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. 2023 Nov 23;14(11):761.
doi: 10.1038/s41419-023-06288-9.

_targeting protein methylation in pancreatic cancer cells results in KRAS signaling imbalance and inhibition of autophagy

María F Montenegro #  1 , Román Martí-Díaz #  2 , Ana Navarro  3 , Jorge Tolivia  3 , Luis Sánchez-Del-Campo  2 , Juan Cabezas-Herrera  4 , José Neptuno Rodríguez-López  5
Affiliations

_targeting protein methylation in pancreatic cancer cells results in KRAS signaling imbalance and inhibition of autophagy

María F Montenegro et al. Cell Death Dis. .

Abstract

Pancreatic cancer cells with mutant KRAS require strong basal autophagy for viability and growth. Here, we observed that some processes that allow the maintenance of basal autophagy in pancreatic cancer cells are controlled by protein methylation. Thus, by maintaining the methylation status of proteins such as PP2A and MRAS, these cells can sustain their autophagic activity. Protein methylation disruption by a hypomethylating treatment (HMT), which depletes cellular S-adenosylmethionine levels while inducing S-adenosylhomocysteine accumulation, resulted in autophagy inhibition and endoplasmic reticulum stress-induced apoptosis in pancreatic cancer cells. We observed that by reducing the membrane localization of MRAS, hypomethylation conditions produced an imbalance in KRAS signaling, resulting in the partial inactivation of ERK and hyperactivation of the PI3K/AKT-mTORC1 pathway. Interestingly, HMT impeded CRAF activation by disrupting the ternary SHOC2 complex (SHOC2/MRAS/PP1), which functions as a CRAF-S259 holophosphatase. The demethylation events that resulted in PP2A inactivation also favored autophagy inhibition by preventing ULK1 activation while restoring the cytoplasmic retention of the MiT/TFE transcription factors. Since autophagy provides pancreatic cancer cells with metabolic plasticity to cope with various metabolic stress conditions, while at the same time promoting their pathogenesis and resistance to KRAS pathway inhibitors, this hypomethylating treatment could represent a therapeutic opportunity for pancreatic adenocarcinomas.

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Conflict of interest statement

