RETRACTED ARTICLE: LATS2 overexpression attenuates the therapeutic resistance of liver cancer HepG2 cells to sorafenib-mediated death via inhibiting the AMPK–Mfn2 signaling pathway

In the present study, HepG2 liver cancer cells, purchased from Shanghai Cancer Institute (China), were used to explore the influences of LATS2 on cancer cell phenotype. These cells were incubated under Dulbecco’s Modified Eagle’s medium (DMEM; GIBCO BRL, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; GIBCO BRL) in a humidified incubator at 37 °C and 5% CO₂ [25]. Different doses of sorafenib was added into the medium of HepG2 cells according to a previous study [26]. FCCP (5 μm for 40 min, Selleck Chemicals, Houston, TX, USA) was added in order to activated mitophagy based on a recent report [27].

Cell death was measured via the TUNEL assay using an In Situ Cell Death Detection Kit (Roche, Indianapolis, IN, USA). The TUNEL kit stains nuclei that contain fragmented DNA. After treatment, the cells were fixed with 3.7% paraformaldehyde for 30 min at room temperature. An equilibration buffer, nucleotide mix and rTdT enzyme were subsequently incubated with the samples at 37 °C for 60 min. A saline-sodium citrate buffer was then used to stop the reaction. After loading with DAPI, the samples were visualized via fluorescence microscopy (Olympus BX-61). In addition, the MTT assay was performed to analyze the cell viability according to the methods described in a previous study [28]. The absorbance at 570 nm was determined. The relative cell viability was recorded as a ratio with the control group. The experiments were performed in triplicate and repeated three times with similar results.

The samples were washed with cold-PBS three times and then permeabilized using 0.1% Triton X-100, followed by neutralization with NH4Cl buffer for 45 min. The samples were subsequently incubated overnight with the following primary antibodies: cyt-c (1:1000; Abcam; #ab90529), Tom20 (mitochondrial marker, 1:1000, Abcam, #ab186735), LAMP1 (lysosome marker, 1:1000, Abcam, #ab24170). The immunofluorescence images were recorded under an inverted microscope (BX51; Olympus Corporation, Tokyo, Japan) [29].

The Mitochondrial Membrane Potential Detection Kit (JC-1) (Beyotime Institute of Biotechnology, China) was used to observe changes in the mitochondrial potential. Briefly, 5 mg/ml JC-1 working solution was added to the medium and incubated for 30 min at 37 °C with CO₂. The cells were subsequently washed with PBS to remove the JC-1 probe, and images were obtained via fluorescence microscopy (Olympus BX-61) [30]. The ratio of red to green fluorescence was analyzed using Image Pro Plus version 4.5 (Media Cybernetics, Inc., Rockville, MD, USA). The LDH release assay was used to observe cell death according to the manufacturer’s guidelines [31]. The relative LDH release was recorded as the ratio to that of the control group. The experiments were performed in triplicate and repeated three times with similar results.

Cytosolic and mitochondrial fractions were used for the Western blotting assays. Proteins (40–60 μg) were loaded for immunodetection. The samples were resolved by 10% SDS-PAGE and then transferred to PVDF membranes (85 V for 60 min) [32]. Then, 5% nonfat dried milk in Tris-buffered saline was used to block the membranes, which were incubated with primary antibodies overnight at 4 °C. The membranes were subsequently incubated with a secondary antibody for 45 min at room temperature. The membranes were washed with TBST at least three times. The immunoblots were then detected using an enhanced chemiluminescence substrate (Applygen Technologies, Inc.). The primary antibodies used in the present study were as follows: LATS2 (1:1000, Abcam, #ab135794), AMPK (1:1000, Abcam, #ab131512), p-AMPK (1:1000, Abcam, #ab23875), Mfn2 (1:1000, Abcam, #ab56889), ATG5 (1:1000, Cell Signaling Technology, #12994), Beclin1 (1:1000, Cell Signaling Technology, #3738), LC3II (1:1000, Cell Signaling Technology, #3868), Bcl2 (1:1000, Cell Signaling Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), Bad (:1000; Abcam; #ab90435), Cyclin D (1:1000, Abcam, #ab134175), CDK4 (1:1000, Abcam, #ab137675). GAPDH (rabbit polyclonal, ab9485) was served as a loading control protein for cytoplasmic protein detection. The experiments were performed in triplicate and repeated three times with similar results.

