Nuclear MAST4 Suppresses FOXO3 through Interaction with AKT3 and Induces Chemoresistance in Pancreatic Ductal Carcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with a 5-year survival rate of less than 10%. Furthermore, the acquisition of anticancer drug resistance makes PDAC treatment difficult. We established MIA-GEM cells, a PDAC cell line resistant to gemcitabine (GEM), a first-line anticancer drug, using the human PDAC cell line-MIA-PaCa-2. Microtubule-associated serine/threonine kinase-4 (MAST4) expression was increased in MIA-GEM cells compared with the parent cell line. Through inhibitor screening, dysregulated AKT signaling was identified in MIA-GEM cells with overexpression of AKT3. MAST4 knockdown effectively suppressed AKT3 overexpression, and both MAST4 and AKT3 translocation into the nucleus, phosphorylating forkhead box O3a (FOXO3) in MIA-GEM cells. Modulating FOXO3 target gene expression in these cells inhibited apoptosis while promoting stemness and proliferation. Notably, nuclear MAST4 demonstrated higher expression in GEM-resistant PDAC cases compared with that in the GEM-sensitive cases. Elevated MAST4 expression correlated with a poorer prognosis in PDAC. Consequently, nuclear MAST4 emerges as a potential marker for GEM resistance and poor prognosis, representing a novel therapeutic target for PDAC.

Keywords: FOXO3; MAST4; gemcitabine resistance; pancreatic ductal adenocarcinoma; stemness.

Figure 1

GEM-resistant cell line derived from human PDAC cell line—MIA-PaCa-2. (A) GEM sensitivity of MIA-P and MIA-GEM cells. (B–G) Characteristics of MIA-GEM cells in comparison with MIA-P cells: morphology (B), cell growth (C), GEM-induced apoptosis (D), in vitro invasion (E), sphere formation (F), and expression of stemness-related genes (G) with the semi-quantification (H). Scale bar, 50 μm. Error bars represent the standard deviations from three independent trials. Statistical differences were calculated using Student’s t-test. PDAC, pancreatic ductal adenocarcinoma; MIA-P, parental MIA-PaCa-2; MIA-GEM, gemcitabine-resistant MIA-PaCa-2; GEM, gemcitabine; NS, nucleostemin.

Figure 2

MAST4 expression in PDAC cells. (A) MAST4 expression in GEM-resistant human PDAC cells. (B) Relationship between MAST4 expression and GEM IC50. (C) MAST4 protein in GEM-treated MIA-GEM cells. (D) MAST4 knockdown by AS. Right panel, semi-quantification of (C,D). (E) GEM sensitivity in MAST4-knocked down MIA-GEM cells. (F) GEM sensitivity in MAST4-knocked down PDAC cells. (G) Expression of known GEM-resistant genes. Error bar, standard deviation from three independent trials. Statistical differences were calculated using Student’s t-test. PDAC, pancreatic ductal adenocarcinoma; MIA-P, parental MIA-PaCa-2; MIA-GEM, GEM-resistant MIA-PaCa-2; GEM, gemcitabine; MAST4, microtubule-associated serine/threonine kinase family member 4; MIAA, MIAB; GEM-resistant MIA-PaCa-2 [8]; Panc1G, GEM-resistant Panc1 [8]; Capan2G, GEM-resistant Capan2 [8]; IC50, 50% inhibition concentration; C, sense S-oligonucleotide; AS, antisense S-oligonucleotide; RPM, NB-ARC domain-containing disease resistance protein; MRP, melittin-derived peptide; MDR, multiple drug resistance; hENT, human equilibrative nucleoside transporter; dCK, deoxycytidine kinase.

Figure 3

Multiple drug resistance in MIA-GEM cells. (A–E) Drug sensitivity of MIA-GEM cells to 5FU (A), CDDP, L-OHP, CPT-11, and PTX. (B). (F) Growth of subcutaneous tumors in nude mice. (G) Effect of MAST4 knockdown on tumor growth. Red arrows indicate GEM administration (F,G). Error bar, standard deviation from three independent trials or five mice. Statistical differences were calculated using Student’s t-test. PDAC, pancreatic ductal adenocarcinoma; MIA-P, parental MIA-PaCa-2; MIA-GEM, GEM-resistant MIA-PaCa-2; GEM, gemcitabine; MAST4, microtubule-associated serine/threonine kinase family member 4; C, sense S oligonucleotide; AS, antisense S oligonucleotide; Tx, GEM treatment; 5FU, 5-fluorouracil; CDDP, cisplatin; L-OHP, oxaliplatin; CPT-11, irinotecan; PTX, paclitaxel.

