Targeting PP2A with lomitapide suppresses colorectal tumorigenesis through the activation of AMPK/Beclin1-mediated autophagy

We screened an FDA-approved small-molecule library upon HCT116 cells, and identied lomitapide as a novel CRC anticancer compound. Then we conrmed the activities of lomitapide on CRC cells by WST-1 assay, colony formation, and ow cytometry. RNA sequencing and GO analysis were used to investigate the mechanisms underlying the anticancer effects of lomitapide. LiP-SMap was introduced to search for the potential targets of lomitapide. The in vivo experiment was conducted to conrm the therapeutic eciency and safety of lomitapide as an anticancer agent. with indicated concentrations of lomitapide for 48 h. Colocalization of blue DAPI puncta and MitoTracker red was observed by confocal microscopy, and it was quantied through the determination of altered mitochondrial morphology. b Intracellular ATP levels were detected by bioluminescence assay. c Intracellular ROS levels were evaluated by DHE assay. d Immunoblot analysis of AMPK, p-AMPK, and p-ACC expression in HCT116 and HT29 cells. e The interaction of Beclin1 with Vps34 or ATG14 after lomitapide treatment was determined by coimmunoprecipitation assay. All the data are presented as the means±SD. *P < 0.05, **P < 0.01, ***P < 0.001. error data b, c SDS-PAGE results of total protein, PP2A-GST and PP2A as displayed by Coomassie brilliant blue staining (b). The interaction between PP2A and lomitapide was determined by representative surface plasmon resonance (SPR) sensorgram (c). d Immunoblots showing that PP2A was knocked out in the HCT116 and HT29 cells. e Immunoblots of p-ACC and p-AMPK in WT and PP2A-knockout CRC cells with or without lomitapide for 48 h. f-h WT and PP2A-knockout CRC cells were treated with vehicle or lomitapide for 48 h. Cell tumorigenesis was determined by colony formation (f). The apoptotic cell rate was analyzed by Annexin V-FITC/PI double-staining (g), and cleaved caspase-3 and cleaved PARP expression levels were compared (h). All the data are presented as the means±SD. *P < 0.05, **P < 0.01, ***P < 0.001.


Results
Lomitapide exhibited remarkable antitumor properties in vitro and in vivo, while activated autophagy is characterized by GO analysis as a key biological process in lomitapide-induced CRC repression. Moreover, lomitapide stimulated mitochondrial dysfunction-mediated AMPK activation, resulting in increased AMPK phosphorylation and enhanced Beclin1/Atg14/Vps34 interactions, provoking autophagy induction. LiP-SMap analysis showed that PP2A was the direct target of lomitapide, and the bioactivity of lomitapide was attenuated in PP2A-de cient cells, suggesting that the anticancer effect of lomitapide occurs in a PP2A-dependent manner.

Conclusions
Our results indicate that lomitapide activates AMPK-regulated autophagy to inhibit the proliferation and tumorigenesis of CRC cells by directly targeting PP2A, and can be a novel therapeutic agent for the treatment of CRC patients.