Total RNA was extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology, China). cDNA was then reverse-transcribed according to the methods described in a previous study [33]. The mRNA expression was measured via qRT-PCR (SYBR Green method). The SYBR Green reagent was obtained from Solarbio (Beijing, China). The relative mRNA levels were normalized to GAPDH and calculated using the 2^−ΔΔCt method as described in a previous study. The following primers were used in the present study: CXCR4: 5′-TCAGTGGCTGACCTCCTCTT-3′, reverse 5′-CTTGGCCTTTGACTGTTGGT-3′; CXCR7: 5′-TGGGCTTTGCCGTTCCCTTC-3′ and 5′-TCTTCCGGCTGCTGTGCTTC-3′; and GAPDH: 5′-AGATCATCAGCAATGCCTCC-3′ and 5′-GTGGCAGTGATGGCATGGAC-3′.

The caspase-3 and caspase-9 activities were determined using commercial kits (Beyotime Institute of Biotechnology) [34]. The levels of antioxidant factors, including GPX, SOD, and GSH, were measured with ELISA kits purchased from the Beyotime Institute of Biotechnology [35]. The experiments were performed in triplicate and repeated three times with similar results.

Cellular proliferation was detected via EdU staining. Cells were fixed with 4% paraformaldehyde for 15 min. The cells were subsequently incubated with EdU staining solution (FluoProbes^®, catalog number FP-MM9829) for 20 min in the dark [36]. After being washed with PBS to avoid a strong background, the cells were labeled by DAPI. Images were obtained using fluorescence microscopy [37].

Transwell assays were performed using 24-well transwell chambers that contain an insert with an 8-micron pore size. Approximately 2.5 × 10⁴ cells suspended in 50 μl of L-DMEM were seeded in the upper chamber, and l-DMEM supplemented with 5% FBS was added to the lower chamber. After 24 h, nonmigrated cells on the upper surface were removed from the membranes, and the membranes were with 4% paraformaldehyde for 10 min at room temperature [38]. The membranes were then stained with crystal violet staining solution (Sigma-Aldrich) for 1 h at room temperature. The membranes were then photographed, and the number of migrated cells was counted [39].

The pDC315-LATS2 vector was purchased from Shanghai Gene-Pharma Co. (Shanghai, China). The plasmid (3.0 μg per 1 × 10⁴ cells/well) was used to transfect HEK293 cells. Subsequently, the supernatant was collected after cells had been detached from the plates; the viral supernatant was then amplified to obtain adenovirus-LATS2 (Ad-LATS2), which was used to infect HepG2 cells at 37 °C in a humidified atmosphere containing 5% CO₂. Transfections were performed using Opti-MEM supplemented with Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol [40].

All results presented in this study were acquired from at least three independent experiments. The statistical analyses were performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). All results in the present study were analyzed with one-way analysis of variance, followed by Tukey’s test. p < 0.05 was considered statistically significant.

