Asparaginyl endopeptidase improves the resistance of microtubule-targeting drugs in gastric cancer through IQGAP1 modulating the EGFR/JNK/ERK signaling pathway

Figures & data

Table 1 Clinical characteristics of patients

Characteristics	Number of patients (N=120)
Age (years)
Median	60.5 (28–76)
Gender, n (%)
Male	64 (53.3)
Female	56 (46.7)
ECOG PS, n (%)
0	6 (5.0)
1	88 (73.3)
2	26 (21.7)
Pathological type, n (%)
Highly/moderately differentiated adenocarcinoma	38 (31.7)
Less/poorly differentiated adenocarcinoma	82 (68.3)
Prior therapy, n (%)
Surgery only	6 (5.0)
Surgery and adjuvant chemotherapy	34 (28.3)
None	80 (66.7)

Figure 1 The analysis of survival time.

Notes: (A) The median PFS was 5.0 months (95% CI: 4.61–5.39 months). (B) The median OS was 12.0 months (95% CI: 11.10–12.89 months).
Abbreviations: CI, confidence interval; PFS, progression-free survival time; OS, overall survival time.

Figure 2 The analysis of survival time according to AEP expression.

Notes: PFS (A) and OS (B) were longer in AEP low-expression patients than in AEP high-expression patients (P<0.01).
Abbreviations: AEP, asparaginyl endopeptidase; PFS, progression-free survival time; OS, overall survival time.

Table 2 The expression level of AEP in the serum of gastric cancer patients’ peripheral blood with different clinical response

	AEP (pg/mL)	P-value
Responder (n=42)	276.85±165.12	0.001
Nonresponder (n=78)	382.51±161.47

Note: Data presented as mean ± standard deviation.

Abbreviation: AEP, asparaginyl endopeptidase.

Table 3 The expression of AEP in the serum of peripheral blood at baseline and the survival time

	PFS (months, 95% CI)	OS (months, 95% CI)
> median (n=60)	5.0 (4.51–5.49)	10 (9.06–10.94)
≤ median (n=60)	6.0 (5.05–6.95)**	14 (11.83–16.16)**

Notes: The median value of AEP was 334.80 pg/mL.

** P<0.01.

Abbreviations: AEP, asparaginyl endopeptidase; CI, confidence interval; OS, overall survival time; PFS, progression-free survival time.

Figure 3 Construction of stable AEP knockout and overexpressive gastric cancer cell lines and the sensitivity to different chemotherapeutic drugs.

Notes: (A and B) AEP was suppressed in AEP knockout cells and increased in AEP overexpressive cells by Western blot assay. (C and D) The cell viability was investigated when oxaliplatin, irinotecan, 5-FU, paclitaxel, docetaxel, and T-DM1 treated NC and AEP-KO gastric cancer cells. **P<0.01.
Abbreviations: AEP-KO, asparaginyl endopeptidase knockout; AEP-OE, AEP overexpression; NC, negative control; 5-FU, 5-fluorouracil.

Figure 4 Analysis of AEP interacting proteins.

Notes: (A) The pathways were associated with AEP by KEGG_pathway analysis. (B) The interacting proteins including >10 peptides were shown in the Venn diagram, and the overlapping proteins were listed.
Abbreviations: AEP, asparaginyl endopeptidase; MAPK, mitogen-activated protein kinase.

Figure 5 IQGAP1 could interact with AEP in gastric cancer cells.

Notes: (A and D) IQGAP1 was one of the interacting proteins when used M2-FLAG beads to pull down the proteins. Five percent of the total lysate purified FLAG-tagged proteins were loaded as input. (B and E) IQGAP1 could be detected when AEP monoclonal antibody was used to extract the endogenous interacting proteins. (C and F) IQGAP1 was increased when AEP was knocked out.
Abbreviations: AEP, asparaginyl endopeptidase; KO, AEP knockout.

Figure 6 AEP and MDR1 were decreased at mRNA level when AEP was knocked out, both in SGC7901 (A) and MKN45 (B) gastric cancer cell lines.

Notes: Glyceraldehyde 3-phosphate dehydrogenase was used for an endogenous reference to standardize the mRNA expressive level. **P<0.01.
Abbreviations: AEP, asparaginyl endopeptidase; NC, negative control; KO, AEP knockout.

Figure 7 JNK and ERK signaling pathways and MDR1 were inhibited in AEP knockout gastric cancer cell lines, and vice versa.

