Small-Molecule MMRi62 Induces Ferroptosis and Inhibits Metastasis in Pancreatic Cancer via Degradation of Ferritin Heavy Chain and Mutant p53

Abstract

High frequency of KRAS and TP53 mutations is a unique genetic feature of pancreatic ductal adenocarcinoma (PDAC). TP53 mutation not only renders PDAC resistance to chemotherapies but also drives PDAC invasiveness. Therapies targeting activating mutant KRAS are not available and the outcomes of current PDAC treatment are extremely poor. Here, we report that MMRi62, initially identified as an MDM2-MDM4-targeting small molecule with p53-independent pro-apoptotic activity, shows anti-PDAC activity in vitro and in vivo. We show that MMRi62 inhibits proliferation, clonogenic, and spheroid growth of PDAC cells by induction of cell death. MMRi62-induced cell death in PDAC is characteristic of ferroptosis that is associated with increased autophagy, increased reactive oxygen species, and lysosomal degradation of NCOA4 and ferritin heavy chain (FTH1). In addition to induced degradation of FTH1, MMRi62 also induces proteasomal degradation of mutant p53. Interestingly, MMRi62-induced ferroptosis occurs in PDAC cell lines harboring either KRAS and TP53 double mutations or single TP53 mutation. In orthotopic xenograft PDAC mouse models, MMRi62 was capable of inhibiting tumor growth in mice associated with downregulation of NCOA4 and mutant p53 in vivo. Strikingly, MMRi62 completely abrogated metastasis of orthotopic tumors to distant organs, which is consistent with MMRi62's ability to inhibit cell migration and invasion in vitro. These findings identified MMRi62 as a novel ferroptosis inducer capable of suppressing PDAC growth and overcoming metastasis.

Fig.1. selectively inhibits proliferation and induces cell death in (QM-PDA) subtypes pancreatic cancer lines.

A, Top, chemical structure of MMRi62. Middle, growth curves of pancreatic cell lines in the presence of MMRi62 in 72h proliferation assay. Bottom, the IC 50 concentrations for each cell line and their RAS and TP53 status of each cell line was shown. B, Top, MMRi62 induces pancreatic cancer cell death. Representative images of trypan blue exclusion assay in MMRi62-treated Panc1 and BxPc3 cells at indicated concentrations for 24h. C, nontreated control. Bottom, Quantitative results of live cell fractions for Panc1 (left) and BxPc3 (right). *P < 0.05. C, Effect of MMRi62 on clonogenic growth. Cells were treated with indicated concentrations of MMRi62 for 24h followed culturing in the absence of the drug for 14 days before violet blue staining. D, Left, the shapes of spheroids of Panc1 and BxPc3 after 4mM MMRi62 for continuous treatment under light microscope at various days (D1 to D10) d and the HE staining of the final day cultures were shown. E, Quantification of the average spheroid diameter (SD in mm) at various time points for Panc1 (upper) and BxPC-3 (lower)

Fig.2. induces autophagic cell death associated with downregulation of p53.

A, MMRi62 induces downregulation of p53 expression in pancreatic cancer cells. WB analysis of p53, MDM2 and MDM4 in Panc1 and BxPc3 treated for 24h with indicated concentrations of MMRi62 (μM). B, MMRi62 induces increased autophagy and apoptosis. WB analysis of p53, cleaved PARP and LC3 in Panc1 or BxPC3 cells treated with MMRi62 at indicated concentrations for 72h. C, IF staining of p53 and LC3 in Panc1 and BxPc3 after 72h treatment with 2mM MMRi62. D. MMRi62 treatment induces increase in cellular ROS after MMRi62 treatment at two concentrations for 72h. Left, Panc1, p=0.0003 and <0.0001 for C vs 2 and 4 μM MMRi62 respectively; right, BxPc3, p=<0.0001 for C vs 2 or 4 μM MMRi62 respectively.

Fig.3. induces lysosomal degradation of FTH1 and NCOA4 and proteasomal degradation of mut-p53 in PDAC cells.

