Pulmonary Adenocarcinoma In Situ and Minimally Invasive Adenocarcinomas in European Patients Have Less KRAS and More EGFR Mutations Compared to Advanced Adenocarcinomas.

Pulmonary adenocarcinoma (ADC) is a very diverse disease, both genetically and histologically, which displays extensive intratumor heterogeneity with numerous acquired mutations. ADC is the most common type of lung cancer and is believed to arise from adenocarcinoma in situ (AIS) which then progresses to minimally invasive adenocarcinoma (MIA). In patients of European ethnicity, we analyzed genetic mutations in AIS (n = 10) and MIA (n = 18) and compared the number of genetic mutations with advanced ADC (n = 2419). Using next-generation sequencing, the number of different mutations detected in both AIS (87.5%) and MIA (94.5%) were higher (p < 0.001) than in advanced ADC (53.7%). In contrast to the high number of mutations in Kirsten rat sarcoma virus gene (KRAS) in advanced ADC (34.6%), there was only one case of AIS with KRAS G12C mutation (3.5%; p < 0.001) and no cases of MIA with KRAS mutation (p < 0.001). In contrast to the modest prevalence of epidermal growth factor receptor (EGFR) mutations in advanced ADC (15.0%), the fraction of EGFR mutant cases was higher in both in AIS (22.2%) and MIA (59.5%; p < 0.001). The EGFR exon 19 deletion mutation was more common in both MIA (50%; n = 6/12) and ADC (41%; n = 149/363), whereas p.L858R was more prevalent in AIS (75%; n = 3/4). In contrast to pulmonary advanced ADC, KRAS driver mutations are less common, whereas mutations in EGFR are more common, in detectable AIS and MIA.

Filamin A increases aggressiveness of human neuroblastoma.

Background: The actin-binding protein filamin A (FLNA) regulates oncogenic signal transduction important for tumor growth, but the role of FLNA in the progression of neuroblastoma (NB) has not been explored. Methods: We analyzed FLNA mRNA expression in the R2 NB-database and FLNA protein expression in human NB tumors. We then silenced FLNA expression in human SKNBE2 and IMR32 NB cells by lentiviral vector encoding shRNA FLNA and assayed the cells for proliferation, migration, colony, spheroid formation, and apoptosis. SKNBE2 xenografts expressing or lacking FLNA in BALB/c nude mice were analyzed by both routine histopathology and immunohistochemistry. Results: We observed shorter patient survival with higher expression of FLNA mRNA than patients with lower FLNA mRNA expression, and high-risk NB tumors expressed higher FLNA levels. Overexpression of FLNA increased proliferation of SH-SY5 NB cells. NB cell lines transfected with siRNA FLNA proliferated and migrated less, expressed lower levels of phosphorylated AKT and ERK1/2, formed smaller colonies and spheroids, as well as increased apoptosis. After inoculation of SKNBE2 cells infected with lentivirus expressing shRNA FLNA, size of NB tumors and number of proliferating cells were decreased. Furthermore, we identified STAT3 as an interacting partner of FLNA. Silencing FLNA mRNA reduced levels of NF-κB, STAT3 and MYCN, and increased levels of p53 and cleaved caspase 3. Conclusion: Inhibition of FLNA impaired NB cell signaling and function and reduced NB tumor size in vivo, suggesting that drugs targeting either FLNA or its interaction with STAT3 may be useful in the treatment of NB.

Filamin A Regulates Cardiovascular Remodeling.