Notes: (A) The expression of MDR-1, p-EGFR, p-JNK, p-ERK and p-Rac1/cdc42 were decreased in AEP-KO cells, but the expression of p-p38 and total EGFR, JNK, ERK, Rac1/cdc42 and p38 did not change. (B) The expressions of MDR1, p-EGFR, p-JNK, p-ERK, and p-Rac1/cdc42 were increased in AEP-OE cells. Vinculin and tubulin were used as an internal control. (C) The AEP-OE cells were treated with varying concentrations of SP600125 or PD98059, and both could inhibit the expressions of MDR1, p-JNK, and p-ERK. (D–F) The IC₅₀ of docetaxel, paclitaxel, and T-DM1 in AEP-OE cells was calculated with the treatment of SP600125 or PD98059 for 24 h. *P<0.05; **P<0.01.
Abbreviations: AEP-KO, asparaginyl endopeptidase knockout; AEP-OE, AEP overexpression; IC₅₀, half maximal inhibitory concentration.

Figure S1 Expression of genes in EGFR/mitogen-activated protein kinase signaling pathway at mRNA level. There was no change in these genes when AEP was knocked out.

Abbreviations: AEP, asparaginyl endopeptidase; NC, negative control; KO, AEP knockout.

Table S1 The concentration of chemotherapeutic agents and targeting drugs

Concentration (μg/mL)	SGC7901	MKN45
Oxaliplation	3.67	1.99
Irinotecan	7.35	5.83
5-Fluorouracil	11.53	6.19
Paclitaxel	3.14	1.01
Docetaxel	4.49	1.13
T-DM1	0.047	0.035

Table S2 The primer sequence of real-time polymerase chain reaction

Gene name	5′ to 3′
GAPDH	Forward: TGAACGGGAAGCTCACTGG
	Reverse: GCTTCACCACCTTCTTGATGTC
AEP	Forward: CACCGGTTCGTCAGGAATCCCATTG
	Reverse: AAACCAATGGGATTCCTGACGAACC
EGFR	Forward: CGCCCACAACCTTTCTTCAA
	Reverse: ATCCGACCTAATGACGCCTT
IQGAP1	Forward: TTTATTCGGGCAAACAAAGC
	Reverse: TGATAACCTCTTCCCGCATC
H-RAS	Forward: GGACGAATACGACCCCACTA
	Reverse: ATGGCAAACACACACAGGAA
K-RAS	Forward: TGTCATCTTGCCTCCCTACC
	Reverse: TTCTCTTGAGCCCTGAGGAA
N-RAS	Forward: ACAAAACAAGCCCACGAACT
	Reverse: CCCTGAGTCCCATCATCACT
B-RAF	Forward: CTGCCATTCCGGAGGAG
	Reverse: GTGGATTATGCTCCCCACC
MEK1	Forward: TCCAAAATGCCCAAGAAGAA
	Reverse: CCTCCAGCTTCTTCTGCAAG
MEK2	Forward: GCTTCTACGGGGCCTTCTAC
	Reverse: GCTGACTTTCCCCAGGATCT
ERK1	Forward: GGGAGATCCAGATCCTGCT
	Reverse: GGTCAGTCTCCATCAGGTCC
ERK2	Forward: ACCAACCTCTCGTACATCGG
	Reverse: CTGGCAGTAGGTCTGGTGCT
MDR1	Forward: GTGGGGCAAGTCAGTTCATT
	Reverse: TCTTCACCTCCAGGCTCAGT
MRP1	Forward: GGCTTATTTCGGATCAACGA
	Reverse: TGAATGGGTCCAGGTTCATT
GST-π	Forward: ACCTCCGCTGCAAATACATC
	Reverse: TCAGCGAAGGAGATCTGGTC

Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; AEP, asparaginyl endopeptidase.

Table S3 The interacting proteins involving >10 peptides in SGC7901 gastric cancer cell line

