Krüppel-like Factor 6 Suppresses the Progression of Pancreatic Cancer by Upregulating Activating Transcription Factor 3

Background: As a member of the Krüppel-like factor (KLFs) family, Krüppel-like factor 6 (KLF6) plays a critical role in regulating key cellular functions. Presently, scholars have proved the important role of KLF6 in the tumorigenesis of certain cancers through a large number of experiments. However, gaps still remain in our knowledge of the role of KLF6 in pancreatic cancer (PAAD). Therefore, this paper mainly investigates the role of KLF6 in the progression of pancreatic cancer. Methods: The expression pattern of KLF6 in pancreatic cancer was explored in pancreatic cancer tissues and cell lines. Then, we investigated the prognostic value of KLF6 in pancreatic cancer by immunohistochemical assays. Next, Cell Counting Kit-8 (CCK8) and clone information assays were employed to explore the proliferation of PAAD affected by KLF6. The metastasis and epithelial-mesenchymal transition (EMT) abilities affected by KLF6 were identified through transwell invasion as well as migration assays and western blots. Finally, the TRRUST tool was used to analyze the potential targeted genes of KLF6. The results were verified by Quantificational Real-time Polymerase Chain Reaction (qRT-PCR), western blot and rescue assays. Results: KLF6 expresses lowly in pancreatic cancer compared to corresponding normal tissues and relates to poor survival times. Overexpression of KLF6 inhibits the proliferation, metastasis, and EMT progression in pancreatic cancer cells. Further studies suggest that KLF6 could upregulate ATF3 in PAAD. Conclusions: Our results suggest that KLF6 can be a useful factor in predicting the prognosis of PAAD patients and that it inhibits the progression of pancreatic cancer by upregulating activating transcription factor 3 (ATF3).


Introduction
The Krüppel-like factor 6 (KLF6) gene, a member of the Krüppel-like factors (KLFs) family and located on chromosome 10, directly interacts with the deoxyribonucleic acid (DNA) sequence by either a GC box promoter component or a CACCC motif in its response promoter region [1,2]. Previous studies have proposed that the KLF6 gene is involved in the regulation of diverse forms of cellular progression covering cellular differentiation and growth [3][4][5], immunological and inflammatory responses [6,7], and tissue damage and repair [2,8]. Genetic alterations and the aberrant expression of KLF6 are correlated with the formation and progression of cancer [9]. In 2001, Narla et al. found that KLF6 was frequently inactivated in sporadic prostate cancer [10]. Subsequently, increasing studies have revealed that KLF6 could work as a tumor suppressor gene. For example, KLF6 was found to be inactivated and/or expression-downregulated in lung cancer [11], colorectal cancer [12], liver cancer [13], and some other cancers [14,15]. Further studies have revealed that knockdown KLF6 had positive effects on tumor progression, while KLF6 overexpression resulted in decreased tumorigenicity [16][17][18]. Nevertheless, an oncogenic

PAAD Clinical Samples
We collected 57 formalin-fixed, paraffin-embedded cancer specimens from pancreatic cancer patients who underwent surgical excision between 2014 and 2018 at West China Hospital, Sichuan University (Chengdu, China). Clinical characteristics including age, gender, TNM stage (according to the 2018 eighth edition of the National Comprehensive Cancer Network staging criteria), tumor grade, and preoperative CA199 and CEA values were all collected. In total, the patients in our sample comprised 36 males and 21 females, with a mean age of 60.2 years, ranging from 43 to 76 years old. Within the sample, 1 case was well differentiated, 24 cases were moderately differentiated, and 32 cases were poorly differentiated. 2 patients were classified as stage I, 52 were classified as stage II, 1 was classified as stage III, and 2 were classified as stage IV. Follow-up data containing survival time and performance status were collected until September 2019. The median overall survival time of these 57 patients was 14 months, ranging from a minimum of 1 month to a maximum of 44 months. In addition to this sample, we collected 7 paired frozen pancreatic cancer and normal tissues from West China Hospital, Sichuan University. Patients were diagnosed clinically and pathologically with pancreatic ductal adenocarcinoma. None of these patients had been treated with radiotherapy or/and chemotherapy before surgery. This study was approved by the Institutional Ethics Committee of West China Hospital, Sichuan University, with authorized informed consent secured from all patients.

