Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors
<p>The AKT degrader outcompeted AKT inhibitors in impeding cell growth. (<b>A</b>) The colony formation assay compared the effect on colony formation of the AKT degrader INY-05-040 and inhibitor GDC0068 in the human pancreatic cancer cell line PANC-1, low-passage patient-derived PDAC cell UM5, and mouse PDAC cell line KPC. A total of 50 cells were seeded into each well of 6-well plates and grew in indicated conditions for 9 days, 16 days, or 20 days, respectively, before crystal violet staining. Media were replaced every 4 days once drug treatment started. Left, pictures of the colonies. Middle and right, the quantifications of colony sizes and numbers of the colony formation assay. Data were analyzed with a one-way ANOVA, followed by multiple comparisons with the Tukey’s method. ns not significant, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, **** <span class="html-italic">p</span> < 0.0001. (<b>B</b>–<b>D</b>) Immunoblot comparing the AKT degrader and inhibitor treatment in the human pancreatic cancer cell line PANC-1, low-passage patient-derived PDAC cell UM5, and mouse KPC cell line FC1245, respectively. Cells were cultured as in (<b>A</b>).</p> "> Figure 2
<p>Generation of KPC cell lines deficient in <span class="html-italic">Akt</span> isoforms by CRISPR-Cas9 genome editing. (<b>A</b>) Schematic of the strategy to genetically delete all three <span class="html-italic">Akt</span> paralogous genes. (<b>B</b>) Immunoblot of AKT proteins in knockout cell lines. <b>(C</b>) Immunoblot comparing AKT signaling between KPC vs. <span class="html-italic">Akt</span>-deficient cell lines. (<b>D</b>) Immunoblot using indicated phospho-antibodies comparing MEK/ERK signaling between KPC vs. <span class="html-italic">Akt</span>-deficient cell lines. Both blots were probed with anti-HSP90 antibodies as an additional loading control.</p> "> Figure 3
<p>Loss of <span class="html-italic">Akt1/2/3</span> slowed KPC cell growth in vitro. (<b>A</b>) The growth curve of <span class="html-italic">Akt</span>-deficient KPC cell lines indicated <span class="html-italic">Akt1/2/3</span> KO cells grew the slowest. A total of 50,000 cells were seeded in each well of 6-well plates for a four-day growth assay (<span class="html-italic">n</span> = 3). The experiments were repeated three times with different passage numbers of the cell lines. The cell numbers were plotted as mean ± SD; the cell numbers on day 4 were analyzed by a one-way ANOVA (<span class="html-italic">p</span> < 0.0001). Šídák’s multiple comparisons test was conducted between every possible pair, but only comparisons with <span class="html-italic">p</span> < 0.05 are shown. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001. (<b>B</b>) The colony formation assay of <span class="html-italic">Akt</span>-deficient cell lines. A total of 50 cells were seeded into each well of 6-well plates and grew for 7 days before evaluation. On the left are the images of the colonies of the indicated cell lines in technical replicates (<span class="html-italic">n</span> = 3). On the right is the quantification of the colony numbers and sizes. The colony numbers of parental KPC and <span class="html-italic">Akt1/2/3</span>KO cells were compared with the <span class="html-italic">t</span>-test. The colony sizes of all the lines were compared by a nested one-way ANOVA (<span class="html-italic">p</span> < 0.0001). Multiple comparisons were performed with Tukey’s method between KPC and every other cell line, but only comparisons with <span class="html-italic">p</span> < 0.05 are shown. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001. See also <a href="#app1-cells-13-01061" class="html-app">Supplementary Figures S1 and S3</a>.</p> "> Figure 4
<p>Genetic ablation of <span class="html-italic">Akt1/2/3</span> slowed the progression of KPC tumors in a syngeneic orthotopic implantation mouse model. (<b>A</b>) Representative IVIS images for indicated time points of surviving mice with orthotopic cancer cell implantation. B6 mice were implanted with pancreatic cancer cells of the indicated genotypes. Mice were imaged using IVIS one day post-implantation and every week thereafter. The sample sizes are labeled in (<b>B</b>). (<b>B</b>) Quantification of the total flux from IVIS data of all the mice. As there were missing values, a mixed-effects analysis was conducted between KPC and <span class="html-italic">Akt1/2/3</span> KO with repeated measures, followed by Tukey’s multiple comparisons test. The statistical insignificance between KPC and <span class="html-italic">Akt1/2/3</span> KO of the 2-week