3.1 Establishment of lentivirus-mediated G-cleave LC3B biosensor in MDA-MB-231 breast cancer cells
Autophagy is a cellular mechanism with dual roles that regulate tumor initiation, progression, and chemoresistance [26]. It promotes the therapeutic resistance of cancer cells to toxic stress or activates cell death mechanisms to increase treatment efficiency in tumor cells [27]. Several methods and pathways have been reported for detecting autophagy in cancer cells [28]. Still, a responsive and rapid autophagy detection with high-throughput potential in a cell lineage-specific manner remains to be established. To effectively monitor autophagy induction in cells, we modified the previous NIADS sensor [20-22] and inserted three repeats of the autophagy cleavage sequence (3X TFGMKLS) between pepA-N’luc and pepB-C’luc fusion proteins, with the repeats of glycine serving as a linker [23,29]. Next, to utilize this G-cleave LC3B autophagy biosensor (LC3BpepABLuc) for cell normalization, we fused the enhanced green fluorescent protein (EGFP) gene with a self-cleaving peptide P2A/T2A at the amino termini of pepABLC3B (pEGFP-LC3BpepABLuc, Figure 1A).
After introducing the pEGFP-LC3BpepABLuc sensor into MDA-MB-231 breast cancer cells using a lentivirus delivery system, we observed strong EGFP signals from fluorescence microscopy (Figure 1B). Next, the protein expression of EGFP and luciferase was further confirmed by immunoblot analysis (Figure 1C), indicating EGFP as a transfection indicator of the pEGFP-LC3BpepABLuc sensor in MDA-MB-231 cells with luciferase fusion protein expression. We also assessed the enzyme activity of luciferase by adding luciferin as the substrate in MDA-MB-231 cells expressing pEGFP-LC3BpepABLuc. The IVIS detection showed strong luciferase activity in MDA-MB-231 cells transfected with the pEGFP-LC3BpepABLuc sensor, compared to the parental MDA-MB-231 cells (Figure 1D). Based on the above result, we displayed a brief schematic model of how this G-cleave LC3B biosensor functions in MDA-MB-231 cells (Figure 1E). First, EGFP is released from the pepABLC3B fusion protein via cells self-cleaving at P2A/T2A. Second, autophagy signaling cleaves the specific autophagy cleavage sequences and enables the formation of cleaved (N’luc2 and C’luc2) due to high protein-protein interaction affinity (pepA and pepB). Finally, luciferin exposure activates the luciferase protein, and bioluminescence represents the autophagy LC3B cleavage activity during autophagy events.
It is well known that the LC3B lipidation and autophagy cargo protein (SQSTM1) degradation are commonly used to assess autophagy events [30]. To understand whether the pEGFP-LC3BpepABLuc sensor might activate autophagy, we measured the LC3B lipidation and cargo protein degradation of pEGFP-LC3BpepABLuc in MDA-MB-231 and parental cells treated with/without the autophagy degradation inhibitor chloroquine (CQ) (Figure 1F). Immunoblot analysis revealed that the expression of pEGFP-LC3BpepABLuc did not alter the levels of LC3B lipidation or SQSTM1 degradation in MDA-MB-231 cells compared to the parental cells, indicating that this pEGFP-LC3BpepABLuc biosensor construct does not interfere with endogenous autophagy process. This finding also suggests that the pEGFP-LC3BpepABLuc (G-cleave LC3B biosensor) in MDA-MB-231 breast cancer cells is ideal for exploring autophagy activation without intruding into endogenous autophagy activity.