Immunohistochemistry (IHC)
As reported before [24], fixed tissue samples were subjected to paraffin embedding, sectioning, deparaffinating, rehydrating, and antigen retrieval. Following this, sections were incubated with a 3% hydrogen peroxide solution for 10 min at room temperature. Slides were then incubated with a KLF6 polyclonal antibody (Abcepta, Suzhou, China) at a dilution of 1:150 overnight at 4 • C. The next day, goat anti-rabbit antibody (Jackson, New York, NY, USA) at a dilution of 1:250 was added to the slides and incubated for 40 min at room temperature. A SignalStain DAB Substrate Kit (Cell Signaling Technology, Danvers, MA, USA) was used to for colorimetric detection. The stained sections were analyzed using a microscope (Pannoramic MIDI). The staining intensity was classified into four categories: 0, no staining; 1, weak; 2, moderate; 3, strong. The staining proportion was scored as follows: 0 indicating 0-5%; 1 indicating 6-25%; 2 indicating 26-50%; 3 indicating > 50%. The results of the intensity times the proportion of each slide were calculated, and a product > 3 was considered a high KLF6 expression, while a product ≤ 3 was considered a low KLF6 expression.
2.4. RNA Extraction, Reverse Transcription, and Quantitative Polymerase Chain Reaction (qRT-PCR) RNA was isolated from cell lines and frozen tissues using a trizol reagent (Invitrogen, Thermofisher, Waltham, MA, USA) according to the manufacturer's instructions. Reverse transcription was performed using a PrimeScript™ RT Master Mix (TaKaRa, Shiga, Japan). NovoStart ® SYBR qPCR SuperMix Plus (Novoprotein, Suzhou, China) was applied to conduct real-time quantitative PCR. The amplification procedure was set as follows: An initial denaturation at 95 • C for 30 s, followed by 40 cycles at 95 • C for 5 s, 60 • C for 30 s, and 72 • C for 30 s. The related target gene expression was normalized against β-actin by the relative quantification (2 −∆∆Ct ) method.

Plasmid Construction, Lentiviral Transduction, and Generation of Stable Cell Lines
To establish stable KLF6-overexpression cell lines, human cDNA encoding full-length KLF6 gene was obtained by PCR amplification. KLF6 cDNA were subcloned into pLVX-NEO-3xFlag vector kindly provided by Dr. Zhou Zhao at Sichuan University. For the generation of lentiviral particles, the lentiviral vector and packaging DNA were co-transfected into 293T cells for 48-72 h, and the media containing lentiviruses were harvested and transduced to target cells. SiRNA was purchased from the Youkangjianxing Biotechnology Company (Chengdu, China). 5 nmol siRNA (siNC or siATF3) was transfected into pancreatic cancer cells by lipofectamine 3000 agents (Thermo) according to instructions.

CCK8 Assay and Clone Formation Assay
Briefly, cells (2 × 10 3 /well) were seeded onto 96-well plates and incubated for 0, 2, 4, and 6 days. 10 microliters of CCK8 reagent (TargetMol) per well were added and incubated at 37 • C for 2 h. Absorbance was measured at 450 nm under a multifunctional enzyme marker. For clone formation assays, transfected cells (2 × 10 3 /well) were cultured in 12-well plates for 10-14 days. Cells were fixed by application of 4% formaldehyde in PBS and stained with crystal violet.

Migration and Invasion Assays
The migration and invasive capability of pancreatic cancer cells were determined using transwell cell culture chambers with an 8-µm pore size polycarbonate membrane (Corning). In detail, cells (6 × 10 4 /well) with different treatments in 200 µL of serumfree medium were seeded into the upper chamber, while 800 µL of complete medium containing 10% FBS was placed in the lower chamber as a chemoattractant. As for the invasion assay, the membrane was coated with Matrigel™ (Corning, Corning, NY, USA) diluted at a 1:15 ratio with medium. After incubation at 37 • C for 24 h, the filters were fixed in 4% paraformaldehyde for 15 min and stained with crystal violet for 20 min. The cells on the upper side of the filter were wiped off gently by a cotton swab.

Databases
TRRUST v2 (https://www.grnpedia.org/trrust/, accessed on 1 May 2022), an online dataset exploring the regulating network between transcription factor and genes [25], was used to find the targets of KLF6. Additionally, the GEPIA2 database (http://gepia2.cancerpku.cn, accessed on 1 May 2022), an online tool for exploring gene expression pattern and interactive analyses [26], was utilized to analyze the relationship between the expression of KLF6 and target gene in this study.

