Tumor promoting effect of PDLIM2 downregulation involves mitochondrial ROS, oncometabolite accumulations and HIF-1α activation

Abstract

Background: Cancer is characterized by dysregulated cellular metabolism. Thus, understanding the mechanisms underlying these metabolic alterations is important for developing targeted therapies. In this study, we investigated the pro-tumoral effect of PDZ and LIM domain 2 (PDLIM2) downregulation in lung cancer growth and its association with the accumulation of mitochondrial ROS, oncometabolites and the activation of hypoxia-inducible factor-1 (HIF-1) α in the process.

Methods: Databases and human cancer tissue samples were analyzed to investigate the roles of PDLIM2 and HIF-1α in cancer growth. DNA microarray and gene ontology enrichment analyses were performed to determine the cellular functions of PDLIM2. Seahorse assay, flow cytometric analysis, and confocal microscopic analysis were employed to study mitochondrial functions. Oncometabolites were analyzed using liquid chromatography-mass spectrometry (LC-MS). A Lewis lung carcinoma (LLC) mouse model was established to assess the in vivo function of PDLIM2 and HIF-1α.

Results: The expression of PDLIM2 was downregulated in lung cancer, and this downregulation correlated with poor prognosis in patients. PDLIM2 highly regulated genes associated with mitochondrial functions. Mechanistically, PDLIM2 downregulation resulted in NF-κB activation, impaired expression of tricarboxylic acid (TCA) cycle genes particularly the succinate dehydrogenase (SDH) genes, and mitochondrial dysfunction. This disturbance contributed to the accumulation of succinate and other oncometabolites, as well as the buildup of mitochondrial reactive oxygen species (mtROS), leading to the activation of hypoxia-inducible factor 1α (HIF-1α). Furthermore, the expression of HIF-1α was increased in all stages of lung cancer. The expression of PDLIM2 and HIF-1α was reversely correlated in lung cancer patients. In the animal study, the orally administered HIF-1α inhibitor, PX-478, significantly reduces PDLIM2 knockdown-promoted tumor growth.

Conclusion: These findings shed light on the complex action of PDLIM2 on mitochondria and HIF-1α activities in lung cancer, emphasizing the role of HIF-1α in the tumor-promoting effect of PDLIM2 downregulation. Additionally, they provide new insights into a strategy for precise targeted treatment by suggesting that HIF-1α inhibitors may serve as therapy for lung cancer patients with PDLIM2 downregulation.

Keywords: HIF-1α; HIF-1α inhibitor; Mitochondria; PDLIM2; Succinate; Succinate dehydrogenase.

Fig. 1

PDLIM2 expression in human cancers. Real-time PCR analysis of PDLIM2 levels was performed using a TissueScan cancer survey panel, including tissues from colon, kidney, liver, lung, ovary, prostate, thyroid gland tumors, and their corresponding non-tumor tissues. The expression levels were normalized to β-actin mRNA levels. Data represent means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the normal tissues

Fig. 2

Significance of PDLIM2 expression in lung cancer pathogenesis and clinical outcomes. A Kaplan–Meier survival curve depicting the overall survival (OS), first progression (FP), and post-progression survival (PPS) of lung cancer patients (data sourced from Kaplan–Meier Plotter). B Expression levels of PDLIM2 in normal tissue compared to different stages of lung adenocarcinoma from patients, utilizing the TCGA dataset. Data and p-values (*** p < 0.001) are provided by the UALCAN database. C Analysis of PDLIM2 expression in TissueScan lung cancer cDNA arrays, comparing normal tissue (n = 15) and tumor biopsies (n = 81) across various tumor stages

Fig. 3

PDLIM2 downregulation impairs expression of genes for mitochondrial functions. A Gene ontology enrichment analysis of microarray data by comparing shEmpty and shPDLIM2 LLC cells. Analysis focused on changes with -log fold-changes exceeding 2 or falling below -2. B Fold change in the expression of TCA cycle-related genes derived from the mitochondrial term in microarray analysis. The genes encoding subunits of SDH complex are boxed with red color. C, D mRNA expression levels of SDH subunits, TNF-α, IL-1β, and MMP9 were measured by real-time PCR following stable PDLIM2 knockdown in LLC and A549 cells. E, F Protein levels of SDH subunits in PDLIM2 knockdown LLC and A549 cells were measured by immunoblotting. Data represent means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the shEmpty group

