BRSK2 induced by nutrient deprivation promotes Akt activity in pancreatic cancer via downregulation of mTOR activity

Abstract

Neoplastic cells in pancreatic ductual adenocarcinoma (PDAC) survive in an energy-deprived milieu, and hyper-activation of Akt is thought to contribute to the neoplastic cell survival in PDAC. Kras activating mutations, common in PDAC, was believed to be the major driver of Akt activation. However, the inhibitor to Kras was not therapeutic for PDAC patients. This implied that PDAC cells might harbor an intrinsic merit that strengthens Akt activity. Here we showed that BRSK2, a serine/threonine-protein kinase of AMPK family, was induced by nutrient deprivation in PDAC cells and suppressed mTORC1 activity via phosphorylation of tuberous sclerosis complex 2 (TSC2). The suppression of mTORC1 activity in PDAC results in a dominant loss of feedback inhibition on Akt activity by mTORC1, consequently enhancing cell survival. This finding indicates that the intrinsic molecular merit that BRSK2 provides is a survival advantage to PDAC cells and strengthens the invasiveness of these neoplastic cells in energy-deprived environments.

Keywords: AMPK; Akt; BRSK2; PDAC; mTOR.

Figure 1. BRSK2 was upregulated in PDAC and IPMN tumors

(A) BRSK2 expression patterns in pancreas and IPMN/PDAC tumor (tumor: i, IPMN; ii &iii, PDAC). Comparison of BRSK2 expression in PDAC and IPMN with non-tumor exocrine ducts. scale bar: 20 μm. (B) BRSK2 expression in an array of human cells including PANC-1, AsPC-1, H1299, U2OS, SK-Hep-1, HepG2, Focus, HuH-7, 7721, and PC-3.

Figure 2. BRSK2 expression enhances cancer cells survival in vitro

(A) Cell survival ability of different cells upon nutrient deprivation. Different cells were subjected to no-glucose culturing media, and survival assay (MTS/PMS) was performed at appropriate time points (24-144 Hours). (B) Overexpression of BRSK2 increased cell survival under conditions of nutrient deprivation. PANC-1 cells were transiently transfected with series amount of HA-BRSK2 constructs. After 72 hours of no-glucose treatment, cells were stained with PI and subjected to FACS for survival assay. And the cell survival rate increased along with the amount of exogenously expressed BRSK2. (C) Effects of BRSK2 on cell survival during nutrient deprivation. Cells were transiently transfected with constructs exogenously expressing BRSK2 (or kinase-dead mutant BRSK2KD). Then the cells were subjected to no-glucose culturing media for 72 hours before performing the cell survival assay (FACS assay). (D) Knock-down of endogenous BRSK2 expression decreased cell survival upon nutrient deprivation. PANC-1 cells were transiently transfected with siRNA fragment specifically targeting BRSK2, and the cells were recorded with microscope 48 hours after no-glucose treatment. (E) Stable over-expression of BRSK2 increased cell survival under conditions of nutrient deprivation. Mono-clone PANC-1 cell line stably expressing HA BRSK2 was constructed (neo as the control cell line). The stable cell lines were seeded in 96-well plates, and were subjected to no-glucose culturing media. The survival assay (MTS/PMS) was performed at appropriate time points (n=6).

Figure 3. BRSK2 expression strengthened cell survival in vivo and activated Akt

(A, B) Histology showed the necrosis in the core of xenograft tumors from PANC-1 highly expressing BRSK2 and control PANC-1. Scale bar: 40 μm. (C, D) IHC of pmTOR (Ser 2448) and pAkt substrate in xenograft tumors from BRSK2-overexpressing PANC-1 and control PANC-1. Scale bar: 20 μm.

Figure 4. The relationship of BRSK2, pAkt substrate and pmTORS (2448) in cancer cells of human PDAC tissues

(A) BRSK2 and pAkt substrate expression pattern in PDAC and the correlation of BRSK2 and pAkt substrate expression by IHC. Scale bar, 100μm. (B) The detection of BRSK2 and pAkt substrate expression levels in PDAC tissues with western blotting. The expression levels of BRSK2 and Akt activity were correlated in PDAC tissues (n=12). (C) BRSK2, pAkt substrate and pmTOR (Ser2448) expression pattern in consecutive sections of PDAC tissues. Scale bar, 200μm.

Figure 5. BRSK2 in PDAC cells strengthens Akt activity via TSC2-dependent mTORC1 repression

(A) Effect of 2-DG treatment on BRSK2 and other components of AKT/mTOR signaling pathway. PANC-1 cells were treated with series concentrations of 2-DG for three hours before analysis. (B) Overexpression of BRSK2 inhibited S6K and 4EBP-1 phosphorylation. PANC-1 cells were transiently co-transfected with pCMV-HA-BRSK2 and pCMV-HA-S6K (or pFLAG-CMV4-4EBP1) constructs. (C) Inhibition of S6K phosphorylation upon energy deprivation was alleviated when BRSK2 was knocked-down. PANC-1 cells were first transiently transfected with BRSK2 specific siRNA fragment or control fragment. 3 days after transfection, cells were treated with regular media plus 2-DG or no glucose media for three hours. (D) BRSK2 phosphorylated TSC2 on Ser1387 in vitro. (E) BRSK2 expression levels are positively correlated with the levels of TSC2 (Ser1387) in human PDAC. We have co-immunostained the consecutive frozen sections of human PDAC samples (n=14) with pTSC2 (Ser1387) or BRSK2 and Cytokeratin. Scale bar: 50 μm. (F) Activation of TSC2 phosphorylation upon energy deprivation was impeded when BRSK2 was knocked-down. PANC-1 cells were first transiently transfected with BRSK2 specific siRNA fragment or control fragment. 3 days after transfection, cells were treated with regular media plus 2-DG for three hours. (G) Scheme of BRSK2's role on Akt activation in PDAC. BRSK2 was significantly upregulated in the nutrient deprivation micro-environment, which inhibited mTOR via phosphorylating TSC2. This repression of mTOR may cause a loss of feedback inhibition on Akt activation. And hyperactivation of Akt possibly would attribute to increased survival in PDAC.