Background
Colorectal cancer (CRC) is the third leading cause of cancer mortality worldwide. With advances in diagnostic techniques and therapeutic strategies, the survival time for patients with CRC has increased in recent decades, yet the mortality rate of CRC remains high due to limited e cacy and signi cant side effects of treatments [1] . Hence, novel drugs for CRC treatment are urgently needed. Currently, drug repurposing has gradually emerged as an alternative approach to cancer treatment. This is a promising strategy for investigating novel antineoplastic medicines,because it capitalizes on previous investments while derisking clinical trials. In this study, a drug library consisting of 1056 U.S. Food and Drug Administration (FDA)-approved medications was tested for compounds with anticancer indications.
Lomitapide, an inhibitor of mitochondrial trifunctional protein (MTP), which is usually used for the treatment of hypercholesterolemia in the clinic [2] , was found to signi cantly suppress colorectal tumorigenesis. To the best of our knowledge, the anticancer properties of lomitapide have not been reported, and the mechanism involved is not clear.
Autophagy is an evolutionarily conserved process that maintains cellular metabolism and homeostasis [3] and is subjected to a range of cellular stresses, including oxidative stress, mitochondrial abnormalities and abnormal protein accumulation [4][5][6] . Several studies have indicated that autophagy can act to suppress tumor survival and growth in advanced cancers [7] . In addition, silencing of some key autophagy-related genes has been reported to facilitate tumor progression in colon, gastric, breast, and prostate cancers [8][9][10] . However, the regulatory role of autophagy in colorectal tumorigenesis remains ambiguous. Many studies have revealed that the modulation of autophagy by the mTOR, AMPK, and MAPK pathways plays a critical role in CRC [11][12][13] . Targeting autophagy via key signaling pathways has been recognized as an effective approach to CRC therapy. In this study, RNA sequencing was performed with lomitapide-treated CRC cells, and pathway enrichment analyses of differentially expressed genes suggested that autophagy activation may contribute to the antitumor effects of lomitapide. The direct target of lomitapide in cancer cells is important to its clinical implications but remains to be elucidated.
Quantitative proteomic analysis coupled with bioinformatics analysis has been identi ed as a favorable strategy for exploring the molecular mechanism underlying the actions of small molecules [14] . In previous studies, we successfully used drug a nity responsive target stability (DARTS) technology to demonstrate that ADP-ribosylation factor 1 (ARF1) acts as a direct target and mediates the anticancer effect of azelastine [15] . In contrast to DARTS technology, the optimized limited proteolysis-mass spectrometry (LiP-SMap) method takes advantage of high-resolution LC-MS/MS and high-throughput quantitative proteomic technology to identify increased peptide levels upon limited proteolysis. Moreover, LiP-SMap relies on the binding a nity between a small molecule and its target protein(s), providing more consistent peptide detection and accurate proteome quanti cation [16] . Here, by performing an optimized LiP-SMap, we found that protein phosphatase 2A (PP2A), which plays a crucial role in various biological processes [17] , is a direct target of lomitapide in cancer cells. Although PP2A was originally characterized as a tumor suppressor, recent studies have revealed that targeting PP2A can achieve therapeutic bene ts in certain malignancies via cell cycle arrest, apoptosis induction and disruption of DNA repair [18][19][20] . In the present study, a series of functional assays were performed to investigate whether lomitapide activates AMPK-regulated autophagy to inhibit the proliferation and tumorigenesis of CRC cells by directly targeting PP2A.
Methods manufacturer's instructions [22] . The results were obtained by measuring the absorbance with an automated microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) at 490 nm.

Colony formation assay
To analyze long-term survival, HCT116 and HT29 cells at a density of 500 or 800 cells per well were seeded in 6-well plates, and treated with lomitapide for 14 days. The colonies were xed with 75% ethanol, stained with 1% crystal violet, and counted for analysis.

Flow cytometric analysis
The cell apoptosis was detected using an Annexin V-FITC/PI apoptosis detection kit, and analyzed by a BD FACSCelesta ow cytometer (BD Biosciences, San Diego, CA). For intracellular ROS level measurement, cells were stained with 10 µM dichloro-dihydro-uorescein diacetate (DCFH-DA) dye in serum-free culture medium at 37 °C for 20 min in the dark and washed with PBS, and the relative DCFH uorescence of the cells was analyzed with a BD FACSCelesta ow cytometer.

ATP production assay
For measurements of ATP levels, cell lysates were centrifuged at 12,000 g for 5 min, and the supernatant was mixed with an ATP working dilution (Beyotime Biotechnology). Luminance was measured using a monochromator microplate reader.

Immuno uorescence (IF) staining
Laser scanning confocal microscopy (Carl Zeiss AG, Jena, Thuringia, Germany) was carried out as reported previously [23] . In brief, the lomitapide-treated or control cells were xed with 4% paraformaldehyde, permeabilized with 0.1% TrintonX-100, and blocked with 5% blocking buffer. Then cells were stained with anti-LC3B antibody, and counterstained with DAPI (Thermo Fisher Scienti c, Waltham, MA, USA). Images were visualized and pictures in the same panel were taken under the same excitation conditions.

Detection of mitochondrial morphology
Morphology of mitochondria was determined with the MitoTracker Mitochondrion-Selective Probe (Invitrogen, Gaithersburg, MD, USA). Cells were incubated with the MitoTracker® probe (100 nM) for 30 min at room temperature, and xed with 4% formaldehyde and then stained with DAPI. Confocal microscopy was performed and the obtained images were analyzed by ImageJ software with the mitochondrial network analysis (MiNA) toolset.

