The Candidate Tumor Suppressor p29ING4 Is Down-Regulated in Human Myeloma Cells and Involved in Their Pro-Angiogenic Properties.

Multiple Myeloma (MM) cells grow into the bone marrow (BM) mainly through the interactions with the microenvironment. The marked induction of angiogenesis by myeloma cells suggests that this process has a pivotal role in the progression of MM. However the molecular mechanisms underlying the regulation of angiogenesis remain unresolved. The new candidate tumor suppressor gene “inhibitor of growth family member 4” (p29ING4) has been recently implicated in solid tumors as a repressor of angiogenesis and tumor growth through the association with p65 subunit of NF-kB and the suppression of angiogenic related gene as IL-8 (Garkavtsev I et al., Nature 2004).

In this study first we have evaluated the mRNA expression of p29ING4 in human myeloma cell lines (HMCLs): U266, RPMI-8226, XG-1, XG-6, OPM-2 and JJN3, in EBV+ lines HS-SULTAN and ARH-77 and in purified CD138+ plasmacells (purity >90%) obtained from either 32 MM patients or 6 patients with MGUS by both qualitative RT-PCR and quantitative real time PCR. Healthy CD20+ BM B lymphocytes and/or CD138+ plasmacells obtained from reactive tonsils have been used as controls. p29ING4 has been checked at protein level by western blot on nuclear extracts using a polyclonal anti-p29ING4 Ab. In addition, p29ING4 mRNA levels have been compared with IL-8, VEGF, Ang-1 mRNA levels and correlated with BM angiogenesis in MM patients.

The presence of p29ING4 transcript was observed in HMCLs even if p29ING4 mRNA levels were significantly reduced in all HMCLs tested as compared to controls with a median ΔCt p29ING4 (Ct p29ING4-Ct Abl ): 3.2 vs. 0.45 (p<0.05). Consistently a marked down-regulation of p29ING4 protein has been observed in all HMCLs tested compared to controls. The levels of p29ING4 mRNA in HMCLs were independent to the p53 status, whereas, checking the activity of the p50 and p65 subunit of NF-kB by an ELISA-based method, we found that HMCLs with higher p65 activity showed lower levels of p29ING4 mRNA. A significantly negative correlation was observed between p29ING4 and IL-8 mRNA levels (R= −0.84, Spearman 2-tailed test, p=0.04) but not with VEGF (R= −0.07, p=NS) whereas the correlation with ANG-1 did not reach a statistical significance (R=−0.60, p=0.09). Similarly to HMCLs we found that CD138+ MM cells had significantly suppressed levels of p29ING4 mRNA as compared to both normal plasma cells (median ΔCt p29ING4:3.3 vs. 0.47, p=0.03) and MGUS subjects (ΔCt p29ING4:3,3 vs. 0.2 p=0.02) with a mean n-fold expression of 0.11 normalized to controls and 0.09 normalized to MGUS subjects. Consistently lower p29ING4 protein levels were detected in nuclear extracts of CD138+ MM cells as compared to controls. On the other hand we found that IL-8 median levels were significantly higher in BM plasma of MM patients as compared to MGUS subjects (39,3 vs. 25,2 pg/ml p=0.05) and correlated with IL-8 mRNA levels in CD138+ MM cells (R=0.77, p0.041). Interestingly, we observed that MM patients with higher BM IL-8 levels (> 25 pg/ml) have a significantly lower p29ING4 mRNA levels (median ΔCt p29ING4:4.71 vs. 1.9; p=0.04). Finally we found that MM patients with high microvalscular density (MVD) and number of vessels X field have significant lower p29ING4 mRNA levels as compared to those with low MVD (median ΔCt p29ING4:4,8 vs. 2; p=0.04). Our data indicate that the tumor suppressor p29ING4 is down-regulated in myeloma cells and regulate their pro-angiogenic properties.