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Research Article
Fibrinogen Alpha Chain Knockout Promotes Tumor Growth and Metastasis through
Integrin-AKT Signaling Pathway in Lung Cancer
Meng Wang1#, Guangxin Zhang1, Yue Zhang1, Xuelian Cui1, Shuaibin Wang1, Song Gao1,
Yicun Wang1, Ying Liu1, Jeeyoo H. Bae1, Wei-Hsiung Yang2, Lei S. Qi3, Lizhong Wang1,4*,
and Runhua Liu1,4*
1Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama
2Department of Biomedical Sciences, Mercer University, Savannah, GA.
3Department of Bioengineering, Stanford University, Stanford, CA
4Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham,
Alabama
# Current address: Department of Oncology, Cancer Hospital of Harbin Medical University,
Harbin, China
* Correspondence to Runhua Liu, 720 20th Street South, Birmingham, AL 35294. Phone:
205-934-7308; email: [email protected] or Lizhong Wang, [email protected]
Running Title: Role of FGA in lung cancer
No potential conflicts of interest were disclosed.
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Abstract
Fibrinogen is an extracellular matrix protein composed of three polypeptide chains with
fibrinogen alpha (FGA), beta (FGB) and gamma (FGG). While fibrinogen and its related
fragments are involved in tumor angiogenesis and metastasis, their functional roles
areincompatible. A recent genome-scale screening reveals that loss of FGA affects the
acceleration of tumor growth and metastasis of lung cancer, but the mechanism remains
elusive. We used CRISPR/Cas9 genome editing to knockout (KO) FGA in human lung
adenocarcinoma (LUAD) cell lines A549 and H1299. By colony formation, transwell
migration and matrix invasion assays, FGA KO increased cell proliferation, migration, and
invasion but decreased the expressions of epithelial-mesenchymal transition marker E-
cadherin and cytokeratin 5/8 in A549 and H1299 cells. However, administration of FGA
inhibited cell proliferation and migration but induced apoptosis in A549 cells. Of note,
FGA KO cells indirectly co-cultured by transwells with FGA wild-type cells increased
FGA in the culture medium, leading to decreased migration of FGA KO cells. Furthermore,
our functional analysis identified a direct interaction of FGA with integrin α5 as well as
FGA-integrin signaling that regulated the AKT-mTOR signaling pathway in A549 cells.
In addition, we validated that FGA KO increased tumor growth and metastasis through
activation of AKT signaling in an A549 xenograft model.
Implications: These findings demonstrate that that loss of FGA facilities tumor growth
and metastasis through integrin-AKT signaling pathway in lung cancer.
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Introduction
Lung cancer is the leading cause of cancer deaths around the world (1,2). About 80% to
85% of lung cancers are non-small cell lung cancers (NSCLC), including lung
adenocarcinoma (LUAD, 40% of lung cancers) and lung squamous cell carcinoma (LUSC,
25% to 30% of lung cancers) (3). The majority of lung cancers are diagnosed at advanced
stages and are inoperable (4). However, biologic risk factors of lung cancer aggressiveness
and metastasis remain elusive. Fibrinogen is an extracellular matrix protein involved in
blood clot formation, but also a key biologic factor associated with tumor angiogenesis and
metastasis (5,6). Fibrinogen is composed of fibrinogen alpha chain (FGA), beta chain
(FGB), and gamma chain (FGG) encoded by a compact gene cluster, and each chain
contributes two copies to the functional fibrinogen hexamer joined by disulfide bridging
(7,8). Fibrinogen is expressed primarily in hepatocytes (9) and mutations in any of the three
genes (FGA, FGB, and FGG) cause dysfibrinogenemias. Specifically, FGA mutations can
lead to hereditary systemic amyloidosis (10).
Early studies identified the role of fibrinogen and related fragments in tumor
angiogenesis and metastasis. Fibrinogen and its breakdown products modulate the overall
angiogenic potential of the solid tumors (6). Specifically, fibrinogen binds growth factors
to stimulate endothelial cells and promotes an angiogenic phenotype (6). Fibrinogen is also
cleaved by thrombin to form fibrin in conjunction with growth factors, extracellular matrix
(ECM) proteins, and integrin α5β3 to promote angiogenesis (6). In animal models, lung
metastasis after intravenous injection of lung carcinoma and melanoma cell lines is
substantially reduced in fibrinogen-deficient mice (11). Recent clinical studies revealed
that pretreatment of plasma fibrinogen is associated with poor disease-free survival in
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various cancers, including lung cancer (12). However, the degradation of fibrinogen yields
fragments that affect angiogenic and metastatic processes. Fibrinogen fragments, caused
by the degradation of FGB, have been shown to inhibit endothelial cell migration and
tubule formation (13,14). Of note, FGA interacts with HBsAg to promote apoptosis in
HepG2 cells (15). Thus, fibrinogen and its polypeptide chains or yielded fragments may
play different roles in tumor angiogenesis and metastasis.
Gene knockout (KO) for different parts of the fibrinogen molecule is now
warranted to elucidate their role in angiogenesis and metastasis. A recent study used a
genome-scale CRISPR screening library with 67,405 single guide RNAs (sgRNAs) to
mutagenize a non-metastatic mouse cell line of lung cancer (16). Once the mutant cells are
transplanted into immunocompromised mice, resulting metastases are generated quickly.
Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a
small set of genes, suggesting specific loss-of-function mutations drive tumor growth and
metastasis (16). Individual sgRNAs and a small pool of 624 sgRNAs that target the top
scoring genes from the primary screen dramatically accelerate metastasis (16). Of note,
mouse Fga is one of the most frequent targets with enriched sgRNAs in metastatic lung
tumors compared with that in primary tumors (16). Human FGA encodes 610 amino acid
residues, which is a plasma glycoprotein with a crucial role in the coagulation cascade
through its conversion to fibrin (7). In the present study, to address the role of FGA in
tumor growth and metastasis of lung cancer cells, we generated an FGA KO in two LUAD
cell lines A549 and H1299 using CRISPR/Cas9 genome editing. Using these cell models,
we investigated the effect of FGA on tumor growth and metastasis as well as in underlying
signaling pathways.
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MATERIALS AND METHODS
Cell lines, antibodies, and reagents
Human LUAD cell lines A549 and H1299, breast cancer cell lines MBA-MB-231 and
MCF7, prostate cancer cell lines LNCaP, PC3, and DU145, and hepatocellular carcinoma
cell line HepG2 were obtained from the American Type Culture Collection (Manassas,
VA). Cells freshly amplified and frozen after obtention from the ATCC were used every 5
months. Cell line was authenticated by examination of morphology and growth
characteristics and was confirmed to be mycoplasma-free. Cells were maintained in
Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum (Thermo
Fisher Scientific, Waltham, MA) and cultured for less than 6 months. Specific primary
antibodies for Western blots or immunohistochemistry (IHC) were used to detect the
following proteins: FGA, Integrin α5, CK5, CK8, Ki67, E-cadherin, Vimentin, BCL2,
BCL-XL, MCL1, cleaved-caspase3, AKT, p-AKTT308, p-AKTS473, S6, p-S6S235/236, 4EBP1,
p-4EBP1S65, and p-4EBP1T37/46 as shown in Supplementary Table S1. Western Blotting
Detection Kit was purchased from Millipore (Billerica, MA). Recombinant human FGA
(Zeye Biotechnology, Shanghai, China), mutant recombinant human FGA (Cloud-Clone
Corp., Katy, TX), and Fibrinogen (Sigma, St. Louis, MO) were used for the treatment of
cells. pCMV3-FGA-Flag vector was ordered from SinoBiological (Cat#: HG16000-CF,
Wayne, PA) used for the over-expression of FGA in A549 cells.
