Fibrinogen Alpha Chain Knockout Promotes Tumor Growth And

Published OnlineFirst March 23, 2020; DOI: 10.1158/1541-7786.MCR-19-1033

MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS

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

ABSTRACT ◥ Fibrinogen is an extracellular matrix protein composed of istration of FGA inhibited cell proliferation and migration but three polypeptide chains with fibrinogen alpha (FGA), beta induced apoptosis in A549 cells. Of note, FGA KO cells indirectly (FGB) and gamma (FGG). Although fibrinogen and its related cocultured by transwells with FGA wild-type cells increased FGA fragments are involved in tumor angiogenesis and metastasis, in the culture medium, leading to decreased migration of FGA their functional roles are incompatible. A recent genome-scale KO cells. Furthermore, our functional analysis identified a direct screening reveals that loss of FGA affects the acceleration of interaction of FGA with integrin a5aswellasFGA–integrin tumor growth and metastasis of lung cancer, but the mechanism signaling that regulated the AKT–mTOR signaling pathway in remains elusive. We used CRISPR/Cas9 genome editing to A549 cells. In addition, we validated that FGA KO increased knockout (KO) FGA in human lung adenocarcinoma (LUAD) tumor growth and metastasis through activation of AKT signal- cell lines A549 and H1299. By colony formation, transwell inginanA549xenograftmodel. migration and matrix invasion assays, FGA KO increased cell proliferation, migration, and invasion but decreased the expres- Implications:These findings demonstrate that that loss of FGA sions of epithelial–mesenchymal transition marker E-cadherin facilities tumor growth and metastasis through the integrin–AKT andcytokeratin5/8inA549andH1299 cells. However, admin- signaling pathway in lung cancer.

Introduction functional fibrinogen hexamer joined by disulfide bridging (7, 8). Fibrinogen is expressed primarily in hepatocytes (9) and mutations in Lung cancer is the leading cause of cancer-related deaths around the any of the three genes (FGA, FGB, and FGG) cause dysfibrinogenemia. world (1, 2). About 80% to 85% of lung cancers are non–small cell lung Specifically, FGA mutations can lead to hereditary systemic cancers (NSCLC), including lung adenocarcinoma (LUAD, 40% of amyloidosis (10). lung cancers) and lung squamous cell carcinoma (LUSC, 25%–30% of Early studies identified the role of fibrinogen and related fragments lung cancers; ref. 3). The majority of lung cancers are diagnosed at in tumor angiogenesis and metastasis. Fibrinogen and its breakdown advanced stages and are inoperable (4). However, biologic risk factors products modulate the overall angiogenic potential of the solid of lung cancer aggressiveness and metastasis remain elusive. Fibrin- tumors (6). Specifically, fibrinogen binds growth factors to stimulate ogen is an extracellular matrix protein involved in blood clot forma- endothelial cells and promotes an angiogenic phenotype (6). Fibrin- tion, but also a key biologic factor associated with tumor angiogenesis ogen is also cleaved by thrombin to form fibrin in conjunction with and metastasis (5, 6). Fibrinogen is composed of fibrinogen alpha chain growth factors, extracellular matrix (ECM) proteins, and integrin (FGA), beta chain (FGB), and gamma chain (FGG) encoded by a a5b3 to promote angiogenesis (6). In animal models, lung metastasis compact gene cluster, and each chain contributes two copies to the 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 1Department of Genetics, University of Alabama at Birmingham, Birmingham, associated with poor disease-free survival in various cancers, including 2 Alabama. Department of Biomedical Sciences, Mercer University, Savannah, lung cancer (12). However, the degradation of fibrinogen yields Georgia. 3Department of Bioengineering, Stanford University, Stanford, fragments that affect angiogenic and metastatic processes. Fibrinogen California. 4Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama. fragments, caused by the degradation of FGB, have been shown to inhibit endothelial cell migration and tubule formation (13, 14). Of Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). note, FGA interacts with HBsAg to promote apoptosis in HepG2 cells (15). Thus, fibrinogen and its polypeptide chains or yielded Current address for M. Wang: Department of Oncology, Cancer Hospital of fragments may play different roles in tumor angiogenesis and Harbin Medical University, Harbin, China. metastasis. Corresponding Authors: Runhua Liu, University of Alabama at Birmingham, 720 Gene knockout (KO) for different parts of the fibrinogen molecule is 20th Street South, Kaul 705, Birmingham, AL 35294. Phone: 205-934-7308; now warranted to elucidate their role in angiogenesis and metastasis. A Fax: 205-975-5689; E-mail: [email protected]; and Lizhong Wang, [email protected] recent study used a genome-scale CRISPR screening library with 67,405 single guide RNAs (sgRNAs) to mutagenize a nonmetastatic Mol Cancer Res 2020;XX:XX–XX mouse cell line of lung cancer (16). Once the mutant cells are doi: 10.1158/1541-7786.MCR-19-1033 transplanted into immunocompromised mice, resulting metastases 2020 American Association for Cancer Research. are generated quickly. Enriched sgRNAs in lung metastases and late-