Coimmunoprecipitation and immunoblotting
For coimmunoprecipitation (co-IP) assay, HCT116 and HT29 cells were transfected with the indicated plasmids for 24 h, following by the lomitapide treatment for another 48 h. The collected cells were lysed with ice-cold RIPA buffer containing 2% phenylmethyl sulfonyl uoride and 10% phosphatase inhibitor for 40 min. After centrifugation for 30 min at 134,000 g at 4 °C, about 10% of the supernatant was collected as input, and the rest cell lysate was incubated with the indicated antibodies overnight at 4 °C. Then cell lysate was incubated with Protein A/G plus agarose at 4 °C for 4 h, and washed with lysis buffer 5 times.
The immunoprecipitated proteins were detected by Western blot analysis and visualized with the enhanced chemiluminescent (ECL) reagent (Bio-Rad, Hercules, CA, USA). The images were obtained using a Tanon 5200 Multi chemiluminescent imaging system (Tanon Science & Technology Co. Ltd., Shanghai, China).

Protein expression and puri cation
The pGEX-4T-1 vector was used to construct the pGEX-4T-1-PP2A plasmids expressing (GST)-tagged PP2A fusion proteins, as previously described [24] . The expression plasmids were transformed into Escherichia coli BL21, and the cells grown in LB medium at 37 °C until the optical density reached 0.6-0.8 at 600 nm. To induce target protein expression, 0.5 mM isopropyl--d-thiogalactoside (IPTG) was added to the LB medium and cultured with the bacteria for an additional 12 h at 20 °C. Bacteria were then harvested, resuspended in binding buffer (20 mM Na3PO4·12 H 2 O and 0.5 M NaCl, Ph = 7.4) and sonicated for 20 min followed by centrifugation at 12,000 g for 30 min. GST-a nity column (Glutathione Sepharose 4B, GE Healthcare) was used to collect the protein with GST-tag. After washing with elution buffer (10 mM GSH and 50 mM Tris-HCl) thoroughly, the target protein was harvested with a centrifugal lter (10 K, Merck Millipore). Afterwards, the GST-tag was removed by overnight digestion with thrombin (GE Healthcare) at room temperature.
Surface plasmon resonance (SPR) SPR was conducted on an OpenSPR system (Nicoya Lifesciences Inc., Kitchener, Canada) to evaluate the binding a nity of lomitapide to PP2A. In brief, protein was immobilized on sensor chips, and lomitapide solution was introduced into the sensor chip.

Nude mice xenograft model
The animal study in this research was approved by the Ethics Committee for Animal Experiments of Jinan University. Tumor-bearing mice were established by subcutaneous injection of 2 x 10 6 HT29 cells suspended in a 1:1 mixture of PBS: Matrigel (Corning Incorporated, Tewksbury, MA, USA). The mice were treated with 20 mg/kg lomitapide or vehicle by gavage, every two days. The tumor volume were measured every 3 days with the formula: V = 0.5 x length x width 2 . At the end of the experiment, tumors were harvested for TUNEL assay and Ki67 staining, together with livers, kidneys, and lungs were collected for histologic analyses.

Statistical analysis
All in vitro experiments were performed in triplicate. Two-tailed Student's tests, one-way ANOVA, two-way ANOVA, and Chi-square test were performed using GraphPad Prism software v.5.01 (San Diego, CA, USA). All values were expressed as the means ± SD, and P < 0.05 was considered statistically signi cant. To obtain novel anti-cancer agents, a drug library of 1056 FDA-approved medications was tested for antitumor effects. HCT116 cells were intervened with the 1056 compounds individually at an identical concentration of 10 μM for 48 h, and the inhibitory effects of small-molecules on cells were evaluated by WST-1 assay. As shown in Fig. 1a, lomitapide, an agent widely used for treating hypercholesterolemia, was one of the most potent compounds in suppressing the proliferation of HCT116 cells, indicating that it may be a potential anticancer agent.
To further validate whether lomitapide exerts antitumor actions against CRC, we rstly tested the cell viabilities of different human CRC cell lines post lomitapide treatment. As shown in Fig. 1b, lomitapide signi cantly decreased the growth rate of the HCT116 and HT29 CRC cell lines. The in vitro tumorigenicity of these CRC cells was signi cantly impaired under lomitapide treatment, as veri ed by the reduced colony formation in the lomitapide-treated cells (Fig. 1c). To investigate whether apoptosis is involved in lomitapide-induced suppression of CRC cells viabilities, we conducted ow cytometry assays and detected obvious apoptotic CRC cells resulting from lomitapide treatment (Fig. 1d). In addition, lomitapide treatment led to increased levels of cleaved caspase-3 and cleaved PARP in the CRC cells (Fig.  1e). Collectively, the above results implied that lomitapide may exert anticancer effect in CRC cells in vitro.