Gene accession	Gene name
P11021	78 kDa glucose-regulated protein OS=Homo sapiens GN=HSPA5 PE=1 SV=2–[GRP78_HUMAN]
P46940	Ras GTPase-activating-like protein IQGAP1 OS=H. sapiens GN=IQGAP1 PE=1 SV=1–[IQGA1_HUMAN]
O14744	Protein arginine N-methyltransferase 5 OS=H. sapiens GN=PRMT5 PE=1 SV=4–[ANM5_HUMAN]
P22626	Heterogeneous nuclear ribonucleoproteins A2/B1 OS=H. sapiens GN=HNRNPA2B1 PE=1 SV=2–[ROA2_HUMAN]
P08238	Heat shock protein HSP 90-beta OS=H. sapiens GN=HSP90AB1 PE=1 SV=4–[HS90B_HUMAN]
P11142	Heat shock cognate 71 kDa protein OS=H. sapiens GN=HSPA8 PE=1 SV=1–[HSP7C_HUMAN]
P07900	Heat shock protein HSP 90-alpha OS=H. sapiens GN=HSP90AA1 PE=1 SV=5–[HS90A_HUMAN]
Q3V6T2	Girdin OS=H. sapiens GN=CCDC88A PE=1 SV=2–[GRDN_HUMAN]
Q15208	Serine/threonine-protein kinase 38 OS=H. sapiens GN=STK38 PE=1 SV=1–[STK38_HUMAN]
P07437	Tubulin beta chain OS=H. sapiens GN=TUBB PE=1 SV=2–[TBB5_HUMAN]
P38646	Stress-70 protein, mitochondrial OS=H. sapiens GN=HSPA9 PE=1 SV=2–[GRP75_HUMAN]
P68371	Tubulin beta-4B chain OS=H. sapiens GN=TUBB4B PE=1 SV=1–[TBB4B_HUMAN]
Q00839	Heterogeneous nuclear ribonucleoprotein U OS=H. sapiens GN=HNRNPU PE=1 SV=6–[HNRPU_HUMAN]
Q9BQE3	Tubulin alpha-1C chain OS=H. sapiens GN=TUBA1C PE=1 SV=1–[TBA1C_HUMAN]
P98175	RNA-binding protein 10 OS=H. sapiens GN=RBM10 PE=1 SV=3–[RBM10_HUMAN]
P61978	Heterogeneous nuclear ribonucleoprotein K OS=H. sapiens GN=HNRNPK PE=1 SV=1–[HNRPK_HUMAN]
Q9BQA1	Methylosome protein 50 OS=H. sapiens GN=WDR77 PE=1 SV=1–[MEP50_HUMAN]
O75688	Protein phosphatase 1B OS=H. sapiens GN=PPM1B PE=1 SV=1–[PPM1B_HUMAN]
P08107	Heat shock 70 kDa protein 1A/1B OS=H. sapiens GN=HSPA1A PE=1 SV=5–[HSP71_HUMAN]
P54105	Methylosome subunit pICln OS=H. sapiens GN=CLNS1A PE=1 SV=1–[ICLN_HUMAN]
P14625	Endoplasmin OS=H. sapiens GN=HSP90B1 PE=1 SV=1–[ENPL_HUMAN]
P08195	4F2 cell-surface antigen heavy chain OS=H. sapiens GN=SLC3A2 PE=1 SV=3–[4F2_HUMAN]
P19338	Nucleolin OS=H. sapiens GN=NCL PE=1 SV=3–[NUCL_HUMAN]
P19474	E3 ubiquitin-protein ligase TRIM21 OS=H. sapiens GN=TRIM21 PE=1 SV=1–[RO52_HUMAN]
Q9Y4E8	Ubiquitin carboxyl-terminal hydrolase 15 OS=H. sapiens GN=USP15 PE=1 SV=3–[UBP15_HUMAN]
P09651	Heterogeneous nuclear ribonucleoprotein A1 OS=H. sapiens GN=HNRNPA1 PE=1 SV=5–[ROA1_HUMAN]
P38159	RNA-binding motif protein, X chromosome OS=H. sapiens GN=RBMX PE=1 SV=3–[RBMX_HUMAN]
Q9Y2H1	Serine/threonine-protein kinase 38-like OS=H. sapiens GN=STK38L PE=1 SV=3–[ST38L_HUMAN]
P08670	Vimentin OS=H. sapiens GN=VIM PE=1 SV=4–[VIME_HUMAN]

Table S4 The interacting proteins involving >10 peptides in MKN45 gastric cancer cell line

