PKM2 allosteric converter: a self-assembly peptide for suppressing renal cell carcinoma and sensitizing chemotherapy

doi:10.21203/rs.3.rs-1598044/v1

Download PDF

Article

PKM2 allosteric converter: a self-assembly peptide for suppressing renal cell carcinoma and sensitizing chemotherapy

https://doi.org/10.21203/rs.3.rs-1598044/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Stronger intrinsic Warburg effect and resistance to chemotherapy are the responses to high mortality of renal cell carcinoma (RCC). Pyruvate kinase M2 (PKM2) plays an important role in this process. Promoting PKM2 conversion from dimer to tetramer is a critical strategy to inhibit Warburg effect and reverse chemotherapy resistance. Herein, a PKM2 allosteric converter (PAC) is constructed based on the “in vivo self-assembly” strategy, which is able to continuously stimulate PKM2 tetramerization. The PAC contains three motifs, a serine site that is protected by enzyme cleavable β-N-acetylglucosamine, a self-assembly peptide and a aggregation-induced emission (AIE) motif. Once PAC nanoparticles reach tumor site via the enhanced permeability and retention (EPR) effect, the protective and hydrophilic β-N-acetylglucosamine will be removed by over-expressed O-GlcNAcase (OGA), causing self-assembled peptides to transform into nanofibers with large serine (PKM2 tetramer activator) exposure and long-term retention, which promotes PKM2 tetramerization continuously. Our results show that PAC-induced PKM2 tetramerization inhibits aberrant metabolism mediated by Warburg effect in cytoplasm. In this way, tumor proliferation and metastasis behavior could be effectively inhibited. Meanwhile, PAC induced PKM2 tetramerization impedes the nuclear translocation of PKM2 dimer, which restores the sensitivity of cancer cells to first-line anticancer drugs. Collectively, the innovative PAC effectively promotes PKM2 conversion from dimer to tetramer, and it might provide a novel approach for suppressing RCC and enhancing chemotherapy sensitivity.

Renal cell carcinoma

Self-assembly peptide

PKM2

Metabolism

Warburg effect

Chemotherapy resistance

Renal cell carcinoma (RCC) is characterized by the highly metastatic potential and chemotherapy resistance peculiarity, which is regarded as the primary causes of treatment failure¹. Over 30% of the RCC cases have metastases at the time of diagnosis and miss out the optimal surgical timing². For chemotherapy, initial responders to therapy will eventually develop resistance to sunitinib (the first-line therapeutic agent) within 10–14 months³. It is known that metastasis and chemotherapy-resistance are caused by aberrant biochemical metabolism and gene transcription^{4, 5}. Meanwhile, Warburg effect is essential for tumor nutrient supplement and tumor progression⁶. The lactic acid generated during Warburg effect is highly related to a variety of biological functions of tumors, including tumor cell proliferation, metastasis and promotion of angiogenesis⁷. This confers the advantage of rapid proliferation of tumor cells and induces tumor metastasis, further leading to malignant tumor progression. In addition, due to the aberrant expression of VHL (in > 80% RCC cases), RCC possess stronger intrinsic Warburg effect than other tumors^{8, 9}. Hence, suppression of Warburg effect is a critical strategy for the treatment on proliferation and metastasis in RCC.

Pyruvate kinase M2 (PKM2), a vital regulator of Warburg effect and transcriptional activation, is crucial for tumor progression, tumor metastasis, and chemotherapy resistance¹⁰. The dimer and tetramer of PKM2 can transform mutually¹¹ and exist mostly as dimer in a variety of cancer¹². PKM2 dimer possesses lower pyruvate kinase activity than tetramer, thereby stronger direct Warburg effect¹³. Meanwhile, PKM2 tetramer is limited in the cytoplasm whereas dimer can be translocated into nucleus to participate in transcriptional activation and induce chemotherapy resistance finally¹⁴. The dynamic balance of PKM2 dimer-tetramer can be regulated by multiple ways¹⁵. Serine, as the natural ligand of PKM2, has been proven to activate PKM2 tetramerization, while it failed to maintain the durability of PKM2 allosterism¹⁶. Hence, continuously stimulate PKM2 tetramerization is crucial for cancer suppression. In this work, we developed a PKM2 allosteric converter (PAC) based on “in vivo self-assembly” strategy for PKM2 tetramerization continuously. As depicted in Scheme 1a, this design includes three modules: i) an effect motif composed of serine protected by β-N-acetylglucosamine as an allosteric activator of PKM2 promoting the conversion from dimer to tetramer; ii) a self-assembly peptide motif (KLVFF) derived from β-amyloid for the formation of fibrous structures¹⁷; iii) a signal motif composed of aggregation-induced emission (AIE). Accordingly, the actions of PAC include the following steps. First, PAC targets RCC by EPR effect¹⁸. Second, overexpressed OGA cleaved β-N-acetylglucosamine to expose serine simultaneously enhancing hydrophobicity. And then serine binds to PKM2 and promotes the tetramerization. The break of overall hydrophilic and hydrophobic balance of PAC induces self-assembly peptide form nanofibers, contributing to maintain long-term serine exposure for sustained tetramerization. In this way, PAC not only inhibits the proliferation and metastasis of RCC by restraining Warburg effect, but also produces synergism with sunitinib in the treatment of sunitinib-resistant RCC. Thus, PAC as a novel anti-RCC therapeutic strategy, acts by dual mechanisms to tackle simultaneously two major problems in the treatment of RCC, metastasis and chemotherapy-resistance, and validation of this approach should greatly promote the application of nano-medicine in cancer therapy.