Autophagy induction contributes to the antitumor activity of lomitapide in CRC cells
Accumulating evidence has highlighted the potential implication of autophagy-regulating drugs for use in cancer therapy [25,26] . In this study, RNA sequencing (RNA-seq) was used to compare the gene pro les of lomitapide-treated CRC cells and control cells, and pathway enrichment analysis of the differentially expressed genes in lomitapide-treated cells suggested that the anticancer effect of lomitapide in CRC cells may be attributed to autophagy activation (Fig. 2a). In order to investigate the regulatory role of lomitapide on autophagy in CRC cells, western blot data showed that lomitapide treatment not only facilitated the turnover of LC3-I to LC3-II, but also augmented the accumulation of autophagic vesicles (LC3 puncta) in a dose-dependent manner (Fig. 2b, Supplementary Figure S1a).
To investigate the crucial role of autophagy in the anticancer effect of lomitapide, ba lomycin A1 (Baf-A1), an autophagy inhibitor, was applied to CRC cells. As shown in Supplementary Figure S1b, Baf-A1 restored lomitapide-induced cell proliferation inhibition. A similar increase in cell proliferation was also detected in lomitapide-treated CRC cells coupled with Baf-A1, as further validated by colony formation assay (Fig. 2c). Moreover, the increased apoptosis rate, as well as the apoptotic proteins, of lomitapidetreated CRC cells was markedly attenuated by Baf-A1 treatment (Fig. 2d, Supplementary Figure S1c). Taken together, these data demonstrated that lomitapide may have exerted its anticancer actions in the CRC cells through activation of autophagy.

Lomitapide triggers AMPK-regulated autophagy through mitochondrial ATP depletion and ROS accumulation in CRC cells
Mitochondrial ssion and fusion play critical roles in maintaining functional mitochondria, while the disruption of mitochondrial dynamics leads to altered mitochondrial morphology, increased amounts of ROS and a decreased ATP synthesis rate, thus fueling autophagy [27,28] . To investigate whether lomitapide-induced autophagy is attributable to mitochondrial dysfunction, we performed immuno uorescence staining to detect the morphology of the mitochondrial network, and an obviously higher ratio of tubular mitochondria was observed in lomitapide-treated HCT116 and HT29 cells, implying the disruption of mitochondrial balance by lomitapide (Fig. 3a). This nding was con rmed by reduced ATP levels and enhanced ROS levels in lomitapide-treated CRC cells (Fig. 3b and 3c). We proposed that the impaired mitochondrial dynamics induced by lomitapide contribute to the activation of autophagy in CRC cells; however, the molecular mechanisms involved remain unclear.
Since various mitochondrial signals, including ATP levels and ROS production, can activate AMPK [29][30][31] , we next examined whether AMPK may respond to the indicated stressors arising from lomitapide-induced mitochondrial dysfunction. The results showed that p-AMPK and p-ACC were upregulated in lomitapidetreated CRC cells (Fig. 3d). The mechanism underlying the effect of lomitapide on AMPK-regulated autophagy was further studied. Since AMPK has been reported to phosphorylate multiple tyrosine sites of Beclin1, leading to enhanced association of Beclin1 with pro-autophagy proteins such as Vps34 and Atg14 [32,33] , we postulated that lomitapide might facilitate the formation of the Beclin1-Atg14-Vps34 complex via AMPK signaling. To verify this assumption, we performed a coimmunoprecipitation assay to analyze the interaction of Beclin1 with other proteins in lomitapide-treated CRC cells. As shown in Fig. 3e, the interaction of Beclin1 with Atg14 and Vps34 was signi cantly enhanced in the presence of lomitapide. Collectively, these results revealed that mitochondrial dysfunction-mediated AMPK phosphorylation was required for lomitapide-induced autophagy in these CRC cells.

