Background
The successful invention of Proteolysis-targeting chimeras (PROTACs) has brought targeted cancer therapy into a new stage. Compared with small molecule chemotherapy drugs, monoclonal antibodies and siRNA [
1,
2], PROTACs can solve the problem of drug resistance in cancer treatment to a certain extent, while having good tissue and intracellular permeability like small molecules [
3,
4]. Also, PROTACs contains ligands that can specifically bind to the target protein, resulting in better targeting properties against pathogenic proteins [
5,
6], especially for the degradation of enzymes [
7,
8]. Moreover, new targeted protein degradation technologies such opto-PROTACs [
9], peptide-based PROTACs (p-PROTAC) [
10], and antibody-based PROTACs [
11] are also emerging, providing more accurate and controllable treatment schemes for the treatment of cancer and other diseases. The p-PROTAC possess high specificity and low toxicity when compared with small molecule PROTAC, and can avoid the limitations of shallow binding pockets through large interacting surfaces, resulting in improving the degradation efficiency of E3 ubiquitin ligase [
12].
FOXM1, one of the essential transcription factors in the forked head protein family, is ubiquitous in proliferating cells. It has been found to be highly expressed in various cancers such as liver cancer [
13], lung cancer [
14], breast cancer [
15], and so on, and is closely related to the poor prognosis of patients [
16,
17]. As a typical proliferation-related transcription factor, FOXM1 can be phosphorylated and activated by cell cycle complexes CDK4/6, promoting cell cycle transition and tissue regeneration by regulating the expression of related genes [
18,
19]. It can also activate Wnt/beta-catenin [
20], Raf/MEK/ERK [
21], and other signal pathways to promote cell growth, metastasis, and EMT. Subsequently, it was found that FOXM1 overexpression in hepatoma cells can promote the cell cycle transition from S phase to G2/M phase and accelerate the cell cycle by regulating cell cycle genes such as, CDC25B [
22] and CyclinB1 [
23]. The high metabolic level of cancer cells also suggests that the expression level of GLUT1 on the cell membrane of cancer cells is higher than that of normal cells, which results in an increase in cancer cell proliferation and is consistent with the poor prognosis of cancer patients [
24,
25]. Additionally, clinical data analysis shows that the expression of PD-L1 is highly correlated with the expression of FOXM1 and GLUT1 [
26,
27]. However, currently there is a lack of experimental data showing a direct link between the regulation of cell proliferation by FOXM1 and the immune suppression caused by PD-L1 expression.
Here, we develop a peptide-based PROTAC, which achieves the purpose of degrading FOXM1 by facilitating the recruitment of E3 ubiquitin to FOXM1. Phage display technology was used to screen peptides in high-throughput libraries, with the peptides are effectively transported to cells by adding cell-penetrating peptide sequences. Finally, the efficient peptide is linked with the protein degradation ligand pomalidomide through appropriate PEG, forming a FOXM1-targeted protein targeting chimera named FOXM1-PROTAC. The two segments of FOXM1-PROTAC, in the presence of FOXM1 and E3 ubiquitin ligase, assist the ubiquitination and degradation the FOXM1 protein, thereby inhibiting the proliferation of liver cancer cells, further decreasing the expression of GLUT1 and PD-L1, and reducing glucose metabolism. In vivo studies confirmed that the molecule has no apparent toxicity while successfully suppressing tumors. This work suggests that FOXM1-PROTAC may become a promising cancer treatment drug.