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stage primary tumors were found to target a small set of genes, Cell growth assay suggesting specific loss-of-function mutations drive tumor growth Cells were seeded into 12-well plates at a density of 1.5 104 cells/ and metastasis (16). Individual sgRNAs and a small pool of 624 well and were grown in complete medium containing 10% FBS. The sgRNAs that target the top scoring genes from the primary screen viable cells were stained by 0.4% trypan blue solution (Sigma), and the dramatically accelerate metastasis (16). Of note, mouse Fga is one of cells were counted in triplicate every day using a hemocytometer as the most frequent targets with enriched sgRNAs in metastatic lung described previously (20). tumors compared with that in primary tumors (16). Human FGA encodes 610 amino acid residues, which is a plasma glycoprotein with a Transwell migration assay crucial role in the coagulation cascade through its conversion to After starvation of cells for 24 hours, 105 cells with 200 mL serum- fibrin (7). In the current study, to address the role of FGA in tumor free DMEM were seeded into the upper chamber in Transwell chamber growth and metastasis of lung cancer cells, we generated an FGA KO in (8-mm pore size; Millipore), and 500 mL DMEM with 10% FBS was two LUAD cell lines A549 and H1299 using CRISPR/Cas9 genome added into the lower chamber. After 24 hours, nonmigrated cells on editing. Using these cell models, we investigated the effect of FGA on the filter side of the upper chamber were cleansed with a cotton swab, tumor growth and metastasis as well as in underlying signaling and the polycarbonate membrane on the Transwell chamber was fixed pathways. with 10% formalin 800 mL for 15 minutes, rinsed with PBS 3 times, and stained with 50 mL DAPI for 10 minutes in the dark. The Transwell membrane was covered with cover glass by Fluoromount G (Thermo Materials and Methods Fisher Scientific). The migrated cells were counted under an immu- Cell lines, antibodies, and reagents nofluorescent microscope. Human LUAD cell lines A549 and H1299, breast cancer cell lines MBA-MB-231 and MCF7, prostate cancer cell lines LNCaP, PC3, Colony formation assay and DU145, and hepatocellular carcinoma cell line HepG2 were Three hundred cells/well were seeded into 6-well plates. After obtained from the ATCC. Cells freshly amplified and frozen after colony formation for 12 days, the plates were washed twice with cold obtention from the ATCC were used every 5 months. Cell line was PBS buffer, fixed with 4% paraformaldehyde for 10 minutes, and then authenticated by examination of morphology and growth charac- stained with 0.2% (w/v) crystal violet for 30 minutes. The colonies were teristics and was confirmed to be Mycoplasma free. Cells were quantified by using the software of Image J. maintained in DMEM supplemented with 10% FBS (Thermo Fisher Scientific) and cultured for less than 6 months. Specific primary Soft agar colony formation assay antibodies for Western blots or IHC were used to detect the Cells are harvested and pipetted well to become single-cell suspen- following proteins: FGA, Integrin a5, CK5, CK8, Ki67, E-cadherin, sion in complete culture media in 1 106/mL. A mixture of 0.9 mL 4% Vimentin, BCL2, BCL-XL, MCL1, cleaved caspase-3, AKT, soft agar (Sigma) with 4.1 mL prewarmed 10% FBS DMEM was added p-AKTT308,p-AKTS473,S6,p-S6S235/236, 4EBP1, p-4EBP1S65,and into a 60-mm culture dish to make the bottom layer. The top layer p-4EBP1T37/46 as shown in Supplementary Table S1. Western contained 3 104 cells in 3 mL of 10% FBS DMEM and 0.36% agar. Blotting Detection Kit was purchased from Millipore. Recombinant The soft agar colony dish was marked and placed at a 37C incubator human FGA (Zeye Biotechnology), mutant recombinant human for 3 weeks. FGA (Cloud-Clone Corp.), and Fibrinogen (Sigma) were used for the treatment of cells. pCMV3-FGA-Flag vector was ordered from Cell apoptosis assay SinoBiological (Cat #: HG16000-CF, Wayne, PA) used for the Apoptosis was assessed by flow cytometry based on cell binding to overexpression of FGA in A549 cells. Annexin V (BD Biosciences). For apoptosis induction by FGA, cells were treated with 100 mg/mL recombinant human FGA for 1 hour. Generation of FGA KO cell line For FGA KO, the single guide RNAs (sgRNA) were designed using Western blotting and coimmunoprecipitation the online CRISPR design tool (Benchling, https://benchling.com). Western blotting was performed as described previously (21, 22). The exon 2 region of FGA was selected to be targeted by CRISPR/ For coimmunoprecipitation, cells were lysed in ice-cold buffer Cas9 genome editing. A ranked list of sgRNAs was generated with [20 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1 mmol/L EDTA, specificity and efficiency scores. The pair of oligos for two targeting and 1% NP-40] supplemented with complete protease inhibitors sites was annealed and ligated to the Bbs I-digested pSpCas9(BB)-2A- (Sigma) on ice for 10 minutes. Lysates were aliquoted into two tubes GFP (PX458) vector (Addgene) referencing a previously published and incubated with the designated antibody or an appropriate IgG protocol (17, 18). The pX458 plasmids containing each target sgRNA control for 16 hours at 4C. Protein A/G agarose (Thermo Fisher sequences were transfected into cells with Lipofectamine 3000 Scientific) was used to precipitate antibody–protein complexes (23) (Thermo Fisher Scientific). After flow cytometry sorting with GFP, þ 100 GFP cells were seeded into each well of a 96-well plate. After the IHC selection of single colonies, FGA KO colonies were determined by The ABC detection system (Vectastain Elite ABC Kit, Vector Labs) Sanger sequencing with isolated genomic DNA, and FGA expression was used for immunostaining according to the manufacturer's pro- levels in each clone were validated by Western blot analysis. All tocol as described previously (21, 22). The results were determined to sgRNAs were accessed using the online, off-target searching tool be negative if <10% of cells within tumor areas were stained or positive (Cas-OFFinder; http://www.rgenome.net/cas-offinder; ref. 19). To if 10% to 100% were stained. The percentage of positive tumor cells per avoid an off-target effect, potential off-target regions were selected slide (10%–100%) was multiplied by the dominant intensity pattern of and subjected to PCR and Sanger sequence analysis. As previously staining (1, weak; 2, moderate; 3, intense); therefore, the overall score described, the sgRNAs and primers for CRISPR design are shown in ranged from 10 to 300 H-scores (24). All slides were examined by two Supplementary Table S2 (18). pathologists in a blinded fashion.

