L1cam promotes tumor progression and metastasis and is an independent unfavorable prognostic factor in gastric cancer

Abstract

Background: Previous reports have demonstrated that L1cam is aberrantly expressed in various tumors. The potential role of L1cam in the progression and metastasis of gastric cancer is still not clear and needs exploring.

Methods: Expression of L1cam was evaluated in gastric cancer tissues and cell lines by immunohistochemistry and Western blot. The relationship between L1cam expression and clinicopathological characteristics was analyzed. The effects of L1cam on cell proliferation, migration and invasion were investigated in gastric cancer cell lines both in vitro and in vivo. The impact of L1cam on PI3K/Akt pathway was also evaluated.

Results: L1cam was overexpressed in gastric cancer tissues and cell lines. L1cam expression was correlated with aggressive tumor phenotype and poor overall survival in gastric cancer patients. Ectopic expression of L1cam in gastric cell lines significantly promoted cell proliferation, migration and invasion whereas knockdown of L1cam inhibited cell proliferation, migration and invasion in vitro as well as tumorigenesis and metastasis in vivo. The low level of phosphorylated Akt in HGC27 cells was up-regulated after ectopic expression of L1cam, whereas the high level of phosphorylated Akt in SGC7901 cells was suppressed by knockdown of L1cam. Moreover, the migration and invasion promoted by L1cam overexpression in gastric cancer cells could be abolished by either application of LY294002 (a phosphoinositide-3-kinase inhibitor) or knockdown of endogenous Akt by small interfering RNA.

Conclusions: Our study demonstrated that L1cam, overexpressed in gastric cancer and associated with poor prognosis, plays an important role in the progression and metastasis of gastric cancer.

Figure 1

L1cam is overexpressed in gastric cancer cell lines and primary tumor tissues. (A) L1cam mRNA expression levels in gastric cancer cell lines compared with normal gastric cancer tissue, (*P < 0.005). (B) L1cam protein expression in gastric cancer cell lines and normal gastric cancer tissue. (C) L1cam mRNA expression levels in 30 paired gastric cancer tissues and adjacent non-cancerous tissues. (D) L1cam protein expression level in paired gastric cancer tissues and adjacent non-cancerous tissues (the representative ones are shown).

Figure 2

The prognostic significance of L1cam in gastric cancer patients. (A) Representative photos of L1cam expression in 156 gastric cancer patients, a, negative staining of L1cam in adjacent normal tissues; b, weak staining of L1cam in well differentiated gastric cancer tissues; c, moderate staining of L1cam in cancer tissues; d, strong staining of L1cam in cancer tissues, amplification (×100). (B) Kaplan-Meier analysis of overall survival based on L1cam expression in all 156 patients. (C) and (D) Kaplan-Meier analysis of overall survival based on L1cam expression in stage I-II (C) and stage III-IV gastric cancer patients (D).

Figure 3

L1cam promotes cell proliferation, migration and invasion in gastric cancer cell lines. (A) L1cam protein level is increased after over-expression of L1cam in HGC27 cells and decreased after knockdown of L1cam in SGC7901 cells. (B) and (C) Ectopic expression of L1cam stimulates cell proliferation in HGC27 cells whereas knockdown of L1cam inhibits cell proliferation in SGC7901 cells as determined by MTT assays (B) and colony formation assays (C). (D) and (E) Ectopic expression of L1cam stimulates cell motility whereas knockdown of L1cam inhibits cell motility in gastric cancer cells as determined by migration assays (D) and invasion assays (E). Bars represented mean ± SD of three independent tests, all *P < 0.05.

Figure 4

L1cam affects the reponsiveness to oxaliplatin of gastric cancer cells. (A) SGC7901 cells were transfected with sh-L1cam or sramble lentivirus. Cells were left untreated or administrated with different concentrations of oxaliplatin and cell cycle analyses were performed. Graph indicates the percentage of cells in sub G1/G0 phase (apoptotic cells), *P <0.05. (B) HGC27 cells were transfected with L1cam or negative control vectors. Cells were left untreated or administrated with different concentrations of oxaliplatin and cell cycle analyses were performed. Graph indicates the percentage of cells in sub G1/G0 phase (apoptotic cells), *P <0.05.

Figure 5

L1cam promotes gastric cancer cell growth and metastasis in vivo. (A) SGC7901/Scramble and SGC7901/sh-L1cam cells (1 × 10⁶cells/mouse) were injected subcutaneously into the left and right dorsal flanks of the nude mice (n = 6), tumor volumes were measured on the indicated days. Data points are presented as mean volume ± SD. (B) Histopathology of xenograft tumors, the tumor sections were under HE staining and IHC staining for L1cam and Ki-67. (C) SGC7901/Scramble and SGC7901/sh-L1cam cells (2 × 10⁶cells/mouse) were injected into the tail vein of two groups of nude mice (ten for each group). Six weeks post injection, the mice were killed and the lungs and livers were removed and paraffin embedded.

Figure 6

PI3K/Akt signaling is involved in L1cam stimulated gastric cancer cell migration and invasion. (A) Western blot analysis of whole- cell lysates using anti-phospho-Akt (Ser473) or anti-Akt antibody. (B) Western blot analysis in from HGC27 cells expressing L1cam or empty vector after pretreatment with 20 μM LY294002 for 30 min, using anti-phospho-Akt (Ser473) or anti-Akt antibody. (C) Migratory and invasive abilities of HGC27 cells expressing L1cam or the empty vector evaluated by Transwell assay after pretreatment with LY294002. (D) Western blot analysis of whole-cell lysates from HGC27 cells expressing L1cam or the empty vector 24 h after transfection with AKT siRNA or a scrambled siRNA, using anti-phospho-Akt (Ser473) or anti-Akt antibody. (E) Migratory and invasive abilities of HGC27 cells expressing L1cam or the empty vector as evaluated by Transwell assay after transfection with Akt siRNA or the scrambled siRNA. (F) SGC7901 cells were injected into the flank of nude mice, LY294002 (25 mg/kg) were intraperitoneally injected into the nude mice every four days, tumor volumes were measured at indicated days. (G) Tumors formed by different cells (SGC7901/Scramble or SGC7901/sh-L1cam, SGC7901/DMSO or SGC7901/LY294002) were collected and protein was extracted. Western blot analysis was conducted to detect the Akt and phospho-Akt level. Photomicrographs are at 100×. Bars correspond to mean ± SD of three independent experiments.