PKM2 and OGA are overexpressed in patients with renal cell carcinoma

The PKM2 allosterism between dimer and tetramer plays an important role in cancer progression, and OGA is highly expressed in a variety of cancers¹⁹. Therefore, PKM2 and OGA were analyzed in 538 RCC cancer samples and 72 normal samples from The Cancer Genome Atlas (TCGA) database. TCGA database shown that PKM2 and OGA are highly expressed in cancer tissues compared to normal tissues (Fig. 1a, b). In addition, we performed immunohistochemistry to examine the expression of PKM2 and OGA in 64 cases of RCC tissues and 16 cases of normal renal tissues (Fig. 1c). The results demonstrated PKM2 and OGA are more frequently up-regulated in cancer tissues than in normal tissues (Fig. 1d, e, Supplementary Fig. 1, patients characteristics are described in Table 1).

The Synthesis and Identification of PAC and PAC-C

PAC (S(GlcNAc)-K(TPA-1)LVFF) and its non-transformable control peptide PAC-C (S(GlcNAc)-K(TPA-1)AAGG) were synthesized according to the solid-phase peptide synthesis method, and the molecular structures of PAC and PAC-C were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Supplementary Fig. 2). Both PAC and PAC-C contain the GlcNAc-motif and AIE-motif simultaneously, accompanying with OGA-hydrolyzing and signal properties, but PAC-C lacks transformable ability. First, the AIE aggregation fluorescence of PAC and PAC-C in mixed solvents was analyzed by a fluorescence spectrometer. With increasing water content, the fluorescence intensity became stronger (Fig. 2a, Supplementary Fig. 3). The transformation process of PAC was monitored by the time-dependent turbidity curve (Fig. 2b, Supplementary Fig. 4). The results indicated that transformation of PAC was induced in the presence of OGA and no significant change of the turbidity was observed in the absence of OGA. By contrast, PAC-C did not cause any detectable changes whether OGA existed or not. We further validated the formation of β-sheet structure of PAC via amyloid sensor dye thioflavin T (ThT)²⁰. The PAC + OGA group was the only one that accumulated and enhanced ThT fluorescence signals, the intensity of fluorescence increased when β-sheet structure formed (Fig. 2c). To analyze the molecular arrangement mode and chemical composition, we conducted Fourier transform infrared (FTIR) spectroscopy. Following cleavage of PAC by OGA, the peak at 1636 cm^-1in the FTIR spectra exhibited the antiparallel β-sheet formation²¹. However, the peak at 1647 cm^-1 of PAC was identified as oligomer similar to PAC-C (Fig. 2d, Supplementary Fig. 5). These results suggested the β-sheet structure existed in PAC with OGA exposure. Subsequently, transmission electron microscopy (TEM) was used to detect morphological changes. PAC exhibited spherical structures with the mean diameter of 26.4 ± 4.2 nm. After incubation with OGA for 2 h, PAC transformed into nanofibers (Fig. 2e). In contrast, PAC-C (without KLVFF motif) did not show the morphological transformation, indicating that PAC, but not PAC-C, possesses self-assembly property conferred by the KLVFF peptide (Supplementary Fig. 6). The positive circular dichroism signal at 203 nm and negative signal at 220 nm presented the typical characteristics of β-sheet secondary structure (Fig. 2f, Supplementary Fig. 7), implying the KLVFF sequence being the β-sheet stacking structure²².

In order to further explore the binding of PAC to PKM2, we verified the PAC monomer without GlcNAc by molecular docking experiments, and the crystal structure of PKM2 (PDB number: 6B6U) was used for molecular docking. As shown in Fig. 2g, PAC monomer can selectively bind to PKM2, which is attributed to the formation of hydrogen bonding to LEU-353, ASP-353, and LYS-311 of PKM2 protein. At the A-A' interface of the PKM2 tetramer, we observed the binding of PAC monomer to N-terminal domain of PKM2 (Fig. 2h), which is consistent to the activator of PKM2 tetramerization²³.

