Cells deficient for Krüppel-like factor 4 exhibit mitochondrial dysfunction and impaired mitophagy

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Research paper

Cells deficient for Krüppel-like factor 4 exhibit mitochondrial dysfunction and impaired mitophagy William M. Rosencransa,b,1, Zachary H. Walsha,c,1, Nadia Houerbia, Andrew Bluma, Mezmur Belewa,d, Changchang Liua,e, Brian Chernaka,f, Philip R. Brauera,g, Angel Trazoa, Anna Olsona, Engda Hagosa,* a

Department of Biology, Colgate University, Hamilton, NY, 13346, USA Section on Molecular Transport, National Institute of Childhood Health and Human Development, Bethesda, MD, 20814, USA c Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, 80045, USA d Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90007, USA e Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA f SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA g School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Mitochondria Krüppel-like factor 4 Mitophagy Reactive oxygen species Bnip3

Krüppel-like factor 4 (Human Protein: KLF4; Human Gene: Klf4; Murine Protein: KLF4; Murine Gene: Klf4) is a zinc finger-containing transcription factor with diverse regulatory functions. Mouse embryonic fibroblasts (MEFs) lacking Klf4 exhibit genomic instability, increased reactive oxygen species (ROS), and decreased autophagy. Elevated ROS is linked to impairments in mitochondrial damage recovery responses and is often tied to disruption in mitochondrial-targeted autophagy known as mitophagy. In this study, we sought to identify a mechanistic connection between KLF4 and mitophagy. Using flow cytometry, we found that Klf4-null MEFs have diminished ability to recover mitochondrial health and regulate ROS levels after mitochondrial damage. Confocal microscopy indicated decreased localization of autophagy protein LC3 to mitochondria following mitochondrial damage in Klf4-null cells, suggesting decreased mitophagy. Western blotting and RT-PCR revealed decreased mRNA and protein expression of the mitophagy-associated protein Bnip3 and antioxidant protein GSTα4 in Klf4-null cells, providing a rationale for their impaired mitophagy and ROS accumulation. Inducing Bnip3 expression in these cells recovered mitophagy but did not decrease ROS accumulation. Our findings suggest that in Klf4-null cells, decreased Bnip3 expression impairs mitophagy and is associated with increased mitochondrial ROS production after mitochondrial damage, providing a rationale for their genomic instability and supports a tumor suppressive role for KLF4 in certain tumors as previously observed.

1. Introduction Krüppel-like factor 4 (KLF4) belongs to the Krüppel-like factor family of zinc-finger transcription factors and has diverse regulatory functions in cellular proliferation, differentiation, apoptosis, invasion and migration (Brauer et al., 2018; Garrett-Sinha et al., 1996; Shields et al., 1996). In many human cancers, KLF4 is regarded as a tumor suppressor. Its expression is downregulated in pancreatic, lung, colorectal, gastric, and prostate cancers (Hu et al., 2009; Wang et al., 2010; Wei et al., 2005; Wrzosek et al., 2013; Zammarchi et al., 2011). Previous research in our laboratory demonstrated that knockdown of Klf4 in mouse embryotic fibroblasts (MEFs) was associated with increased

reactive oxygen species (ROS), DNA damage, and overall genomic instability (El-Karim et al., 2013; Hagos et al., 2009; Liu et al., 2015b). ROS are a class of damaging chemicals produced during cell metabolism, mainly in the mitochondria, known to cause DNA damage and disrupt membrane integrity within cells (Ames et al., 1993; Gores et al., 1989; Zorov et al., 2000). ROS released by mitochondria can subsequently damage other mitochondria with in the cell and cause a chain reaction of ROS production known as ROS induced ROS release (RIRR) (Zorov et al., 2000). Despite ample contextual evidence of KLF4’s role in cancer development, the specific molecular mechanisms targeted remain unclear. One possible mechanism that we have previously investigated is

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Corresponding author at: Department of Biology, Colgate University, 13 Oak Drive, Hamilton, NY 13346-1398, USA. E-mail address: [email protected] (E. Hagos). 1 Co-First authors, equal contribution to this work. https://doi.org/10.1016/j.ejcb.2019.151061 Received 29 July 2019; Received in revised form 9 November 2019; Accepted 28 November 2019 0171-9335/ © 2019 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: William M. Rosencrans, et al., European Journal of Cell Biology, https://doi.org/10.1016/j.ejcb.2019.151061

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Fig. 1. KLF4 increases the ability of cells to recover from mitochondrial damage. (A) MEFs were treated with a CCCP recovery assay. Cells were treated with either DMSO or CCCP for 12 h and then collected, and a third group was allowed 24 h to recover after CCCP treatment (CCCP Recovery). N = 3. (B) RKO cells were grown for 72 h in DMEM containing either PA or EtOH control, then treated with a CCCP recovery assay. All cells in A and B were stained with TMRE and analyzed using flow cytometry. N = 3. (C) MEFs were treated with a CCCP recovery assay. All cells were stained with Mitotracker Deep Red and imaged with confocal microscopy. (D) Quantification of mitochondrial network size. N = 3 (**** P < 0.0001, *** P < 0.001, ** P < .01, * P < .05).

indicator of mitochondrial health (Liao et al., 2015; Wang et al., 2018). A possible mediator of KLF4 regulation of mitochondrial homeostasis is Bnip3 (Bcl-2/E1B-19K-interacting protein 3). Bnip3 is a crucial mitophagy related protein (MRP) that is involved in recruiting mitochondria for destruction by autophagosomes (Springer and Macleod, 2016; Tracy et al., 2007). Bnip3 attaches to damaged mitochondria, and through binding to LC3, targets the mitochondria for destruction via autophagy (Hanna et al., 2012). More recently, it has shown that phosphorylation of Bnip3’s LC3 binding domain (LIR), which is regulated by ROS levels and other apoptotic proteins, activates mitophagy (Choe et al., 2015). Bnip3 is considered a tumor suppressor in several cancer types (Chourasia et al., 2015a; Murai et al., 2005). Deficiencies in Bnip3 result in reductions in mitophagy, impaired mitochondrial function, impaired mitochondrial energetics, and ROS production (Chourasia et al., 2015b; Glick et al., 2012). In induced