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In vivo xenogeneic transplantation and analyzed with SPSS (version 24; IBM), and StatView (version 5.0.1, For tumor growth, wild-type (WT) and FGA KO A549 cells (2 SAS Institute Inc.). 106 cells in 200 mL PBS) were injected subcutaneously into the right flanks of immunodeficient BALB/c nude mice 8 weeks old. Xeno- graft tumor size was measured every other day and a tumor volume Results formula was used (volume ¼ (width (2) length)/2) for caliper Characterization of genetic alterations and expression profiling measurements. Mice were sacrificed at week 8 after tumor cell of FGA in human lung cancers injection, and metastatic sites were checked by histologic analysis. We performed a genetic analysis of FGA in human lung cancers with All animal experiments were conducted in accordance with accept- the most commonly used TCGA dataset for LUAD and LSCC and ed standards of animal care and approved by the Institutional other public multiple datasets for small cell lung cancer (SCLC). As Animal Care and Use Committee of Harbin Medical University shown in Supplementary Fig. S1A–S1C, in these datasets with more Cancer Hospital (Harbin, China). 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 In vivo tumor metastasis assay alterations of FGA mainly compose of gene deletions and mutations, A total of 1 104 control A549 WT cells or KO cells were implanted including several truncating mutations and few gene amplification. intravenously into 8-week-old immunodeficient BALB/c nude mice. Furthermore, we analyzed the mRNA expression of FGA and its At 4 weeks after implantation, the mice were euthanized for histologic relationship with patient survival in the TCGA dataset. Our analysis examination and expression analysis. The number of surface lesions showed a significant 2.8-fold decreased mRNA expression of FGA in over all lobes of the liver and lungs was scored before pathologic LUAD tissues compared with normal lung tissues (Supplementary analysis. Tumor burden in the lungs was quantified in two-step Fig. S2A). Although 27-fold decreased mRNA expression of FGA was sections from each lobe (lung left two lobes and right three lobes) in also evident in LUSC tissues compared with normal lung tissues, there a blinded fashion by calculating the area of tumor tissue as a percentage was no statistical significance (Supplementary Fig. S2B). Of note, of the total tissue area as described previously (23). survival analysis showed that high mRNA expression of FGA was likely to be associated with poor prognosis for patients with LUSC but Human tissue specimens not LUAD (Supplementary Fig. S2C and S2D). In addition, DNA Fifty formalin-fixed and paraffin-embedded human lung cancer hypermethylation in the promoter region of FGA was evident in both specimens were obtained from the Harbin Medical University Cancer LUAD and LUSC as compared with normal lung tissue controls Hospital. The tumor specimens were collected from 50 patients with (Supplementary Fig. S3A) and was negatively correlated with mRNA lung cancer who underwent primary surgery between January 2012 expression of FGA in LUAD but not LUSC (Supplementary Fig. S3B and June 2018. All had histologically confirmed lung cancer with and S3C). These data suggest that genetic alterations of FGA are most information on the histologic type and tumor stage (AJCC, American likely to be an infrequent event, but a low mRNA expression is a Joint Committee on Cancer) and grade (Supplementary Table S3). common event in human lung cancers, which may be through DNA This study, involving the use of human lung tumor specimens, was hypermethylation in the promoter region of FGA in LUAD. approved by the Institutional Review Board (IRB) of the Harbin Medical University Cancer Hospital. For all specimens, written FGA KO promotes cell proliferation, migration, and invasion in informed consent was obtained from all subjects in accordance with human LUAD cells the requirements of the IRB. Fibrinogen is generated primarily in hepatocytes (9), but it is also synthesized and secreted from epithelial cells, such as a LUAD cell line Datasets, analysis of gene alteration and expression data, and A549 (29) and breast cancer cell line MCF-7 and MDA-MB-231 (30). annotation We next examined the protein levels of FGA in multiple human cancer The Cancer Genome Atlas (TCGA) Data Portal was used to cell lines. As shown in Fig. 1A, the expression level of FGA protein was download the data from samples of LUAD, lung squamous cell the highest in hepatocellular carcinoma cell line HepG2, and the carcinoma (LSCC), and normal lung controls. The TCGA data analysis median expression was found in two LUAD cell lines A549 and was performed using cBioPortal (http://www.cbioportal.org; H1299 but not in two breast cancer cell lines MBA-MB-231 and refs. 25, 26) for genetic alteration analysis, UALCAN (http://ualcan. MCF7 and three prostate cancer cell line LNCaP, PC3, and DU145. path.uab.edu/index.html; ref. 27) for gene expression and survival Furthermore, using CRISPR/Cas9 genome editing, we knocked out analysis, and MethHC (http://methhc.mbc.nctu.edu.tw; ref. 28) for FGA in A549, and H1299 cells, respectively, and the FGA KO cells were DNA methylation analysis. Gene-level normalized expression data confirmed by Sanger sequencing (Supplementary Fig. S4) and Western were used in Partek Genomic Suite (PGS) for additional normaliza- blotting (Fig. 1B). In A549 and H1299 cells, cell proliferation and tion, statistics, and annotation. FDR corrections (Benjamini– colony numbers were increased in FGA KO cells compared with that in Hochberg methods) were applied to test multiple hypotheses. WT cells (Fig. 1C–G). Likewise, cell migration and invasion were increased in FGA KO cells by transwell migration assay (transferred Statistical analyses cell numbers, P < 0.001, KO vs. WT; Fig. 1H and I) and matrix invasion Continuous variables were summarized using mean, SD, and assay (colony spheroid area, P < 0.001, KO vs. WT; Fig. 1J–M), median values. In samples with normal distributions, the means of respectively. In addition, to test whether FGA KO-increased cell the variables were compared using a two-tailed t test between two migration and invasion are related to the epithelial–mesenchymal groups. In samples with nonnormal distributions, the medians of the transition (EMT), we further analyzed the expressions of EMT mar- variable between two groups were compared by a Mann–Whitney U kers by Western blotting. As shown in Fig. 1N, expressions of test. ANOVA, one- and two-way, were used to test for overall cytokeratin (CK5 and CK8), and E-cadherin were reduced in FGA differences, followed by Dunnett post hoc test for differences between KO cells compared with that in WT cells, suggesting an increased EMT groups. All data were entered into an access database using Excel 2016 by FGA KO in LUAD cells.