PAC promotes the PKM2 transformation from dimer to tetramer.

To confirm the ability to promote PKM2 tetramerization of PAC, the cytotoxicity of PAC and PAC-C was examined firstly in RCC cell lines (786-O, ACHN) and normal cells (HK-2) at different concentrations from 0 to 200 μM. PAC exhibited functional inhibition at 50 μM to RCC cells and biological toxicity to normal cells HK-2 at 200 μM. Hence, 50 μM dose was carried out in following experiments (Fig. 3a, Supplementary Fig. 8). PAC was designed to be a PKM2 allosteric converter, which promotes PKM2 transformation from dimer into tetramer. However, maintain stability of PKM2 tetramerization in complex intracellular dynamic environment is a crucial strategy. Therefore, we further investigated the allosteric and sustained stimulative effects of PAC in RCC. Confocal Laser Scanning Microscopy (CLSM) was used to observe the retention of PAC and PAC-C in cells. 786-O and ACHN cells were incubated with PAC and PAC-C respectively, and then maintained for 1, 4, 8, 12 and 24 h. CLSM images showed that PAC-C fluorescence began to decay rapidly after 8 h and nearly disappeared at 24 h. By comparison, the fluorescence of PAC was maintained for 24 h at least (Fig. 3b, Supplementary Fig. 9). These results indicated that the application of self-assembly strategy is helpful for PAC on long-term retention, which may play a critical role in long-lasting tumor suppression.

PKM2 dimer transfers into the nucleus to regulate transcription exerting cancer-promoting action. The content of PKM2 dimer in the nucleus indirectly indicates the degree of abnormal transcription in tumor cells²⁴. Hence, nuclear translocation of PKM2 was detected by western blot. The results indicated that PAC reduced nuclear translocation (Fig. 3c, Supplementary Fig. 10). For detecting the effect of PKM2 allosterism induced by PAC, PKM2 polymers were further examined. The results showed that PAC remarkably stimulated PKM2 dimer-tetramer transition(Fig. 3d, Supplementary Fig. 11).

PAC effectively inhibits the Metastasis and Warburg effect via promoting PKM2 tetramer formation in RCC.

The carcinogenic effect of PKM2 dimer has been confirmed and the change of PKM2 allosteric structure from dimer to tetramer is considered an aggressive tumor therapy²⁵ (Fig. 4a). Our present experiments have showed that PAC promoted PKM2 tetramerization. Therefore, we performed Transwell assay to investigate the effects of PAC on the migration and invasion of 786-O and ACHN cells. As illustrated in Fig. 4b, the migration and invasion of 786-O were significantly mitigated by PAC by 71.9±4.6 % and 64.6±3.5 % respectively and the same situation was observed in ACHN cells (Fig. Supplementary Fig. 12, 13). Meanwhile, PKM2 as a type of pyruvate kinase, its tetramer can well reduce Warburg effect²⁶. Thus, the activity of pyruvate kinase was measured, it showed that PAC increased the activity of pyruvate kinase markedly (Fig. 4c, Supplementary Fig. 14). Since Warburg effect could enhance secretion of lactic acid by consuming glucose²⁷, we detected the glucose consumption and lactic acid production. As expected, PAC decreased 49.3±15% glucose consumption and 64.8 ± 20.2% lactic acid production in 786-O cells and these same changes occured in ACHN cells simultaneously, indicating that PAC could inhibit Warburg effect (Fig. 4d, e, Supplementary Fig. 15, 16). For metabolism and bioenergetic parameters, PAC significantly increased basal and maximal oxygen consumption rates (OCR) and decreased extracellular acidification rates (ECAR) in 786-O and ACHN cells (Fig. 4f, g, Supplementary Fig. 17). These findings suggested that PAC induced PKM2 transformation inhibited metastasis and Warburg effect in RCC.

PAC effectively inhibits tumor growth and metastasis in vivo.

Tumor-bearing mouse model was established by subcutaneous injection with 786-O cells. PAC and PAC-C were injected via the tail vein when the tumor reached 50 mm³. Then, the targeting and retention ability of PAC and PAC-C in the mice were detected by in vivo imaging system (IVIS). PAC enhanced fluorescence signal within 24 h and maintained strong signal for 72 h, but PAC-C group decreased gradually after injection (Supplementary Fig. 18). The biological distribution in the major organs of mice showed that PAC or PAC-C was mainly metabolized by the liver and accumulated in the tumor site, indicating that PAC or PAC-C can effectively target the tumor site in vivo (Supplementary Fig. 19). The findings suggest that the formation of nanofibers might be a mechanism that maintained the long-term retention of PAC in tumor tissue.

