Therapeutic Targeting of Aldolase a Interactions Inhibits Lung Cancer Metastasis

Author Manuscript Published OnlineFirst on July 29, 2019; DOI: 10.1158/0008-5472.CAN-18-4080 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Therapeutic Targeting of Aldolase A Interactions Inhibits Lung Cancer Metastasis

2 and Prolongs Survival

4 Yu-Chan Chang 1, Jean Chiou1, Yi-Fang Yang2, Chia-Yi Su1, Yuan-Feng Lin3, Chia-Ning Yang4,

5 Pei-Jung Lu5, Ming-Shyan Huang 6, Chih-Jen Yang7* and Michael Hsiao 1,8*

7 1. Genomics Research Center, Academia Sinica, Taipei, Taiwan.

8 2. Translational Research Center, Kaohsiung Medical University Hospital, Kaohsiung Medical

9 University, Kaohsiung, Taiwan.

10 3. Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei,

11 Taiwan.

12 4. Department of Life Sciences, National University of Kaohsiung, Kaohsiung, Taiwan.

13 5. Institute of Clinical Medicine, Medical College, National Cheng Kung University, Tainan, Taiwan

14 6. Department of Internal Medicine, E-DA Cancer Hospital, School of Medicine, I-Shou University,

15 Kaohsiung, Taiwan.

16 7. Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical

17 University, Kaohsiung, Taiwan.

18 8. Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung,

19 Taiwan.

21 *To whom correspondence should be addressed:

22 Dr. Michael Hsiao, Genomics Research Center, Academia Sinica, 128 Academia Rd., Sec. 2,

23 Nankang-Dist., Taipei, Taiwan. Tel: +886-2-2787-1243, Fax: +886-2-2789-9931, E-mail:

24 [email protected]

25 Or to

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2019; DOI: 10.1158/0008-5472.CAN-18-4080 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Dr. Chih-Jen Yang, Department of Internal Medicine, Kaohsiung Municipal Ta-Tung Hospital,

2 Kaohsiung Medical University, No. 68 Chunghwa 3rd Road, Cianjin District, 80145 Kaohsiung City,

3 Taiwan. Tel: +886-7-320-8159, Email: [email protected]

5 Competing financial interest

6 The authors declare that they have no competing interests.

9 Running Title: Targeting of ALDOA inhibits lung cancer metastasis

1 Abstract

2 Cancer metabolic reprogramming promotes tumorigenesis and metastasis, however, the underlying

3 molecular mechanisms are still being uncovered. In this study, we show that the glycolytic enzyme

4 aldolase A (ALDOA) is a key enzyme involved in lung cancer metabolic reprogramming and metastasis.

5 Overexpression of ALDOA increased migration and invasion of lung cancer cell lines in vitro and

6 formation of metastatic lung cancer foci in vivo. ALDOA promoted metastasis independent of its

7 enzymatic activity. Immunoprecipitation and proteomic analyses revealed gamma-actin binds to

8 ALDOA; blocking this interaction using specific peptides decreased metastasis both in vitro and in vivo.

9 Screening of clinically available drugs based on the crystal structure of ALDOA identified raltegravir, an

10 anti-retroviral agent that targets HIV integrase, as a pharmacological inhibitor of ALDOA-gamma-actin

11 binding that produced anti-metastatic and survival benefits in a xenograft model with no significant

12 toxicity. In summary, ALDOA promotes lung cancer metastasis by interacting with gamma-actin,

13 targeting this interaction provides a new therapeutic strategy to treat lung cancer metastasis.

15 Significance

16 This study demonstrates the role of aldolase A and its interaction with γ-actin in the metastasis of

17 non-small lung cancer and that blocking this interaction could be an effective cancer treatment.

19 Key word: Aldolase A, non-glycolytic function, raltegravir

1 Introduction

2 Lung cancer is the most common cancer in terms of mortality and incidence rates worldwide, and

3 non-small cell lung cancer (NSCLC) accounts for 80% in these cohorts (1). Metastasis remains a major

4 cause of disease mortality and failure for NSCLC after treatment. Genetic alteration events involved in

5 the aggressive progression of lung cancer have been recently reported, but the precise molecular

6 mechanisms for this progression remain unclear (2). Additionally, metabolic variation has been

7 demonstrated as a mechanism for tumorigenesis (3). In fact, Otto Warburg demonstrated that

8 malignancies turn over glycolysis in the absence of oxygen concentration to produce adenosine

9 triphosphate (ATP), the so-called Warburg effect, for progressive development (4). Physiologically,

10 glycolysis is the initial step for glucose metabolism, and the subsequent intermediates can be converted

11 to synthesize lipid acid, amino acids or nucleotides (5). Recent studies reported that the glycolytic

12 enzymes at each step of glycolysis has been aberrantly over-activated under hypoxia or oncogenic

13 stimulation (6). Therefore, several studies have been performed to identify promising glycolytic

14 enzymes or metabolic pathways through proteomic or high-throughput biochemical approaches to

15 identify novel biomarkers for diagnosis or druggable targets for cancer therapy (7-9). Although several

16 inhibitors of glycolytic enzymes have been developed as anti-cancer agents, their combination with

17 other chemotherapeutics or identification of cancer-specific glycolytic enzymes has still been pursued to

18 reduce off-target effects (10).

19 Fructose-bisphosphate aldolase is a member of the family of the glycolytic enzymes that catalyzes

20 the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde-3-phosphate (GAP) and

21 dihydroxyacetone phosphate (DHAP) (11). The family comprises 3 members: ALDOA, ALDOB and

22 ALDOC. Differences indicate that aldolases A, B, and C are distinct proteins, and the products of this

23 family show developmentally regulated expression and location (12). ALDOA is highly conserved from

24 rabbits to humans, and it is the most extensively studied isozyme of the aldolase family (13). ALDOA

25 protein has demonstrated higher expression in various types of tumor cells (14-21). However, recent

1 studies have shown that the only well-established effect of ALDOA is keratinocyte migration following

2 the induction of lamellipodia formation (11) or epithelial-mesenchymal transition (EMT) (22).

