MicroRNAs (miRs) are non-coding RNAs that inhibit expression of their targets in a sequence-specific manner and play crucial roles during oncogenesis. Here we show that miR-7 inhibits p21-activated kinase 1 (Pak1) expression, a widely upregulated signaling kinase in multiple human cancers including breast and gliomas, by targeting the 3′-UTR of Pak1 mRNA. We noticed an inverse correlation between the levels of endogenous miR-7 and Pak1 expression in human cancer cells. We discovered that endogenous miR-7 expression is positively regulated by a homeodomain transcription factor HoxD10, loss of which leads to an increased invasiveness. The HoxD10 directly interacts with the miR-7 chromatin. Accordingly, the levels of Pak1 protein are progressively upregulated while that of miR-7 and its upstream activator HoxD10 are progressively downregulated in a cellular model of breast cancer progression from low to highly invasive phenotypes. Furthermore, HoxD10 expression in highly invasive breast cancer cells resulted in an increased expression of miR-7 but a reduced Pak1 3′UTR-luciferase activity as well as reduced Pak1 protein. Finally, we show that miR-7 introduction inhibits the motility, invasiveness, anchorage-independent growth and tumorigenic potential of highly invasive breast cancer cells. Collectively, these findings establish for the first time that Pak1 is a target of miR-7 and that HoxD10 play a regulatory role in modifying the expression of miR-7, and consequently, functions of miR7 - Pak1 pathway in human cancer cells.

SUMMARY Chromatin dynamics play a central role in maintaining genome integrity, but how this is achieved remains largely unknown. Here, we report that microrchidia CW-type zinc finger 2 (MORC2), an uncharacterized protein with a derived PHD finger domain and a conserved GHKL-type ATPase module, is a physiological substrate of p21-activated kinase 1 (PAK1), an important integrator of extracellular signals and nuclear processes. Following DNA damage, MORC2 is phosphorylated on serine 739 in a PAK1 dependent manner, and phosphorylated MORC2 regulates its DNA-dependent ATPase activity to facilitate chromatin remodeling. Moreover, MORC2 associates with chromatin and promotes gamma-H2AX induction in a PAK1 phosphorylation-dependent manner. Consequently, cells expressing MORC2-S739A mutation displayed a reduction in DNA repair efficiency and were hypersensitive to DNA-damaging agent. These findings suggest that the PAK1-MORC2 axis is critical for orchestrating the interplay between chromatin dynamics and the maintenance of genomic integrity through sequentially integrating multiple essential enzymatic processes.

Breast cancer transcriptome acquires a myriad of regulation changes, and splicing is critical for the cell to “tailor-make” specific functional transcripts. We systematically revealed splicing signatures of the three most common types of breast tumors using RNA sequencing: TNBC, non-TNBC and HER2-positive breast cancer. We discovered subtype specific differentially spliced genes and splice isoforms not previously recognized in human transcriptome. Further, we showed that exon skip and intron retention are predominant splice events in breast cancer. In addition, we found that differential expression of primary transcripts and promoter switching are significantly deregulated in breast cancer compared to normal breast. We validated the presence of novel hybrid isoforms of critical molecules like CDK4, LARP1, ADD3, and PHLPP2. Our study provides the first comprehensive portrait of transcriptional and splicing signatures specific to breast cancer sub-types, as well as previously unknown transcripts that prompt the need for complete annotation of tissue and disease specific transcriptome.

