RKIP reduction enhances radioresistance by activating the Shh signaling pathway in non-small-cell lung cancer

Figures & data

Figure 1 RKIP reduction is correlated with NSCLC radiation resistance and poor overall survival.

Notes: (A) Representative results of RKIP staining in radiosensitive (a), and radioresistant (b) and negative control (c) NSCLC tissues. Original magnification 400×. (B) Kaplan–Meier survival analysis of NSCLC patients based on RKIP expression level. Patients with lower RKIP expression have clearly worse overall survival than patients with higher RKIP expression.
Abbreviations: NSCLC, non-small-cell lung cancer; RKIP, Raf kinase inhibitor protein.

Table 1 Correlation between RKIP expression and clinicopathological characteristics in NSCLC (n=93, χ² test)

Variables	N	RKIP		P-value
		Low	High
Gender				0.058
Male	75	23	52
Female	18	10	8
Age				0.520
≤60	46	18	28
>60	47	15	32
Pathological types				0.164
SCC	63	19	44
ADC	30	14	16
Primary tumor (T) stage				<0.001
T1–2	58	12	46
T3–4	35	21	14
Lymph node (N) metastasis				0.334
N0–1	25	11	14
N2–3	68	22	46
Clinical tumor/node/metastasis stage				0.800
II	21	8	13
III	72	25	47
Radiation response				<0.001
Radioresistant	44	24	20
Radiosensitive	49	9	40

Notes: P<0.05 by χ² test, RKIP expression vs NSCLC primary tumor (T) stage; RKIP expression vs NSCLC radiation response.

Abbreviations: ADC, adenocarcinoma; NSCLC, non-small-cell lung cancer; RKIP, Raf kinase inhibitor protein; SCC, squamous cell carcinoma.

Table 2 Univariate and multivariate analyses of prognostic factors for overall survival using Cox proportional hazards regression model (N=93)

Variables	Overall survival	HR (95%) CI
	Univariate analysis
	P-value
Gender
Male vs female	0.35	1.316 (0.740–2.341)
Age
≤60 vs >60	0.955	0.986 (0.609–1.598)
Pathological types
SCC vs ADC	0.824	0.943 (0.562–1.583)
Primary tumor (T) stage
T1–2 vs T3–4	0.020*	0.549 (0.331–0.911)
Lymph node (N) metastasis
N0–1 vs N2–3	0.172	0.699 (0.418–1.168)
Clinical tumor/node/metastasis stage
II vs III	0.354	0.772 (0.446–1.335)
RKIP expression
Low vs high	0.003*	1.810 (0.617–5.313)
Variables	Overall survival	HR (95%) CI
	Multivariate analysis
	P-value
Primary tumor (T) stage
T1–2 vs T3–4	0.386	0.767 (0.421–1.398)
RKIP expression
Low vs high	0.042#	1.830 (1.023–3.276)

Note:

* P<0.05 by univariate Cox proportional hazards regression model,

# P<0.05 by multivariate Cox proportional hazards regression model.

Abbreviations: ADC, adenocarcinoma; HR, hazard ratio; RKIP, Raf kinase inhibitor protein; SCC, squamous cell carcinoma.

Figure 2 Analysis of RKIP expression levels in transfected NSCLC cell lines by Western blots.

Notes: (A) Western blots showed RKIP expression levels in stable transfected cell lines and the corresponding control cell lines. β-actin was used as a loading control. (B) Histograms show relative expression levels of RKIP in these cell lines by grayscale analysis. Data are presented as the mean ± SD. *P<0.05 vs A549; ^#P<0.05 vs SK-MES-1. All experiments were performed in triplicate.
Abbreviations: A549, A549 cells without transfection; A-EV, A549 cells transfected with empty vector; A/RKIP-KD, A549 cells transfected with RKIP-knockdown vector; EV, empty vector; KD, knockdown; NSCLC, non-small-cell lung cancer; RKIP, Raf kinase inhibitor protein; SK-MES-1, SK-MES-1 cells without transfection; SK-EV, SK-MES-1 cells transfected with empty vector; SK/RKIP-KD, SK-MES-1 cells transfected with RKIP-knockdown vector.

Figure 3 RKIP reduction enhances radioresistance of NSCLC cells in vitro.

