Fer governs mTORC1 regulating pathways and sustains viability of pancreatic ductal adenocarcinoma cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a high percentage of morbidity. The deciphering and identification of novel targets and tools for intervening with its adverse progression are therefore of immense importance. To address this goal we adopted a specific inhibitor of the intracellular tyrosine kinase Fer, whose expression level is upregulated in PDAC tumors, and is associated with poor prognosis of patients. Subjecting PDAC cells to the E260-Fer inhibitor, unraveled its simultaneous effects on the mitochondria, and on a non-mitochondrial ERK1/2 regulatory cascade. E260 caused severe mitochondrial deformation, resulting in cellular- aspartate and ATP depletion, and followed by the activation of the metabolic sensor AMPK. This led to the phosphorylation and deactivation of the bona fide AMPK substrate, RAPTOR, which serves as a positive regulator of the mTORC1 metabolic hub. Accordingly, this resulted in the inhibition of the mTORC1 activity. In parallel, E260 downregulated the activation state of the ERK1/2 kinases, and their ability to neutralize the mTORC1 suppressor TSC2, thereby accentuating the inhibition of mTORC1. Importantly, both activation of AMPK and downregulation of ERK1/2 and mTORC1 were also achieved upon the knockdown of Fer, corroborating the regulatory role of Fer in these processes. Concomitantly, in PDAC tumors and not in healthy pancreatic tissues, the expression levels of Fer demonstrate moderate but statistically significant positive correlation with the expression levels of mTOR and its downstream effector LARP1. Finally, targeting the Fer driven activation of mTORC1, culminated in necrotic death of the treated PDAC cells, envisaging a new intervention tool for the challenging PDAC disease.

Keywords: AMPK; E260; Fer; mTORC1; mitochondria; pancreatic ductal adenocarcinoma.

Figure 1

Death profiles evoked by E260 in PDAC cells. (A) PANC-1 cells were subjected to 5 µM E260 for 24, 48, and 72 hr. The number of live cells in each time point was determined using an automatic cell counter after the addition of Trypan blue to the samples. (B) The percentage of live cells is also shown. (C, D) Similar experiments and analyses were carried out with the SU.86.86 cells that were subjected to 2.5 µM, for 24, 48, and 72 hr. Data represent average values of three independent experiments that gave similar results. Standard deviations and P values are presented.

Figure 2

E260 evokes necrotic death in PDAC cells. (A) PANC-1 (I-III), and (B) SU.86.86 (I, II) cells were left untreated or subjected to 5 µM, or 2.5 µM E260, respectively, for 48 hr. Cells were stained with Annexin V-FITC and PI. Staining was quantified by FACS ARIAIII. All data were analyzed using FlowJo software. AIII and BII present respective average values of PANC-1 and SU.86.86 cell cohorts, obtained from three independent experiments that gave similar results. Standard deviations and P values are presented.

Figure 3

(A) TEM morphology images of PANC-1 cells exposed to E260 for 24 hr. Treated and fixed cells were inspected under TEM after vehicle treatment alone (I, III), or 2µM E260 for 24 hr (II, IV). Red arrows indicate mitochondria. Nu - cell nucleus. Magnification bars are given in the bottom of each image. (B) TEM morphology images of PANC-1 cells after 48 hr exposure to E260. PANC-1 inspected under TEM after vehicle treatment alone (I, III), or E260 treatment for 48 hr (II, IV). Red arrows indicate mitochondria. Nu - cell nucleus. Magnification bars are given in the bottom of each image. (C) TEM morphology images of PANC-1 cells after 72 hr exposure to E260. PANC-1 cell inspected under TEM after vehicle treatment alone (I, III), or E260 treatment for 72 hr (II, IV). Red arrows indicate mitochondria. Purple arrows denote autophagosomes, and blue arrows indicate disruption of the cells plasma membrane. Nu - cell nucleus. Magnification bars are given in the bottom of each image. (D) Cells were left untreated or subjected to E260 for 48 and 72 hr. Whole cell lysates were resolved in SDS-PAGE and reacted with: anti- NDUFA, and anti-ATP5A, in a WB analysis. (E) Lysates prepared from the cells in (D) were also taken for aspartate levels determination. Each panel of the above represent one out of three independent experiments that gave similar results is presented.

