Cancer Res Treat.  2021 Oct;53(4):944-961. 10.4143/crt.2020.466.

Correlation of NUF2 Overexpression with Poorer Patient Survival in Multiple Cancers

• Jiang X
• Jiang Y
• Luo S
• Sekar K
• Koh CKT
• Deivasigamani A
• Dong Q
• Zhang N
• Li S
• Hao F
• Goh BKP
• Ooi LL
• Wang Y
• Hui KM

• KMID: 2521571
• DOI: http://doi.org/10.4143/crt.2020.466

Abstract

Purpose
NUF2 has been implicated in multiple cancers recently, suggesting NUF2 may play a role in the common tumorigenesis process. In this study, we aim to perform comprehensive meta-analysis of NUF2 expression in the cancer types included in the Cancer Genome Atlas (TCGA).
Materials and Methods
RNA-sequencing data in 31 cancer types in the TCGA data and 11 independent datasets were used to examine NUF2 expression. Silencing NUF2 using targeting shRNAs in hepatocellular carcinoma (HCC) cell lines was used to evaluate NUF2’s role in HCC in vitro and in vivo.
Results
NUF2 up-regulation is significantly observed in 23 out of the 31 cancer types in the TCGA datasets and validated in 13 major cancer types using 11 independent datasets. NUF2 overexpression was clinically important as high NUF2 was significantly associated with tumor stages in eight different cancers. High NUF2 was also associated with significantly poorer patient overall survival and disease-free survival in eight and six cancers, respectively. We proceeded to validate NUF2 overexpression and its negative association with overall survival at the protein level in an independent cohort of 40 HCC patients. Compared to the non-targeting controls, NUF2 knockdown cells showed significantly reduced ability to grow, migrate into a scratch wound and invade the 8 μm porous membrane in vitro. Moreover, NUF2 knockdown cells also formed significantly smaller tumors than control cells in mouse xenograft assays in vivo.
Conclusion
NUF2 up-regulation is a common feature of many cancers. The prognostic potential and functional impact of NUF2 up-regulation warrant further studies.

Keyword

NUF2; Pan-cancer; Overall survival; Disease-free survival; Cell cycle

Figure

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  Fig. 1 NUF2 expression in tumor versus non-tumor samples in 31 cancer types. (A) Boxplots showing significant overexpression of NUF2 in tumor samples compared to non-tumor samples in 23 cancer types. (B) Boxplots showing significant under-expression of NUF2 in tumor samples compared to non-tumor samples in LAML and TGCT. (C) Boxplots showing NUF2 expression is not statistically significantly different between tumor samples and non-tumor samples in six cancer types. Each dot indicates a sample. The red box on the left represents tumor samples (T) while the grey box on the right represents non-tumor (N) samples. The asterisks indicate statistically significant NUF2 differential expression between T and N samples with unpaired Student’s t test p < 0.05. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; TPM, transcripts per milion; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma.

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  Fig. 2 High NUF2 level is consistently observed in multiple cancer patient datasets and cell lines. (A) Overexpression of NUF2 transcript in tumors versus non-tumor tissues is validated in BLCA (GSE40355), BRCA (GSE42568), CESC (GSE27678), CHOL (GSE26566), COAD (GSE110225), HNSC (GSE107591), LGG & GBM (GSE16011), LUAD & LUSC (GSE30219), PAAD (GSE28735), and SKCM (GSE15605). Relative NUF2 expression is expressed as normalized probe intensity in Log2 scale for microarray data or RPKM for RNA-sequencing data. (B) Normalized NUF2 transcript expression in 20 normal tissues (left panel) and 48 cancer cell lines from 24 cancer types. NUF2 transcript expression is measured using RT-qPCR normalized against GAPDH in 20 normal tissues and MCF7, HepG2, HCT116WT, Huh7, and THP1 cell lines. NUF2 transcript expression of 48 cancer cell lines is retrieved from Cancer Cell Line Encyclopedia (CCLE) and normalized against the average factor according to the five common cell lines. BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; N, non-tumor; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; RPKM, reads per kilo base per million mapped reads; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; T, tumor; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma.

