Additional file 1 of Alpha-B-Crystallin overexpression is sufficient to promote tumorigenesis and metastasis in mice
Creators: name: Rashidieh, Behnam
name: Bain, Amanda Louise
name: Tria, Simon Manuel
name: Sharma, Sowmya
name: Stewart, Cameron Allan
name: Simmons, Jacinta Ley
name: Apaja, Pirjo M.
name: Duijf, Pascal H. G.
name: Finnie, John
name: Khanna, Kum Kum
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 1. Figure S1. CRYAB is frequently gained copy number in many cancer types and these copy number changes are correlated with Crystalin expression in some cancers. (a) Chart shows the percentages of copy number amplification (red) or copy number gain (pink) of CRYAB across various cancers using the cBioPortal for Cancer Genomics. (b) Correlation between CRYAB copy number and expression level across cancer types. Comparison between CRYAB copy number (x-axis) and CRYAB expression level (y-axis) in tumour samples from The Cancer Genome Atlas (TCGA) across 14 cancer types, as indicated. X-axis labels respectively refer to: CRYAB copy number deep loss (-2), loss (-1), copy number neutral (0), gain (1) and amplification (2). P values and p value abbreviations: Pearson correlation. Figure S2.CRYAB higher expression is associated with poor survival in multiple cancer types. Kaplan-Meier survival analysis for TCGA cancer type indicated below the chart for the overall survival. Patients were split into two groups of high (red) and low (black) CRYAB expression levels. Numbers of patients at risk are shown at the bottom. Figure S3. CRYAB overexpression correlates with deregulation of signaling, oncogenic features and tumor infiltration across some cancers. (a–d) The heatmaps show the correlations between CRYAB gene expression levels and variables (determined via the integration of multi-platform data from several types of ‘omics’ analyses) listed on the y-axis in up to 32 cancer types, whose abbreviations are listed on the top x-axis based on the methodology previously described 9 ; for all analysis (a-d): each tile in the heatmaps shows the Spearman correlation between the gene expression levels (mRNA levels from The Cancer Genome Atlas (TCGA) RNAseq datasets) and the variables listed below. The color of each tile reflects the Spearman correlation coefficient (r), as indicated by the color key on the right. Each tile shows the Spearman p value abbreviations in the following format: blank tiles: p &gt; 0.05; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001; ****, p &lt; 0.0001. Cancer type abbreviations: ACC, Adrenocortical Cancer; BLCA, Bladder Cancer; BRCA, Breast Cancer; CESC, Cervical Cancer; CHOL, Cholangiocarcinoma (bile duct cancer); COAD, Colon Cancer; DLBC, Large B-cell Lymphoma; ESCA, Esophageal Cancer; GBM, Glioblastoma; HNSC, Head and Neck Cancer; KICH, Kidney Chromophobe; KIRC, Kidney Clear Cell Carcinoma; KIRP, Kidney Papillary Cell Carcinoma; LGG, Lower Grade Glioma; LIHC, Liver Cancer; LUAD, Lung Adenocarcinoma; LUSC, Lung Squamous Cell Carcinoma; MESO, Mesothelioma; OV, Ovarian Cancer; PAAD, Pancreatic Cancer; PCPG, Pheochromocytoma &amp; Paraganglioma; PRAD, Prostate Cancer; READ, Rectal Cancer; SARC, Sarcoma; SKCM, Melanoma; STAD, Stomach Cancer; TGCT, Testicular Cancer; THCA, Thyroid Cancer; THYM, Thymoma; UCEC, Endometrioid Cancer; UCS, Uterine Carcinosarcoma; UVM, Ocular melanoma. Figure S4. Protein alignment and mouse model target validation. (a) Alignment of human (NP_001876, upper in red) and mouse (NP_034094.1, lower in blue) ɑB-Crystallin protein sequences. The spaces indicate sequence mismatch and a + indicates a positive match on the scoring matrix and ɑB-crystallin domain is underlined in the consensus. (b) PCR genotyping showing CryabWt/Tg (Het), CryabWt/Wt (Wt) and CryabTg/Tg (Tg) genotypes. (c) Immunoblot analysis of Crystallin protein expression of CryabWt/Wt, CryabWt/Tg, and CryabTg/Tg mouse tissues as indicated in the figure. β-actin was used as a loading control. (d) Immunoblot analysis of Crystallin