fibrinogen alpha chain knockout promotes tumor growth and ... · 3/21/2020 · fga-integrin...

1

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.

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

mailto:[email protected]


http://mcr.aacrjournals.org/

2

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.




3

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




4

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.




5

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



https://benchling.com/


6

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



http://www.rgenome.net/cas-offinder


7

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%




8

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-




9

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,



http://www.cbioportal.org/

http://ualcan.path.uab.edu/index.html

http://methhc.mbc.nctu.edu.tw/


10

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




11

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




12

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




13

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




14

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




15

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,




16

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




17

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-




18

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




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




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-




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/.



http://cancergenome.nih.gov/


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.




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.




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.




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




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.




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




28

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




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




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




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.




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




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




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




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




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



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




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



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

Updated version

10.1158/1541-7786.MCR-19-1033doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2020/03/21/1541-7786.MCR-19-1033.DC1

Access the most recent supplemental material at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/early/2020/03/21/1541-7786.MCR-19-1033To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-19-1033

http://mcr.aacrjournals.org/content/suppl/2020/03/21/1541-7786.MCR-19-1033.DC1

http://mcr.aacrjournals.org/cgi/alerts


http://mcr.aacrjournals.org/content/early/2020/03/21/1541-7786.MCR-19-1033


fibrinogen alpha chain knockout promotes tumor growth and ... · 3/21/2020 · fga-integrin...

Documents