Overexpression of CBX3 in Pancreatic Adenocarcinoma Promotes Cell Cycle Transition-Associated Tumor Progression

Abstract

Background: Previous studies showed that Chromobox protein homolog 3 (CBX3) was overexpressed in several types of human cancers, however its pattern and role in pancreatic adenocarcinoma (PAAD) has not yet been understood. The aim of this study was to identify the expression and function of CBX3 in PAAD.

Methods: Data of transcriptomic and protein expression of CBX3 in PAAD were collected from different databases and analyzed. The in vitro and in vivo role of CBX3 in PAAD was examined.

Results: CBX3 was overexpressed in human PAAD tissues, which was associated with poor prognosis of overall and disease-free survival of the patients. Overexpression of CBX3 induced the in vitro proliferation, anchorage-free growth, migration and invasion of the PAAD cells, and led to in vivo growth of orthotoptic PAAD tumors in mice. GO and KEGG pathway analysis, as well as experimental observation showed that CBX3 may be associated with cell cycle transition of PAAD cells, and cyclin-dependent kinase 1 (CDK1) and proliferating cell nuclear antigen (PCNA) may mediate the tumor-promoting action of CBX3. CDK1 knockdown attenuated the cell cycle transition, proliferation and invasion of CBX3-overexpressing PAAD cells.

Conclusion: Our findings suggest the tumor-promoting role of CBX3 in PAAD to be targeted by novel therapeutic strategies.

Keywords: CBX3; CDK1; cell cycle regulation; pancreatic adenocarcinoma; tumor progression.

Figure 1

CBX3 was overexpressed in PAAD and predicted poor prognosis. (a) Data of CBX3 expression in human PAAD and non-tumor tissues were extracted from GEO dataset GDS4336 and GDS4103. It was shown that CBX3 was highly expressed in PAAD tissue. *** p < 0.001 when comparison was made between groups; (b) Protein expression of CBX3 was accessed from Human Protein Atlas project. Expression of HP1γ in human PAAD slides was significantly increased, as evidence of positive staining of the protein (brown dots). The black arrows showed cells with strong expression of HP1γ; (c) Data of expression of CBX3 and survival time of corresponding patients were extracted from TCGA database. KM plots showed that patients with CBX3 expression higher than median level had shorter overall survival. The area between the upper and lower blue/red dash lines indicated areas within 95% confident intervals (CIs); (d) Data of expression of CBX3 and survival time of corresponding patients were extracted from TCGA database. KM plots showed that patients with CBX3 expression higher than median level had shorter disease-free survival. The area between the upper and lower blue/red dash lines indicated areas within 95% CIs; (e) Data were collected from Gepia database. The results showed that CBX3 was increase during the disease progression of PAAD.

Figure 2

CBX3 overexpression increased in vitro proliferation and invasion of PAAD cells. (a) Expression of HP1γ was increased in CBX3-overexpressing KP3L and PANC-1 cells; (b) KP3L and PANC-1 cells with or without CBX3 overexpression were seeded at the density of 10⁴/well and allowed proliferation. Cell number was counted at Day 3, 6 and 9 after seeding. Overexpression of CBX3 significantly accelerated the proliferation of cells; (c) Expression of HP1γ was knocked down in PANC-1 cells with CBX3 siRNA; (d) Knockdown of CBX3 in PANC-1 cells reduced the proliferation rate of the cells; (e) Overexpression of CBX3 maintain the anchorage-free growth of KP3L and PANC-1 cells; (f) Overexpression of CBX3 induced the migration of KP3L and PANC-1 cells towards the center of the wound; (g) Overexpression of CBX3 promoted KP3L and PANC-1 cell invasion through extracellular matrix. In all panels, * p < 0.05, ** p < 0.01 and *** p < 0.001 when comparison was made between groups.

Figure 3

CBX3 overexpression promoted in vivo tumor progression of PAAD cells. (a) Luciferase-tagged KP3L cells were implanted to the pancreas of nude mice (n = 5). Luciferase was checked once per week for four weeks; (b) Increased luciferase intensity could be observed throughout the experiment, and CBX3 overexpressing KP3L cells showed a more rapid increase of luciferase intensity; (c) Size of pancreatic tumor formed by CBX3-overexpressing KP3L cells at the end of experiment was larger; (d) H&E staining revealed that more cells undergoing mitosis, indicating accelerating proliferation of the tumor cells. The black arrows highlighted cells undergoing rapid proliferation. In all panels, ** p < 0.01 when comparison was made between groups.