Statistical Analysis
GraphPad Prism 5 and SPSS Statistics 26 were used for statistical analysis. Each experiment was repeated at least three times. Data were represented as mean ± SD, and Student's t-test was applied for calculating p values. For studies of clinicopathologic parameters and for IHC signal quantification, chi-square tests and Fisher's exact tests were applied to detect statistically significant associations with KLF6 expression. The Kaplan-Meier method was used to draw survival curves and p values were calculated using a log-rank test. Cox proportional hazards models were applied to conduct univariate and multivariate survival analyses. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Evaluating the Expression Patterns and Prognostic Value of KLF6 in PAAD Patients and Pancreatic Cancer Cell Lines
Compared to paired normal tissues, tumor tissues express low protein and mRNA levels of KLF6 ( Figure 1A,B). Additionally, protein levels of KLF6 are markedly different among pancreatic cancer cells ( Figure 1C). Compared to an hTERT-HPNE cell, a normal pancreatic duct cell line, the mRNA of KLF6 is highly expressed in AsPc-1, while it is lowly expressed in BxPC-3, MIA PaCa-2, and PANC-1 ( Figure 1D). To evaluate the prognostic potential for KLF6 expression in PAAD, we analyzed clinical specimens by IHC and found that the KLF6 immunostained signals in the PAAD tissue were markedly different among samples ( Figure 1E). Furthermore, KM analysis revealed that PAAD patients with lower expression levels for KLF6 are positively associated with poor overall survival ( Figure 1F). and found that the KLF6 immunostained signals in the PAAD tissue were markedly different among samples ( Figure 1E). Furthermore, KM analysis revealed that PAAD patients with lower expression levels for KLF6 are positively associated with poor overall survival ( Figure 1F). Next, we sought to analyze KLF6 expression profiles and clinicopathologic features in an independent collection of pancreatic cancer samples. As shown in Table 1, of 57 Next, we sought to analyze KLF6 expression profiles and clinicopathologic features in an independent collection of pancreatic cancer samples. As shown in Table 1, of 57 PAAD specimens, 28 (49.1 %) were considered high KLF6 expression, and 29 (50.9 %) were consid-ered low KLF6 expression. Statistical analysis showed that KLF6 expression is independent of age (p = 0.896), sex (p = 0.470), TNM stage (p = 0.611), tumor grade (p = 0.223), preoperative CA199 value (p = 0.525), and preoperative CEA value (p = 0.631). In Table 2, univariate analysis with the Cox proportional hazards model identified low KLF6 expression (p = 0.017) and tumor grade with poor differentiation (p = 0.020) as statistically significant risk factors influencing the clinical survival of PAAD patients. Finally, we performed a multivariate analysis including TNM stage, which is considered an essential factor related to patient survival clinically, and discovered that both KLF6 expression (HR = 2.254, p = 0.033) and tumor grade (HR = 2.313, p = 0.026) were informative as prognostic markers for clinical outcome in patients ( Table 3), indicating that KLF6 may be a potential prognosticator of PAAD prognosis.

KLF6 Overexpression Inhibits Proliferation of Pancreatic Cancer Cells
Owing to the different expression of KLF6 between pancreatic normal cells and cancer cells, we further explored the potential function of KLF6 in PAAD. Overexpression KLF6 in AsPc-1, CFPAC-1, and PANC1 cell lines was performed. The results of the western blot test, as shown in Figure 2A

KLF6 Overexpression Inhibits Proliferation of Pancreatic Cancer Cells
Owing to the different expression of KLF6 between pancreatic normal cells and cancer cells, we further explored the potential function of KLF6 in PAAD. Overexpression KLF6 in AsPc-1, CFPAC-1, and PANC1 cell lines was performed. The results of the western blot test, as shown in Figure 2A, revealed that the protein level of KLF6 is overexpressed successfully. Then, CCK8 and clone formation assays were conducted to investigate the role of KLF6 in PAAD cell proliferation. As a result, overexpression of KLF6 in AsPc-1, CFPAC-1, and PANC1 cells decreases the growth of pancreatic cancer cells (Figure 2B-E).