Generation of FGA KO cell line
For FGA KO, the single guide RNAs (sgRNAs) were designed using the online CRISPR
design tool (Benchling, San Francisco, CA, https://benchling.com). The exon 2 region of
FGA was selected to be targeted by CRISPR/Cas9 genome editing. A ranked list of
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sgRNAs was generated with specificity and efficiency scores. The pair of oligos for two
targeting sites was annealed and ligated to the Bbs I-digested pSpCas9(BB)-2A-GFP
(PX458) vector (Addgene, Cambridge, MA) referencing a previously published protocol
(17,18). The pX458 plasmids containing each target sgRNA sequences were transfected
into cells with Lipofectamine 3000 (Thermo Fisher Scientific). After flow cytometry
sorting with GFP, 100 GFP+ cells were seeded into each well of a 96-well plate. After the
selection of single colonies, FGA KO colonies were determined by Sanger sequencing with
isolated genomic DNA, and FGA expression levels in each clone were validated by
Western blot. All sgRNAs were accessed using the online, off-target searching tool (Cas-
OFFinder, Daejeon, South Korea, http://www.rgenome.net/cas-offinder) (19). To avoid an
off-target effect, potential off-target regions were selected and subjected to PCR and
Sanger sequence analysis. As previously described, the sgRNAs and primers for CRISPR
design are shown in Supplementary Table S2 (18).
Cell growth assay
Cells were seeded into 12-well plates at a density of 1.5×104 cells/well and were grown in
complete medium containing 10% fetal bovine serum (FBS). The viable cells were stained
by 0.4% trypan blue solution (Sigma), and the cells were counted in triplicate every day
using a hemocytometer as previously described (53)
Transwell migration assay
After starvation of cells for 24 hours, 105 cells with 200µl serum-free DMEM were seeded
into the upper chamber in Transwell chamber (8-μM pore size; Millipore), and 500μl
DMEM with 10% FBS was added into the lower chamber. After 24 hours, non-migrated
cells on the filter side of the upper chamber were cleansed with a cotton swab, and the
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polycarbonate membrane on the Transwell chamber was fixed with 10% formalin 800μl
for 15 minutes (mins), rinsed with PBS 3 times, and stained with 50μl DAPI for 10 mins
in the dark. The Transwell membrane was covered with cover glass by Fluoromount G
(Thermo Fisher Scientific). The migrated cells were counted under an immunofluorescent
microscope.
Colony formation assay
Three hundred cells/well Cells were seeded into 6 wells plates. After colony formation for
12 days, the plates were washed twice with cold PBS buffer, fixed with 4%
paraformaldehyde for 10 mins, and then stained with 0.2% (w/v) crystal violet for 30 mins.
The colonies were quantified by using the software of Image J.
Soft–agar colony formation assay
Cells are harvested and pipetted well to become single-cell suspension in complete culture
media in 1x 106/ml. A mixture of 0.9 ml 4% soft-agar (Sigma) with 4.1 ml pre-warmed 10%
FBS DMEM was added into a 60-mm culture dish to make the bottom layer. The top layer
contained 3 x 104 cells in 3 ml of 10% FBS DMEM and 0.36% agar. The soft-agar colony
dish was marked and placed at a 37 °C incubator for 3 weeks.
Cell apoptosis assay
Apoptosis was assessed by flow cytometry based on cell binding to Annexin V (BD
Biosciences). For apoptosis induction by FGA, cells were treated with 100 μg/mL
recombinant human FGA for 1 hour.
Western blotting and co-immunoprecipitation (co-IP)
Western blotting was performed as previously described (20,21). For co-IP, cells were
lysed in ice-cold buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 1%
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NP-40] supplemented with complete protease inhibitors (Sigma) on ice for 10 mins.
Lysates were aliquoted into two tubes and incubated with the designated antibody or an
appropriate IgG control for 16 hours at 4°C. Protein A/G agarose (Thermo Fisher Scientific)
was used to precipitate antibody-protein complexes (23)
Immunohistochemistry (IHC)
The ABC detection system (Vectastain Elite ABC kit, Vector Labs, Burlingame, CA) was
used for immunostaining according to the manufacturer’s protocol as described previously
(20,21). The results were determined to be negative if <10% of cells within tumor areas
were stained or positive if 10%-100% were stained. The percentage of positive tumor cells
per slide (10% to 100%) was multiplied by the dominant intensity pattern of staining (1,
weak; 2, moderate; 3, intense); therefore, the overall score ranged from 10 to 300 H-scores
(22). All slides were examined by two pathologists in a blinded fashion.
In vivo xenogeneic transplantation
For tumor growth, wild-type (WT) and FGA KO A549 cells (2 × 106 cells in 200μl PBS)
were injected subcutaneously into the right flanks of immunodeficient BALB/c nude mice
8 weeks old. Xenograft tumor size was measured every other day and a tumor volume
formula was used (volume = (width (2) × length)/2) for caliper measurements. Mice were
sacrificed at week 8 after tumor cell injection, and metastatic sites were checked by
histologic analysis. All animal experiments were conducted in accordance with accepted
standards of animal care and approved by the Institutional Animal Care and Use Committee
of Harbin Medical University Cancer Hospital.
In vivo tumor metastasis assay
A total of 1 × 104 control A549 WT cells or KO cells were implanted intravenously into 8-
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week-old immunodeficient BALB/c nude mice. At 4 weeks after implantation, the mice
were euthanized for histologic examination and expression analysis. The number of surface
lesions over all lobes of the liver and lungs was scored before pathologic analysis. Tumor
burden in the lungs was quantified in two-step sections from each lobe (lung left two lobes
and right three lobes) in a blinded fashion by calculating the area of tumor tissue as a
percentage of the total tissue area as previously described (23).
Human tissue specimens
Fifty formalin-fixed and paraffin-embedded human lung cancer specimens were obtained
from the Harbin Medical University Cancer Hospital. The tumor specimens were collected
from 50 patients with lung cancer who underwent primary surgery between January 2012
and June 2018. All had histologically confirmed lung cancer with information on the
histologic type and tumor stage (AJCC, American Joint Committee on Cancer) and grade
(Supplementary Table S3). This study, involving the use of human lung tumor specimens,
was approved by the Institutional Review Board of the Harbin Medical University Cancer
Hospital. For all specimens, written informed consent was obtained from all subjects in
accordance with the requirements of the Institutional Review Board.
Datasets, analysis of gene alteration and expression data, and annotation
The TCGA Data Portal was used to download the data from samples of LUAD, lung
squamous cell carcinoma (LSCC), and normal lung controls. The TCGA data analysis was
performed using cBioPortal (24,25) (http://www.cbioportal.org) for genetic alteration
analysis, UALCAN (26) (http://ualcan.path.uab.edu/index.html) for gene expression and
survival analysis, and MethHC (27) (http://methhc.mbc.nctu.edu.tw) for DNA methylation
analysis. Gene-level normalized expression data were used in Partek Genomic Suite (PGS,
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St. Louis, MO) for additional normalization, statistics, and annotation. False discovery rate
(FDR) corrections (Benjamini-Hochberg methods) were applied to test multiple
hypotheses.
Statistical analyses
Continuous variables were summarized using mean, standard deviation (SD), and median
values. In samples with normal distributions, the means of the variables were compared
using a two-tailed t-test between two groups. In samples with non-normal distributions, the
medians of the variable between two groups were compared by a Mann–Whitney U test.
Analysis of variance (ANOVA), one- and two-way, were used to test for overall differences,
followed by Dunnett's post hoc test for differences between groups. All data were entered
into an access database using Excel 2016 and analyzed with SPSS (version 24; IBM,
Armonk, NY), and StatView (version 5.0.1, SAS Institute Inc., Cary, NC).
RESULTS
Characterization of genetic alterations and expression profiling of FGA in human
lung cancers
We performed a genetic analysis of FGA in human lung cancers with the most commonly
used The Cancer Genome Atlas (TCGA) dataset for LUAD and LSCC and other public
multiple datasets for small cell lung cancer (SCLC). As shown in Supplementary Figures.