To verify the role of LATS2 in HCC, Western blotting was used to observe changes in LATS2 in response to different doses of sorafenib treatment. As shown in Fig. 1a, b, compared to the control group, the expression of LATS2 was significantly reduced after exposure to sorafenib treatment. This finding was further supported via analyzing the transcription of LATS2. As shown in Fig. 1c, compared to the control group, the transcription of LATS2 decreased in response to sorafenib treatment. Therefore, the above data indicated that sorafenib treatment repressed LATS2 expression in HepG2 liver cancer cells. MTT assay was used to observe the cell viability in response to sorafenib treatment. Compared to the control group, the cell viability was significantly reduced by sorafenib treatment (Fig. 1d). To verify whether overexpression of LATS2 could further enhance the anticancer effects of sorafenib, adenovirus-loaded LATS2 was transfected into HepG2 cells. The overexpression efficiency was confirmed via Western blotting (Fig. 1e, f). The minimal fatal effect of sorafenib occurred at a concentration of 5 μM, and therefore, this concentration was used in the following studies (Fig. 1d). Subsequently, cell death was evaluated using LDH release assays. As shown in Fig. 1g, compared to the control group, the content of LDH rapidly increased in the medium of HepG2 cells after exposure to sorafenib. Notably, LATS2 overexpression further enhanced sorafenib-mediated LDH release, indicative of the synergistic effects of LATS2 overexpression and sorafenib treatment on HepG2 cell apoptosis. This finding was further validated via analyzing the activity of caspase-3. As shown in Fig. 1h, compared to the control group, sorafenib treatment increased the activity of caspase-3. Interestingly, sorafenib treatment combined with LATS2 overexpression further elevated caspase-3 activity, indicating that LATS2 overexpression enhanced sorafenib-mediated cancer death in HepG2 cells. To this end, TUNEL assay was used to quantify cell apoptosis induced by sorafenib treatment and LATS2 overexpression. As shown in Fig. 1i, j, compared to the control group, the number of TUNEL-positive cells was rapidly increased in response to sorafenib treatment. Interestingly, sorafenib treatment in combination with LATS2 overexpression further elevated the ratio of TUNEL-positive cells. Altogether, the above data indicated that LATS2 was inhibited by sorafenib, and overexpression of LATS2 could further augment sorafenib-mediated HepG2 cell death in vitro.

Subsequently, cell proliferation and migration were determined in response to sorafenib treatment and LATS2 overexpression. First, EdU staining was used to quantify cell proliferation. As shown in Fig. 2a, b, compared to the control group, sorafenib treatment reduced the number of EdU positive HepG2 liver cancer cells. Notably, combination of sorafenib and LATS2 overexpression further repressed cell proliferation, as evidenced by the decreased ratio of EdU-positive cells. This finding was further supported via analyzing the expression of cell cyclin proteins using Western blotting. As shown in Fig. 2c–e, compared to the control group, the expression of Cyclin D1 and CDK4 was significantly downregulated in response to sorafenib treatment. Interestingly, overexpression of LATS2 in the presence of sorafenib treatment further reduced the levels of Cyclin D1 and CDK4 in HepG2 liver cancer cells. Therefore, the above data indicated that cell proliferation was negatively regulated by sorafenib, and its antiproliferative ability could be augmented via overexpression of LATS2.

Subsequently, transwell assays were used to observe the cell migration response. As shown in Fig. 2f, g, compared to the control group, the number of migrated cells was rapidly reduced. Interestingly, combination of sorafenib treatment and LATS2 overexpression further repressed the HepG2 cell mobilization, as evidenced by the decreased ratio of migrated cells. This finding was further supported via analyzing the transcription of chemotactic factors such as CXCR4 and CXCR7. As shown in Fig. 2h, compared to the control group, the transcription of CXCR4 and CXCR7 was rapidly downregulated in response to sorafenib treatment, and this alteration could be further enhanced by LATS2 overexpression. Therefore, the above information indicated that HepG2 liver cancer cell movement and proliferation were inhibited by sorafenib, and these effects could be further enhanced by LATS2 overexpression.

