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. Author manuscript; available in PMC: 2017 Mar 14.
Published in final edited form as: Cancer Cell. 2016 Mar 14;29(3):324–338. doi: 10.1016/j.ccell.2016.02.005

KLF4 is essential for induction of cellular identity change and acinar-to-ductal reprograming during early pancreatic carcinogenesis

Daoyan Wei 1, Liang Wang 1, Yongmin Yan 1, Zhiliang Jia 1, Mihai Gagea 2, Zhiwei Li 1, Xiangsheng Zuo 3, Xiangyu Kong 1, Suyun Huang 4, Keping Xie 1
PMCID: PMC4794756  NIHMSID: NIHMS760224  PMID: 26977883

SUMMARY

Understanding the molecular mechanisms of tumor initiation has significant impact on cancer early detection and intervention. To define the role of KLF4 in pancreatic ductal adenocarcinoma initiation, we used molecular biological analyses and mouse models of klf4 gain- and loss-of-function and mutant Kras. KLF4 is upregulated in and required for acinar-to-ductal metaplasia. Klf4 ablation drastically attenuates the formation of pancreatic intraepithelial neoplasia induced by mutant KrasG12D, whereas upregulation of KLF4 does the opposite. Mutant KRAS and cellular injuries induce KLF4 expression, and ectopic expression of KLF4 in acinar cells reduces acinar lineage- and induces ductal lineage-related marker expression. These results demonstrate that KLF4 induces ductal identity in PanIN initiation and may be a potential target for prevention of PDA initiation.

Graphic Abstract

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INTRODUCTION

Pancreatic ductal adenocarcinoma (PDA) remains one of the most deadly human malignancies, largely because PDA is usually diagnosed at an advanced stage (Siegel et al., 2015). Early detection methods and effective preventive strategies are urgently needed; for these to be developed, it is essential to identify the determinant factors that control tumor initiation and understand the underlying molecular mechanisms.

Invasive PDA is believed to arise from a spectrum of preneoplastic mucinous lesions with ductal morphology (Hezel et al., 2006), among which pancreatic intraepithelial neoplasia (PanIN) is the most common and best characterized at the anatomopathological and molecular levels (Hruban et al., 2005). During disease progression, accumulation of genetic mutations in these lesions leads to an increasing degree of cellular atypia and ultimately PDA (Hezel et al., 2006). The earliest detectable mutations found in preneoplastic lesions are activating mutations of the Kras gene (Kanda et al., 2012), which occur in about 40% of cases of human PanIN1A/B and more than 90% of cases of human PDA. The significance of Kras mutation for disease initiation and maintenance has been demonstrated in mouse models (Hingorani et al., 2003).

Over the past few decades, tremendous efforts have been made to define the pancreatic cell types responsible for tumor initiation. Expression of the oncogenic Kras transgene in ductal cell lineage under the control of the cytokeratin-19 (CK-19) promoter failed to induce PanIN or did so at an extremely low frequency (Brembeck et al., 2003; Ray et al., 2011). However, recent studies have suggested that transformation of pancreatic ductal cells can also give rise to PDA (Pylayeva-Gupta et al., 2012; Ray et al., 2011; von Figura et al., 2014), and pancreatic ductal organoids expressing oncogenic Kras is sufficient to induce preinvasive neoplasms (Boj et al., 2015). Alternatively, selective expression of an endogenous KRasG12V oncogene in embryonic cells of acinar/centroacinar lineage resulted in PanIN and invasive PDA, suggesting that PDA originates through differentiation of acinar/centroacinar cells or their precursors into ductal-like cells (Stanger et al., 2005). Although inconsistencies and contradictions remain, accumulating evidence suggests that PDA is primarily derived from pancreatic acinar cells (Gidekel Friedlander et al., 2009; Habbe et al., 2008; Ji et al., 2009; Kopp et al., 2012; Morris et al., 2010; Sawey et al., 2007; Wu et al., 2014).

Mechanistically, in KrasG12D-expressing mice, PanIN formation coincides with or is preceded by acinar-to-ductal metaplasia (ADM). ADM is also observed in chronic pancreatitis, which is a significant risk factor for PDA in humans (Lowenfels et al., 1993) and accelerates KrasG12D-mediated PanIN and PDA formation in mice (Guerra et al., 2007; Morris et al., 2010). These findings suggest that ADM may be a mechanism for initiation of PanIN. However, acinar cells and particularly, the insulin-expressing endocrine cells in adult mice become refractory to K-RasG12V-induced PanIN and PDA unless they are exposed to chronic pancreatitis (Gidekel Friedlander et al., 2009; Guerra et al., 2007). These observations suggest that, in addition to genetic events (e.g., somatic KRas mutation), other nongenetic insults (e.g., inflammation or tissue injury) are also required for the development of PanIN and PDA during adulthood. However, many fundamental concepts remain unclear, such as the underlying mechanisms for the intrinsic differences in susceptibility of different pancreatic lineage cells to Kras activation (Gidekel Friedlander et al., 2009; Kopp et al., 2012), how genetic and non-genetic signaling converge to cell fate-determinant factors to drive the initiation of PanIN from acinar cells. Given the difficulty of targeting mutant Kras, identifying the genetic and/or non-genetic determinant factors that can sense tissue damage and mutant KRAS signaling to drive PanIN initiation will have a substantial impact on PDA prevention and treatment.

The Krüppel-like factor 4 (KLF4) transcription factor was initially identified as an important regulator of cell fate decisions such as cell differentiation, cell cycle progression, and apoptosis (Wei et al., 2006). The discovery of KLF4 as one of the four Yamanaka factors (Oct3/4, Sox2, c-Myc, and Klf4) required for stem cell reprogramming (or inducible pluripotent stem cells, iPS) (Park et al., 2008; Takahashi et al., 2007) and its essential role in maintenance of genomic stability (El-Karim et al., 2013) further substantiate its cell fate determinant function. Additionally, functioning as an early response gene, KLF4 is widely involved in host response to inflammation, stress, and injury (Liao et al., 2011; Talmasov et al., 2015), and plays an important role in homeostasis of tissues and/or organs. Thus, KLF4 has drawn extensive attention over recent years.