Methods
Screening of FOXM1 targeting peptides
150 μl of 100 μg/ml FOXM1 full-length recombinant protein (Abnova) dissolved in NaHCO3 (0.1 M, pH 8.6) was added at the 96-well microplate, The well plate was then rotated until the surface is completely entirely wetted and then incubated overnight in a humidified container at 4 °C with agitation. The next day, an ER2738 individual clone was put into 30 ml lysogeny broth (LB) medium and cultured under vigorous shaking at 37 °C. Simultaneously, we discarded the coating liquid in the microtiter plate, shook off the remaining liquid, filled the plate with the blocking solution immediately, and incubated at 4 °C for 1 h. Afterward, the blocking solution was discarded, and TBST (50 mM pH 7.5 Tris-HCl, 150 mM NaCl, 0.1% Tween-20) buffer was used to wash the plate six times. 10 μl of (2 × 1011 PFU) Ph.D.™ phage library (BioLabs) was added to 100 μl TBST, transferred to a microplate, and shaken at room temperature for 1 h. The nonbinding phages were discarded, and the microplate was washed ten times with TBST buffer. After removing residual water from the plates, 100 μl Glycine-HCl (0.2 M, pH 2.2) was added and the plate was rocked gently for 15 minutes to obtain the binding phages. 15 μl Tris-HCl (1 M, pH 9.1) was used to neutralize the eluate. 2 μl of the eluate was taken out, and the remaining eluate was put into 20 ml of ER2738 bacterial solution (approximate OD600 value of 0.5), shaken vigorously at 37 °C for 5 h. The culture was transferred to a clean centrifuge tube and centrifuged at 4 °C, 10,000 rpm for 10 minutes. The supernatant was transferred to a fresh centrifuge tube where PEG/NaCl (20% PEG-8000, 2.5 M NaCl) solution was added at approximately 1/6th the volume of the supernatant and was then allowed to precipitate overnight at 4 °C. The precipitate was then centrifuged at 4 °C, 10,000 rpm for 15 min. The pellet was resuspended in 1 ml TBST and centrifuged at 4 °C, 10,000 rpm for 5 minutes. Another 1/6th volume of PEG/NaCl was added to the pellet and incubated on ice for 1 h, then centrifuged at 4 °C, 10,000 rpm for 10 minutes. The precipitate was then resuspended in 200 μl TBS as the amplification eluate, and could be used for the second round of screening.
Meanwhile, the spare 2 μl eluate was diluted 101–104 fold separately, and 10 μl eluate of different dilutions were added to 200 μl of ER2738 bacterial solution (approximate OD600 value of 0.5), mixed, and incubated at room temperature for 5 minutes. The infected cells were added to 3 ml top agar preheated to 45 °C, mixed, and immediately poured into the LB/IPTG/Xgal plate preheated to 37 °C. The plate was incubated overnight at 37 °C after cooling. The plates with approximately 100 plaques had the plaque counted and then multiplied by the dilution factor to obtain the phage titer in plaque forming unit (PFU) titer of phage per 10 μl. According to this value, the addition amount of corresponding 2 × 1011 PFU was calculated, and an additional two or three rounds of screening were carried out according to the above steps. In the second and third rounds of screening, 15 clones were selected from the plates with less than 100 plaques. After amplification according to the above amplification steps, phage sheath DNA was extracted with M13 phage DNA Extraction Kit (Omega Bio-tek) and sequenced.
Synthesis of peptides and proteolysis-targeting chimeras
The three peptides obtained from the phage library were further conjugated by solid-phase synthesis (Apeptide, Shanghai). Specifically, the final three peptides were conjugated with FITC and TAT (GRKKRRQRRRPPQQ) cell-penetrating peptides at the N-terminus and C-terminus. The synthesized peptides were purified to a purity greater than 95% using HPLC. Lastly, MS detection was performed. When used the peptide needs to be resuspended in sterile double-distilled water, and then stored at − 20 °C. The pomalidomide-PEG2-COOH was synthesized chemically (Biochempartner, Shanghai), purified to a purity greater than 98% using HPLC, and subjected to MS detection. FOXM1-PROTAC was also synthesized via chemical methods (Apeptide, Shanghai), purified to a purity greater than 95% using HPLC, and tested by MS. When ready for use, it was resuspended in sterile double-distilled water and stored at − 20 °C.
Cell lines and cell culture
HepG2, A549, MDA-MB-231 and HCT116 cells were obtained from ATCC (Manassas, USA). All cells were grown in Dulbecco’s Modified Eagle Media (Gibco) medium and supplemented with 10% fetal bovine serum (Gibco). Unless otherwise specified, all cell cultures were grown at 5% CO2, 37 °C.
