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 3 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* 6 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. 20 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 1 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] 4 5 Competing financial interest 6 The authors declare that they have no competing interests. 7 8 9 Running Title: Targeting of ALDOA inhibits lung cancer metastasis 10 11 2 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 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. 14 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. 18 19 Key word: Aldolase A, non-glycolytic function, raltegravir 20 3 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 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 4 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 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. 5 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript