Abstract

   Aldehyde dehydrogenase 3A1 (ALDH3A1) is an NAD^+-dependent enzyme that
   is closely related to tumor development. However, its role in
   non-small-cell lung cancer (NSCLC) has not been elucidated. This study
   aimed to clarify the mechanism of ALDH3A1 and identify potential
   therapeutic targets for NSCLC. Here, for the first time, we found that
   ALDH3A1 expression could be induced by a hypoxic environment in NSCLC.
   ALDH3A1 was highly expressed in NSCLC tissue, especially in some
   late-stage patients, and was associated with a poor prognosis. In
   mechanistic terms, ALDH3A1 enhances glycolysis and suppresses oxidative
   phosphorylation (OXPHOS) to promote cell proliferation by activating
   the HIF-1α/LDHA pathway in NSCLC. In addition, the results showed that
   ALDH3A1 was a target of β-elemene. ALDH3A1 can be downregulated by
   β-elemene to inhibit glycolysis and enhance OXPHOS, thus suppressing
   NSCLC proliferation in vitro and in vivo. In conclusion,
   hypoxia-induced ALDH3A1 is related to the energy metabolic status of
   tumors and the efficacy of β-elemene, providing a new theoretical basis
   for better clinical applications in NSCLC.

   graphic file with name 41419_2023_6142_Figa_HTML.jpg

   Subject terms: Cancer metabolism, Non-small-cell lung cancer

Introduction

   Hypoxic tumor microenvironments induce the glycolytic phenotype in many
   cancer cells [[44]1]. While this hypoxic-induced increase in glycolytic
   flux is beneficial for promoting cell growth during hypoxic injury and
   is therefore adaptive in nature, the mechanisms surrounding this
   increased glycolytic flux can also be manipulated by cancer cells to
   promote their growth, even in the presence of adequate oxygen levels, a
   property of tumor cells known as the “Warburg effect” [[45]2, [46]3].
   The “Warburg effect” aims to meet the need of tumor cells for unlimited
   proliferation, which is characterized by active glycolysis, the
   accumulation of lactic acid, and a weakened oxidative phosphorylation
   process of mitochondria. These changes in energy metabolism are also
   called the energy metabolism reprogramming of tumors. Numerous studies
   have shown that the reprogramming of the energy metabolism of tumor
   cells can affect disease progression and efficacy [[47]4–[48]7];
   therefore, new therapeutic strategies based on the reprogramming of
   energy metabolism in tumor cells have attracted significant attention.

   Aldehyde dehydrogenase 3A1 (ALDH3A1) is an NAD^+-dependent enzyme that
   oxidizes various endogenous and exogenous aldehydes to carboxylic acids
   [[49]8]. Studies have demonstrated that ALDH3A1 expression is closely
   related to changes in the biological behavior of tumor cells, such as
   epithelial mesenchymal transition (EMT), metastasis, and cancer stem
   cell expansion, impairing immune surveillance [[50]9–[51]11]. In
   addition, one study indicated that the knockdown of ALDH3A
   significantly reduced ATP production and induced apoptosis in gastric
   cancer [[52]12]. Exosomes carrying ALDH3A1 from irradiated lung cancer
   cells contribute to the motility of recipient cells by accelerating
   glycolysis [[53]13]. A bioinformatics analysis showed that the
   glycolytic and gluconeogenic metabolic pathway mediated by ALDH3A1 was
   associated with p53 mutants and prognosis in lung adenocarcinoma
   [[54]14]. Another study showed that the increased expression of ALDH3A1
   was associated with drug resistance in NSCLC [[55]15]. The exploration
   of ALDH3A1’s pathogenic mechanism in NSCLC has contributed to the
   discovery of effective therapeutic drugs targeting ALDH3A1 and
   combatting drug resistance, providing new clues for the identification
   of new precision therapeutic targets.

   β-elemene is a monomeric antitumor compound extracted from turmeric.
   The previous studies of our team found that β‐elemene enhances the
   sensitivity of tumor cells to chemotherapeutic agents and has the
   potential to be a novel drug for multidrug-resistant cells, effectively
   preventing the proliferation, apoptosis, and metastasis of cancer
   [[56]16–[57]19]. Another study suggested that β-elemene could also
   inhibit breast cancer metastasis by inhibiting the translocation of
   pyruvate kinase M2 (a key glycolytic enzyme) to the nucleus [[58]20].
   However, the exact target of β-elemene is still undefined, leading to
   limitations on drug discovery and development.