Figure 4

E260 leads to ATP depletion and activation of AMPK in PDAC cells. SU.86.86 and PANC-1 cells were left untreated or subjected to 2.5 µM E260 for 48 hr, in MEM supplied with 2mM L-glutamine. Cells were then harvested, and their lysates were, (A) subjected to ATP analysis, or (B) resolved in SDS-PAGE and reacted with: anti-phospho-AMPK (Thr. 172), anti-AMPK α1, anti-phospho-RAPTOR (Ser 792), anti-Raptor, and anti-tubulin, antibodies, in a WB analysis. One out of three independent experiments that gave similar results is presented.

Figure 5

E260 inhibits ERK1/2 signaling cascade and mTORC1 activation in PDAC cells. SU.86.86 and PANC-1 cells were left untreated or subjected to 2.5 µM E260 for 48 hr, in MEM supplied with 2mM L-glutamine. Cells were then harvested, and their lysates were resolved in SDS-PAGE and subjected to (A) anti-pospho-ERK1/2 (Thr.202/Tyr204), anti-ERK1/2, anti-phspho-TSC2 (Ser 664), anti-TSC, and anti-Tubulin. (B) anti-phospho-mTOR (Ser 2448), anti-mTOR, and anti-Tubulin, in a WB analysis. (C) Asparagine alleviates the death level evoked by E260 in PDAC cells. SU.86.86 cells were left untreated or treated with 2.5 µM E260, in the absence or presence of 4 mM asparagine, for 48 hr. The percentage of viable cells in each sample was determined using an automatic cell counter after the addition of Trypan blue to the sample. Data represent average values of three independent experiments that gave similar results. Standard deviations and P values are presented. (D) Lysates from each sample were resolved in SDS-PAGE and reacted with: anti-phospho-mTOR (Ser 2448), anti-mTOR (left panel), anti- phosphor-S6K (Thr389), anti-S6K (right panel), and anti-Tubulin, in a WB analysis. In each panel, one out of three independent experiments that gave similar results is presented.

Figure 6

Knockdown of Fer activates AMPK and downregulates ERK1/2 in PDAC cells. (A) SU.86.86. cells were transfected with control siRNA (siRNAc), or fer-targeting siRNA (siRNAfer) and incubated in MEM supplied with 2 mM L-glutamine, for 72 hr. Cells were then harvested, and their lysates were resolved in SDS-PAGE and subjected to: anti-Fer (SH2), anti-pospho-ERK1/2 (Thr.202/Tyr204), anti-ERK1/2, anti-phspho-TSC2 (Ser 664), anti-TSC, anti-phospoh-AMPK (Thr172), anti-AMPK, anti-phospho-mTOR (Ser 2448), anti-mTOR, and anti-Tubulin, in a WB analysis. One out of three independent experiments that gave similar results is presented. (B) Summarizing scheme depicting the two regulatory pathways affected by E260 and converging to the downregulation of mTORC1 and mitochondrial function, in PDAC cells.

Figure 7

The expression level of Fer shows positive correlation with the expression level of mTOR and LARP1- in PDAC tumors. Scatter plot of Fer vs mTOR expression (A), and Fer vs LARP1 expression levels (B), in PDAC tumors taken from TCGA, and in normal human pancreatic tissue taken from GTEX. The Pearson correlation r and the p values are given. (C) SU.86.86 cells were left untreated or subjected to 2.5 µM E260 for 72 hr. Lysates resolved in SDS-PAGE were subjected to anti-mTOR, and anti-Tubulin, in a WB analysis. One out of three independent experiments that gave similar results is presented.