Furthermore, both experimental and clinical evidence has demonstrated that KLF4 has a tumor suppressor function in many cancers including gastric, colorectal, lung cancers and leukemia (Faber et al., 2013; Katz et al., 2005; Wei et al., 2005; Wei et al., 2006; Yu et al., 2015), and targeted activation of KLF4 for therapeutic intervention of advanced solid tumors has been approved for clinical trial (NCT01281592) (Cercek et al., 2015). However, KLF4 was also reported to have oncogenic function in breast and other cancers (Rowland et al., 2005; Wei et al., 2006), although controversy remains (Akaogi et al., 2009). In the pancreas, KLF4 was found to be expressed in pancreatic ductal epithelial cells and critical for regulating the expression of CK-19, a specific pancreatic ductal epithelial cell marker (Brembeck et al., 2001; Brembeck and Rustgi, 2000). KLF4 was also shown to have a tumor-suppressive function in PDA (Shi et al., 2014; Wei et al., 2008). However, other reports showed that KLF4 mRNA expression was increased in human and mouse PanIN lesions (Maier et al., 2013; Prasad et al., 2005).

These previous findings not only prompt us to ask whether targeted activation of KLF4 is safe and effective for PDA intervention, but also highlight the high necessity to defining the definitive role of KLF4 in PDA initiation given its critical role in cell fate determination and cell reprogramming. In the current study, we examined the impact of KLF4 deficiency and overexpression on ADM and PanIN formation and investigated the underlying molecular mechanisms.

RESULTS

KLF4 Is Overexpressed in Human and Mouse Pancreatic Premalignant PanIN

Although KLF4 has been previously shown to have a tumor-suppressive function in PDA (Wei et al., 2008), there are reports showing that KLF4 mRNA expression was increased in human and mouse PanIN cells (Maier et al., 2013; Prasad et al., 2005) compared with normal ductal cells or normal pancreatic tissues. To determine whether KLF4 is involved in PDA initiation, we first performed immunohistochemical (IHC) analysis of KLF4 expression in pancreatic tissues in a tissue microarray, and the specificity of KLF4 antibody was further confirmed by IHC analyses using intestinal tissue samples derived from Villin-Cre;klf4f/f and klf4f/f mice (Figures S1A and S1B). Increased KLF4 expression was found in human PanIN cells compared with adjacent normal cells (Figures 1A–1C; Figures S1C and S1D), suggesting that elevated KLF4 expression be associated with PDA initiation. To provide additional evidence, we examined KLF4 expression in pancreatic tissues derived from KrasG12D mutant mice (Pdx-Cre;LSL-KrasG12D, referred to as PR mice here, which are equivalent to the KC mice) (Hingorani et al., 2003), and found strong KLF4 staining in PanIN epithelial cells compared with no or very weak KLF4 staining in adjacent normal acinar or ductal cells (Figures 1D and 1E; Figures S1E and S1F). Consistently, increased KLF4 expression was found at both the mRNA level, determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the protein level, determined by Western blot analysis, compared with pancreatic tissues from LSL-KrasG12D-negative littermate control mice (Figures 1F and 1G). More strikingly, elevated KLF4 expression in PanIN cells was observed in pancreatic tissues derived from another mouse model of PDA (Ela-CreERT;cLGL-KrasG12V) (Ji et al., 2009) (Figures 1H and 1I; Figures S1G and S1H). In addition, increased KLF4 expression in PanIN cells was closely correlated with expression of CK-19 and Muc5ac, 2 specific markers associated with PanIN lesions (Figures S1I–S1T). These results indicate that overexpression of KLF4 may be required for the initiation of pancreatic premalignant lesions.

Figure 1. KLF4 Is Overexpressed in Human and Mouse Premalignant Pancreatic Lesions.

Figure 1

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(A–C) Immunohistochemistry (IHC) staining for KLF4 and hematoxylin counterstain in a tissue microarray spotted with human pancreatic tissue cores. Representative images show increased KLF4 expression in PanIN cells (black arrow) compared with adjacent acinar cells (yellow arrow; A and B); the percentage of tissue cores displaying no, weak, or strong KLF4 staining intensity in normal pancreas and PanIN lesions (C).

(D–G) KLF4 expression in pancreatic tissues derived from Pdx-Cre;LSL-KrasG12D or control mice at the age of 3 months. Representative images of IHC staining for KLF4 show increased KLF4 staining in PanIN cells (black arrow) compared with adjacent normal acinar cells (yellow arrow) or ductal cells (red arrow; D and E). Quantitative reverse transcription polymerase chain reaction (qPCR) analysis of KLF4 mRNA expression was performed in pancreatic tissues from Pdx-Cre;LSL-KrasG12D (PR) or Pdx-Cre;LSL-KrasG12D-negative (CTR) mice (n=3; F), and Western blot analysis of KLF4 protein expression was performed in pancreatic tissues from individual mice in the PR and CTR groups, and the quantitated data expressed as relative ratio of KLF4 to β-actin were shown as numbers in italic font under individual blots (G).

(H and I) IHC staining for KLF4 and hematoxylin counterstain in mouse pancreatic tissues from Ela-CreERT;cLGL-KrasG12V mice at the age of 2 months. Representative images show increased KLF4 expression in PanIN cells (black arrow, I) compared with adjacent relatively normal ductal cells (red arrow, H).

Values in graphs are means±SD. *p<0.05, **p<0.01. Scale bars: 100 µm (A, D, and H) and 50 µm (B, E, and I).

See also Figure S1.