3.2 Activation of autophagy in MDA-MB-231 breast cancer cells expressing G-cleave LC3B biosensor
Autophagy initiation is a multi-step process comprising nucleation, elongation, fusion, and degradation. To elucidate the duration and efficiency of autophagy detection using the G-cleave LC3B biosensor, we evaluated the activation of autophagy in MDA-MB-231 cells expressing the biosensor by nutrient depletion (Figure 2A). We examined the levels of LC3B lipidation and SQSTM1 degradation under EBSS or serum starvation and found that these two canonical autophagy stimuli time-dependently increased the lipidation of LC3B (LC3B-II expression). The time-dependent degradation of SQSTM1 also occurred in MDA-MB-231 cells expressing the G-cleave LC3B biosensor under either EBSS or serum starvation, except the SQSTM1-related compensation of autophagic degradation eventuated 48 hours after the serum starvation treatment. To ensure the increase of LC3B-II expression is not due to inhibition of autophagy degradation, we co-treated cells with EBSS/nutrient depletion and CQ. We found that the induction of LC3B lipidation upon either EBSS (Figure 2B, left panel) or serum starvation (Figure 2B, right panel) was further enhanced by adding CQ. The enhancement of SQSTM1 degradation by both stimuli was slightly inhibited by the addition of CQ (Figure 2B), indicating that the strategies of EBSS/nutrient depletion-mediated autophagy can be used to investigate G-cleave LC3B biosensor in MDA-MB-231 cells.
To monitor the autophagic flux by visualizing the autophagosomes fusion with lysosomes to form autophagolysosomes, we observed the fluorescent punctate signals in MDA-MB-231 cells transduced with mCherry-EGFP-LC3B. The formation of mCherry (red) and EGFP (green) puncta was visualized in the structure of autophagosomes, shown as yellow/orange puncta in the merged images. Due to the pH sensitivity of the EGFP fluorescence signals, the fluorescence was quenched in the structure of autophagolysosomes, resulting in the appearance of red puncta in the merged images. Microscope images showed that nutrient depletion increased the number of yellow/orange puncta, indicating autophagosome formation. Furthermore, the green puncta, which represent the inhibition of autophagosome-lysosome fusion, was particularly evident in the cells incubated with CQ compared to its counterpart (Figure 2C), indicating that EBSS/nutrient depletion activated autophagic flux in MDA-MB-231 cells. Taken together, our results support the evidence that nutrient depletion activates LC3B lipidation, SQSTM1 degradation, and autophagic flux in breast cancer cells. The level of autophagy induction under nutrient depletion can be observed in either MDA-MB-231 cells or the cells expressing the G-cleave LC3B biosensor by traditional immunoblot- and fluorescent-based autophagy assays.
3.3 EBSS/nutrient depletion stimulates luciferase activity of G-cleave LC3B biosensor in a proteasome degradation-dependent manner.
To investigate whether nutrient depletion efficiently activates the G-cleave LC3B biosensor, we employed a bioluminescence-based assay to monitor the luciferase degradation activity of the biosensor in MDA-MB-231 cells. We revealed that EBSS and serum starvation significantly decreased the bioluminescence activity in MDA-MB-231 cells (Figure 3A), which refers to the previous finding that autophagy elevated LC3B lipidation and cargo protein degradation. Notably, the luciferase degradation activity (showed in autophagy activity) of the G-cleave LC3B biosensor was found to reach the maximum level at 4 hours after EBSS/serum starvation, with no further acceleration observed upon prolonged treatment (Supplementary Figure 1). To assess the rapid detection capability of luciferase degradation activity, MDA-MB-231 cells expressing G-cleave LC3B biosensor were treated with EBSS/serum starvation for less than 4 hours. The results indicated that both treatments led to a time-dependent activation of luciferase degradation activity (showed in autophagy activity) within 4 hours (Figure 3B and 3C), which was further confirmed by IVIS imaging system with quantification analysis of the photon flux at 4 hours post-treatment (Figure 3A, right panel), demonstrating the effectiveness of this bioluminescence-based autophagy biosensor.
