RBM5 (RNA-binding motif protein 5) is a nuclear RNA binding protein containing 2 RNA recognition motifs. The RBM5 gene is located at the tumor suppressor locus 3p21.3. Deletion of this locus is the most frequent genetic alteration in lung cancer, but is also found in other human cancers. RBM5 is known to induce apoptosis and cell cycle arrest but the molecular mechanisms of RBM5 function are poorly understood. Here, we show that RBM5 is important for the activity of the tumor suppressor protein p53. Overexpression of RBM5 enhanced p53-mediated inhibition of cell growth and colony formation. Expression of RBM5 augmented p53 transcriptional activity in reporter gene assays and resulted in increased mRNA and protein levels for endogenous p53 target genes. In contrast, shRNA-mediated knockdown of endogenous RBM5 led to decreased p53 transcriptional activity and reduced levels of mRNA and protein for endogenous p53 target genes. RBM5 affected protein, but not mRNA, levels of endogenous p53 after DNA damage suggest that RBM5 contributes to p53 activity through posttranscriptional mechanisms. Our results show that RBM5 contributes to p53 transcriptional activity after DNA damage and that growth suppression and apoptosis mediated by RBM5 are linked to activity of the tumor suppressor protein p53.RBM5 (also known as LUCA-15 or H37) is a nuclear RNA binding protein widely distributed in mammalian tissues. 1 RBM5 was initially cloned as a putative tumor suppressor gene mapping to the human chromosomal locus 3p21.3 2-4 that is frequently deleted in human cancers, including lung, renal and breast cancer. 5,6 Deletion at chromosome 3p21.3 is the most frequent genetic alteration in lung cancer suggesting that the region contains 1 or more tumor suppressor genes. 6 Accumulating evidence suggests that RBM5 can function as a tumor suppressor protein by inhibiting tumor transformation and progression. 7,8 The expression of the RBM5 transcript and protein is decreased in 70-80% of lung cancers. 9 RBM5 is also downregulated in human schwannomas 10 and in ras-transformed rat embryonic fibroblastic cells. 11 Ectopic expression of the RBM5 gene suppresses growth of human lung cancer, breast cancer, 9 fibrosarcoma 11 and leukemic cells. 12 These observations suggest that RBM5 has antiproliferative properties. Furthermore, decrease of RBM5 expression is part of a 17 gene expression signature predictive of metastasis and poor clinical outcome for various types of human cancers. 13 Most of the functional studies for RBM5 have focused on its ability to induce cell cycle arrest and apoptosis. 7,8 Overexpression of RBM5 inhibits cell proliferation by extending the G1 phase of the cell cycle in leukemic cells. 12 RBM5 also inhibits tumor growth of lung cancer cells both in vitro and in vivo with antitumor mechanisms involving G1 cell cycle arrest and apoptosis. The extended G1 phase correlated with reduced levels of cyclin A and of phosphorylated Rb. 14

RNA-binding motif 5 is a putative tumor suppressor gene that modulates cell cycle arrest and apoptosis. We recently demonstrated that RNA-binding motif 5 inhibits cell growth through the p53 pathway. This study evaluated the clinical significance of RNA-binding motif 5 expression in gastric cancer and the effects of altered RNA-binding motif 5 expression on cancer biology in gastric cancer cells. RNA-binding motif 5 protein expression was evaluated by immunohistochemistry using the surgical specimens of 106 patients with gastric cancer. We analyzed the relationships of RNA-binding motif 5 expression with clinicopathological parameters and patient prognosis. We further explored the effects of RNA-binding motif 5 downregulation with short hairpin RNA on cell growth and p53 signaling in MKN45 gastric cancer cells. Immunohistochemistry revealed that RNA-binding motif 5 expression was decreased in 29 of 106 (27.4%) gastric cancer specimens. Decreased RNA-binding motif 5 expression was correlated with histological differentiation, depth of tumor infiltration, nodal metastasis, tumor-node-metastasis stage, and prognosis. RNA-binding motif 5 silencing enhanced gastric cancer cell proliferation and decreased p53 transcriptional activity in reporter gene assays. Conversely, restoration of RNA-binding motif 5 expression suppressed cell growth and recovered p53 transactivation in RNA-binding motif 5-silenced cells. Furthermore, RNA-binding motif 5 silencing reduced the messenger RNA and protein expression of the p53 target gene p21. Our results suggest that RNA-binding motif 5 downregulation is involved in gastric cancer progression and that RNA-binding motif 5 behaves as a tumor suppressor gene in gastric cancer.