Klf4 Deficiency Reveals No Major Pancreatic Abnormalities

Previous studies showed that KLF4 is highly expressed in gut epithelial cells and is essential for homeostasis of gastric and colonic tissues (Katz et al., 2005; Katz et al., 2002; Li et al., 2012b; Wei et al., 2005). In the pancreas, KLF4 is expressed in pancreatic ductal epithelial cells and is critical for regulation of CK-19 expression (Brembeck et al., 2001; Brembeck and Rustgi, 2000). However, it is unclear whether KLF4 is required for pancreatic development and normal function, partially because germline Klf4 KO mice die shortly after birth (Segre et al., 1999). To this end, we crossed previously described Klf4loxp/loxp mice with Pdx-Cre mice (Figure S2A) to generate pancreatic Klf4-deficient Klf4f/f;Pdx-Cre mice (referred to as PK mice). PK mice were viable and fertile and born at the expected Mendelian ratio. Successful Cre-mediated recombination was assessed by PCR (Figure S2B). IHC staining revealed loss of KLF4 protein expression in pancreatic cells from PK mice compared to littermate Klf4f/f mice (Figures S2C and S2D).

No significant weight or size alteration was observed in PK mouse pancreata (Figures S2E and S2F), and a grossly normal morphology was observed at ages up to 6 months compared with age-matched Klf4f/f littermate mice (Figures S2G–S2J). Nevertheless, we observed a low incidence of hyperplasia in ductal epithelial cells associated with the large or main pancreatic duct in PK mice older than 10 months (Figures S2K and S2L), which is consistent with previous observations that KLF4 plays an important role in ductal epithelial cell differentiation (Brembeck et al., 2001; Brembeck and Rustgi, 2000). Survival duration did not differ between the 2 groups of mice up to 18 months of age (data not shown). Thus, we concluded that deletion of Klf4 did not result in any major pancreatic abnormalities.

Ablation of Klf4 Drastically Attenuates KrasG12D-Mediated PanIN Induction

To examine the role of KLF4 in the initiation of PDA, we compared pancreatic lesions among groups of mice with intact pancreatic Klf4 or Klf4 gene ablation in the presence or absence of mutant KrasG12D. First, we measured pancreas weight in mice aged 10 weeks and found that pancreata in Pdx-Cre;LSL-KrasG12D;Klf4f/f mice (referred to as PRK mice) were much smaller than in Pdx-Cre;LSL-KrasG12D;Klf4+/+ mice (referred to as PR mice), but no significant differences were observed between PRK mice and age-matched control (PK) mice (Figures 2A and 2B).

Figure 2. Klf4 Ablation Attenuates Mutant KrasG12D-Induced PanIN Formation.

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(A and B) Pancreas weight in Pdx-Cre;LSL-KrasG12D;Klf4f/f (PRK), Pdx-Cre;LSL-KrasG12D;Klf4+/+ (PR), and Pdx-Cre;Klf4f/f (PK) mice at the age of 3 months (n=5; A) and representative images showing pancreas size in each group (B).

(C–K) Histopathologic lesions observed in pancreas of mice from three different groups (PK, PR, and PRK, respectively). Representative images show the results of hematoxylin and eosin (HE; C–E), Alcian blue (F–H), and Sirius red staining (I–K).

(L and M) Percentage of the pancreatic area in each group affected by PanIN (L) and staining positive for Alcian blue (M).

(N) Percentage of mice in each group with different PanIN grades.

(O) Percentage of mice in each group with inflammation and fibroplasia of the pancreas.

Values in graphs are means±SD. *p<0.05, **p<0.01. Scale bars: 500 µm.

See also Figure S2.

Next, we analyzed histologic changes of pancreas by comparing H&E (Figures 2C–2E, and 2L), Alcian blue (Figures 2F–2H, and 2M), and Sirius red staining (Figures 2I–2K) and PanIN grade (Figure 2N) among the 3 groups of mice at the age of 3 to 3.5 months. Ablation of Klf4 drastically attenuated KrasG12D-mediated PanIN initiation and impaired the development of PanIN lesions, as indicated by the presence of fewer and lower grades of PanIN lesions in PRK mice compared with the other groups (Figure 2N). The PanIN lesions in PRK mice also had no or lower incidence of inflammation or fibroplasia associated (Figures 2I–2K, and 2O; note: Panel O is arranged at the top-right corner). No significant histologic alterations were observed in age-matched control (PK) mice (Figures 2C, 2F, 2I, and 2L–2O). These results suggest that KLF4 is critical for KrasG12D-induced PanIN initiation.

In contrast, the incidence of pancreatic cystic papillary neoplasia (CPN) and hyperplasia of islet cells (HLC) was increased in PRK mice compared with PR mice (Figures S2M–S2S). Additionally, unlike PR mice, all PRK mice developed multiple severe skin neoplastic lesions, particularly on both sides of the face, as early as age 3–4 months (Figures S2T and S2U), and the affected mice had to be euthanized, which did not allow us to continue observation of the mice beyond the age of 5 months. These data also clearly demonstrate a context-dependent function of KLF4 in carcinogenesis.

KLF4 Expression Is Increased in ADM

Given that PanIN is primarily derived from acinar cells and ADM is a precursor to PanIN, we sought to determine whether KLF4 is required for the induction of ADM. To do this, we used the ductal ligation assay, which is a quick, reliable, and widely used experimental model for inducing ADM (Hamamoto et al., 2002). Ductal ligation in Klf4 wild-type mice resulted in typical ADM formation, as demonstrated by histologic examination (Figures 3A–3C) and immunofluorescent staining for amylase expression (Figures 3D–3I). We found that drastic increases in KLF4 expression were restricted to cells with ductal-like morphology (Figures 3J–3O), which coincided with overexpression of CK-19, a ductal epithelial-specific marker (Figures 3P–3U). This observation was further confirmed by co-immunofluorescence staining of both KLF4 and CK-19 (Figures S3A–S3H). These results suggest that KLF4 overexpression is involved in the pathogenesis of ADM.

Figure 3. KLF4 Expression in Experimental ADM Lesion Induced by Ductal Ligation.

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(A–C) Histologic changes of exocrine pancreas from Klf4 wild-type mice subjected to pancreatic ductal ligation at postsurgical day 5. Representative images of H&E stained sections show both normal (red square) and ligated pancreatic tissues (green square) (B); Higher magnification images show normal pancreas (A) versus ADM of pancreatic acini (yellow arrows) in the area with pancreatic ductal ligation (C).

(D–I) Immunofluorescent analysis for amylase staining in pancreatic sections. There is intense cytoplasmic red staining in normal pancreatic tissues (E and F); while the pancreas with ductal ligation shows many ductal-like structures (G, white arrows) and only a few small foci of red staining for amylase consistent with remaining patches of pancreatic acini (I, yellow arrowheads).