At the molecular level, cell viability, proliferation and migration are highly modulated by mitochondria. Accordingly, we investigated whether LATS2 overexpression could further cause mitochondrial dysfunction in the presence of sorafenib. First, mitochondrial function was evaluated via observing mitochondrial membrane potential using a JC-1 probe. As shown in Fig. 3a, b, normal mitochondria exhibited red fluorescence, which is indicative of healthy mitochondrial membrane potential. Interestingly, sorafenib treatment reduced mitochondrial membrane potential, as evidenced by decreased red fluorescence and increased green fluorescence. Notably, LATS2 overexpression combined with sorafenib treatment further reduced mitochondrial membrane potential, as evidenced by the lower ratio of red-to-green fluorescence intensity (Fig. 3a, b). In addition to mitochondrial membrane potential, mitochondrial ROS production was also evaluated using flow cytometry. As shown in Fig. 3c, d, compared to the control group, mitochondrial ROS production was significantly elevated by sorafenib treatment. Interestingly, sorafenib treatment combined with LATS2 overexpression further promoted ROS production in HepG2 cells. We also found that the levels of cellular antioxidants, such as SOD, GPX and GSH, were rapidly downregulated in sorafenib-treated cells (Fig. 3e–g). Interestingly, the levels of cellular antioxidants were further inhibited by cotreatment with Ad-LATS2 and sorafenib (Fig. 3e–g). This information indicates sorafenib-mediated oxidative injury could be enhanced by LATS2 overexpression. The primary effect of mitochondria is to produce ATP, which is required for cellular metabolism. However, the production of ATP was rapidly downregulated in response to sorafenib treatment (Fig. 3h). Interestingly, in sorafenib-treated cells, transfection of Ad-LATS2 further repressed ATP production. Altogether, the above data indicated that sorafenib-mediated mitochondrial damage could be further exacerbated by LATS2 overexpression.

Excessive mitochondrial dysfunction initiates the mitochondrial apoptosis pathway, which features mitochondrial cyt-c release, mPTP opening, and mitochondrial apoptotic protein upregulation. In the present study, we investigated whether LATS2 overexpression could augment sorafenib-mediated mitochondrial apoptosis in HepG2 liver cancer cells. First, Western blotting was used to observe the alterations of mitochondrial apoptosis-related proteins. As shown in Fig. 4a–e, compared to the control group, the expression of Bax and Bad were rapidly increased in response to sorafenib treatment. In contrast, the levels of Bcl2 and survivin, the mitochondrial antiapoptotic proteins, were significantly downregulated after exposure to sorafenib treatment. Notably, combined sorafenib treatment and LATS2 overexpression further repressed the antiapoptotic protein content and elevated the levels of pro-apoptotic factors (Fig. 4a–e).

In addition to proapoptotic protein upregulation, immunofluorescence assay was used to observe the cyt-c translocation from mitochondria into the nucleus [41, 42]. As shown in Fig. 4f, g, compared to the control group, sorafenib treatment increased the expression of nuclear cyt-c, and this effect was enhanced by LATS2 overexpression. Besides, the mitochondrial permeability transition pore (mPTP) opening rate was markedly augmented in response to sorafenib treatment, which was further amplified via overexpression of LATS2 (Fig. 4h). To the end, caspase-9 activity, the hallmark of mitochondrial apoptosis [43, 44], was rapidly elevated after exposure to sorafenib treatment (Fig. 4i). Notably, with overexpression of LATS2, the sorafenib-mediated caspase-9 activation was further enhanced in HepG2 cells. Therefore, the above data indicated that sorafenib and LATS2 overexpression have synergistic effects in promoting HepG2 cell apoptosis.

Previous studies have reported that mitophagy is one of the therapeutic resistance mechanisms involved in cancer treatment [45, 46]. Mitophagy helps promote the removal of damaged mitochondria, and this process sustains mitochondrial quality and quantity. In the present study, we asked whether mitophagy was involved in LATS2-augmented cell death and mitochondrial damage in the presence of sorafenib. First, Western blotting analysis was used to observe the parameters related to mitophagy. As shown in Fig. 5a–d, compared to the control group, the expression of mito-LC3II, ATG5, and Beclin1 were rapidly increased in response to sorafenib, indicative of mitophagy activation in sorafenib-treated cells. Interestingly, the markers of mitophagy were markedly repressed by LATS2 overexpression, indicating that sorafenib-activated mitophagy could be suppressed by LATS2 overexpression (Fig. 5a–d). Therefore, the above data validate our hypothesis that protective mitophagy is activated by sorafenib due to LATS2 downregulation.