Metastasis-associated protein 1 (MTA1), a component of the nucleosome remodeling and histone deacetylation (NuRD) complex, is widely upregulated in human cancers. However, the mechanism for regulating its protein stability remains unknown. Here we report that MTA1 is an ubiquitinated protein and targeted by the RING-finger E3 ubiquitin-protein ligase constitutive photomorphogenesis protein 1 (COP1) for degradation via the ubiquitin-proteasome pathway. Induced expression of wild-type COP1 but not its RING motif mutants promotes the ubiquitination and degradation of MTA1, indicating that the ligase activity is required for the COP1-mediated proteolysis of MTA1. Conversely, depletion of endogenous COP1 resulted in a marked decrease in MTA1 ubiquitination, accompanied by a pronounced accumulation of MTA1 protein. MTA1, in turn, destabilizes COP1 by promoting its autoubiquitination, thus creating a tight feedback loop that regulates both MTA1 and COP1 protein stability. Accordingly, disruption of the COP1-mediated proteolysis by ionizing radiation leads to MTA1 stabilization, accompanied by an increased coregulatory function of MTA1 on its target. Furthermore, we discovered that MTA1 is required for optimum DNA double-strand break repair after ionizing radiation. These findings provide novel insights into the regulation of MTA1 protein and reveal a novel function of MTA1 in DNA damage response.coregulator ͉ DNA repair ͉ ubiquitination R egulation of fundamental cellular processes demands dynamic coordinated participation of transcription factors and their coregulators at the target gene chromatin (1, 2), and deregulation of such processes plays a critical role in the development of malignant phenotypes. One emerging family of ubiquitously expressed chromatin modifiers is the metastasisassociated protein (MTA) family, which has an integral role in nucleosome remodeling and histone deacetylation (NuRD) complexes that modify DNA accessibility for cofactors (2, 3). MTA1, the founding member of the MTA family, is widely upregulated in human cancers and plays an important role in tumorigenesis and tumor aggressiveness, especially tumor invasion and metastasis (4-6). MTA1 functions not only as a transcriptional repressor of estrogen receptor ␣ (7), but also as a transcriptional activator on certain promoters, such as the breast cancer-amplified sequence 3 (BCAS3) promoter (8). In this context, MTA1 is acetylated at lysine 626 (K626) by histone acetyltransferase p300; such modification allows MTA1 to recruit RNA polymerase II (Pol II) on the BCAS3 enhancer region and confers its coactivator function upon BCAS3 (8). MTA1 is also a mechanistic mediator of c-Myc-regulated transformation as a downstream target of the oncogene c-Myc (9). Although a paramount role of MTA1 in cancer and coregulator biology, the mechanism for regulating its protein stability remains unknown.Constitutive photomorphogenic 1 (COP1; also known as RFWD2, RING finger and WD repeat domain protein 2), an evolutionarily conserved RING-finger ubiquitin-protei...

Breast cancer is a heterogeneous disease with a poorly defined genetic landscape, which poses a major challenge in diagnosis and treatment. By massively parallel mRNA sequencing, we obtained 1.2 billion reads from 17 individual human tissues belonging to TNBC, Non-TNBC, and HER2-positive breast cancers and defined their comprehensive digital transcriptome for the first time. Surprisingly, we identified a high number of novel and unannotated transcripts, revealing the global breast cancer transcriptomic adaptations. Comparative transcriptomic analyses elucidated differentially expressed transcripts between the three breast cancer groups, identifying several new modulators of breast cancer. Our study also identified common transcriptional regulatory elements, such as highly abundant primary transcripts, including osteonectin, RACK1, calnexin, calreticulin, FTL, and B2M, and “genomic hotspots” enriched in primary transcripts between the three groups. Thus, our study opens previously unexplored niches that could enable a better understanding of the disease and the development of potential intervention strategies.