(J–O) Immunofluorescent analysis for KLF4 staining in pancreatic sections. These images show increased KLF4 expression (green staining) of newly formed ductal-like structures (M, white arrows; O, yellow arrowheads) in the pancreas with ductal ligation (M–O).

(P–U) Immunofluorescent analysis for CK-19 expression in pancreatic tissue. Representative images show increased CK-19 staining (red staining) of newly formed ductal-like structures (S, white arrows; U, yellow arrowheads) in pancreas with ductal ligation (S–U).

Scale bars: 500 µm (B) and 50 µm (A, C, and D–U).

See also Figure S3.

KLF4 Is Required for ADM Formation

To further determine whether KLF4 is required for ADM formation, we examined the impact of Klf4 ablation on ADM formation in vivo and in vitro. Ductal ligation resulted in severe damage/injury to acinar cells, as indicated by the breakdown of acini into smaller pieces, shown by immunofluorescent staining for amylase (Figures 4A–4H), and no or only a few ductal-like structures with associated KLF4/CK-19 expression were observed in the pancreatic tissues from PK mice (Figures 4K and 4L) compared with that from Klf4f/f mice, in which numerous typical ADM lesions were observed (Figures 4D and 4J). Similar observations were confirmed by H&E staining (Figures S4A–S4E) and IHC analysis (Figures S4F–S4M).

Figure 4. KLF4 Is Required for ADM Formation.

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(A–H) Immunofluorescent staining for amylase in normal and ductal ligated pancreatic tissues from Klf4 wild-type (Klf4f/f; A–D) or Pdx-Cre;Klf4f/f (PK; E–H) mice. Representative images show areas of amylase staining (C and D) in pancreatic tissues of Klf4f/f mice, indicating intact residual acini (D, yellow arrowheads); while G and H images show scattered smaller pieces of amylase staining appear in pancreatic tissues of PK mice, indicating the breakdown of acini (H, arrows).

(I–L) Co-immunofluorescence staining for KLF4 and CK-19 confirmed increased KLF4 and associated CK-19 expression in ductal-like structures induced by ductal ligation in pancreatic tissues from Klf4f/f mice (I and J, arrowheads) but not in pancreatic tissue from PK mice (K and L).

(M–T) Representative images of explanted acinar cells collected from 3-week-old Klf4f/f (M–P) or PK (Q–T) mice after 1 day (M, N, Q, and R) or 5 days (O, P, S, and T) of culturing in the presence or absence of TGF-α. Ductal-like cell clusters were formed in the Klf4f/f mouse cell culture treated with TGF-α (P, arrowheads) but not in the PK mouse culture (T).

(U–Y) Immunofluorescence analysis confirmed that ductal-like cell clusters with increased CK-19 expression appeared in the culture from Klf4f/f mice (U and V, arrows) but not in the culture from PK mice (W and X). Quantification of CK-19-positive ductal-like cell clusters is shown in Y.

**p<0.01. Scale bars: 50 µm.

See also Figure S4.

To further establish the critical role of KLF4 in mediating acinar-to-ductal epithelial reprograming, we isolated pancreatic acinar cells from both PK and Klf4f/f mice for explant culture and treated the cells with or without TGF-α. Consistently, Klf4 ablation drastically blocked the formation of ductal-like structures of explanted cultured acinar cells (Figures 4T, 4X, and 4Y) compared with acinar cells with an intact Klf4 gene (Figures 4P, 4V, and 4Y). These results suggest that KLF4 is essential for acinar-to-ductal epithelial reprograming.

KLF4 Is Critical for ADM and PanIN Formation after Pancreatic Injury in the Presence of Mutant Kras

Previous studies have demonstrated that pancreatic injury, including chronic pancreatitis, an etiologic factor for PDA, can significantly accelerate the development of ADM and PanINs in the presence of mutant Kras (Guerra et al., 2007; McAllister et al., 2014). Consistent with these findings, we found that ablation of Klf4 in the presence of oncogenic Kras drastically attenuated KrasG12D-mediated changes in pancreatic morphology and PanIN development (Figure 2). However, it remains unclear whether KLF4 is also involved in promoting ADM and PanIN formation after pancreatic injury. To address this question, we examined pancreata for ADM and PanIN formation after treatment with caerulein among different groups of mice. Comparing with Pdx-Cre;Klf4+/+ control mice (Figures 5E and 5U), we observed significant reduction of ADM-like lesions in the pancreata of PK mice on day 4 after the initial injection of caerulein (Figures 5F and 5U), and almost complete morphologic recovery of pancreata with no significant differences could be distinguished between the 2 groups on day 20 after the initial injection of caerulein (Figures 5I and 5J). In contrast, in the presence of mutant Kras, less ADM or PanIN lesions were observed on days 4 and 20 after the initial injection of caerulein in PRK mice (Figures 5H, 5L, 5P, 5T, 5U, and 5V) than in PR mice, as determined by histologic examination, immunofluorescent staining, and Alcian blue staining (Figures 5G, 5K, 5O, 5S, 5U, and 5V). These results suggest that KLF4 is critical for ADM and PanIN formation after pancreatic injury in the presence of mutant KrasG12D.

Figure 5. KLF4 Is Critical for ADM and PanIN Formation after Pancreatic Injury in the Presence of Mutant Kras.

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(A–L) Six-week-old transgenic and control mice were treated with 1 set of caerulein or saline injections for 2 consecutive days and analyzed 4 or 20 days (d) later. Saline-treated mice were analyzed at 20 days post-treatment (A–D). Pancreatic tissues from the mice were collected and processed for different staining. Representative images show ADM-like lesions of pancreatic acini at 4d after caerulein-induced acute pancreatitis (E vs F; G vs H, arrow), and persistent ADM (yellow arrow) with concurrent PanIN lesions (arrowhead) in pancreatic tissues at 20d after caerulein treatment in Pdx-Cre;LSL-KrasG12D;Klf4+/+ (PR) and Pdx-Cre;LSL-KrasG12D;Klf4f/f (PRK) mice (K and L).