3 Additionally, multiple growth factor signaling pathways crosslink with alternative metabolism,

4 including the EGFR/MAPK, MEK-ERK or PI3K/AKT-mTOR pathways (23-25). However, the link

5 between ALDOA and metabolism in tumorigenesis pathways is still unknown.

6 The nonglycolytic functions of enzymes involved in glycolysis have recently been identified to be

7 predominantly associated with the mechanisms of cancer development (26-31). For example, pyruvate

8 kinase M2 (PKM2) is extensively upregulated in tumors, allowing for high lactate production via

9 aerobic glycolysis, the so-called Warburg effect, thereby promoting tumor growth (32). Moreover,

10 PKM2 acts as a phosphotyrosine-binding protein (33) and appears to be phosphorylated by epidermal

11 growth factor receptor (EGFR)-activated ERK2 directly via a protein-protein interaction (PPI) before

12 translocation into the nucleus, where PKM2 acts as a coactivator of -catenin (34) or HIF-1α (35) or as

13 a kinase to phosphorylate histone H3 (36) to promote gene transcription for tumor growth. Despite the

14 above evidence, the PPIs of other glycolytic enzymes, apart from their role in glucose metabolism, may

15 reprogram cellular signaling networks and gene transcription, thereby facilitating cancer progression.

16 Therefore, identifying the cancer-associated PPIs of glycolytic enzymes is valuable to further develop

17 new anti-cancer agents with fewer side effects.

18 Using an RNA interference (RNAi)-based screen, we have determined that, compared with other

19 glycolytic enzymes, aldolase A (ALDOA) is a key molecule that mediates the in vitro

20 migration/invasion abilities of lung cancer cells. Notably, upregulated ALDOA forms a

21 cancer-associated PPI with -actin, irrespective of enhanced glycolysis, and ultimately promotes lung

22 cancer metastasis. Raltegravir, which blocks the binding of ALDOA with -actin, reduces the metastasis

23 ability in vitro and prolongs survival rate in vivo. These findings demonstrate a novel therapeutic

24 potential for ALDOA and a new aspect of targeting nonglycolytic PPIs for cancer therapy.

1 Materials and Methods

2 Cell Culture and Stable Clone Establishment

3 The human lung adenocarcinoma cell lines H1355, CL1-0 and CL1-5 and the large cell carcinoma

4 cell line H661 were grown in RPMI 1640 medium supplemented with 10% FBS (Invitrogen, Carlsbad,

5 CA, USA). The human lung adenocarcinoma cell lines A549 and PC14 and the large cell carcinoma cell

6 line H1299 were grown in DMEM supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA). All

7 cells were incubated under a humidified atmosphere of 5% CO2 at 37°C. The pGIPZ lentiviral

8 shRNAmir system (Thermo, Waltham, MA, USA) and ALDOA sequence were used to establish stable

9 cell lines. Lentiviruses were used to infect the cells for two days. Stable clones were selected with 1

10 μg/ml of puromycin (Sigma, St. Louis, MO, USA) for 2 weeks. The cell lines CL1-0 and CL1-5 were

11 established and provided as a gift from Dr. Pan-Chyr Yang (National Taiwan University, Taipei, Taiwan).

12 The cell lines A549, H1355 and H1299 were purchased from the American Type Culture Collection

13 (ATCC, Manassas, VA, USA) cell bank.

14 RT-PCR Analysis

15 The cells were lysed in TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA was

16 extracted according to the manufacturer’s protocol. The amount of RNA was measured using a

17 Nanodrop spectrophotometer (Thermo, Waltham, MA, USA). Reverse transcription-PCR (RT-PCR) was

18 performed using the SuperScript III kit (Invitrogen, Carlsbad, CA, USA) according to the

19 manufacturer’s protocol. The expression levels of target genes were normalized to those of the

20 ribosomal protein 26S, which was used as an internal control. The specific primer sequences and

21 template conditions are provided in the key resources table.

22 Western Blot Analysis

23 Western blot analysis was performed with primary antibodies directed against ALDOA (Cat No.

24 T0891; Epitomics, Cambridge, MA, USA), ACTG1 (Cat No. GTX1794; GeneTex, Hsinchu City,

25 Taiwan), ACTG2 (Cat. No GTX 2926; GeneTex, Hsinchu City, Taiwan), His-tag (Cell Signaling,

1 Beverly, MA, USA) or α-tubulin (Sigma, St. Louis, MO, USA). Immunoreactive bands were visualized

2 using an enhanced chemiluminescence (ECL) system (Amersham ECL Plus™; GEHealthcare Life

3 Sciences, Chalfont, St. Giles, UK).

4 Pull-down Assay and Mass Spectrometry Analysis

5 Whole-cell lysates (2 mg) from cells with the forced expression of a vector with or without the

6 His-tagged ALDOA gene were incubated overnight with 20 μl of Ni-NTA beads (GE Healthcare Life

7 Sciences, Chalfont St. Giles, UK). ALDOA-interacting proteins were purified according to the

8 manufacturer’s protocol. After the protein mixtures were in-solution digested with trypsin, they were

9 subjected to protein identification by mass spectrometry.

10 Case Selection

11 In total, 107 patients diagnosed with non-small cell lung cancer at the Kaohsiung Medical University

12 Hospital in Taiwan from 1991 to 2007 were included in this study. All cases were staged according to

13 the 7th edition of the Cancer Staging Manual of the American Joint Committee on Cancer (AJCC), and

14 the histological cancer type was classified according to the World Health Organization (WHO) 2004

15 classification. Patients who received preoperative chemotherapy or radiation therapy were excluded.

