Fig 1: Elaiophylin exploits MAPK activation as a vulnerability in drug-resistant ovarian cancer. a Left panel: representative light microscopy images of doxycycline (DOX)-inducible iKRAS cells cultured in the absence or presence of DOX (2 μg/mL) for 48 h and exposed to 0.75 µM elaiophylin for 6 h. Arrows indicate cytoplasmic vacuoles. Scale bar: 50 µm. Right panel: proportion of cells displaying vacuoles was scored by visually examining at least 100 cells. Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t test, **p < 0.01). b Assessment of indicated protein levels using western blotting in iKRAS cells cultured in the absence or presence of DOX (2 μg/mL) for 48 h and then exposed to 0.75 µM elaiophylin for 6 h. c The viability of iKRAS cells cultured in the absence or presence of DOX (2 μg/mL) for 48 h and then exposed to 0.75 µM elaiophylin for 24 h. Data represent three independent experiments and are shown as mean ± SD (Two-tailed unpaired Student’s t test, ***p < 0.001). d A schematic diagram of elaiophylin-induced MAPK activation in parental and resistant cells. e Assessment of indicated protein levels using western blotting in indicated parental and resistant cells exposed to 0.5 µM elaiophylin for 6 h. f The viability of indicated parental and resistant cells exposed to 0.5 µM elaiophylin for 36 h. The results are presented as percentages of the control. Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t test, *p < 0.05, **p < 0.01). g Female immune-deficient BALB/c nu/nu (nude) mice were inoculated subcutaneously with C13* cells and were treated with vehicle, cisplatin (10 mg/kg/2d), oxaliplatin (10 mg/kg/2d), or elaiophylin (2 mg/kg/d) (n = 5 in each group). From the beginning of treatment, tumor volumes were measured to plot tumor growth curves. Data are mean ± SEM (Two-tailed unpaired Student’s t test, ***p < 0.001). h Female immune-deficient BALB/c nu/nu (nude) mice were inoculated subcutaneously with OVCAR8 (taxane-resistant) cells and were treated with vehicle, paclitaxel (30 mg/kg/2d), docetaxel (30 mg/kg/2d), or elaiophylin (2 mg/kg/d) (n = 5 in each group). From the beginning of treatment, tumor volumes were measured to plot tumor growth curves. Data are mean ± SEM (Two-tailed unpaired Student’s t test, *p < 0.05, ***p < 0.001). i Female immune-deficient BALB/c nu/nu (nude) mice were inoculated subcutaneously with A2780 (PARPi-resistant) cells and were treated with vehicle, olaparib (50 mg/kg/d), talazoparib (0.33 mg/kg/d), or elaiophylin (2 mg/kg/d) (n = 5 in each group). From the beginning of treatment, tumor volumes were measured to plot tumor growth curves. Data are mean ± SEM (Two-tailed unpaired Student’s t test, **p < 0.01, ***p < 0.001). j Sections of tumors from (i) were stained using p-SHP2 Tyr542 antibody (red), p-ERK1/2 antibody (green), and DAPI (blue). Representative sections are shown. Scale bar: 20 µm. k Sections of tumors from (i) were stained using CHOP antibody (red), DAPI (blue), and using TUNEL assay (green). Representative sections are shown. Scale bar: 20 µm. l Quantification of the mean fluorescence intensity and TUNEL-positive proportion in (j) and (k). Four randomly selected viewing fields were individually evaluated per section and five sections were assessed per group. Data are mean ± SEM (Two-tailed unpaired Student’s t test, **p < 0.01, ***p < 0.001)
Fig 2: CRISPR library screening identifies PTPN11 as a critical gene for elaiophylin-induced paraptosis. a A diagram for the genome-wide CRISPR/Cas9 knockout library screening. b Elaiophylin selection identified potential target genes. The top 10 positive selected genes were ranked by p value. c Top: SKOV3 cells were exposed for 24 h to DMSO or 0.5 µM elaiophylin and subject to CETSA. Bottom: the intensity of the SHP2 bands or PTPRM bands was quantified to exhibit the binding affinity of elaiophylin to SHP2 or PTPRM in SKOV3 cells. d SPR analysis showing direct interaction between elaiophylin and human recombinant SHP2 protein at the molecular level. e Pattern diagram of SHP2/SOS1/MAPK pathway. f Assessment of indicated protein levels using western blotting in SKOV3 cells exposed to 0.5 µM elaiophylin. g Left panel: representative light microscopy images of SKOV3 and OVCAR8 cells exposed to 0.5 µM elaiophylin for 9 h after PTPN11 knockdown. Arrows indicate cytoplasmic vacuoles. Scale bar: 50 µm. Right panel: proportion of cells displaying vacuoles was scored by visually examining at least 100 cells. Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t test, *p < 0.05, ***p < 0.001). h Assessment of indicated protein levels using western blotting in SKOV3 cells exposed to 0.5 µM elaiophylin for 9 h after PTPN11 knockdown. i The viability of SKOV3 and OVCAR8 cells exposed to 0.5 µM elaiophylin for 36 h after PTPN11 knockdown. Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t test, **p < 0.01, ***p < 0.001)
Fig 3: Elaiophylin-induced paraptosis partially contributes to autophagy inhibition by impairing lysosomal function. a Assessment of indicated protein levels using western blotting in SKOV3 cells exposed to 0.5 µM elaiophylin for 9 h after ATG7 or ULK1 knockdown. b Assessment of indicated protein levels using western blotting in SKOV3 cells exposed to 0.5 µM elaiophylin for 24 h after PTPN11 knockdown. c Assessment of indicated protein levels using western blotting in SKOV3 cells exposed to 0.5 µM elaiophylin for 24 h alone, or in combination with CHX (5 µM pretreatment for 4 h), U0126 (15 µM), SHP099 (30 µM), BAY293 (5 µM), respectively. d FACS analysis of LysoTracker Deep Red in SKOV3 cells exposed to 0.5 µM elaiophylin for 24 h after PTPN11 knockdown. e Mean fluorescence intensities in (d) were quantified. Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t test, ***p < 0.001). f FACS analysis of LysoTracker Deep Red in SKOV3 cells exposed to 0.5 µM elaiophylin for 24 h alone, or in combination with CHX (5 µM pretreatment for 4 h), U0126 (15 µM), SHP099 (30 µM), BAY293 (5 µM), respectively. g Mean fluorescence intensities in (f) were quantified. Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t-test, **p < 0.01, ***p < 0.001). h, i SKOV3 cells were exposed to 0.5 µM elaiophylin for 36 h after PTPN11 knockdown, and subject to enzymatic activity determination of CTSB (h) and CTSD (i). Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t test, **p < 0.01, ***p < 0.001). j, k SKOV3 cells were exposed to 0.5 µM elaiophylin alone for 36 h, or in combination with CHX (5 µM pretreatment for 4 h), U0126 (15 µM), SHP099 (30 µM), BAY293 (5 µM), respectively, and subject to enzymatic activity determination of CTSB (j) and CTSD (k). Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001). l A schematic diagram of paraptosis and consequent autophagy inhibition induced by elaiophylin. Chemical structure depiction of elaiophylin was obtained from NCBI, PubChem Compound Summary for CID 6444206, Elaiophylin. Retrieved 2 January, 2022 from https://pubchem.ncbi.nlm.nih.gov/compound/Elaiophylin
Fig 4: SHP2 inhibition attenuates the effects of elaiophylin in PDX models. a PDX models were established on female NOG mice using the tumor tissues from a high-grade serous ovarian cancer patient, and were treated with vehicle, elaiophylin (2 mg/kg/d), SHP099 (30 mg/kg/d), or the combination of elaiophylin (2 mg/kg/d) and SHP099 (30 mg/kg/d) (n = 5 in each group). From the beginning of treatment, tumor volumes were measured to plot tumor growth curves. Data are mean ± SEM (Two-tailed unpaired Student’s t test, ***p < 0.001). b Photograph of resected tumor tissues from PDX models. Scale bar: 1 cm. c Quantification of tumor weight in (b). Data are mean ± SEM (Two-tailed unpaired Student’s t test, ***p < 0.001). d Representative images of immunohistochemical staining with indicated antibodies in tumor specimens from (b). Scale bar: 25 µm. e Quantification of immunohistochemical scores in (d). Five sections were assessed per group and the mean of four randomly selected viewing fields were evaluated for every section. Data are mean ± SEM (Two-tailed unpaired Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001). f, g Enzymatic activity of CTSB (f) and CTSD (g) in tumor tissues from (b). The results are presented as percentages of the vehicle group. Data are mean ± SEM (Two-tailed unpaired Student’s t test, n = 5, *p < 0.05, **p < 0.01, ***p < 0.001)
Supplier Page from Abcam for Recombinant Human SHP2 protein (His tag C-Terminus)