RANKL/RANK pathway abrogates cetuximab sensitivity in gastric cancer cells via activation of EGFR and c-Src

Figures & data

Figure 1 The expression of RANKL in GC tissues.

Notes: (A) (I and III) EGFR and RANKnegative staining and (II and IV) EGFR and positive staining, respectively (in brown). Magnification ×400. (B) The expression of EGFR and RANK in SGC-7901, MGC-803, BGC-823, MKN-45 and KATO-III was detected by Western blot, using actin as a loading control.
Abbreviation: GC, gastric cancer.

Table 1 Correlation between the expression of EGFR or RANK and the clinicopathological factors in primary GC patients

Characteristics	Cases	EGFR			RANK
		Low (%)	High (%)	P-value	Low (%)	High (%)	P-value
Sex				0.903			0.118
	Male	38 (72)	15 (28)		25 (47)	28 (53)
	Female	11 (73)	4 (27)		10 (67)	5 (33)
Age (years)				0.907			0.335
	<60	24 (73)	9 (27)		19 (58)	14 (42)
	≥60	25 (71)	10 (29)		16 (46)	19 (54)
pTNM stage				0.019*			0.017*
	I + II	16 (94)	1 (6)		13 (76)	4 (24)
	III	33 (65)	18 (35)		22 (43)	29 (57)
T stage				0.046*			0.093
	T1–2	9 (100)	0 (0)		7 (78)	2 (22)
	T3–4	40 (68)	19 (32)		28 (48)	31 (52)
N stage				0.013*			0.136
	N0	17 (94)	1 (6)		12 (67)	6 (33)
	N1–3	32 (64)	18 (36)		23 (46)	27 (54)
Lauren grade				0.545			0.693
	Intestinal	18 (67)	9 (33)		12 (44)	15 (56)
	Diffuse	17 (77)	5 (23)		14 (64)	8 (36)
	Mixed	14 (74)	5 (26)		9 (47)	10 (53)

Note:

* P<0.05.

Abbreviations: GC, gastric cancer; pTNM, pathological tumor–node–metastasis.

Table 2 Spearman’s correlations between EGFR and RANK expression in primary GC patients

EGFR	Cases (%)	RANK		Spearman’s r	P-value
		Low (%)	High (%)
Low (%)	49 (72)	33 (67)	16 (33)	0.510	<0.001
High (%)	19 (28)	2 (10)	17 (90)

Abbreviation: GC, gastric cancer.

Figure 2 RANKL stimulated EGFR phosphorylation and downstream signaling in GC cells.

Notes: (A) Two RAS wide-type GC cell lines (SGC-7901 and MGC-803) were treated with 1 μg/mL sRANKL for the indicated times. The expression of EGFR, c-Src, AKT and ERK and phosphorylation levels were analyzed by Western blot. (B) SGC-7901 and MGC-803 cells were respectively treated with sRANKL at 0.1 μg/mL and 1 μg/mL for 48 hours. Cell viability was assessed by MTT assay. The results from three independent experiments are shown.
Abbreviation: GC, gastric cancer.

Figure 3 The effect of RANKL on sensitivity to cetuximab in GC cells.

Notes: (A) SGC-7901 and MGC-803 cells were pretreated with 1 μg/mL sRANKL for 1 hour and then incubated with 10 μg/mL cetuximab for 48 hours, followed by MTT assay. *Comparisons between the cetuximab-treated cells and the combined-treated cells, P<0.05. (B) SGC-7901 and MGC-803 cells were pretreated with 1 μg/mL sRANKL for 1 hour and then incubated with 10 μg/mL cetuximab for 14 days, and the clones were counted. *Comparisons between the cetuximab-treated cells and the combined-treated cells, P<0.05.
Abbreviation: GC, gastric cancer.

Figure 4 The effect of RANKL/RANK pathway on cetuximab sensitivity in SGC-7901 cells.

Notes: (A) SGC-7901 cells were transiently transfected with RANK siRNA for 24 hours and then pretreated with 1 μg/mL sRANKL for 1 hour, followed by 10 μg/mL cetuximab for 48 hours. Cell viability was assessed by MTT assay. *Comparisons between the cetuximab-treated cells and the combined-treated cells in the NS control arm, P<0.05. (B) After transient transfection with RANK siRNA for 48 hours, SGC-7901 cells were pretreated with 1 μg/mL sRANKL for 1 hour, followed by 10 μg/mL cetuximab for 48 hours. Expression of RANK, EGFR, AKT, ERK, and phosphorylation levels were analyzed by Western blot. (C) SGC-7901 cells were preincubated with 10 μg/mL rOPG for 4 hours and then pretreated with 1 μg/mL sRANKL for 1 hour, followed by 10 μg/mL cetuximab for 48 hours. Cell viability was assessed by MTT assay. *Comparisons between the cetuximab-treated cells and the combined-treated cells in the control arm, P<0.05. (D) After preincubating with 10 μg/mL rOPG for 4 hours, SGC-7901 cells were pretreated with 1 μg/mL sRANKL for 1 hour, followed by 10 μg/mL cetuximab for 48 hours. Expression of EGFR, AKT, and ERK and phosphorylation levels were analyzed by Western blot.
Abbreviations: NS, non-silencing; siRNA, small interfering RNA.

Figure 5 The effect of dasatinib on RANKL-induced cetuximab resistance in SGC-7901 cells.

Notes: (A and B) SGC-7901 and MGC-803 cells were treated with 100 nM dasatinib or 10 μM PP2 for the indicated times. Expression of c-Src, EGFR, AKT, and ERK and phosphorylation levels were analyzed by Western blot. (C) SGC-7901 cells were treated with 100 nM dasatinib for 24 hours, followed by 1 μg/mL sRANKL for 1 hour and then 10 μg/mL cetuximab for 48 hours. The cell viability was assessed by MTT assay. *Comparisons between the cetuximab-treated cells and the combined-treated cells in the control arm, P<0.05. (D) SGC-7901 cells were treated with or without 100 nM dasatinib for 2 hours and then preincubated with 1 μg/mL sRANKL for 1 hour, followed by 10 μg/mL cetuximab for 48 hours. The expression of c-Src, EGFR, AKT, and ERK and phosphorylation levels were analyzed by Western blot.

Figure 6 The effect of RANKL/RANK pathway on sensitivity to cetuximab in Caco-2 cells.

Notes: (A) Caco-2 cells were treated with 1 μg/mL sRANKL for the indicated times. The expression of EGFR, AKT, ERK, and c-Src and phosphorylation levels were analyzed by Western blot. (B) Caco-2 cells were pretreated with 1 μg/mL sRANKL for 1 hour and then incubated with 10 μg/mL cetuximab for 48 hours, followed by MTT assay. *Comparisons between cetuximab-treated arm and combined-treated arm, P<0.05. (C) Caco-2 cells were transiently transfected with RANK siRNA for 24 hours and then pretreated with 1 μg/mL sRANKL for 1 hour, followed by 10 μg/mL cetuximab for 48 hours. The cell viability was assessed by MTT assay. *Comparisons between the cetuximab-treated cells and the combined-treated cells in the NS control arm, P<0.05.
Abbreviations: NS, non-silencing; siRNA, small interfering RNA.