KLF4’s regulation of autophagy (Liu et al., 2015a). Autophagy is a conserved intracellular process that involves the degradation of cytoplasmic components inside lysosomes (Yang and Klionsky, 2010). It is induced in response to stress and nutrient deprivation and preserves cellular homeostasis by removing and recycling misfolded proteins and damaged organelles (Yang and Klionsky, 2010). Autophagy specifically targeted to mitochondria is known as mitophagy (Kim et al., 2007). Impairments in mitophagy are associated with a wide variety of diseases including Parkinson’s and cancer (Deas et al., 2011; Springer and Macleod, 2016). Furthermore, dysregulation of mitophagy results in accumulation of impaired mitochondria that are known to increase DNA damage (Kurihara et al., 2012). KLF4 has been demonstrated to regulate mitochondrial homeostasis in myocytes and regulate mitochondrial health in glioblastoma. In these studies, KLF4 was also shown to positively correlate with increased respirational capacity, an

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Fig. 2. Klf4-null MEFs exhibit reduced levels of mitophagy after mitochondrial damage. (A) Wild-type and Klf4-null MEFs were treated with a CCCP recovery assay and analyzed for TOM20 using western blotting. (B) Quantification of TOM20 protein expression in (A). N = 3. (C) Wild-type and Klf4-null MEFs were transfected with either GFP or LC3-GFP. All cells were stained with Mitotracker then treated with CCCP in conjunction with the latestage autophagy inhibitor BafA for 6 h. Localization of the GFP tag was compared with localization of the mitochondria. (D) Magnification of cells from each group showing LC3 foci. White arrows indicate LC3 foci with high mitochondrial colocalization, indicating mitophagy. Pink arrows indicate foci with low relative mitochondrial colocalization. (E) Weighted colocalization coefficient quantification of GFP with Mitotracker. In each group, at least 30 cells were analyzed per experimental replicate. N = 3 (*** P < .001).

2. Results

pluripotent stem cells (iPSCs), where KLF4 is one of the reprogramming factors, the Bnip3 family member NIX (Bnip3L) was shown to be activated and induce mitophagy during the reprogramming process (Xiang et al., 2017). In this study, we sought to understand the role of KLF4 in maintenance of mitochondrial health, mitophagy, and ROS production. We found that KLF4 plays an integral role in the ability of cells to recover from mitochondrial damage. We demonstrated that one mechanism through which KLF4 performs this function is by promoting the transcription of Bnip3, a protein which targets autophagosomes to damaged mitochondria, facilitating mitophagy. Cells lacking Klf4 showed reduced mitochondrial recovery, as well as impaired mitophagy, which could be improved by inducing expression of Bnip3. Inability of Klf4null MEFs to recover following mitochondrial damage led to further increased levels of ROS. We further showed that increased ROS in the absence of Klf4 may also be caused in part by the decreased transcription and expression of an antioxidant protein, GSTα4.

2.1. KLF4 increases the ability of cells to recover from mitochondrial damage First, we evaluated the ability of Klf4-deficient cells to recover from mitochondrial damage. By treating cells with the electron transport chain (ETC) uncoupling drug CCCP, we damaged mitochondria in order to induce mitochondrial stress (Georgakopoulos et al., 2017). We treated cells with CCCP or DMSO control for 12 h and analyzed these groups of cells immediately following treatment. In another group, referred to as CCCP recovery, we washed cells after a 12 -h CCCP treatment and allowed 24 h of recovery time before analysis. Moreover, to determine mitochondrial damage, we assessed mitochondrial membrane potential (MMP) using TMRE, a dye which is sequestered only in healthy, polarized mitochondria, in conjunction with flow cytometry (Perry et al., 2011). We also assessed the expression kinetics of KLF4 with DMSO, CCCP, and CCCP Recovery treatment (Fig. S3). Both wildtype and Klf4-null MEFs exhibited similar levels of basal TMRE 3

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Fig. 3. KLF4 regulates the expression of the mitophagy-associated protein Bnip3. (A) MEFs were treated with a CCCP recovery assay and analyzed for Bnip3 expression via western blotting. (B) Quantification of (A), protein expression has been normalized to the WT DMSO condition, N=5. (C) MEFs were treated with a CCCP recovery assay and analyzed for NIX expression using western blotting. (D) Quantification of NIX expression in (C). N = 3. (E) MEFs were treated with either DMSO or CCCP in conjunction with BafA for 6 h, then collected for western blot analysis of Bnip3 expression. (F) Wild-type and Klf4-null MEFs were transfected with either GFP or Klf4-GFP and analyzed for Bnip3 expression using western blotting. (G) Quantification of Bnip3 expression in (F), N = 3. (H) Wild-type and Klf4-null MEFs were transfected with either GFP or Klf4-GFP and analyzed for Bnip3 mRNA expression, N = 3. (I) RKO cells were treated in either PA or EtOH control for 72 h and then analyzed for Bnip3 expression using western blotting. (J) Quantification of Bnip3 expression in (I). N = 5. (K) qRT-PCR was used to assess Bnip3 mRNA levels in PA- and EtOH-treated RKO cells, N = 3. (**** P < 0.0001, *** P < 0.001, ** P < .01, * P < .05). For all western blots, cropped images are represented for clarity in figures. The crop is delineated by a black border. Full, uncropped images of each blot are included in supplemental data.

fluorescence (Fig. 1A, S2A). CCCP treatment reduced the fluorescence of TMRE in both cell types, confirming that TMRE fluorescence was reduced in non-polarized mitochondria (Fig. 1A, S2A). However, only wild-type MEFs were able to recover MMP within 24 h after removal of

