JCM, Vol. 12, Pages 200: Krüppel-like Factor 6 Suppresses the Progression of Pancreatic Cancer by Upregulating Activating Transcription Factor 3

Conceptualization, Q.X. and Z.Z.; methodology, Y.Y.; validation, Q.X., Y.X., and Y.Y.; formal analysis, Y.X.; investigation, Q.X., Y.Z. (Ying Zhou), and S.Z.; resources, J.L.; data curation, Y.Z. (Ying Zheng); writing—original draft preparation, Q.X.; writing—review and editing, Q.Z.; visualization, Z.Z.; supervision, Q.Z.; project administration, Q.Z.; funding acquisition, Q.Z. All authors have read and agreed to the published version of the manuscript.

Figure 1. The expression pattern and prognostic value of KLF6 in pancreatic cancer. (A) KLF6 protein expression level comparison between tumor and paired normal tissues from 6 patients with pancreatic ductal adenocarcinoma. N: normal; T: tumor. (B) Relative KLF6 mRNA expression level comparison between tumor and paired normal tissues from 7 patients with pancreatic ductal adenocarcinoma. P: patient. Data represent mean ± SD; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, ns means no significance. (C) KLF6 protein expression level in pancreatic cancer cell lines. (D) Relative mRNA expression of KLF6 in pancreatic cancer cell lines and a normal pancreatic ductal cell line. Data represent mean ± SD; n = 3. * p < 0.05, ** p < 0.01, ns means no significance. (E) Representative immunohistochemical staining images of low KLF6 expression and high KLF6 expression in pancreatic ductal adenocarcinoma tissues (original magnification of left pictures ×100; original magnification of right pictures ×1260; scale bar of left pictures 200 μm; scale bar of right pictures 20 μm). (F) Kaplan-Meier survival curves of patients with pancreatic ductal adenocarcinoma (n = 57).

Figure 1. The expression pattern and prognostic value of KLF6 in pancreatic cancer. (A) KLF6 protein expression level comparison between tumor and paired normal tissues from 6 patients with pancreatic ductal adenocarcinoma. N: normal; T: tumor. (B) Relative KLF6 mRNA expression level comparison between tumor and paired normal tissues from 7 patients with pancreatic ductal adenocarcinoma. P: patient. Data represent mean ± SD; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, ns means no significance. (C) KLF6 protein expression level in pancreatic cancer cell lines. (D) Relative mRNA expression of KLF6 in pancreatic cancer cell lines and a normal pancreatic ductal cell line. Data represent mean ± SD; n = 3. * p < 0.05, ** p < 0.01, ns means no significance. (E) Representative immunohistochemical staining images of low KLF6 expression and high KLF6 expression in pancreatic ductal adenocarcinoma tissues (original magnification of left pictures ×100; original magnification of right pictures ×1260; scale bar of left pictures 200 μm; scale bar of right pictures 20 μm). (F) Kaplan-Meier survival curves of patients with pancreatic ductal adenocarcinoma (n = 57).

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Figure 2. Overexpression of KLF6 inhibits proliferation of pancreatic cancer cells. (A) Western blot test results showing KLF6 protein expression level (KLF6-3xFlag) after KLF6-overexpression plasmid transfection of AsPc-1, CFPAC-1, and PANC-1 cells. (B) Cell proliferation of AsPc-1, CFPAC-1, and PANC-1 cells after KLF6 overexpression measured by CCK8 assays. Data represent mean ± SD; n = 6. *** p < 0.001. (CE) Cell proliferation of AsPc-1, CFPAC-1, and PANC-1 cells after KLF6 overexpression measured by clone formation assays. Data represent mean ± SD; n = 3. *** p < 0.001.

Figure 2. Overexpression of KLF6 inhibits proliferation of pancreatic cancer cells. (A) Western blot test results showing KLF6 protein expression level (KLF6-3xFlag) after KLF6-overexpression plasmid transfection of AsPc-1, CFPAC-1, and PANC-1 cells. (B) Cell proliferation of AsPc-1, CFPAC-1, and PANC-1 cells after KLF6 overexpression measured by CCK8 assays. Data represent mean ± SD; n = 6. *** p < 0.001. (CE) Cell proliferation of AsPc-1, CFPAC-1, and PANC-1 cells after KLF6 overexpression measured by clone formation assays. Data represent mean ± SD; n = 3. *** p < 0.001.

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Figure 3. Overexpression of KLF6 inhibits metastasis of pancreatic cancer cells. (A) Cell migration ability of AsPc-1, CFPAC-1, and PANC-1 cells after KLF6 overexpression, respectively. Data represent mean ± SD; n = 4. *** p < 0.001. (B) Cell invasion ability of AsPc-1, CFPAC-1, and PANC-1 cells after KLF6 overexpression, respectively. Data represent mean ± SD; n = 3. *** p < 0.001.

Figure 3. Overexpression of KLF6 inhibits metastasis of pancreatic cancer cells. (A) Cell migration ability of AsPc-1, CFPAC-1, and PANC-1 cells after KLF6 overexpression, respectively. Data represent mean ± SD; n = 4. *** p < 0.001. (B) Cell invasion ability of AsPc-1, CFPAC-1, and PANC-1 cells after KLF6 overexpression, respectively. Data represent mean ± SD; n = 3. *** p < 0.001.

