Targeting Cancer Metabolism Plasticity with JX06 Nanoparticles via Inhibiting PDK1 Combined with Metformin for Endometrial Cancer Patients with Diabetes

Abstract Diabetes is closely related to the occurrence of endometrial cancer (EC) and its poor prognosis. However, there is no effective clinical treatment for EC patients with diabetes (patientEC+/dia+). To explore new therapeutic targets, Ishikawa is cultured with high glucose (IshikawaHG) mimicking hyperglycemia in patientEC+/dia+. Subsequently, it is discovered that IshikawaHG exhibits glucose metabolic reprogramming characterized by increased glycolysis and decreased oxidative phosphorylation. Further, pyruvate dehydrogenase kinase 1 (PDK1) is identified to promote glycolysis of IshikawaHG by proteomics. Most importantly, JX06, a novel PDK1 inhibitor combined metformin (Met) significantly inhibits IshikawaHG proliferation though IshikawaHG is resistant to Met. Furthermore, a reduction‐sensitive biodegradable polymer is adopted to encapsulate JX06 to form nanoparticles (JX06‐NPs) for drug delivery. It is found that in vitro JX06‐NPs have better inhibitory effect on the growth of IshikawaHG as well as patient‐derived EC cells (PDC) than JX06. Additionally, it is found that JX06‐NPs can accumulate to the tumor of EC‐bearing mouse with diabetes (miceEC+/dia+) after intravenous injection, and JX06‐NPs combined Met can significantly inhibit tumor growth of miceEC+/dia+. Taken together, the study demonstrates that the combination of JX06‐NPs and Met can target the cancer metabolism plasticity, which significantly inhibits the growth of EC, thereby provides a new adjuvant therapy for patientsEC+/dia+.

substances. Protein concentration of the solution was determined by a BCA protein quantitation method. The solution containing 50 µg protein was disulfide reduced with 5 mM tris -(2-chloroethyl) -phosphoric acid (TCEP) (room temperature, 30 minutes) and alkylated with 10 mm IAA (room temperature, 30 minutes in the dark).
Then trichloroacetic acid (TCA) was added to precipitate the protein. The precipitated protein was separated by centrifugation at 14000 g for 30 min, and the particles were washed three times in acetone to remove residual TCA. The dried protein was then reconstituted in 50 mM Tris-HCl (pH 8.2) containing 8 M urea solution. The solution was diluted to a targeted urea concentration of 2 M by 50 mM Tris HCl, and the protein was digested at a 1 / 50 (w / w) enzyme-protein ratio at 37 °C overnight. The same amount of peptide samples was first cleaned with C18 column (based on the monolithic technology of Merck KGaA company in Darmstadt, Germany). Then the obtained peptide sample was loaded onto the column. The liquid passing through the column was labeled as FT, which contained peptides that did not bind or weakly bound to the C18 beads. The column was washed with 300 μl ddH 2 O, eluted with 200 μl methanol, and vacuum-dried for the LC-MS/MS analysis. Each sample was separated by HPLC liquid phase system Easy nLC with nanoliter flow rate. The samples were separated by chromatography and analyzed by Q-Exactive mass spectrometer. Data analysis: the original data of mass spectrometry analysis was a raw file, and MaxQuant software was used for database identification and quantitative analysis.

Western blot
The expressions of PDK1, P-AKT and P-GSK3β proteins were detected by Western blot. Cell lysate (RIPA: protease inhibitor: phosphorylated protease inhibitor = 100:2:1) was added to the cell dish to extract the all proteins in the cell. The protein was quantified by the Coomassie brilliant blue protein quantitation method. An appropriate separation gel was selected according to the molecular weight. 40 μg of protein sample was added per lane, and the samples were separated by SDS-PAGE gel electrophoresis. Then the protein was transferred to the NC membrane. 5% milk or 5% BSA was used for blocking for one hour. Anti-PDK1, P-AKT, P-GSKβ and β-catenin were diluted with antibody diluents at 1000:1 and incubated at 4 ℃ overnight. The next day, after washing the NC membrane, secondary antibody was incubated, and the scanning quantitative analysis was carried out on an Odyssey infrared fluorescence scanning imaging system.

Lactate measurement
We seeded 3×10 5 ishikawa cells in six-well plates for 24 h and then replaced the medium with 3 mL of fresh medium with different treatments. After treatments for 24h, cell supernatant was collected, and then lactate production of cells was measured by infinite M200 multifunctional enzyme-labeling instrument according to the instructions of a Lacate Assay Kit (Njjcbio, A019-2-1).