CCCP, while Klf4-null MEFs were not (Fig. 1A, S2A). To understand the effect of KLF4 overexpression on MMP recovery in transformed human cells, we repeated the CCCP recovery assay and TMRE analysis in the RKO-EcR-KLF4 cell line (further referred to as 4

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2.3. KLF4 regulates the expression of the mitophagy-related protein Bnip3

RKO), a human colorectal cancer cell line in which KLF4 overexpression can be induced by treating with the insect hormone Ponasterone-A (PA), and is known to reduce tumorigenicity (Dang et al., 2003). We first validated that PA treatment induces Klf4 expression in RKO-EcRKLF4 cells, using ethanol (EtOH) treatment as the vehicle control (Fig. S1C). Next, we found that while RKOs treated with EtOH and PA were both able to recover mitochondrial membrane potential following CCCP treatment, cells treated with PA demonstrated an accentuated recovery, resulting in MMP that was significantly greater than basal levels (Fig. 1B, S2B). Previously it has also been reported KLF4 is important in mitochondrial network formation, a process which has been shown to be important in maintaining mitochondrial health (Wang et al., 2018). We sought to determine if KLF4 played a role in modifying mitochondrial networking in response to CCCP-induced mitochondrial damage. Using Mitotracker Deep Red dye, which is permanently sequestered in polarized mitochondria, we assessed the extent of mitochondrial networking morphology during the CCCP treatment and recovery process (Valente et al., 2017). Consistent with previous studies, we found that Klf4-null MEFs exhibit significantly smaller average mitochondrial network sizes, as quantified by average number of mitochondria per network, when compared to wild-type MEFs (Fig. 1C–D) (Wang et al., 2018). Upon addition of CCCP, wild-type MEFs exhibited a more fragmented morphology with a smaller average network size, while Klf4null cells showed no significant change. After recovery, wild-type cells average network returned to pre-CCCP morphology, while Klf4-null MEFs remained fragmented (Fig. 1C–D).

We next sought to determine a molecular mechanism to connect KLF4 to mitophagy. Previous studies have shown that deletion of KLF4 does prevent expression of the mitophagy protein PINK1 (Liao et al., 2015). We confirmed these findings in MEFs using western blotting (Fig. S4A). Bnip3 is an alternative MRP that functions independently of PINK1 and Parkin by directly recruiting LC3 to the mitochondria (Kubli et al., 2015). Reductions in Bnip3 expression could therefore explain impairments in mitophagy despite the preservation of PINK1 signaling in Klf4-null MEFs. Using western blotting, we found a significant reduction in basal expression of Bnip3 in Klf4-null MEFs (Fig. 3A–B). After CCCP treatment, the expression of Bnip3 was significantly reduced in wild-type cells but returned to basal levels after a recovery period. Klf4-null MEFs did not demonstrate flux in Bnip3 in response to CCCP treatment and recovery (Fig. 3A-B). These findings were consistent when the recovery assay was performed using serum starvation instead of CCCP treatment, and we observed a similar trend with expression of the autophagy-related protein ULK1 (Fig. S4B–C). We hypothesized that the reduction in Bnip3 expression in wild-type MEFs resulted from Bnip3 being cleared along with damaged mitochondria during mitophagy. To evaluate this, we examined Bnip3 expression when co-treating MEFs with the late-stage autophagy inhibitor BafA. We found that the depletion of Bnip3 observed in wildtype MEFs after CCCP treatment (Fig. 3A) was inhibited by BafA cotreatment (Fig. 3E). Wild-type MEFs which were treated with CCCP but did not receive BafA (Fig. 3A) served as an appropriate control for the CCCP-BafA co-treatment. This finding indicated that loss of Bnip3 in these cells during CCCP treatment was specifically due to removal by mitophagy. We also tested for differences in expression of the Bnip3 family protein NIX, which is known to play a similar role to Bnip3 in mediating mitophagy (Fig. 3C–D) (Zhang and Ney, 2009). Western blotting for NIX during the CCCP recovery assay revealed slightly lower basal levels of NIX in Klf4-null MEFs. After CCCP treatment, NIX expression in wildtype cells decreased to 25 % of the original levels, whereas an insignificant drop in NIX was observed in the Klf4-null MEFs. After the recovery period, NIX levels returned to baseline in both the wild-type and Klf4-null MEFs (Fig. 3C–D). To determine whether KLF4 regulates Bnip3 expression at the transcriptional level, we used two-step quantitative RT-qPCR. We found that Bnip3 mRNA expression was decreased more than two-fold in Klf4null MEFs (Fig. 3H), suggesting that KLF4 acts as a positive transcriptional regulator of Bnip3. Next, we sought to determine whether introduction of GFP-tagged KLF4 to Klf4-null MEFs could drive increased Bnip3 protein expression and transcription. The success of Klf4-GFP transfection was confirmed by the appearance of the KLF4-GFP fusion protein at the appropriate molecular weight (Fig. S1B), and by RT-qPCR for Klf4 (Fig. S1E). After the addition of Klf4-GFP to Klf4-null MEFs, Bnip3 protein expression was significantly increased compared to cells transfected with GFP alone, suggesting that KLF4 expression induces expression of Bnip3 (Fig. 3F–G). Furthermore, Klf4-GFP transfection led to a slight, but non-significant increase in Bnip3 mRNA expression in Klf4-null MEFs when compared to the GFP-transfected control (Fig. 3H). While Bnip3 transcription and protein expression in Klf4-GFP transfected Klf4-null MEFs increased compared to control GFP-transfected cells, expression levels were not fully recovered to those of wildtype MEFs (Fig. 3F–H). Low transfection efficiency of the Klf4-GFP construct, as indicated by fluorescence microscopy, provided a rationale for this partial recovery (Fig. S1D). Next, we sought to generalize our findings by investigating the expression of Bnip3 in KLF4-inducible RKO human colorectal cancer cells. We found that with three days of exposure to PA, which induced KLF4 expression, RKO cells demonstrated increased Bnip3 protein expression by approximately 50 % (Fig. 3I–J), as well as increased Bnip3 mRNA expression (Fig. 3K), further supporting the role of KLF4 as a regulator

