Fig 1: Validation of ALDH1A3 as a tumor-specific biomarker of pro-invasive behavior ARepresentative images of ALDH1A3 expression on pro-invasive (Ren13 and Ren86) and non-pro-invasive (Ren28 and Ren50) tumors.BConfirmation of ALDH1A3 mRNA levels of gene expression in pro-invasive (Ren13 and Ren86) and non-pro-invasive (Ren28 and Ren50) tumors. Average ± SD of 4–6 independent replicates per group were analyzed by Mann–Whitney test *P < 0.05 **P < 0.01.C, DAssociation of pre-treatment ALDH1A3 protein levels by IHC on a series of Ren-PDOX models treated with sunitinib. (C) Representation of ALDH1A3 expression on pro-invasive and non-pro-invasive Ren-PDOX after sunitinib treatment (**P < 0.01 by Chi-Square test). (D) Correlation of pre-treatment ALDH1A3 protein levels by IHC with increased tumor invasion found after sunitinib treatment. n = 7, Spearman’s non-parametric correlation, P = 0.0095.EPatient characteristics of a series of 15 metastatic ccRCC patients specifically annotated for their type of progression after antiangiogenic therapy. 60% progressed in preexisting lesions (non-pro-aggressive) while 40% progressed with new lesions (pro-aggressive).FRepresentation of ALDH1A3 score (0 = absent, 1 = low, 2 = high expression) on pre-treatment samples of pro-aggressive and non-pro-aggressive ccRCC patients described above. n = 15, Chi-square test for independence P = 0.015, and Chi-square test for trend P = 0.030 (*).GROC curve for the ALDH1A3 score on 15 ccRCC patients. ROC variation method was used due to categorical predictor factor. AUC = 80.6 ± 13.8, Sensitivity 83.3% [95%CI: 35.9–99.5], Specificity 33.3% [95%CI: 7.5–70.0], empirical P = 0.008 by Bootstrapping method (n = 1,000).HLevels of ALDH1A3 differentiate between non-pro-invasive and pro-invasive tumors. High levels of ALDH1A3 in samples before treatment are associated with more invasive tumors after antiangiogenic treatment. Source data are available online for this figure.
Fig 2: Selective binding of MCI-INI-3 to ALDH1A3 in glioma stem cell lysates.a Scatter plots of statistical significance versus the change in relative protein abundance for 1810, 1822, and 1545 protein groups were determined by label-free differential mass spectrometry for cellular thermal stability experiments conducted at 45, 50, and 55 °C, respectively. Quantification of unique protein groups was accomplished with the MaxQuant software program and outliers were removed with peptide occupancy filtering. b Box and whisker plots of ALDH1A3 and CPOX protein abundance for vehicle and MCI-INI-3 treated (10 µM) lysates following heating at 55 °C and centrifugation at 25,000 × g. Each of the eight technical replicates (n = 8) was digested with trypsin and subjected to LC-MS/MS based proteomic analysis.
Fig 3: Crystal structure analysis of ALDH1A3 in complex with MCI-INI-3.a Ribbon representation of the ALDH1A3 monomer structure. The ligands NAD+ and MCI-INI-3 are shown as gray and black sticks. b Zoom-in of MCI-INI-3 binding site. The inhibitor is shown as black sticks and the residues defining the binding pockets as pink sticks. c Omit Fo–Fc electron density map covering MCI-INI-3. The omit electron density map is shown in orange contoured at 2.5 standard deviations. d Superposition of the ALDH1A3 and ALDH1A1 structural elements responsible for MCI-INI-3 selectivity for ALDH1A3: N469 and T315 in ALDH1A3 (pink sticks) superimposed to the structurally equivalent G458 and I304 in ALDH1A1. MCI-INI-3 is shown in black sticks. All the images were prepared using the program PyMol.
Fig 4: Analysis of upregulated gene expression levels in SK-BR-3 vs. MDA-MB-231 cells. (A) Upregulated gene expression levels in SK-BR-3 vs. MDA-MB-231 cells. The expression levels of ALDH1A3, ALDH3B2, CD24, CD164 and EpCAM were markedly higher in SK-BR-3 cells. FPKM value represents gene expression level. (B) Analysis of upregulated mRNA expression levels in MDA-MB-231, SK-BR-3, MCF-7 and MCF-10A cell lines by RT-qPCR. All cell lines were normalized to the relative expression levels of MDA-MB-231. GADPH was used as an internal reference gene. ***P<0.001. Error bars represent standard deviation. RT-qPCR was performed in triplicate. (C) Protein expression levels of ALDH1A1, ALDH1A3, CD24, CD44, CD109, CD164 and EpCAM were detected in MCF-10A, SK-BR-3, MDA-MB-231 and MCF-7 cell lines by western blotting. ß-actin served as a control. ALDH, aldehyde dehydrogenase; EpCAM, epithelial cell adhesion molecule; FPKM, Fragments Per Kilobase of transcript per Million mapped reads; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Fig 5: MIR600HG overexpression in combination with Oxaliplatin inhibits tumour relapse(A,B) Tumour volume and weight in xenografts treated with Oxaliplatin, MIR600HG or both at days 29, 32 and 36. Caco2 cells and injected s.c. into nude mice (n=9/group, 1 × 104 cells/mouse). (C) The level of CD133 mRNA derived from xenograft model tumour analysis by RT-PCR. (D) Ki-67 immunohistochemistry assay show that combination of MIR600HG inhibition and Oxaliplatin treatment more significantly inhibits cell proliferation in xenograft tumour. (E) A schematic model of MIR600HG target ALDH1A3 inhibited CRC metastasis and chemosensitivity. NC, negative control oligonucleotides; inhibitor, MIR600HG inhibitor; Oxa, Oxaliplatin. *, P<0.05; **, P<0.01.
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