S100A4 alters metabolism and promotes invasion of lung cancer cells by up-regulating mitochondrial complex I protein NDUFS2

Abstract

It is generally accepted that alterations in metabolism are critical for the metastatic process; however, the mechanisms by which these metabolic changes are controlled by the major drivers of the metastatic process remain elusive. Here, we found that S100 calcium-binding protein A4 (S100A4), a major metastasis-promoting protein, confers metabolic plasticity to drive tumor invasion and metastasis of non-small cell lung cancer cells. Investigating how S100A4 regulates metabolism, we found that S100A4 depletion decreases oxygen consumption rates, mitochondrial activity, and ATP production and also shifts cell metabolism to higher glycolytic activity. We further identified that the 49-kDa mitochondrial complex I subunit NADH dehydrogenase (ubiquinone) Fe-S protein 2 (NDUFS2) is regulated in an S100A4-dependent manner and that S100A4 and NDUFS2 exhibit co-occurrence at significant levels in various cancer types as determined by database-driven analysis of genomes in clinical samples using cBioPortal for Cancer Genomics. Importantly, we noted that S100A4 or NDUFS2 silencing inhibits mitochondrial complex I activity, reduces cellular ATP level, decreases invasive capacity in three-dimensional growth, and dramatically decreases metastasis rates as well as tumor growth in vivo Finally, we provide evidence that cells depleted in S100A4 or NDUFS2 shift their metabolism toward glycolysis by up-regulating hexokinase expression and that suppressing S100A4 signaling sensitizes lung cancer cells to glycolysis inhibition. Our findings uncover a novel S100A4 function and highlight its importance in controlling NDUFS2 expression to regulate the plasticity of mitochondrial metabolism and thereby promote the invasive and metastatic capacity in lung cancer.

Keywords: NADH:ubiquinone oxidoreductase core subunit S2 (NDUFS2); S100 calcium-binding protein A4 (S100A4); S100 proteins; cellular respiration; energy metabolism; fibroblast-specific protein-1; glycolysis; invasion; lung cancer; metastasin-1; metastasis; mitochondria; mitochondrial complex I; mitochondrial respiratory chain complex; non-small cell lung cancer (NSCLC).

Figure 1.

Knockdown of S100A4 decreases cellular respiration and shifts cell metabolism toward glycolysis. A, cellular respiration in A549 cells with S100A4 knockdown (shS100A4) or control (shCont) was analyzed by a mitochondrial stress test using a Seahorse XF96 analyzer. OCR was measured prior to and after injection of 1 μm oligomycin, 0.6 μm carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 μm antimycin A and rotenone combination (A&R). Representative data are shown. B, OCR for basal respiration, maximal respiration, ATP production, proton leak, and nonmitochondrial respiration from A was analyzed using Wave 2.1 and normalized to protein concentration. C and D, H460 cells were cultured in regular media for 5 days and assessed visually (C), or medium pH was measured using a pH meter (D). E and F, glycolysis in A549 shCont and shS100A4 cells was assessed by glycolysis stress test using a Seahorse XF96 Analyzer. ECAR was measured prior to and after injection of 10 mm glucose, 1 mm oligomycin, and 100 mm 2-DG. Representative data are shown in E. ECAR for glycolysis, glycolysis capacity, and glycolysis reserve capacity was analyzed using Wave 2.1 and normalized to protein concentration (F). G and H, cells (5 × 10⁵), as indicated, were seeded into a 6-well plate, and media were changed after 5 h and collected the next day for glucose and lactate assessment. Cells were counted, and the amount of glucose consumption (G) and lactate production (H) was normalized to cell number. *, p < 0.05. Error bars, S.E. (A, B, E, and F) or S.D. (D, G, and H).

Figure 2.

S100A4 regulates NDUFS2 expression. NDUFS2 and S100A4 expression was assessed in shCont and shS100A4 cells as indicated in A–D or in H1299 stably expressing GFP only or GFP-S100A4 (E and F) by Q-PCR (A, C, D, and E) or immunoblot assessment (B and F). *, p < 0.05. Error bars, S.D.

Figure 3.

