A Novel ER Stress Mediator TMTC3 Promotes Squamous Cell Carcinoma Progression by Activating GRP78/PERK Signaling Pathway

Abstract

During tumor progression, tumor cells are exposed to various stress conditions, which result in endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to restore ER homeostasis. Accumulating evidence reported the orchestrating role of ER stress in epithelial-mesenchymal transition (EMT) progress, but the detailed mechanism was unclear. Here, we identified ectopic expression of TMTC3 in cells undergoing ER stress and verified the association with EMT markers through the cellular model of ER stress and database analysis. TMTC3 was abnormally highly expressed in squamous cell carcinomas (SCCs), and regulated by TP63, an SCCs-specific transcription factor. Biological function experiments indicated that TMTC3 promoted a malignant phenotype in vitro, and accelerated tumor growth and metastasis in vivo. RNA-seq analyses and further experiments revealed that TMTC3 promoted the expression of EMT markers via interleukin-like EMT inducer (ILEI, FAM3C). Further studies on the mechanism showed that TMTC3 disrupted the interaction between PERK and GRP78 to activate the PERK pathway and promote the nuclear translocation of ATF4, which increased the transcriptional activity of ILEI. These findings indicated that TMTC3 activates GRP78/PERK signaling pathway during ER stress-induced EMT, which might serve as a potential therapeutic target in SCCs.

Keywords: EMT; ER stress; TMTC3; squamous cell carcinoma.

Figure 1

Identification of TMTC3 in cells under ER stress. A. Heatmap of DEGs by RNA-seq of human ESCC cell lines treated with complete medium or medium without amino acids for 48 h. B. Pathways associated with ER were altered in cells cultured without amino acid supplementation. C. Volcano plot for genes involved in ER stress, red for upregulated genes and blue for down-regulated genes. D. The protein levels of ER sensors and TMTC3 in SCCs cells, including ESCC, LUSC, HNSC. E. The FPKM of TMTC3 in transcriptome sequencing data in 91 paired ESCC tumors and adjacent normal tissues. F. Cross correlation analysis of reads between TMTC3 and EMT markers from transcriptome sequencing data in 91 paired ESCC tissues. G. The knockdown efficiency of TMTC3 by siRNA in KYSE180, KYSE410 and KYSE450 cells detected by Western blot. H. Representative IF staining images showing the expression of TMTC3 and Vimentin in KYSE410 and KYSE450 cells. The scale bar represents 30 μm.

Figure 2

Ectopic expression of TMTC3 in SCCs tissues. A. Representative IHC images of TMTC3 in the ESCC tissue array. B. IHC staining score for each ESCC tissue spot was calculated based on the intensity and area of the staining for TMTC3. C. High expression of TMTC3 was associated with poor survival in ESCC patients. D and E. Univariate (D) and multivariate (E) Cox regression survival analysis of correlations between overall survival and other clinicopathological features. *, p < 0.05. **, p < 0.01. F and G. Representative IHC images (F) and IHC staining score for TMTC3 (G) in lung cancer tissue array. H and I. Representative IHC images (H) and IHC staining score for TMTC3 (I) in HNSC tissue array.

Figure 3

TMTC3 was regulated by ΔNp63 at the transcriptional level. A. Schematic diagram depicting the positions of the binding sites of TP63 in the TMTC3 promoter from the JASPAR database. TSS, transcription starting site. B. ChIP-qPCR analysis of the interaction between ΔNp63 and the TMTC3 promoter. IgG was used as a negative control. *, p < 0.05, **, p < 0.01. C. The luciferase activity of TMTC3 after stimulation with ΔNp63. ***, p < 0.001. D. The mRNA expression of TMTC3 after overexpressing ΔNp63. E. The ΔNp63 levels in SCCs cells treated with complete medium or with amino acid and FBS-deficient medium for 24 h. F and G. The correlation between TP63 and TMTC3 in EC (F) and other SCCs, including CESC, HNSC, LUSC (G) from GEPIA2 database.

