ZNF750 is a lineage-specific tumour suppressor in squamous cell carcinoma

Abstract

ZNF750 controls epithelial homeostasis by regulating epidermal-differentiation genes, a role underscored by its pathogenic mutations in esophageal squamous cell cancers (SCCs). However, the precise role of ZNF750 in SCC cell biology remains unclear. In this study, we report that ZNF750 is exclusively deleted, mutated and underexpressed in human SCCs, and low ZNF750 expression is associated with poor survival. Restoration of wildtype, but not mutant ZNF750 protein uniquely inhibited the malignant phenotypes of SCC cells both in vitro and in vivo. Notably, ZNF750 promoted the expression of a long non-coding RNA (TINCR), which mediated both cancer-inhibition and differentiation-induction effects of ZNF750. In addition, ZNF750 potently suppressed cell migration by directly inhibiting the transactivation of LAMC2. Together, our findings characterize ZNF750 as a crucial SCC-specific suppressor and uncover its novel anticancer-associated functions.

Figure 1

ZNF750 is exclusively disrupted in squamous cell carcinomas. (a) Analysis of ZNF750 somatic mutations in CSCC, HNSCC, LSCC from TCGA (see URL). Results in ESCC were summarized from published studies.^, Different types of mutations and their location and frequency are shown. (b) Location of ZNF750 mutations in CSCC, HNSCC, LSCC and ESCC. (c) Summary of ZNF750 mutations across different tumour types from TCGA. Numbers in the parentheses indicate the number of cases sequenced. CSCC/EA, CSCC and endocervical adenocarcinoma; CA, colorectal adenocarcinoma; BIC, breast invasive carcinoma; LA, lung adenocarcinoma; BLGG, brain lower grade glioma; AML, acute myeloid leukemia; PA, prostate adenocarcinoma; GM, glioblastoma multiforme; OSC, ovarian serous cystadenocarcinoma; TC, thyroid carcinoma; UC, uterine carcinosarcoma.

Figure 2

ZNF750 is under-expressed in squamous cell carcinomas. (a) ZNF750 mRNA expression across normal tissues examined from GEO (series GSE7307). Data show mean±s.d. (b) ZNF750 protein expression across normal tissues from Human Protein Atlas (see URL). IHC was performed using anti-ZNF750 antibody (HPA021573, Sigma-Aldrich, St Louis, MO, USA). (c) Representative IHC photos (left panel) and IHC scores (right panel) of ZNF750 protein expression in CSCC and HNSCC from Human Protein Atlas. IHC was performed using anti-ZNF750 antibody (HPA023012, Sigma-Aldrich). (d) In-house IHC assay of ZNF750 expression in CSCC, HNSCC and LSCC samples. Normal tissues are from cervical tissues (× 10 magnification). (e) Low ZNF750 expression (mRNA expression z-Scores (RNA Seq V2 RSEM)>mean+1.8 SD or 2.0 SD) was associated with poor disease-free survival of HNSCC and LSCC patients in the TCGA cohorts.

Figure 3

ZNF750 is a p63-dependent transcription factor and controls KLF4 expression in SCCs. (a) qRT-PCR analysis of ZNF750 mRNA in SCC cells following p63 depletion. Data show mean±s.d. N=3. *P<0.05. (b) Western blot analysis of ZNF750 levels in p63-silenced SCC cells. (c, d) Western blot analysis (c) and co-expression analysis (d) of p63 and ZNF750 protein (40 μg/lane) levels in various SCC cell lines. (e) Co-expression analysis of ZNF750 mRNA and p63 mRNA in 86 squamous-type cell lines from CCLE (see URL). (f) Summary of correlation coefficients between ZNF750 mRNA and p63 mRNA across different tumour types from TCGA datasets. (g, h) qRT-PCR analysis of KLF4 mRNA in ZNF750 overexpressed SCC cells (g) and ZNF750 depleted SCC cells (h). Data show mean±s.d. N=3. *P<0.05. (i) Co-expression analysis of KLF4 mRNA and ZNF750 protein (40 μg/lane) levels in various SCC cell lines. (j) Co-expression analysis of ZNF750 and KLF4 mRNA based on TCGA dataset. r; spearman score.

Figure 4

Tumour-suppressive properties of ZNF750 in SCCs. (a–c) Short-term proliferation assay (MTT) (a), foci formation assay (b) and the quantification of SCC cell growth (c) either with ectopic expression of GFP control (CTL), wildtype or C2H2 mutant ZNF750. Data show mean±s.d. N=3. *P<0.05. (d) UMSCC1 cells expressing indicated vectors (upper panel) were injected subcutaneously on the upper flanks of NOD-SCID mice, and tumour weights were measured. Data represent mean±s.d. *P<0.05. (e) Representative samples of Ki67 and IVL expression detected by IHC in dissected xenograft tumours. (f–g) SCC cells expressing indicated vectors were subjected to migration assay (f) and adhesion assay (g). Data show mean±s.d. N=3 (f) and 4 (g). *P<0.05. (h-i), Dead cells (trypan-blue positive) (h) and cleaved-PARP (i) in SCC cells expressing indicated vectors upon anoikis challenge. Data show mean±s.d. N=4 (h). *P<0.05.