S1A-C, in these datasets with more than 1,000 cases, genetic alterations of FGA were
present in 4% of LUAD cases, 5% of LSCC cases, and 5% of SCLC cases. The genetic
alterations of FGA mainly compose of gene deletions and mutations, including several
truncating mutations and few gene amplification. Furthermore, we analyzed the mRNA
expression of FGA and its relationship with patient survival in the TCGA dataset. Our
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analysis showed a significant 2.8-fold decreased mRNA expression of FGA in LUAD
tissues compared with normal lung tissues (Supplementary Figure S2A). Although 27-fold
decreased mRNA expression of FGA was also evident in LUSC tissues compared with
normal lung tissues, there was no statistical significance (Supplementary Figure S2B). Of
note, survival analysis showed that high mRNA expression of FGA was likely to be
associated with poor prognosis for patients with LUSC but not LUAD (Supplementary
Figures S2C-D). In addition, DNA hypermethylation in the promoter region of FGA was
evident in both LUAD and LUSC as compared to normal lung tissue controls
(Supplementary Figure S3A) and was negatively correlated with mRNA expression of
FGA in LUAD but not LUSC (Supplementary Figures S3B-C). These data suggest that
genetic alterations of FGA are most likely to be an infrequent event, but a low mRNA
expression is a common event in human lung cancers, which may be through DNA
hypermethylation in the promoter region of FGA in LUAD.
FGA KO promotes cell proliferation, migration, and invasion in human LUAD cells
Fibrinogen is generated primarily in hepatocytes (9), but it is also synthesized and secreted
from epithelial cells, such as a LUAD cell line A549 (28) and breast cancer cell line MCF-
7 and MDA-MB-231 (29). We next examined the protein levels of FGA in multiple human
cancer cell lines. As shown in Figure 1A, the expression level of FGA protein was the
highest in hepatocellular carcinoma cell line HepG2, and the median expression was found
in two LUAD cell lines A549 and H1299 but not in two breast cancer cell lines MBA-MB-
231 and MCF7 and three prostate cancer cell line LNCaP, PC3, and DU145. Furthermore,
using CRISPR/Cas9 genome editing, we knocked out FGA in A549, and H1299 cells,
respectively, and the FGA KO cells were confirmed by Sanger sequencing (Supplementary
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Figure S4) and Western blotting (Figure 1B). In A549 and H1299 cells, cell proliferation
and colony numbers were increased in FGA KO cells compared with that in WT cells
(Figures 1C-G). Likewise, cell migration and invasion were increased in FGA KO cells by
transwell migration assay (transferred cell numbers, p < 0.001, KO vs. WT; Figures 1H-I)
and matrix invasion assay (colony spheroid area, p < 0.001, KO vs. WT; Figures 1J-M),
respectively. In addition, to test whether FGA KO-increased cell migration and invasion
are related to the epithelial-mesenchymal transition (EMT), we further analyzed the
expressions of EMT markers by Western blotting. As shown in Figure 1N, expressions of
cytokeratin (CK5 and CK8), and E-cadherin were reduced in FGA KO cells compared with
that in WT cells, suggesting an increased EMT by FGA KO in LUAD cells.
Administration of FGA induces cell apoptosis in human LUAD cells
We next determined the effect of FGA on apoptosis of A549 and H1299 cells. Although a
decreased apoptosis was observed in FGA KO cells compared with that in FGA WT cells,
no statistical significance was found (Figures 2A-B), To address whether FGA induces
apoptosis, we added FGA (10 μg/mL) into the culture medium to treat FGA KO A549 and
H1299 cells, respectively. At 6, 12, and 24 hours after treatment with FGA, apoptosis was
gradually elevated upon FGA stimulation in both FGA KO A549 and H129 cells (Figures
2C-D), suggesting that FGA induces apoptosis in LUAD cells. BCL-2 family-regulated
activation of caspase along with apoptosis in cancer cells contain several different signaling
pathways. (30). To elucidate the molecular mechanism underlying FGA-mediated cell
apoptosis, expressions of BCL-2 family members and related proteins, such as BCL2,
BCLXL, MCL1, and cleaved-caspase3 were determined by Western blot in A549 cells. As
shown in Figure 2E, expressions of BCL2 and MCL1 were gradually decreased but
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BCLXL was not changed after FGA treatment for 12 hours in FGA KO A549 cells, whereas
expression of cleaved-caspase3 was also gradually increased after FGA treatment for 24
hours in both FGA KO A549 and H1299 cells (Figure 2F), suggesting FGA-induced
apoptosis in LUAD cells.
Administration of FGA inhibits cell proliferation and migration in human LUAD
cells
We first measured the secreted protein levels of FGA in the culture medium of human
A549 cells using ELISA. FGA in culture medium was dramatically reduced in FGA KO
cells compared with that in WT cells (Figure 3A). To determine the effect of FGA on cell
proliferation, we added the recombinant human FGA (10 μg/mL) into the culture medium
of A549 FGA KO cells and found a strong inhibition of cell proliferation by FGA (Figure
3B), respectively. Cell colony assays further identified similar effects of FGA KO on tumor
growth (Figures 3C-D). Likewise, we observed similar effects of FGA on the suppression
of cell migration (Figures 3E-F). Furthermore, we used FGA WT and FGA KO cells,
respectively, to co-culture with FGA KO cells separated by Transwell chambers, and then
counted migrated cells in the lower chamber (Figure 3G). As shown in Figure 3H, a
decreased number of transferred FGA KO cells was observed by transwell migration assay
in FGA KO cells co-cultured with FGA WT cells as compared to those with FGA KO cells.
Likewise, an increased FGA in the culture medium was also confirmed by ELISA in FGA
KO cells co-cultured with FGA WT cells (Figure 3I). These results implicate a suppressive
role of FGA in cell growth and migration of LUAD cells. Next, we treated the WT A549
cells with fibrinogen (10 μg/mL) or fibrinogen (10 μg/mL) plus FGA (10 μg/mL),
respectively. Significant induction of cell proliferation was observed in the A549 cells after
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fibrinogen treatment, but this induction was partly reduced by addition of FGA (Figure 3J).
Likewise, this observation was confirmed by cell colony assay (Figures 3K-L) and
transwell migration assay (Figures 3M-N). These results suggest an opposite or
competitive role of fibrinogen and FGA in cell growth and migration of LUAD cells.
FGA-integrin α5 interaction regulates the AKT-mTOR signaling pathway in human
LUAD cells
To investigate the FGA-mediated molecular mechanism in LCAD cells, we performed a
co-expression analysis of FGA in human LUAD using the TCGA dataset. The mRNA
expression levels of FGA was positively correlated with that of 192 genes and negatively
correlated with that of 156 genes (Spearman correlation coefficient r ≥ 0.3 or r ≤ -0.3, p <
0.05; Figure 4A and Supplementary Table S4). Of note, mRNA expression levels of FGA
was highly co-expressed with that of FGG (r = 0.92, p < 0.001; Figure 4B). Next, using
these co-expression genes, we performed the KEGG pathway enrichment analysis. The top
3 enriched KEGG pathways, including focal adhesion, PI3K-AKT signaling, and
riboflavin metabolism pathways, were significantly associated with FGA expression in
human LUAD (Adjusted p < 0.05; Figure 4C).
PI3K-AKT signaling plays a critical role in tumorigenesis of NSCLC (31,32).
Consequently, we determined the effect of FGA on the top 2 signaling pathway in A549
cells. As shown in Figure 4D, while expression levels of total AKT were not changed, both
p-AKTT308 and p-AKTS473 were dramatically increased in FGA KO A549 cells compared
with in FGA WT A549 cells. Likewise, as critical downstream effectors of AKT-mTOR
signaling, p-4EBP1 and p-S6 were simultaneously upregulated after FGA KO in A549 cells
(Figure 4D), identifying an FGA loss-induced AKT-mTOR signaling. In addition, we used
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FGA to treat both FGA WT and KO A549 cells. Western blot analysis revealed that FGA
did not induce p-AKT in WT cells, whereas FGA suppressed phosphorylation of both
AKTT308 and AKTS473 in FGA KO cells (Figure 4E). However, in FGA KO A549 cells,
phosphorylation of AKTT308 was increased in treatment with both recombinant integrin 5α
and FGA as compared to those with FGA alone (Supplementary Figure S5), suggesting
that integrin 5α may compete with FGA to block the FGA-mediated suppression of AKT
activation in LUAD cells.