Figure 4

CBX3 may regulate the cell cycle transition of PAAD cells. (a) Correlated genes were retrieved from TCGA data and processed with GO annotation. Biological process (BP), Molecular function (MF) and cell component (CC) of the correlated genes were predicted. It was shown that CBX3-correlated genes were mostly related to cell cycle regulation and DNA replication. Items tagged with red asterisk were related to DNA replication and cell cycle regulation; (b) Common genes among cell cycle regulations from BP, MF and CC annotation was extracted by Venn diagram. Sixteen genes were shared in common; (c) KEGG pathway analysis with CBX3-correlated genes. It was consistently shown that cell cycle regulation may be involved; (d) Common genes between GO annotation and KEGG analysis revealed that CDK1 and PCNA may be the core element in the regulation of cell cycle by CBX3; (e) Expression of CBX3, CDK1 and PCNA in PAAD tissue was extracted, and correlation was analyzed by linear regression. It was shown that CDK1 and PCNA expression in PAAD tissue was positively correlated to CBX3 expression.

Figure 4

CBX3 may regulate the cell cycle transition of PAAD cells. (a) Correlated genes were retrieved from TCGA data and processed with GO annotation. Biological process (BP), Molecular function (MF) and cell component (CC) of the correlated genes were predicted. It was shown that CBX3-correlated genes were mostly related to cell cycle regulation and DNA replication. Items tagged with red asterisk were related to DNA replication and cell cycle regulation; (b) Common genes among cell cycle regulations from BP, MF and CC annotation was extracted by Venn diagram. Sixteen genes were shared in common; (c) KEGG pathway analysis with CBX3-correlated genes. It was consistently shown that cell cycle regulation may be involved; (d) Common genes between GO annotation and KEGG analysis revealed that CDK1 and PCNA may be the core element in the regulation of cell cycle by CBX3; (e) Expression of CBX3, CDK1 and PCNA in PAAD tissue was extracted, and correlation was analyzed by linear regression. It was shown that CDK1 and PCNA expression in PAAD tissue was positively correlated to CBX3 expression.

Figure 5

CBX3 overexpression promoted G2/M cell cycle transition. (a) mRNA expression of CDK1 and PCNA was increased in CBX3-overexpressing KP3L cells; (b) Protein expression of CDK1 and PCNA was increased in CBX3-overexpressing KP3L cells; (c) KP3L cells were arrested at G2/M phase by treatment of 5 μM Ro-3306 for 12 h. Medium was then replaced with fresh full medium and allow cell cycle progression for another 12 h. It was found that treatment of Ro-3306 accumulated cells at G2/M phase, and after release CBX3-overexpressing cells showed a more rapid transition of cell cycle as evidence of low G2/M portion; (d) KP3L cells were fixed and stained with DAPI. Cells with condensed nuclear were counted as mitotic cells under fluorescence microscope. Percentage of mitotic cells out of total viable cells was calculated as mitotic index. CBX3-overexpressing cells showed higher mitotic index than WT cells. In all panels, * p < 0.05, ** p < 0.01 and *** p < 0.001 when comparison was made between groups.

Figure 6

CDK1 mediated the tumor-promoting effect of CBX3 in PAAD cells. (a) Expression of CDK1 in CBX3-overexpressing cells was knockdown by siRNA and validated by Immunoblotting. (b) Knockdown of CDK1 increased G2/M accumulation of CBX3-overexpressing KP3L cells. Knockdown of CDK1 reduced cell: (c) mitotic index; (d) proliferation; (e) anchorage-free growth; (f) migration; and (g) invasion of the CBX3-overexpressing KP3L cells. In all panels, * p < 0.05 and ** p < 0.01 when comparison was made between groups.

Figure 7

Overall regulatory mechanism involved in the action of CBX3 in PAAD. Red arrows indicated the changes of trend of protein expression and cellular activity when CBX3 is upregulated.