Cancer Stem Cell Formation Induced and Regulated by Extracellular ATP and Stanniocalcin-1 in Human Lung Cancer Cells and Tumors
<p>eATP induced migration, invasion, and expression of EMT- and CSC-related genes in A549 cells. Migration and invasion rates were compared between human NSCLC A549 cells, treated with 0.5 mM eATP at select timepoints, and untreated A549 cells. In addition, eATP treated cells were analyzed by RNAseq and RT-PCR, to identify changes in the expression of EMT- and CSC-related transcription factors or CSC-related genes. TGF-β (10 ng/mL) was used as a gene induction control for comparison. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001. (<b>a</b>) eATP treatment increased cell migration. (<b>b</b>) eATP treatment increased invasion. (<b>c</b>) Pie graphs show percentages of CSC-related genes induced by eATP or TGF-β treatment at two treatment time points in an RNAseq study. (<b>d</b>) RT-PCR of selected CSC-related transcription factor (TF) genes induced by eATP or TGF-β. (<b>e</b>) Selected EMT-related TF genes induced by eATP or TGF-β.</p> "> Figure 2
<p>eATP induced CSC-like changes at protein, cell, and functional levels in A549 cells. A549 cells were treated with eATP at various concentrations, and then examined for colony formation and time- and dose-dependent responses in expression of CSC-related transcription factors (TFs) and CSC marker proteins. A549 cells without eATP treatment served as controls. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001. (<b>a</b>) Anchor-independent soft agar assay: eATP increased colony formation, confirming its CSC inducing activity. (<b>b</b>) Quantification of the soft agar assay: colony number changes in the assay (from (<b>a</b>)). (<b>c</b>) Quantification of number of colonies formed in the soft agar assay. colony size (area) changes in the soft agar assay from (<b>a</b>,<b>d</b>). Western blot analysis: eATP induced expression changes of CSC-related protein markers at different doses and different induction times (6 and 12 h). (<b>e</b>) Quantifications of eATP-induced CSC-related protein markers (from (<b>d</b>)).</p> "> Figure 3
<p>eATP induced CSC formation and CSC-related markers. Human NSCLC A549 and H1299 cells were treated with eATP at various concentrations and at different times, and then analyzed for CSC subpopulation and CSC surface protein markers. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001. (<b>a</b>) Cell sorter study: eATP increased the number of CD44+/CD166+ cells in the general cell population in both A549 and H1299 cells. Quantification of CSC changes in the cell sorter study for A549 cells (<b>b</b>) and for H1299 cells (<b>c</b>). (<b>d</b>) Protein analysis and quantification of CSC-related cell surface protein markers in A549 cells treated with different concentrations of eATP for 6 h. (<b>e</b>) Fluorescence microscopy of A549 cells treated with 0.5 mM eATP for 6 h. eATP-treated cells show significantly increased intensity of CSC markers CD44 and CD166 proteins. (<b>f</b>) Time-dependent expression and quantification of CSC-related cell surface protein markers in 0.5 mM eATP treated A549 cells. (<b>g</b>) Dose-dependent expression and quantification of CSC-related cell surface protein markers in H1299 cells, following 6 h eATP treatment.</p> "> Figure 4
