CBX3 Regulated By YBX1 Promotes Smoking-induced Pancreatic Cancer Progression via Inhibiting SMURF2 Expression

Abstract

As a key reversible and heritable mechanism of transcriptional regulation, the epigenetic modification plays a crucial role in tumorigenesis. Of note, tobacco smoking induces epigenetic modifications to promote pancreatic cancer development. Chromobox protein homolog 3 (CBX3) acts as an epigenetic regulator, modulating gene expression of downstream targets via chromatin modifications. To date, the relationship between CBX3 and smoking in pancreatic cancer remains unknown. This study aimed to uncover the specific role and underlying mechanism of CBX3 in smoking-related pancreatic cancer. The bioinformatics analyses were conducted to identify CBX3 as a key player in tobacco-induced pancreatic cancer. The abnormal upregulation of CBX3 was associated with poor prognosis in pancreatic cancer patients. Moreover, cigarette smoke extract (CSE) exposure promoted the overexpression of Y-box-binding protein 1 (YBX1), which consequently led to upregulated CBX3 in pancreatic cancer cells. We also revealed that CBX3 enhanced pancreatic cancer progression, likely by inhibiting the expression of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) and promoting the activation of TGF-β signaling. In summary, the YBX1/CBX3/SMURF2 signaling axis may be a promising therapeutic target in patients with smoking-related pancreatic cancer.

Keywords: CBX3; Pancreatic cancer; SMURF2; Smoking; YBX1.

Fig 1

Smoking-induced CBX3 upregulation is correlated with poor prognosis in pancreatic cancer. (A) Venn diagrams showed numbers of differentially expressed CBX family genes in current smokers compared to former smokers in TCGA-PAAD (Pancreatic adenocarcinoma), TCGA-LUAD (Lung adenocarcinoma) and TCGA-LUSC (Lung squamous cell carcinoma) datasets. (B) Box plot showed CBX3 was upregulated in current smokers compared to former smokers or never smokers in TCGA-PAAD dataset. (C and D) PANC-1 and BxPC-3 cells were treated with CSE (5%) for 0, 24, 48 and 72 hours as indicated, and then were collected for Western blot (C) and RT-qPCR assay (D). (E and F) PANC-1 and BxPC-3 cells were treated with a serial concentration of CSE for 72 hours, and then were collected for Western blot (E) and RT-qPCR assay (F). (G) The tissue-wise expression of CBX3 in Pancreatic adenocarcinoma (PAAD) tissues and non-tumor tissues were analyzed by the GEPIA web tool. P < 0.01*. (H) The mRNA and protein levels of CBX3 were examined. (I and J) CBX3-related Disease Free Survival (I) and Overall Survival (J) were determined by the GEPIA web tool. (K and L) CBX3-related Recurrence Free Survival (K) and Overall Survival (L) were checked in the ICGC-PACA-AU dataset. Data in (D), (F) and (H) are presented as mean ± SD (n = 3). P < 0.05 *; P < 0.01 **; P < 0.001 ***.

Fig 2

YBX1 increases the expression of CBX3 through transcriptional regulation in smoking related pancreatic cancer. (A) Enrichment analysis by ChIP-Atlas predicted a large amount of YBX1 bound to promoter region of CBX3 within ±1000bp from TSS. (B) PANC-1 and BxPC-3 cells were treated with CSE (5%) for 0, 24, 48 and 72 hours as indicated, and then were collected for Western blot. (C) PANC-1 and BxPC-3 cells were treated with a serial concentration of CSE for 72 hours, and then were collected for Western blot. (D and E) PANC-1 and SW1990 cells were transfected with indicated shRNAs for 72 hours. After puromycin selection, cells were harvested for the Western blot (D) and RT-qPCR assay (E). The protein level of CBX3 was quantified by the Image J software. (F and G) CFPAC1 and BxPC-3 cells were transfected with indicated plasmids for 24 hours, and then were harvested for Western blot (F) and RT-qPCR assay (G). The protein level of CBX3 was quantified by the Image J software. (H) The ChIP-seq data of YBX1 indicated that there were binding peaks in the promoter region of CBX3. (I) The ChIP-qPCR assay of CBX3 by using the IgG or YBX1 antibodies in the PANC-1 and BxPC-3 cells. (J) PANC-1 cells were transfected with indicated shRNAs for 72 hours, and then were harvested for the ChIP-qPCR assay by using the IgG or YBX1 antibodies. (K) The double luciferase report assay was conducted to investigate whether YBX1 could regulate the promoter activities of CBX3 (Fig. 2K). (L and M) TIMER2.0 showed purity-adjusted correlation between CBX3 and YBX1 in various cancer types (K) and PAAD, exclusively (L). Data in (D), (E), (F), (H), (I) and (J) are presented as mean ± SD (n = 3). Not significant ^Ns; P < 0.05 *; P < 0.01 **; P < 0.001 ***.