16 Clinical information and pathology data were collected via a retrospective review of patient medical

17 records. Follow-up data were available in all cases, and the longest clinical follow-up time was 190

18 months. The study was performed with the approval of the institutional review board and with

19 permission from the ethics committee of the institution involved (KMUH-IRB-2011-0286).

20 Requirement for informed consent was waived by the Institutional Review Boards of Kaohsiung

21 Medical University Hospital of Taiwan. All data were analyzed anonymously, and no identifying

22 information related to the participants were included. Overall survival and disease-free survival were

23 defined as the intervals from surgery to death caused by non-small cell lung cancer and recurrence or

24 distant metastasis, respectively.

1 Lung Metastatic Foci Assay

2 Age-matched, nonobese, diabetic-severe, combined immune deﬁcient gamma

3 (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ JAX®, NOD-SCID γ) male mice (age: 6-8 weeks; body weight:

4 20-25 g) were used. All animal experiments were conducted in accordance with a protocol approved by

5 the Academia Sinica Institutional Animal Care and Utilization Committee (IACUC). To evaluate lung

6 colony-forming ability, 5×106 (for CL1-5 cells) or 1×106 (for CL1-0 cells) cells were resuspended in 100

7 μl of PBS and were injected into the lateral tail vein. Lung nodule formation was quantified after H&E

8 staining using a dissecting microscope at the experiment endpoint.

9 Statistical Analysis

10 The non-parametric Mann-Whitney U-test was used to analyze the statistical significance of

11 results from three independent experiments. Statistical analyses were performed using SPSS (Statistical

12 Package for the Social Sciences) 17.0 software (SPSS, Chicago, IL, USA). The association between

13 clinicopathological categorical variables and the ALDOA IHC expression levels were analyzed by

14 Pearson’s chi-square test. Estimates of the survival rates were calculated using the KM method and were

15 compared using the log-rank test. Univariate and multivariate analyses were performed using Cox

16 proportional hazards regression analysis with and without an adjustment for the ALDOA IHC

17 expression level, tumor stage, lymph node stage, and metastasis. Paired t-test was performed to compare

18 the ALDOA IHC expression levels in cancer tissues and corresponding normal adjacent tissues. The

19 follow-up time was censored if the patient was lost during the follow up. For all analyses, a P value

20 <0.05 was considered significant.

22 Other materials and methods are provided in the online supplement.

1 Results

2 ALDOA is a critical glycolytic enzyme that promotes the metastatic progression of lung cancer

3 cells and predicts poor clinical outcomes

4 To determine the critical glycolytic enzyme(s) (Figure 1A) whose upregulation likely correlates with the

5 migration/invasion abilities of lung cancer cells, we analyzed the gene expression levels of these

6 enzymes using previously published microarray results (37) obtained from the transcriptional profiling

7 of CL1-5 (highly invasive) lung cells and their counterparts, CL1-0 (poorly invasive) lung cancer cells

8 (Figure 1B). We found that the gene expression levels of several glycolytic enzymes, including aldolase

9 A (ALDOA), were causally associated with the progressive invasiveness of these sublines from human

10 lung adenocarcinoma (38). Additionally, we observed high gene expression levels of several lipid

11 synthesis-related enzymes in CL1-5 compared with those in CL1-0 cells. We proposed that glycolytic

12 enzymes that trigger lipid biosynthesis and phospholipase production for lipid raft creation shift cells

13 toward further cancer metastasis. Therefore, we determined the knockdown efficiency by two

14 independent shRNA clones and evaluated the migration/invasion abilities of the highly invasive lung

15 cancer cell lines CL1-5 and H1299 (30) after an RNA interference (RNAi)-based high-throughput

16 screening assay against these enzymes (Figure 1C and Supplemental Figure 1). Among the identified

17 glycolytic enzymes, we found that ALDOA knockdown most significantly suppressed the

18 migration/invasion abilities of the highly invasive CL1-5 and H1299 lung cancer cells (Figure 1C).

19 Moreover, our data showed that the endogenous protein level of ALDOA (Figure 1D) was positively

20 correlated with cellular invasion activity in a panel of lung cancer cell lines (Figure 1E). We also

21 examined the endogenous protein level of ALDOB and ALDOC in the lung cancer cell panel. Our data

22 showed that rare cancer cell lines have ALDOB/ALDOC expression (Supplemental Figure 2). The

23 ectopic expression of an exogenous ALDOA gene in the less invasive CL1-0 lung cancer cells

24 dramatically enhanced the migration/invasion abilities of these cells (Figure 1F). By contrast, the

25 migration/invasion abilities of the highly invasive cell lines CL1-5 was decreased by ALDOA

1 knockdown in vitro (Figure 1G). Furthermore, the functional phenotypes of in vivo studies were

2 consistent with in vitro studies (Figure 1H and 1I). Similar results were also found in other lung cancer

3 cell lines, H1355 and H1299 (30) (Supplemental Figure 3A-3B). Our evidence also supports the dual

4 role of ALDOA in the control of glycolysis pathways and lipid biosynthesis.