KLF6 Overexpression Inhibits Metastasis of Pancreatic Cancer Cells
To evaluate the effect of KLF6 on cell metastasis capability, we performed transwell migration and invasion assays. The number of AsPc-1, CFPAC-1, and PANC1 cells invading the lower side of the membrane decreased dramatically in those cells transfected with KLF6-overexpression plasmid compared to wild-type cells ( Figure 3A). As for invasion ability, KLF6 overexpression markedly decreased the invasive ability of AsPc-1, CFPAC-1, and PANC1 cells as well ( Figure 3B). These data suggest that KLF6 could effectively inhibit the malignant behaviors of pancreatic cancer cells.

KLF6 Overexpression Inhibits Metastasis of Pancreatic Cancer Cells
To evaluate the effect of KLF6 on cell metastasis capability, we performed transwell migration and invasion assays. The number of AsPc-1, CFPAC-1, and PANC1 cells invading the lower side of the membrane decreased dramatically in those cells transfected with KLF6-overexpression plasmid compared to wild-type cells ( Figure 3A). As for invasion ability, KLF6 overexpression markedly decreased the invasive ability of AsPc-1, CFPAC-1, and PANC1 cells as well ( Figure 3B). These data suggest that KLF6 could effectively inhibit the malignant behaviors of pancreatic cancer cells.

Overexpression of KLF6 Impairs EMT Progression and Works by Upregulating ATF3 in Pancreatic Cancer Cell Lines
Next, the expressions of EMT marker proteins including E-cadherin (epithelial), Ncadherin, MMP2, and vimentin (mesenchymal) were investigated by western blot assays. The results demonstrated that the overexpression of KLF6 increased E-cadherin expression, simultaneously decreasing N-cadherin, MMP2, and vimentin expression ( Figure  4A). The data above suggest that KLF6 overexpression reduced EMT progression in PAAD. To explore the potential mechanism of KLF6 in pancreatic cancer, the TRRUST database was used to determine possible targets of KLF6 and the regulatory relationship of KLF6 among them ( Figure 4B). We determined that KLF6 can activate ATF3 (Activating Transcription , and TXNIP (Thioredoxin Interacting Protein) are predicted to be regulated by KLF6. We have previously reported that decreased ATF3 expression is correlated with poor differentiation and shorter overall survival in PAAD patients, indicating that ATF3 is essential to the progression of pancreatic cancer (14). Therefore, we selected ATF3 for further analysis. Using the GEPIA2 tool, we

Overexpression of KLF6 Impairs EMT Progression and Works by Upregulating ATF3 in Pancreatic Cancer Cell Lines
Next, the expressions of EMT marker proteins including E-cadherin (epithelial), Ncadherin, MMP2, and vimentin (mesenchymal) were investigated by western blot assays. The results demonstrated that the overexpression of KLF6 increased E-cadherin expression, simultaneously decreasing N-cadherin, MMP2, and vimentin expression ( Figure 4A). The data above suggest that KLF6 overexpression reduced EMT progression in PAAD. To explore the potential mechanism of KLF6 in pancreatic cancer, the TRRUST database was used to determine possible targets of KLF6 and the regulatory relationship of KLF6 among them ( Figure 4B). We determined that KLF6 can activate ATF3 (Activating Transcription , and TXNIP (Thioredoxin Interacting Protein) are predicted to be regulated by KLF6. We have previously reported that decreased ATF3 expression is correlated with poor differentiation and shorter overall survival in PAAD patients, indicating that ATF3 is essential to the progression of pancreatic cancer (14). Therefore, we selected ATF3 for further analysis. Using the GEPIA2 tool, we found that the expression of KLF6 is positively associated with ATF3 (R = 0.57, p = 6.8 × 10 −17 : Figure 4C). Moreover, after KLF6 overexpression in AsPc-1, CFPAC-1, and PANC1 cells, the mRNA and protein expression levels of ATF3 are elevated ( Figure 4D-G), suggesting that KLF6 may affect the progression of PAAD by upregulating ATF3. found that the expression of KLF6 is positively associated with ATF3 (R = 0.57, p = 6.8 × 10 −17 : Figure 4C). Moreover, after KLF6 overexpression in AsPc-1, CFPAC-1, and PANC1 cells, the mRNA and protein expression levels of ATF3 are elevated ( Figure 4D-G), suggesting that KLF6 may affect the progression of PAAD by upregulating ATF3.  To determine whether KLF6 is involved in pancreatic cancer progression through ATF3, we performed rescue experiments ( Figure 5A,B). The CCK8 results showed that silencing ATF3 could partially or completely rescue the decreased cell proliferation induced by KLF6 overexpression in AsPc-1, CFPAC-1, and PANC-1 cells ( Figure 5C-E). The migration result showed that knockdown of ATF3 partially rescued the decreased cell migration induced by KLF6 overexpression in PANC-1 cells ( Figure 5F). These results suggest that KLF6 is involved in the progression of pancreatic cancer through upregulating ATF3.