Filamin A (FLNA) is a large actin-binding cytoskeletal protein that is important for cell motility by stabilizing actin networks and integrating them with cell membranes. Interestingly, a C-terminal fragment of FLNA can be cleaved off by calpain to stimulate adaptive angiogenesis by transporting multiple transcription factors into the nucleus. Recently, increasing evidence suggests that FLNA participates in the pathogenesis of cardiovascular and respiratory diseases, in which the interaction of FLNA with transcription factors and/or cell signaling molecules dictate the function of vascular cells. Localized FLNA mutations associate with cardiovascular malformations in humans. A lack of FLNA in experimental animal models disrupts cell migration during embryogenesis and causes anomalies, including heart and vessels, similar to human malformations. More recently, it was shown that FLNA mediates the progression of myocardial infarction and atherosclerosis. Thus, these latest findings identify FLNA as an important novel mediator of cardiovascular development and remodeling, and thus a potential target for therapy. In this update, we summarized the literature on filamin biology with regard to cardiovascular cell function.

Lack of RAC1 in macrophages protects against atherosclerosis.

The Rho GTPase RAC1 is an important regulator of cytoskeletal dynamics, but the role of macrophage-specific RAC1 has not been explored during atherogenesis. We analyzed RAC1 expression in human carotid atherosclerotic plaques using immunofluorescence and found higher macrophage RAC1 expression in advanced plaques compared with intermediate human atherosclerotic plaques. We then produced mice with Rac1-deficient macrophages by breeding conditional floxed Rac1 mice (Rac1fl/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter (LC). Atherosclerosis was studied in vivo by infecting Rac1fl/fl and Rac1fl/fl/LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Rac1fl/fl/LC macrophages secreted lower levels of IL-6 and TNF-α and exhibited reduced foam cell formation and lipid uptake. The deficiency of Rac1 in macrophages reduced the size of aortic atherosclerotic plaques in AdPCSK9-infected Rac1fl/fl/LC mice. Compare with controls, intima/media ratios, the size of necrotic cores, and numbers of CD68-positive macrophages in atherosclerotic plaques were reduced in Rac1-deficient mice. Moreover, we found that RAC1 interacts with actin-binding filamin A. Macrophages expressed increased RAC1 levels in advanced human atherosclerosis. Genetic inactivation of RAC1 impaired macrophage function and reduced atherosclerosis in mice, suggesting that drugs targeting RAC1 may be useful in the treatment of atherosclerosis.

Can a Peritoneal Conduit Become an Artery?

OBJECTIVE: Current vascular grafts all have limitations. This study examined peritoneum as a potential graft material and the in vivo transfer of peritoneum into a functional artery like conduit after end to end anastomosis into the common carotid artery of sheep. The aim was to investigate whether implantation of a peritoneal tube into the arterial tree results in a structure with function, histological findings, and gene expression like an artery, and whether such arterialisation occurs through a conversion of the phenotype of peritoneal cells or from host cell migration into the implant. METHODS: Peritoneum with adherent rectus aponeurosis from sheep was used to form tubular vascular grafts that were implanted into the common carotid artery of six sheep, then removed after five months. Two sheep received allogenic peritoneal grafts and four sheep received autologous peritoneal grafts. RESULTS: One sheep died shortly after implantation, so five of the six sheep were followed. Five months after implantation, four of the five remaining grafts were patent. Three of four patent grafts were aneurysmal. The four patent grafts had developed an endothelial layer indistinguishable from that of the adjacent normal artery, and a medial layer with smooth muscle cells with a surrounding adventitia. The new conduit displayed vasomotor function not present at the time of implantation. DNA genotyping showed that the media in the new conduit consisted of recipient smooth muscle cells. Little difference in mRNA expression was demonstrated between the post-implantation conduit and normal artery. CONCLUSION: During a five month implantation period in the arterial system, peritoneum converted into a tissue that histologically and functionally resembled a normal artery, with a functional genetic expression that resembled that of an artery. Single nucleotide polymorphism analysis indicated that this conversion occurs through host cell migration into the graft.

Targeting Filamin A Reduces Macrophage Activity and Atherosclerosis.

