RETRACTED ARTICLE: Large tumor suppressor kinase 2 overexpression attenuates 5-FU-resistance in colorectal cancer via activating the JNK-MIEF1-mitochondrial division pathway

SW480 colorectal cancer cells (ATCC^® CCL-228™) were purchased from the National Infrastructure of Cell Line Resource (Beijing, China). All these cells were grown in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS, Gibco, Grand Island, NY, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. SW480 cells were treated with different doses of 5-FU for 12 h according to a previous study [15]. The siRNA against MIEF1 and the pDC315-LATS2 vector were obtained from GenePharm (Shanghai, China). Meanwhile, transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer’s instructions [16]. After 6 h, the cells were transferred to complete growth medium, and 48 h later, the cells were harvested and used for further experiments. The siRNA knockdown efficiency and overexpression efficiency were confirmed via western blotting [17].

Total protein was extracted by RIPA (R0010, Solarbio Science and Technology, Beijing, China), and the protein concentration of each sample was detected with a bicinchoninic acid (BCA) kit (20201ES76, Yeasen Biotech Co., Ltd, Shanghai, China) [18]. Deionized water was added to generate 30-µg protein samples for each lane. A 10% sodium dodecyl sulphate (SDS) separation gel and concentration gel were prepared [19]. The following diluted primary antibodies were added to the membrane and incubated overnight: Bcl2 (1:1000, Cell Signaling Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase-9 (1:1000, Cell Signaling Technology, #9504), survivin (1:1000, Cell Signaling Technology, #2808), Cyclin D (1:1000, Abcam, #ab134175), CDK4 (1:1000, Abcam, #ab137675), complex III subunit core (CIII-core2, 1:1000, Invitrogen, #459220), complex II (CII-30, 1:1000, Abcam, #ab110410), complex IV subunit II (CIV-II, 1:1000, Abcam, #ab110268), Drp1 (1:1000, Abcam, #ab56788), Fis1 (1:1000, Abcam, #ab71498), Opa1 (1:1000, Abcam, #ab42364), Mfn2 (1:1000, Abcam, #ab56889), Mff (1:1000, Cell Signaling Technology, #86668), LATS2 (1:1000, Abcam, #ab135794), MIEF1 (1:1000, Abcam, #ab89944), JNK (1:1000; Cell Signaling Technology, #4672), p-JNK (1:1000; Cell Signaling Technology, #9251) [20].

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA from cells. Subsequently, the Reverse Transcription kit (Kaneka Eurogentec S.A., Seraing, Belgium) was applied to transcribe RNA (1 μg in each group) into cDNA at room temperature (~ 25 °C) for 30 min [21]. The qPCR was performed with primers using SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, Inc. Cat. No. 4309155) [22].

The Caspase 9 Activity Assay Kit (Beyotime, China, Cat. No: C1158) was used to measure the activity of caspase-9 according to the manufacturer’s instructions [23]. The concentrations of GSH, SOD and GPX were evaluated using commercial kits (Cellular Glutathione Peroxidase Assay Kit, Beyotime, China, Cat. No: S0056; Glutathione Reductase Assay Kit, Beyotime, China, Cat. No: S0055; Total Superoxide Dismutase Assay Kit, Beyotime, China, Cat. No: S0101, respectively) [24]. ATP production was measured using a luciferase-based ATP assay kit (Beyotime Institute of Biotechnology) with a microplate reader [25].

Cellular proliferation was evaluated via EdU assay. Cells were seeded onto a 6-well plate, and the Cell-Light™ EdU Apollo^®567 In Vitro Imaging Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA; Catalogue No. A10044) was used to observe the EdU-positive cells according to the manufacturer’s instructions [26]. MTT assay was used to observe the cellular viability. Cells were seeded onto a 96-well plate, and the MTT was then added to the medium (2 mg/ml; Sigma-Aldrich) [27]. Subsequently, the cells were cultured in the dark for 4 h, and DMSO was added to the medium. The OD of each well was observed at A490 nm via a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA) [28].

To perform the TUNEL assay, cells were fixed in 4% paraformaldehyde at room temperature for 30 min [29]. After that, a TUNEL kit (Roche Apoptosis Detection Kit, Roche, Mannheim, Germany) was used on the slices according to the instructions [30]. Finally, the sections were amplified to 400×; the apoptotic cells in at least 10 fields were randomly chosen. The apoptotic index was the proportion of apoptotic cells to total cells according to a previous study [31].

Transwell units were used to evaluate the migratory response of cells. A total of 1 × 10³ cells were added to the upper chamber inside the transwell units [32]. Then, RPMI-1640 supplemented with 10% foetal bovine serum was added to the lower chamber inside the transwell unites. After 12 h, the upper chamber was isolated, and the migrated cells were fixed with 4% paraformaldehyde at room temperature for 30 min [33]. Subsequently, migrated cells were treated with 0.1% crystal violet at room temperature for 30 min. Finally, the migrated cells in at least 10 fields were randomly chosen and recorded with a digital microscope system (IX81, Olympus) [34].

