This study provides the first evidence that LncRNA ARHGAP5-AS1 was downregulated in human breast cancer specimens. Furthermore, ARHGAP5-AS1 could inhibit cell migration via suppression of stress fibers in breast cancer cells. More importantly, to our knowledge it is the first lncRNA could interact with SMAD7, which is an important inhibitory Smads that negatively control TGFβ signaling pathway. The present study demonstrated that ARHGAP5-AS1 impaired the degradation of SMAD7 via interaction with its PY motif, blocking the interation with its E3 ligase (SMURF1 & SMURF2) and thus decreased its ubiquitination. Knockdown of SMAD7 mimicked the promotion of cell migration by knockdown of ARHGAP5-AS1. Meanwhile, ectopic expression of SMAD7 blocked the increase in migration and stress fiber formation by knockdown of ARHGAP5-AS1. Moreover, ARHGAP5-AS1 inhibited the formation of stress fiber and hence migration via suppression of TGFβ signaling by stabilized SMAD7.
ARHGAP5-AS1 located on the human chromosome 14 and was first identified as a transcriptional isoform of ARHGAP5 in gastric cancer by large-scale sequencing [23]. Non-coding RNA was considered as transcription “noise” in the past. However, more and more studies showed an emerging and diverse role in cancer pathways [9]. For example, lncRNA NKILA suppresses breast cancer metastasis by interaction with p65 and blocking IκB phosphorylation [24]. Here we illustrated that ARHGAP5-AS1 was a long non-coding RNA and its expression was downregulated in breast cancer tissues, which data were achieved from TCGA database. Meanwhile, the downregulation of ARHGAP5-AS1 was further evidenced in the collected human breast cancer tissues (n = 22), compared to the corresponding adjacent normal tissues, suggesting a tumor-suppressor role in breast caner. Thus, we investigated the role of ARHGAP5-AS1 in initiation and progression of breast cancer. As a result, ARHGAP5-AS1 inhibited migration of breast cancer cells. Moreover, stress fiber formation was reduced by ARHGAP5-AS1. It’s a pity that we have not studied the regulatory mechanism of ARHGAP5-AS1 expression in breast cancer cells. We thought there could be a possibility that the expression of ARHGAP5-AS1 is regulated by some transcription factors which expression are quite different in breast cancer tissues.
LncRNA functions as protein scaffold, which are already well established [25]. We demonstrated that ARHGAP5-AS1 interacted with PY-motif of SMAD7. PY motif is responsible for the interaction of SMAD7 with WW domain of E3 ligase [20]. Indeed, ARHGAP5-AS1 occupied PY motif and blocked the ubiquitination of SMAD7 by E3 ligase (SMRUF1 & SMURF2), so that reduced the degradation of SMAD7 protein. SMAD7 is a powerful inhibitor of TGFβ signaling, antagonizing TGFβ signaling via multiple mechanisms [14]. TGFβ signaling pathway exerts tumor-suppressive effects in cancer initiation, yet it promotes epithelial to mesenchymal transition (EMT), cell invasion as well as migration in cancer progression [26, 27]. The dual role of TGFβ in cancer has long been noted, but its mechanistic basis, operating logic, and clinical relevance have remained elusive. TGFβ induces cytoskeleton reorganization by upregulation of RhoGEFs, such as NET1 [28] and GEFH1 [29]. While SMAD7 inhibits TGFβ induced actin reorganization and RhoA activation [22]. It is expectable that TGFβ induced stress fibers and cell migration could be blocked by ARHGAP5-AS1 due to its interaction with SMAD7 and increased the stability of the latter. However, the function of ARHGAP5-AS1 in breast cancer cells was detected without exogenous TGFβ stimulation. Tumor cells themselves are able to produce TGF-β ligands [30, 31], which might be a reasonable explanation. Indeed, knockdown of SMAD7 mimicked the promotion effect of cell migration by knockdown of ARHGAP5-AS1. Furthermore, ectopic expression of SMAD7 was able to block the promotion of cell migration by knockdown of ARHGAP5-AS1. Above results were also achieved without TGFβ stimulation. To verify the effects of ARHGAP5-AS1 on TGFβ stimulated signaling, we further knockdown the expression of ARHGAP5-AS1 in MDA-MB-231 cells and these cells were starved for 12 h and treated with 0.3 ng/mL TGFβ1. Prolonged activation of TGFβ signaling was demonstrated by upregulation of TGFβ R1 and increased phosphorylation of SMAD2 compared to the control cells. Besides, four fragments of SMAD-binding element (SBE) were cloned into pGL4.27 vector. ARHGAP5-AS1 overexpression showed reduced luciferase activity with stimulation of TGFβ, proving the inhibitory role on TGFβ signaling.
In this study, we give a clear interpretation that ARHGAP5-AS1, which is downregulated in breast cancer tissue, suppresses the cell migration and stress fiber formation in breast cancer cells. Furthermore, SMAD7, which is an important inhibitory Smads that negatively control TGFβ signaling pathway, is demonstrated to be interacted with ARHGAP5-AS1 and its protein level is regulated by ARHGAP5-AS1. Thus, our findings provide valuable clues toward understanding the mechanisms of human breast cancer progression and present an opportunity to develop more effective clinical therapies in the future.