Figure 4

TMTC3 promoted EMT progress via ILEI. A. The representative images of the Transwell assay in ESCC cells transfected with siRNA for TMTC3. The scale bar represents 500 μm. B. Effects of TMTC3 siRNA on ESCC cell growth via RTCA assays. C. Heatmap of DEGs by RNA-seq in KYSE180 cells stably infected with Sh-TMTC3 or Sh-NC. D. The significantly enriched KEGG pathways and GO terms for DEGs from RNA-seq in TMTC3-knockdown KYSE180 cells. E. Venn diagram of TMTC3 downstream genes from two RNA-seq analysis and 11 genes were selected. F. Western blot analysis for E-cadherin (epithelial marker), N-cadherin and Vimentin (mesenchymal marker) and ILEI in Sh-TMTC3 cells. G. The FPKM of ILEI in transcriptome sequencing data in 91 paired ESCC tumors and adjacent normal tissues. H. Cross correlation analysis of reads between ILEI and EMT markers from transcriptome sequencing data in 91 paired ESCC tissues. I. The positive correlation between TMTC3 and ILEI from transcriptome sequencing data in 91 paired ESCC tissues. J and K. The Pearson's correlation coefficient between TMTC3 and ILEI in EC (J) and other SCCs, including CESC, HNSC, LUSC (K) from GEPIA2 database. L. The correlation between TMTC3 and ILEI in LUAD.

Figure 5

TMTC3 enhanced ER stress by disturbing the interaction between GRP78 and PERK. A. Colocalization of TMTC3 and GRP78 or PERK in cell lines by IF. Scale bar, 30 µm. B and C. Expression of ER sensors in ESCC negative control and Si-TMTC3 (B) or Sh-TMTC3 (C) cells. D and E. Co-immunoprecipitation of TMTC3, GRP78 and PERK in cell lines. F. Co-immunoprecipitation and Western blot analysis for GRP78 and PERK in Sh-TMTC3 or Sh-NC cells.

Figure 6

ATF4 increased the transcriptional activity of ILEI to promote EMT in ESCC. A. ATF4 protein expression in fractionated cells with stably expressed Sh-TMTC3 and Sh-NC (left). The quantification of ATF4 intensity in WB (right). Nuc, nuclear. Cyto, cytoplasm. β-tubulin is a cytoplasmic control, LaminB1 is a nuclear control. B. IF images for the localization of ATF4 in TMTC3 cells transfected with siRNA and negative control. Scale bar, 30 µm. C. Binding of ATF4 to the ILEI promoter region in KYSE410 and KYSE450 cells was analyzed by ChIP-qPCR. IgG was used as a nonspecific binding control. **, p < 0.01. ***, p < 0.001. D. The relative luciferase reporter activity of ATF4 on the ILEI promoter in KYSE180 and KYSE450 cells. ***, p < 0.001. n.s., no significance. E. The relative dual-luciferase activity of the ILEI promoter in TMTC3 cells with stable knockdown. ***, p < 0.001. F. Model of the ATF4-ILEI axis in regulating EMT in SCCs.

Figure 7

TMTC3 silencing inhibited tumor metastasis and growth in vivo. A-C. Hematoxylin and eosin (H&E) staining of metastasis to the lung (A), lymph node (B) and liver (C). The scale bar represents 100 μm. The incidence of metastasis including lung, lymph node and liver metastasis, after tail injection. Additionally, the metastatic nodule numbers in the liver were counted. D. Curve analysis of xenograft tumor growth for bearing lenti-Sh-TMTC3 cells and negative control cells. **, p < 0.01. E. The volume of xenograft tumors after mice were sacrificed. **, p < 0.01. F. The xenograft tumor weight of lenti-Sh-TMTC3 cells compared with Sh-NC cells. **, p < 0.01. G. The IHC staining of xenograft tumor for TMTC3, Ki-67, PERK, ATF4, ILEI and N-cadherin. Scale bar, 100 μm.

Figure 8

Model of TMTC3 regulating SCCs metastasis via the PERK/ATF4/ILEI axis.