BACKGROUND: The actin-binding protein FLNA (filamin A) regulates signal transduction important for cell locomotion, but the role of macrophage-specific FLNA during atherogenesis has not been explored. METHODS: We analyzed FLNA expression in human carotid atherosclerotic plaques by immunofluorescence. We also produced mice with Flna-deficient macrophages by breeding conditional Flna-knockout mice ( Flna o/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter ( LC). Atherosclerosis in vivo was studied by transplanting bone marrow from male Flna o/fl/ LC mice to atherogenic low-density lipoprotein receptor-deficient ( Ldlr-/-) mice; and by infecting Flna o/fl and Flna o/fl/ LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Furthermore, C57BL/6 mice were infected with AdPCSK9 and then treated with the calpain inhibitor calpeptin to inhibit FLNA cleavage. RESULTS: We found that macrophage FLNA expression was higher in advanced than in intermediate human atherosclerotic plaques. Flna o/fl/ LC macrophages proliferated and migrated less than controls; expressed lower levels of phosphorylated AKT and ERK1/2; exhibited reduced foam cell formation and lipid uptake; and excreted more lipids. The deficiency of Flna in macrophages markedly reduced the size of aortic atherosclerotic plaques in both Ldlr-/-BMT: Flnao/fl/LC and AdPCSK9-infected Flna o/fl/ LC mice. Intima/media ratios and numbers of CD68-positive macrophages in atherosclerotic plaques were lower in Flna-deficient mice than in control mice. Moreover, we found that STAT3 interacts with a calpain-cleaved carboxyl-terminal fragment of FLNA. Inhibiting calpain-mediated FLNA cleavage with calpeptin in macrophages reduced nuclear levels of phosphorylated STAT3, interleukin 6 secretion, foam cell formation, and lipid uptake. Finally, calpeptin treatment reduced the size of atherosclerotic plaques in C57BL/6 mice infected with AdPCSK9. CONCLUSIONS: Genetic inactivation of Flna and chemical inhibition of calpain-dependent cleavage of FLNA impaired macrophage signaling and function, and reduced atherosclerosis in mice, suggesting that drugs targeting FLNA may be useful in the treatment of atherosclerosis.

Deficiency of filamin A in endothelial cells impairs left ventricular remodelling after myocardial infarction.

AIMS: Actin-binding protein filamin A (FLNA) regulates signal transduction important for cell locomotion, but the role of FLNA after myocardial infarction (MI) has not been explored. The main purpose of this study was to determine the impact of endothelial deletion of FLNA on post-MI remodelling of the left ventricle (LV). METHODS AND RESULTS: We found that FLNA is expressed in human and mouse endothelial cells (ECs) during MI. To determine the biological significance of endothelial expression of FLNA, we used mice that are deficient for endothelial FLNA by cross-breeding adult mice expressing floxed Flna (Flna(o/fl)) with mice expressing Cre under the vascular endothelial-specific cadherin promoter (VECadCre+). Male Flna(o/fl) and Flna(o/fl)/VECadCre+ mice were subjected to permanent coronary artery ligation to induce MI. Flna(o/fl)/VECadCre+ mice that were deficient for endothelial FLNA exhibited larger and thinner LV with impaired cardiac function as well as elevated plasma levels of NT-proBNP and decreased secretion of VEGF-A. The number of capillary structures within the infarcted areas was reduced in Flna(o/fl)/VECadCre+ hearts. ECs silenced for Flna mRNA expression exhibited impaired tubular formation and migration, secreted less VEGF-A, and produced lower levels of phosphorylated AKT and ERK1/2 as well as active RAC1. CONCLUSION: Deletion of FLNA in ECs aggravated MI-induced LV dysfunction and cardiac failure as a result of defective endothelial response and increased scar formation by impaired endothelial function and signalling.

Targeting filamin A reduces K-RAS-induced lung adenocarcinomas and endothelial response to tumor growth in mice.

BACKGROUND: 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. 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. METHODS: First, 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. RESULTS: Knockout 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. CONCLUSIONS: We 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.