S100A4 impacts mitochondrial activity, which is required for invasive growth. A–C, A549 cells were grown in 3D Matrigel and treated with either DMSO (Control) or 0.5 μm rotenone (Rot) for 6 days. Representative phase-contrast images are shown in A. The percentage of colonies with invasive growth is shown in B. The diameter of colonies from randomly chosen fields was measured, quantified for average individual colony volume, and presented as mean ± S.E. (error bars) in C. Mitochondrial complex I activity (D, F, and H) and cellular ATP level (E, G, and I) were measured in shCont and shS100A4 A549 cells (D and E) and H460 cells (F and G) or in H1299 cells stably expressing GFP only or GFP-S100A4 (H and I). Representative data are shown. Scale bar in A, 100 μm. *, p < 0.05; **, p < 0.001.

Figure 4.

Effect of S100A4 on the expression of select genes in glycolysis pathway in lung cancer cells. Cell lysates from H460 shCont and shS100A4 (shA4) cells or H1299 cells stably expressing GFP only or GFP-S100A4 (GFP-A4) were evaluated by immunoblotting for the noted proteins involved in the glycolysis pathway (A), or the expression of hexokinases in cells as indicated was assessed by Q-PCR (B). C, mitochondria from stable knockdown of S100A4 (A4) in H460 and control cells (Cont) were immunoblotted with antibodies as indicated. Cox-IV was used as internal control for mitochondrial proteins, and actin was used as loading control for cytosol protein. *, p < 0.05. Error bars, S.D.

Figure 5.

NDUFS2 is critical to S100A4-driven biological effects. NDUFS2 was knocked down in A549 cells by three separate shRNAs, and the expression level of NDUFS2 was assessed by immunoblotting (A) or Q-PCR (B) and compared with control cells (Cont). ECAR (C), glucose consumption (D), lactate production (E), OCR (F), mitochondrial complex I activity (G), cellular ATP levels (H), cell proliferation (I), and 3D Matrigel culture colony size measurement (J) were assessed on cells as indicated. H460 shS100A4 cells transfected with the NDUFS2 construct were sorted by GFP and compared with H460 shCont and shS100A4 cells for glucose consumption (L) and 3D growth (M). The diameter of colonies (>100 colonies) from randomly chosen fields was measured, quantified for average individual colony volume. Data presented as mean ± S.E. (error bars) (C, F, J, and M) or S.D. (D, E, G, H, and L). *, p < 0.05; **, p < 0.001.

Figure 6.

Knockdown of S100A4 and NDUFS2 in A549 cells decreases lung metastases in vivo. A, cells (1 × 10⁶), as indicated, in a 1:1 mixture of PBS and growth factor–reduced Matrigel were implanted subcutaneously into NSG mice. Tumor growth was monitored, and volumes were calculated every 2 days. Mice were sacrificed at week 12 (shCont and shS100A4 1st, blue arrows) and week 14 (shS100A4 2nd and shNDUFS2, black arrow). Three-color straight solid lines are fitted tumor volume profiles, and the corresponding curved lines are observed mean tumor volume for each group. B, primary tumors from the indicated cell line–injected groups were collected, snap-frozen, and homogenized. Tissue lysates (400 μg) were used for the mitochondrial complex I activity assay. C and D, representative images from histologic assessments of primary tumor sections (C) or lung sections (D) with H&E staining or immunohistochemical staining with antibodies against S100A4 and Ki67. Magnification is ×400 for H&E and S100A4 staining (scale bars, 100 μm) and ×200 for Ki67 staining (scale bars, 200 μm) in C. Magnification is ×100 for images in D. Arrows in D indicate tumor foci in the lung. Scale bars, 400 μm in D. The total number of tumor foci was recorded based on histology assessment. The incidence of tumor foci was calculated based on the observation time and presented in (E). The size of the tumor foci in the indicated cell injection groups was measured by a digital micrometer. The average size of tumor foci in each group is presented in F. *, p < 0.05. Error bars, S.D.

Figure 7.

Effect of glycolysis and S100A4 inhibition by niclosamide and 2-DG on cell survival. A, A549 shCont and shS100A4 cells were plated in a 6-well plate and treated with 5 mm 2-DG for 16 h, and then cells were counted and the amount of lactate production was measured and normalized to cell number. B, A549 shCont and shS100A4 cells were treated with either medium only or 5 mm 2-DG for 3 days, and then cell number was quantified by a cell counter (B). C, A549 cells were treated with DMSO (Cont), 0.25 μm niclosamide (nic), or 5 mm 2-DG, or both niclosamide and 2-DG for 3 days. A₅₉₀ was measured after crystal violet staining. *, p < 0.002; **, p < 0.0001. Error bars, S.D.