(M–P) Co-immunofluorescence staining for KLF4 and CK-19 confirmed accelerated formation of ADM-like lesions in pancreatic tissues from PR mice (O) compared with PRK mice (P) on day 4 after treatment with caerulein.

(Q–T) Alcian blue/eosin staining shows PanIN lesions in pancreatic tissues from PR and PRK mice (S and T, arrowheads).

(U and V) Quantification of ADM-like lesions (U) and areas staining positive for Alcian blue (V) in pancreatic tissues on days 4 (U) and 20 (V) after treatment with caerulein (n=4–5).

Values in graphs are means±SD. **p<0.01. Scale bars: 100 µm (A–L and Q–T) and 50 µm (M–P).

Overexpression of KLF4 Induces Acinar-to-Ductal Reprogramming and Potentiates Mutant Kras-Induced PanIN Formation

To further define the role of KLF4 in ADM and PDA initiation, we generated a gain-of-function Klf4 transgenic mouse model (referred as KLF4OE), in which CAG promoter-driven EGFP expression was restricted to acinar cells but not ductal and islet cells in the absence of Crerecombinase (Figures S5). These mice served as a very good model for us to address the effect of KLF4 overexpression in acinar cells on ADM and PanIN formation. Overall, no apparent abnormalities were observed in KLF4OE transgenic mice without Cre recombinase (Figures 6A–6C); most of the pancreatic tissues looked normal with only scattered acini or small patched pancreatic acinar clusters with dilated lumens, and no significant increase in Alcian blue and Sirius red staining was observed in the pancreata of Pdx-Cre;KLF4OE mice without Kras mutation at 9 weeks of age (Figures 6D–6F and 6M–6O). In contrast, in the presence of mutant Kras, KLF4 overexpression significantly promoted PanIN formation affecting large areas of the pancreas that had extensive Alcian blue and Sirius red positive staining (Figures 6J–6L, and 6M–6O) in PR;KLF4OE mice compared with that in PR mice (Figures 6G–6I, and 6M–6O). These results suggest that KLF4 overexpression alone is not sufficient to induce PanIN initiation (see Figures 7E–7L), but significantly potentiates the development of PanIN formation in the presence of mutant Kras.

Figure 6. Overexpression of KLF4 Potentiates Mutant Kras-Induced PanIN Formation.

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(A–L) Pancreatic tissues collected from 9-week-old mice were stained for hematoxylin and eosin (HE; A, D, G, and J), Alcian blue/fast red (B, E, H, and K), and Sirius red (C, F, I, and L); representative images are shown.

(M–O) Quantification of areas of the pancreas affected by PanIN (M), staining positive for Alcian blue (N), and staining positive for Sirius red (O) in each mouse group, revealing that KLF4OE potentiates PanIN formation in the presence of mutant Kras (n=6 per group).

Values in graphs are means±SD. *p<0.05, **p<0.01. Scale bars: 500 µm.

See also Figures S5.

Figure 7. KLF4 Induces Ductal Cell-Specific but Reduces Acinar Cell-Specific Gene Expression in Acinar Cells.

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(A) Western blot analysis of related protein expression in 266-6 acinar cells 48 hr after transduction of different doses of the KLF4 gene, and the quantitated data expressed as relative ratio of KLF4 to α-tubulin were shown as numbers in italic font under individual blots.

(B) Western blot analysis reveals specific regulatory effects of KLF4 on acinar- and ductal-related gene expression in 266-6 acinar cells 48 hr after mock transfection (Mock) or transfection with control (Ctr) vector, wild-type KLF4 (WT), or KLF4 mutant (Mut) vector.

(C and D) qPCR analysis of related gene expression in 266-6 acinar cells 48 hr after transduction of different doses of the KLF4 gene mediated by adenoviral infection.

(E) qPCR analysis of CK-19 and amylase in whole pancreatic RNA from control KLF4OE/+, Pdx-Cre;KLF4OE/+, and Pdx-Cre;KLF4OE/OE mice (n=3).

(F–L) Co-immunofluorescence staining for amylase and CK-19 shows increased intensity of CK- 19 staining associated with increased ductal-like structures in pancreatic sections from Pdx-Cre;KLF4OE/OE mice (J and K) at postnatal day (p) 10 compared with Pdx-Cre;KLF4OE/+ (H and I) and KLF4OE/+ control (F and G) mice. Pancreatic cellular density was lower in Pdx-Cre;KLF4OE/OE mice (J and K) than in Pdx-Cre;KLF4OE/+ (H and I) and control (F and G) mice, and reduced pancreatic cellular density was correlated with reduced pancreas weight (n=3; L).

Values in graphs are means ± SD. *p<0.05, **p<0.01. Scale bars: 100 µm (F, H, and J) and 50 µm (G, I, and K).

See also Figure S6.

Overexpression of KLF4 in Acinar Cells Induces Ductal Cell-Specific Gene Expression but Reduces Acinar Cell-Specific Gene Expression

To understand the molecular mechanisms by which KLF4 impacts ADM induction and PanIN initiation, we sought to determine whether ectopic expression of KLF4 in acinar cells regulates cell-lineage related gene expression in the absence of oncogenic Kras. We found that overexpression of KLF4 mediated by gene transduction in 266-6 acinar cells significantly downregulated acinar-specific amylase and Ptf1a but upregulated ductal-specific CK-19 and CA-II protein expression (Figure 7A). Consistent with previous report (Wei et al., 2008), ectopic expression of KLF4 in 266-6 acinar cells significantly induced the expression of p27 and p21 (Figure 7A), 2 critical negative regulators of the cell cycle that are required for cell differentiation (Deschenes et al., 2001).