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Figure 1. Effects of FGA KO on cell proliferation, migration, and invasion in A549 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 analysis. B, Protein expression of FGA in A549 and H1299 cells before and after CRISPR/Cas9 genome editing. C and D, Cell proliferation of FGA WT and KO cells for 7 days. Data, means SD. , P < 0.05 by two-way ANOVA test versus the WT control group. E, Cell morphology in FGA WT and KO cells. F and 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 10 different 10 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 post hoc test versus the WT control group. J and K, Cell invasion in A549 and H1299 cells for 12 days determined by in vitro soft agar colony formation assay. L and M, Quantifying areas of cell invasion in the cells. Data, means SD. , P < 0.05 by two-tailed t test versus the WT control group. N, Protein expression of CK5/8 and E-cad in the cells measured by Western blot. KO, knockout; CK5/8, cytokeratin 5/8; E-cad, E-cadherin. Data, means SD. , P < 0.05 by one- way ANOVA test, followed by Dunnett post hoc test versus the WT control group. KO, knockout. All experiments were repeated three times.

Administration of FGA induces cell apoptosis in human LUAD expressions of BCL-2 family members and related proteins, such as cells BCL2, BCLXL, MCL1, and cleaved caspase-3 were determined by We next determined the effect of FGA on apoptosis of A549 and Western blot analysis in A549 cells. As shown in Fig. 2E, expressions of H1299 cells. Although a decreased apoptosis was observed in FGA KO BCL2 and MCL1 were gradually decreased but BCLXL was not cells compared with that in FGA WT cells, no statistical signiﬁcance changed after FGA treatment for 12 hours in FGA KO A549 cells, was found (Fig. 2A and B), To address whether FGA induces whereas expression of cleaved caspase-3 was also gradually increased apoptosis, we added FGA (10 mg/mL) into the culture medium to after FGA treatment for 24 hours in both FGA KO A549 and H1299 treat FGA KO A549 and H1299 cells, respectively. At 6, 12, and cells (Fig. 2F), suggesting FGA-induced apoptosis in LUAD cells. 24 hours after treatment with FGA, apoptosis was gradually elevated upon FGA stimulation in both FGA KO A549 and H129 cells (Fig. 2C Administration of FGA inhibits cell proliferation and migration and D), suggesting that FGA induces apoptosis in LUAD cells. BCL-2 in human LUAD cells family-regulated activation of caspase along with apoptosis in cancer We ﬁrst measured the secreted protein levels of FGA in the culture cells contain several different signaling pathways (31). To elucidate the medium of human A549 cells using ELISA. FGA in culture medium molecular mechanism underlying FGA-mediated cell apoptosis, was dramatically reduced in FGA KO cells compared with that in WT