Confocal fluorescence microscopy analysis of cell uptake and localization of FIP-1
HepG2 cells were seeded in a 12-well plate at 5 × 104 cells/well with cell climbing films (NEST) and allowed to culture overnight. The next day, the medium was discarded and the cell climbing films were washed three times with cold PSB. The FITC-labeled peptide was diluted with water to make a 1000 μM mother solution according to the molecular weight, and with further dilution a final peptide concentration of 10 μM was added to the plates, and incubated at 37 °C for 6 h. Subsequently, the culture medium was discarded, rinsed three times with cold PBS, and fixed with 4% paraformaldehyde for 10 minutes at room temperature. The cells were first washed three times with 0.01% PBST (PBS, 0.01% Triton-100), and then 0.2% PBST (PBS, 0.01% Triton-100) was added and the cells were incubated for 10 minutes at room temperature. Then the cells were blocked with 2% BSA (PBS, 2% BSA) for 30 minutes at room temperature. After discarding the blocking solution, the cells were washed three times with 0.01% PBST. The FOXM1 antibody (CST) was then added according to the appropriate proportion and the cells were incubated at room temperature for 2 hours. Excess antibody was discarded, and the cells were washed three times with 0.01% PBST. Fluor 555-labeled fluorescent secondary antibody (CST) was added and the cells were incubated for 1 h at room temperature. Excess secondary antibody was aspirated, and then the cells were washed thrice with PBS, 3 μM DAPI (Invitrogen) was then added, and the cells were incubated afterwards for 10 minutes at room temperature. Cell climbing films were taken out, and was fixed with a fixative containing anti-fluorescence quencher (Corning). The final images were taken under a two-photon fluorescence microscope (Nikon).
Cell viability
HepG2 cells were seeded at 5 × 103 per well in 96-well plates and cultured overnight. The following day, the medium was then replaced, and different concentrations of peptides or FOXM1-PROTAC were added. The culture plate was incubated in the incubator for 48 h, and then 10 μl of CCK-8 solution (Abcam) was added to each well. After incubating for 1–4 h, and the absorbance at 450 nm was measured with a microplate reader. The cell viability was calculated according to the following formula, and the inhibition curve also was drawn. Cell viability (%) = [A (dosing)-A (blank)]/[A(0dosing)-A (blank)] × 100, A (dosing): the OD value of the wells with cells, CCK-8 solution, and drugs. A (0 dosing): the OD value of the wells with cells and CCK-8 solution but no drug solution, A (blank): the OD of the wells without cells.
HepG2 hepatocellular carcinoma cells were seeded in a 6-well cell culture plate at 100 cells/well, with a medium volume of 2 ml per well, and were cultured for 24 h, after culturing, 20 μM FOXM1-PROTAC and the appropriate amount of fresh medium were added. The peptides were introduced to the experimental groups while no drugs were added to the control group. The medium and drugs were replaced every 2 days, and the cells were cultured continuously until each cell cluster contained more than 50 cells. After fixing the cells in the experimental and control groups with methanol, the cells in both were stained with 0.1% crystal violet (Macklin). The number of clones formed in each group was then counted.
Scratch assay
HepG2 was inoculated into a new 6-well cell culture plate with a ratio of 1:2. 2 ml of medium was added to each well. After 24 h of culture, the cells were scratched with a wall, and the floating cells and debris were washed away with PBS. The scratch distance was photographed and recorded with a microscope, FOXM1-PROTAC with a concentration of 20 μM was added to the plate and an appropriate amount of serum-free fresh medium was used as the control group. After 48 hours, the floating cells were washed off, and the scratch distance was recorded by microscope.
Transwell
One hour before the experiment, Matrigel (Corning) was mixed with medium according to the protocol, and then added to the upper well and cultured at 37 °C, 5% CO2 for 1 h. After gel coagulation, HepG2 and MDA-MB-231 cells were diluted with serum-free DMEM to obtain 5 × 105 cells in each well. FIP-1 and FOXM1-PROTAC with a concentration of 20 μM were added. At the same time, DMEM with 20% serum was added to the lower well, and was incubated at 37 °C, 5% CO2. 24 h later, wash with PBS to remove the residual medium, fix with paraformaldehyde for 20 minutes, and gently wipe the non-transferred cells on the membrane with a cotton swab. After crystal violet staining for 20 minutes, wash with PBS until the background is colorless, take photos under the microscope, and count.
Protein extraction and Western blotting
HepG2 hepatocellular carcinoma cells were seeded at 5 × 105 cells/well in a 6-well cell culture plate, and 2 ml medium was added to each well to culture overnight. Different concentrations of FIP-1 or FOXM1-PROTAC were added, and the cells were collected 24 h later; in the time group, 20 μM FIP-1 and FOXM1-PROTAC were added, and the cells were collected at set times. The collected cells were extracted with the total cell protein extraction kit (Thermo Fisher), and the total protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific). The 10% SDS-PAGE was prepared in advance with the PAGE Gel Fast Preparation Kit (Omni), and then the sample was analyzed. FOXM1, GLUT1, and PD-L1 antibodies (CST) were used to detect the corresponding proteins, and β-actin (CST) was used as an internal reference for quantitative analysis.