AMPK silencing impairs lomitapide-induced autophagic cell death
To validate whether activation of AMPK, not the modulation of other signaling pathways, accounts for the lomitapide-induced autophagic cell death of CRC cells, we examined the anticancer effect of lomitapide in AMPK-knockdown CRC cells. As shown in Fig. 4a-d, AMPK silencing profoundly counteracted the toxicity induced by lomitapide in HCT116 and HT29 cells, as evidenced by enhanced cell proliferation and reduced apoptosis rates. Moreover, the elevated number of autophagic vacuoles, as represented by the LC3-II levels and GFP-LC3 puncta in the lomitapide-treated CRC cells, was markedly abrogated when the cells were transfected with siRNAs against AMPK ( Fig. 4e and Supplementary Figure  S2e). Consistently, knockdown of AMPK attenuated the promoting effect of lomitapide on the formation of the Beclin1-Atg14-Vps34 complex. Taken together, these data suggested that AMPK/Beclin1-mediated autophagy was critical for lomitapide-induced CRC suppression (Fig. 4f).

Targeting PP2A decreases lomitapide-induced AMPK phosphorylation and CRC suppression.
To identify potential targets of lomitapide, LiP-SMap was introduced to search for the proteins that could directly bind to lomitapide. Among the candidate proteins, PP2A, a well-known upstream regulator of AMPK phosphorylation [34] , drew our attention and became our focus in this study. We puri ed the PP2A protein, and the binding of lomitapide with PP2A was validated by surface plasmon resonance assay ( Fig. 5b and 5c).
We proposed that PP2A may be a direct target for mediating AMPK-regulated cell death of lomitapidetreated CRC cells. To con rm this hypothesis, we generated PP2A-knockout HCT116 and HT29 cell lines (PP2A-KO) using the CRISPR/Cas9 system and observed that lomitapide failed to stimulate the phosphorylation of AMPK and ACC in the PP2A-de cient cells (Fig. 5d and 5e). Then, we further investigated whether lomitapide-induced cell death required PP2A-dependent activation of AMPK. As shown in Supplementary Figure S3a and Fig. 5f, PP2A knockout attenuated lomitapide-induced growth inhibition of the CRC cells, as presented by WST-1 and colony formation assays. Moreover, in contrast to the PP2A-expressing cell line (Figure 1d and 1e), moderate apoptosis rates of the HCT116-PP2A-KO and HT29-PP2A-KO cells exposed to lomitapide were observed (Fig. 5g and 5h). Collectively, these ndings suggested that PP2A was important for lomitapide-induced p-AMPK upregulation and proliferation suppression in these CRC cells.
3.6 Lomitapide exhibits antitumor effects against CRC in vivo As shown in Fig. 6a, mice treated with lomitapide exhibited a lower growth comparing to the vehicletreated group mice. In addition, either tumor volumes or tumor weights were signi cantly reduced in the lomitapide-treated mice (Fig. 6b and 6c). Additionally, a TUNEL assay and Ki-67 immunohistochemical analysis were performed to detect apoptosis and proliferation indices. As indicated in Fig. 6d and 6f, xenografts from lomitapide-treated mice presented an increased cell apoptosis rate and decreased cell proliferation rate. Western blot data showed that lomitapide activated the AMPK pathway and autophagy in tumor tissues, indicated by increased expression levels of p-AMPK and LC3 I/II (Fig. 6h). Notably, the lomitapide treatment induced no toxic effect on the animals, as indicated by unchanged body weight and pathological morphology of major organs (Fig. 6e and 6g). Collectively, these results veri ed the therapeutic e ciency and safety of lomitapide as an anticancer agent.

