KIAA1522 overexpression promotes tumorigenicity and metastasis of esophageal cancer cells through potentiating the ERK activity

Figures & data

Figure 1 Representative IHC staining for the KIAA1522 protein in ESCC tumors and adjacent normal esophageal squamous epithelium.

Note: Original magnification: 100× and 400×.
Abbreviations: ESCC, esophageal squamous cell carcinoma; IHC, immunohisto-chemistry.

Table 1 Association of KIAA1522 levels with clinicopathological features of 342 patients with ESCC

Clinicopathological features	KIAA1522 expression		Total	P-value
	Negative (%)	Positive (%)
All patients	185 (54)	157 (46)	342
Age (years)				0.632
<60	92 (55.4)	74 (44.6)	166
≥60	93 (52.8)	83 (47.2)	176
Gender				0.837
Male	135 (55.4)	113 (44.6)	248
Female	50 (53.2)	44 (46.8)	94
TNM classification
pT				0.625*
T1	8 (66.7)	4 (33.3)	12
T2	105 (51.5)	99 (48.5)	204
T3	40 (56.3)	31 (43.7)	71
T4	32 (58.2)	23 (41.8)	55
pN				0.361
N0	37 (49.3)	38 (50.7)	75
N1	147 (55.5)	118 (44.5)	265
Histology grade				0.030
Good (G1)	42 (66.7)	45 (33.3)	87
Moderate (G2)	106 (51.5)	96 (48.5)	202
Poor (G3)	36 (56.3)	15 (43.7)	51
AJCC6 stage				0.921
I	14 (56.0)	11 (44.0)	25
II	101 (53.2)	89 (46.8)	190
III	69 (55.2)	56 (44.8)	125

Note:

* Fisher’s exact test.

Abbreviations: ESCC, esophageal squamous cell carcinoma; TNM, tumor, regional lymph node, distant metastasis; pT, pathologic T stage; pN, lymph node metastases; AJCC6, American Joint Committee on Cancer (Sixth Edition).

Figure 2 Inhibition of KIAA1522 expression suppresses the proliferation and colony formation ability of ESCC cells.

Notes: The ESCC cell lines KYSE510 and KYSE150 were transiently transfected with two independent KIAA1522-specific siRNA (si-A and si-C) or negative control siRNA (siCtrl) for 48 h. (A) The expression levels of KIAA1522 protein were detected by immunoblotting. The arrow indicates the specific band of KIAA1522 protein located around 170 kDa. Relative ratios of absorbance for KIAA1522 to GAPDH were plotted. The data are presented as the mean ± SEM. ^***P<0.001. (B) Cell proliferation was determined by CCK-8 assay. Data are mean ± SEM. ^***P<0.001. (C) Colony formation assay. Representative results are shown, and the colony number was plotted. The data are presented as the mean ± SEM from three separate experiments. ^***P<0.001.
Abbreviations: CCK-8, cell counting kit-8; Ctrl, control; ESCC, esophageal squamous cell carcinoma; GAPDH, glyceral dehyde 3-phosphate dehydrogenase; P, parental; SEM, standard error of the mean; si, siRNA; si-A, siKIAA-A; si-c, siKIAA-C.

Figure 3 Knockdown of KIAA1522 expression reduces the tumorigenicity of esophageal carcinoma cells in nude mice.(p)(p)Notes: KYSE510 and KYSE150 cells were infected with lentiviruses expressing KIAA1522 shRNA (shKIAA) or negative control shRNA (shCtrl), and stable cell clones were selected with puromycin. (A) Western blot analysis of KIAA1522 expression levels in stable cell clones. The arrow indicates the position of KIAA1522 specific band. Relative ratios of absorbance for KIAA1522 to GAPDH were plotted. The data are presented as the mean ± SEM. ^***P<0.001. (B) Colony formation assay. (C–E) KYSE150-shKIAA or shCtrl stable cell clones were implanted subcutaneously into nude mice (n=10 per group), the tumor volumes were measured every 3 days for 3 weeks, and sc tumors were excised 21 days after inoculation. (C) Tumor growth curves. The data are presented as the mean ± SEM. ^**P<0.01, ^***P<0.001. (D) Tumor weight. ^***P<0.001; (E) Photographs of sc tumors. Scale bar =1 cm.(p)(p)Abbreviations: Ctrl, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; sc, subcutaneous; sh, shRNA.