Next, we evaluated the suppressive effect of PAC and PAC-C on tumor growth and metastasis in vivo. To maintain an effective concentration of materials, PAC and PAC-C were injected per 48 h for 5 times. PAC markedly reduced 786-O tumor weight by 74.7 ± 6.1% and volume by 62.5±15.8% (Fig. 5a, b, Supplementary Fig. 20) with no significant effects (Fig. 5c). Then, the lung metastasewas determined by counting the number of metastatic nodules. Compared with the PBS group, the number of lung metastases decreased by 64.4 ± 13.8% in the PAC group (Fig. 5d). Additionally, PAC significantly improved the survival rate (Fig. 5e). Consistent with the results in vitro, the activity of pyruvate kinase was increased 1.82 ± 0.32 folds via PAC treatment (Fig. 5f). Additionally, the systemic toxicity of the materials was verified at 48 h after the 5th injection. Then, H&E staining and blood routine indices were performed in major organs and blood of mice. No obvious toxicity induced by PAC was observed in mice (Supplementary Fig. 21, 22). Above results indicate that PAC could effectively inhibit the growth and metastasis with high biosafety in vivo.

PAC reverses chemotherapy resistance in RCC.

Sunitinib is the first-line anticancer drug for metastatic RCC and often develops the chemotherapy-resistance within 10-14 months³. PKM2 is a novel mediator of chemotherapy resistance via intranuclear transcriptional activation in RCC cells¹⁴. To verify the role of PAC and PAC-C on chemosensitivity, we created sunitinib-resistant cell line (786O-R) (Fig. 6a)²⁸. PKM2 transformation from dimer to tetramer was remarkably stimulated by PAC in 786O-R cells (Fig. 6b) and intranuclear dimer were decreased significantly (Fig. 6c). Pyruvate kinase activity was enhanced by 2.37 ± 0.41 folds with PAC treatment (Fig. 6d). Moreover, PAC reduced glucose consumption and lactic acid synthesis by 47.8 ± 18.2% and 49.1 ± 10.8% respectively in 786O-R cells (Fig. 6e, f). Finally, PAC induced suppression and chemosensitivity in 786O-R were verified by CCK-8 assay (Fig. 6g).

In vivo, we constructed a drug-resistant tumor-bearing mouse model according to the reported method²⁹ (Fig. 6h). PAC and PAC-C were administrated by the intravenous injection every 48 hours and sunitinib was given simultaneously through oral gavage. PAC alone and combined administration obviously reduced the volume, and weight of tumor (Fig. 6i, j, Supplementary Fig. 23) in resistant tumor xenografts. Combined administration hardly influenced body weights of mice compared with PBS group (Supplementary Fig. 24). Meanwhile, PAC injection resulted in a significant prolongation of overall survival (Fig. 6k). Combined administration clearly reduced carcinogenesis compared with each agent alone. Overall, PAC present inhibitive effects in chemotherapy resistant RCC and increased cell sensitivity to sunitinib in vitro.

The high Warburg effect and insensitive to chemotherapy promote RCC progression and metastasis^{30, 31}. PKM2 dimer, as a co-regulatory factor of these major characteristics of cancer, has been widely concerned³². Here, we successfully developed a novel self-assembly converter named PAC to promote PKM2 conformational change from dimer to tetramer. In this work, over-expressed OGA cleaves β-N-acetylglucosamine unit from PAC and then contributes to exposing serine. This process breaks the balance between hydrophobic and hydrophilic, and initiate the self-assembly peptide with β-sheet structure, forming nanofibers. In this way, transformed nanofibers maintain PKM2 long-term allosteric activation by serine residues, which promotes PKM2 transformation from dimer to tetramer to suppress Warburg effect and sensitize chemotherapy. In vitro, PAC maintains high concentrations in RCC cells for ≥ 24 h. Eventually, the insufficient of PKM2 dimer suppress Warburg effect and chemotherapy-resistance collectively in vitro. Besides, PAC reduces tumor grow and metastasis, and prolongs overall survival. Meanwhile, PAC significantly improved the therapeutic effect of sunitinib in chemotherapy-resistant mice in vivo. Thus, PAC is an innovative technique for enhancing RCC therapy and promoting translational research of nanomedicine.

General Methods. All reagents were purchased from commercially available sources. Phosphate-Buffered Saline (PBS), Fetal Bovine Serum (FBS), DMEM (Dulbecco's Modified Eagle Medium), and DMEM-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) were obtained from Gibco Invitrogen (CA, USA). 786-O cell lines, ACHN cell lines and HK2 cell lines were purchased from the cell culture center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). 100 mm Culture Dishes, Transwell 24-well permeable support, 6-well culture plates and 96-well culture plates were purchased from Coning Company (NY, USA). CCK-8(Cell counting kit-8) was purchased from TransGen Biotech (Beijing, China). Thioflavin T (ThT) was purchased from Sigma-Aldrich (MO, USA). Matrigel matrix was purchased from BD Bioscience (Franklin Lakes, USA). Serine was purchased from MedChem Express (New Jersey, USA). DAPI (D8200) and Nuclear Protein Extraction Kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Sunitinib was purchased from Selleck (Shanghai, China). Female BALB/c mice (6–8 weeks, 16–18 g) were purchased from Vital River laboratory animal technology Co., Ltd. (Beijing, China).