The proteolytic processing of LC3 by the ATG4 cysteine protease is essential for the initiation of autophagy membrane conjugation [31] and the ubiquitin-mediated autophagic degradation [32]. To determine whether the autophagy-triggered reduction in G-cleave LC3B bioluminescence in MDA-MB-231 cells requires a protease degradation process, we exposed cells to MG132, a potent proteasome inhibitor to examine the bioluminescence changes upon EBSS/serum starvation treatments. We found that the high autophagy activity of EBSS/serum starvation treatments was significantly reduced during MG132 exposure, whereas MG132 treatment only maintained low autophagy activity (Figures 3D and 3E). The ubiquitin–proteasome system (UPS) is the primary mechanism that degrades the short-lived protein [32]; we, therefore, proposed that the activated luciferase (a cleaved form of LC3BpepABLuc) may be degraded through a proteasome-associated pathway shortly after autophagy activation. Indeed, immunoblot results showed that both EBSS (Figure 3F) and serum starvation (Figure 3G) increased the degradation of the cleavage form of luciferase, with no significant changes in protein expression in the full-length luciferase. In contrast, these cleaved forms of luciferase degradation were further inhibited in cells pre-treated with MG132. To investigate whether autophagy degradation responsible for the long-lived protein is involved in this luciferase degradation process, we added the autophagy lysosome-fusion degradation inhibitor CQ for 24 hours to the G-cleave LC3B biosensor. We examined the effect of serum starvation on luciferase degradation. Unlike the effects of MG132, the degradation of the cleaved forms of luciferase in response to either EBSS or serum starvation was not attenuated during CQ exposure (Figure 3H). At the same time, the long-term autophagic degradation was inhibited based on the accumulation of LC3B in cells co-treated with CQ (Figure 3H). This result indicates that the autophagy lysosome-mediated long-term degradation pathway is not the limiting step of the G-cleave LC3B biosensor.
Taken together, the above results provide evidence that autophagic stimuli such as EBSS and serum starvation trigger the rapid degradation of short-lived cleaved-luciferase and increase the autophagy activity of the G-cleave LC3B biosensor in MDA-MB-231 cells primarily via proteasome-related degradation mechanisms (Figure 3I).
3.4 LC3B conjugating enzyme ATG4B is required for nutrient depletion-mediated activation of autophagy activity in G-cleave LC3B biosensors.
The post-translational modification of LC3B is an essential step in autophagy, in which the C-terminus of the soluble LC3-I is cleaved by ATG4B, an autophagy-related cysteine protease, to expose the C-terminal glycine residue required for the formation of the membrane-bound LC3-II [33]. The G-cleave LC3B biosensor used in this study was designed to incorporate an autophagy cleavage sequence that contains the critical C-terminal glycine residue needed for LC3B-PE conjugation [23,29]. To investigate the role of ATG4B in regulating the luciferase degradation (autophagy activity) of G-cleave LC3B biosensor, we used lentivirus-mediated CRISPR-Cas9 gene editing to establish ATG4B gene-edited MDA-MB-231 cells with pEGFP-LC3BpepABLuc sensor (Figure 4A). Through Tracking of Indels by DEcomposition (TIDE) analysis, we noticed that the average indels rate was 91.2% in ATG4B gene edited pool MDA-MB-231 cells with pEGFP-LC3BpepABLuc sensor (Figure 4B), whereas the most abundant indel was +1 nucleotide insertion with 42.9% of all population (Figure 4C). On the other hand, ATG4B protein expression was completely abolished in the ATG4B gene-edited pool MDA-MB-231 cells with pEGFP-LC3BpepABLuc sensor, compared with SC parental cells (Figure 4D). In addition, we found that the EBSS- and serum starvation-triggered autophagy activity of the G-cleave LC3B biosensor was significantly inhibited in ATG4B gene-edited cells (Figure 4E), indicating that the autophagic lipidation enzyme ATG4B is essential for initiating autophagy process and downstream proteasome-related degradation of G-cleave LC3B biosensor during autophagic stimulation.