Figure 5

Transcriptome analysis of ZNF750-regulated genes and processes. (a) GO analysis of ZNF750-activated and -repressed genes measured by RNA-seq comparing cells over-expressing GFP versus ZNF750 protein. (b) GSEA of ZNF750-induced genes versus indicated gene signatures. (c) Venn diagram illustrating the overlap between LncRNAs regulated by ZNF750 in UMSCC1, and the ones with differential expression patterns during epidermal differentiation reported by Kretz et al. (d) Venn diagram illustrating the overlap between ZNF750-dependent repressed genes and epidermal progenitor signature genes reported by Vanessa et al.

Figure 6

ZNF750 regulates epidermal differentiation in a variety of SCC cells. (a) qRT-PCR analysis of differentiation related genes and (b) basal cell marker K14 gene in SCC cells expressing indicated vectors. Data show mean±s.d. N=3. *P<0.05. (c) Western blot analysis of IVL and K14 levels in SCC cells expressing indicated vectors. (d) qRT-PCR analysis of differentiation related genes in SCC cells treated with either scrambled siRNAs (siCTL) or ZNF750 siRNAs (siZNF750). Data show mean±s.d. N=3. *P<0.05. (e) Western blot analysis of IVL and K14 levels in SCC cells treated with indicated siRNA. (f) Co-expression analysis of ZNF750 mRNA and epidermal differentiation gene mRNA based on TCGA dataset. r; spearman score. (g) Summary of correlation coefficient between the mRNA levels of ZNF750 and indicated differentiation genes across different tumour types from TCGA. (h) Western blot analysis of ZNF750 protein levels in SCC cells under proliferative condition (cells<50% confluent) (P) or differentiation condition (super confluent) (D) in vitro. (i) qRT-PCR analysis of differentiation related genes including KLF4 in non-SCC cell line HaCaT treated with scrambled siRNAs (siCTL) or ZNF750 siRNAs (siZNF750). Data show mean±s.d. N=3. *P<0.05.

Figure 7

LncRNA TINCR mediates the function of ZNF750. (a) qRT-PCR analysis of TINCR in SCC cells either with ectopic expression of GFP control (CTL) or wildtype ZNF750 and (b) in ME180 cells (CSCC) transfected with either scrambled siRNAs (siCTL) or ZNF750 siRNAs (siZNF750). Data show mean±s.d. N=3–6. *P<0.05. (c) Co-expression analysis of TINCR transcripts and ZNF750 protein (40 μg/lane) levels in variety of SCC cell lines. (d) Human H1 embryonic stem cells were differentiated to keratinocyte progenitors in vitro, and the global RNA expression profile during differentiation were analysed using RNA-seq. Sequencing reads after normalization spanning ZNF750 gene are shown. Numbers indicate FPKM value (Fragments Per Kilobase of Exon per Million Fragments Mapped). (e) Co-expression analysis of ZNF750 mRNA and TINCR transcripts from TCGA dataset. (f) Summary of correlation coefficient between ZNF750 mRNA and TINCR transcripts across different tumour types from TCGA dataset. (g) SCC cells expressing either GFP (CTL, control) or wildtype ZNF750 were transfected with either scrambled or TINCR siRNAs and then subjected to foci formation assay (representative photos), and (h) foci were quantified. Data show mean±s.d. N=4. *P<0.05. (i) SCC cells with indicated treatment were subjected to migration assay. (j) Effects of TINCR knockdown on differentiation-related gene expression; heatmaps show average fold change measured by qRT-PCR (N=3). (k) Low TINCR expression is associated with poor disease-free survival of HNSCC patients on TCGA cohort.

Figure 8

ZNF750 represses LAMC2 transactivation. (a) qRT-PCR analysis of LAMC2 mRNA levels in SCC cells expressing indicated vectors. Data show mean±s.d. N=3. *P<0.05. (b) Western blot analysis of LAMC2 levels in SCC cells expressing indicated vectors. (c) ZNF750 motifs present in R2, R3 and R4 DNA fragments 5' upstream of LAMC2 (R1: nucleotide (nt) −3701/−3528; R2: nt −2506/−2273; R3: nt −737/−570; R4: nt −346/−182) (lower). (d) Chip-PCR analysis of DNA fragments pulled down by an anti-HA antibody. IgG was used as a negative control. Representative data are shown. N=3. (e) 293 T cells transfected with the reporter constructs pGL3-(R2), renilla, and either GFP or wildtype ZNF750. Renilla and luciferase activity was analysed. Data show mean±s.d. N=3. *P<0.05. (f) Western blot analysis of LAMC2 and ZNF750 levels in SCC cells in indicated conditions. (g) SCC cells in indicated conditions were subjected to migration assay. Data show mean±s.d. N=3. *P<0.05.