It has been shown that ADM reprograming requires embryonic or regenerative signaling (Carriere et al., 2007; Huch et al., 2013; Jensen et al., 2005; Kopp et al., 2012; Means et al., 2005; Sawey et al., 2007; Siveke et al., 2008), we therefore examined the effect of KLF4 on related gene expression in 266-6 cells after KLF4 gene transduction. We found that KLF4 significantly inhibited the expression of acinar-specific transcription factor Ptf1a and upregulated the embryonic/progenitor marker Nestin, and to a less extend, Lgr5 but did not have a significant effect on Hes1 or Sox9 expression (Figure 7A). The specific effect of KLF4 on pancreatic lineage-specific gene expression was further demonstrated by Western blot results showing that the regulatory effects of KLF4 on amylase and CK-19 expression were lost when 266-6 cells were transfected with a mutant KLF4 gene with deletion of 3 zinc-finger DNA binding domain (Figure 7B). In addition, KLF4 expression occurred with increased CK-19 or decreased amylase expression in 266-6 cells after transfection with the WT KLF4 gene (Figure S6A–S6D). Consistently, transduction of KLF4 upregulated or downregulated relevant gene expressions at the mRNA level (Figures 7C and 7D), suggesting that a transcriptional mechanism is involved in KLF4 regulation of expression of these genes. This is also supported by the fact that the KLF4 zinc-finger DNA binding domain is required for the regulatory effect of KLF4 on gene expression (Figure 7B).

To validate the in vitro findings, we examined relevant gene expression in pancreatic tissues derived from KLF4OE mice at postnatal day 10. We found that KLF4OE gene dose-dependently correlated with increased CK-19 and decreased amylase expression at the mRNA level (Figure 7E). These results were also consistent with the results of immunofluorescent staining for CK-19 and amylase expression (Figures 7F–7K; Figures S6E–S6S), as well as the significant pancreatic histologic changes indicated by an increased number of ductal-like structures and reduced density of acini (Figures 7F–7K) and reduced pancreatic weight (Figure 7L). However, these changes were gradually recovered as the mice aged, and only marginal histologic changes could be detected in mice older than 5 weeks, including reduced pancreatic cell density, irregular arrangement of acinar cells (Figures S6E–S6J and S6Q–S6S), scattered acinar cells or acini with increased CK-19 expression (Figures S6K–S6S), and foci of acini with dilated lumens (Figure 6D), but no PanIN lesion was observed (Figures 6E–6F and 6M–6O). These results suggest that KLF4 overexpression induces ductal-cell lineage gene expression and acinar-to-ductal reprogramming but is not sufficient to induce PanIN formation.

KLF4 Expression Is Induced by Mutant Kras and Stress Signaling

Given that KLF4 is an early response gene and plays an important role in homeostasis, we sought to determine whether KLF4 expression is induced under pathophysiologic conditions associated with pancreatic injury and carcinogenesis. We found that KLF4 protein and mRNA expression were induced in 266-6 acinar cells after transduction of the mutant KrasG12D gene (Figures 8A and 8B), which was consistent with a previous report showing that mutant Kras can induce KLF4 expression in pancreatic ductal epithelial cells (Qian et al., 2005).

Figure 8. Mutant Kras and Stress Signaling Induce KLF4 Expression.

Figure 8

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(A and B) Western blot and qPCR analyses of KLF4 protein (A) and mRNA expression (B) in 266-6 acinar cells 48 hr after mutant KrasG12D gene transduction.

(C and D) Western blot and qPCR analyses of KLF4 protein (C) and mRNA (D) expression in 266-6 and PANC-1 cells 12 hr after treatment with TNF-α.

(E) Western blot analysis of KLF4 protein expression in 266-6 and PANC-1 cells 3 hr after treatment with H2O2.

(F) qPCR analyses of KLF4 mRNA expression in 266-6 and PANC-1 cells 2.5 hr after treatment with H2O2 at 0, 0.1 and 0.3 mM, respectively.

(G–H) Western blot analysis of KLF4 protein expression in 266-6 and PANC-1 cells 2 hr after treatment with caerulein (CA; G) and in whole pancreatic proteins from wild-type Klf4 mice after 4 consecutive hourly CA or saline injections (H).

Quantitated data for Western blot expressed as relative ratio of KLF4 or Kras to α-tubulin were shown under individual blots. Values in graphs are means±SD. **p<0.01.

Because caerulein-induced pancreatitis can significantly accelerate the development and progression of ADM and PanIN, and TNF-α and H2O2 are 2 critical mediators in pancreatitis and inflammation, we investigated the effects of TNF-α and H2O2 on KLF4 expression. Treatment with TNF-α in 266-6 and PANC-1 cells led to significant induction of KLF4 expression at both the mRNA and the protein level (Figures 8C and 8D). Similarly, treatment with H2O2 induced KLF4 protein and mRNA expression in both 266-6 and PANC-1 cells (Figures 8E and 8F).

Finally, we examined the effect of caerulein treatment on KLF4 expression in vitro and in vivo. We observed a quick and significant induction of KLF4 protein expression in both 266-6 and PANC-1 cells in vitro (Figure 8G), and more strikingly, in mouse pancreatic tissues (Figure 8H) after caerulein treatment. These results provide a mechanistic basis for the requirement or potentiating effect of KLF4 in ADM and PanIN formation associated with pancreatic injury or inflammation and carcinogenesis.

DISCUSSION

Our findings provide several lines of evidence supporting the critical role of KLF4 in the induction of ADM and initiation of PanIN. First, the expression of KLF4 was significantly increased in both human and mouse PanIN. Second, KLF4 expression was induced in and required for ADM formation, and ectopic expression of KLF4 in acinar cells suppressed acinar-related biomarker expression but induced ductal epithelial-related marker expression and, to a certain level, ADM formation. Third, genetic ablation of klf4 drastically attenuated PanIN formation in mouse pancreata, whereas overexpression of KLF4 did the opposite when mutant Kras was present. Fourth, Klf4 ablation attenuated the formation of ADM and PanIN after pancreatic injury in the presence of mutant Kras. Fifth, KLF4 expression was induced by mutant KrasG12D gene transfection or treatment with caerulein or the inflammatory mediators TNF-α and H2O2 in pancreatic acinar or cancer cells. Collectively, these findings identify KLF4 as an important inducer of ductal identity and a sensor and executor of injury signaling, thus playing a critical role in acinar-to-ductal reprogramming and initiation of PanIN in the presence of mutant KRAS.