The University of Murcia holds a patent on the methods used to synthesize and the therapeutic uses of TMCG and the combination therapy with DIPY. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. HMT inhibits basal autophagy and decreases the methylation capacity of pancreatic cancer cells.
A Western blot experiment showing a time-course analysis of LC3-II/LC3-I. PANC1 cells were treated with vehicle or HMT for 48 h followed by treatment with CQ (40 µM) for the indicated times. *P < 0.05. ns = not significant. Differences between vehicle and HMT-treated samples at each time were found significant (#P < 0.05). B Pancreatic cancer cells were treated with vehicle or HMT for 48 h followed (or not) by treatment with CQ (40 µM) for an additional 24 h. Accumulated autophagosomes were evaluated by LC3 staining. *P < 0.05 when compared with vehicle-treated cells without CQ (w/o). C Accumulation of p62 was analyzed by confocal microscopy (left) and Western blot (right) in PANC1 cells after 72 h of treatment with vehicle or HMT. D Effect of HMT and individual treatments [TMCG (10 µM) or DIPY (5 µM)] on the methylation capacity of PANC1 cells. Metabolite determination was performed 72 h after treatment. E Effect of HMT and AdOx (20 µM) on the methylation status (at L309) of the catalytic PP2Ac subunit. The histogram shows the effect of HMT on PP2A activity. Protein and activity analysis was carried out 72 h after each indicated treatment. In this figure, blots are representative of three independent experiments. Error bars show the mean ± SD. *P < 0.05 compared with vehicle-treated controls.
Fig. 2
Fig. 2. HMT inhibited nuclear translocation of MiT/TFE proteins in PDAC.
A In normal cells and under nutrient-rich conditions, MiT/TFE proteins are repressed by mTORC1 via phosphorylation and remain in the cytosol; however, upon starvation, mTORC1 is inactivated and MiT/TFE proteins enter the nucleus, where they activate autophagy-lysosome gene transcription. Interestingly, this mechanism was found to be decoupling in PDAC [21]. Although overexpression of the nucleocytoplasmic transporter importin (IPO-7/8) was proposed to facilitate the nuclear translocation of MiT/TFE, the high PP2A activity found in autophagic PDAC cells could also explain the MiT/TFE factors decoupling mechanism. The possible mechanism by which HMT inhibits basal autophagy in PDCA is also depicted. B Efficient PP2Ac silencing in PANC1 cells promotes cytosolic accumulation of MITF and TFE3 factors (*P < 0.05 when compared with siControl transfected cells). Blots were overexposed (TFE3over) for cytosolic TFE3 quantification. C Fluorescence microscopy showing the effect of PP2Ac silencing on the localization of TFE3 in Hs766T cells. The line bar (30 µm) was used as a template for fluorescence intensity determination. Relative occupancy of TFE3 in the nucleus (Nu) and cytosol (Cyt) was calculated by analysis of the fluorescence intensity under the curve (IUC) in each compartment. *P < 0.05 when compared with siControl transfected cells. D HMT promotes cytosolic accumulation of MITF and TFE3 factors in PANC1 cells (*P < 0.05 when compared with vehicle-treated cells). Blots were overexposed (TFE3over) for cytosolic TFE3 quantification. E HMT avoids nuclear translocation of TFE3 and MITF in Hs766T cells. Cells were treated with vehicle or HMT (72 h) or rapamycin (0.2 µM; 120 min). Cell extracts were analyzed by Western blotting for the indicated proteins (left panel). Nuclear localization of MITF and TFE3 was also evaluated by confocal microscopy (right panels) in Hs766T cells treated with vehicle or HMT (72 h). F Relative expression of proteins 72 h after each indicated treatment. Histogram represents the relative amount of proteins with respect to actin. *P < 0.05 when compared with vehicle-treated cells. G HMT treatment (72 h) causes aberrant lysosomal morphology and increased size as shown by LysoTracker staining. Inset: magnified view. Graph (right) quantification of lysosome diameter in HMT (N = 173) and vehicle-treated cells (N = 169). Bar, mean. H Confocal microscopy showing lysosome number and distribution in autophagic cells and HMT-treated cells. Lysosomes were stained with LysoTracker-Red DND-99 and an iABP Pan cathepsin probe (green). Differences in the number of lysosomes were significant (*P < 0.01) when compared with untreated autophagic cells (graph, right). I TEM showing lysosome localization in autophagic cells and HMT-treated cells. The arrow indicates the position of lysosomes. The boxed area contains representative lysosomes at 60,000× magnification. Lysosomes were randomly distributed in the cytoplasm of autophagic PANC1 cells, but HMT-treated cells showed grouped lysosomes.
Fig. 3
Fig. 3. HMT activates AKT/mTORC1 and prevents ULK1 activation in PANC1 cells.
A Western blot showing the effects of HMT on AKT and S6K phosphorylation under basal autophagy. B Western blots showing the effect of HMT on AKT phosphorylation after rapamycin (0.2 µM) treatment. Before rapamycin treatment, the cells were treated with vehicle or HMT for 72 h. C Western blots showing the effects of HMT on S6K and ULK1 phosphorylation under rapamycin-induced autophagy. The graph represents the level of phosphorylated ULK1 (at S637) at the indicated times after rapamycin (0.2 µM) exposure. Before rapamycin treatment, the cells were treated with vehicle or HMT for 72 h. Error bars show the mean ± SD. D Western blots showing the effects of HMT and HMT/IAKT on S6K and ULK1 phosphorylation under rapamycin-induced autophagy (0.2 µM). Before rapamycin treatment, the cells were treated with vehicle, HMT, or a combination of HMT and IAKT (10 µM) for 72 h. In this figure, the blots shown are representative of three independent experiments.
Fig. 4
Fig. 4. Effects of HMT on MAPK signaling pathway.