TCGA data. FPKM data of 539 patients with renal clear cell carcinoma and 72 surrounding normal tissue samples were downloaded from TCGA database (https://tcga-data.nci.nih.gov/tcga/). After standardization, the differential expressions of OGA and PKM2 were observed in tumor tissue and normal tissue, and the statistical significance was determined by T test.

Molecule synthesis and characterization. Two kinds of glycopeptides were synthesized by the previously reported solid-phase synthesis method³³, including PAC (S(GlcNAc)-K(TPA-1)LVFF) and its control group PAC-C (S(GlcNAc)-K(TPA-1)AAGG). The glycopeptides molecules were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS, Bruker Daltonics). The AIE aggregation fluorescence of PAC and PAC-C in mixed solvents was analyzed by Fluorescence Spectrophotometer.

Molecular docking simulation. Molecular docking study is the most reliable method for analyzing and predicting best-fit protein-ligand confirmation. The crystal structure of PKM2 (PDB number: 6B6U) was used for molecular docking, where the position of the co-crystallized ligand was set as the binding site. AutoDock is a suite of automated protein-ligand docking tools commonly used to predict drug molecules and substrates. The interaction of ligand molecules at the specified target site of the protein was analyzed by AutoDock. All ligands were docked 256 times to obtain sufficient sampling.

CD spectroscopy aqueous solutions. PAC, PAC-C, PAC + OGA and PAC-C + OGA were diluted to 100 µM in aqueous solutions, then the secondary structures of PAC, PAC-C, PAC + OGA and PAC-C + OGA were recorded by CD spectroscopy (JASCO Corporation, JC-1500) at room temperature.

Fourier Transform Infrared (FTIR) Spectra. The aqueous solutions of PAC, PAC-C, PAC + OGA and PAC-C + OGA were first detected by an FTIR spectrometer to analyze their secondary structures. Then the liquid samples were freeze-dried and obtained by allowing static for 12 h after.

ThT assay. The solution of PAC, PAC-C, PAC + OGA and PAC-C + OGA were added to 96-well plates for co-incubation, respectively. 1 h later, ThT (20 µM) was added and cultured for 30 minutes. The fluorescence intensity was observed using Fluorescent microplate reader (Infinite 200 Pro, Tecan, Switzerland).

The Confirmation of Morphology. PAC, PAC-C, PAC + OGA and PAC-C + OGA were first dispersed to 100 µM with distilled water. 10 µL of the solution was then placed on a copper mesh, followed by filter paper to remove excess droplets. Subsequently, the samples were stained with uranyl acetate for 10 min and further washed with water (10 µL) for 1 min. Finally, the dried samples were observed by TEM.

The Effects of PAC on Cell Viability. Cell viability of 786-O, ACHN and HK-2 cells was evaluated by CCK-8 assay. Cells were treated with PBS, PAC-C, and PAC at different concentration for 24 h, respectively, which seeded in 96-well plates at a concentration of 1×10⁴ cells/well. Then wash three times with PBS. Subsequently, 10 µL of CCK-8 solution and 90µL of serum-free medium was added to each well and cultured for 2 h. Finally, use microplate reader (Epoch, BioTek, Vermont, USA) to measure the absorbance at 450nm.

Western Blot. The determination of protein content was completed by the BCA kit (Beyotime, China). Each protein sample (50µg) was subjected to 10% SDS-PAGE, then transferred onto PVDF membrane (Millipore, USA). Blots were blocked in a blocking buffer containing 5% (wt/v) non-fat milk then incubated overnight with PKM2 antibody (Proteintech, 15822-1-AP), PCNA (Proteintech, 10205-2-AP) and β- actin antibody (Proteintech, 66009-1-Ig) at 4°C on a shaker. Next washed 3 times with PBST and incubated with the corresponding secondary antibody for 1 hour at room temperature on a shaker. After washed as above described, PVDF membrane was scanned on Odyssey (LI-COR, USA) .

PKM2 Oligomerization Assay. Dilute 50% glutaraldehyde (Macklin, G810414) with sterile distilled water to 0.025%.

The whole-cell lysate was cross-linked with diluted 0.025% glutaraldehyde for 5 minutes at 37°C, and the reaction was terminated with Tris-HCl (pH 8.0, 50 mM). Subsequently, being cross-linked protein samples were analyzed by Western blotting with PKM2 antibodies.