PDA is a very complicated disease. For example, each pancreatic tumor has an average of 63 genetic alterations (Jones et al., 2008), which makes it difficult to develop targeted therapy. What makes it more complicated is that we found a stage-dependent function of KLF4 in PDA. Previously, both clinical and experimental studies have suggested that KLF4 has a tumor-suppressive function in PDA, as evidenced by frequent loss of KLF4 gene and protein expression and marked inhibition of cell proliferation and tumorigenicity after ectopic expression of KLF4 in PDA cells (Shi et al., 2014; Wei et al., 2008). However, in the present study, we found that KLF4 was overexpressed in both human and mouse PanINs. In investigating the role of KLF4 in ADM formation and PDA initiation, we generated mouse models with pancreatic Klf4 deficiency or overexpression. Surprisingly, we found that genetic ablation of Klf4 drastically attenuated PanIN formation, whereas overexpression of KLF4 potentiated PanIN formation in the presence of mutant Kras. These results suggest that KLF4 has a stage-dependent function in PDA; i.e., KLF4 has a protumorigenic function in PDA initiation but a tumor-suppressive function in developed PDA.

Our findings also suggest that KLF4 has cell lineage-dependent functions in the pancreas. For example, low incidence of hyperplastic lesions in pancreatic ductal epithelia associated with large or main pancreatic ducts was observed in aged Klf4-deficient mice and increased incidence of mucinous cystic neoplasia associated with large or main ducts was observed in PRK mice. This observation is consistent with previous findings showing that KLF4 is associated with pancreatic ductal epithelial differentiation (Brembeck et al., 2001). Additionally, we observed a relatively high level of KLF4 expression in pancreatic islet cells compared with cells of other lineages in the pancreas. Interestingly, we also found that Klf4 ablation could lead to hyperplasia of islet cells in PRK mice. These data suggest that KLF4 may maintain a tumor-suppressive function in cells of ductal and islet epithelial lineages, whereas overexpression of KLF4 in acinar cells may result in acinar-to-ductal reprogramming, which potentiates the initiation of PanIN in the presence of mutant KRAS. However, Klf4 deletion was found to synergize with mutant KRas to facilitate carcinoma development in mouse tongue and lung (Abrigo et al., 2014). Of note, we also observed accelerated development of skin neoplastic lesions in PRK mice compared with PR mice, and specific Klf4 gene ablation was detected in the skin tumor tissues derived from PRK mice, which is consistent with previous studies showing that Pdx-1 is expressed in skin epidermal cells (Mazur et al., 2010), and Klf4 deficiency promoted the skin tumorigenesis in a classical DMBA/TPA mouse skin cancer model (Li et al., 2012a). Therefore, our findings further support the context-dependent function of KLF4 in carcinogenesis (Rowland et al., 2005).

How KLF4 becomes a stage-dependent protumorigenic or tumor-suppressor gene in the pancreas remains unclear. Evidently, the fundamental function of KLF4 in inducing cell differentiation and suppressing cell cycle progression may remain active when KLF4 acts like a tumor suppressor in later stages of PDA. For examples, transduction of KLF4 into human PDA cells induces cell cycle arrest, ductal epithelial marker expression, and mesenchymal-to-epithelial transition (MET) phenotype, whereas knockdown of KLF4 expression does the opposite. Transduction of KLF4 into a sub-population of Pdx1-positive SCA1-KrasG12D p53KO tumorigenic pancreatic cells resulted in the cell differentiation into quasi-normal cells with suppressed tumorigenicity and metastasis (Ischenko et al., 2014). Clinically, loss of KLF4 expression is associated with poor differentiation of PDA, epithelial-to-mesenchymal transition, and metastasis (Shi et al., 2014). Likewise, the fundamental function of KLF4 in inducing cell differentiation and suppressing cell cycle progression may remain active when KLF4 acts like a protumorigenic gene in the initiation of PanIN. For examples, KLF4 overexpression in human islet-derived mesenchymal stromal cells induces MET and redifferentiation towards pancreatic cell types with re-expression of insulin and transcription factors specific to β-cells (Muir et al., 2015). In the present study, KLF4 overexpression in acinar cells upregulates ductal-specific molecular markers and the expression of negative cell cycle-regulator genes, and induces acinar-to-ductal reprogramming, thus promoting PanIN formation under KRas mutant condition. Therefore, those fundamental functions of KLF4 in regulation of cell cycle and differentiation may fate cells to different directions with different biological outcomes, depending upon cell lineage and functional status. The detailed molecular basis for such stage-dependent or context-dependent protumorigenic or tumor-suppressor function of KLF4 in PDA and other cancers warrants further investigations.

Another possible mechanism responsible for the KLF4 functional switch is the level (or activity) of KLF4 expression. Physiologically, KLF4 expression is finely tuned in different lineages of cells. For example, the level of KLF4 expression in pancreatic ductal epithelial cells is much lower than in gastric and colorectal epithelial cells and barely detectable in acinar cells. Previous studies showed that physiologic expression of KLF4 is required for esophageal squamous epithelial differentiation (Tetreault et al., 2010b), whereas KLF4 overexpression activates epithelial cytokines and inflammation-mediated esophageal squamous cell cancer in mice (Tetreault et al., 2010a). Considering that KLF4 is an early stress response gene and plays an important role in maintaining homeostasis and genomic stability, and that any adverse stress and damage to acinar cells may result in the release of many digestive enzymes that could cause severe negative consequences, it is possible that increased KLF4 expression leading to suppression of acinar-specific genes such as those related to digestive enzymes and upregulating cell cycle arrest, including p21 and p27, which are also required for cell differentiation (Deschenes et al., 2001), may represent a host intrinsic defense mechanism to protect acinar cells from damage under adverse conditions such as acute pancreatitis.

