The function of RASSF1C has not been as extensively studied as that of RASSF1A. Initial reports in the literature suggested that RASSF1C might function as a tumor suppressor in ovarian, prostate, renal cancer cells [15
]. Recently, RASSF1C has been shown to interact with DAXX, a protein involved in apoptosis and transcriptional repression. It has been suggested that RASSF1C may contribute to the activation of Stress-Activated Protein-kinase/c-jun N-terminal kinase [17
]. In contrast, we recently demonstrated that RASSF1C promotes lung cancer cell proliferation [19
]. We previously showed that RASSF1C plays a role in promoting osteoblast cell proliferation through interaction with Insulin like Growth Factor Binding protein (IGFBP)-5 [18
]. Consistent with our hypothesis, another group has recently shown that RASSF1C interacts with βTrCP (the receptor subunit of the SCFβTrCP
ubiquitin ligase that recruits signaling proteins and cell cycle regulators for proteosomal degradation) [41
]. Through this mechanism RASSF1C over-expression in the human lung cancer cell line A549 promotes the accumulation β-catenin, an oncogene and a key player in the Wnt signaling pathway, leading to increased transcriptional activation and cell proliferation [41
In this study, we demonstrated that reduction of RASSF1C mRNA in breast cancer cells correlated with a small but statistically significant decrease in cell proliferation compared to control cells that express RASSF1C. The reduction in RASSF1C did not affect cell viability as judged by trypan blue staining (data not shown). Overall our results are consistent with those we obtained in osteosarcoma (TE85, MG63, U2) and lung cancer (NCI H1299) cells [18
]. In the studies with osteosarcoma cells, the effects of silencing RASSF1C in cells that express both RASSF1A and RASSF1C (TE85) were very similar to those observed in cells that express only RASSF1C (MG63 and U2), clearly indicating that RASSF1A does not modulate the effect of RASSF1C on cell proliferation and survival [18
]. Based on these observations we think that, unlike RASSF1A, RASSF1C is not a tumor suppressor at least in breast, bone, and lung. Instead, it may function as a growth-stimulating factor.
To further determine the effect(s) of RASSF1C on breast cancer cell proliferation, we used a tetracycline-regulated MLV-based vector to stably over-express RASSF1C in MDA-MB231 and T47D. Tetracycline-regulated over-expression of RASSF1C did not inhibit cell proliferation. In fact, over-expression of RASSF1C caused reproducible and statistically significant increase in cell proliferation, further suggesting that RASSF1C is not a growth suppressor gene. In support of this, we have found that expression of RASSF1C is elevated in breast cancer cell lines compared to primary mammary epithelial cells, consistent with our hypothesis that RASSF1C may act as a potential growth and survival factor in breast cancer.
As mentioned above, a recent study suggested that regulated over-expression of RASSF1C may inhibit the growth of prostate cancer (LNCaP) and renal cell carcinoma (KRC/Y) cells [15
]. However, we have now shown that RASSF1C over-expression does not inhibit cell growth but rather significantly increases proliferation of osteosarcoma [18
], lung cancer [19
], and breast cancer (this study) cells. The different findings related to the stimulatory function of RASSF1C in human osteosarcoma, breast cancer, and lung cancer cells [18
], and the inhibitory function of RASSF1C in prostate, kidney, and ovarian cancer cells [15
] may be due to tissue-specific actions of RASSF1C. It is well known that some proteins such as IGFBP-5 and FHIT (Fragile Histidine Triad) exert opposite effects in different tissues [42
]. Our silencing and over-expression studies of RASSF1C underscores a potential growth promoting function at least in breast, lung, and osteosarcoma cell lines.
To begin to address the effect of RASSF1C on cellular growth, we carried out a microarray study to identify novel RASSF1C target genes. We found that RASSF1C over-expression interestingly modulated the expression of a number of genes that are involved in cancer development, cell growth/proliferation, cell cycle, cell death, and apoptosis. We validated the expression of several RASSF1C target genes by QRT-PCR and have also validated the expression of caspase 3, CXCR4, GHR, and TGM2 by Western blot analysis. We also found that cancer cells over-expressing RASSF1C exhibited increased phosphorylation of ERK1/2 compared to control cells (but no increase in total ERK1/2 levels), suggesting that RASSF1C may exert its activities in part through the activation of the ERK1/2 pathway. We also show that breast cancer cells over-expressing RASSF1C showed enhanced invasion/migration, while cells with knockdown of RASSF1C expression had reduced invasion/migration compared to control cells.