time point was in part caused by the reduced sample size due to the deaths of the mice. (<b>C</b>) The Kaplan–Meier survival curve of mice implanted with the indicated cell line. Log-rank test of the survival of animals was performed (<span class="html-italic">p</span> < 0.0001). The multiple comparisons between mice implanted with KPC and each of the single-, double-<span class="html-italic">Akt-</span>deficient cell lines, and <span class="html-italic">Akt1/2/3</span>KO and between <span class="html-italic">Akt1/2/3</span>KO and each of the double-<span class="html-italic">Akt</span>-deficient groups were made with Bonferroni correction, and only comparisons beyond the threshold (α<sub>bonferroni</sub> = 0.05/10 = 0.005) are shown. ns not significant, * <span class="html-italic">p</span> < 0.005 ** <span class="html-italic">p</span> < 0.001, *** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 5
<p><span class="html-italic">Akt1/2/3</span> was required for growth-promoting IGF-1 signaling in KPC cells. (<b>A</b>) Colony formation assay of parental KPC and <span class="html-italic">Akt1/2/3</span>KO cells cultured in the indicated conditions. A total of 10,000 cells were seeded in a 96-well plate and treatment started on day 2 by replacing the media with either DMEM with PBS, DMEM containing 100 ng/mL EGF or 100 ng/mL IGF-1 or 100 ng/mL EGF plus 100 ng/mL IGF-1, or 10% FBS. The media were replaced every day and cells were stained with crystal violet on day 5. The quantification of the crystal-violet-positive area with Image J is shown on the right; a two-way ANOVA was performed followed by Šídák’s multiple comparisons test. *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.001. (<b>B</b>) Heatmap of all genes by RNA-seq of KPC and <span class="html-italic">Akt1/2/3</span>KO (<span class="html-italic">n</span> = 3). The experiment was conducted with triplicates of three different passages. The color scale for the z-score is shown on the right. (<b>C</b>) GSEA pathway enrichment analysis of the differentially expressed genes between KPC and <span class="html-italic">Akt1/2/3</span>KO cell lines, and subsequent visualization by EnrichmentMap in Cytoscape showed a cluster of interconnected pathways related to cholesterol metabolism. (<b>D</b>) The transcript levels of key genes involved in the cholesterol synthesis pathway. <span class="html-italic">t</span>-tests were performed. ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001. <span class="html-italic">Srebp2</span> is a master regulator of cholesterol metabolism. <span class="html-italic">Hmgcs1</span> and <span class="html-italic">Hmgcr</span> encode the rate-limiting enzymes in the mevalonate pathway. <span class="html-italic">Ldlr</span> encodes the low-density lipoprotein receptor, responsible for cholesterol uptake. (<b>E</b>) Protein-mass-normalized whole-cell cholesterol levels were measured by the Amplex Red assay of KPC and <span class="html-italic">Akt1/2/3</span>KO cells in the indicated conditions. Two-way ANOVA with uncorrected Fisher’s LSD test. ns not significant, ** <span class="html-italic">p</span> < 0.01.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. AKT Degrader and Inhibitor Treatment
2.3. Crispr-Cas9-Mediated Genome Editing
2.4. Immunoblotting
2.5. Cell Counting and Colony Formation Assay
2.6. Three-Dimensional Culture
2.7. Apoptosis Assay and Cell Cycle Analysis
2.8. Orthotopic Implantation and IVIS Imaging
2.9. RNA-Seq Analysis
2.10. Amplex Red Cholesterol Measurement Assay
2.11. RPPA
3. Results
3.1. AKT Degrader Was Superior to AKT Kinase Inhibitor in Slowing Pancreatic Cancer Cell Growth
3.2. Generation of KPC Cell Lines Deficient in AKT Isoforms by CRISPR-Cas9 Genome Editing
3.3. Genetic Deletion of Akt1/2/3 Significantly Impeded In Vitro PDAC Cell Growth
3.4. Genetic Ablation of Akt1/2/3 Slowed KPC Pancreatic Tumor Progression in a Syngeneic Orthotopic Implantation Mouse Model
3.5. Growth Effects of IGF-1-AKT Signaling in the KPC Cell Line and Role of Cholesterol Metabolism
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Chen, C.; Jiang, Y.-P.; You, I.; Gray, N.S.; Lin, R.Z. Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors. Cells 2024, 13, 1061. https://doi.org/10.3390/cells13121061
Chen C, Jiang Y-P, You I, Gray NS, Lin RZ. Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors. Cells. 2024; 13(12):1061. https://doi.org/10.3390/cells13121061
Chicago/Turabian StyleChen, Chuankai, Ya-Ping Jiang, Inchul You, Nathanael S. Gray, and Richard Z. Lin. 2024. "Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors" Cells 13, no. 12: 1061. https://doi.org/10.3390/cells13121061