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Figure 2. Effect of FGA on cell apoptosis in A549 and H1299 cells. A and B, Quantitative cell apoptosis of FGA WT and KO cells. C and D, Quantitative cell apoptosis of FGA KO cells after treatment with FGA for 24 hours. Data, means SD. , P < 0.05 by one-way ANOVA test followed by Dunnett post hoc test or two-tailed t test versus the WT control group. E, Protein expression of apoptotic-related proteins in the cells after treatment with FGA for 12 hours was measured by Western blot analysis in FGA KO A549 cells. F, Protein expression of cleaved caspase-3 determined by Western blot analysis after treatment with FGA for 24 hours in FGA KO cells. KO, knockout; A549 KO, FGA KO A549 cells; H1299 KO, FGA KO H1299 cells. All experiments were repeated three times. cells (Fig. 3A). To determine the effect of FGA on cell proliferation, we this observation was confirmed by cell colony assay (Fig. 3K and L) added the recombinant human FGA (10 mg/mL) into the culture and transwell migration assay (Fig. 3M and N). These results suggest medium of A549 FGA KO cells and found a strong inhibition of cell an opposite or competitive role of fibrinogen and FGA in cell growth proliferation by FGA (Fig. 3B), respectively. Cell colony assays further and migration of LUAD cells. identified similar effects of FGA KO on tumor growth (Fig. 3C and D). Likewise, we observed similar effects of FGA on the suppression of cell FGA–integrin a5 interaction regulates the AKT–mTOR signaling migration (Fig. 3E and F). Furthermore, we used FGA WT and FGA pathway in human LUAD cells KO cells, respectively, to coculture with FGA KO cells separated by To investigate the FGA-mediated molecular mechanism in LCAD Transwell chambers, and then counted migrated cells in the lower cells, we performed a coexpression analysis of FGA in human LUAD chamber (Fig. 3G). As shown in Fig. 3H, a decreased number of using the TCGA dataset. The mRNA expression levels of FGA transferred FGA KO cells was observed by transwell migration assay in were positively correlated with that of 192 genes and negatively FGA KO cells cocultured with FGA WT cells as compared with those correlated with that of 156 genes (Spearman correlation coefficient with FGA KO cells. Likewise, an increased FGA in the culture medium r ≥ 0.3 or r ≤ 0.3, P < 0.05; Fig. 4A; Supplementary Table S4). Of was also confirmed by ELISA in FGA KO cells cocultured with FGA note, mRNA expression levels of FGA were highly coexpressed with WT cells (Fig. 3I). These results implicate a suppressive role of FGA in that of FGG (r ¼ 0.92, P < 0.001; Fig. 4B). Next, using these cell growth and migration of LUAD cells. Next, we treated the WT coexpression genes, we performed the KEGG pathway enrichment A549 cells with fibrinogen (10 mg/mL) or fibrinogen (10 mg/mL) plus analysis. The top 3 enriched KEGG pathways, including focal adhe- FGA (10 mg/mL), respectively. Significant induction of cell prolifer- sion, PI3K–AKT signaling, and riboflavin metabolism pathways, ation was observed in the A549 cells after fibrinogen treatment, but this were significantly associated with FGA expression in human LUAD induction was partly reduced by addition of FGA (Fig. 3J). Likewise, (adjusted P < 0.05; Fig. 4C).

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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 and D, Colony formation of FGA WT, KO, and FGA-treated KO cells for 14 days. E and F, The cell migration rate of FGA WT, KO, and FGA-treated KO cells for 24 hours by trans-well migration assay. G and 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 and L, Colony formation of FGA WT A549 cells after treatment with Fibrinogen or Fibrinogen plus FGA for 14 days. M and N, The cell migration rate of FGA WT A549 cells after treatment with Fibrinogen or Fibrinogen plus FGA for 12 hours. Data, means SD. , P < 0.05 by two-way ANOVA test, one-way ANOVA test followed by Dunnett post hoc test or two-tailed t test versus the WT control group. KO, knockout. All experiments were repeated three times.

