, Invitrogen) supplemented with 1% BSA and 100 U/ml DNase at 37°C for 15 min with gentle agitation as described earlier [17]. Rat anti-CD31 (BD Pharmingen) antibody-coated magnetic beads (Dynabeads M-450, sheep anti-Rat IgG, Dynal) were added, and after incubation at 4°C for 30 min with gentle agitation, pulmonary endothelial cells were isolated with a magnetic particle concentrator (MPC, Dynal) and washed three times with HBSS supplemented with 1% BSA. Subcutaneous tumor cell inoculation in mice Five-week-old VE-CadCre/+Flnao/fl and control VECadCre/+Flnao/+ male mice were given subcutaneous injections of 1 × 106 of T241 mouse fibrosarcoma or B16 melanoma cells in the dorsal back region as described earlier [18]. Differences between experiment groups were analyzed for statistical significance (P < 0.05) by two-way ANOVA or Student’s t test
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