Focal adhesion is the top ranking pathway associated with FGA expression in
human LUAD (Figure 4C). In focal adhesion, integrins are α/β heterodimeric adhesion
glycoprotein receptors that regulate a wide variety of dynamic cellular processes, including
cell growth, migration, and phagocytosis (33), through major downstream signal pathways,
such as PI3K-AKT signaling pathway, in lung cancer progression (34-36). Fibrinogen is a
ligand for integrin α5β1 on endothelial cells (37). Thus, FGA may regulate PI3K-AKT
signaling through integrins in LUAD cells. To test this possibility, we immunoprecipitated
FGA from A549 cells and probed them with an anti-integrin α5 monoclonal antibody. As
shown in Figure 4F, anti-FGA co-precipitated integrin α5, whereas anti-integrin α5 co-
precipitated FGA. In previous studies, crystal structure analysis identified the extracellular
segment of integrin α5β3 in complex with an Arg-Gly-Asp (RGD) sequences (38), and
functional analysis demonstrated that integrin α5β3 binds two specific RGD sequences
(amino acids 114-116 and 590-593) of FGA (39). We used a mutant recombinant human
FGA (amino acids 124-214) without the RGD sequences to test the binding of mutant FGA
to integrin α5 in FGA knockout A459 cells. As shown in Supplementary Figures S6A and
S6B, there was no specific binding of mutant FGA to integrin α5 in the cells. In addition,
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we treated the WT A549 cells with fibrinogen or fibrinogen plus FGA, respectively.
Western blot analysis revealed that fibrinogen induced phosphorylation of both AKTT308
and AKTS473 in A549 cells, whereas FGA dramatically suppressed the p-AKT in the cells
regardless of fibrinogen treatment (Figure 4G), indicating that FGA-mediated suppression
of p-AKT may be independent to fibrinogen in LUAD cells. These data suggest that FGA
inhibits PI3K-AKT signaling through a direct interaction of FGA with integrin 5α in
LUAD cells (Figure 4H).
FGA KO facilitates tumor growth and metastasis in human LUAD cells in vivo
To determine the effect of FGA on tumor growth in vivo, FGA WT and KO A549 cells
were subcutaneously injected, respectively, into both male and female immunodeficient
BALB/c nude mice. Xenograft tumor growth was faster in mice with FGA KO A549 cells
compared with in WT A549 cells (Figures 5A-B) up to 4 weeks after injection. Likewise,
tumor weights were increased in mice with FGA KO A549 cells than in WT A549 cells at
day 28 (Figure 5C). Increased protein expression of Ki67 but decreased protein expression
of E-cadherin were evident in FGA KO xenograft tumors compared with the WT xenograft
tumors (Figures 5D-F). Likewise, protein expressions of p-AKTS473 were also increased in
FGA KO xenograft tumors compared with the WT xenograft tumors (Figure 5D). In
addition, we conducted a xenograft assay with FGA over-expressed A459 cells in both
male and female immunodeficient nude mice. As shown in Supplementary Figures S7A-
C, tumor growth and weights were decreased in mice with FGA over-expressed A549 cells
as compared to those with WT A459 cells.
Pulmonary metastases of the A549-derived LUAD xenograft tumors have been
observed in nude mice (40). However, we did not observe lung metastasis in the nude mice
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at 6 weeks after subcutaneous injection with FGA WT or KO A549 cells. Thus, to test the
role of FGA in tumor metastasis in vivo, we intravenously injected FGA WT or KO A549
cells into male and female immunodeficient BALB/c nude mice. At 4 weeks after injection,
a significant increase in the tumor number and burden of lung metastases were observed in
the mice injected with FGA KO cells as compared to those with WT A549 cells (Figures
5G-I). Significant increases of Ki67 and p-AKTS473 in the xenografted metastatic tumors
were also detected FGA KO cells (Figures 5J-K). These data suggest that FGA KO
promotes A549 cell growth and colonization in vivo.
An inverse relationship between protein expression of FGA and p-AKT in human
primary lung cancer specimens
We next evaluated, by IHC, the protein expressions of FGA and p-AKTS473 and their
relationship in 50 human primary lung cancer tissues, including LUAD, LSCC, lung
adenosquamous carcinoma and SCLC (Supplementary Table S3). The protein expression
of FGA in tumor cells was found in 36% (18/50) of total cases, including 41% (9/22) of
LUAD, 32% (6/19) LSCC and 29% (2/7) SCLC cases (Supplementary Table S3 and Figure
7A). However, expression of p-AKTS473 was found in 66% (33/50) of total cases, including
59% (13/22) of LUAD, 79% (15/19) LSCC and 57% (4/7) SCLC cases (Supplementary
Table S3 and Figure 6A). H-score quantitative analysis showed a negative correlation of
protein expressions of FGA with p-AKTS473 (Figure 6B). Furthermore, no expression of
FGA or expression of p-AKTS473 was likely to be associated with poor 5-year disease-free
survival, but these differences were not statistically significant (Figures 6C-D). However,
expression of FGA without expression of p-AKTS473 was significantly associated with a
better disease-free survival as compared to no expression of FGA with the expression of p-
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AKTS473 (Figure 6E). In addition, the expression of FGA was not associated with the
histologic type and tumor stages and grades (Supplementary Tables S3). These data suggest
that the downregulation of FGA with the upregulation of p-AKT is likely to be a poor
prognostic factor in human lung cancer.
Discussion
Based on our bioinformatics analysis, genetic alterations of FGA are unlikely to be a
frequent event in human lung cancers. However, our expression pattern analysis showed a
low expression of FGA in human lung cancer tissues, including LUAD and LUSC. Of note,
DNA hypermethylation in the promoter region of FGA is correlated with low expression
of FGA in human LUAD tissues, suggesting an epigenetic mechanism in the transcriptional
regulation of FGA in LUAD. Furthermore, in our experimental data, FGA KO promotes
but the administration of FGA inhibits LUAD cell growth, migration, and invasion, as well
as tumor colonization in the lung. Our functional analysis revealed the FGA-mediated
regulation of tumor growth and metastasis through apoptosis and EMT is also involved in
the integrin-AKT signaling pathway in LUAD cells in vitro and xenograft tumor model in
vivo. These data suggest that FGA plays a suppressive role in the growth and metastasis of
LUAD cells.
Fibrinogen, fibrin and their degradation products are involved in blood clotting,
inflammation, angiogenesis and tumor metastasis (6,41). Fibrinogen is thought to originate
from exudation of plasma fibrinogen and subsequent deposition into the tumor stroma and
is converted to fibrin polymers, resulting in the inflammatory response within the tumor
microenvironment (TME) (42). The fibrin matrix may help induce angiogenesis to promote
tumor growth and metastasis, while fibrinogen-depletion can result in a reduction in tumor
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19
colonization in the lung (11,43,44). Various fibrinogen-derived peptide fragments also
modulate the migration, proliferation, and differentiation of endothelial cells to affect
tumor growth and metastasis (6). The products of fibrin degradation (E and D fragments)
can stimulate the proliferation, migration, and differentiation of endothelial cells,
contributing to tumor vasculature, progression, and metastasis (41). However, as
polypeptide chains of fibrinogen, FGA-derived fragment (a 15-amino acid peptide) inhibits
endothelial cell migration, adhesion, and tubule formation to reduce tumor growth (45).