PI3K–AKT signaling plays a critical role in tumorigenesis of However, in FGA KO A549 cells, phosphorylation of AKTT308 was NSCLC (32, 33). Consequently, we determined the effect of FGA increased in treatment with both recombinant integrin 5a and on the top 2 signaling pathway in A549 cells. As shown in Fig. 4D, FGA as compared with those with FGA alone (Supplementary although expression levels of total AKT were not changed, both Fig. S5), suggesting that integrin 5a may compete with FGA p-AKTT308 and p-AKTS473 were dramatically increased in FGA KO to block the FGA-mediated suppression of AKT activation in A549 cells compared with in FGA WT A549 cells. Likewise, as LUAD cells. critical downstream effectors of AKT–mTOR signaling, p-4EBP1 Focal adhesion is the top ranking pathway associated with FGA and p-S6 were simultaneously upregulated after FGA KO in A549 expression in human LUAD (Fig. 4C). In focal adhesion, integrins cells (Fig. 4D), identifying an FGA loss-induced AKT–mTOR are a/b heterodimeric adhesion glycoprotein receptors that regulate signaling. In addition, we used FGA to treat both FGA WT and a wide variety of dynamic cellular processes, including cell growth, KO A549 cells. Western blot analysis revealed that FGA did not migration, and phagocytosis (34), through major downstream induce p-AKT in WT cells, whereas FGA suppressed phosphor- signal pathways, such as PI3K–AKT signaling pathway, in lung ylation of both AKTT308 and AKTS473 in FGA KO cells (Fig. 4E). cancer progression (35–37). Fibrinogen is a ligand for integrin a5b1

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Figure 4. FGA–integrin interaction and its regulated signaling pathways in A549 cells. A, Heatmap of coexpression of FGA withitsrelatedgenesinmRNAexpression levels. B, Coexpression of FGG with FGA in mRNA expression levels. C, Top signaling pathways related to FGA expression in human LUAD samples using the dataset from TCGA. D, Expression levels of key proteins on the AKT–mTOR signaling pathway determined by Western blot analysis 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, Coimmunoprecipitation of FGA and Integrin a5 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; KO, knockout. All experiments were repeated three times. on endothelial cells (38). Thus, FGA may regulate PI3K–AKT that FGA-mediated suppression of p-AKT may be independent to signaling through integrins in LUAD cells. To test this possibility, fibrinogen in LUAD cells. These datasuggestthatFGAinhibits weimmunoprecipitatedFGAfromA549cellsandprobedthem PI3K–AKT signaling through a direct interaction of FGA with with an anti-integrin a5mAb.AsshowninFig. 4F,anti-FGA integrin 5a in LUAD cells (Fig. 4H). coprecipitated integrin a5, whereas anti-integrin a5 coprecipitated FGA. In previous studies, crystal structure analysis identified the FGA KO facilitates tumor growth and metastasis in human LUAD extracellular segment of integrin a5b3incomplexwithanArg-Gly- cells in vivo Asp (RGD) sequences (39), and functional analysis demonstrated To determine the effect of FGA on tumor growth in vivo, FGA WT that integrin a5b3 binds two specific RGD sequences (amino acids and KO A549 cells were subcutaneously injected, respectively, into 114–116 and 590–593) of FGA (40). We used a mutant recombinant both male and female immunodeficient BALB/c nude mice. Xenograft human FGA (amino acids 124–214) without the RGD sequences to tumor growth was faster in mice with FGA KO A549 cells compared test the binding of mutant FGA to integrin a5inFGA knockout with in WT A549 cells (Fig. 5A and B) up to 4 weeks after injection. A459 cells. As shown in Supplementary Fig. S6A and S6B, there was Likewise, tumor weights were increased in mice with FGA KO A549 no specific binding of mutant FGA to integrin a5 in the cells. In cells than in WT A549 cells at day 28 (Fig. 5C). Increased protein addition, we treated the WT A549 cells with fibrinogen or fibrin- expression of Ki67 but decreased protein expression of E-cadherin ogen plus FGA, respectively. Western blot analysis revealed that was evident in FGA KO xenograft tumors compared with the WT fibrinogen induced phosphorylation of both AKTT308 and AKTS473 xenograft tumors (Fig. 5D–F). Likewise, protein expressions of in A549 cells, whereas FGA dramatically suppressed the p-AKT in p-AKTS473 were also increased in FGA KO xenograft tumors com- the cells regardless of fibrinogen treatment (Fig. 4G), indicating pared with the WT xenograft tumors (Fig. 5D). In addition, we