6 ALDOA plays a nonenzymatic function role in lung cancer metastasis

7 To determine whether ALDOA-regulated lung cancer metastasis ability occurred through an

8 accelerated rate of glycolysis by an increased final product based on enzyme activity, we performed a

9 functional assay in medium containing low (5.5 mM) and high (22 mM) glucose concentrations

10 compared with the normal glucose concentration (11 mM). The results showed that ALDOA could

11 promote the migration/invasion abilities of lung cancer cells in each condition (Figure 2A). We further

12 recruited 4-deoxyglucose (4-DG), an inhibitor that partially inhibited aldolase enzyme activity in our

13 cell models (10). 4-DG dose-dependently inhibited enhanced ALDOA activity but showed no obvious

14 inhibitory effects on the promotion of migration/invasion abilities in CL1-0 and H1355 cells with forced

15 expression of wild-type ALDOA (Figure 2B and 2C). Similarly, the migration/invasion abilities in

16 CL1-5 and H1299 cells were failed to be inhibited after we efficiently suppressed the enzyme activity of

17 ALDOA. (Figure 2D and Supplemental Figure 4A-4B). However, our results indicated that 4-DG has no

18 significant effect on cell viability (Supplemental Figure 4C). Furthermore, we performed site-directed

19 mutagenesis of residues 33 (D to A), 361 (H to Y) and 363 (Y to S), which are at the catalytic site of

20 ALDOA and are thought to be critical for the catalytic activity of ALDOA (39). While the forced

21 expression of exogenous ALDOA mutants did not increase intracellular ALDOA activity (Figure 2E and

22 2F), surprisingly, these mutants could enhance the migration/invasion abilities (Figure 2E, 2F,

23 Supplemental Figure 4D) of CL1-0 or H1355 cells. Additionally, we purchased another inhibitor,

24 2,3-butanedione, which was previously reported to partially inhibit aldolase expression or enzyme

25 activity (40,41).In our model, we observed the consistent trends between 2,3-butanedione and 4-DG that

1 ALDOA promotes the migration/invasion ability in lung cancer cells with or without enzyme activity

2 (Supplemental Figure 5A-5C). Therefore, we found that ALDOA plays a novel nonenzymatic role to

3 promote lung cancer metastasis.

5 ALDOA forms a protein-protein interaction with γ-actin to enhance the metastatic potential of

6 lung cancer cells nonenzymatically

7 To elucidate the possible nonglycolytic function of ALDOA, we next forced the expression of a

8 hexameric histidine (6xHis)-tagged ALDOA construct in CL1-0 cells before performing pull-down

9 assays. Mass spectrometry analysis revealed that the γ-actin subtypes 1 and2 (ACTG1/2) are relatively

10 abundant in the ALDOA-pull-down protein mixtures (Figure 3A). Because the binding of ALDOA with

11 γ-actin has been previously observed (42) , further experiments were performed to determine the

12 importance of their PPI in cancer metastasis. Our data revealed that both subtypes of γ-actin could bind

13 to ALDOA (Figure 3B) and that the knockdown of γ-actin compromises ALDOA

14 overexpression-enhanced migration/invasion abilities in CL1-0 cells (Figure 3C and Supplemental

15 Figure 6A). Utilizing the previous report (43) that γ-actin binds to ALDOA through intermolecular

16 interactions with residues K288, K293, and K341 of ALDOA (Figure 3D), we next performed

17 site-directed mutagenesis of residue 293 (K to A) in ALDOA. Compared with wild-type ALDOA and

18 the D33A mutant, the forced expression of K293A clearly blocked the PPI of ALDOA with γ-actin in

19 CL1-0 cells (Figure 3E). The K293A mutation did not affect the ALDOA overexpression-enhanced

20 enzymatic activity of ALDOA (Figure 3F) and glycolysis as judged by increased glucose uptake and

21 lactate production, whereas the D33A mutation compromised these activities (Supplemental Figure 6B)

22 in CL1-0 cells. Nevertheless, the K293A mutant, but not the D33A mutant, dramatically suppressed the

23 ALDOA overexpression-promoted migration/invasion abilities (Figure 3F) in CL1-0 cells. We also

24 assessed the R42A mutant form of ALDOA that was identified as the critical mutation to disrupt actin

25 binding (44,45). The results showed the consistency trend with the ALDOA K293A mutant form that

1 decreased the metastasis ability and F-actin/G-actin ratio without enzyme activity in R42A/K293A

2 overexpression stable cells compared with the wild-type in CL1-0 cells (Supplemental Figure 7A-7C).

3 Furthermore, we investigated the interaction between ALDOA and γ-actin in normal cells and cancer

4 cell lines. We found that the interaction with γ-actin was specific to ALDOA, not ALDOB or ALDOC.

5 The interaction was cancer cell specific and strongly associated with invasion ability (Figure 3G and

6 3H). Additionally, we detected β-actin by the ALDOA antibody pull-down assay in cell models. Our

7 data indicated that ALDOA does not directly bind to β-actin in various primary cells and lung cancer

8 cell lines (Supplemental Figure 8A-8B). Moreover, we observed actin filament accumulation when

9 ALDOA directly binds to γ-actin using the F-actin/G-actin ratio assay (Figure 3I) (46,47), with

10 phalloidin as a marker for immunofluorescence (Supplement Figure 9A). Although fluorescent

11 phalloidin stained F-actin in cells in a single step at the nanomolar level, phalloidin may only represent

12 actin polymerization that does not fully reflect the binding of directional aldolase to γ-actin or β-actin.

13 Therefore, we performed immunofluorescence assays, and the results showed data consistent with

14 immunoprecipitation assay data. We detected the protein expression of ALDOA combined to γ-actin or

15 β-actin in CL1-0 stable cell models by immunofluorescence assays, indicating the colocalization status

16 between ALDOA and γ-actin (Supplement Figure 9B). On the other hand, β-actin appears to have no

17 significant role in the ALDOA-induced lung cancer metastasis model (Supplement Figure 9C). The

18 evidence showed that the interaction between ALDOA and γ-actin regulated dynamic actin

19 polymerization in cancer metastasis.