A, WB analysis of NCOA4 and FTH1in Panc1 and BxPc3 cells treated with indicated concentrations of MMRi62 (μM) for 24h. Tubulin and GAPDH were used as loading controls. B, MMRi62 promotes lysosomal degradation of FTH1and NcoA4 and proteasomal degradation of mut-p53. Upper, WB analysis of the indicated proteins in Panc1 treated with indicated concentrations of MMRi62 for 24h in the presence of either 50nM of Bafilomycin B1 (BAF) or 200 nM Carfizomib (CFZ). Lower, the same as in Upper except for using BxPC3 cells., C. In vivo ubiquitnation of mut-p53 in MMRi62-treated Panc1 cells. Panc1 cells were transfected with His-tag-ubiquitin expression plasmid followed by MMRi62 treatment and his-tag pulldown and WB for p53 and polyubiquitin. Ub-p53 and polyubiquitin adducts were indicated. D, Knockdown of mut-p53 increases FTH1 expression. WB analysis of indicated proteins in BxPc3 or HPAFII cells after infection with lentiviral expressing control shRNA (shSer) or shp53. GAPDH serves as loading control. E, IC50 changes in the indicated cells.

Fig.4. inhibits growth of pancreatic tumor in orthotopic xenograft mouse models associated with downregulation of p53 and NCOA4 expression.

A, Top, Panc1luc cells were implanted orthotopically into the pancreas of SCID mice followed by MMRi62 treatment via intraperitoneal injection at doses of 25 mg/kg at every other day for 5 injections, 2 days/week for 2 weeks. The dorsal luciferase signals in the mice bearing Panc1luc orthotopic tumors were captured in different days during and after treatment. Middle, mouse body weight (left) and tumor BLI signal in Flux (Right) measured in indicated days in vehicle-control (V) mice and MMRi62-treated (M) mice were shown. Bottom, At the termination day of the experiments, median tumor weight in gram and median tumor size in cm in the two groups of mice were shown. *P =0.0002 and <0.00001 for Panc1 tumor weight and tumor size between V and M groups; and *P =0.001 and <0.0001 for BxPc3 tumor weight and tumor size between V and M groups. B, the same in A except for BxPc3luc cells. C, HE staining of Panc1luc and BxPc3luc orthotopic tumors collected on Day 37 in MMRi62-treated (M) as compared with vehicle-treated mice (V). D, WB analysis of the resected tumor tissues on Day 37 for the expression of p53 and NCOA4 in V and M group of mice. E, Immunofluorescence staining of activate caspase 3 (AC3) for in Panc1luc and BxPc3luc orthotopic tumors collected on Day 37 in MMRi62-treated (M) as compared with vehicle-treated mice (V).

Fig.5. prevents invasion in vitro and metastasis of pancreatic cancer cells in vivo.

A, MMRi62 treatment inhibits cell migration in wound-healing assay. Confluent monolayers of Panc1 (Left) and BxPc3 cells (right) were scratched with a 1mL pipette tip at experimental time zero and then were cultured with or without presence of MMRi62 (4mmol/L). The images of cell monolayers were taken at 24, 48 and 72h after the scratch and representative images were shown. Quantification of migration distance (MD in μm) were shown in histograms on left. B, MMRi62 inhibits Panc1 cell invasion in transwell assay. Left, a representative image of Panc1 cells transpassed to the other side of membrane when cells are cultured in medium without (C) or with 4mM MMRi62 (M) for 24h. Right, quantitative analysis of the transpassed cell numbers in this assay. C, MMRi62 inhibits BxPc3 cell invasion in transwell assay. The experiment was done similarly as in B except with BxPc3 cells. D, MMRi62 inhibits metastasis in orthotopic PDAC tumors. On the termination day of the in vivo efficacy experiments, mice bearing orthotopic tumors of Panc1 (left) and BxPc3 (Right) were examined for tumor metastasis to various organs. The numbers of mice with metastasis to the peritoneum, liver, and lungs were counted and presented. E, HE staining of liver metastasis in mice bearing Panc1 and BxPc3 metastasis orthotopic tumors in vehicle control (C) and MMRi62-treated (M) mice were shown. Dark and dense areas are metastatic tumors in C group.

Fig.6.. Gemcitabine resistant PDAC cells remain sensitive to MMRi62 in vitro.

A, Combination index (CI) plots for Panc1 cells (top) and BxPc3 cells (bottom) derived from dose-effect data of MMRi62 combination with gemcitabine in 72h proliferation assay. Gem, gemcitabine. CI <1, synergism, CI=1, additive, CI>1, antagonism. B, drug response curves of BxPc3 and Gem-R-BxPc3 cells to gemcitabine (top) and to MMRi62 (bottom, IC50=2.38±0.88 and 3.72±0.36μM, respectively for BxPc3 and Gem-R-BxPc3). C, drug response curves of HPAFII and Gem-R-HPAFII cells to gemcitabine (top) and to MMRi62 (bottom, IC50=4.04±0.59 and 6.62±0.52μM, respectively for HPAFII and Gem-R-HPAFII).