Figure 4 KIAA1522 depletion enhances anoikis in ESCC cells.

Notes: (A) KYSE510 and KYSE150 cells were cultured in suspension for the indicated times, and the KIAA1522 protein levels were detected by Western blotting. Relative ratios of absorbance for KIAA1522 to GAPDH were plotted. The data are presented as the mean ± SEM. ^**P<0.01. The arrows in images A and B indicate the position of KIAA1522 specific band. (B–D) KYSE150 and KYSE510 cells were transiently transfected with KIAA1522 siRNA for 48 h and then cultured on polyHEMA-coated dishes for 20 h. (B) KIAA1522 expression was determined by Western blot. (C) Anoikis was measured by Annexin V-FITC/PI staining and flow cytometry analysis. Representative results are shown (upper panel), and the percentages of apoptotic cells were plotted (lower panel). The data are presented as the mean ± SEM. ^**P<0.01, ^***P<0.001.
Abbreviations: Ctrl, control; ESCC, esophageal squamous cell carcinoma; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, non-silencing; P, parental; PI, propidium iodide; polyHEMA, polyhydroxylethylmethacrylate; si, siRNA; si-A, siKIAA-A; si-B, siKIAA-B; si-c, siKIAA-C.

Figure 5 KIAA1522 promotes lung metastasis of KYSE150 cells in NOD/SCID mice.

Notes: (A) KYSE510 and KYSE150 cells transfected with shKIAA or shCtrl were cultured on polyHEMA-coated dishes for 20 h and subjected to Annexin V-FITC/PI staining followed by flow cytometry analysis. Representative results are shown, and degrees of apoptosis were plotted. The data are presented as mean ± SEM. ^*P<0.05. (B–D) Nude mice were injected with 2×10⁶ KYSE150-shCtrl or KYSE150-shKIAA stable cell clones via the tail vein. After 4 weeks, the mice were sacrificed. (B) The lungs were subjected to Bouin’s fixation and photographed. Scale bar =1 cm. (C) Visible lung metastases nudes were counted and plotted. (D) The sections of the lungs were stained with H&E. Scale bar =200 μm.
Abbreviations: Ctrl, control; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; polyHEMA, polyhydroxylethylmethacrylate; sh, shRNA.

Figure 6 KIAA1522 activates ERK signaling pathway in ESCC cells.

Notes: (A) The ESCC cell lines KYSE510 and KYSE150 were transiently transfected with KIAA1522-specific siRNA or control non-silencing siRNA for 48 h. (B) Transiently transfected cells were cultured on polyHEMA-coated dishes for 20 h, and the cell lysates were immunoblotted for the indicated proteins. The arrows indicate the position of KIAA1522 specific band. Relative ratios of absorbance for KIAA1522 to GAPDH, pERK to total ERK were plotted. The data are presented as the mean ± SEM. ^**P<0.01, ^***P<0.001.
Abbreviations: Ctrl, control; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, non-silencing; P, parental; pERK, phosphorylated ERK; polyHEMA, polyhydroxylethylmethacrylate; si, siRNA; si-A, siKIAA-A; si-c, siKIAA-C.

Figure S1 Kaplan–Meier overall survival curve according to the KIAA1522 levels in ESCC patients.

Notes: Overall survival is shown for the 115 patients with ESCC based on KIAA1522 levels as measured by immunohistochemistry. P-values were calculated by log-rank test.

Abbreviation: ESCC, esophageal squamous cell carcinoma.

Figure S2 Effects of KIAA1522 knockdown on the apoptosis and cell cycle distribution of ESCC cells.

Notes: KYSE510 and KYSE150 cells were transiently transfected with KIAA1522-specific siRNA or control non-silencing siRNA. Cells were collected after 48 h and subjected to the analysis. The results of a flow cytometry analysis of apoptosis (A) and cell cycle distribution (B) are shown.

Abbreviations: Ctrl, control; ESCC, esophageal squamous cell carcinoma; si, siRNA.

Figure S3 KIAA1522 depletion inhibits cell–cell adhesion.

Notes: KYSE150 and KYSE510 cells were transiently transfected with KIAA1522 siRNA or control non-silencing siRNA for 48 h and then seeded on polyHEMA-coated dishes. The cell morphology was observed under the phase. Scale bars =50 μm.

Abbreviations: Ctrl, control; polyHEMA, polyhydroxylethylmethacrylate; si, siRNA.