Figure 4

AKT3 in MIA-GEM cells. (A) Heat map of MIA-GEM cell-inhibitory effect of signal blockers. Inhibitors unrelated to AKT are shown in the left column, and AKT-related inhibitors are shown in the right column. Heatmap values indicate cell growth inhibition rate. (B) Effect of MAST4 knockdown on protein expression of AKT family. (C) Effect of AKT3 knockdown on mRNA expression (upper) and protein (lower) of MAST4 and AKT family. (D) GEM sensitivity in AKT3-knocked down MIA-GEM cells. (E) Phosphorylation levels of AKT3 signal proteins. (F) Protein levels of AKT family in GEM-resistant PDAC cells. (G) GEM sensitivity in AKT3-knocked down PDAC cells. (H) Effect of AKT3 knockdown on other anti-cancer drugs. Error bar, standard deviation from three independent trials. Statistical differences were calculated using Student’s t-test. PDAC, pancreatic ductal adenocarcinoma; MIA-P, parental MIA-PaCa-2; MIA-GEM, GEM-resistant MIA-PaCa-2; MIAA, MIAB; GEM-resistant MIA-PaCa-2 [8]; Panc1G, GEM-resistant Panc1 [8]; Capan2G, GEM-resistant Capan2 [8]; GEM, gemcitabine; MAST4, microtubule-associated serine/threonine kinase family member 4; C, sense S-oligonucleotide; AS, antisense S-oligonucleotide; siC, control short interfering RNA; siAKT3, short interfering RNA to AKT3; GSK3β, glycogen synthase kinase 3β; TOR, target of rapamycin; PRAS40, proline-rich AKT substrate of 40-kDa; 5FU, 5-fluorouracil; CDDP, cisplatin; L-OHP, oxaliplatin; CPT-11, irinotecan; PTX, paclitaxel.

Figure 5

Role of miR-582-5p on MAST4 and AKT3 expression. (A) Expression of miR-582-5p in MIA-GEM cells. (B) Effect of inhibition of miR-582-5p on expression of MAST4 and AKT3. Upper, mRNA, lower, protein. (C) Effect of inhibition of miR-582-5p on GEM sensitivity in MIA-GEM cells. Error bar, standard deviation from three independent trials. Statistical differences were calculated using Student’s t-test. PDAC, pancreatic ductal adenocarcinoma; MIA-P, parental MIA-PaCa-2; MIA-GEM, GEM-resistant MIA-PaCa-2; GEM, gemcitabine; MAST4, microtubule-associated serine/threonine kinase family member 4; miR-I, miR-582-5p inhibitor.

Figure 6

Intranuclear association between MAST4 and AKT3. (A) Intracellular localization of MAST4. (B) Nuclear localization of MAST4 (left) and proximity assay of MAST4 and AKT3 (right). Scale bar, 20 μm. (C) Physical interaction between MAST4 and AKT3 in the nuclei. (D) Phosphorylation of FOXO3 in the nuclei. (E) Effect of inhibition of MAST4 kinase by AX13587 on AKT3 phosphorylation. (F) Effect of MAST4 knockdown on phosphorylation of FOXO3 in the nuclei in PDAC cells. (G) Expression of apoptosis-associated genes of FOXO3 targets. (H) Effect of MAST4 knockdown on GEM-induced apoptosis. (I) Expression of stemness-associated genes of FOXO3 targets. Error bar, standard deviation from three independent trials. Statistical differences were calculated using Student’s t-test. PDAC, pancreatic ductal adenocarcinoma; MIA-P, parental MIA-PaCa-2; MIA-GEM, GEM-resistant MIA-PaCa-2; MIAA, MIAB; GEM-resistant MIA-PaCa-2 [8]; Panc1G, GEM-resistant Panc1 [8]; Capan2G, GEM-resistant Capan2 [8]; GEM, gemcitabine; MAST4, microtubule associated serine/threonine kinase family member 4; WCL, whole cell lysate; cytosol, cytosol fraction; nuclear, nuclear fraction; ip, immunoprecipitant, C, sense S-oligonucleotide; AS, antisense S-oligonucleotide; CB, Coomassie blue; FOXO3, forkhead box protein O3; pFOX3, phosphorylated FOXO3; pAKT3, phosphorylated AKT3; BIM, BCL2 like 11; PUMA, p53 upregulated modulator of apoptosis; FLIP, Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein; BCL2, B cell lymphoma 2; NS, nucleostemin.

Figure 7

MAST4 expression in human PDAC cases. (A) Immunohistochemical detection of MAST4 in the nuclei of PDAC cases. Insets show high magnification. Middle panels, MAST4 immunohistochemistry images in mouse tumors of MIA-P (negative control) and MIA-GEM (positive control). Lower panels, MAST4 immunohistochemistry images in colon mucosa (negative control) and bladder urothelium (positive control). (B–D) Survivals between MAST4-high and -low cases; overall (B), stage II (C), and stage III (D). The MAST4-high and MAST4-low groups were divided based on the median MAST4 expression. Survival curves were calculated using the Kaplan–Meier method. Statistical differences were calculated using the log-rank test. PDAC, pancreatic ductal adenocarcinoma; MIA-P, parental MIA-PaCa-2; MIA-GEM, GEM-resistant MIA-PaCa-2; GEM, gemcitabine; MAST4, microtubule-associated serine/threonine kinase family member 4.