Discussion
Autophagy has been extensively studied in cancers, and it is now universally recognized that autophagy has dual, contradictory roles in cancer progression [35] . Intracellular autophagy can be enhanced by a series of stressors, such as oxidative stress, nutrient starvation, and misfolded protein accumulation.
Once the process is activated, a portion of the cytoplasm and organelles will enter the autophagic vesicles in succession, responding to the cellular stresses [36] . Owing to sustained autophagic stimulators, excessive autophagy leads to the depletion of organelles and key proteins and eventually leads to cell death [37][38][39] . Recently, several autophagy regulators, such as rapamycin, chloroquine, and hydroxychloroquine, have been widely used in cancer therapy [25,[40][41][42][43] . Thus, the development of novel autophagy regulators has presented remarkable potential in CRC treatment.
In recent years, drug repositioning strategies have gained considerable momentum, with approximately one-third of new drug approvals corresponding to repurposed drugs [44] . For example, metformin, a rstline medication for the treatment of type 2 diabetes, has been developed as an anti-cancer agent and is currently under phase II/phase III clinical trials [45] . A novel use of aspirin against prostate cancer has also been reported [46] . Our previous studies also identi ed several traditional drugs that exhibit potential anticancer actions [15,22,24,47,48] . In the present study, lomitapide, an inhibitor of MTP routinely applied in the management of hypercholesterolemia, was found to suppress CRC growth through activation of autophagy, further implying the signi cance of drug repurposing in the development of cancer therapy.
Our study provides the rst evidence that lomitapide exhibits a strong antitumor effect against CRC cells, as validated by WST-1, colony formation and ow cytometry assays (Fig. 1). Lomitapide induces activation of AMPK by interfering with mitochondrial dynamics and intracellular ATP and ROS levels (Fig. 3). Mechanistically, our study demonstrates that lomitapide promotes the recruitment of the Beclin1-Atg14-Vps34 complex, which is required for autophagosome formation, thus inducing AMPK-regulated autophagy in colorectal cancer cells (Figs. 2 and 3). AMPK regulates autophagy by interacting with the Beclin1 complex. Phosphorylated AMPK participates in the direct phosphorylation of Beclin1 at multiple tyrosine sites, contributing to enhanced binding of Beclin1 with pro-autophagic proteins such as Vps34 and Atg14. Thus, formation of the Beclin1-Atg14-Vps34 complex is promoted, and autophagy is initiated [32,49,50] . Our results demonstrate that lomitapide-mediated interaction of Beclin1 with Vps34 and Atg14, as well as autophagy induction, is compromised by AMPK silencing. Furthermore, the anticancer property of lomitapide is abrogated in AMPK-knockdown CRC cells (Fig. 4). These results, therefore, indicate that the suppressive activity of lomitapide in CRC cells is dependent on AMPK/Beclin1-mediated autophagy.
Interestingly, our study also reveals that lomitapide can directly target PP2A, thus activating AMPKregulated autophagy to inhibit CRC tumorigenesis. PP2A, an essential Ser/Thr phosphatase modulating numerous signaling pathways by dephosphorylating key components of these pathways [51][52][53] in cells, is involved in the regulation of a broad range of cancer hallmarks. It has also been reported that PP2A physically interacts with AMPK and reduces its activity via Thr172 dephosphorylation [54] . Another study indicated that nilotinib, a novel TKI, induced autophagic cell death in HCC via PP2A-regulated AMPK dephosphorylation [55] . Here, we demonstrate that lomitapide can directly bind to PP2A, and more importantly, PP2A knockout signi cantly abrogates the effect of lomitapide on AMPK phosphorylation and its downstream proteins in CRC cells, indicating that the regulation of AMPK phosphorylation by lomitapide is partially dependent on PP2A (Fig. 5). Moreover, lomitapide does not exert its anticancer effect in PP2A-de cient CRC cells (Fig. 6). Herein, we discovered that lomitapide may directly impair PP2A-mediated AMPK inactivation, leading to autophagic CRC cell death. Notably, more than 300 substrates can be dephosphorylated by PP2A [51] , and whether other molecules are involved in the therapeutic mechanisms of lomitapide warrants further investigation.
Colorectal cancer (CRC) is among the most lethal and prevalent malignancies worldwide; it accounted for nearly 935,000 cancer-related deaths in 2020 [56] . After decades of development, great efforts have been devoted to updating CRC therapeutic drugs for better patient responses, fewer adverse events and more individualized treatment strategies. However, these regimens exhibit compromised clinical outcomes due to side effects or drug resistance, and the overall prognosis for CRC patients remains poor [57] . Therefore, the identi cation of novel targets and the renewal of effective drugs are in great demand for CRC patients. In summary, the current ndings demonstrate that lomitapide suppresses CRC growth via induction of AMPK/Beclin1-mediated autophagic cell death by directly targeting PP2A without observed side effects. These data shed light on the therapeutic targets and mechanism of action of lomitapide and suggest the potential application of lomitapide in the management of CRC.