<p>RNAseq and knockdown (KD) studies: eATP and TGF-β upregulated gene expression, and <span class="html-italic">STC1</span> gene knockdown led to EMT- and CSC-related changes in A549 and H1299 cells. A549 cells were treated with eATP or TGF-β for 2 or 6 h, and polyA mRNA was isolated. RNA samples were sent to a commercial service for RNAseq, and the data were analyzed. The gene <span class="html-italic">STC1</span>, identified from the RNAseq data, was investigated by KD studies in A549 and H1299 NSCLC cells. In these studies, untreated A549 cells served as negative controls. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span>< 0.001. (<b>a</b>) Heatmap of 11 consistently and significantly upregulated genes by both eATP and TGF-β after 2 and 6 h of treatment. (<b>b</b>,<b>c</b>) Comparison of overall survival rates (<b>b</b>) and disease-free survival rates (<b>c</b>) of high and low <span class="html-italic">STC1</span>-expressing human lung cancer patients. Higher <span class="html-italic">STC1</span> levels were associated with lower survival rate. These data were generated from GEPIA (<a href="http://gepia.cancer-pku.cn/index.html" target="_blank">http://gepia.cancer-pku.cn/index.html</a>). (<b>d</b>) <span class="html-italic">STC1</span> gene expression was knocked down, and <span class="html-italic">STC1</span> protein was reduced, as analyzed by Western blot. Cofilin was used as the protein loading control. (<b>e</b>) Overexpression of <span class="html-italic">STC1</span> protein was induced by 0.5 mM eATP treatment for 6 h. (<b>f</b>) Viability assay of A549 cells with <span class="html-italic">STC1</span> gene knocked down. (<b>g</b>) Viability assay: drug resistance in A549 cells following <span class="html-italic">STC1</span> gene KD and 24-h treatment with target drug sunitinib (20 μM) in the presence or absence of ATP. Cell samples treated with sunitinib alone were used as controls to normalize other experimental samples in this resazurin assay. (<b>h</b>) Invasion assay: invasion of A549 cells with <span class="html-italic">STC1</span> gene KD. (<b>i</b>) Anchor-independent colony formation assay: Effects of <span class="html-italic">STC1</span> KD on the number of colonies formed; quantification of colonies. (<b>j</b>) Analysis of <span class="html-italic">STC1</span> protein, following siRNA KD of <span class="html-italic">STC1</span> in H1299 cells as examined by Western blot. Cofilin was used as the protein loading control; quantification shown in <a href="#app1-ijms-23-14770" class="html-app">Figure S1</a>. (<b>k</b>) Effect of <span class="html-italic">STC1</span> KD on the invasion of H1299 cells. Left images show transwell invasion assay; quantification shown on the right. Wildtype H1299 cells were used as controls for this assay.</p> "> Figure 5
<p>KO studies in vitro: KO of <span class="html-italic">STC1</span> downregulated CSC-like properties in A549 cells. The <span class="html-italic">STC1</span> KO in A549 cells was achieved with CRISPR-cas9 technology. KO cell clones were selected, identified, and further characterized by functional assays, in which the healthy A549 cells served as controls. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001. (<b>a</b>) <span class="html-italic">STC1</span> protein was absent from <span class="html-italic">STC1</span> KO (A549stc1ko) cells, as analyzed by Western blot. (<b>b</b>) KO of <span class="html-italic">STC1</span> resulted in slower cell proliferation rate compared with control A549 cells. (<b>c</b>) KO of <span class="html-italic">STC1</span> led to reduced iATP levels, and eATP treatment partially restored the iATP level. (<b>d</b>) Seahorse metabolic analysis: Mito stress test of A549 and A549stc1ko cells indicated that the relative OCR rate was reduced in A549stc1ko cells, in comparison to A549 cells. (<b>e</b>) OCR changes in the presence or absence of eATP: eATP partially restored OCR. ATP-linked respiration rate was significantly reduced in A549stc1ko cells and eATP treatment partially restored the ATP-linked respiration rate. (<b>f</b>) ECAR vs. OCR graph (energy map). eATP led to drastic changes in energetic metabolism in A549 and A549stc1ko cells as shown in an energy map. ATP treatment resulted in the left-to-right and lower-to-higher shifts of energy status for both A549 and A549stc1ko cells.</p> "> Figure 6