In vivo study
BALb/c nude mice, 5-week of age, were purchased from the animal laboratory of the People's Hospital of Peking University (Beijing, China) and raised under SPF conditions. The animal experiment was approved by the ethics committee of Peking University People's hospital (2020PHE093), and the ethical requirements of experimental animals and the animal welfare law were strictly adhered to during the experimental operations. BALB/ c nude mice were divided into two major groups, including a diabetic group (Mice Dia+ ) and a non-diabetic group (Mice Dia-), First, nude mice in the Mice Dia+ group received intraperitoneal injection of streptozotocin (1% w/V solution in fresh cold sodium citrate buffer, pH 4.5) and high fat feed. Nude mice in the Mice Diagroup were given the same volume of citric acid buffer and normal feed. The blood glucose level of mice was detected by blood sugar device. When any random blood glucose concentration went over 11.1mmol/l, the modeling was considered successfully. Ishikawa cell line, of 100 μl which roughly included 3×10 6 cells, was inoculated subcutaneously in nude mice, then ishikawa bearing BALB/c mice with diabetes (mice EC+/dia+ ) and non-diabetes (mice EC+/dia-) were constructed.
The Ishikawa cells in Mice Dia+/sh-NC group were transfected with a PDK1 negative control vector, and the cells in the Mice Dia+/sh-PDK1 group were transfected with shRNA-PDK1. When the tumor volume reached about 500 mm 2 , the mice EC+/diain saline group were treated with saline (n=5) and in Met group were treated with Met dissolved in drinking water (500 mg/L) daily (n=5). The mice EC+/dia+ in saline group were treated with saline (n=5) and in Met group were treated with Met dissolved in drinking water (500 mg/L) daily (n=5). The mice EC+/diain JX06 group were injected with JX06 1.5 mg/kg (n=5), in JX06-NPs group were injected with JX06-NPs (equivalent to 1.5 mg/kg JX06) (n=5), in JX06+Met group were treated with Met and JX06 1.5 mg/kg, and in JX06-NPs+Met group were treated with Met and JX06-NPs (equivalent to 1.5 mg/kg JX06). The tumor size was measured every 3 days, and the formula used for the calculation of tumor volume was v = a × b 2 × 0.52, in which a represents the long diameter of the tumor and b represents the short diameter of the tumor. After the experiment, nude mice were killed, and the subcutaneous tumors were removed, photographed, and recorded. In addition, tissue samples were fixed, paraffin-embedded and sectioned for follow-up studies.

Immunohistochemical staining
Immunohistochemical staining was performed on EC tissue arrays, purchased from Superbiotek, Shanghai, including 118 EC tissue samples (patient EC+ ) and 17 normal control endometrial tissue samples adjacent to EC specimens (patient NC ).
Immunohistochemical staining was also performed on the tumor tissues from mice EC+/dia+ after treatments. The paraffin sections were dewaxed. High temperature and pressure repair method was used for antigen repair. Antigen repair solution (pH6.0 citric acid) was added to the pressure cooker and boiled at a high temperature.
The slices were soaked in 3% H 2 O 2 for 30 min, removing endogenous peroxidase, and were washed by PBS for 5min for 3 times. Rabbit anti-PDK1 antibody (1:50) or rabbit anti-PDHA1 (Phospho S293) (1:400) was added to the section and put into the wet box at 4 ℃ overnight. The next day, after rewarming for 30min, it was again rinsed with PBS for 5 min, which was repeated 3 times. HRP-labeled Goat anti-rabbit IgG was added to the sections and incubated at room temperature for 30 min and 3 times washed with PBS for 5 min. DAB solution was added to the tissue, and the color was observed under the microscope. The slides were immersed in water to stop further staining. The slides were counterstained with hematoxylin and differentiated by hydrochloric acid alcohol solution. The slides were dehydrated with 100% ethanol, sealed, observed, scanned, and photographed under a microscope. The results were analyzed by the Fromowitz comprehensive scoring method ①staining intensity score: 0 for no stain, 1 for light yellow particles significantly standing out from the background, 2 for light brown-yellow particles, and 3 for a large number of dark brown particles; ② Score of positive cells: the number of positive cells in 500 cells randomly counted in each piece, < 5% is 0, 5% ~ 25% is 1, 26% ~ 50% is 2, 51% ~ 75% is 3, and > 75% is 4. The sum of the score for staining intensity and the proportion of positive cells was calculated and graded: < 2 points was considered negative (-), 2 ~ 3 points was considered weakly positive (+), 4 ~ 5 points was moderately positive (+), 6 ~ 7 points were highly positive (+ + +), and + ~ + + + were