Conclusions
Our ndings demonstrate that Lomitapide could inhibit CRC by inducing AMPK-dependent autophagy, and lomitapide could directly impair PP2A-regulated AMPK dephosphorylation while phosphorylated AMPK participates in the direct phosphorylation of Beclin1, contributing to enhanced binding of Beclin1 with pro-autophagic proteins such as Vps34 and Atg14, and leading to autophagic CRC cell death (Fig. 6i). Our study provides a preclinical rationale for development of lomitapide in the future treatment of CRC.

Consent for publication
All the authors agree to publish this paper. Screening of a FDA-approved small-molecule library led to the identi cation of lomitapide as a novel CRC anticancer compound. a Flow chart for the FDA-approved drug library screening used to identify compounds with CRC anticancer activities. b, c HCT116 and HT29 cells were treated with the indicated concentrations of lomitapide. Cell viability was assessed by WST-1 assay (b). Cell tumorigenesis was determined by colony formation (c). d, e Cells were treated with the indicated concentrations of lomitapide for 48 h, apoptotic cells were analyzed by Annexin V-FITC/PI double-staining (d), and cleaved caspase-3 and cleaved PARP expression levels were compared (e). All the data are presented as the means±SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2
Autophagy induction contributes to the antitumor activity of lomitapide in CRC cells. a Identi cation of lomitapide-regulated biological process by bioinformatics analysis. b The accumulation of endogenous LC3 puncta was quantitated by immuno uorescent analysis in the absence or presence of the indicated concentrations of lomitapide for 48 h. c HCT116 and HT29 cells were treated with Baf-A1 (0.5 nM) in the presence or absence of lomitapide (2.5 μM) for 48 h. Cell tumorigenesis was determined by colony formation. d The apoptotic effects induced by lomitapide were analyzed by Annexin V-FITC/PI doublestaining. All the data are presented as the means±SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3
Lomitapide triggers AMPK-regulated autophagy through mitochondrial ATP depletion and ROS accumulation in CRC cells. a HCT116 cells and HT29 cells were treated with indicated concentrations of lomitapide for 48 h. Colocalization of blue DAPI puncta and MitoTracker red was observed by confocal microscopy, and it was quanti ed through the determination of altered mitochondrial morphology. b Intracellular ATP levels were detected by bioluminescence assay. c Intracellular ROS levels were evaluated by DHE assay. d Immunoblot analysis of AMPK, p-AMPK, and p-ACC expression in HCT116 and HT29 cells. e The interaction of Beclin1 with Vps34 or ATG14 after lomitapide treatment was determined by coimmunoprecipitation assay. All the data are presented as the means±SD. *P < 0.05, **P < 0.01, ***P < 0.001. AMPK silencing prevents lomitapide-induced autophagic cell death. a HCT116 and HT29 cells were treated with siScramble or siAMPK in the presence or absence of lomitapide (2.5 μM) for 48 h. b-d Cells were treated as in a. Cell tumorigenesis was detected by colony formation (b). Apoptotic cells were analyzed by Annexin V-FITC/PI double-staining (c) and cleaved caspase-3 and cleaved PARP expression levels were compared (d). e, f Cells were treated as in a. The accumulation of endogenous LC3 puncta was quantitated by immuno uorescent analysis in the absence or presence of lomitapide (e). The interaction of Beclin1 with Vps34 or Atg14 was determined by coimmunoprecipitation assay (f). All the data are presented as the means±SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5
Targeting PP2A decreases lomitapide-induced AMPK phosphorylation and CRC suppression. a Flow chart depicting the LiP-SMap assay. Freshly prepared whole-cell lysates were treated with or without lomitapide