2.2. Klf4-null MEFs exhibit reduced levels of mitophagy after mitochondrial damage We next sought to evaluate whether KLF4 could facilitate mitochondrial recovery following stress by inducing mitophagy. As mitophagy is characterized by degradation and recycling of mitochondria, we examined flux of total mitochondrial mass during the CCCP stress recovery assay by assessing expression of the mitochondrial import receptor protein TOM20. TOM20 expression is an established surrogate for cellular mitochondrial mass (Baldelli et al., 2014). We found that wild-type MEFs exhibited a significant decrease in TOM20 expression following CCCP treatment, while Klf4-null MEFs demonstrated a nonsignificant increase (Fig. 2A–B). This finding suggested that stress-induced mitochondrial degradation or recycling was impaired in MEFs lacking Klf4. Inability of Klf4null MEFs to decrease mitochondrial mass in response to CCCP treatment or recover mitochondrial membrane potential following a recovery period suggested that they may have impairments in mitophagy. Thus, we sought to specifically determine if mitophagy was reduced in Klf4-null MEFs following mitochondrial damage. To assess this, we first transfected cells with either GFP or LC3-GFP plasmids. LC3 is known to sequester cellular components to the autophagosome, and thus LC3 foci indicate the presence of localized autophagy (Kabeya et al., 2000). We then treated MEFs with CCCP in conjunction with Bafilomycin A (BafA), a drug which blocks the last phase of autophagy, thereby protecting the LC3-GFP fusion protein from autophagosome degradation (Ding and Yin, 2012). Finally, we assessed LC3 colocalization with mitochondria as a marker of mitochondrial-directed autophagy. In MEFs transfected with GFP control alone, GFP fluorescence was found to be diffuse throughout the entire cell (Fig. 2C). In LC3-GFP transfected MEFs, GFP foci were observed in both cell types, indicating the presence of autophagosomes (Fig. 2C). Co-localization of LC3-GFP with mitochondria increased in both cell types compared to those transfected with only GFP. However, overlap of LC3-GFP was significantly greater in wild-type MEFs than in Klf4-null MEFs (Fig. 2D–E). This finding demonstrates reduced levels of mitophagy in the absence of Klf4.

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Fig. 4. Addition of Bnip3 to Klf4-null cells rescues defects in mitophagy. (A) MEFs were transfected with either GFP control or Bnip3-myc, treated with BafA for 12 h, and then analyzed for Bnip3 and LC3 II/I expression using western blotting. (B) Quantification of ratio of LC3-II to LC3-I in (A). N = 3. (C) MEFs were either Bnip3 or mock transfected and then subjected to CCCP and BafA treatment for 6 h. Localization of LC3 was then compared with localization of mitochondria using immunofluorescence microscopy. (D) Magnified regions of images in C. White arrows denote regions of LC3-mitochondrial overlap, indicating mitophagy. (E) Analysis of microscopy data in C. Manders overlap coefficient of LC3 and Mitotracker Deep Red was measured in Klf4-null cells which were either Bnip3 or mock transfected. In each group, at least 0 cells were analyzed per experimental replicate. N = 3 (** P < 0.01).

Bnip3-Myc, Bnip3 localized to mitochondria and LC3 aggregated into intense foci localized with the Myc tag (Fig. 4C-D). To quantitatively measure the frequency of mitophagy, we determined the Manders overlap coefficient between LC3 and mitochondria (Fig. 4E). In mock transfected cells, the LC3-mitochondria overlap coefficient was consistent with the overlap previously measured in Klf4-null MEFs transfected with LC3-GFP (Fig. 2C). In Bnip3-Myc transfected cells, the overlap coefficient between LC3 and mitochondria increased significantly, confirming that Bnip3 can rescue mitophagy defect in Klf4null cells (Fig. 4E). Bnip3-Myc transfection was also performed on wildtype cells and drove similar increases in LC3-mitochondria colocalization, confirming that Bnip3-mediated mitophagy induction was not unique to Klf4-null cells (Fig. S5).

of Bnip3. 2.4. The addition of Bnip3 to Klf4-null MEFs rescues defects in mitophagy Since we find that Bnip3 expression was reduced in Klf4-null MEFs, we sought to determine if the addition of Bnip3 to Klf4-null MEFs could rescue their observed defects in mitophagy. To test this, we transfected Klf4-null MEFs with either Bnip3-Myc or GFP control in conjunction with BafA treatment to prevent autophagy-related protein removal. Western blotting for both Bnip3 and the Myc tag demonstrated the success of the transfection (Fig. 4A). We first evaluated the expression of the autophagy protein LC3 in MEFs treated with BafA to determine whether Bnip3 transfection impacted global autophagy in Klf4-null MEFs. LC3 I is converted to LC3 II when autophagy is active (Klionsky et al., 2016). A small, but insignificant increase in LC3 II to LC3 I ratio was observed following Bnip3-Myc transfection (Fig. 4B). Next, we evaluated whether Bnip3-Myc transfection could specifically induce mitophagy in Klf4-null MEFs treated with CCCP and BafA using immunostaining (Fig. 4C). In mock-transfected cells, LC3 expression was diffuse and few foci were visible. In cells positive for

2.5. KLF4 regulates ROS production through mitochondrial damage control and GSTα4 Finally, we sought to determine if defects in mitochondrial recovery following stress contributed the high levels of ROS observed in Klf4-null cells. We used flow cytometry and immunofluorescence microscopy 6