3.5 Screening of autophagy-modulating drugs by G-cleave LC3B biosensor in MDAMD231 breast cancer cells
To identify potent drugs that regulate autophagy in breast cancer, we performed a drug screening assay using the G-cleave LC3B biosensor, including clinical anti-breast cancer drugs, autophagy modulators, and flavonoids. The drug details used to validate the luciferase degradation activity (autophagy activity) of the biosensor in MDA-MB-231 cells are listed in Supplementary Table 2. We found that EBSS and serum starvation increased the autophagy activity of the biosensor by 2.82±0.48 and 2.04±0.19 folds, respectively (Supplementary Table 1). The autophagy-inducing peptide Tat-Beclin1 L11, which specifically activates autophagy via interaction with the autophagy suppressor GAPR-1/GLIPR2 [34], increased the autophagy activity by 3.89 folds (Supplementary Table 2). The autophagy inhibitors CQ and 3-MA did not alter the autophagy activity of the biosensor in MDA-MB-231 cells. Additionally, we found that RSV, a natural anti-tumor phenol isolated from grapes, achieved the highest autophagy activity (4.28 folds) compared to other drugs as determined by the G-cleave LC3B biosensor in MDA-MB-231 cells. The IVIS assay also demonstrated significant biosensor degradation after RSV treatment (Figure 4F).
Based on the drug pre-screening results (Supplementary Table 2), we found that RSV, a natural phenol known for its anti-tumoral properties [35], increased the autophagy activity of MDA-MD-231cells much more than other drugs. It was evident that RSV treatment at 10 to 100 µM increased autophagy activity dose-dependently (Figure 4G). Additionally, 24 hours after RSV exposure, MDA-MB-231 cells maintained autophagy characteristics, such as LC3B lipidation and SQSTM1 degradation in cells with or without CQ (Figure 4H). Furthermore, RSV treatment was able to increase autophagy flux, as observed by the formation of yellow/orange (autophagosome) puncta in pmCherry-EGFP-LC3B introduced MDA-MB-231 and green puncta (autophagolysosome) in cells with CQ exposure (Figure 4I). These results indicate the efficacy and precision of the G-cleave LC3B biosensor in identifying potential autophagy-based drugs for further treatment of TNBC.
3.6 RSV enhances the drug-sensitivity of DOX in MDA-MB-231 breast cancer cells
The anti-tumoral role of autophagy has also been reported in breast cancer [36]. Dysregulation of autophagy caused by the loss of the autophagy-related gene BECN1 facilitates tumor formation and progression in TNBC. Despite numerous studies aimed at verifying potential cancer therapeutic drug combinations by inhibiting or stimulating autophagy, no authorized pharmaceuticals are currently designed to manipulate autophagy for addressing TNBC. To explore whether selected autophagy agents synergize with clinical chemotherapy agents to improve the anti-cancer effect on TNBC, we combined RSV and DOX to treat MDA-MB-231 cells. The cytotoxicity determination showed that the IC50 concentration of RSV and DOX were 78.5 µM (Supplementary Figure 2A) and 2.0 µM (Supplementary Figure 2B), respectively. Since the development of drug resistance in malignant breast tumors is frequently observed, the combination of DOX with other anti-neoplastic agents is therefore required in clinical settings [37-38]. Hence, we evaluated the anti-cancer potential of RSV in combination with DOX to suppress breast cancer cell survival and growth.
RSV is a natural compound that exhibited health benefits wildly, including its anti-cancer properties. The effective concentration of resveratrol for the anti-tumor study can vary depending on the specific type of cancer, the experimental conditions, and the study design. According to the current anti-cancer literature, the RSV concentration range from 50 µM to 100 µM are commonly utilized in the anti-cancer study due to higher concentration of RSV for long-term treatment usually represent better anti-tumor properties when counteracts with other drugs [39-40]. In addition, RSV has been known to inhibits the mechanistic target of rapamycin complex 1 (mTORC1) to activate early autophagosome formation by facilitating unc-51 like autophagy activating kinase 1 (ULK1)/ class III phosphatidylinositol 3-kinase (PtdIns3K) complex interaction [41-43]. While RSV activate autophagy through inhibition of mTORC1, the over-activated PI3K/Akt/mTOR signaling cascade that associated with DOX chemo-resistance was concomitantly prevented and leads to apoptosis during combination treatment [42,44]. Thus, RSV may promote DOX-mediated apoptosis through mTOR-dependent autophagy activation to suppress aggressive breast cancer progression.