Subsequently, immunofluorescence assays were conducted to observe the mitophagy activity via containing mitochondria and lysosomes [47, 48]. As shown in Fig. 5e, f, several mitochondria were contained in lysosome in the normal cells. Interestingly, sorafenib treatment significantly increased the interaction between mitochondria and lysosomes, and this alteration could be prevented by LATS2 overexpression. Therefore, this result reconfirmed that sorafenib treatment activated mitophagy via LATS2 in HepG2 liver cancer cells.

Next, experiments were performed to analyze the role of mitophagy in cancer cell viability. FCCP, an activator of mitophagy, was used to recall mitophagy in cells transfected with Ad-LATS2. Then, cell viability was determined via analyzing caspase-9 and caspase-3 activities. As shown in Fig. 5g, h, compared to the control group, caspase-9/3 viabilities were significantly increased by sorafenib treatment. However, cotreatment with sorafenib and Ad-LATS2 further elevated caspase-9/3 viabilities, and these effects were negated by FCCP treatment. Therefore, the above information indicated that activated mitophagy sustained cancer cell viability in the presence of sorafenib and/or LATS2 overexpression.

At the molecular level, mitophagy has been found to be modulated by MFN2 in gastric cancer. AMPK has been associated with cancer metastasis and mitochondrial homeostasis [49, 50]. Accordingly, we asked whether AMPK was involved in MFN2-mediated mitophagy in HepG2 liver cancer cells. Western blotting analysis demonstrated that the expression of MFN2 and p-AMPK was rapidly elevated in sorafenib-treated cells. Interestingly, LATS2 overexpression repressed the content of MFN2 and p-AMPK (Fig. 6a–c). To understand whether AMPK was involved in MFN2 regulation, Compound C (CC), an AMPK pathway inhibitor, was added into the medium of HepG2 cells. After treatment with CC, the expression of MFN2 was rapidly downregulated in sorafenib-treated cells (Fig. 6a–c), similar to the results obtained via overexpression of LATS2, indicating that the AMPK pathway was the upstream activator of MFN2. Overall, the above data illustrated that mitophagy is activated by sorafenib via upregulating the AMPK–MFN2 pathway. LATS2 overexpression repressed sorafenib-mediated AMPK activation and MFN2 upregulation, finally inhibiting mitophagy activity in HepG2 liver cancer.

Although we found that the AMPK–MFN2 pathway is involved in sorafenib-modulated mitophagy, it remains unclear whether the AMPK–MFN2 pathway is associated with cell viability and mitochondria homeostasis [51, 52]. Subsequently, experiments were performed to analyze the alterations of cell death and mitochondrial function in response to AMPK inhibition. As shown in Fig. 6d, LDH release assays demonstrated that sorafenib-mediated LDH release could be further enhanced by AMPK inhibition, similar to the results obtained via overexpression of LATS2. Therefore, the above data indicated that the AMPK pathway was also implicated in the viability of sorafenib-mediated cell death.

In addition to cell death, mitochondrial function was further measured to figure out the detailed role played by AMPK in modulating mitochondria energy metabolism and apoptosis. ATP production was repressed by sorafenib (Fig. 6e), and this effect was further validated by AMPK inhibition, similar to the result obtained via overexpression of LATS2. With respect to mitochondrial apoptosis, cyt-c release assay was confirmed via immunofluorescence. As shown in Fig. 6f, g, compared to the control group, sorafenib increased the nuclear expression of cyt-c. Interestingly, AMPK inhibition and/or LATS2 overexpression further elevated the content of nuclear cyt-c. Altogether, the above data indicated that blockade of the AMPK pathway augmented sorafenib-mediated death in HepG2 liver cancer cells in vitro.