21 Targeting ALDOA blocks lung cancer metastasis via the protein-protein interaction of ALDOA

22 with -actin involving a shift in the actin cytoskeleton

23 To block the interaction of ALDOA with -actin in cancer cells, we designed a specific peptide to

24 target sites 286-302aa in the ALDOA structure (Figure 4A) that included the previously reported

25 binding sites 288 and 293 (43,48). Initially, we utilized the peptide to validate whether the interaction

1 between ALDOA and -actin was blocked, and we found that the peptide treatment resulted in a

2 dramatic decrease in the interaction between ALDOA and -actin in CL1-5 cells and CL1-0 ALDOA

3 cells (Figure 4B). The peptide could also reverse the phenotype of ALDOA-induced metastatic ability in

4 cells expressing high levels of ALDOA and in an ALDOA overexpression model (Figure 4C and 4D).

5 Next, we analyzed glucose consumption, lactate production and aldolase enzyme activity under peptide

6 treatment, and we did not observe any significant differences compared with the sham group

7 (Supplemental Figure 10). Consistent with these results, in an immunofluorescence assay, phalloidin

8 staining revealed that the actin filaments were significantly reduced by peptide treatment and that the

9 ALDOA/γ-actin interaction in the cytoskeleton was blocked by a mutant form of ALDOA (Figure 4E).

10 Using an actin filament assay, we investigated whether there was a dynamic shift in the cytoskeleton in

11 the ALDOA model upon peptide treatment. We found that ALDOA could promote F-actin

12 polymerization by interacting with γ-actin, and this polymerization was reversed to G-actin in response

13 to peptide treatment (Figure 4F). Additionally, we also confirmed that the peptide did not induce

14 cytotoxicity in a dose-dependent experiment using normal Beas2B cells and the cancer cell lines CL1-0

15 and CL1-5 (Figure 4G). Finally, we established the metastatic in vivo model through orthotopic injection.

16 Our data showed that the peptide could block invasion from the left to the right lobes of the lung and

17 that it prevented distant metastasis to the liver and other organs. (Figure 4H). The peptide treatment

18 further significantly prolonged animal survival in the peptide group (mean: 68 days) compared with

19 sham group (mean: 41 days) (Figure 4I).

21 Identification and characterization of a small-molecular inhibitor to reverse the ALDOA/-actin

22 phenotype

23 To confirm the ALDOA-specific peptide for potential clinical use, we processed virtual screening

24 on ALDOA/γ-actin interaction sites. This action allows the identification of several drug candidates for

25 further evaluation. We dissected the monomer (363 a.a.) of the hALDOA homotetramer structure

1 (PDB:1ALD) with a monomer (374 a.a.) of the hF-actin structure by cryo-EM (PDB:5JLH). The data

2 showed that the K293-base pocket in ALDOA acted as a more significant linkage point for small

3 molecules but not as a competitive manner with ALDOA-γ-actin. We further predicted and selected

4 candidate compounds from DrugBank that were recruited and previously approved drugs for clinical use

5 (07-01-2016:1861 drugs). Through a series of selected formulas, including LidDock, LigandFit and

6 CDOCKER, we validated several compounds in the top ranking for further experimentation (Figure 5A).

7 We processed these in a high-throughput screening for malignant lung cancer cells and an

8 ALDOA-overexpressed model. The results showed that RAL could compromise the migration ability

9 induced by ALDOA (Figure 5B). We further determined raltegravir (RAL) to be the most significant in

10 terms of efficiency (Supplemental Figure 11A) to reverse the G-actin/F-actin ratio induced by ALDOA

11 compared with other ranking drugs (Figure 5C and Supplemental Figure 11B). We also confirmed that

12 RAL could dose-dependently inhibit the migration ability in an ALDOA-overexpression model and

13 malignancy. Additionally, RAL did not produce strong toxicity in lung cancer cells (Supplemental

14 Figure 12A-12B). Therefore, we evaluated RAL efficiency in vivo using a subcutaneous and a lung

15 orthotopic model. Our results showed that RAL indeed significantly inhibited tumorigenesis (Figure 5D)

16 and nodule formation ability (Figure 5E and 5F). Moreover, RAL could prolong the mouse survival rate

17 (mean: 73 days) >2-fold relative to the control group (mean: 39 days) (Figure 5G). Combining all of this

18 evidence, RAL had a similar potential as the specific peptide against ALDOA/γ-actin binding in

19 metastatic lung cancer.

21 ALDOA overexpression predicts poor clinical outcomes in lung cancer patients

22 We performed global meta-analysis of the RNA-sequencing results from The Cancer Genome Atlas

23 (TCGA) database. The data showed that the transcriptional activities of ALDOA were positively

24 correlated with several cancer types, including lung, GBM and liver cancer (Supplemental Figure 13A).

25 The results showed strong consistency with the Kaplan-Meier (KM) Plotter database

1 (http://kmplot.com/analysis/). We also found that high ALDOA gene expression, which was detected by

2 2 different probes on the microarray, was significantly correlated with poor overall and first

3 progression-free (Figure 6A and Supplemental Figure 13B) survival probabilities in clinical patients

4 with lung cancer. Furthermore, we performed global meta-analysis of the ALDOA gene using the

5 PrognoScan database (http://www.prognoscan.org/). We performed IHC staining in clinical lung cancer

6 tissues using an ALDOA-specific antibody (Figure 6B and Supplemental Table 1) to validate these

7 findings. The IHC results demonstrated that an increased ALDOA protein level predicted poorer overall

8 and disease-free survival rates in patients with lung cancer (Figure 6C) and correlated with several

9 clinicopathologic parameters, including N and M stage (Supplemental Table 2). Using the defined

10 scoring criteria, we found that ALDOA expression was significantly correlated with the lymph node

11 status and acted as an independent predictive factor for unfavorable outcomes (Supplemental Table 2) in

12 lung cancer. (Figure 6D, 6E and 6F). A previous study noted the coordination of glycolysis and

13 cytoskeletal remodeling by PI3K signaling (44). PI3K activation accelerates actin dynamics via Rac

14 increasing the level of free, cytoplasmic aldolase, thereby coordinating the generation of ATP and

15 biomass with the energy-intensive process of cytoskeletal remodeling. Moreover, we demonstrated that

16 ALDOA can directly interact with the inverse phenotype of γ-actin, which promotes actin

17 polymerization for cancer metastasis, with or without the enzymatic activity of lung cancer (Figure 7).