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conducted a xenograft assay with FGA overexpressed A459 cells in Fibrinogen, fibrin, and their degradation products are involved in both male and female immunodeficient nude mice. As shown in blood clotting, inflammation, angiogenesis, and tumor metasta- Supplementary Fig. S7A–S7C, tumor growth and weights were sis (6, 42). Fibrinogen is thought to originate from exudation of plasma decreased in mice with FGA overexpressed A549 cells as compared fibrinogen and subsequent deposition into the tumor stroma and is with those with WT A459 cells. converted to fibrin polymers, resulting in the inflammatory response Pulmonary metastases of the A549-derived LUAD xenograft within the tumor microenvironment (TME) (43). The fibrin matrix tumors have been observed in nude mice (41). However, we did not may help induce angiogenesis to promote tumor growth and metas- observe lung metastasis in the nude mice at 6 weeks after subcutaneous tasis, while fibrinogen depletion can result in a reduction in tumor injection with FGA WT or KO A549 cells. Thus, to test the role of colonization in the lung (11, 44, 45). Various fibrinogen-derived FGA in tumor metastasis in vivo, we intravenously injected FGA WT peptide fragments also modulate the migration, proliferation, and or KO A549 cells into male and female immunodeficient BALB/c nude differentiation of endothelial cells to affect tumor growth and metas- mice. At 4 weeks after injection, a significant increase in the tumor tasis (6). The products of fibrin degradation (E and D fragments) can number and burden of lung metastases were observed in the mice stimulate the proliferation, migration, and differentiation of endothe- injected with FGA KO cells as compared with those with WT A549 lial cells, contributing to tumor vasculature, progression, and metas- cells (Fig. 5G–I). Significant increases of Ki67 and p-AKTS473 in the tasis (42). However, as polypeptide chains of fibrinogen, FGA-derived xenografted metastatic tumors were also detected in FGA KO cells fragment (a 15-amino acid peptide) inhibits endothelial cell migration, (Fig. 5J and K). These data suggest that FGA KO promotes A549 cell adhesion, and tubule formation to reduce tumor growth (46). Likewise, growth and colonization in vivo. FGB-derived fragment (a beta 43–63-amino acid peptide) is an inhibitor of activated endothelial cells and reduces tumor vasculari- An inverse relationship between protein expression of FGA and zation but induces the formation of tumor necrosis (14). Thus, p-AKT in human primary lung cancer specimens fibrinogen, components, and their derivatives appear to play different We next evaluated, by IHC, the protein expressions of FGA and roles in endothelial cells within the TME. Currently, in tumor cells, the p-AKTS473 and their relationship in 50 human primary lung cancer role of fibrinogen, components, and their derivatives are not suffi- tissues, including LUAD, LSCC, lung adenosquamous carcinoma, ciently understood. Fibrinogen can directly be synthesized and secret- and SCLC (Supplementary Table S3). The protein expression of FGA ed by breast cancer cells and assembles into the extracellular matrix in tumor cells was found in 36% (18/50) of total cases, including 41% and reduces cancer cell migration (5). In lung cancer cells, fibrinogen (9/22) of LUAD, 32% (6/19) LSCC, and 29% (2/7) SCLC cases augments tumor cell proliferation through interaction with fibroblast (Supplementary Table S3; Supplementary Fig. S7A). However, expres- growth factor-2 (47) but blocks tumor cell migration (48). In the sion of p-AKTS473 was found in 66% (33/50) of total cases, including current study, FGA KO induces proliferation, migration, and EMT of 59% (13/22) of LUAD, 79% (15/19) LSCC, and 57% (4/7) SCLC cases LUAD cells in vitro and promotes LUAD xenograft tumor growth and (Supplementary Table S3; Fig. 6A). H-score quantitative analysis lung metastasis in vivo, but administration of FGA inhibits LUAD cell showed a negative correlation of protein expressions of FGA with growth, migration, and invasion, supporting an inhibiting role of FGA p-AKTS473 (Fig. 6B). Furthermore, no expression of FGA or expres- against LUAD cells. In addition, fibrinogen binds integrin a5b3 sion of p-AKTS473 was likely to be associated with poor 5-year disease- through two specific RGD sequences of FGA but not FGB or free survival, but these differences were not statistically significant FGG (39, 40). Although FGA KO promotes tumor growth and (Fig. 6C and D). However, expression of FGA without expression of metastasis through integrin a5, FGB or FGG unlikely has similar p-AKTS473 was significantly associated with a better disease-free effects on LUAD. survival as compared with no expression of FGA with the expression The mechanism by which functional roles are different between of p-AKTS473 (Fig. 6E). In addition, the expression of FGA was not fibrinogen and FGA in tumor growth and metastasis remains associated with the histologic type and tumor stages and grades unknown. In the current study, we observed a significant induction (Supplementary Table S3). These data suggest that the downregu- of cell proliferation and migration in LUAD cells by fibrinogen lation of FGA with the upregulation of p-AKT is likely to be a poor treatment, but this induction was partly reduced by the addition of prognostic factor in human lung cancer. 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 Discussion cells, but this induction was blocked by the addition of FGA in a On the basis of our bioinformatics analysis, genetic alterations of fibrinogen-independent manner. These data suggest that FGA may FGA are unlikely to be a frequent event in human lung cancers. compete with fibrinogen to inhibit LUAD cell growth and migration However, our expression pattern analysis showed a low expression through AKT signaling, but also has a fibrinogen-independent effect of FGA in human lung cancer tissues, including LUAD and LUSC. Of on AKT signaling through a direct interaction of FGA with integrin a5 note, DNA hypermethylation in the promoter region of FGA is in LUAD cells. However, the mechanism by which fibrinogen and FGA correlated with low expression of FGA in human LUAD tissues, interact or compete against tumor cells or TME cells in vivo remains to suggesting an epigenetic mechanism in the transcriptional regulation be elucidated by further studies. of FGA in LUAD. Furthermore, in our experimental data, FGA KO Many soluble secretory proteins released from cancer cells into the promotes but the administration of FGA inhibits LUAD cell growth, extracellular space are involved in inflammation and angiogenesis migration, and invasion, as well as tumor colonization in the lung. Our during tumor growth and metastasis (49, 50). As a secretory protein, functional analysis revealed the FGA-mediated regulation of tumor fibrinogen binds integrins (e.g., a5b1, a2bb3, and a5b3; ref. 51) in growth and metastasis through apoptosis and EMT is also involved in endothelial cells to promote tumor growth and metastasis. Fibrinogen the integrin–AKT signaling pathway in LUAD cells in vitro and has critical roles in tumor metastasis by facilitating the adhesion of xenograft tumor model in vivo. These data suggest that FGA plays pancreatic tumor cells to endothelial cells and transendothelial migra- a suppressive role in the growth and metastasis of LUAD cells. tion and extravasation (52). However, fibrinogen polypeptide chains,

