Identification of SLAMF3 (CD229) as an Inhibitor of Hepatocellular Carcinoma Cell Proliferation and Tumour Progression

<div><p>Although hepatocellular carcinoma (HCC) is one of the most common malignancies and constitutes the third leading cause of cancer-related deaths, the underlying molecular mechanisms are not fully understood. In the present study, we demonstrate for the first time that hepatocytes express signalling lymphocytic activation molecule family member 3 (SLAMF3/CD229) but not other SLAMF members. We provide evidence to show that SLAMF3 is involved in the control of hepatocyte proliferation and in hepatocellular carcinogenesis. SLAMF3 expression is significantly lower in primary human HCC samples and HCC cell lines than in human healthy primary hepatocytes. In HCC cell lines, the restoration of high levels of SLAMF3 expression inhibited cell proliferation and migration and enhanced apoptosis. Furthermore, SLAMF3 expression was associated with inhibition of HCC xenograft progression in the nude mouse model. The restoration of SLAMF3 expression levels also decreased the phosphorylation of MAPK ERK1/2, JNK and mTOR. In samples from resected HCC patients, SLAMF3 expression levels were significantly lower in tumorous tissues than in peritumoral tissues. Our results identify SLAMF3 as a specific marker of normal hepatocytes and provide evidence for its potential role in the control of proliferation of HCC cells.</p></div

SLAMF3 is expressed by hepatocytes.

<p>(A) SLAMF3 expression was analysed by flow cytometry analysis in HHPHs and in Huh-7, HepG2 and Hep3B human HCC cell lines. SLAMF3 expression in Jurkat lymphocytes was used as a positive control; SLAMF3 staining (in grey) is overlaid by the negative control (in white) and corresponds to one representative of five independent experiments. (B) Western blot analysis of proteins extracted from Huh-7 cells, HepG2 cells or HHPHs, with a mAb (K12) against SLAMF3′s first extracellular domain (D1) or an anti-actin antibody as a control. One of four independent experiments is presented here. (C) Expression of SLAMF3 transcripts in hepatocytes. After reverse transcription, SLAMF3 cDNA was amplified by PCR using specific primers. GAPDH was amplified as a control gene and pure H₂O was used as a PCR control. The Daudi and Jurkat human lymphocyte cell lines were used as positive controls and the monkey kidney COS-7 cell line was used as a negative control. One of three independent experiments is shown here; (D) SLAMF3 mRNA was assayed by Q-PCR in (i) HHPHs, Huh-7 cells and HepG2 cells, (ii) Daudi B lymphocytes and Jurkat T lymphocytes (positive controls) and (iii) the green monkey kidney COS-7 cell line (a negative control). Results are presented as the mean ± SD (n = 6) ***<i>p<0.005, **p<0.01</i>).</p

SLAMF3 expression is repressed in tumour cells from ten resected HCC patients.

<p>(A) SLAMF3 mRNA expression was analysed in hepatocytes from resected HCC patients. Specific amplification of SLAMF3 mRNAs using PCR (representative patients #2, #7 and #12) and quantitative PCR in tumour tissues (T) and peritumoral tissues (pT) presented separately (B) and as median (**<i>p<0.05</i>) (C); (D) Immunohistochemical staining of SLAMF3 in T and pT areas from representative patients #3 and #12 (×20).</p

The effect of SLAMF3 expression on the growth of HCC xenografts in nude mice.

<p>(A) Tumour volumes were measured 6, 7, 8 and 9 weeks after injection in nude mice. Four out of ten animals developed a tumour on the left flank (after injection of mock-transfected Huh7 cells) but none of the ten developed a tumour on the right flank (after injection of SLAMF3-overexpressing Huh7 cells) and represented as mean ± SD (*<i>p<0.05</i>); (B) photographs of tumour progression on left flanks (mock-transfected Huh7 cells) and right flanks (SLAMF3-overexpressing Huh7 cells) (mice #2, #3, #5 and #6).</p

Restoration of SLAMF3 expression in HCC cells inhibits MAPK ERK1/2, JNK and mTOR pathways and induces caspase-dependent apoptosis.

<p>(A) SLAMF3 expression was confirmed by Western blot analysis in SLAMF3-over-expressing Huh-7 and mock cells at 48 h post-transfection. ERK1/2, JNK, p38, AKT and mTOR proteins were detected as controls. The activation levels of MAPK ERK1/2 (Thr 202/Tyr 204), JNK (Thr 183/Tyr 185), p38 (Thr 180/Tyr 183), PI3K (p85/p110 Thr 467/Tyr 199), AKT (Ser 473, Thr 308) and mTOR (Ser 2448, Ser 2481) are represented. One of three representative experiments is shown here. (B) The results of an active caspase assay based on cell-permeable fluorochrome inhibitor of caspases (FLICA). Caspase activity was evaluated in HCC cells (Huh-7 and HepG2 lines) at the indicated times after the ectopic introduction of SLAMF3 vector. Caspase activity is shown in the SLAMF3^neg subpopulation (in white) and the SLAMF3^pos (overlaid in grey) subpopulation from one representative of two independent experiments.</p

Correlation between HCC cell SLAMF3 expression and cell motility.

<p>Cell migration activities in Huh-7 (A) and HepG2 (B) cells overexpressing SLAMF3 and in mock cells were compared in a wound-healing assay. Same areas of culture plate were photographed at the indicated time points. The migratory index corresponds to the percentage of wound closure (estimated using Image J software) and presented as the mean ± SD from three independent experiments with Huh-7 cells (C) (statistical significance: *<i>p<0.05</i> ***<i>p<0.005</i>) and from one experiment for Hep3B cells (D).</p