1 Discussion

2 Here, we demonstrated that ALDOA upregulation enhances metastatic ability in lung cancer cells.

3 Notably, changing the external glucose concentration or overexpressing an enzyme-dead construct of

4 ALDOA did not alter the metastatic potential of lung cancer cells, indicating the existence of a

5 non-glycolytic mechanism of ALDOA overexpression-enhanced lung cancer metastasis. By performing

6 a His-tagged ALDOA pull-down assay followed by mass spectrometry analysis, we further identified

7 the PPI between ALDOA and -actin to regulate cellular invasion by binding to actin and modulating

8 actin polymerization (46,49). Collectively, these findings support the hypothesis that the non-glycolytic

9 PPI of ALDOA with -actin not only modulates the dynamics of actin filaments but also accelerates

10 glycolysis and metabolic reprogramming.

11 Here, we have reported that metastatic lung cancer cells had a specific interaction between

12 ALDOA and -actin. However, this interaction was not found in normal cells, including fibroblast,

13 epithelial and vascular endothelial cells. Our designed peptide can block the ALDOA/-actin interaction

14 to inhibit orthotopic lung cancer metastasis and suppress tumor growth. However, this peptide treatment

15 did not induce toxicity in normal lung epithelial Beas2B cells. These results suggest further

16 development of compounds that block ALDOA/-actin interaction can be beneficial to lung cancer

17 patients with minimal toxicity (Supplemental Figure 14). Moreover, ALDOA overexpression has been

18 found extensively in other metastatic cancers—e.g., breast cancer. These findings highlight the

19 therapeutic value of targeting the ALDOA/-actin complex to combat malignant cancers. Notably, a

20 previous report revealed that an alanine mutation at aldolase residue R42, K107 or R148 diminishes the

21 PPI between aldolase and F-actin (42) and that the positively charged surface regions of aldolase

22 (residues Lys13, 27, 288, 293, and 341 and Arg257) are attracted to the negatively charged amino

23 terminus (Asp1 and Glu2 and4) and to other patches (Asp24, 25, and 363 and Glu361, 364, 99, and 100)

24 of actin subunits in a computational simulation (43). Certainly, our data showed that the alanine

25 mutation at Lys293 disrupts the PPI of ALDOA with -actin without affecting the enzymatic activity of

1 ALDOA and ultimately diminishes the metastatic ability of lung cancer cells. These results may provide

2 useful information regarding the feasibility of developing a PPI inhibitor for the ALDOA/-actin

3 complex in cancer therapy. Over time, we searched several clinical cases of ALDOA mutation events in

4 cancer patients from the cBioPortal website. However, rare cases have been reported in patients and

5 cancer cell lines. For example, the NCI-60 cell line panel revealed HCT15 and MOLT-4 cells show

6 genetic change, including T125I/D196G and S336N, respectively. On the other hand, a combined study

7 only showed 0.4% of cases in lung cancer patients (n=3146). However, we identified the interaction

8 ability between ALDOA and γ-actin in clinical specimens. ALDOA and γ-actin may be strongly

9 correlated with poor survival or N/M stage in cancer patients.

10 Combining this study with a previously reported study (14), we validated that ALDOA

11 overexpression may occur through protein-protein interactions with γ-actin or the HIF1α-MMP9 axis.

12 HIF-1α could be stabilized through lactate production, could suppress prolyl hydroxylase (PHDs)

13 enzyme activity and could further form a positive feedback loop in lung tumorigenesis (14). We further

14 identified the characteristic of the promoter region of ALDOA through directly targeting HIF-1α.

15 Additionally, we postulated that the non-enzymatic function of ALDOA regulates cancer metastasis.

16 Combining all the data, when ALDOA receives signals from HIF-1α or PI3K/Akt (44) , it performs both

17 enzyme-dependent and -independent functions in tumorigenesis. Through multivariate analysis, we

18 confirmed the correlation of the ALDOA expression level with lymph node invasion and distal

19 metastasis in lung cancer patients. Therefore, we can preferentially select the metastasis status or high

20 expression level of target genes in clinical specimens by immunohistochemistry. We anticipate the

21 ability to identify eligible clinical patients for further pre-clinical trials. Indeed, raltegravir is a good

22 inhibitor option for HIV treatment and may inhibit the ALDOA/-actin interaction status in cancer

23 patients. Until now, only the pharmacokinetics and pharmacology of raltegravir have been summarized

24 in several clinical studies (50) . However, those studies focused only on antiviral therapy, not cancer

25 treatment. In our laboratory, we are modifying several functional groups of raltegravir and using in

1 silico prediction to evaluate its synergy with other candidate small-molecule compounds for virtual

2 screening or drug repurposing. In further prospective studies, we will re-measure the drug delivery and

3 half-life of raltegravir for combined therapy.

4 In conclusion, this study demonstrates the relatively abundant levels of the ALDOA/-actin

5 complex in malignant tumors, and the knowledge regarding intermolecular interactions between

6 aldolase and F-actin offers an opportunity to develop a more selective PPI inhibitor against this complex

7 to combat clinical malignancies.