Real-time PCR
After treatment with 20 μM peptide and FOXM1-PROTAC for 24 h, the total RNA of the cells was extracted with TRIzol™ Plus RNA Purification Kit I (Thermo Scientific). Immediately, total RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific) in a 42 °C metal bath. The DyNAmo Color Flash SYBR Green qPCR kit (Thermo Scientific) was used to perform quantitative real-time PCR using the Bio-Rad machine, using the FOXM1 primers (5′-ATACGTGGATTGAGGACCACT-3′5’-TCCAATGTCAAGTAGCGGTTG-3′), and the experimental results were analyzed.
Flow cytometry analysis of the cell cycle and PD-L1 expression
About 2 × 106 HepG2 cells were seeded in 6 cm plates and cultured overnight. Then the medium was removed, FIP-1 or FOXM1-PROTAC at a final concentration of 20 μM were added to the fresh medium, and the cells were cultured for another 24 h. After that, the cells were processed according to the Cell Cycle and Apoptosis Analysis Kit (Beyotime) protocol, and the cell cycle was detected using the flow cytometry (Beckman). Similarly, HepG2 cells, treated with FIP-1 or FOXM1-PROTACs, were stained with PD-L1–488 (CST) antibody and analyzed by flow cytometry.
Lactate analysis
1 × 106 HepG2 cells were seeded in 3.5 cm plates and cultured overnight. Then the medium was removed and the plates were washed twice with cold PBS, replaced with fresh medium and FIP-1 or FOXM1-PROTAC at a final concentration of 20 μM were added. After culturing for 24 h, the cells were collected, 200 μl of Lactate Assay Buffer was added, and then the cells were well shaken and mixed. Then the collected cells were centrifuged at 12000 g for 5 minutes at 4 °C, and the supernatant was drawn for lactate analysis. The detection reagents were configured according to the CheKine™ Lactate Assay Kit (Abbkine). The OD value at 450 nm was measured at the start and 30 minutes later, and the lactate metabolism level was analyzed.
2-NBDG uptake assays
2 × 106 HepG2 cells were inoculated in 6 cm plate. After culturing overnight, the medium was replaced with fresh medium, and FIP-1 or FOXM1-PROTAC at a final concentration of 20 μM were added. After 24 h of treatment, the medium was discarded and washed twice with cold PBS. A sugar-free and serum-free medium was used, and 2-NBDG with a final concentration of 300 μM was added to the plate. After 30 minutes of treatment, the medium was aspirated and washed with cold PBS until there was no residual 2-NBDG. The uptake of glucose was recorded by microscope (Nikon). At the same time, the cells were collected and resuspended in 200 μl of pre-cooled PBS and then the propidium iodide (PI) concentration was adjusted to 1 μg/ml, and kept at 4 °C for 30 minutes. Flow cytometry analysis was then performed.
The 96-well plate was seeded with 2 × 104 cells per well, an appropriate amount of medium was added, and the cells were allowed to culture overnight. The next day, the medium was replaced, and the experimental groups were treated with 20 μM FIP-1 or FOXM1-PROTAC. The control group was instead treated with the sterile water and cultured. After culturing for 24 h, the medium was aspirated and the Glycolysis Assay (Extracellular acidification) kit (Abcam) was used according to the set procedures. The prepared plate was then put into the fluorescent plate reader preset to a temperature of 37 °C. The glycolysis test signal was measured every 1.5 minutes for at least 2 h, using excitation and emission wavelengths of Ex/Em = 380/615 nm.
Tumorigenesis assays in nude mice
We purchased 21 female BALB/c nude mice (female, 6-week old) from Guangdong Medical Laboratory Animal Center. HepG2, a human hepatocellular carcinoma cell line, was used in a subcutaneous tumor model. Each nude mouse was injected subcutaneously with 5 × 106 HepG2 cells for tumor growth (approximately one week). When the tumor volume exceeded 50 mm3, mice were randomly assigned, seven per group, into the control group, FIP-1 group, and FOXM1-PROTAC group. The FIP-1 group and FOXM1-PROTAC group were injected with 20 mg/kg FIP-1 and FOXM1-PROTAC, respectively, through the caudal vein once a day for 14 days. The tumor volumes and nude mice body weights were measured every 2 days. The tumor volumes and nude mice weight continued to be monitored the week after halting treatment. Then, after collecting blood from the mice via retro-orbit, the tumor and several organs were removed and fixed in 4% paraformaldehyde. Tissue sectioning, immunohistochemistry, and immunofluorescence experiments were outsourced to a company (Servicebio). The blood was stored at 4 °C overnight and then centrifuged at 4 °C, 8000 rpm for 5 minutes. The supernatant was collected, and a kit was used to detect the liver index.