A Western blots showing the effects of HMT with and without EGF stimulation, and U0126 (10 µM; 24 h) on several components of the MAPK pathway in PANC1 cells. B Effect of PP2A (left panel) or SHOC2 (right panel) silencing on CRAF phosphorylation at S259 in PANC1 cells. The silencing effect was determined according to the corresponding siControl. C HMT reduces coimmunoprecipitation of MRAS with SHOC2 and CRAF. Total PANC1 extracts (1 mg) were immunoprecipitated using anti-MRAS, and bound proteins were immunoblotted with SHOC2 or CRAF antibodies. D A model for CRAF activation following dephosphorylation of S259 by the SHOC2 complex. E HMT modulates the localization of MRAS in PANC1 and Hs766T cells as determined by confocal microscopy (line bars were used as a template for fluorescence intensity determination) and Western blot analysis. F The effect of HMT on CRAF/BRAF heterodimerization in the absence or presence of MG132. For assays without MG132, PANC1 cells were treated with vehicle or HMT for 72 h. For assays in the presence of MG132 (10 µM), cells were treated with vehicle or HMT for 67 h, followed by treatment with MG132 for an additional 5 h. Total cell extracts (1 mg) were immunoprecipitated using anti-BRAF, and bound proteins were immunoblotted with a CRAF antibody. G Effect of HMT on BRAF stability in PANC1 cells in the presence or absence of MG132 (10 µM) as indicated in the previous panel. Total cell extracts were used for BRAF quantification. *P < 0.05 compared with vehicle-treated cells; **P < 0.05 compared with HMT-treated cells in the absence of MG132.
Fig. 5
Fig. 5. HMT suppresses MAPK but activates PI3K activity.
A Accumulated autophagosomes in PANC1 cells subjected to the indicated treatments were evaluated by LC3 staining. For these assays, PANC1 cells were treated with vehicle or U0126 (10 µM) for 24 h followed by treatment with CQ (40 µM) for an additional 6 h. For combined HMT/U0126 treatment, HMT-pretreated cells (24 h) were treated with U0126 and CQ as described above. *P < 0.05 compared with vehicle-treated cells. **P < 0.05 compared with U0126-treated cells. B Effect of HMT on U0126-induced protective autophagy. Representative Western blot of LC3B and p62 from PANC1 cells treated with vehicle, U0126, or HMT/U0126 as indicated above and then treated with CQ (40 µM) for an additional 6 h. C Western blots showing the effects of U0126 and/or HMT treatments on AKT phosphorylation. For individual treatments, PANC1 cells were treated with U0126 (10 µM; 24 h) or HMT (48 h). For combined HMT/U0126 treatment, HMT-pretreated cells (24 h) were treated with U0126 (10 µM) for an additional 24 h. D The effect of HMT on RAS activation in PANC1 cells was determined by two independent methods. The cells were treated with vehicle or HMT for 72 h and then stimulated or not with EGF. Left panel: the active form of RAS was carefully isolated from endogenous lysates using a KRAS activation assay kit that makes use of CRAF RBD agarose beads. Using a polyclonal anti-KRAS antibody, Western blot analysis was used to identify the precipitated GTP-KRAS. Error bars show the mean ± SD. *P < 0.05 compared with vehicle-treated cells. Right panel: after EGF stimulation, RAS activation was assayed using an ELISA-based assay for the quantification of GTP-bound RAS and normalized to its level in unstimulated cells. Error bars indicate the SD. *P < 0.05 compared with the respective unstimulated cells. **P < 0.05 compared with vehicle + EGF-treated cells.
Fig. 6
Fig. 6. HMT induces ER stress-dependent apoptosis in PANC1 cells.
A TEM image showing ribosomes in autophagic (vehicle treatment) and HMT-treated (72 h) PANC1 cells. The boxed area contains representative ribosomes at ×60,000 magnification. Ribosomes were quantified using ImageJ as indicated in the “Methods” section. *P < 0.05 compared with untreated controls. B PANC1 cells were treated with HMT for 72 h and then analyzed by TEM. They exhibited vacuolation of the cell cytoplasm due to swelling of the ER cisternae. C Western blots showing the expression of the indicated proteins in PANC1 cells after 72 h of vehicle or HMT treatment. The histograms show the relative amounts of Bcl-2 and p-Bcl-2 versus β-actin and Bcl-2, respectively, under different treatments. *P < 0.05 compared with untreated controls. D Schematic representation of HMT inducing ER stress-dependent apoptosis. E Confocal microscopy showing Bcl-2 and mitochondrial localization in PANC1 cells. F HMT significantly decreased cell viability in PANC1 cells compared with individual treatments [TMCG (10 µM) or DIPY (5 µM)] and those including U0126 (10 µM), wortmannin (WORT; 0.2 µM), or methotrexate (MTX, 10 µM). Cells were subjected to the indicated treatment for 72 h. Following HMT treatment, a reduction in cell viability was accompanied by an increase in apoptosis (as determined by TUNEL) and the cleavage of PARP and caspase 3 (as indicated by a time-dependent reduction in full-length caspase 3). Histograms represent the apoptotic factor (assuming an apoptotic factor of 1 for PANC1 untreated cells) evaluated using a DNA fragmentation assay. *P < 0.05 compared with untreated controls. G Luciferase imaging of vehicle (control) and HMT-treated mice at 21 days post-tumor cell injection (N = 3). Firefly luciferin (120 mg/kg) was injected intraperitoneally.
Fig. 7
Fig. 7. Proposed mechanism(s) by which impaired protein methylation inhibits autophagy in KRAS-mutated PDAC tumors.
PDAC cell growth depends on high basal autophagy [18]. In addition to mTORC1 inactivation, starvation also causes an increase in PP2A activity toward ULK1, a mTORC1 substrate whose dephosphorylation at S637 is required for autophagy induction. Strong PP2A activity toward MiT/TFE proteins and ULK1 can explain the contradictory coexistence of strong basal autophagy activity and intact mTORC1 function in KRAS-mutated PDAC tumors. Under such conditions, activation of MRAS by protein methylation maintains and operates the RAS–MEK–ERK pathway to ensure cell proliferation, growth and survival. However, mechanisms for maintaining autophagy and cell survival are disrupted under hypomethylating conditions (HMT). Impaired protein methylation induced a KRAS signaling imbalance that resulted in attenuation of the MAPK pathway but hyperactivation of the PI3K/AKT pathway. The high levels of AKT activation (by phosphorylation at S473) in the presence of HMT failed to inhibit mTORC1 even in the presence of its specific inhibitor rapamycin. Therefore, by activating mTORC1 while inhibiting the methylation/activation of PP2A, HMT induced strong autophagy inhibition, which has been found to protect KRAS-mutated PDAC tumors from drugs designed to specifically _target the MAPK pathway [–8]. Residual ERK2 activation maintained by SHOC2-independent mechanisms together with the disruption of PP2A-dependent control of anti-apoptotic Bcl-2 rendered the pancreatic cancer cells unable to defend themselves against cumulative damage resulting from HMT-induced ER stress.
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