Gene accession	Gene name
Q13813	Spectrin alpha chain, nonerythrocytic 1 OS=Homo sapiens GN=SPTAN1 PE=1 SV=3–[SPTN1_HUMAN]
Q01082	Spectrin beta chain, nonerythrocytic 1 OS=H. sapiens GN=SPTBN1 PE=1 SV=2–[SPTB2_HUMAN]
Q15149	Plectin OS=H. sapiens GN=PLEC PE=1 SV=3–[PLEC_HUMAN]
P05187	Alkaline phosphatase, placental type OS=H. sapiens GN=ALPP PE=1 SV=2–[PPB1_HUMAN]
P80723	Brain acid soluble protein 1 OS=H. sapiens GN=BASP1 PE=1 SV=2–[BASP1_HUMAN]
P09923	Intestinal-type alkaline phosphatase OS=H. sapiens GN=ALPI PE=1 SV=2–[PPBI_HUMAN]
P46940	Ras GTPase-activating-like protein IQGAP1 OS=H. sapiens GN=IQGAP1 PE=1 SV=1–[IQGA1_HUMAN]
P11021	78 kDa glucose-regulated protein OS=H. sapiens GN=HSPA5 PE=1 SV=2–[GRP78_HUMAN]
P10809	60 kDa heat shock protein, mitochondrial OS=H. sapiens GN=HSPD1 PE=1 SV=2–[CH60_HUMAN]
O14744	Protein arginine N-methyltransferase 5 OS=H. sapiens GN=PRMT5 PE=1 SV=4–[ANM5_HUMAN]
P08238	Heat shock protein HSP 90-beta OS=H. sapiens GN=HSP90AB1 PE=1 SV=4–[HS90B_HUMAN]
Q9NYL9	Tropomodulin-3 OS=H. sapiens GN=TMOD3 PE=1 SV=1–[TMOD3_HUMAN]
Q14247	Src substrate cortactin OS=H. sapiens GN=CTTN PE=1 SV=2–[SRC8_HUMAN]
P22626	Heterogeneous nuclear ribonucleoproteins A2/B1 OS=H. sapiens GN=HNRNPA2B1 PE=1 SV=2–[ROA2_HUMAN]
O15020	Spectrin beta chain, nonerythrocytic 2 OS=H. sapiens GN=SPTBN2 PE=1 SV=3–[SPTN2_HUMAN]
Q15208	Serine/threonine-protein kinase 38 OS=H. sapiens GN=STK38 PE=1 SV=1–[STK38_HUMAN]
P02545	Prelamin-A/C OS=H. sapiens GN=LMNA PE=1 SV=1–[LMNA_HUMAN]
P29966	Myristoylated alanine-rich C-kinase substrate OS=H. sapiens GN=MARCKS PE=1 SV=4–[MARCS_HUMAN]
Q9UHB6	LIM domain and actin-binding protein 1 OS=H. sapiens GN=LIMA1 PE=1 SV=1–[LIMA1_HUMAN]
Q9Y608	Leucine-rich repeat flightless-interacting protein 2 OS=H. sapiens GN=LRRFIP2 PE=1 SV=1–[LRRF2_HUMAN]
Q04695	Keratin, type I cytoskeletal 17 OS=H. sapiens GN=KRT17 PE=1 SV=2–[K1C17_HUMAN]
O75128	Protein cordon-bleu OS=H. sapiens GN=COBL PE=1 SV=2–[COBL_HUMAN]
Q92945	Far upstream element-binding protein 2 OS=H. sapiens GN=KHSRP PE=1 SV=4–[FUBP2_HUMAN]
P08670	Vimentin OS=H. sapiens GN=VIM PE=1 SV=4–[VIME_HUMAN]
Q16643	Drebrin OS=H. sapiens GN=DBN1 PE=1 SV=4–[DREB_HUMAN]
P62158	Calmodulin OS=H. sapiens GN=CALM1 PE=1 SV=2–[CALM_HUMAN]
P07437	Tubulin beta chain OS=H. sapiens GN=TUBB PE=1 SV=2–[TBB5_HUMAN]
P68371	Tubulin beta-4B chain OS=H. sapiens GN=TUBB4B PE=1 SV=1–[TBB4B_HUMAN]
P29692	Elongation factor 1-delta OS=H. sapiens GN=EEF1D PE=1 SV=5–[EF1D_HUMAN]
P52272	Heterogeneous nuclear ribonucleoprotein M OS=H. sapiens GN=HNRNPM PE=1 SV=3–[HNRPM_HUMAN]
P68104	Elongation factor 1-alpha 1 OS=H. sapiens GN=EEF1A1 PE=1 SV=1–[EF1A1_HUMAN]
P07900	Heat shock protein HSP 90-alpha OS=H. sapiens GN=HSP90AA1 PE=1 SV=5–[HS90A_HUMAN]
P68363	Tubulin alpha-1B chain OS=H. sapiens GN=TUBA1B PE=1 SV=1–[TBA1B_HUMAN]

Table S5 Resensitizing AEP-OE MKN45 cells to taxanes and T-DM1 using inhibitors of JNK or ERK signaling pathway

IC50	Control	10 μM	20 μM	30 μM
Docetaxel (nM)
SP60925	1,443.5±193.03	1,061.5±85.33	546.47±33.71	262.37±14.37
PD98095	1,373.3±187.43	994.93±15.94	683.47±12.30	354.63±33.94
Paclitaxel (nM)
SP60925	1,181.14±98.16	826.37±19.46	556.13±19.43	264.07±23.11
PD98095	1,235.0±17.67	944.87±20.38	605.93±18.75	356.60±21.90
T-DM1 (nM)
SP60925	0.229±0.014	0.154±0.010	0.077±0.015	0.041±0.003
PD98095	0.303±0.017	0.233±0.020	0.155±0.012	0.066±0.003

Note: Data presented as mean ± standard deviation.

Abbreviations: AEP, asparaginyl endopeptidase; IC₅₀, half maximal inhibitory concentration; OE, AEP overexpression.