Discussion
The human KLF genes, encoding Krüppel-like factors with zinc finger DNA-binding proteins, have been reported to play a vital role in cellular processes [1,2,9]. Playing a central role in modulating these processes, the human KLF6 gene has been reported to influence tumorigenesis and the development of cancer [9]. Previously, several studies identified KLF6 as a tumor-suppressor gene due to frequent somatic inactivation of the KLF6 gene and/or downregulation of KLF6 expression in prostate carcinoma, glioblastoma, colorectal tumors, gastric cancer, hepatocellular carcinoma, and lung cancer [10,11,17,[27][28][29]. In contrast to these studies, several groups proposed that genetic alterations of KLF6 were infrequently observed and overexpressed in some types of cancer [30][31][32][33][34]. In this research, we found that KLF6 expresses lowly in pancreatic cancer samples and that survival analysis demonstrates that lower KLF6 expression is linked to a worse survival prognosis in PAAD. This indicates that KLF6 is a promising molecular biomarker for clinical survival prognosis. We also verified that KLF6 inhibits growth and metastasis in pancreatic cancer, indicating that KLF6 plays a tumor-suppressor role in pancreatic cancer. It should be noted that the KLF6 analyzed in our research was the longest isoform. Studies have reported that a splice variant of KLF6, KLF6-SV1, is a cancer-promoting gene in ovarian and prostate cancer models [15,35]. In 2008, Hartel et al. reported that enhanced alternative splicing of the KLF6 gene positively correlates with better prognosis in patients with pancreatic cancer samples, and the increased alternative splicing of KLF6 was primarily due to enhanced splice form expression rather than reduced KLF6 full length mRNA, indicating alternative splicing of KLF6 as a growth-promoting mechanism in human cancer. Our study identifies the tumor-suppressor role of KLF6 in pancreatic cancer, yet the role of splicing isoforms of KLF6 in pancreatic cancer needs further research.
KLF6, as a transcription factor, can interact with other proteins and regulate the transcription of its downstream targets to participate in the tumorigenesis development. Additionally, the mRNA of KLF6 receives the regulation of microRNA. For example, KLF6 can be targeted by miRNA-630 to regulate the growth and invasion in ovarian cancer [36]. Recent studies have illustrated the ability of KLF6 to recruit and interact with other transcription factors, such as p53 (Tumor Protein P53), RUNX1 (runt-related transcription factor 1), E2F1 (E2F Transcription Factor 1), and HDAC3 (histone deacetylase 3) [19,[37][38][39]. Additionally, KLF6 could regulate the expression of ATF3, which is a member of the ATF/CREB family of transcription factors and which is involved in many cellular functions, including cell proliferation and metastasis [40]. For example, Huang et al. found that KLF6 can induce apoptosis in prostate cancer by modulating ATF3 [41]. Our results showed that knockdown ATF3 partially or completely rescues the reduced cell proliferation and migration induced by KLF6 overexpression, suggesting that KLF6 functions partly through ATF3. However, the specific and detailed molecular mechanisms of how ATF3 is regulated by KLF6 and other potential mechanisms in pancreatic cancer need more research. Meanwhile, these results also indicate the potential function of ATF3 on pancreatic cancer cell. In a word, this study demonstrates that KLF6 inhibits the progression of pancreatic cancer through upregulating ATF3 and may serve as a potential therapeutic target. Nevertheless, more preclinical studies are needed before this is possible.

Conclusions
Our work investigated the essential role of KLF6 in pancreatic cancer. The results indicate that KLF6 is lowly expressed in tumor samples when compared to normal samples, and its expression is closely correlated with clinical prognosis in pancreatic cancer. Furthermore, KLF6 can inhibit the progression of pancreatic cancer by upregulating ATF3. These findings may better clarify the potential value of KLF6 in tumorigenesis and progression, and provide a novel perspective for a more precise treatment of pancreatic cancer in the future.