1 Acknowledgments

2 We would like to thank Ms. Tracy Tsai for her technical support in the pathological and

3 immunohistochemical analyses. We would also like to thank the GRC Instrument Core Facilities for

4 their support for the Affymetrix microarray, IP-MASS spectrometry, IVIS spectrum, and Aperio digital

5 pathology analyses. This research was supported by Academia Sinica grants AS-SUMMIT-108 to

6 Michael Hsiao.

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Figures and Figure Legends

Figure 1. ALDOA-involved cancer metabolism promotes the metastatic abilities of lung cancer cells in vitro and in vivo. (A) Heatmap of the microarray results (GSE42407) of the mRNA expression of glycolytic and lipid biosynthesis enzymes in CL1-0 (poorly invasive) and CL1-5 (highly invasive) lung cancer cells. (B) The flowchart describes the enzymes that are involved and the intermediates that are yielded in glycolysis and lipid biosynthesis pathways. The red color represents the enzyme name in the glycolysis and lipid biosynthesis pathways. (C) The migration/invasion abilities of CL1-5 cells after glycolysis enzyme expression was knocked down in an RNA interference (RNAi)-based high-throughput screen. (D) Western blot analysis of ALDOA and tubulin protein expression in various lung cancer cells. Tubulin was used as an internal control for protein loading. (E) Correlation between ALDOA protein expression and cellular invasion ability in various lung cancer cells and nontumor Beas2B cells. The significance of the correlation was analyzed using the nonparametric Spearman method. (F) Western blot analysis and migration and invasion abilities of ALDOA and tubulin protein expression after ALDOA overexpression in CL1-0 cells. (G) Western blot analysis and migration and invasion abilities of ALDOA and tubulin protein expression after ALDOA knockdown with control nonsilencing shRNA (NS) in CL1-5 cells. (H) Lung nodule appearance (right panel) and colony formation [12.5× magnification and 100× magnification], as indicated by arrows, in mice (n=5) implanted with control (vector control, VC) or ALDOA-overexpressing CL1-0 cells through tail vein injection. Quantified nodule number (left panel). (I) Lung nodule appearance (right) and colony formation [12.5× magnification and 100× magnification], as indicated by arrows, in mice (n=5) implanted with control (nonsilencing shRNA, NS) or ALDOA-silenced CL1-5 cells through tail vein injection. Quantified nodule number (left panel). The data from three independent experiments are presented as the means  SEM. The significance of the difference was analyzed using the nonparametric Mann-Whitney U-test. The significance of the differences in E, F, G, H and I was analyzed using Student’s t-test. In C, F and G, the blue and green columns represent cellular migration and invasion abilities, respectively.

Figure 2. ALDOA plays a nonenzymatic role to promote lung cell metastasis. (A) Migration/invasion abilities of CL1-0 cells with or without forced expression of an exogenous ALDOA gene in the presence of increasing concentrations of extracellular glucose. The blue and green columns represent cellular migration and invasion abilities, respectively. (B) Representative Giemsa staining to estimate the migration/invasion abilities of CL1-0 and H1355 cells with forced expression of the vector

control (VC) or exogenous ALDOA in the presence or absence of the competitive inhibitor 4-DG at the designated concentrations. (C) Intracellular ALDOA activity and migration/invasion abilities of CL1-0 cells with forced expression of the vector control (VC) or an exogenous mutant or wild-type ALDOA gene in the absence or presence of 4-DG at the designated concentrations. (D) Intracellular ALDOA activity and cellular migration/invasion abilities of H1299 cells treated with or without the ALDOA competitive inhibitor 4-DG at the designated concentrations. (E) Intracellular ALDOA activity and cellular migration/invasion abilities of CL1-0 cells with forced expression of the VC or an exogenous mutant or the wild-type ALDOA gene. (F) Intracellular ALDOA activity and cellular migration/invasion abilities of CL1-0 cells with forced expression of the VC or an exogenous mutant or the wild-type ALDOA gene. The data from three independent experiments are presented in A, C, D, E and F as the means ± SEM. The significance of the difference was analyzed using the nonparametric Mann-Whitney U-test. In A, C, D, E and F, the blue and green columns represent cellular migration and invasion abilities, respectively.

Figure 3. The protein-protein interaction between ALDOA and -actin regulates lung cancer cell metastasis ability. (A) Mass spectrometric analysis of ALDOA pull-down protein mixtures in CL1-0 cells with overexpression (OE) of an exogenous His-tagged ALDOA gene. The proteins identified in the mass spectrometric analysis were ranked by Mascot scores. (B) Western blot analysis of γ-actin subtypes 1 (ACTG1) and 2 (ACTG2) and His-tagged protein in the Ni-NTA pull-down protein mixture derived from CL1-0 cells with forced expression of the VC or an exogenous pull-down assay. (C) Migration/invasion abilities of control (vector only) or ALDOA-overexpressing CL1-0 cells with or without γ-actin knockdown (KD). (D) The locations of residues K288, K293 and K341 surrounding the -actin binding site in the crystal structure (PDB accession no. 3B8D) of ALDOA. (E) Pull-down assay for whole-cell lysates derived from CL1-0 cells with forced expression of exogenous wild-type (wt) or mutant ALDOA using Ni-NTA beads, followed by Western blot analysis for -actin and His-tagged ALDOA proteins. (F) Migration/invasion abilities of CL1-0 cells after forced expression of exogenous wild-type or mutant ALDOA. (G) Two-way immunoprecipitation assay for whole-cell lysates derived from Beas2B, CL1-0 and CL1-5 cells using protein A/G beads followed by Western blot analysis for -actin and aldolase family member proteins. Light chain was used as the internal control. (H) Two-way immunoprecipitation assay for whole-cell lysates derived from human umbilical vein endothelial cells (HUVECs), human pulmonary fibroblasts (HPFs) and human bronchial epithelial cells (HBEpic) using beads, followed by Western blot analysis for -actin and aldolase family member proteins. Light chain was used as the internal control. (I) G-actin/F-actin assay of the CL1-0 cells after forced expression of

exogenous wild-type ALDOA. S: supernatant, G-actin. P: pellet, F-actin. Quantitation of the expression of the F-actin level versus its corresponding G-actin level by ImageJ software. The data from three independent experiments are presented in C, F and I as the means ± SEM. The significance of the difference was analyzed using the nonparametric Mann-Whitney U-test. In C and F, the blue and green columns represent cellular migration and invasion abilities, respectively.