Generally, compared with ductal and islet cells, acinar cells are much more sensitive to stress or inflammation-induced damage, and the intrinsically high plasticity of acinar cells provides a means for them to cope with stress or adverse conditions for overall organ homeostasis and health (Houbracken et al., 2011), during which KLF4 plays an important role. This assumption is supported by our results and those of others showing that short-term pancreatic injury via treatment with caerulein results in quick upregulation of KLF4 expression in the pancreas and temporary dedifferentiation of acinar cells that is quickly resolved when the cells reassume an acinar identity (Jensen et al., 2005). However, sustained and high levels of KLF4 expression in acinar cells, particularly in the presence of mutant KRAS plus stress or injury, may lead to irreversible cellular identity change (transdifferentiation) and progression to PanIN. Consequently, the original protective function of KLF4 in acinar cells for homeostasis switches to a protumorigenic function through induction of acinar-to-ductal reprogramming. Although this is supported by our findings, other questions still remain unanswered, such as how KLF4 expression is finely tuned in different cell lineages, whether the differences in KLF4 expression level in different pancreatic lineage cells are responsible for their discrepancy in intrinsic susceptibility to oncogenic Kras-induced transformation (Gidekel Friedlander et al., 2009), how KLF4 drives acinar cell transdifferentiation, and how stress and KRAS signaling activate KLF4 expression. The answers to these questions may help in the development of novel strategies that can properly manipulate or regulate KLF4 expression or activity to best utilize its intrinsic protective function while avoid any adverse effects of PDA prevention and therapy.

Both the present and previous studies highlight the critical role of cellular identity change in the initiation of PDA, and cellular identity can be shaped by genetic, epigenetic, and microenvironment factors. In the present study, we identified KLF4 as a determinant factor in ADM and PanIN formation and provided the molecular basis for its action. First, KLF4 is an inducer of ductal identity in pancreatic acinar cells, which has profound effects on the expression of genes affecting a broad range of cellular lineages (for example, KLF4 induces ductal CK-19 and CA2 but reduces acinar amylase and ptf1a expression). Second, both genetic (e.g., mutant Kras) and epigenetic or environmental (e.g., inflammation, injury, stress, reactive oxygen species) signaling can converge to activate KLF4 expression; and consequently, overexpression of KLF4 results in acinar-to-ductal reprograming, which promotes the initiation of PanIN in the presence of mutant Kras. Given the untargetable nature of mutant KRAS in PDA, our findings suggest that KLF4 might represent a potential target for the development of preventive strategies to block early tumor-initiating events.

In summary, our findings identify the cell fate determinant KLF4 as a ductal identity inducer and a critical mediator in ADM process and initiation of PanIN induced by mutant KRAS; and suggest that KLF4 may be a potential target for preventing early PDA-initiating events. Significantly, our demonstration of cell lineage-dependent outcome of KLF4 functioning in pancreas provides new insight into the context-dependent pro-tumorigenic or tumor-suppressor function of KLF4, and also raise concerns of potential risks and/or limitations of targeting KLF4 for preventive and/or therapeutic intervention of cancers.

EXPERIMENTAL PROCEDURES

Mouse Procedures

All animal experiments described here were approved by MD Anderson Cancer Center Institutional Animal Care and Use Committee. The sources and generation for mouse strains as well as genotyping strategies are described in the Supplementary Experimental Procedures.

Cell Lines and Culture Conditions

The human PDA cell lines AsPC-1 and PANC-1 and mouse pancreatic acinar cell line 266-6 were purchased from American Type Culture Collection (Manassas, VA) and cultured as described previously (Wei et al., 2010).

Histology, Immunohistochemical and Immunofluorescence Analysis

Paraffin-embedded or frozen sections were subjected to hematoxylin (Mayers or Harris formulations), eosin, Alcian blue, nuclear fast red, Sirius red (IHC WORLD, Woodstock, MD), immunohistochemical or immunofluorescence staining as described (Li et al., 2012b; Wei et al., 2010). The use of archived human specimens was approved by the institutional review board. Detailed procedures for histologic and morphometric analyses, as well as a list of primary and secondary antibodies can be found in the Supplemental Experimental Procedures.

Statistical Analysis

Statistical significance was determined by Student’s t test (2-tailed), or analysis of variance (two-way) with Bonferroni Post test (for quantitative data); or by Fisher’s exact tests or Pearson Chi-square test (for categorical variables), when appropriate. All significance was defined at the p<0.05 or p<0.01 level.

Supplementary Material

Highlights.

KLF4 induces ductal but represses acinar identity in pancreatic acinar cells.

KLF4 is required for acinar-to-ductal cell reprogramming.

Mutant KRas, injury, and stress signaling converge to activate KLF4 expression.

KLF4 synergizes with mutant KRas in initiation of PanIN.

Significance.

PDA, usually diagnosed at an advanced stage, has a hopelessly dismal prognosis. Identifying the determinants in tumor initiation is essential for the development of early detection methods and effective preventive and therapeutic modalities. By using genetically engineered mouse models of mutant KrasG12D and klf4 alterations, and molecular biology analyses, we demonstrate that KLF4 is a ductal fate determinant and plays a critical role in acinar-to-ductal cell reprograming. Moreover, injury and stress signaling can converge to induce KLF4 expression in acinar cells, which potentiates the initiation of premalignant pancreatic intraepithelial neoplasia (PanIN) induced by mutant Kras. Therefore, KLF4 is a potential target for designing effective intervention strategies to block early tumor-initiating events.

Acknowledgments

We thank Erica Goodoff for editorial comments. We are grateful to Dr. Craig Logsdon’s Lab for the gifts of pCAGGS-cLGL-KrasG12V vector and pancreatic tissue samples from Ela-CreERT;cLGL-KrasG12V mice. This work was supported in part by NCI grants R01CA129956, R01CA148954, R01CA152309, R01CA172233 and R01CA195651 (to K.X.); and the American Institute for Cancer Research grant (10A073) and NCI grant R03CA124523 (to D.W.).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflicts of interest

All authors disclose no conflicts of interest.

SUPPLEMENTAL INFORMATION

Supplemental information includes ten figures, one table, and Supplemental Experimental Procedures.

AUTHOR CONTRIBUTIONS

K.X., D.W., and S.H. conceived experiments, analyzed results, and wrote the manuscript, K.X. and D.W. secured funding. D.W., L.W., Y.Y., Z.J., M.G., Z.L., and X.Z. performed experiments. X.K and S.H. provided reagents, expertise and feedback.

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