Likewise, FGB-derived fragment (a beta 43-63-amino acid peptide) is an inhibitor of
activated endothelial cells and reduces tumor vascularization but induces the formation of
tumor necrosis (14). Thus, fibrinogen, components, and their derivatives appear to play
different roles in endothelial cells within the TME. Currently, in tumor cells, the role of
fibrinogen, components, and their derivatives are not sufficiently understood. Fibrinogen
can directly be synthesized and secreted by breast cancer cells and assembles into the
extracellular matrix and reduces cancer cell migration (5). In lung cancer cells, fibrinogen
augments tumor cell proliferation through interaction with fibroblast growth factor-2 (46)
but blocks tumor cell migration (47). In the present study, FGA KO induces proliferation,
migration, and EMT of LUAD cells in vitro and promotes LUAD xenograft tumor growth
and lung metastasis in vivo, but administration of FGA inhibits LUAD cell growth,
migration, and invasion, supporting an inhibiting role of FGA against LUAD cells. In
addition, fibrinogen binds integrin α5β3 through two specific RGD sequences of FGA but
not FGB or FGG (38,39). While FGA KO promotes tumor growth and metastasis through
integrin α5, FGB or FGG unlikely has similar effects on LUAD.
The mechanism by which functional roles are different between fibrinogen and
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20
FGA in tumor growth and metastasis remains unknown. In the present study, we observed
a significant induction of cell proliferation and migration in LUAD cells by fibrinogen
treatment, but this induction was partly reduced by the addition of FGA, suggesting a
potential competition between fibrinogen and FGA in cell growth and migration of LUAD
cells. Likewise, fibrinogen induced phosphorylation of both AKTT308 and AKTS473 in
LUAD cells, but this induction was blocked by the addition of FGA in a fibrinogen-
independent manner. These data suggest that FGA may compete with fibrinogen to inhibit
LUAD cell growth and migration through AKT signaling, but also has a fibrinogen-
independent effect on AKT signaling through a direct interaction of FGA with integrin α5
in LUAD cells. However, the mechanism by which fibrinogen and FGA interact or
compete against tumor cells or TME cells in vivo remains to be elucidated by further studies.
Many soluble secretory proteins released from cancer cells into the extracellular
space are involved in inflammation and angiogenesis during tumor growth and metastasis
(48,49). As a secretory protein, fibrinogen binds integrins (e.g., α5β1, α2bβ3, and α5β3)
(50) in endothelial cells to promote tumor growth and metastasis. Fibrinogen has critical
roles in tumor metastasis by facilitating the adhesion of pancreatic tumor cells to
endothelial cells and transendothelial migration and extravasation (51). However,
fibrinogen polypeptide chains, FGA, FGB, and FGG are downregulated during EMT of
lung cancer cells (52). In HepG2 cells, knockdown of FGA promotes cell apoptosis,
suggesting an FGA-mediated inhibition of apoptosis in cells, and expression of BCLXL
and MCL1 are likely to be decreased, but BCL2 is increased in the FGA knockout-downed
cells (15). However, FGA-derived fragment induces apoptosis and blocks tube formation
of endothelial cells in gastric cancer, suggesting an apoptotic role of FGA in anti-
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21
angiogenesis (45). In the present study, the administration of FGA induces apoptosis
through downregulation of BCL2 and MCL1 but not BCLXL in LUAD cells. Of note, FGA
directly binds to integrin α5 and stimulates AKT-mTOR signaling, suggesting a functional
FGA-integrin-AKT axis in LUAD cells. Likewise, in TCGA dataset, survival analysis also
showed an opposing role of FGA between patients with liver cancer and renal cancer
(Supplementary Figures S8). Thus, the role of FGA in apoptosis is likely to be different
between various cell types, but the underlying mechanism remains to be elucidated by
further studies.
In conclusion, FGA may play a suppressive role in LUAD cells to inhibit tumor
growth and metastasis through induction of apoptosis and inhibition of EMT. In LUAD
cells, FGA can bind integrin α5 and reduce phosphorylation of AKT, leading to an
inhibition of mTOR signaling. Administration of FGA may provide a new therapeutic
approach to inhibit LUAD cell growth and metastasis. However, FGA may also affect TME
cells in vivo, such as endothelial cells, leading to the various roles of FGA in tumor growth
and metastasis.
ACKNOWLEDGMENTS
We thank Dr. Jonathan Leavenworth for editorial assistance in preparing this manuscript.
This work was supported by grants from the Mike Slive Foundation for Prostate Cancer
Research (L. Wang and R. Liu), the Breast Cancer Research Foundation of Alabama (L.
Wang), and the Mercer University Seed Grant (W.H. Yang). Results are based, in part,
upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/.
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22
References
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin
2018;68(1):7-30 doi 10.3322/caac.21442.
2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer
statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for
36 cancers in 185 countries. CA Cancer J Clin 2018 doi 10.3322/caac.21492.
3. Key Statistics for Lung Cancer. About Non-Small Cell Lung Cancer. American
Cancer Society 2016.
4. Gajra A, Jatoi A. Non-small-cell lung cancer in elderly patients: a discussion of
treatment options. J Clin Oncol 2014;32(24):2562-9 doi
10.1200/JCO.2014.55.3099.
5. Simpson-Haidaris PJ, Rybarczyk B. Tumors and fibrinogen. The role of
fibrinogen as an extracellular matrix protein. Ann N Y Acad Sci 2001;936:406-
25.
6. Staton CA, Brown NJ, Lewis CE. The role of fibrinogen and related fragments in
tumour angiogenesis and metastasis. Expert Opin Biol Ther 2003;3(7):1105-20
doi 10.1517/14712598.3.7.1105.
7. Tiscia GL, Margaglione M. Human Fibrinogen: Molecular and Genetic Aspects
of Congenital Disorders. Int J Mol Sci 2018;19(6) doi 10.3390/ijms19061597.
8. Huang S, Cao Z, Chung DW, Davie EW. The role of betagamma and
alphagamma complexes in the assembly of human fibrinogen. J Biol Chem
1996;271(44):27942-7.
9. Matsuda M, Sugo T. Hereditary disorders of fibrinogen. Ann N Y Acad Sci
2001;936:65-88.
10. Benson MD. Ostertag revisited: the inherited systemic amyloidoses without
neuropathy. Amyloid 2005;12(2):75-87 doi 10.1080/13506120500106925.
11. Palumbo JS, Kombrinck KW, Drew AF, Grimes TS, Kiser JH, Degen JL, et al.
Fibrinogen is an important determinant of the metastatic potential of circulating
tumor cells. Blood 2000;96(10):3302-9.
on April 5, 2020. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 23, 2020; DOI: 10.1158/1541-7786.MCR-19-1033
23
12. Perisanidis C, Psyrri A, Cohen EE, Engelmann J, Heinze G, Perisanidis B, et al.
Prognostic role of pretreatment plasma fibrinogen in patients with solid tumors: A
systematic review and meta-analysis. Cancer Treat Rev 2015;41(10):960-70 doi
10.1016/j.ctrv.2015.10.002.
13. Bootle-Wilbraham CA, Tazzyman S, Marshall JM, Lewis CE. Fibrinogen E-
fragment inhibits the migration and tubule formation of human dermal
microvascular endothelial cells in vitro. Cancer Res 2000;60(17):4719-24.
14. Krajewska E, Lewis CE, Chen YY, Welford A, Tazzyman S, Staton CA. A novel
fragment derived from the beta chain of human fibrinogen, beta43-63, is a potent
inhibitor of activated endothelial cells in vitro and in vivo. Br J Cancer
2010;102(3):594-601 doi 10.1038/sj.bjc.6605495.
15. Li P, Xiao L, Li YY, Chen X, Xiao CX, Liu JJ, et al. Fibrinogen alpha chain acts
as a HBsAg binding protein and their interaction promotes HepG2 cell apoptosis.
Current Proteomics 2014;11(1):48-54.
16. Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, et al. Genome-wide
CRISPR screen in a mouse model of tumor growth and metastasis. Cell
2015;160(6):1246-60 doi 10.1016/j.cell.2015.02.038.
17. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome
engineering using the CRISPR-Cas9 system. Nat Protoc 2013;8(11):2281-308 doi
10.1038/nprot.2013.143.
18. Wang Y, Li X, Liu W, Li B, Chen D, Hu F, et al. MicroRNA-1205, encoded on
chromosome 8q24, targets EGLN3 to induce cell growth and contributes to risk of
castration-resistant prostate cancer. Oncogene 2019 doi 10.1038/s41388-019-
0760-3.
19. Bae S, Park J, Kim JS. Cas-OFFinder: a fast and versatile algorithm that searches
for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics
2014;30(10):1473-5 doi 10.1093/bioinformatics/btu048.
20. Wang L, Liu R, Ye P, Wong C, Chen GY, Zhou P, et al. Intracellular CD24
disrupts the ARF-NPM interaction and enables mutational and viral oncogene-
mediated p53 inactivation. Nat Commun 2015;6:5909 doi 10.1038/ncomms6909.
on April 5, 2020. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 23, 2020; DOI: 10.1158/1541-7786.MCR-19-1033
24
21. Zhang W, Yi B, Wang C, Chen D, Bae S, Wei S, et al. Silencing of CD24
Enhances the PRIMA-1-Induced Restoration of Mutant p53 in Prostate Cancer
Cells. Clin Cancer Res 2016;22(10):2545-54 doi 10.1158/1078-0432.CCR-15-
1927.
22. Wu L, Yi B, Wei S, Rao D, He Y, Naik G, et al. Loss of FOXP3 and TSC1
Accelerates Prostate Cancer Progression through Synergistic Transcriptional and
Posttranslational Regulation of c-MYC. Cancer Res 2019;79(7):1413-25 doi
10.1158/0008-5472.CAN-18-2049.
23. Liu R, Liu C, Chen D, Yang WH, Liu X, Liu CG, et al. FOXP3 Controls an miR-
146/NF-kappaB Negative Feedback Loop That Inhibits Apoptosis in Breast
Cancer Cells. Cancer Res 2015;75(8):1703-13 doi 10.1158/0008-5472.CAN-14-
2108.
24. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio
cancer genomics portal: an open platform for exploring multidimensional cancer
genomics data. Cancer Discov 2012;2(5):401-4 doi 10.1158/2159-8290.CD-12-
0095.
25. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al.
Integrative analysis of complex cancer genomics and clinical profiles using the
cBioPortal. Sci Signal 2013;6(269):pl1 doi 10.1126/scisignal.2004088.
26. Chandrashekar DS, Bashel B, Balasubramanya SAH, Creighton CJ, Ponce-
Rodriguez I, Chakravarthi B, et al. UALCAN: A Portal for Facilitating Tumor
Subgroup Gene Expression and Survival Analyses. Neoplasia 2017;19(8):649-58
doi 10.1016/j.neo.2017.05.002.
27. Huang WY, Hsu SD, Huang HY, Sun YM, Chou CH, Weng SL, et al. MethHC: a
database of DNA methylation and gene expression in human cancer. Nucleic
Acids Res 2015;43(Database issue):D856-61 doi 10.1093/nar/gku1151.
28. Haidaris PJ. Induction of fibrinogen biosynthesis and secretion from cultured
pulmonary epithelial cells. Blood 1997;89(3):873-82.
on April 5, 2020. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 23, 2020; DOI: 10.1158/1541-7786.MCR-19-1033
25
29. Costantini V, Zacharski LR, Memoli VA, Kisiel W, Kudryk BJ, Rousseau SM.
Fibrinogen deposition without thrombin generation in primary human breast
cancer tissue. Cancer Res 1991;51(1):349-53.
30. Hata AN, Engelman JA, Faber AC. The BCL2 Family: Key Mediators of the
Apoptotic Response to Targeted Anticancer Therapeutics. Cancer Discov
2015;5(5):475-87 doi 10.1158/2159-8290.CD-15-0011.
31. Westcott PM, Halliwill KD, To MD, Rashid M, Rust AG, Keane TM, et al. The
mutational landscapes of genetic and chemical models of Kras-driven lung
cancer. Nature 2015;517(7535):489-92 doi 10.1038/nature13898.
32. Cancer Genome Atlas Research N. Comprehensive genomic characterization of
squamous cell lung cancers. Nature 2012;489(7417):519-25 doi
10.1038/nature11404.
33. Arnaout MA, Goodman SL, Xiong JP. Structure and mechanics of integrin-based
cell adhesion. Curr Opin Cell Biol 2007;19(5):495-507 doi
10.1016/j.ceb.2007.08.002.
34. Gotz R. Inter-cellular adhesion disruption and the RAS/RAF and beta-catenin
signalling in lung cancer progression. Cancer Cell Int 2008;8:7 doi 10.1186/1475-
2867-8-7.
35. Yan P, Zhu H, Yin L, Wang L, Xie P, Ye J, et al. Integrin alphavbeta6 Promotes
Lung Cancer Proliferation and Metastasis through Upregulation of IL-8-Mediated
MAPK/ERK Signaling. Transl Oncol 2018;11(3):619-27 doi
10.1016/j.tranon.2018.02.013.
36. Whitlock BB, Gardai S, Fadok V, Bratton D, Henson PM. Differential roles for
alpha(M)beta(2) integrin clustering or activation in the control of apoptosis via
regulation of akt and ERK survival mechanisms. J Cell Biol 2000;151(6):1305-
20.
37. Suehiro K, Gailit J, Plow EF. Fibrinogen is a ligand for integrin alpha5beta1 on
endothelial cells. J Biol Chem 1997;272(8):5360-6.
38. Xiong JP, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, et al. Crystal
structure of the extracellular segment of integrin alpha Vbeta3 in complex with an
on April 5, 2020. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 23, 2020; DOI: 10.1158/1541-7786.MCR-19-1033
26
Arg-Gly-Asp ligand. Science 2002;296(5565):151-5 doi
10.1126/science.1069040.
39. Springer TA, Zhu J, Xiao T. Structural basis for distinctive recognition of
fibrinogen gammaC peptide by the platelet integrin alphaIIbbeta3. J Cell Biol
2008;182(4):791-800 doi 10.1083/jcb.200801146.
40. Jakubowska M, Sniegocka M, Podgorska E, Michalczyk-Wetula D, Urbanska K,
Susz A, et al. Pulmonary metastases of the A549-derived lung adenocarcinoma
tumors growing in nude mice. A multiple case study. Acta Biochim Pol
2013;60(3):323-30.
41. Kolodziejczyk J, Ponczek MB. The role of fibrinogen, fibrin and fibrin(ogen)
degradation products (FDPs) in tumor progression. Contemp Oncol (Pozn)
2013;17(2):113-9 doi 10.5114/wo.2013.34611.
42. Hassan-Kadle MA, Osman MS, Ogurtsov PP. Epidemiology of viral hepatitis in
Somalia: Systematic review and meta-analysis study. World J Gastroenterol
2018;24(34):3927-57 doi 10.3748/wjg.v24.i34.3927.
43. Palumbo JS, Potter JM, Kaplan LS, Talmage K, Jackson DG, Degen JL.
Spontaneous hematogenous and lymphatic metastasis, but not primary tumor
growth or angiogenesis, is diminished in fibrinogen-deficient mice. Cancer Res
2002;62(23):6966-72.
44. Camerer E, Qazi AA, Duong DN, Cornelissen I, Advincula R, Coughlin SR.
Platelets, protease-activated receptors, and fibrinogen in hematogenous
metastasis. Blood 2004;104(2):397-401 doi 10.1182/blood-2004-02-0434.
45. Zhao C, Su Y, Zhang J, Feng Q, Qu L, Wang L, et al. Fibrinogen-derived
fibrinostatin inhibits tumor growth through anti-angiogenesis. Cancer Sci
2015;106(11):1596-606 doi 10.1111/cas.12797.