Notes: (A) Colony formation assays show the radioresponse of A/RKIP-KD, SK/RKIP-KD and the corresponding control cell lines, A-EV and SK-EV. Cells were treated with different doses (0–8 Gy) of irradiation, and the colonies formed after 12 days of incubation were fixed and counted. (B) Cell proliferation assays show the viability of A/RKIP-KD and SK/RKIP-KD after different doses of irradiation as well as the viability of the corresponding control cell lines A-EV and SK-EV. Data are presented as the mean ± SD (P<0.05). All experiments were performed in triplicate.
Abbreviations: EV, empty vector; KD, knockdown; NSCLC, non-small-cell lung cancer; RKIP, Raf kinase inhibitor protein; A-EV, A549 cells transfected with empty vector; A/RKIP-KD, A549 cells transfected with RKIP-knockdown vector; SK-EV, SK-MES-1 cells transfected with empty vector; SK/RKIP-KD, SK-MES-1 cells transfected with RKIP-knockdown vector.

Figure 4 RKIP reduction enhances NSCLC cell migration and invasion abilities.

Notes: (A) A representative result showing the migration and invasion abilities of A/RKIP-KD, SK/RKIP-KD and of their corresponding control cell lines A-EV and SK-EV, depicted in Transwell chambers. The cells were incubated in the upper chamber for 48 h. (B) Histograms show numbers of cells of A/RKIP-KD, SK/RKIP-KD and their corresponding control cell lines A-EV and SK-EV penetrating into the lower chambers. **P<0.01. (C) Histograms show the relative wound closure (present vacant area/original vacant area) of the NSCLC cell lines in wound-healing assays at different time points. Data are presented as the mean ± SD (P<0.05). All experiments were performed in triplicate.
Abbreviations: EV, empty vector; KD, knockdown; NSCLC, non-small-cell lung cancer; RKIP, Raf kinase inhibitor protein; A-EV, A549 cells transfected with empty vector; A/RKIP-KD, A549 cells transfected with RKIP-knockdown vector; SK-EV, SK-MES-1 cells transfected with empty vector; SK/RKIP-KD, SK-MES-1 cells transfected with RKIP-knockdown vector.

Figure 5 Effects of RKIP reduction on NSCLC cell apoptosis and cell cycle changes.

Notes: (A) Histograms show apoptotic rates of A/RKIP-KD, SK/RKIP-KD and the corresponding control cell lines A-EV and SK-EV under different doses (0–8 Gy) of irradiation. **P<0.01. (B) Histograms show cell cycle phase distributions of A/RKIP-KD, SK/RKIP-KD and the corresponding control cell lines A-EV and SK-EV with and without irradiation. Data are presented as the mean ± SD (P<0.05). (C) Representative images of A/RKIP-KD, SK/RKIP-KD and the corresponding control cell lines A-EV and SK-EV under 4 Gy irradiation using TUNEL assays. Original magnification 200×. *P<0.01. Histograms show apoptotic rates of A/RKIP-KD, SK/RKIP-KD and the corresponding control cell lines A-EV and SK-EV under 4 Gy irradiation. All experiments were performed in triplicate.
Abbreviations: EV, empty vector; KD, knockdown; NSCLC, non-small-cell lung cancer; RKIP, Raf kinase inhibitor protein; A-EV, A549 cells transfected with empty vector; A/RKIP-KD, A549 cells transfected with RKIP-knockdown vector; SK-EV, SK-MES-1 cells transfected with empty vector; SK/RKIP-KD, SK-MES-1 cells transfected with RKIP-knockdown vector.

Figure 6 RKIP reduction enhances Shh–Gli1 signaling and increases CD133 expression in NSCLC cell lines.

Notes: (A) Representative Western blot results show the expression levels of RKIP, Smo, Gli1 and CD133 in A/RKIP-KD, SK/RKIP-KD and the corresponding control cell lines A-EV and SK-EV; the results when these cell lines were treated with N-shh and cyclopamine are also shown. β-actin was used as a loading control. (B) Histograms show the relative expression levels of RKIP, Smo, Gli1 and CD133 in the cells by grayscale analysis. Data are presented as the mean ± SD. ⁺P<0.05 vs cells transfected with empty vectors; *P<0.05 vs cells transfected with empty vectors; ^#P<0.05 vs cells with RKIP knockdown. All experiments were performed in triplicate.
Abbreviations: EV, empty vector; KD, knockdown; NSCLC, non-small-cell lung cancer; RKIP, Raf kinase inhibitor protein; A-EV, A549 cells transfected with empty vector; A/RKIP-KD, A549 cells transfected with RKIP-knockdown vector; SK-EV, SK-MES-1 cells transfected with empty vector; SK/RKIP-KD, SK-MES-1 cells transfected with RKIP-knockdown vector.