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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 ¼ 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 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 Ki67þ cells as an indicator of proliferating cells among the xenograft tumor tissues. At least five 40 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 ¼ 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 Ki67þ cells as an indicator of proliferating cells among the lung metastatic tissues. Data, means SD. , P < 0.05 by two-way ANOVA test or two-tailed t test versus the WT control group. KO, knockout; CK5/8, cytokeratin 5/8; E-cad, E-cadherin. All in vivo experiments were repeated twice.

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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 and 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-AKTS473. All experiments were repeated twice.

FGA, FGB, and FGG are downregulated during EMT of lung cancer and reduce phosphorylation of AKT, leading to an inhibition of cells (53). In HepG2 cells, knockdown of FGA promotes cell apoptosis, mTOR signaling. Administration of FGA may provide a new suggesting an FGA-mediated inhibition of apoptosis in cells, and therapeutic approach to inhibit LUAD cell growth and metastasis. expression of BCLXL and MCL1 are likely to be decreased, but BCL2 However, FGA may also affect TME cells in vivo,suchasendothelial is increased in the FGA knockout-downed cells (15). However, FGA- cells, leading to the various rolesofFGAintumorgrowthand derived fragment induces apoptosis and blocks tube formation of metastasis. endothelial cells in gastric cancer, suggesting an apoptotic role of FGA in antiangiogenesis (46). In the current study, the administration of Disclosure of Potential Conﬂicts of Interest ﬂ FGA induces apoptosis through downregulation of BCL2 and MCL1 No potential con icts of interest were disclosed. but not BCLXL in LUAD cells. Of note, FGA directly binds to integrin Authors’ Contributions a5 and stimulates AKT–mTOR signaling, suggesting a functional – – Conception and design: R. Liu FGA integrin AKT axis in LUAD cells. Likewise, in TCGA dataset, Development of methodology: S. Gao, W.-H. Yang survival analysis also showed an opposing role of FGA between Acquisition of data (provided animals, acquired and managed patients, provided patients with liver cancer and renal cancer (Supplementary Fig. S8). facilities, etc.): M. Wang, Y. Zhang, Y. Liu Thus, the role of FGA in apoptosis is likely to be different between Analysis and interpretation of data (e.g., statistical analysis, biostatistics, various cell types, but the underlying mechanism remains to be computational analysis): X. Cui, S. Wang, J.H. Bae, R. Liu elucidated by further studies. Writing, review, and/or revision of the manuscript: X. Cui, R. Liu Administrative, technical, or material support (i.e., reporting or organizing data, In conclusion, FGA may play a suppressive role in LUAD cells to constructing databases): Y. Wang, L.S. Qi, L. Wang inhibit tumor growth and metastasis through induction of apoptosis Study supervision: R. Liu and inhibition of EMT. In LUAD cells, FGA can bind integrin a5 Other (carried out the experiments): G. Zhang, S. Gao

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Acknowledgments The costs of publication of this article were defrayed in part by the We thank Dr. Jonathan Leavenworth for editorial assistance in preparing this payment of page charges. This article must therefore be hereby marked manuscript. This work was supported by grants from the Mike Slive Foundation advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate for Prostate Cancer Research (L. Wang and R. Liu), the Breast Cancer this fact. 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 Received October 18, 2019; revised February 28, 2020; accepted March 18, 2020; Research Network: http://cancergenome.nih.gov/. published ﬁrst March 23, 2020.

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Fibrinogen Alpha Chain Knockout Promotes Tumor Growth and Metastasis through Integrin−AKT Signaling Pathway in Lung Cancer

Meng Wang, Guangxin Zhang, Yue Zhang, et al.

Mol Cancer Res Published OnlineFirst March 23, 2020.

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