protein expression of tumor and adjacent tumor tissues from two lungs of CryabTg/Tg mice. Vinculin was used as a loading control. Figure S5. CRYAB overexpression increases carcinogen (DMBA)-induced tumorigenesis (a) Chart shows number of nodules from dissected lungs of DMBA-treated CryabWt/Wt, CryabWt/Tg, and CryabTg/Tg mice, the outliers were removed using the ROUT method (Q = 1%) based on the False Discovery Rate (FDR). (b) representative image of Bouin's solution fixed lungs from each genotype, the visible lung tumors (nodules) were counted by checking the surface of each lung lobe (upper) and H&amp;E stained tissue from CryabWt/Wt and CryabTg/Tg was used to validate the tumor counting ( lower), arrows show the nodules or tumors, the bigger size of tumors is evident in CryabTg mice.. Figure S6. IHC on tumors and normal tissues and targeted mass spectrometry (MS). (a) IHC staining compares the expression level of indicated markers in malignant tissue of CryabTg/Tg with age and tissue matched control from CryabWt/Wt, (b) IHC staining compares expression of indicated markers in Crystallin high and low expression regions of malignant tissue from CryabTg mice. (c) Chart shows the peak area of indicated peptides (obtained based on targeted-MS) ɑ-SMA (VAPEEHPTLLTEAPLNPK), Vimentin (FADLSEAANR ) and PDGFR (YGDLVDYLHR) in each group of CryabWt liver (black), normal liver tissue from CryabTg (blue), liver tumor from CryabTg (red). All protein lysates obtained from FFPE tissue sections annotated by pathologist for tumor and normal tissues. (d) IHC staining compares the expression level of indicated markers: Crystallin (green), F4/80 (macrophages, yellow) DAPI (nucleus, blue) in Crystallin high and low expression regions of liver hepatocellular carcinoma section (left) and lung adenocarcinoma sections of CryabTg/Tg mice(right), Boxed areas are shown at higher magnification where indicated (Scale bars, 100 µm). Figure S7. Genotyping and WB on MEFs. (a) PCR genotyping showing CryabWt/Wt (Wt) and CryabTg/Tg (Tg) E1A/Ras transformed MEFs genotypes (b) Immunoblot analysis of Crystallin protein expression of CryabWt/Wt, and CryabTg/Tg E1A/Ras transformed MEFs after heat-shock (43̊ C for 1 hour) following by time course incubation as indicated in the figure. GAPDH was used as a loading control. Figure S8. Biological process annotation. (a) Functional annotation and pathway enrichment analysis of differentially regulated proteins of the proteomic data comparing Cryab Tg E1ARas-transformed MEFs to CryabWT MEFs for either upregulated ( green) or downregulated (yellow), Data were normalized to the enrichment ratio. Data was plotted using default parameters of DAVID (the database for annotation, visualization and integrated discovery) (b) The table shows the top five highest upregulated genes. (c) co-occurance analysis of CRYAB gene and the top five highest upregulated genes from TCGA pan-cancer. Figure S9. CRYAB expression is associated with metastasis in human tumors. (a) CRYAB expression levels in normal cervix and early-stage cervical tumors. (b) CRYAB expression levels in primary and metastatic renal cell carcinoma. (c-f) CRYAB expression levels in primary tumors without and with metastasis in, respectively: (c) hepatocellular carcinoma, (d) pancreatic cancer, (e) breast cancer and (f) colorectal cancer. (g) CRYAB expression levels in pancreatic cancer locally metastasized to pancreas or distantly metastasized to muscle. (h) CRYAB expression levels in prostate cancer locally metastasized to lymph node or distantly metastasized to liver. (i) CRYAB expression levels in lung cancer locally metastasized to lymph node or distantly metastasized to brain. Error bars indicate means and standard errors. P values: two-sided t tests. (j) Immunoblot analysis of siRNA mediated knockdown of Basigin was performed on E1A/Ras-transformed MEF using Scrambled as a control and two siRNA sequences targeting exon 2 and exon 6 of Basigin, vinculin served as a loading control.