   In this study, we identified that ALDH3A1 is consistently upregulated
   and a biomarker of a poor prognosis in NSCLC. Interestingly,
   environmental hypoxia induced ALDH3A1 expression and ALDH3A1 mediated
   hypoxia-induced functions. Furthermore, ALDH3A1 induced energy
   metabolism reprogramming, promoted tumor growth, and was identified,
   for the first time, as a target of β-elemene in vivo and in vitro. In
   conclusion, this study suggests that hypoxia-induced ALDH3A1 is
   associated with the energy metabolic status of tumors and the efficacy
   of β-elemene, providing a new theoretical basis for better clinical
   applications.

Materials and methods

Antibodies and reagents

   β-Elemene (molecular formula C15H24), whose molecular weight is 204.35,
   was provided by Jingang Pharmaceuticals (#081152, Dalian, China). The
   antibody of HIF-1α (#3716 S), LDHA (2012S), was obtained from Cell
   Signalling Technology (Danvers, MA, USA). The antibody GLUT1 (2646S)
   was purchased from NOVUS (USA), and the antibody PFKL (55028-1-AP) was
   purchased from Proteintech. Anti-PDK1 (ab226963) and Anti-PHD1
   (ab113077) were purchased from Abcam. Anti-Ki67 antibody was purchased
   from Fuzhou Maixin Biological Technology (Fujian, China). ALDH3A1
   (sc-137168), Cyclin B1 (166757), Cyclin D1 (8396), Cyclin E
   (sc-377100), and ACTIN (sc-47778) were purchased from Santa Cruz
   Biotechnology (Santa Cruz, CA, USA). 2-Deoxy-D-glucose (2-DG) was
   purchased from MedChemExpress (HY-13966, USA). The Cell Counting Kit-8
   (CCK-8) was purchased from APExBIO (Catalog No. K1018, USA).

Bioinformatics analysis

   [59]GSE18842 and [60]GSE30979 were collected from Gene Expression
   Omnibus (GEO) ([61]https://www.ncbi.nlm.nih.gov/gds/). The Limma R
   package was used to detect differentially expressed ALDH3A1. The
   correlation of ALDH3A1 with the overall survival of NSCLC cases was
   analyzed using the R package survminer. Lung adenocarcinoma data were
   downloaded from The Cancer Genome Atlas (TCGA,
   [62]https://www.cancer.gov/tcga), which was used to perform Gene Set
   Enrichment Analysis (GSEA). The molecular docking was performed using
   PubChem ([63]https://pubchem.ncbi.nlm.nih.gov/), the PDB
   ([64]https://www.rcsb.org/) online website, and Schrodinger Suites
   software.

Immunohistochemistry and immunofluorescence staining assay

   Tumor histopathology specimens from 100 patients with lung
   adenocarcinoma were obtained from surgical specimens from the
   Department of Pathology of the Second Hospital of China Medical
   University from 2010 to 2013 and were approved by the Ethics Committee
   of China Medical University for use in the study (CMU2021037). The
   tumor sample preparation methods, as well as the staining intensity and
   staining area calculation methods, are described in our previous study
   [[65]21]. The staining was evaluated by scanning the entire tissue
   specimen under low magnification (×10) and confirmed under high
   magnification (×20 and ×40). The protein expression was visualized and
   classified based on the percentage of positive cells and the intensity
   of staining. From each section, five visual fields were randomly
   selected. The degree of protein expression was based on the percentage
   of positive cells and the intensity of staining. Staining intensity was
   scored as 0 (no staining), 1 (low staining), 2 (intermediate staining),
   and 3 (high staining). For the staining area, ≤5%, 5–25%, 26–50, 51–75%
   and >75% were recorded as 0, 1, 2, 3 and 4 points, respectively.
   Histological score = staining intensity × staining area. A score 0 was
   classified as negative (−), 1–4 points as weakly positive (+) and 6–12
   points as a strong positive (++). Final scores were assigned by three
   independent pathologists.

   A total of 100 samples of NSCLC and adjacent normal tissues were
   acquired from patients who underwent surgical resection at the second
   hospital of China Medical University. Clinical information was
   collected. The participants were informed about the study’s ethical
   guidelines through written consent.

   The immunofluorescence staining references are provided in our previous