Estrogen/ERα signaling is critical for breast cancer progression and therapeutic treatments. Thus, identifying new regulators of this pathway will help to develop new therapeutics to overcome chemotherapy resistance of the breast cancer cells. Here, we report Ajuba directly interacts with ERα to potentiate ERα target gene expression, and biologically Ajuba promotes breast cancer cell growth and contributes to tamoxifen resistance of these cells. Ajuba constitutively binds the DBD and AF2 regions of ERα, and these interactions can be markedly enhanced by estrogen treatment. Mechanistically, Ajuba recruits DBC1 and CBP/p300 and forms a ternary complex to co-activate ERα transcriptional activity and concomitantly enhances ERα acetylation. Moreover, components of this complex can be found at endogenous promoters containing functional ERα responsive elements. Taken together, these data demonstrate that Ajuba functions as a novel co-activator of ERα and that Ajuba/DBC1/CBP/p300 ternary complex may be a new target for developing therapeutics to treat breast cancer.

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Damage-regulated autophagy modulator gene (DRAM) was first identified by Crighton et al. in 2006 when they treated Saos-2 cells with doxycycline and adriamycin after p53 induction and analyzed gene expression by using microarray technology [1]. The DRAM gene locates on the human chromosomal 12q23.2 [2] and encodes a 238 amino acid lysosomal protein whose role in autophagy is phylogenetically conserved. As a direct target of P53, DRAM is a critical modulator in p53-induced autophagy and programed cell death [3]. Nevertheless, the exact mechanism through which DRAM modulates autophagy has not been clarified. Sequence analysis predicted that the DRAM protein contains six transmembrane domains and several other highly conversed domains [1], therefore understanding the exact role of each domain of DRAM in autophagy may help to clarify the exact mechanism.To get a preliminary understanding of the functions of DRAM and its different transmembrane domains, we constructed eukaryotic expression plasmids of DRAM, pEGFP-DRAM and of its deletants, including pEGFP-DN1, pEGFP-DN2-3, pEGFP-DN4-5, pEGFP-DN4, pEGFP-DN5, and pEGFP-DN6 according to the prediction results [1] (Fig. 1A), and transfected them into HepG2 cells. The empty vector pEGFP-N1 was used as the negative control. The mRNA levels of DRAM or its deletants were identified by real-time PCR at 24 h after transfection (Fig. 1B). The protein levels for the two deletants ( pEGFP-DN4, pEGFP-DN5) were further determined by western blot analysis ( Supplementary Fig. S1). Primers used are listed in Supplementary Table S1. As DRAM is an autophagy modulator and autophagy is involved in apoptosis as well as cell proliferation under different conditions [4-6], we detected apoptosis, cell proliferation, and colony formation ability of these transfected cells.The results showed that expression of DRAM alone was not sufficient to affect apoptosis ( Supplementary Fig. S2), but it was able to promote cell proliferation ( Fig. 2A) and colony formation (Fig. 2B) of HepG2 cells, which are consistent with the observation of Kerley-Hamilton et al. [2] in Saos cell lines stably expressing DRAM. By comparing the results in the cell proliferation and colony formation assays ( Fig. 2A,B), we found that the deletion of the fourthfifth transmembrane domain significantly impaired DRAM's ability in promoting proliferation and colony formation of HepG2 cells, indicating that this region is crucial for the function of DRAM.To determine which of the fourth and the fifth transmembrane domains is more important than the other, the deletants of pEGFP-DN4 and pEGFP-DN5 were further analyzed. It was found that both regions were important in DRAM's ability of promoting cell proliferation (Fig. 2C), but the effect of the fourth transmembrane domain was more obvious than the fifth transmembrane domain. The soft agar colony formation assay revealed that DRAM could still cause a significant increase in the clonogenic potential of HepG2 cells when the fifth transmembrane domain was deleted. However, the delet...

LIM protein-domain containing protein Ajuba (encoded by AJUBA) functions as a scaffold protein to regulate protein-protein interactions, signalling transduction and genes transcription. AJUBA expression is higher in colorectal cancer (CRC) tissues than normal tissues, but its specific molecular function in CRC progression is still not very clear. Here, we found that, in CRC cancer cell lines, overexpression of AJUBA decreased p53 levels, whereas knock-down of AJUBA significantly increased p53 levels. Although the presence of Ajuba did not influence p53 transcription, it formed a complex with p53 and MDM2 to promote the degradation of p53. AJUBA overexpression reduced the sensitivity of cancer cells to chemotherapeutic drugs and vice versa. In addition, chemotherapeutic drugs significantly induced AJUBA expression, which was largely dependent on the presence of p53. Therefore, Ajuba formed a negative feedback loop to regulate p53 expression and activity. In conclusion, as a novel p53-negative regulator, Ajuba inhibits the apoptosis of CRC cells induced by chemotherapeutic drugs and it may be a new therapeutic target for CRC treatment.

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