46. Sahni A, Simpson-Haidaris PJ, Sahni SK, Vaday GG, Francis CW. Fibrinogen
synthesized by cancer cells augments the proliferative effect of fibroblast growth
factor-2 (FGF-2). J Thromb Haemost 2008;6(1):176-83 doi 10.1111/j.1538-
7836.2007.02808.x.
on April 5, 2020. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 23, 2020; DOI: 10.1158/1541-7786.MCR-19-1033
27
47. Schneider G, Bryndza E, Poniewierska-Baran A, Serwin K, Suszynska M, Sellers
ZP, et al. Evidence that vitronectin is a potent migration-enhancing factor for
cancer cells chaperoned by fibrinogen: a novel view of the metastasis of cancer
cells to low-fibrinogen lymphatics and body cavities. Oncotarget
2016;7(43):69829-43 doi 10.18632/oncotarget.12003.
48. Dimou E, Nickel W. Unconventional mechanisms of eukaryotic protein secretion.
Curr Biol 2018;28(8):R406-R10 doi 10.1016/j.cub.2017.11.074.
49. Rabouille C. Pathways of Unconventional Protein Secretion. Trends Cell Biol
2017;27(3):230-40 doi 10.1016/j.tcb.2016.11.007.
50. Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding to integrins. J
Biol Chem 2000;275(29):21785-8 doi 10.1074/jbc.R000003200.
51. Huang C, Li N, Li Z, Chang A, Chen Y, Zhao T, et al. Tumour-derived
Interleukin 35 promotes pancreatic ductal adenocarcinoma cell extravasation and
metastasis by inducing ICAM1 expression. Nat Commun 2017;8:14035 doi
10.1038/ncomms14035.
52. Wang H, Meyer CA, Fei T, Wang G, Zhang F, Liu XS. A systematic approach
identifies FOXA1 as a key factor in the loss of epithelial traits during the
epithelial-to-mesenchymal transition in lung cancer. BMC Genomics 2013;14:680
doi 10.1186/1471-2164-14-680.
53. Liu, R., L. Wang, C. Chen, Y. Liu, P. Zhou, Y. Wang, X. Wang, J. Turnbull, B.A.
Minassian, and P. Zheng. Laforin Negatively Regulates Cell Cycle Progression through
Glycogen Synthase Kinase 3- Dependent Mechanisms. Molecular and Cellular Biology
2008 28(23): 7236.
Figure legends
Figure 1. Effects of FGA KO on cell proliferation, migration, and invasion in A549
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and H1299 cells. (a) Protein expression levels of FGA in A549, H1299, HepG2, MDA-
MB-231, MCF7, LNCaP, PC3, and DU145 cells measured by Western blot. (b) Protein
expression of FGA in A549 and H1299 cells before and after CRISPR/Cas9 genome
editing. (c, d) Cell proliferation of FGA WT and KO cells for 7 days. Data are presented
as means ± standard division (SD). * p < 0.05 by two-way ANOVA test vs. the WT control
group. (e) Cell morphology in FGA WT and KO cells. (f, g) Colony formation of FGA WT
and KO cells for 14 days. (h) Cell migration rate in A549 and H1299 cells for 24 hours
determined by in vitro trans-well assay. Images of ten different 10x fields were captured
from each membrane, and the number of migratory cells was counted by fluorescence
microscopy. (i) Quantifying rates of cell migration in the cells. Columns, mean of three
independent experiments; bars: SD. * p < 0.05 by one-way ANOVA test, followed by
Dunnett's post hoc test vs. the WT control group. (j, k) Cell invasion in A549 and H1299
cells for 12 days determined by in vitro soft agar colony formation assay. (l, m) Quantifying
areas of cell invasion in the cells. Data are presented as means ± SD. * p < 0.05 by two-
tailed t-test vs. the WT control group. (n) Protein expression of CK5/8 and E-cad in the
cells measured by Western blot. WT, wild-type; KO, knockout; CK5/8, cytokeratin 5/8; E-
cad, E-cadherin. Data are presented as means ± SD. * p < 0.05 by one-way ANOVA test,
followed by Dunnett's post hoc test vs. the WT control group. WT, wild-type; KO,
knockout. All experiments were repeated three times.
Figure 2. Effect of FGA on cell apoptosis in A549 and H1299 cells. (a, b) Quantitative
cell apoptosis of FGA WT and KO cells. (c, d) Quantitative cell apoptosis of FGA KO cells
after treatment with FGA for 24 hours. Data are presented as means ± SD. * p < 0.05 by
one-way ANOVA test followed by Dunnett's post hoc test or two-tailed t-test vs. the WT
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29
control group. (e) Protein expression of apoptotic-related proteins in the cells after
treatment with FGA for 12 hours was measured by Western blot in FGA KO A549 cells.
(f) Protein expression of cleaved caspase3 determined by Western blot after treatment with
FGA for 24 hours in FGA KO cells. WT, wild-type; KO, knockout; A549 KO, FGA KO
A549 cells; H1299 KO, FGA KO H1299 cells. All experiments were repeated three times.
Figure 3. Administration of FGA and its effects on cell proliferation and migration in
A549 cells. (a) Protein expression levels of FGA in culture medium measured by ELISA.
(b) Cell proliferation of FGA KO cells after treatment with or without FGA for 7 days. (c,
d) Colony formation of FGA WT, KO, and FGA-treated KO cells for 14 days. (e, f) The
cell migration rate of FGA WT, KO, and FGA-treated KO cells for 24 hours by trans-well
migration assay. (g, h) The cell migration rate of FGA KO cells co-cultured with FGA WT
or KO cells for 36 hours. (i) Protein expression levels of FGA in culture medium in the co-
cultured cells. (j) Cell proliferation of FGA WT A549 cells after treatment with Fibrinogen
or Fibrinogen plus FGA for 7 days. (k, l) Colony formation of FGA WT A549 cells after
treatment with Fibrinogen or Fibrinogen plus FGA for 14 days. (m, n) The cell migration
rate of FGA WT A549 cells after treatment with Fibrinogen or Fibrinogen plus FGA for
12 hours. Data are presented as means ± SD. * p < 0.05 by two-way ANOVA test, one-
way ANOVA test followed by Dunnett's post hoc test or two-tailed t-test vs. the WT control
group. WT, wild-type; KO, knockout. All experiments were repeated three times.
Figure 4. FGA-integrin interaction and its regulated signaling pathways in A549 cells.
(a) Heatmap of co-expression of FGA with its related genes in mRNA expression levels.
(b) Co-expression of FGG with FGA in mRNA expression levels. (c) Top signaling
pathways related to FGA expression in human LUAD samples using the dataset from
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30
TCGA. (d) Expression levels of key proteins on the AKT-mTOR signaling pathway
determined by Western blot in FGA WT and KO A549 cells. (e) Phosphorylation and
expression of AKT in FGA WT and KO A549 cells after treatment with FGA for 6 hours.
(f) Co-immunoprecipitation of FGA and Integrin α5 in A549 cells after treatment with
FGA for 6 hours. (g) Phosphorylation and expression of AKT and S6 in the FGA WT A549
cells after treatment with Fibrinogen or Fibrinogen plus FGA for 6 hours. (h) Diagram of
FGA-integrin-AKT signaling in LUAD cells. r, Pearson correlation coefficient; WT, wild-
type; KO, knockout. All experiments were repeated three times.