Cells were plated on glass slides in a 6-well plate at a density of 1 × 10⁶ cells per well. Subsequently, cells were fixed in ice-cold 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 2% gelatine in PBS at room temperature [35]. The cells were then incubated with the primary antibodies: Tom20 (1:1000, Abcam, #ab186735), MIEF1 (1:1000, Abcam, #ab89944), p-JNK (1:1000; Cell Signaling Technology, #9251) and cyt-c (1:500; Abcam; #ab90529) [36].

Mitochondrial membrane potential was measured with JC-1 assays (Thermo Fisher Scientific Inc., Waltham, MA, USA; Catalogue No. M34152). Cells were treated with 5 mM JC-1 and then cultured in the dark for 30 min at 37 °C [37]. Subsequently, cold PBS was used to remove the free JC-1, and DAPI was used to stain the nucleus in the dark for 3 min at 37 °C. The mitochondrial membrane potential was observed under a digital microscope (IX81, Olympus) [38]. Cellular ATP content was measured according to a previous report via ELISA assay. Cells were washed with PBS and then collected at room temperature. Subsequently, a luciferase-based ATP assay kit (Celltiter-Glo Luminescent Cell Viability assay; Promega, Madison, WI, USA; Catalogue No. A22066) was used according to the instructions [39].

Cell suspensions were collected. The liquor (50 g, digested two times) was collected, centrifuged for 2 min with the supernatant removed, supplemented with the MitoSOX red mitochondrial superoxide indicator (Molecular Probes, USA) [40], incubated at room temperature for 10 min, centrifuged, and washed with PBS. The cells were resuspended by adding binding buffer (1×) in the dark [41]; then, the cells were incubated at room temperature for 30 min and filtered with a nylon mesh (40 µm well). The ROS production was measured by fluorescence-activated cell sorting (FACS) [42].

SPSS 21.0 software (IBM Corp., Armonk, New York, USA) was applied for data analysis. All experiments were repeated 3 times in each group. The mean value of the measurement data was expressed as the mean and SEM. Comparisons among groups were by one-way analysis of variance (ANOVA), and multiple comparisons between the average number of samples were performed by LSD analysis according to several previous studies [43, 44]. p < 0.05 indicated that the difference was statistically significant.

To examine the function of LATS2 in 5-FU resistance, western blotting was performed to observe changes in LATS2 in response to 5-FU treatment. As shown in Fig. 1a, b, compared to the control group, LATS2 was significantly downregulated in SW480 cells after exposure to 5-FU. This finding was also validated by qPCR. As shown in Fig. 1c, compared with the control group, 5-FU treatment markedly reduced the transcription of LATS2 in SW480 cells. Besides, cell viability was measured via MTT assay. As shown in Fig. 1d, compared to the control group, SW480 cell viability was reduced after exposure to 5-FU treatment. Because minimal 5-FU toxicity was observed at a concentration of 2 μg/ml, this concentration was used in the following studies. To further examine the effect of LATS2 loss in SW480 cells, adenovirus-loaded LATS2 was transfected into SW480 cells to reverse LATS2 expression in the presence of 5-FU treatment. The overexpression efficiency was confirmed via western blotting. As shown in Fig. 1e, f, compared to the 5-FU group, Ad-LATS2 transfection significantly reversed LATS2 expression in SW480 cells. Subsequently, cell viability was measured via an LDH release assay. As shown in Fig. 1g, compared to the control group, the content of LDH in the medium was rapidly increased in response to 5-FU treatment, indicating induction of cell membrane breakage by 5-FU. Interestingly, this alteration was enhanced by LATS2 overexpression.

To further examine whether this cell death resulted from increased apoptosis, ELISA was used to analyse caspase-3 activity. Compared to the control group, the activity of caspase-3 increased rapidly in response to 5-FU treatment, and this effect was further enhanced by overexpression of LATS2 (Fig. 1h). This finding was further supported by TUNEL staining. As shown in Fig. 1i, j, compared to the control group, 5-FU treatment increased the ratio of TUNEL-positive cells. Interestingly, overexpression of LATS2 in the presence of 5-FU further elevated the number of TUNEL-positive cells, indicating that 5-FU-mediated cell apoptosis could be augmented via upregulation of LATS2 (Fig. 1i, j). Taken together, the above data indicated that the anti-apoptotic effect of 5-FU could be enhanced by LATS2 overexpression in SW480 cells.