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Fig. 5. KLF4 regulates ROS production through mitochondrial damage control and antioxidant protein expression. (A) MEFs were treated with a CCCP recovery assay, stained with H2DCF-DA, and analyzed using flow cytometry. N=4. (B) DCF fluorescence was measured in MEFs using flow cytometry following transfection with mock or Klf4. N=3 (C) Klf4-null MFEs were either Bnip3 or mock transfected, then treated with a CCCP recovery assay, then stained with H2DCF-DA and analyzed using flow cytometry. N = 3. (D) GSTα4 mRNA expression in MEFs was assessed using qRT-PCR. N = 3. (E) Western blot of GSTα4 and KLF4 expression in RKO cells treated with either PA or EtOH control. (F) Western blot of GSTa4 expression in MEFs during CCCP recovery assay. (**** P < 0.0001, *** P < 0.001, ** P < .01, * P < .05).

reduce ROS production. Success of the KLF4 transfection was confirmed using western blotting (S1A). We found that transfection of Klf4 reduced ROS production in the Klf4-null cells (Fig. 5B). To determine whether KLF4 regulated ROS levels in a Bnip3-dependent manner, we added Bnip3 to Klf4-null MEFs and examined ROS production. Addition of Bnip3-myc to Klf4-null MEFs elevated ROS levels at basal conditions (Fig. 5C). Following CCCP recovery, both Bnip3 and mock transfected cells demonstrated increased ROS production compared to basal and CCCP-treated cells. Because Bnip3 did not reduce ROS during CCCP recovery, we investigated whether another Klf4 target, the antioxidant gene Gstα4,

with the H2DCF-DA ROS stain to analyze the production of ROS in a CCCP recovery assay. Klf4-null MEFs demonstrated higher basal levels of ROS than wild-type MEFs, consistent with our previously published research (Fig. 5A, S6) (Liu et al., 2015b). After CCCP treatment, ROS increased in the wild-type MEFs, suggesting that damaged mitochondria were producing elevated levels of ROS (Fig. 5A, Fig. S6). Interestingly, Klf4-null cells treated with CCCP exhibited a drop in ROS production. After recovery, wild-type ROS levels remained constant while Klf4-null MEFs demonstrated an increase in ROS production (Fig. 5A). We next evaluated whether addition of Klf4 to Klf4-null MEFs would 7

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Interestingly, while LC3 foci were clearly observed aggregating with the Bnip3-Myc tag, only slight increases in the LC3 II/I ratio were observed via western blotting (Fig. 4A–B). We interpret this finding to indicate that Bnip3 is only partially able to increase the rate of general autophagy in Klf4-null MEFs, but can direct activated LC3 II to the mitochondria, increasing the rate of mitophagy. It is possible that ATG proteins such as ATG7 are necessary to fully recover autophagy in Klf4null MEFs, which may also be evaluated in future experiments (Liu et al., 2015a). Interestingly, when examining ROS production, we found that the burst of ROS seen in wild-type MEFs following CCCP treatment was not seen in Klf4-null MEFs (Fig. 5A). A similar result has been observed in cells lacking NIX, a protein closely related to Bnip3, wherein CCCP failed to initiate a ROS burst and mitochondrial depolarization (Ding et al., 2010). Likewise, lack of Bnip3 in Klf4-null MEFs could prevent the CCCP-induced ROS burst. Interestingly, the increase of ROS after mitochondrial damage is thought to drive the activation of mitophagy and autophagy in a negative feedback loop (Scherz-Shouval and Elazar, 2011). Bnip3, like NIX, has long been known to induce increased ROS upon expression (Velde et al., 2000). Because NIX levels do not vary significantly between the two cell types, lack of Bnip3 could explain the absence of a ROS burst in Klf4-null MEFs (Xiang et al., 2017) (Fig. 3D). Indeed, transfecting Klf4-null MEFs with Bnip3-myc led to an increase of basal ROS and increased ROS production upon CCCP treatment (Fig. 5C). The large increase in ROS production observed after recovery in Klf4-null MEFs suggests that damaged mitochondria could not be cleared by mitophagy, resulting in ROS accumulation (Fig. 5A) which could not be rescued by Bnip3-myc transfection (Fig. 5C). This finding suggests that other proteins regulated by KLF4 are likely also involved in mitigating ROS accumulation. Our previous work suggested that the antioxidant protein GSTα4 could be involved in KLF4-mediated regulation of ROS, and showed decreased Gsta4 mRNA in Klf4-null MEFs (Liu et al., 2015b). GSTα4 can target the mitochondria to prevent oxidative damage (Haider et al., 2002; Raza, 2011). Impairments in GSTα4 have been previously shown to increase levels of mitochondrial ROS and impair mitochondrial function (Curtis et al., 2010; Yang et al., 2008). Greater levels of GSTα4 in wild-type MEFs, RKOs treated with PA, and increased Gstα4 mRNA in Klf4-null MEFs transfected with Klf4, confirm this regulation (Fig. 5D–F). However, the similar expression pattern during CCCP treatment and recovery across wild-type and Klf4-null MEFs suggests that KLF4-mediated regulation of GSTα4 does not fully explain differences in ROS production during mitochondrial damage (Fig. 5F). Instead, decreased Bnip3 expression and subsequent failure to recycle damaged mitochondria may be the dominant contributor to aberrant ROS production observed in Klf4-null MEFs under mitochondrial stress. Importantly, while our study provides strong evidence that KLF4 regulates ROS production and DNA damage through induction of GSTα4 expression, Bnip3 expression, and mitophagy, a majority of our findings have been demonstrated in mouse embryonic fibroblasts rather than in transformed human cancer cells. To address this, we extended our study to human colorectal cancer, demonstrating that KLF4 overexpression also drives increased Bnip3 and GSTα4 expression in RKO human colorectal cancer cells (Figs. 3 and 4). These promising findings, along with other recent studies identifying KLF4 as a mitophagy regulator in other human cell types suggest that loss of KLF4 expression may drive impaired mitophagy and increased ROS levels in human cancer (Liao et al., 2015). However, it will be imperative for future studies to assess a broad panel of human cancers and cell lines in order to confirm this mechanism in a broader range of human cancers.