<p>Tumor formation studies using <span class="html-italic">STC1</span> KO cells. A549stc1ko or A549 cells were subcutaneously injected into the flanks of nude mice to generate tumors. The tumor sizes were measured weekly for four weeks. After euthanasia, tumors were surgically removed, weighed, photographed, fixed, and then further studied by Western blot analysis and immunofluorescence microscopy. A549 tumors served as negative controls in these studies. ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001. (<b>a</b>) Flow chart of the animal study. KO of <span class="html-italic">STC1</span> drastically reduced tumor sizes (<b>b</b>), tumor growth rate (<b>c</b>), and tumor weight (<b>d</b>). (<b>e</b>) KO of <span class="html-italic">STC1</span> significantly reduced expression of CSC markers CD44 and CD166 in A549stc1ko tumors compared with A549 tumors. (<b>f</b>) KO of <span class="html-italic">STC1</span> altered expression levels of proteins involved in EMT, CSC formation, and cell growth signaling as determined by Western blots. Quantification in <a href="#app1-ijms-23-14770" class="html-app">Figure S2</a>. (<b>g</b>) Hypothetical model of eATP-induced CSCs and <span class="html-italic">STC1</span> function in CSC formation in A549 cells. Based on our previous and current studies on eATP induced EMT and CSC in A549 and other NSCLC cells, we propose a hypothetical model for eATP’s activities. First, eATP functions as an extracellular messenger, binding and activating purinergic receptors (PRs) [<a href="#B27-ijms-23-14770" class="html-bibr">27</a>], which triggers Raf-MEK-ERK signal transduction pathway. Activated Raf-MEK-ERK signaling results in the activation of transcription factors and transcriptional changes in genes related to EMT [<a href="#B37-ijms-23-14770" class="html-bibr">37</a>] and CSC. Meanwhile, eATP is also internalized by macropinocytosis/macropinosomes and is released intracellularly to drastically increase intracellular ATP (iATP) levels [<a href="#B28-ijms-23-14770" class="html-bibr">28</a>,<a href="#B29-ijms-23-14770" class="html-bibr">29</a>]. The increased iATP in turn downregulates AMPK and upregulates mTOR, leading to accelerated cell growth. During this process, one of the upregulated genes, <span class="html-italic">STC1</span>, functions to upregulate mitochondrial energy metabolism, facilitate cell growth, mediate EMT, and induce CSC formation. Multiple questions remain to be answered in this model and additional studies are needed to elucidate the complete mechanisms of eATP in these processes.</p> ">
Abstract
:1. Introduction
2. Results
2.1. eATP Increased Rates of Cell Migration and Invasion and Altered Expression of Genes Involved in CSCs
2.2. eATP Induced Time- and Dose-Dependent Changes in Colony Formation and Levels of Proteins Involved in CSC Formation
2.3. eATP Increased CSC Surface Markers and CSC Subpopulations in NSCLC Cells
2.4. eATP Induced Genes Involved in EMT and CSC, and KD of STC1 Reduced CSC Phenotypic Changes
2.5. Knockout of STC1 Lowered iATP Levels, Oxygen Consumption Rate, and Mitochondrial ATP Synthesis
2.6. eATP-Treated A549 Cells Formed More Tumors at Lower Cell Injection Numbers, and Tumors Formed from A549stc1ko Cells Grew Slower and With Fewer CSCs
2.6.1. First Animal Study
2.6.2. Second Animal Study
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Cell Culture
4.2. Transwell Migration and Invasion Assays
4.3. qRT-PCR
4.4. Colony Formation Assay
