CD47 and IFT57 Are Colinear Genes That Are Highly Coexpressed in Most Cancers and Exhibit Parallel Cancer-Specific Correlations with Survival

Abstract

An association between high CD47 expression and poor cancer survival has been attributed to its function on malignant cells to inhibit phagocytic clearance. However, CD47 mRNA expression in some cancers lacks correlation or correlates with improved survival. IFT57 encodes an essential primary cilium component and is colinear with CD47 across amniote genomes, suggesting coregulation of these genes. Analysis of The Cancer Genome Atlas datasets identified IFT57 as a top coexpressed gene with CD47 among 1156 human cancer cell lines and in most tumor types. The primary cilium also regulates cancer pathogenesis, and correlations between IFT57 mRNA and survival paralleled those for CD47 in thyroid and lung carcinomas, melanoma, and glioma. CD47 ranked first for coexpression with IFT57 mRNA in papillary thyroid carcinomas, and higher expression of both genes correlated with significantly improved overall survival. CD47 and IFT57 mRNAs were coordinately regulated in thyroid carcinoma cell lines. Transcriptome analysis following knockdown of CD47 or IFT57 in thyroid carcinoma cells identified the cytoskeletal regulator CRACD as a specific target of IFT57. CRACD mRNA expression inversely correlated with IFT57 mRNA and with survival in low-grade gliomas, lung adenocarcinomas, and papillary thyroid carcinomas, suggesting that IFT57 rather than CD47 regulates survival in these cancers.

Keywords: CD47; RNA sequencing; capping protein inhibiting regulator of actin dynamics (CRACD); glioma; intraflagellar transport-57 (IFT57); lung adenocarcinoma; microsynteny; papillary thyroid carcinoma; primary cilium.

Figure 1

Coexpression of IFT57 with CD47 in TCGA tumors and cancer cell lines. (a) Scatter plot showing coexpression of CD47 and IFT57 for lung adenocarcinomas (n = 517). (b) Scatter plot showing coexpression of CD47 and IFT57 for lung squamous carcinomas (n = 501). (c) Scatter plot showing coexpression of CD47 and IFT57 for papillary thyroid carcinomas (n = 509). (d) Scatter plot showing coexpression of CD47 and IFT57 for cell lines in cancer cell lines from the CCLE (n =1156).

Figure 2

IFT57 and CD47 mRNA expression have tumor-specific parallel correlations with overall survival. (a) Decreased trend for overall survival for lung squamous carcinomas with CD47 mRNA expression greater than the mean (n = 501). (b) Decreased overall survival for lung squamous carcinomas with IFT57 mRNA expression greater than the mean. (c) Decreased trend for overall survival for low-grade gliomas with CD47 mRNA expression greater than the mean (n = 530). (d) Decreased overall survival for low-grade gliomas with IFT57 mRNA expression greater than the mean. (e) Improved overall survival for lung adenocarcinomas with CD47 mRNA expression greater than the mean (n = 517). (f) Improved overall survival for lung adenocarcinomas with IFT57 mRNA expression greater than the mean.

Figure 3

Higher IFT57 or CD47 mRNA expression correlates with improved survival in papillary thyroid carcinomas. (a) Overall survival for tumors segregated based on CD47 mRNA expression greater than or less than the mean (n = 509). (b) Disease-free survival for tumors segregated based on CD47 mRNA expression greater than or less than the mean (n = 509). (c) Overall survival for tumors segregated based on IFT57 mRNA expression greater than or less than the mean (n = 509). (d) Disease-free survival for tumors segregated based on IFT577 mRNA expression greater than or less than the mean (n = 509).

Figure 4

IFT57 and CD47 mRNA expression in normal and malignant thyroid epithelial cells and responses to transcriptional regulators. (a–c) MDA-T68 cells were treated with 10 μM JQ1 in DMSO or DMSO control for the indicated times. Expression of IFT57 and the long and short isoforms of CD47 mRNA were analyzed using real-time qPCR. (d) MDA-T68 cells were treated with 10 μM vemurafenib in DMSO or DMSO control for 24 h (n = 3). Expression of IFT57 and the long and short isoforms of CD47 mRNA were analyzed using real-time qPCR. (e) Scatter plot showing linear regression analysis of CD47 and IFT57 mRNA levels in the 5 primary anaplastic thyroid carcinoma cell lines in CCLE.3.2. (f–h) in Nthy-ori 3-1 normal follicular thyroid epithelial cells, the follicular variant of papillary thyroid carcinoma cell line MDA-T68, and 8505C anaplastic thyroid carcinoma cell lines were analyzed for expression of IFT57 and long and short CD47 transcripts (n = 3, (** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant)) using default setting of GraphPad Prism software version 10.0.0..