In addition, we found that RASSF1C over-expressing cells exhibited reduced caspase 3 activity compared to control cells when treated with etoposide. We also found that over-expressing RASSF1C for 14 days did not induce apoptosis as judged by lack of DNA fragmentation in Hs578T, MDA-MB231 and T47D cells, further suggesting that RASSF1C does not promote apoptosis. Taken together, the findings are consistent with our hypothesis that RASSF1C may potentially function as a metastasis promoting and apoptosis attenuating protein in breast cancer cells perhaps through the modulation of the CXCR4 and caspase 3 gene expression, respectively.
Our findings, together with others', are building a case that RASSF1C and RASSF1A have opposing, antagonistic functions at least in breast, lung, and bone cancer cells. For instance, it has been reported that RASSF1A inhibits lung cancer cell migration and promotes cell adhesion [44
]. RASSF1A has also been shown to activate the pro-apoptotic Bax1 gene [45
] which is consistent with its tumor suppressing activities. In this study, we have shown that RASSF1C promotes cell migration and down-regulates pro-apoptotic genes such as Bax, caspase 3, and SRPX. RASSF1C over-expression also up-regulates the expression of inhibitor of differentiation (Id2), which has been shown to be down-regulated by RASSF1A in nasopharyngeal carcinoma [46
]. Also, RASSF1C down-regulates TGM2 gene expression, which is up-regulated by RASSF1A in the non-small cell lung cancer cell line A549 [47
]. Lastly, our RASSF1C studies in breast cancer, lung cancer, and osteosarcoma cells, as well as other reports in the literature [41
] also suggest that RASSF1C functions in an opposing manner to that of RASSF1A. In all of these studies, RASSF1C appears to stimulate cell growth, while RASSF1A is well established to suppress it. Thus, a fine balance of RASSF1A and RASSF1C isoform expression may be critical in determining the neoplastic potential of a cell.
As mentioned above microarray analyses of mRNA in cells subjected to forced expression of RASSF1C stimulates expression and silencing of RASSF1C reduces expression of genes that are associated with cell growth and proliferation. These data provide significant information to propose testable models to explain how RASSF1C exerts it growth promoting activities in breast cancer. For example, up-regulation of CXCR4 by RASSF1C is an interesting discovery suggesting the hypothesis that RASSF1C may play a role in promoting breast cancer metastasis as mentioned above. Recent studies have shown that CXCR4 expression is increased early in the transition from normal to transformed breast epithelium [48
]. CXCR4 is a signaling receptor that interacts with a cognate ligand (SDF-1, also known as CXCL12) that is expressed at higher levels in tissues that attract breast cancer cells and support metastasis (e.g., bone, liver, lung). CXCR4 interaction with SDF-1 activates PI3K and Erk1/2 pathways and promotes cancer cell proliferation and metastasis [48
]. In light of these findings we are proposing a testable model of RASSF1C action (see Figure ) based on the hypothesis that RASSF1C increases the expression of CXCR4 in pre-malignant/malignant breast epithelial cells, thus supporting successful seeding and metastases in sites that express high levels of SDF-1. We plan to test this model and others to delineate the molecular mechanism/pathway(s) through which RASSF1C exerts its growth promoting effects on breast cancer cells using both in vitro
and in vivo
Figure 7 A hypothetical model proposed to explain RASSF1C action in breast cancer cells: we hypothesize that RASSF1C activates the Ras-Raf-ERK1/2 pathway leading to up-regulation of the CXCR4. Breast cancer cells expressing high number of CXCR4 receptor are attracted (more ...)
In conclusion, together with our previous work on RASSF1C in osteosarcoma and lung cancer cells, our present findings in breast cancer cells provide further evidence that RASSF1C is not a tumor suppressor and instead it may promote metastasis and survival of cancer cells. Our studies further suggest that RASSF1C may accomplish this by up-regulation of specific growth promoting genes and down-regulation of specific pro-apoptotic genes.