To verify the role of LATS2 in cell proliferation and mobilization, EdU staining and Transwell assays were performed. As shown in Fig. 2a, b, EdU staining demonstrated that the number of EdU-positive cells was rapidly repressed in response to 5-FU treatment. Interestingly, 5-FU-mediated cell proliferation arrest was further enhanced by LATS2 overexpression, as evidenced by the deceased ratio of EdU-positive cells in SW480 cells (Fig. 2a, b). At the molecular level, cell proliferation is modulated by cyclin proteins such as CDK4 and Cyclin D1. As shown in Fig. 2c, e, compared to the control group, 5-FU treatment inhibited the expression of Cyclin D1 and CDK4. Interestingly, the combination of LATS2 overexpression and 5-FU treatment further repressed the levels of CDK4 and Cyclin D1 in SW480 cells (Fig. 2c, e). Therefore, the above data indicate that cyclin proteins are modulated by 5-FU and this effect is further enhanced by LATS2 overexpression.

In addition to cell proliferation, Transwell assays demonstrated that the number of migrating cells decreased rapidly in response to 5-FU treatment (Fig. 2f, g). However, combining LATS2 overexpression with 5-FU treatment further inhibited the number of migrating cells (Fig. 2f, g). Subsequently, qPCR was used to observe alterations of metastatic genes such as ROCK1 and Rac. As shown in Fig. 2h, i, compared to the control group, ROCK1 and Rac transcription were significantly inhibited by 5-FU treatment, and this effect was enhanced by ad-LATS2 transfection. These results indicated that cellular movement is highly regulated by 5-FU in a LATS2-dependent manner.

Next, experiments were performed to analyse mitochondrial function in response to 5-FU treatment and LATS2 overexpression in SW480 cells. As shown in Fig. 3a, b, mitochondrial membrane potential, as assessed via the JC-1 probe, was rapidly downregulated in response to 5-FU treatment and LATS2 overexpression. Interestingly, the combination of 5-FU treatment and LATS2 overexpression further inhibited mitochondrial membrane potential (Fig. 3a, b), as evidenced by decreased red-to-green fluorescence intensity ratio. At the molecular level, the mitochondrial potential plays a decisive role in ATP production in cancer cells [44, 45]. Accordingly, ATP production was measured in SW480 cells. As shown in Fig. 3c, compared to the control group, 5-FU treatment repressed ATP content in SW480 cells, and this effect was further enhanced by LATS2 overexpression. Notably, the mitochondrial respiratory complex also participates in energy metabolism [46, 47]. Western blotting revealed that the expression of the mitochondrial respiratory complex was rapidly downregulated in response to 5-FU treatment, and this effect was further augmented by LATS2 overexpression (Fig. 3d–g). Taken together, the above data indicate that LATS2 overexpression aggravates 5-FU-mediated mitochondrial dysfunction in SW480 cells.

Excessive mitochondrial malfunction is associated with the initiation of mitochondrial apoptosis [7, 48, 49]. There are three molecular events in mitochondrial apoptosis: mitochondrial ROS accumulation, cyt-c release into the nucleus, and upregulation of mitochondrial apoptosis proteins [50‐52]. Flow cytometry analysis revealed that the production of mitochondrial ROS was significantly increased in response to 5-FU treatment (Fig. 4a, b). However, the pro-oxidant effects of 5-FU were further enhanced by LATS2 overexpression (Fig. 4a, b). In addition to ROS overproduction, cyt-c translocation into the nucleus was evaluated by immunofluorescence assays. As shown in Fig. 4c, d, compared to the control group, a portion of cyt-c was translocated into the nucleus in response to 5-FU treatment. Interestingly, LATS2 overexpression facilitated 5-FU-mediated cyt-c translocation (Fig. 4c, d), as evidenced by the increase in nuclear cyt-c expression upon 5-FU treatment combined with LATS2 overexpression. Finally, western blotting was used to observe the alterations of mitochondrial apoptosis-related proteins. As shown in Fig. 4e–i, compared to the control group, 5-FU treatment elevated the expression of mitochondrial apoptosis-related proteins, as evidenced by increased caspse-9 and Bax. Interestingly, the levels of Bcl-2 and survivin were rapidly downregulated after exposure to 5-FU. Notably, LATS2 overexpression enhanced the upregulation of mitochondrial pro-apoptotic proteins mediated by 5-FU (Fig. 4e–i). Therefore, the above data indicate that 5-FU treatment induces mitochondrial apoptosis and that this effect is further amplified by LATS2 overexpression.