Cytoplasmic protein and nuclear protein extraction. Use the Nuclear Protein Extraction Kit (Solarbio, China) to perform the following operations. Add PMSF to appropriate cytoplasmic protein extraction reagent and nucleoprotein extraction reagent according to the amount to make the final concentration of PMSF 1mM. After collecting the cells, add 200 µl of cytoplasmic protein extraction reagent. Vortex vigorously at high speed for 10 seconds. and then ice bath for 10 minutes. Vortex vigorously for 5 seconds., and centrifuge at 12000–16000 g at 4ºC for 10 minutes. The supernatant was transferred to a precool EP tube, which was the extracted cytoplasmic protein.

Completely removed the remaining supernatant in the above precipitation, add 50 µl additional nucleoprotein extraction reagent. Vortex vigorously for 15–30 seconds to disperse the cell pellet completely, and bath in ice for 10 minutes. Vortex vigorously for another 15–30 seconds. Centrifuge at 12000–16000 g for 10 min at 4ºC, immediately transfer the supernatant to a precool EP tube, which is the nuclear protein extracted.

Cellular Imaging. Observe the fluorescence intensity and residence time of PAC-C and PAC in the cells by confocal laser scanning microscope (Hitachi, Japan). Cells were seeded in confocal microscopy dish and incubated with PAC-C and PAC (50µM, medium:DMSO = 99.5:0.5 v/v) at 37°C. The samples were examined by CLSM at 1, 4, 8, 12 and 24 h respectively.

Glucose Consumption and Lactate Production Assay. Cells (1×10⁶) were planted in a 6-well plate. Collected the medium from cultured cells after the cells were treated with PAC-C and PAC 24 hours for the assay of glucose and lactate. Glucose levels were determined by using a glucose (GO) assay kit (Sigma). The calculation method for glucose consumption was to subtract the glucose concentration measured in the medium from the original glucose concentration. Lactate production level was determined by using Lactate Assay Kit (BioVision) according to the manufacturer’s protocol. The fluorescence intensity was measured under fluorescent microplate reader (Infinite 200 Pro, Tecan, Switzerland).

PKM2 Activity Assay. Cells (1×10⁶/well) were planted in a 6-well plate and treated with PAC-C and PAC for 24 hours to acquired whole-cell lysates. Whole-cell lysates were incubated with reaction buffer. According to the manufacturer’s protocol, the colorimetric based PK activity detection kit (BioVision) was used to measure PK activity. The fluorescence intensity was measured under fluorescent microplate reader (Infinite 200 Pro, Tecan, Switzerland).

Establishment of sunitinib-resistant cell lines. By continuous low-dose stimulation with increasing concentration of sunitinib cultured 786-O cell lines to establish sunitinib-resistant RCC cell lines. The concentration of sunitinib was continuously increased in 786-O medium until 10 µM. And continue to cultivate at this concentration.

Measurement of OCR and ECAR. The OCR and ECAR were respectively in real time determined with the Agilent Seahorse XFp (Seahorse Bioscience, Agilent). Cells (8×10³/well) were seeded in a XFp FluxPak, and then allowed to attach overnight. According to the manufacturer’s protocol, the XFp Cell Mito Stress Test Kit and Seahorse XFp Glycolysis Stress Test Kit (Seahorse Bioscience, Agilent) were used to assessed OCR and ECAR. Both OCR and ECAR measurements were normalized to cell numbers.

Fluorescence Imaging of PAC-C and PAC In Vivo. All animal experiments comply with the NIH Guide for Care and Use of Laboratory Animals. Meanwhile, all experiments were approved by the Committees for Ethical Review of the Fourth Hospital of Harbin Medical University(2021-SCILLSC-21). For the subcutaneous tumor formation assay, 786-O cells (5×10⁶ cells in 200 µL of PBS) subjected to different treatments were injected subcutaneously into the BALB/c nude mice. After the tumor grew to 150 mm³, the mice were injected with 200 µL of PAC-C and PAC (200µM, PBS: DMSO = 98:2 per mouse) through i.v. injection. After injection for 4, 12, 24, 48, and 72h the fluorescence imaging of mice was observed by a CRi Maestro 2 multispectral fluorescence small animal in vivo imaging system. Meanwhile, The biodistribution of major organs was further shown at 24 h post-injection.

Immunohistochemistry. Paraffin-embedded sections of excised ccRCC specimens were immunostained for PKM2 (Proteintech, 15822-1-AP) and OGA protein (Proteintech,14711-1-AP). Staining was performed with the streptavidin biotin peroxidase complex method and according to the manufacturer’s recommendation (Dako, Denmark). The staining was analyzed under a microscope (DP80, Olympus, Japan). Image-Pro Plus (6.0.0.260) was used for quantitative analysis.