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  Fig. 3 NUF2 expression is associated with tumor stages in eight cancer types. Violin plots showing higher NUF2 expression in more advanced stages (III and IV) in adrenocortical carcinoma (ACC) (A), kidney chromophobe (KICH) (B), kidney renal clear cell carcinoma (KIRC) (C), kidney renal papillary cell carcinoma (KIRP) (D), liver hepatocellular carcinoma (LIHC) (E), lung adenocarcinoma (LUAD) (F), lung squamous cell carcinoma (LUSC) (G), and thyroid carcinoma (THCA) (H).

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  Fig. 4 High NUF2 level is significantly associated with poorer patient survival in multiple cancer types. (A) Top: Overall survival map showing log10 (HR) between NUF2 high and NUF2 low tumors based on median NUF2 expression in 31 cancer types. The red box indicates a significant difference at p < 0.05. Bottom: Kaplan-Meier survival curve analysis showing high NUF2 level is significantly associated with poorer overall survival in nine cancer types including adrenocortical carcinoma (ACC), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), brain lower grade glioma (LGG), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD), sarcoma (SARC), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), testicular germ cell tumors (TGCT), thyroid carcinoma (THCA), thymoma (THYM), uterine corpus endometrial carcinoma (UCEC), and uterine carcinosarcoma (UCS). HR, hazard ratio; TPM, transcripts per milion. (B) Top: Disease-free survival map showing log10 (HR) between NUF2 high and NUF2 low tumors based on median NUF2 expression in 31 cancer types. The red box indicates a significant difference at p < 0.05. Bottom: Kaplan-Meier survival curve analysis showing high NUF2 level is significantly associated with poorer disease-free survival in six caner types including ACC, KIRP, LGG, LIHC, PRAD, and SARC.

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  Fig. 5 NUF2 overexpression in HCC. (A) Box dot plot showing significant higher NUF2 transcript in tumor versus non-tumor tissues from LINC-JP dataset in ICGC. (B) Kaplan-Meier survival curve analysis showing high NUF2 transcript level is associated with significantly poorer overall survival in the same LINC-JP dataset. (C) Immunohistochemistry staining using NUF2 specific antibody in a representative HCC patient section from a local cohort of 40 patients. NUF2 protein is found to be significantly higher expressed, compared to the paired adjacent non-tumor section. Scale bars=100 μm. (D) Kaplan-Meier survival curve analysis showing high NUF2 transcript level is associated with significantly poorer overall survival in this cohort of 40 HCC patients. HCC, hepatocellular carcinoma; ICGC, International Cancer Genome Consortium.

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  Fig. 6 NUF2 inhibition significantly reduced HCC cell growth in vitro and in vivo. (A) Significant inhibition of NUF2 transcript level in HepG2 (left) and Huh7 (right) cells stably expressing shRNAs against NUF2 (shNUF2-1 & -2) compared with that of shRNA controls (shControl-1 & -2), measured using quantitative reverse transcription polymerase chain reaction and normalized against endogenous GAPDH. (B) Significant inhibition of in vitro cell growth in HepG2 (left) and Huh7 (right) cells stably expressing shRNAs against NUF2 (shNUF2-1 & -2) compared with that of shRNA controls (shControl-1 & -2), measured using live-cell imaging with Incucyte Zoom. (C) Significant inhibition of in vitro cell migration in Huh7 cells stably expressing shRNAs against NUF2 (shNUF2-1 & -2) compared with that of shRNA controls (shControl-1 & -2), measured using scratch wound healing assay. (D) Significant inhibition of in vitro cell invasion in HepG2 (top) and Huh7 (bottom) cells stably expressing shRNAs against NUF2 (shNUF2-1 & -2) compared with that of shRNA controls (shControl-1 & -2), measured using invasion chamber assay. (E) Significant inhibition of in vivo xenograft tumor growth in Huh7 cells stably expressing shRNAs against NUF2 (shNUF2-1 & -2) compared with that of shRNA controls (shControl-1 & -2), measured using mouse xenograft assay. Data were expressed as wet tumor weight. *p < 0.05. (F) Ingenuity pathway analysis showing the interconnected network formed by the top 100 NUF2-correlated genes in TCGA-LIHC dataset. (G) Gene set enrichment analysis showing cell cycle, DNA replication and p53 signaling pathways as the most enriched pathways associated with NUF2. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas.

Reference

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