could play a role in reducing ROS during mitochondrial recovery. Klf4null MEFs have previously been shown to have significantly decreased Gstα4 mRNA expression (Liu et al., 2015b). GSTα4 is known to reduce the levels of ROS with the cell and prevent against oxidative damage to cellular components (Yang et al., 2008). We found that reintroduction of Klf4 led to a two-fold increase in Gstα4 expression in Klf4-null MEFs (Fig. 5D). Moreover, the presence of KLF4 in both MEFs and RKO cells notably increased GSTα4 protein expression at basal conditions (Fig. 5E–F). To determine whether GSTα4 expression fluctuated during mitochondrial stress, we analyzed MEFs subjected to a CCCP recovery assay for GSTα4 protein expression using western blotting. After the addition of CCCP, GSTα4 levels decreased in both cell types and remained consistently low following a recovery period (Fig. 5F). 3. Discussion In this study, we investigated the role of KLF4 in regulating mitochondrial health and recovery, mitophagy protein expression, and ROS production. We found that Klf4 expression increased mitochondrial networking and was necessary for the recovery of mitochondrial membrane potential following metabolic stress (Fig. 1). We then identified impaired mitophagy in cells lacking Klf4 as a mechanism for their diminished mitochondrial health (Fig. 2B–C). To further define a mechanism by which KLF4 directly contributes to mitophagy, we identified decreased transcription and protein expression of the mitophagyrelated protein Bnip3 in Klf4-null cells (Fig. 3A) and found that introduction of Bnip3 into Klf4-null cells rescued defects in mitophagy (Fig. 4). Finally, we found decreased transcription and protein expression of the antioxidant protein GSTα4 in Klf4-null cells, suggesting that KLF4 may regulate ROS production not only through facilitating mitochondrial recycling, but also by promoting cellular antioxidant activity (Fig. 5). Mitochondrial phenotypes observed in Bnip3-deficient cells are consistent with those observed in our study, including dysfunctional bioenergetics, impaired mitochondrial fragmentation after mitochondrial damage, and impaired mitophagy (Chourasia and Macleod, 2015; Chourasia et al., 2015b). Furthermore, it has been shown previously that colorectal cancers have low basal levels of Bnip3 expression (Murai et al., 2005). The low levels of endogenous KLF4 expression in colorectal cancers, could explain the low expression of Bnip3 (Choi et al., 2006). This is corroborated by our finding that inducing increased KLF4 expression in RKO cells subsequently increased Bnip3 expression (Fig. 3I–J). Our RT-qPCR results indicate that KLF4 is also connected, either directly or indirectly, to transcriptional regulation of Bnip3 (Fig. 3H, K). This regulation presents a mechanism for the impairments in mitochondrial health, mitophagy, and ROS production observed in cells lacking functional Klf4. Furthermore, Bnip3 also plays a role in regulating mTOR by inactivating the upstream activator of mTOR, RHEB, providing another explanation for the regulation of mTOR by KLF4 (Li et al., 2007; Liu et al., 2015a). A 2008 study by Chen et al. used ultrahigh-throughput ChIP-seq to map the locations of transcription factor binding sites, including KLF4, in mouse embryonic stem cells. In this database, they showed a KLF4 binding site at the proximal promoter of Bnip3 (Chen et al., 2008). Alternatively, KLF4 could regulate Bnip3 at the transcriptional level through its activation of the transcription factor p21, a long-known primary target of KLF4 (Zhang et al., 2000) (Gartel and Tyner, 2002). p21 has a diverse set of transcriptional targets and was recently shown to activate Bnip3 expression as well as general autophagy signaling (Capparelli et al., 2012; Manu et al., 2017). In future research, we intend to determine whether KLF4 control of Bnip3 expression is direct or p21-dependent. The addition of Bnip3 to Klf4-null cells treated with CCCP and BafA demonstrated striking increases in LC3 foci formation and mitochondrial localization, indicating increased mitophagy (Fig. 4C–E).

4. Conclusions Together, our findings establish a specific mechanism for KLF4’s role in maintaining cellular integrity, and how cellular dysfunction arises in its absence. In the absence of Klf4, the expression of GSTα4 and 8

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Fig. 6. A model for how Klf4 deficiency causes mitochondrial impairment and ROS accumulation (A) In the presence of Klf4 the transcription of Bnip3 and GSTα4 is increased. Bnip3 binds to damaged mitochondria and targets them for destruction via mitophagy. Autophagy is activated by the increased presence of ULK1, known to be transcribed by Klf4 (Liao et al., 2015). ATG7, also thought to be transcribed by Klf4, induces autophagy by activating LC3 (Liu et al., 2015a). LC3 can bind to Bnip3 and sequester mitochondria for destruction. Furthermore, the cell is protected from accumulated ROS by the antioxidant GSTα4. (B) Without Klf4, Bnip3 and GSTα4 expression is significantly reduced. Damaged mitochondria are allowed to accumulate within the cell due to impairments in mitophagy and autophagy. The buildup of ROS causes DNA damage and increased mitochondrial dysfunction. ROS induced ROS release causes a snowball effect of continued mitochondrial damage (Zorov et al., 2000). Without Klf4, increased ROS production and impaired mitophagy may lead to genomic instability.

induce autophagy. BafA, purchased from Sigma Aldrich (St. Louis, MO), used to inhibit the last stage of autophagy.

Bnip3 are reduced (Figs. 3 and 5). Furthermore, the expression of ATGs ULK1 and ATG7 is decreased (Fig. S4) (Liao et al., 2015; Liu et al., 2015a). As a result, ROS levels are increased, leading to impaired mitochondria, and ultimately oxidative DNA damage (Liu et al., 2015b; Zorov et al., 2000). Damaged mitochondria then accumulate due to reduced transcription of Bnip3 and impaired mitophagy. In an ongoing cycle of cellular dysfunction, the DNA damage incurred as a result of antioxidant and mitochondrial dysfunction can cause genomic instability that previously reported in cell lacking Klf4. (Fig. 6). Together, these findings provide a broader, more compelling explanation for the role of KLF4 as a tumor suppressor.