Migration and Invasion Assay. Cells (1×10⁵/well) were seeded into upper chambers with 200µL non-FBS cell culture medium which contained PBS, PAC-C, and PAC (50µM, medium: DMSO = 99.5:0.5 v/v), respectively. Matrigel invasion assay precoated with matrigel into upper chambers. 700 µL culture medium containing 10% FBS was added to the lower chamber and cultured at 37°C for 24 hours. Cells on the bottom surface of the inserts were fixed with 4% paraformalde hyde for 15 min and stained with 0.05% crystal violet for 20 min. Then, the number of migrated cells in the bottom surface were counted.

Antitumor Efficacy Assay In Vivo. Tumor-bearing mice were treated with PBS, PAC-C, and PAC (10mg/kg) every 2 days via i.v. administration for five times, respectively. The tumor volumes were measured every 7 days during the experiment.

Establishment of sunitinib-resistant experimental animal models. For the subcutaneous tumor formation, 786-O cells (5×10⁶ cells in 200µL of PBS) subjected to different treatments were injected subcutaneously into the BALB/c nude mice. After the tumor grew to 50 mm³, start sunitinib treatment (30mg/kg). When tumor volume returned to before sunitinib treatment, it was proved that mice were resistant to sunitinib.

Statistical Analysis. All the data are reported as the mean ± Standard Error of Mean (SEM). The in vitro and in vivo experiments were performed at least three independent technical replicates. Student’s t-test and one-way ANOVA analysis were applied. The level of significance was defined at *p < 0.05, **p < 0.01, and ***p < 0.001.

Data availability

The data that support the findings of this study are within the Article, Supplementary Information, or available from the corresponding author upon reasonable request.