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Figure 4. The EMT progression affected by overexpression of KLF6 and potential target genes of KLF6 in pancreatic cancer cells. (A) Western blot test results showing EMT protein markers, including E-cadherin, N-cadherin, MMP2, and vimentin, affected by overexpression of KLF6 in AsPc-1, CFPAC-1, and PANC-1 cells. (B) Potential targeted genes of KLF6 predicted by TRRUST database. Font in red refers to genes upregulated by KLF6; font in blue refers to genes downregulated by KLF6. (C) The correlation between KLF6 and ATF3 by GEPIA2 tool. (DF) The mRNA ATF expression levels of ATF3 after overexpression of KLF6. Data represent mean ± SD; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. (G) The protein ATF expression levels of ATF3 after overexpression of KLF6.

Figure 4. The EMT progression affected by overexpression of KLF6 and potential target genes of KLF6 in pancreatic cancer cells. (A) Western blot test results showing EMT protein markers, including E-cadherin, N-cadherin, MMP2, and vimentin, affected by overexpression of KLF6 in AsPc-1, CFPAC-1, and PANC-1 cells. (B) Potential targeted genes of KLF6 predicted by TRRUST database. Font in red refers to genes upregulated by KLF6; font in blue refers to genes downregulated by KLF6. (C) The correlation between KLF6 and ATF3 by GEPIA2 tool. (DF) The mRNA ATF expression levels of ATF3 after overexpression of KLF6. Data represent mean ± SD; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. (G) The protein ATF expression levels of ATF3 after overexpression of KLF6.

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Figure 5. KLF6 inhibits the progression of pancreatic cancer through upregulating ATF3. (A) The relative mRNA expression levels of KLF6 after KLF6 overexpression or knockdown ATF3 in pancreatic cancer cells. Data represent mean ± SD; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) The relative mRNA expression levels of ATF3 after KLF6 overexpression or knockdown ATF3 in pancreatic cancer cells. Data represent mean ± SD; n = 3. ** p < 0.01, *** p < 0.001. (CE) CCK8 assays were performed to measure the proliferation capacity after KLF6 overexpression or knockdown ATF3 in AsPC-1, CFPAC-1, and PANC-1. Data represent mean ± SD; n = 6. * p < 0.05, *** p < 0.001. (F) The migration capacity was measured after KLF6 overexpression or knockdown ATF3 in AsPC-1, CFPAC-1, and PANC-1. Data represent mean ± SD; n = 3. *** p < 0.001.

Figure 5. KLF6 inhibits the progression of pancreatic cancer through upregulating ATF3. (A) The relative mRNA expression levels of KLF6 after KLF6 overexpression or knockdown ATF3 in pancreatic cancer cells. Data represent mean ± SD; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) The relative mRNA expression levels of ATF3 after KLF6 overexpression or knockdown ATF3 in pancreatic cancer cells. Data represent mean ± SD; n = 3. ** p < 0.01, *** p < 0.001. (CE) CCK8 assays were performed to measure the proliferation capacity after KLF6 overexpression or knockdown ATF3 in AsPC-1, CFPAC-1, and PANC-1. Data represent mean ± SD; n = 6. * p < 0.05, *** p < 0.001. (F) The migration capacity was measured after KLF6 overexpression or knockdown ATF3 in AsPC-1, CFPAC-1, and PANC-1. Data represent mean ± SD; n = 3. *** p < 0.001.

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Table 1. Association of KLF6 expression and clinicopathologic parameters.

Table 1. Association of KLF6 expression and clinicopathologic parameters.

ParametersnLow ExpressionHigh Expressionp ValueNumber of patients572829 Age≤602814140.896>60291415SexMale3619170.470Female21912TNM stageI-II5426280.611III-IV321GradeWell and moderate2510150.223Poor321814DiabetesYes11560.786No462323SmokeYes221390.233No351520DrinkingYes171250.035 *No401624Preoperative CA19-9 value≥374724230.525<371046Preoperative CEA value≥5188100.631<5392019

Table 2. Univariate Cox proportional hazards analysis for survival of PAAD patients.

Table 2. Univariate Cox proportional hazards analysis for survival of PAAD patients.

VariableHazard Ratio95% Confidence Intervalp ValueKLF6 expression (low/high)2.4311.166–5.0700.017 *Age (≤60/>60)0.8190.424–1.5810.552Gender (female/male)0.6050.296–1.2360.168TNM stage (I–II/III–IV)0.5030.150–1.6890.266Grade (poor/moderate and well)2.3161.138–4.7110.020 *Preoperative CA19-9 value (<37/≥37)0.5560.213–1.4500.230Preoperative CEA value (<5/≥5)0.5700.272–1.1920.135

Table 3. Multivariate Cox proportional hazards analysis for survival of PAAD patients.

Table 3. Multivariate Cox proportional hazards analysis for survival of PAAD patients.

VariablesHazard Ratio95.0% CIp ValueTNM stage0.3300.094–1.1630.085Grade2.3131.103–4.8500.026 *KLF6 expression2.2541.068–4.7570.033 *

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