5.2. Western blot analysis For all western blots, cropped images are represented for clarity in figures. The crop is delineated by a black border. Full, uncropped images of each blot are included in supplemental data. Proteins were extracted and analyzed using Western blot as previously described (Liu et al., 2015a). Briefly, the membranes were immunoblotted with the following primary antibodies against: β-Actin, TOM20, KLF4, GAPDH, NIX (Cell Signaling, Danvers, MA), Bnip3, PINK1 (Santa Cruz Biotechnology, Dallas TX, USA), ULK1 (ThermoFisher, Waltham, MA), GSTα4 (Abcam, Cambridge, U.K) LC3 (Proteintech, Rosemont, IL). The blots were then incubated with the appropriate horseradish peroxidaseconjugated secondary antibodies for 1 h at room temperature. Antirabbit and anti-mouse secondary antibody were purchased from Cell Signaling (Danvers, MA, USA) and Abcam (Cambridge, MA, USA), respectively. The antibody-antigen complex was visualized by an ImmunStar™ HRP Chemiluminescence Kit and ChemiDoc™ XRS + System (BioRad Laboratories, Hercules, CA). The intensities of the bands were quantified using volume tools in Gel Imager program by normalizing the band intensity of protein of interest to that of control proteins βactin or GAPDH. For each replicate, all conditions were normalized to a reference condition whose value was scaled to one.

5. Methods 5.1. Cell culture, reagents and drug treatments Previously, mice heterozygous for the Klf4 alleles (Klf4+/+) on a C57BL/6 background were crossbred, and MEFs that are wild-type (Klf4+/+), heterozygous (Klf4+/−), or null (Klf4-/-) for Klf4 were derived from day 13.5 embryos (Katz et al., 2002). In the present study, no experiments were performed with live vertebrates. Klf4+/+ and Klf4-/- MEFs were cultured in DMEM supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin at 37 °C in 5 % CO2. Cells were passaged every 3 days at a density of 106 cells per 10-cm dish following the 3T3 protocol. All MEFs experiments performed were spontaneously immortalized primary MEFs post-senescence after passage 20 at 50–70 % confluence. The RKO (RKO-EcR-KLF4) cell line was derived from a human colon cancer cell line and stably transfected with the pAdLoxEGI-KLF4 plasmid, as previously described (Chen et al., 2001). The plasmid contains the ecdysone-inducible promoter (EcRE), which is not naturally expressed by the human cell line, and a fulllength KLF4 gene that is naturally expressed at lower levels (Dang et al., 2001). Therefore, KLF4 is conditionally expressed in RKO cells via the addition of Ponasterone-A (PA) (Sigma-Aldrich, St. Louis, MO, #P3490) and EtOH served as the solvent control(Dang et al., 2003). Cells were grown to appropriate confluency (40–50 %) and then treated with 5 μM PA in EtOH for 3 days. CCCP (Enzo biochem Inc, Farmingdale, NY) at 25 u M in DMSO (Sigma Aldrich St. Louis, MO) was used to artificially induce mitochondria de-polarization and induce mitophagy. Recovery assays were performed by treating the cells for 12 h followed by a DPBS wash and recovery in normal DMEM for 24 h. EBSS (E2888) and Rapamycin was purchased from Sigma Aldrich (St. Louis, MO) and used to

5.3. Quantitative real-time PCR analysis For the examination of GSTα4 and Bnip3 via RT-qPCR, total RNA was isolated using RNeasy® Mini Kit (Qiagen, Germantown, MD) by following the manufacturer’s protocol. Synthesized cDNA was subjected for RT-qPCR analysis using SYBR® Green PCR Master Mix for 40 cycles (Life Technologies, Carlsbad, CA) following manufacturer’s protocol. The expression of both Bnip3 and GSTα4 was normalized to the expression level of β-Actin. Experiments were performed with three biological replicates each in triplicate. PCR reactions were performed using the following primers purchased from Integrated DNA Technologies (Coralville, IA). MBnip3-F: 5′ ATT CCC AGC CCCT TTC TCT TC 3’ MBnip3-R: 5’ CTA ACA CAG ACC AGA AGC CTA 3’; GSTα4-F: 5’ TCA AAC TCC ACT CCA GCC G 3’; GSTα4-R: 5’ CTC GAG TGC CTG GAG ACA A 3’; 3’; β-Actin-F: 5’ ATG GAG GGG AAT ACA GCC C 3’; β-ActinR: 5’ TTC TTT GCA GCT CCT TCG TT 3’. HKLF4-F: 5'-CCA CCG GCC 9

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immunostaining was calculated using the Student’s unpaired t-test [P ≥ 0.05 (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001]. Error bars represent +/- SEM of data.

GGC TGC ACA CGA CT; HKLF4-R: 5'-TCA TCT GAG CGG GCG AAT TTC CAT CCA; HBNIP3-F:5'-ACC AAC AGG GCT TCT GAA AC; HBNIP3-R:5'GAG GGT GGC CGT GCG C. RT-PCR fold changes were calculated using 2^-(deltaCt), where deltaCt is defined as (Ct-Experimental - Ct-Control).

Data availability 5.4. Flow cytometry The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Flow cytometry was used to quantify mitochondrial membrane potential via TMRE (Abcam, Cambridge, UK) and ROS production using CM-H2DCF-DA (Molecular Probes by Life Technologies, Grand Island, NY, USA). Cells were plated in 12-well plates at 105 cells/well. Various treatments were then administered. After treatment, cells were rinsed in PBS, trypsinized, and pelleted. Cells were stained with 200 nM TMRE or 10 μM CM-H2DCF-DA for 30 min. Cells were then washed 3 times with warm PBS and finally suspended in a solution of 1 % BSA in warm PBS. The cells were than analyzed for fluorescence with a BD AccuriTM D Biosciences C6 Flow Cytometer (San Jose, CA, USA). Cellular debris was gated out of the analysis using forward and side scatter. For microscope confirmation of dye cells were instead stained with Hoechst and imaged with an Axio Observer A1 microscope (Carl Zeiss, Thornwood, NY).