Wang Y, Zhang Y, Wang P, Fu X, Lin W. Circular RNAs in renal cell carcinoma: implications for tumorigenesis, diagnosis, and therapy. Mol Cancer 19, 149 (2020).
Marona P, et al. MCPIP1 Downregulation in Clear Cell Renal Cell Carcinoma Promotes Vascularization and Metastatic Progression. Cancer Res 77, 4905–4920 (2017).
D'Costa NM, et al. Y-box binding protein-1 is crucial in acquired drug resistance development in metastatic clear-cell renal cell carcinoma. J Exp Clin Cancer Res 39, 33 (2020).
Yoshino H, et al. PHGDH as a Key Enzyme for Serine Biosynthesis in HIF2alpha-Targeting Therapy for Renal Cell Carcinoma. Cancer Res 77, 6321–6329 (2017).
Torrino S, et al. Mechano-induced cell metabolism promotes microtubule glutamylation to force metastasis. Cell Metab 33, 1342–1357 e1310 (2021).
Icard P, Shulman S, Farhat D, Steyaert JM, Alifano M, Lincet H. How the Warburg effect supports aggressiveness and drug resistance of cancer cells? Drug Resist Updat 38, 1–11 (2018).
Boedtkjer E, Pedersen SF. The Acidic Tumor Microenvironment as a Driver of Cancer. Annu Rev Physiol 82, 103–126 (2020).
Sanchez DJ, Simon MC. Transcriptional control of kidney cancer. Science 361, 226–227 (2018).
Courtney KD, et al. Isotope Tracing of Human Clear Cell Renal Cell Carcinomas Demonstrates Suppressed Glucose Oxidation In Vivo. Cell Metab 28, 793–800 e792 (2018).
Hsu MC, Hung WC. Pyruvate kinase M2 fuels multiple aspects of cancer cells: from cellular metabolism, transcriptional regulation to extracellular signaling. Mol Cancer 17, 35 (2018).
O'Brien CM, Mulukutla BC, Mashek DG, Hu WS. Regulation of Metabolic Homeostasis in Cell Culture Bioprocesses. Trends Biotechnol 38, 1113–1127 (2020).
Sun Y, Connors KE, Yang DQ. AICAR induces phosphorylation of AMPK in an ATM-dependent, LKB1-independent manner. Mol Cell Biochem 306, 239–245 (2007).
Satyanarayana G, et al. Pyruvate kinase M2 regulates fibrosis development and progression by controlling glycine auxotrophy in myofibroblasts. Theranostics 11, 9331–9341 (2021).
Shanmugasundaram K, Nayak BK, Friedrichs WE, Kaushik D, Rodriguez R, Block K. NOX4 functions as a mitochondrial energetic sensor coupling cancer metabolic reprogramming to drug resistance. Nat Commun 8, 997 (2017).
Wang C, et al. Interactome analysis reveals that lncRNA HULC promotes aerobic glycolysis through LDHA and PKM2. Nat Commun 11, 3162 (2020).
Chaneton B, et al. Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature 491, 458–462 (2012).
Hou DY, et al. An activated excretion-retarded tumor imaging strategy towards metabolic organs. Bioact Mater 14, 110–119 (2022).
Hou DY, et al. In Situ Constructed Nano-Drug Depots through Intracellular Hydrolytic Condensation for Chemotherapy of Bladder Cancer. Angew Chem Int Ed Engl 61, e202116893 (2022).
Singh JP, et al. O-GlcNAcase targets pyruvate kinase M2 to regulate tumor growth. Oncogene 39, 560–573 (2020).
An HW, et al. A Near-Infrared Peptide Probe with Tumor-Specific Excretion-Retarded Effect for Image-Guided Surgery of Renal Cell Carcinoma. ACS Nano 14, 927–936 (2020).
Wang Z, et al. Addressable Peptide Self-Assembly on the Cancer Cell Membrane for Sensitizing Chemotherapy of Renal Cell Carcinoma. Adv Mater 31, e1807175 (2019).
Wang L, et al. Transformable Dual-Inhibition System Effectively Suppresses Renal Cancer Metastasis through Blocking Endothelial Cells and Cancer Stem Cells. Small 16, e2004548 (2020).
Li J, et al. Natural Product Micheliolide (MCL) Irreversibly Activates Pyruvate Kinase M2 and Suppresses Leukemia. J Med Chem 61, 4155–4164 (2018).
Lu Z, Hunter T. Metabolic Kinases Moonlighting as Protein Kinases. Trends Biochem Sci 43, 301–310 (2018).
Apostolidi M, et al. Targeting Pyruvate Kinase M2 Phosphorylation Reverses Aggressive Cancer Phenotypes. Cancer Res 81, 4346–4359 (2021).
Wang Y, et al. O-GlcNAcylation destabilizes the active tetrameric PKM2 to promote the Warburg effect. Proc Natl Acad Sci U S A 114, 13732–13737 (2017).
Li L, et al. TAp73-induced phosphofructokinase-1 transcription promotes the Warburg effect and enhances cell proliferation. Nat Commun 9, 4683 (2018).
Chen SK, Wang YC, Lin TY, Wu HJ, Huang CJ, Ku WC. G-Protein-coupled Estrogen Receptor 1 Agonist G-1 Perturbs Sunitinib Resistance-related Phosphoproteomic Signatures in Renal Cell Carcinoma. Cancer Genomics Proteomics 18, 207–220 (2021).
Karam JA, et al. Development and characterization of clinically relevant tumor models from patients with renal cell carcinoma. Eur Urol 59, 619–628 (2011).
Huang J, Zhao X, Li X, Peng J, Yang W, Mi S. HMGCR inhibition stabilizes the glycolytic enzyme PKM2 to support the growth of renal cell carcinoma. PLoS Biol 19, e3001197 (2021).
Bui TO, Dao VT, Nguyen VT, Feugeas JP, Pamoukdjian F, Bousquet G. Genomics of Clear-cell Renal Cell Carcinoma: A Systematic Review and Meta-analysis. Eur Urol, (2022).
Mazurek S, Boschek CB, Hugo F, Eigenbrodt E. Pyruvate kinase type M2 and its role in tumor growth and spreading. Semin Cancer Biol 15, 300–308 (2005).
An HW, et al. A tumour-selective cascade activatable self-detained system for drug delivery and cancer imaging. Nat Commun 10, 4861 (2019).

Acknowledgements

The work was supported by National Key R&D Program of China (2021YFB3801000), National Natural Science Foundation of China (82172000 and 81971135 and U20A20385). All animal and clinical experiments were performed complying with the Committees for Ethical Review of the Fourth Hospital of Harbin Medical University (2021-SCILLSC-21).

Author contributions

^#L. W., B. F., D.-Y. H., Y.-L.-L. and G. Y. contributed equally to this work. W.-H. X., H. W., L.-L. L and L.W. conceived the project and supervised all experiments. B.F., D.-Y. H., designed the experiments and wrote the manuscript. Y.-L. L. and G. Y. performed the experiments. C. L., J.-C. S., and B. K. analyzed the data. C. L. and J.-J. Z. provided support with experimental techniques. L.-B. Z., Y. Q. and H.-L. W. collected clinical samples and data.

Competing interests

The authors declare no competing interests.

Scheme 1 is in the supplementary files section.

There is NO Competing Interest.

SuppleInfoforPKM2.docx
Supplementary Information
Scheme1.docx
Scheme 1

Download PDF

Version 1

posted

You are reading this latest preprint version

PKM2 allosteric converter: a self-assembly peptide for suppressing renal cell carcinoma and sensitizing chemotherapy

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion

Methods

References

Declarations

Schemes

Additional Declarations

Supplementary Files

Status:

Version 1