4.5. Western Blot Analysis
4.6. Immunofluorescence Microscopy
4.7. Flow Cytometry/Cell Sorter Assay
4.8. RNA Sequencing
4.9. Cell Proliferation and ATP Assays
4.10. STC1 Gene Knockdown (KD) and Knockout (KO)
4.11. Cell Growth Curve
4.12. Seahorse Metabolic Studies
4.13. Tumor Studies
4.14. Immunocytochemistry Study of Tumor Sections
4.15. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Log2(FC) Values | |||||
---|---|---|---|---|---|
Gene Symbol | Gene Name | ATP 2h | TGF-β 2h | ATP 6h | TGF-β 6h |
1. FoxO1 | Forkhead box O1 | 2.36 | 1.23 | 0.69 | 1.07 |
2. FOSL1 | FOS like 1, AP-1 transcription factor subunit | 2.44 | 0.58 | 1.24 | 0.14 |
3. FOS | Fos proto-oncogene, AP-1 transcription factor subunit | −4.20 | −3.21 | −1.05 | −3.32 |
4. JUNB | JunB proto-oncogene, AP-1 transcription factor subunit | −1.34 | 2.30 | −0.16 | 2.34 |
5. JUN | Jun proto-oncogene, AP-1 transcription factor subunit | 0.50 | 1.20 | 0.05 | 1.34 |
6. c-Maf/maf | MAF bZIP transcription factor | 0.81 | 3.54 | 0.80 | 4.75 |
7. MITF | Melanocyte inducing transcription factor | 1.10 | −1.00 | 0.16 | −1.16 |
8. NFkB1 | Nuclear factor kappa B subunit 1 | 1.49 | 1.13 | 0.05 | 0.97 |
9. SNAI1/Snail 1 | Snail family transcriptional repressor 1 | 0.21 | 2.85 | 0.50 | 3.13 |
10. SOX8 | SRY-box 8 | 2.01 | 2.72 | 2.94 | 2.91 |
11. SOX4 | SRY-box 4 | 0.62 | 1.28 | 0.10 | 0.93 |
12. SOX21 | SRY-box 21 | −0.51 | −2.00 | 0.28 | −2.91 |
13. SOX2 | SRY-box 2 | −1.90 | −2.90 | −0.73 | −3.39 |
14. Oct-3/4—POU5F1 | POU class 5 homeobox 1 | 0.31 | 0.85 | 0.76 | 0.94 |
15. Nanog | Nanog homeobox | −4.92 | 2.83 | −4.92 | −4.92 |
Log2(FC) Values | |||||
---|---|---|---|---|---|
Gene Symbol | Gene Name | ATP 2h | TGF-β 2h | ATP 6h | TGF-β 6h |
1. BMP7 | Bone morphogenetic protein 7 | −0.09 | 1.36 | −0.66 | 0.69 |
2. E-Cadherin/CDH1 | Cadherin 1 | 0.23 | 0.04 | −0.82 | −1.17 |
3. LMO2 | LIM domain only 2 | 0.99 | 1.20 | 0.96 | 0.56 |
4. NOTCH1 | Notch 1 | −1.45 | 0.24 | 0.28 | 0.17 |
5. Sonic Hedgehog/SHH | Sonic hedgehog signaling molecule | −0.29 | 1.51 | −0.76 | 0.38 |
6. TRA-1-81/PODXL/TRA-1-60 | Podocalyxin like | −0.16 | 0.26 | 0.62 | 1.99 |
7. Vimentin/VIM | Vimentin | −0.10 | 0.36 | 0.18 | 1.06 |
8. CXCR4 | C-X-C motif chemokine receptor 4 | −1.19 | −1.02 | −0.21 | −0.87 |
9. IL6R | Interleukin 6 receptor | −0.10 | −0.23 | 1.12 | −2.30 |
10. Aminopeptidase N/CD13/ANPEP | Alanyl aminopeptidase, membrane | 0.35 | 0.58 | 0.42 | 1.09 |
11. CXCL8/IL-8 | C-X-C motif chemokine ligand 8 | 1.93 | 0.73 | 0.56 | 0.28 |
12. IL6 | Interleukin 6 | 2.10 | 0.99 | 0.60 | 2.08 |
Number of Tumors Generated out of 10 Injections (X/10) | ||
---|---|---|
Number of Cells Injected | ATP Pretreatment | Without ATP Pretreatment |
3 × 106 | 10/10 | 10/10 |
1 × 106 | 10/10 | 9/10 |
3 × 105 | 9/10 8/10 | |
1 × 105 | 8/10 4/10 |
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Song, J.; Qian, Y.; Evers, M.; Nielsen, C.M.; Chen, X. Cancer Stem Cell Formation Induced and Regulated by Extracellular ATP and Stanniocalcin-1 in Human Lung Cancer Cells and Tumors. Int. J. Mol. Sci. 2022, 23, 14770. https://doi.org/10.3390/ijms232314770
Song J, Qian Y, Evers M, Nielsen CM, Chen X. Cancer Stem Cell Formation Induced and Regulated by Extracellular ATP and Stanniocalcin-1 in Human Lung Cancer Cells and Tumors. International Journal of Molecular Sciences. 2022; 23(23):14770. https://doi.org/10.3390/ijms232314770
Chicago/Turabian StyleSong, Jingwen, Yanrong Qian, Maria Evers, Corinne M. Nielsen, and Xiaozhuo Chen. 2022. "Cancer Stem Cell Formation Induced and Regulated by Extracellular ATP and Stanniocalcin-1 in Human Lung Cancer Cells and Tumors" International Journal of Molecular Sciences 23, no. 23: 14770. https://doi.org/10.3390/ijms232314770