In vivo biodistribution
Ten female BALB/c subcutaneous tumor mice were randomly divided into two groups with five animals in each group. Then 20 mg/kg FIP-1 and FOXM1-PROTACs were injected through the caudal vein. Peripheral blood was taken at 6 h, 12 h, 24 h and 48 h respectively, and the tumors and organs were collected after 48 h. They were homogenized and lysed with RIPA buffer added with protease inhibitor cocktail. The distribution of FOXM1-PROTACs in blood and organs was detected with PEG- ELISA kit (Life Diagnostics).
Statistical analysis
Microsoft Excel program or Graphpad Prism 7 was used to calculate the mean ± standard deviation of the sample. For the analysis method, we used unpaired two-tailed student’s t-tests to analyze the differences between the two groups. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used for multiple comparison test. Statistical significance was defined as ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; n.s. = not significant.
Discussion
The principle of PROTAC is that this bifunctional molecule binds the intracellular or nuclear targets at one end, and binds an E3 ligase at the other end, which forms a ternary complex to recruit the cellular ubiquitin proteasome system (UPS) for proteasomal degradation of targets. There are many different protein targets are successfully degraded by PROTAC, including estrogen receptor (ER), androgen receptor (AR), bromodomain-containing protein 4 (BRD4), anaplastic lymphoma kinase (ALK), et al. Currently, the small molecules are usually used as targeting warheads to design the PROTACs, and this type of PROTAC is heavily rely on the binding pockets of targets. Compared with small molecule PROTACs, the design and synthesis of p-PROTACs is simpler, and possess the ability to target “undruggable” targets with high specificity and low toxicity, and can resist to mutation targets. Since GLUT1 levels can correlate with response to FOXM1, there is increasing interest in understanding cell proliferation and glycolysis [
42,
43]. However, while PD-L1 regulation has been extensively studied in cancer cells, the specific link between PD-L1 and FOXM1 has received less attention. Jingtong Zhang, et. Al., have reported that down-regulation of WDR5 will inhibit the expression of FOXM1, and further research shows that it reduced the expression of PD-L1 [
44]. Hence, it is particularly momentous to explore the relationship between FOXM1 and immunosuppressive point PD-L1 from the perspective of glucose metabolism, a vital movement of cells. Also, proteolysis-targeting chimeras was used to degrade targeting protein, aiming to develop new drugs against different diseases, including cancer [
45,
46].
To address unmet needs, we sought to develop a system combining the inhibitory function of peptide inhibitors with the powerful effect of protein degradation associated with proteolysis-targeting chimeras. Additionally, we attempt to clarify the relationship between cell proliferation, metabolism and immunosuppression. Through screening of a phage display library, we successfully obtained FOXM1-binding antagonistic peptide and proved that it has a strong affinity with FOXM1. We observed significant inhibition of cell viability by FIP-1, which is composed of FOXM1-targeted peptide with the addition of a cell-penetrating peptide sequence. We then selected pomalidomide as the E3 ubiquitin ligase ligand and conjugated it with FIP-1 to form a new PROTAC. This novel peptide based FOXM1-PROTAC showed two main advantages: on the one hand, the antagonistic peptide FIP-1 not only can bind with FOXM1 and recruit E3 ubiquitin to degrade FOXM1 through pomalidomide, but also can inhibit the effects of FOXM1 in cancer progression. Even there is still a little amount of FOXM1 is not degraded by FOXM1-PROTAC, the FIP-1 can also suppress the remain of FOXM1 to down-regulated the oncogenes, such as GLUT1 and PD-L1. On the other hand, the TAT used in our FOXM1-PROTAC has successfully improved the permeability of FIP-1 peptide and facilitated FOXM1-PROTAC enter cancer cells to degrade FOXM1. The biodistribution of injected FOXM1-PROTACs tells us that the p-PROTACs was a strong performance of specific targeting, and no substantial side effect was produced in “off-target” tissues.
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