Figure 5

Validation of CD47 and IFT57 mutants of the 8505C anaplastic thyroid carcinoma cell line. (a) Expression of IFT57 mRNA in WT 8505C and the sg2-B6 and sg1-F4 IFT57 knockdown mutants. (b) Expression of CD47 short mRNA in WT 8505C and the sg2-B6 and sg1-F4 IFT57 knockdown mutants. (c) Expression of IFT57 mRNA in WT 8505C and the CD47 knockdown mutant pool. (d) Flow cytometry analysis of cell surface CD47 expression in WT and mutant 8505C cell lines. (e) Western blot analysis of IFT57 and α-tubulin protein expression in WT and mutant 8505C cell lines. (f) Expression of PCNA mRNA in WT 8505C and the sg2-B6 and sg1-F4 IFT57 knockdown mutants. (g) Proliferation of WT 8505C cells and the sg2-B6 and sg1-F4 IFT57 knockdown mutants assessed using the MTS assay (mean ± SD, n = 6), (** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001) using default setting of GraphPad Prism software version 10.0.0.

Figure 6

Analysis of differentially expressed genes identified by RNAseq in IFT57 and CD47 knockdown mutant 8505C cell lines and in TCGA papillary thyroid carcinomas. (a) Venn diagram showing overlap between differentially expressed genes with p < 0.05 in two IFT57 mutants versus WT (red) and the CD47 mutant pool versus WT 8505C cells (blue). (b) Of the 202 genes up-regulated or down-regulated >8-fold in 8505C IFT57 knockdown cells, 48 exhibited coexpression with IFT57 in TCGA papillary thyroid carcinomas with p < 10⁻⁴. Spearman’s correlations for coexpression of these genes with IFT57 and CD47 mRNA expression in TCGA papillary thyroid carcinomas are presented as a scatter plot. (c–g) Real-time qPCR validation of IFT57-dependent genes identified by RNSseq analysis. p-value ≤ 0.05 (*), ≤0.005 (**), ≤0.001 (***) was calculated using default settings of Bio-Rad CFX Maestro software 2.3. (c) Relative expression of RNF180 mRNA in parental 8505C cells (WT), the CD47 knockdown pool, and the indicated IFT57 knockdown clones. (d) Expression of nidogen-2 (NID2) mRNA in parental 8505C cells (WT), the CD47 knockdown pool, and the indicated IFT57 knockdown clones. (e) Expression of neuromedin-B (NMB) mRNA in parental 8505C cells (WT), the CD47 knockdown pool, and the indicated IFT57 knockdown clones. (f) Expression of CRACD mRNA in parental 8505C cells (WT), the CD47 knockdown pool, and the indicated IFT57 knockdown clones. (g) Expression of GAS6 mRNA in parental 8505C cells (WT), the CD47 knockdown pool, and the indicated IFT57 knockdown clones. (h) Intracellular flow cytometry of CRACD protein expression in parental 8505C cells (WT), the CD47 knockdown pool, and the indicated IFT57 knockdown clones.

Figure 7

Correlations for CRACD mRNA expression with survival in IFT57-dependent cancers. (a) Overall survival for papillary thyroid tumors segregated based on CRACD mRNA expression greater than or less than the mean (n = 509). (b) Disease-free survival for papillary thyroid tumors segregated based on CRACD mRNA expression greater than or less than the mean (n = 509). (c) Overall survival for lung adenocarcinomas segregated based on CRACD mRNA expression greater than or less than the mean (n = 517). (d) Overall survival for low-grade gliomas segregated based on RNF180 mRNA expression greater than or less than the mean (n = 530).

Scheme 1

Model for coregulation of CD47 and IFT57 in thyroid carcinoma. Factors engaging enhancers that coregulate the transcription of IFT57 and CD47 in papillary thyroid tumors (a) may account for their ability to regulate the expression of IFT57- and CD47-dependent genes. Factors that post-transcriptionally regulate IFT57 or CD47 protein levels (b,d) or their downstream signaling (c,e) may account for the exclusive dependence of CRACD on IFT57 but not CD47 mRNA expression.