Our combination index determination results showed that RSV synergistically increased the DOX efficacy on MDA-MB-231 cells, whereas 20 µM RSV and 0.5 µM DOX combinational treatment obtained the most significant anti-cancer effect (Figure 5A). In a dose-dependent manner, co-treatment of 25 µM RSV significantly reduced cell viability to 0.5 µM and 1 µM of DOX alone (Figure 5B). On the other hand, in a time-dependent manner, co-treatment of 25 µM and 50 µM RSV significantly reduced cell viability than 0.5 µM DOX alone after 48 hours of drug treatments (Figure 5C), whereas 50 µM RSV and 0.5 µM DOX combination treatment still maintained the significant inhibition of MDA-MB-231 cell viability after 72 hours after drug exposure. Lastly, we confirmed the above anti-cancer finding with immunoblotting. It was evident that the combined treatment with 100 µM of RSV and 0.5 µM DOX significantly induced caspase-3 and PARP cleavage expression on MDA-MB-231 cells (Figure 5D). In addition, we used flow cytometry to confirm the above synergic apoptosis activity of RSV and DOX exposures. After the combination drug treatments for two days, 100 µM of RSV and 0.5 µM DOX obtained the most effective apoptosis event (sub-G1 phase) than DOX monotherapy and control group (Figure 5E). These findings indicate that RSV, the anti-cancer compound identified from the G-cleave LC3B biosensor cells, enhances the cytotoxic and apoptotic effects during DOX exposure to MDA-MB-231, implying this potential anti-cancer drug combination may be used in clinical breast cancer therapy.
The toxicity of DOX is known to decrease in acidic conditions (pH 6.3) due to reduced cell membrane permeability [45]. Additionally, an acidic extracellular environment can hinder the efficacy of anti-cancer drugs by inhibiting autophagy [46]. However, autophagy can help cancer cells survive in acidic environments. All conditions were maintained at pH 7.3 to pH 7.6 to minimize pH interference in our experiments—the impact of acidic tumor microenvironments on drug efficacy identified by our biosensor warrants further investigation. Our findings suggest the potential use of biomaterials to mitigate acidic environmental effects, enhancing autophagy-based therapies in breast cancer.
3.7 The advantage of G-cleave LC3B biosensor in monitoring of autophagy.
The G-cleave LC3B biosensor offers several distinct advantages in the monitoring of autophagy. We summarized these advantages and compared them to traditional autophagy detection methods commonly employed in cancer research (Table 1). Firstly, conventional techniques such as immunoblotting and flow cytometry provide insights into the autophagy process by monitoring changes in specific autophagy-related proteins, allowing researchers to identify the stage of autophagy, whether it involves elongation, fusion, or protein degradation. However, these methods have significant drawbacks, including their time-consuming and expensive cost requirements, primarily due to the need for various antibodies. Secondly, imaging-based approaches, such as puncta formation and electron microscopy (EM), are frequently used to detect the autophagic flux and formation of the double-membrane autophagosome structure. While these techniques are more cost-effective than immunoblotting and flow cytometry, they demand a deep understanding of molecular cloning techniques, access to sophisticated fluorescence or electron microscopes, and, most importantly, the expertise of trained personnel to obtain reliable imaging results. Furthermore, the data obtained from these traditional methods are either non-quantitative or semi-quantitative, limiting their utility for large-scale drug screening due to the substantial sample requirements. In contrast, our study introduces an innovative autophagy detection approach that is quantifiable, rapid and high-throughput. By culturing live cells that carry the G-cleave LC3B biosensor in multi-well plates, applying autophagy-inducing drugs less than 4 hours, researchers can obtain absolute quantitative experimental data using a fluorescence/luminescence microplate reader within 30 minutes. The G-cleave LC3B biosensor minimizes sample requirement and provides high detection capacity in the multi-well plates, make it an efficient tool for autophagy drug screening. This method also holds great promise for advanced applications in precision medicine, enables the screening of autophagy/apoptosis-targeting drugs in clinical cancer patients, paving the way for more personalized and effective cancer treatments.