The dual role of TGF-β1 in tumorigenesis of epithelial tumors is well established. Under physiological conditions, TGF-β1 is an essential cell growth inhibitor and thereby prevents epithelial hyperproliferation and the onset of epithelial tumors. However, TGF-β1 becomes a potent tumor promoter through its capacity to initiate EMT, to enhance cell migration and invasion and to favour chemoresistance [
12,
13], applying also to PDAC [
38‐
40]. Moreover, experimental evidence indicate that this functional switch might occur already at early stages in tumorigenesis when epithelial cells still do not harbour any of the driver mutations or at least only single mutations, such as in the
k-ras oncogene [
37,
39]. This functional switch of TGF-β1 leading to reprogramming of its activated signaling pathways seems to be context dependent, albeit the underlying mechanisms are still not fully understood. One mechanism explaining the TGF-β1 paradox might be the differential activation of MAPK such as Erk in benign and malignant epithelial cells [
12]. Many tumors exhibit constitutive high Erk activation due to a mutation in the
ras oncogene, leading to sustained TGF-β1 synthesis and signaling. In line with this, Gotzmann et al. showed that cooperation of TGF-β1 signaling and oncogenic expression of
Ha-ras promotes a mesenchymal and invasive phenotype in hepatocytes [
41]. Furthermore, sustained MAPK activation can also result from prolonged exposure to a plethora of inflammatory mediators (e.g. IL-6, TNF-α, TGF-β1) upon chronic inflammation [
42,
43]. Thus, our study extends this view by demonstrating that sustained high expression and activity of the antioxidative transcription factor Nrf2 essentially contributes to deregulation of TGF-β1 signaling by reducing Smad3 and p38 activation and augmenting Erk activation resulting in reduced p21 expression and enhanced proliferation of pancreatic ductal epithelial cells. One can speculate that an elevated Nrf2 activity leads to changes in the assembly of TGF-β I (Alk1 and Alk5) and TGF-β II receptors, thereby favouring an altered recruitment of downstream mediators that can initiate Smad independent/non-canonical pathways like Ras-ERKs. Thus, further studies have to unravel in more detail how Nrf2 suppresses TGF-β1 mediated Smad3 and p38 activation and concomitantly enhance Erk signaling. Interestingly, we could observe these effects likewise in benign, premalignant and malignant pancreatic ductal epithelial cells suggesting that high Nrf2 activity can promote this functional switch of TGF-β1 already early in tumorigenesis. Favoring this hypothesis, we could detect enhanced expression of activated (phosphorylated) Nrf2 in nuclei of early PanINs being significantly associated with a higher proliferative activity exemplified by elevated Ki67 expression. Moreover, these data are in line with findings from an endogenous PDAC mouse model showing that oncogenic
kras activation is one key mechanism leading to sustained Nrf2 expression and activity driving pancreatic tumorigenesis [
25]. Hence, pancreata of Nrf2 deficient mice exhibited fewer PanINs with lower proliferative activity than the wildtype mice whereas no differences in the extent of apoptotic cells could be observed [
25]. Thus, activation of the ras-raf-Erk pathway [
25,
44] represents an important mechanism leading to enhanced Nrf2 expression and activity in tumor cells, applying also to pancreatic ductal epithelial cells. Accordingly, we could demonstrate higher basal Nrf2 expression and activity in H6c7-kras and Colo357 cells compared to benign H6c7-pBp cells. Important to note, constitutive Nrf2 activation is caused to a lesser extent by genetic alterations affecting either the Nrf2 inhibitor Keap1 or Nrf2 itself (accounting for 10–15 % in gallbladder and ovarian cancer or in lung cancer up to 30 % of enhanced Nrf2 activation) as well as epigenetic mechanisms accounting for enhanced Nrf2 activity in 20–30 % of tumors [
45,
46]. Accordingly, we previously showed that PDAC cells with different Nrf2 activity exhibit similar Keap1 expression levels [
18] supporting the view that sustained Nrf2 activation in PDAC cells is not caused by epigenetic or genetic alterations of Keap1 and rather than other factors. Besides the above mentioned oncogene induced Nrf2 activation, the exposure of epithelial cells to persistent metabolic and/or oxidative stress, e.g. caused by the presence of ROS releasing macrophages and neutrophils during the course of a chronic inflammation [
47] is a major cause for constitutive elevated Nrf2 activity. Macrophages are not only abundant in chronic pancreatitis and PDAC [
8] but start to accumulate around early PanINs. Hence, the growing inflammatory microenvironment together with oncogenic
kras activation in ductal epithelial cells, which is present in almost all early PanINs [
3], may promote upregulation of both Nrf2 and TGF-β1 activity already at this early stage of PDAC development. Supporting this view, we previously demonstrated that predominantly proinflammatory macrophages releasing ROS at high levels lead to nuclear accumulation of Nrf2 in colonic epithelial cells conferring apoptosis resistance towards TRAIL or the chemotherapeutic drug irinotecan [
47]. This Nrf2 mediated apoptosis resistance relied on Nrf2 enhanced proteasomal gene expression [
47], a mechanism that apparently also operates in PDAC cells [
18].
In contrast to the findings of Lister et al., in our studies the effect of Nrf2
per se on cell growth of pancreatic ductal epithelial cells was minor [
17] which might be explained by different cultivation periods after modulation of Nrf2 expression. Nrf2 essentially impacts on cell survival of pancreatic ductal epithelial cells by antagonizing the growth inhibitory effect of TGF-β1 by diminishing TGF-β1 induced p21 expression and accelerating Erk signaling resulting in increased proliferation. Besides a pro-proliferative function, Nrf2 has been described to confer profound protection from apoptosis by e.g. upregulation of anti-apoptotic proteins such as bcl-2 or bcl-xL [
19,
20] or proteasomal genes such as s5a/psmd4 or α5/psma5 [
18]. Thus, elevated Nrf2 expression is one pivotal mechanism underlying chemoresistance of many tumors, e.g. PDAC [
18‐
21]. Accordingly, in this study the highest Nrf2 expression/activity could be correlated with the lowest apoptosis rate in Colo357 cells and
vice versa. However, overexpression or knock down of Nrf2 in pancreatic ductal epithelial cells only slightly modulated TGF-β1 mediated apoptosis induction. Thus, Nrf2 apparently enhances the growth of pancreatic ductal epithelial cells predominantly by antagonizing the proliferation inhibiting effect of TGF-β1. Since this phenomenon could be observed not only in malignant but also in premalignant and benign epithelial cells, one can speculate that the switch of Nrf2 from tumor suppressor to a tumor promoter might occur quite early in tumorigenesis and in particular in the concomitant presence of TGF-β1.