Author contributions WR and ZW carried out all the experimental studies, participated in the design of the study, and drafted the manuscript. NH participated in the design of the study, qRT-PCR, immunostaining, western blotting, and statistical analysis. AB participated in confocal microscopy, immunostaining, ROS staining, and western blotting. MB participated in the design of the study, western blotting, and transfections. CL participated in western blotting and transfections. PB participated in ROS staining and flow cytometry. BC, AT, and AO participated in western blotting. AT created the graphic for Fig. 6. EH conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.

5.5. Transient transfection and immunofluorescence microscopy Declaration of Competing Interest MEF cells were transfected with 5 μg/μL of DNA, either encoding GFP, Klf4, or Klf4 conjugated to GFP on a EGFP plasmid backbone, or Bnip3-Myc fusion protein. The Lipofectamine 3000 Transfection Reagent Protocol (Thermo Fisher, Waltham, MA, #L3000015) at 70 % confluency was used as previously described (Liu et al., 2015a). The efficiency of the transfection was observed under an Olympus IX51 microscope 24 h post-transfection. To study the role of KLF4 in mitophagy, we transiently transfected Klf4-null MEFs with either GFP or EGFP-LC3 plasmids and stained with Mitotracker Deep Red at 150 nM (Thermo Fisher, Waltham, MA). Cells were then treated with CCCP to induce mitophagy and BafA to inhibit the destruction of mature autophagosomes for 6 h. In the case of Bnip3-Myc transfection, cells were transfected with Bnip3-myc or a mock transfection and then stained with Mitotracker Deep Red. Cells were then treated with CCCP to induce mitophagy and BafA to inhibit the destruction of mature autophagosomes. Cells were then stained with Mouse anti-Myc (Cell Signaling, Danvers, MA) and Rabbit anti-LC3 (Abcam, Cambridge, MA) primary antibodies, followed by secondary staining with Alexa Fluor 555 or 488, respectively, conjugated antibodies (Thermo Fisher, Waltham, MA). Cells were imaged with a Zeiss 710 confocal laser scanning microscope at 100x magnification. The LC3-mitochondria overlap was assessed using Mander’s Overlap coefficient on the Zen image analysis program using 20 cells minimum per condition (Carl Zeiss, Thornwood, NY) (Manders et al., 1993). EGFP-LC3 (plasmid #11546) and Bnip3FLMyc (plasmid #00796) was obtained from Addgene, Incorporated.

The authors declare no competing interests. Acknowledgments We acknowledge the lab of Dr. Vincent W. Yang (Stony Brook Medical School, NY) for providing RKO cells, KLF4-GFP, and GFP constructs. The FLBnip3-Myc plasmid was a gift from Dr. Joseph Gordon (Addgene plasmid #100796). The LC3-GFP plasmid was a gift from Dr. Karla Kirkegaard (Addgene plasmid #11546). The authors would also like to thank Dr. Frank Frey for help with statistical analysis and Jeanne Willard for help with plasmid preparation. Funding This work was supported in part by the Picker Research Fellowship and Major Grant from the Colgate University Research Council. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ejcb.2019.151061. References Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U. S. A. 90, 7915–7922. Baldelli, S., Aquilano, K., Ciriolo, M., 2014. PGC-1α buffers ROS-mediated removal of mitochondria during myogenesis. Cell Death Dis. 5, e1515. Brauer, P.R., Kim, J.H., Ochoa, H.J., Stratton, E.R., Black, K.M., Rosencrans, W., Stacey, E., Hagos, E.G., 2018. Krüppel-like factor 4 mediates cellular migration and invasion by altering RhoA activity. Cell Commun. Adhes. 24, 1–10. Capparelli, C., Chiavarina, B., Whitaker-Menezes, D., Pestell, T.G., Pestell, R.G., Hulit, J., Andò, S., Howell, A., Martinez-Outschoorn, U.E., Sotgia, F., 2012. CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts,“fueling” tumor growth via paracrine interactions, without an increase in neoangiogenesis. Cell Cycle 11, 3599–3610. Chen, X., Johns, D.C., Geiman, D.E., Marban, E., Dang, D.T., Hamlin, G., Sun, R., Yang, V.W., 2001. Krüppel-like factor 4 (gut-enriched Krüppel-like factor) inhibits cell proliferation by blocking G1/S progression of the cell cycle. J. Biol. Chem. 276, 30423–30428. Chen, X., Xu, H., Yuan, P., Fang, F., Huss, M., Vega, V.B., Wong, E., Orlov, Y.L., Zhang, W., Jiang, J., 2008. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117. Choe, S.C., Hamacher-Brady, A., Brady, N.R., 2015. Autophagy capacity and sub-mitochondrial heterogeneity shape Bnip3-induced mitophagy regulation of apoptosis. Cell Commun. Signal 13, 37.

5.6. Mitochondrial network analysis To determine mitochondrial network morphology confocal images of cells stained with Mitotracker Deep Red, images were first converted to a black and white binary using Fiji’s binary function. Next pictures were analyzed using the miNA mitochondrial network analysis program. Mitochondrial network size is quantified by the average number of mitochondrial “branches” per network. 50 cells were analyzed for each group (Valente et al., 2017). 5.7. Statistical analysis All experiments were repeated independently at least three times unless otherwise indicated. Statistical tests were performed with GraphPad Prism version 6.0 for Macintosh (GraphPad Software). Significance of western blotting, qRT-PCR, flow cytometry, and 10

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