Although metastasis-associated protein 1 (MTA1) has recently been shown as a DNA damage responsive protein, the underlying mechanism for its role in DNA double-strand break (DSB) repair remains unknown. Here, we show that MTA1 controls p53 stability through inhibiting its ubiquitination by E3 ubiquitin ligases mouse double minute 2 (Mdm2) and constitutive photomorphogenic protein 1 (COP1). The underlying mechanisms involve the ability of MTA1 to compete with COP1 to bind to p53 and/or to destabilize COP1 and Mdm2. Consequently, MTA1 regulates the p53-dependent transcription of p53R2, a direct p53 target gene for supplying nucleotides to repair damaged DNA. Depletion of MTA1 impairs p53-dependent p53R2 transcription and compromises DNA repair. Interestingly, these events could be reversed by MTA1 reintroduction, indicating that MTA1 interjects into the p53-dependent DNA repair. Given the fact that MTA1 is widely up-regulated in human cancers, these findings in conjunction with our earlier finding of a crucial role of MTA1 in DSB repair suggest an inherent role of the MTA1-p53-p53R2 pathway in DNA damage response in cancer cells.The p53 tumor suppressor is a central component of cellular mechanisms that respond to DNA damage signals to preserve genomic integrity (1, 2). Under physiological conditions, p53 is tightly regulated and normally maintained at low levels by the action of several RING finger E3 ubiquitin ligases, including constitutive photomorphogenic protein 1 (COP1) (3), mouse double minute 2 (Mdm2) (4, 5), and p53-induced protein with a RING H2 domain (Pirh2) (6). All of these ligases are transcriptionally stimulated by the p53 protein and in turn, target p53 for the ubiquitin-dependent proteolysis, thereby creating tight negative feedback loops for controlling p53 protein stability (7). Accordingly, disruption of these autoregulatory feedback loops is a pivotal event for the activation of p53 in response to various genotoxic stresses (8, 9). Following DNA damage, p53 protein is stabilized and activated through post-translational modifications, resulting in a controlled activation of a series of downstream target genes that mediate its functions (10 -12). In addition to its important functions in cell cycle arrest and apoptosis (2, 12), the p53 protein plays a critical role in regulating DNA repair caused by various genotoxic stresses (13-17). Loss of p53 function leads to decreased repair of damaged DNA and is reflected by increased sensitivity to DNA damage agents. Therefore, blocking the p53-induced DNA repair could prove to be an efficient approach to enhance the efficacy of DNA-damaging agents (18).Recently, numerous potential mechanisms have been described as to how p53 functions to regulate DNA repair. p53 directly associates with TFIIH, a nucleotide excision repair component (19) and transactivates genes implicated in DNA repair, such as p53R2, a newly identified subunit of ribonucleotide reductase (20, 21). The ability of p53R2 to supply nucleotides for repairing DNA damage requires the presen...

In spite of a large number of transforming growth factor β1 (TGF-β1)-regulated genes, the nature of its targets with roles in transformation continues to be poorly understood. Here we discovered that TGF-β1 stimulates transcription of metastasis associated protein 1 (MTA1), a dual master coregulator, in epithelial cells, and that MTA1 status is a determinant of TGF-β1-induced epithelial-to-mesenchymal transition phenotypes. In addition, we found that MTA1/Pol II/AP1 coactivator complex interacts with the FosB-gene chromatin and stimulates its transcription, and FosB in-turn, utilizes FosB/HDAC2 complex to repress E-cadherin expression in TGF-β1 stimulated mammary epithelial cells. These findings suggest that TGF-β1 regulates the components of EMT via stimulating the expression of MTA1 which in turn, induces FosB to repress E-cadherin expression and thus, revealed an inherent function of MTA1 as a target and effector of TGF-β1 signaling in epithelial cells.

MicroRNAs (miR) have been identified as posttranscriptional modifiers of target gene regulation and control the expression of gene products important in cancer progression. Here, we show that miR-661 inhibits the expression of metastatic tumor antigen 1 (MTA1), a widely up-regulated gene product in human cancer, by targeting the 3 ¶ untranslated region (UTR) of MTA1 mRNA. We found that endogenous miR-661 expression was positively regulated by the c/EBPA transcription factor, which is down-regulated during cancer progression. c/EBPA directly interacted with the miR-661 chromatin and bound to miR-661 putative promoter that contains a c/EBPA-consensus motif. In addition, we found that the level of MTA1 protein was progressively up-regulated, whereas that of miR-661 and its activator, c/EBPA, were down-regulated in a breast cancer progression model consisting of MCF-10A cell lines whose phenotypes ranged from noninvasive to highly invasive. c/EBPA expression in breast cancer cells resulted in increased miR-661 expression and reduced MTA1 3 ¶UTR-luciferase activity and MTA1 protein level. We also provide evidence that the introduction of miR-661 inhibited the motility, invasiveness, anchorage-independent growth, and tumorigenicity of invasive breast cancer cells. We believe our findings show for the first time that c/EBPA regulates the level of miR-661 and in turn modifies the functions of the miR661-MTA1 pathway in human cancer cells. Based on these findings, we suggest that miR-661 be further investigated for therapeutic use in down-regulating the expression of MTA1 in cancer cells.