Figure 5. Effects of FGA KO on tumor growth and metastasis of A549 cells in vivo. (a)
Tumor growth in nude mice subcutaneously injected with FGA WT and KO A549 cells
(n=10 mice including 5 male and 5 female mice each group). Data are presented as means
± SD of the tumor volumes. Representative images (b) and weights (c) of xenograft tumors
at day 28 after injection. (d) Representative H/E and immunohistochemical (IHC) staining
of FGA, Ki67, and p-AKTS473 in xenograft tumor tissues. (e) Representative
immunofluorescence staining of CK5/8 and E-cad in xenograft tumor tissues. (f) The
percentage of Ki67+ cells as an indicator of proliferating cells among the xenograft tumor
tissues. At least five 40x fields for each mouse were counted. (g) Representative H/E and
IHC staining of vimentin in the lung at day 28 after tumor cell inoculation (n=10 mice
including 5 male and 5 female mice each group). Quantitative lung metastatic tumor
nodules (h) and burden (i) determined by histologic analysis at day 28 after tumor cell
inoculation. Horizontal lines represent the average value. (j) Representative IHC staining
of FGA, Ki67, E-cad, and p-AKTS473 in lung metastatic tumor tissues. (k) The percentage
of Ki67+ cells as an indicator of proliferating cells among the lung metastatic tissues. Data
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31
are presented as means ± SD. * p < 0.05 by two-way ANOVA test or two-tailed t-test vs.
the WT control group. WT, wild-type; KO, knockout; CK5/8, cytokeratin 5/8; E-cad, E-
cadherin. All in vivo experiments were repeated two times.
Figure 6. Expression levels of FGA and p-AKTS473 in human primary lung cancer
samples. (a) IHC analyses with specific antibodies against human FGA and p-AKTS473
were performed for 50 primary lung cancer tissue samples, including LUAD, LSCC, and
SCLC tissue samples. (b) Correlation of the H-scores of FGA and p-AKTS473 staining in
the human lung cancer tissue samples. (c, d) Kaplan–Meier curves of 5-year lung cancer
disease-free survival in tissue samples with protein expressions of FGA and p-AKTS473,
respectively. (e) Kaplan–Meier curves of 5-year lung cancer disease-free survival in tissue
samples with a combination of protein expressions of FGA and p-AKTS473All experiments
were repeated two times.
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Fig. 1
A
B
E WT KO1 KO2
A5
49
H1
299
F
D
C
G
WT KO1 KO2
A5
49
H1
299
* *
* *
FGA
Actin
WT KO1 KO2 FGA
FGA
FGA
FGA
FGA
FGA
A549 H1299
WT KO1 KO2
FGA
Actin
MC
F7
MD
A-M
B-2
31
Hep
G2
H1299
A549
DU
145
PC
3
LN
CaP
A549
* * *
*
400 μm
400 μm
71kDa
45kDa
71kDa
45kDa
H WT KO1 KO2
A5
49
H1
299
FGA
50μm
50μm 50μm 50μm
50μm 50μm
I
* * * *
FGA
J L
WT
K
O
A549 H1299
FG
A 100μm
100μm
100μm
100μm
20μm
20μm
100μm
100μm
20μm
20μm
100μm
100μm
WT
K
O
FG
A
K M
Avera
ge s
ph
ero
id a
rea
(x10
4µ
m2)
Avera
ge s
ph
ero
id a
rea
(x10
4µ
m2)
WT KO WT KO
A549 *
0 2 4 6 8
10 12 14 16 18 H1299 *
0 2
4 6 8
10 12
FGA FGA
H1299
CK8
CK5
WT KO1 KO2
E-cad
N
Actin
FGA
97kDa
45kDa
54kDa
62kDa
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B D
FGA (hours) FGA (hours)
*
* *
*
MCL1
BCLXL
BCL2
Actin
E FGA (hours) 0 6 12
FGA
Cleaved
caspase3
GAPDH
F FGA (hours) H1299 KO A549 KO
0 2 4 8 16 24h 0 2 4 8 16 24h
Fig. 2
H1299
A549
WT KO A C
H1299 K
O
A549 K
O FGA
FG
A
0 6 12 24
FGA (hours)
FGA
40kDa
45kDa
26kDa
30kDa
19kDa
37kDa
17kDa
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G
F
C
E
A
Fig. 3
WT KO+FGA KO
WT KO+FGA KO D
H
WT
KO
*
*
B
*
*
* I
* *
Co
ncen
trati
on
of
FG
A i
n
cu
ltu
re m
ed
ium
(n
g/m
l)
Co
ncen
trati
on
of
FG
A
in c
ult
ure
med
ium
(ng
/ml)
* *
FGA
FGA
FGA
FGA
FGA
FGA
FGA
FGA FGA
*
50μm 50μm
J * *
FGA
K WT Fibrinogen+FGA Fibrinogen FGA
L * *
FGA
M N WT Fibrinogen+FGA Fibrinogen
* *
FGA FGA
50μm 50μm 50μm
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r Gene
Pearson: 0.84
p-value: 1.35E-210
mRNA expression: FGA vs. FGG
Fig. 4
A B
C
FGA
FG
G
D
p-AKT
T308
AKT
p-AKT
S473
p-S6
S235/236
S6
p-4EBP1
S65
p-4EBP1
T37/46
4EBP1
WT KO1 KO2
Actin
FGA − + − + − +
p-AKT
S473
p-AKT
T308
WT KO1 KO2
AKT
E
Integrin 5α
F
H
FGA
FGA
FGA
FGA IgG input input
Integrin 5α IgG input input
FGA
mTOR
AKT
Integrin
Cell
proliferation
and migration
Cell
apoptosis
Fibrino
gen
G WT
Fibrino
gen
Fibrinogen+
FGA
p-AKT
T308
AKT
p-AKT
S473
p-S6
S235/236
S6
Actin
60kDa
32kDa
60kDa
60kDa
18kDa
18kDa
32kDa
18kDa
32kDa
45kDa
60kDa
32kDa
150kDa
45kDa
60kDa
60kDa
71kDa
60kDa
60kDa
60kDa
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WT KO1 KO2
Fig. 5
C
D
WT
K
O1
K
O2
n=10 n=10 n=10
Tu
mo
r v
olu
me (
mm
3)
B A FGA
FG
A
FGA
FGA
FG
A
p-A
KT
S473
* *
H/E
WT
K
O1
KO
2
FG
A
Ki6
7
E DAPI CK8 E-cad Merge
* *
F
n=10 n=10 n=10
n=10 n=10 n=10
* *
% K
i67
+ c
ells
50μm
50μm 50μm
50μm 50μm 50μm
50μm
50μm
50μm
50μm 50μm
50μm
100μm 100μm 100μm
50μm
50μm 50μm
50μm
50μm
50μm 50μm
50μm 50μm
FGA on April 5, 2020. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
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WT KO1 KO2 FGA H
/E
Fig. 5 V
imen
tin
G F
GA
p
-AK
TS
473
K
i67
E
-cad
H
Lu
ng
tu
mo
r n
od
ule
s
Lu
ng
tu
mo
r b
urd
en
% I
* * * *
n=10/each group n=10/each group FGA FGA
J K
* *
FGA n=10/each group
100μm
100μm
100μm
100μm
100μm 100μm
100μm 100μm
100μm
100μm 100μm 100μm
500μm 500μm 500μm
500μm 500μm 500μm
% K
i67
+ c
ells
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LUAD LUSD SCLC Case 1 Case 2 Case 1 Case 2 Case 1 Case 2
FG
A
p-A
KT
S473
Fig. 6
D E C
A
B
1
2
3
4
1 vs. 2 p = 0.060
1 vs. 3 p = 0.085
1 vs. 4 p = 0.774
2 vs. 3 p = 0.036
2 vs. 4 p = 0.507
n=32
n=18
n=17
n=33
n=6
n=12 n=26
n=7
Log-rank test
Log-rank test Log-rank test
p = 0.051 p = 0.108
Pearson correlation
r = .0.392, p = 0.005
-50
0
50
100
150
200
250
300
0 50 100 150 200 250
FGA/pAKT S473
FG
A H
-co
re
pAKT S473 H-core
3 vs. 4 p = 0.229 on April 5, 2020. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
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Published OnlineFirst March 23, 2020.Mol Cancer Res Meng Wang, Guangxin Zhang, Yue Zhang, et al. CancerMetastasis through Integrin-AKT Signaling Pathway in Lung Fibrinogen Alpha Chain Knockout Promotes Tumor Growth and
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