3 34 13 8 32 10 22 28 16 27 7 25 25 27 23 14 36 0 17 10 3 20 4 20 7 5 14 8 15 12 15 12 9 18 p Value 0.30 0.66 0.01 0.18 0.64 0.89 0.62 Zhang et al. BMC Cancer 2011, 11:32 http://www.biomedcentral.com/1471-2407/11/32 Table 1 Association of RRIG1 expression with clinicopathologic features of breast cancer patients RRIG1 expression Positive Negative Variable Patient age (years) ≤ 35 35-55 > 55 Tumor size (cm) ≤ 2 2-5 > 5 Lymph node metastasis + _ Tumor differentiation I II III Estrogen receptor + - Progesterone receptor + - c-erbB-2 + - Discussion In the current study, we analyzed RRIG1 expression in breast tissue specimens and then determined the role of RRIG1 in breast cancer cells. We found that RRIG1 expression is reduced in breast cancer tissues, as reported by us previously [7]. RRIG1 expression was inversely correlated with lymph node metastasis of breast cancer. Furthermore, the restoration of RRIG1 expression in a breast cancer cell line resulted in tumor- suppressor activities, such as inhibition of tumor cell proliferation, colony formation, migration, and invasion. At molecular levels, RRIG1 suppressed expression of p-Erk1/2, p-Akt, total Akt, c-Jun, and MMP9 and RhoA activity. These genes are related to cell growth, adhe- sion, and mobility. However, knockdown of RRIG1 expression in another breast cancer cell line promoted tumor cell proliferation, colony formation, migration, and invasion, but these effects were associated with reg- ulation of p-Stat3, p-Rb, and E2F-1.This study suggests that RRIG1 plays a role in suppressing breast cancer progression or even development of breast cancer. Our previous studies [5-7] demonstrated that RRIG1 is a novel gene and may be a putative tumor suppressor. RRIG1 gene contains 6 exons, with exons 2, 3, and 5 Page 6 of 12 shared with exons 8, 9, and 10 of SH3GLB2 cDNA [7]. However, the open reading frames of these two genes are different, and therefore they are different genes. Moreover, our study also revealed that these two genes were differentially expressed in esophageal and prostate cancer cell lines [5,8] and that benzo[a]pyrene diol epoxide (BPDE) reduced RRIG1 expression but did not alter SH3GLB2 mRNA levels in esophageal cancer cell lines (our unpublished data). These findings indicate that RRIG1 is a novel protein with no similarities to other proteins and that RRIG1 protein contains several putative functional motifs, such as a cadherin signature- like motif, a glycoprotein GG/GX motif, and proline- rich regions that contain SH3 domain-binding motifs (PxxP). The latter motif was able to functionally mediate RRIG1 antitumor activity by suppressing tumor cell via- bility and cyclin D1 expression [7]. Nevertheless, it is unclear whether knockdown of RRIG1 expression using a vector carrying antisense open reading frame of RRIG1 can block expression of SH3GLB2 protein (Because no anti-SH3GLB2 antibody is available, we could not perform such an experiment). However, our current study showed that the vector carrying antisense open reading frame of RRIG1 did not affect expression of SH3GLB2 mRNA (Figure 5). Our current and previous studies clearly demonstrated that reduced or lost expression of RRIG1 is an impor- tant event in the development or progression of human cancers, although the mechanism underlying the loss of expression of the RRIG1 gene remains unknown [5-8]. Moreover, our previous studies [12-14] reported that benzo[a]pyrene diol epoxide (BPDE), a carcinogen pre- sent in tobacco smoke and environmental pollution, as well as bile acid, a tumor promoter in the gastrointest- inal tract, inhibited RRIG1 expression, whereas retinoic acid induced RRIG1 expression, which was associated with downregulation of RAR-b2 expression by these car- cinogens. In addition, previous studies demonstrated a loss of RAR-b2 expression in breast cancer tissues and cells [15-17]. Further study will be needed to determine the molecular mechanism that contributes to the loss of RRIG1 expression and, most importantly, to define RRIG1 as a tumor-suppressor gene in the inhibition of tumor cell growth, invasion, and gene expression. Our current study demonstrated that the restoration ofRRIG1 expression inhibited breast cancer cell prolif- eration,colony formation, migration, and invasion and regulated geneexpression. Inhibition of phosphorylated Erk1/2 and Akt and c-Junexpression by RRIG1 may mediate RRIG1 suppression of cell growth, while reduced MMP9 expression by RRIG1 mediates the reduced tumor cell mobility and invasion capacity. Our previous study showed that MMP9 was upregulated in breast cancer [18]. Erk1/2 and Akt proteins play an