Fig. 4

PDLIM2 downregulation promotes tumor growth and suppresses SDH gene expression. A C57BL/6 mice were injected subcutaneously with 5 × 10⁵ control and PDLIM2 knockdown LLC cells. Tumor volume was measured at the indicated time points, and the mean tumor size was plotted. Mice were euthanized on day 17, and tumors were excised and photographed. B H&E staining of tumor samples to visualize leukocyte infiltrations (left upper panel: 20X, left lower panel: 40X). Leukocyte infiltrations in 20X magnification areas were quantified using ImageJ software (Right panel). C, D Total RNAs were isolated from the tumors using TRIzol reagent. mRNA expression levels of TNF-α, IL-1β, MMP9, and SDH subunits were measured by real-time PCR. The expression levels were normalized to β-actin mRNA levels. Data represent means ± SEM. * p < 0.05, ** p < 0.01 compared with the shEmpty group

Fig. 5

PDLIM2 knockdown impairs mitochondrial functions in lung cancer cells. A Real-time measurement of cellular oxygen consumption rate (OCR) in control and PDLIM2 knockdown LLC and A549 lung cancer cells using the Seahorse XF24 Extracellular Flux Analyzer. Basal respiration, maximal respiration, spare capacity, ATP production, and proton leak were assessed, respectively. B Accumulation of dysfunctional mitochondria in control and PDLIM2 knockdown LLC and A549 lung cancer cells revealed by staining with MitoTracker Green and MitoTracker Red. Representative dot plots from FACS analysis are shown. The comparison between MitoTracker Green⁺ Red⁺ (functional) and MitoTracker Green⁺ Red⁻ (dysfunctional) populations is presented. Data represent means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the shEmpty group. C Confocal microscopy images depict the mitochondrial morphology of control and PDLIM2 knockdown LLC and A549 cells. Green fluorescence represents mitochondria, and blue fluorescence represents Hoechst-labeled nucleus

Fig. 6

Increased accumulation of mitochondrial ROS and oncometabolites in PDLIM2 knockdown lung cancer cells. A, B Mitochondrial ROS in control and PDLIM2 knockdown LLC (A) and A549 (B) lung cancer cells was analyzed by flow cytometry using the MitoSOX Red probe. C, D Analysis of metabolite levels in PDLIM2 knockdown LLC (C), A549 (D) cells and their corresponding controls by LC–MS. Data represent means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the shEmpty group

Fig. 7

Downregulation of PDLIM2 in lung cancer is associated with increased HIF-1α expression. A, B LLC and A549 lung cancer cells were treated with 400 μM H₂O₂, 5 mM succinate, and 20 mM dimethyl succinate (DMS) for 6 h. Protein level of HIF-1α and PHD2 was measured by immunoblotting. C, D Protein level of HIF-1α and PHD2 was measured by immunoblotting in PDLIM2 knockdown LLC and A549 cells. E UALCAN database provided data showing HIF-1α expression in normal and different stages of lung adenocarcinoma from patients in the TCGA dataset, with corresponding p-values (*** p < 0.001). F HIF-1α expression was analyzed in TissueScan lung cancer cDNA arrays with normal tissue (n = 15) and tumor biopsies (n = 81) across various tumor stages. G Co-expression analysis of PDLIM2 and HIF-1α by using cBioportal for cancer genomics. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the shEmpty group, control, and the normal tissue, respectively

Fig. 8

HIF-1α inhibitor abolishes the tumor-promoting effects of PDLIM2 downregulation. A C57BL/6 mice (n = 6) were injected subcutaneously with 5 × 10⁵ control and PDLIM2 knockdown LLC cells. When the tumors reached approximately 300 mm³, the mice were orally administered PX-478 three times per week for 2 weeks. B Tumor volume was measured at the indicated times, and the mean tumor size was plotted. Mice were euthanized on day 22, and tumors were excised and photographed (right panel). C The body weight of each mouse was measured. D Total RNAs from the tumors were isolated using TRIzol reagent, and the mRNA expression levels of HIF-1α and PDLIM2 were measured by real-time PCR. The expression levels were normalized to β-actin mRNA levels. E Protein levels of HIF-1α were measured by immunoblotting in tumor tissues. F Immunohistochemistry of tumor samples for HIF-1α positive tumor cells (left upper panel: 20X, left lower panel: 40X). HIF-1α-positive area scores were evaluated by using ImageJ software at 20X magnification filed (Right panel). Data represent means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the shEmpty group

Fig. 9

Illustrates a pro-tumor mechanism involving the activation of HIF-1α by PDLIM2 downregulation. In lung cancer, decreased PDLIM2 expression leads to NF-κB signaling activation, resulting in impaired SDH gene expression and mitochondrial dysfunction. This dysfunction includes reduced mitochondrial OCR, increased succinate accumulation, elevated mitochondrial fission, and ROS production. Succinate and ROS further inhibit PHD expression, leading to increased HIF-1α activation and ultimately promoting tumor growth. The inhibition of tumor growth by a HIF-1α inhibitor in this study suggests that HIF-1α may be a potential target for treating of cancers resulting from PDLIM2 downregulation