Recent studies have shown that mitochondrial division is closely associated with cancer therapeutic response by increasing mitochondrial oxidative stress and activating caspase-9-related apoptotic pathways. MIEF1 is recently identified as a novel mediator of mitochondrial division. In the present study, we ask whether LATS2 overexpression enhances 5-FU-mediated SW480 cell damage by modifying MIEF1-related mitochondrial division. First, western blotting was performed to analyse the expression of mitochondrial division-related proteins. Drp1 and Mff are pro-division factors, whereas Mfn2 and Opa1 are anti-division proteins. As shown in Fig. 5a–f, compared to the control group, 5-FU treatment elevated the expression of Drp1 and Mff, an effect that was accompanied by an increase in MIEF1. By contrast, the levels of Mfn2 and Opa1 were rapidly downregulated in response to 5-FU treatment (Fig. 5a–f). Interestingly, the 5-FU-mediated imbalance between pro-division proteins and anti-division factors was further amplified by LATS2 overexpression (Fig. 5a–f). These results indicate that mitochondrial fission is initiated by 5-FU and that this effect is further enhanced by LATS2 overexpression.

Subsequently, immunofluorescence assays were used to observe mitochondrial division based on a previous study [53, 54]. As shown in Fig. 5g, h, mitochondria in normal SW480 cells were rod-like, whereas 5-FU treatment caused mitochondrial fragmentation. Interestingly, this alteration was further enhanced by LATS2 overexpression. Then, mitochondrial length was measured as an indicator of mitochondrial division. As shown in Fig. 5g, h, the mitochondrial length decreased from ~ 9.5 μm to ~ 4.5 μm in response to 5-FU treatment. Interestingly, the combination of LATS2 overexpression with 5-FU treatment further reduced the mitochondrial length to ~ 3.3 μm (Fig. 5g, h). The above data indicate that MIEF1-related mitochondrial division is activated by 5-FU and that this effect is augmented by overexpression of LATS2.

To determine if MIEF1-related mitochondrial division is responsible for 5-FU/LATS2-mediated mitochondrial damage and cell death, siRNA against MIEF1 was transfected into 5-FU/LATS2-treated cells (Fig. 6a, b) [55]. The knockdown efficiency was confirmed via western blotting (Fig. 6a, b). Then, mitochondrial function was determined via immunofluorescence [56]. As shown in Fig. 6c, d, compared to the control group, 5-FU in combination with LATS2 overexpression promoted cyt-c translocation into the nucleus, and this effect was reversed by MIEF1 knockdown. In addition to cyt-c translocation, ELISA was performed to observe alterations of cellular antioxidants. As shown in Fig. 6e–g, compared to the control group, the levels of SOD, GSH and GPX were rapidly downregulated in response to 5-FU and LATS2 overexpression. Interestingly, loss of MIEF1 could reverse the levels of cellular antioxidants. Finally, caspase-9 activity was measured as an indicator of mitochondrial damage. As shown in Fig. 6h, compared to the control group, the activity of caspase-9 was significantly elevated in response to 5-FU/LATS2 overexpression, and this effect was negated by MIEF1 knockdown. Therefore, the above data indicate that MIEF1 is required for 5-FU/LATS2-mediated mitochondrial stress.

The role of MIEF1-related mitochondrial division in cell death was analysed by performing LDH release assays [57]. As shown in Fig. 6i, compared to the control group, the content of LDH in the medium was rapidly increased in response to 5-FU/LATS2 overexpression, and this effect was abolished by MIEF1 siRNA. Taken together, our results confirm the essential role of MEIF1-related mitochondrial division in 5-FU/LATS2 overexpression-mediated mitochondrial malfunction and cell death.

We next sought to determine the molecular mechanism by which 5-FU/LAT2 affects MIEF1-related mitochondrial division in SW480 cells. Previous studies have found that the JNK pathway is an upstream mediator of mitochondrial division by controlling mitochondrial division-related proteins such as Drp1, Mff and Opa1 [58, 59]. Accordingly, we examined whether the JNK pathway was also involved in MIEF1 expression induced by 5-FU/LATS2 overexpression. As shown in Fig. 7a–c, compared to the control group, the expression of p-JNK was markedly increased in response to 5-FU/LATS2 overexpression, an effect that was accompanied by an augmentation of MIEF1 expression. Interestingly, inhibition of the JNK pathway using SP600125 significantly repressed p-JNK expression concomitant with a decrease in MIEF1 expression in the presence of 5-FU treatment and LATS2 overexpression. These results indicated that the JNK pathway, activated by 5-FU/LATS2 overexpression, was required for the upregulation of MIEF1 in SW480 cells. This finding was further supported by the results of immunofluorescence assays. As shown in Fig. 7d–f, compared to the control group, the fluorescence intensity of p-JNK and MIEF1 were rapidly elevated in response to 5-FU/LATS2 overexpression; this effect was largely abrogated by SP600125, a specific inhibitor of the JNK pathway. Taken together, the above data indicate that MIEF1 is modulated by 5-FU/LATS2 overexpression via the JNK pathway.