Fig 3

CBX3 is critical for the malignant progression of pancreatic cancer. (A-F) PANC-1 and SW1990 cells were transfected with CBX3 shRNAs for 72 hours. After puromycin selection, cells were harvested for the Western blot (A), MTS assay (B), colony formation (C and D), and transwell assay (E and F). (G-L) CFPAC1 and BxPC-3 cells were transfected with indicated constructs for 72 hours. After puromycin selection, cells were harvested for the Western blot (G), MTS assay (H), colony formation (I and J), and transwell assay (K and L). (M-S) PANC-1 and SW1990 cells were infected with indicated shRNAs for 48 hours. Then, these cells were infected with shRNA-resistant overexpression (CBX3^SR OE) construct for another 72 hours. After puromycin selection, cells were harvested for the colony formation (M and N), transwell assay (O and P), and subcutaneous xenograft assay (Q-S). Data in (B), (D), (F), (H), (J), (L), (N), (P), (R) and (S) are presented as mean ± SD (n = 3); P < 0.001 ***.

Fig 4

CBX3 triggers the hyperactivation of TGF-β signaling pathway in pancreatic cancer. (A and B) GSEA indicated top 10 REACTOME signaling pathways induced by the overexpression of CBX3 in TCGA-PAAD dataset. Multi-GSEA plot showed running enrichment score for gene sets as the analyses walk down the preranked gene list (A). Ridge plot showed density of signaling pathway generated by using the frequency of fold change values per gene within each set (B). (C-F) REACTOME signaling by TGF-beta receptor complex was activated by the overexpression of CBX3 in TCGA-PAAD (C), ICGC-PACA-AU (D), ICGC-PACA-CA (E) and CPTAC-PDAC (F) datasets (all P < 0.05*).

Fig 5

CBX3 represses the SMURF2 expression in pancreatic cancer cells. (A and B) ChIP-seq data of CBX3 and H3K9me3 on promoter region of SMURF2. (C and D) PANC-1 and SW1990 cells were transfected with CBX3 shRNAs for 72 hours. After puromycin selection, cells were harvested for RT-qPCR assay (C) and Western blot (D). (E and F) CFPAC1 and BxPC-3 cells were transfected with indicated constructs for 48 hours, and then were harvested for the RT-qPCR (E) and Western blot (F). (G and H) ChIP-qPCR of SMURF2 by using the CBX3 and H3K9me3 antibodies. (I and J) The tissue microarray of pancreatic cancer was stained with CBX3 and SMURF2. Typical IHC images stained with CBX3 and SMURF2 were shown in panel I. Correlation analysis of these two proteins was shown in panel J. Data in (C), (E), (G), (H) and (J) are presented as mean ± SD (n = 3). Not significant ^Ns; P < 0.05 *; P < 0.01 **; P < 0.001 ***.

Fig 6

The YBX1/CBX3 axis promotes tumor growth via suppressing SMURF2 in pancreatic cancer. (A-D) PANC-1 and SW1990 cells were transfected with indicated shRNAs for 72 hours. After puromycin selection, cells were harvested for the RT-qPCR (A), MTS (B), and colony formation (C and D). (E-G) PANC-1 cells were transfected with indicated shRNAs for 72 hours. After puromycin selection, cells were subcutaneously injected into the nude mice. The image of tumor was shown in panel E. The tumor mass and tumor growth curve were demonstrated in panel F and G, respectively. Data are presented as mean ± SD (n = 5). (H) PANC-1 and BxPC-3 cells were transfected with indicated shRNAs for 72 hours. After puromycin selection, cells were harvested for the Western blot analysis. (I) PANC-1 and BxPC-3 cells were transfected with indicated shRNAs for 48 hours, and then were transfected with indicated constructs for another 24 hours. Cells were harvested for Western blot. (J) A hypothetic model depicted that CSE exposure-induced YBX1 overexpression contributed to the upregulation of CBX3, which inhibited SMURF2 expression to promote the progression of pancreatic cancer. Data in (A), (B) and (D) are presented as mean ± SD (n = 3). Not significant ^Ns; P < 0.001 ***.