Figure 4. Peptide designed to block the protein-protein interaction between ALDOA with -actin to regulate lung cancer cell metastasis ability. (A) Amino acid sequence sites from 286 to 293 of the ALDOA protein. (B) Pull-down assay for whole-cell lysates derived from CL1-0 cells with forced expression of exogenous wild-type (wt) ALDOA using Ni-NTA beads, followed by Western blot analysis for -actin and His-tagged ALDOA proteins in the mock, solvent and peptide treatment groups. (C) Giemsa staining to evaluate the migration/invasion abilities of ALDOA-overexpressing CL1-0 cells and CL1-5 parental cells with pharmaceutical inhibition of the interaction between ALDOA and -actin by specific peptide treatment. (D) Migration abilities of CL1-0 cells after forced expression of exogenous wild-type ALDOA and of CL1-5 cells in response to dose-dependent peptide treatment. (E) Immunofluorescence assay of CL1-0 cells after forced expression of exogenous ALDOA and after peptide treatment in the CL1-0 ALDOA model and in CL1-5 cells. Green: phalloidin; Blue: DAPI. (F) G-actin/F-actin assay of the CL1-5 model in the sham, solvent and peptide treatment groups. S: supernatant, G-actin. P: pellet, F-actin. Quantitation of the expression of the F-actin level versus its corresponding G-actin level by ImageJ software. (G) Alamar blue assay to measure cell viabilities in Beas2B, CL1-0 and CL1-5 cells with specific peptide treatment (0 μM, 1 μM, 3.3 μM, 10 μM and 33 μM). (H) Photon signaling (left), overview of appearance and H/E staining (right) in mice implanted with ALDOA-overexpressing CL1-0 cells by orthotopic injection in the left lung in the sham and peptide treatment groups. (I) Six-week-old NOD/SCID gamma mice were established as a lung orthotopic model and were randomly assigned to a treated sham control (0.2% acetonitrile) or treatment with peptide (33 μM) by tail vein injection. A Kaplan-Meier plot is displayed for the sham or peptide-treated mice considering time to death. The log-rank test was used to calculate the statistical significance. The red color indicates the sham group, and the blue color indicates the peptide treatment group; n=15 for each. Time chart of the animal experimental procedure. The red arrows (↓) indicate the time of peptide or sham treatment. The significance of the difference was analyzed using the nonparametric Mann-Whitney U-test.

Figure 5. Raltegravir as a competitor between ALDOA and -actin to repress lung cancer cell metastasis ability. (A) Chemical structure of raltegravir and predicted model of raltegravir binding to the ALDOA crystal structure. (B) Giemsa staining to evaluate the migration abilities of ALDOA-overexpressing CL1-0 cells and CL1-5 parental cells with pharmaceutical inhibition of the interaction between ALDOA and -actin with raltegravir treatment. (C) G-actin/F-actin assay of the CL1-5 model with each candidate drug treatment. S: supernatant, G-actin. P: pellet, F-actin. Quantitation of the expression of the F-actin level versus its corresponding G-actin level by ImageJ software. (D) CL1-0-ALDOA cells were subcutaneous injected into NOD-SCID mice that were treated over an interval of 2 days with saline only (PBS) and raltegravir. Left: Overview of tumors at the end point. Right: Quantification of the tumor weight (g). (E) Overview of the appearance (upper) and H/E staining (lower) in mice implanted with ALDOA-overexpressing CL1-0 cells by lung orthotopic injection in the left lung in the solvent and raltegravir treatment groups. (F) Quantitation of photon signaling by raltegravir treatment versus solvent treatment using the Vivo Imagine System (IVIS). (G) Six-week-old NOD/SCID gamma mice were established as a lung orthotopic model and were randomly assigned to treatment with the solvent control (PBS) or raltegravir (100 μM) by tail vein injection. A Kaplan-Meier plot is displayed for the sham or RAL-treated mice considering the time to death. A log-rank test was used to calculate statistical significance. The significance of the difference was analyzed using the nonparametric Mann-Whitney U-test.

Figure 6. ALDOA-positive correlation with clinical parameters and survival rate in lung cancer. (A) Kaplan-Meier (KM) analysis of ALDOA gene expression in patients with lung cancer as identified by 2 different probes in a microarray analysis from the KM Plotter database at the endpoint of overall survival (OS) and first progression-free survival (FPS) probabilities. (B) Representative IHC staining intensity of the ALDOA protein in lung cancer tissues. (C) KM analysis of the overall survival and disease-free survival in patients with lung cancer with ALDOA protein expression at low (scores 0 and 1) and high (scores 2 and 3) levels. (D) Quantification of ALDOA expression by immunohistochemistry analysis of lung cancer specimens by each corresponding clinical parameter. (E) Univariate Cox regression hazard ratio for risk of disease-free survival in patients with lung cancer. (F) Multivariate Cox regression hazard ratio for risk of disease-free survival in patients with lung cancer. The significance of the differences in A, C, E and F was analyzed using Student’s t-test.

Figure 7. Schematic model of the ALDOA dual role in lung cancer.

Therapeutic Targeting of Aldolase A Interactions Inhibits Lung Cancer Metastasis and Prolongs Survival

Yu-Chan Chang, Jean Chiou, Yi-Fang Yang, et al.

Cancer Res Published OnlineFirst July 29, 2019.

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