A Large Scale shRNA Barcode Screen Identifies the Circadian Clock Component ARNTL as Putative Regulator of the p53 Tumor Suppressor Pathway

BACKGROUND: The p53 tumor suppressor gene is mutated in about half of human cancers, but the p53 pathway is thought to be functionally inactivated in the vast majority of cancer. Understanding how tumor cells can become insensitive to p53 activation is therefore of major importance. Using an RNAi-based genetic screen, we have identified three novel genes that regulate p53 function. RESULTS: We have screened the NKI shRNA library targeting 8,000 human genes to identify modulators of p53 function. Using the shRNA barcode technique we were able to quickly identify active shRNA vectors from a complex mixture. Validation of the screening results indicates that the shRNA barcode technique can reliable identify active shRNA vectors from a complex pool. Using this approach we have identified three genes, ARNTL, RBCK1 and TNIP1, previously unknown to regulate p53 function. Importantly, ARNTL (BMAL1) is an established component of the circadian regulatory network. The latter finding adds to recent observations that link circadian rhythm to the cell cycle and cancer. We show that cells having suppressed ARNTL are unable to arrest upon p53 activation associated with an inability to activate the p53 target gene p21(CIP1). CONCLUSIONS: We identified three new regulators of the p53 pathway through a functional genetic screen. The identification of the circadian core component ARNTL strengthens the link between circadian rhythm and cancer

Development of Resistance towards Artesunate in MDA-MB-231 Human Breast Cancer Cells

Breast cancer is the most common cancer and the second leading cause of cancer death in industrialized countries. Systemic treatment of breast cancer is effective at the beginning of therapy. However, after a variable period of time, progression occurs due to therapy resistance. Artesunate, clinically used as anti-malarial agent, has recently revealed remarkable anti-tumor activity offering a role as novel candidate for cancer chemotherapy. We analyzed the anti-tumor effects of artesunate in metastasizing breast carcinoma in vitro and in vivo. Unlike as expected, artesunate induced resistance in highly metastatic human breast cancer cells MDA-MB-231. Likewise acquired resistance led to abolishment of apoptosis and cytotoxicity in pre-treated MDA-MB-231 cells. In contrast, artesunate was more cytotoxic towards the less tumorigenic MDA-MB-468 cells without showing resistance. Unraveling the underlying molecular mechanisms, we found that resistance was induced due to activation of the tumor progression related transcription factors NFκB and AP-1. Thereby transcription, expression and activity of the matrix-degrading enzyme MMP-1, whose function is correlated with increased invasion and metastasis, was up-regulated upon acquisition of resistance. Additionally, activation of the apoptosis-related factor NFκB lead to increased expression of ant-apoptotic bcl2 and reduced expression of pro-apoptotic bax. Application of artesunate in vivo in a model of xenografted breast cancer showed, that tumors growth was not efficiently abolished as compared to the control drug doxorubicin. Taken together our in vitro and in vivo results correlate well showing for the first time that artesunate induces resistance in highly metastatic breast tumors

Apoptosis Induced by Piroxicam plus Cisplatin Combined Treatment Is Triggered by p21 in Mesothelioma

BACKGROUND: Malignant mesothelioma (MM) is a rare, highly aggressive tumor, associated to asbestos exposure. To date no chemotherapy regimen for MM has proven to be definitively curative, and new therapies for MM treatment need to be developed. We have previously shown in vivo that piroxicam/cisplatin combined treatment in MM, specifically acts on cell cycle regulation triggering apoptosis, with survival increase. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed, at molecular level, the apoptotic increase caused by piroxicam/cisplatin treatment in MM cell lines. By means of genome wide analyses, we analyzed transcriptional gene deregulation both after the single piroxicam or cisplatin and the combined treatment. Here we show that apoptotic increase following combined treatment is mediated by p21, since apoptotic increase in piroxicam/cisplatin combined treatment is abolished upon p21 silencing. CONCLUSIONS/SIGNIFICANCE: Piroxicam/cisplatin combined treatment determines an apoptosis increase in MM cells, which is dependent on the p21 expression. The results provided suggest that piroxicam/cisplatin combination might be tested in clinical settings in tumor specimens that express p21

Histone Deacetylase 3 Depletion in Osteo/Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat

Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health

Necdin, a p53-Target Gene, Is an Inhibitor of p53-Mediated Growth Arrest

In vitro, cellular immortalization and transformation define a model for multistep carcinogenesis and current ongoing challenges include the identification of specific molecular events associated with steps along this oncogenic pathway. Here, using NIH3T3 cells, we identified transcriptionally related events associated with the expression of Polyomavirus Large-T antigen (PyLT), a potent viral oncogene. We propose that a subset of these alterations in gene expression may be related to the early events that contribute to carcinogenesis. The proposed tumor suppressor Necdin, known to be regulated by p53, was within a group of genes that was consistently upregulated in the presence of PyLT. While Necdin is induced following p53 activation with different genotoxic stresses, Necdin induction by PyLT did not involve p53 activation or the Rb-binding site of PyLT. Necdin depletion by shRNA conferred a proliferative advantage to NIH3T3 and PyLT-expressing NIH3T3 (NIHLT) cells. In contrast, our results demonstrate that although overexpression of Necdin induced a growth arrest in NIH3T3 and NIHLT cells, a growing population rapidly emerged from these arrested cells. This population no longer showed significant proliferation defects despite high Necdin expression. Moreover, we established that Necdin is a negative regulator of p53-mediated growth arrest induced by nutlin-3, suggesting that Necdin upregulation could contribute to the bypass of a p53-response in p53 wild type tumors. To support this, we characterized Necdin expression in low malignant potential ovarian cancer (LMP) where p53 mutations rarely occur. Elevated levels of Necdin expression were observed in LMP when compared to aggressive serous ovarian cancers. We propose that in some contexts, the constitutive expression of Necdin could contribute to cancer promotion by delaying appropriate p53 responses and potentially promote genomic instability

Motif co-regulation and co-operativity are common mechanisms in transcriptional, post-transcriptional and post-translational regulation

A substantial portion of the regulatory interactions in the higher eukaryotic cell are mediated by simple sequence motifs in the regulatory segments of genes and (pre-)mRNAs, and in the intrinsically disordered regions of proteins. Although these regulatory modules are physicochemically distinct, they share an evolutionary plasticity that has facilitated a rapid growth of their use and resulted in their ubiquity in complex organisms. The ease of motif acquisition simplifies access to basal housekeeping functions, facilitates the co-regulation of multiple biomolecules allowing them to respond in a coordinated manner to changes in the cell state, and supports the integration of multiple signals for combinatorial decision-making. Consequently, motifs are indispensable for temporal, spatial, conditional and basal regulation at the transcriptional, post-transcriptional and post-translational level. In this review, we highlight that many of the key regulatory pathways of the cell are recruited by motifs and that the ease of motif acquisition has resulted in large networks of co-regulated biomolecules. We discuss how co-operativity allows simple static motifs to perform the conditional regulation that underlies decision-making in higher eukaryotic biological systems. We observe that each gene and its products have a unique set of DNA, RNA or protein motifs that encode a regulatory program to define the logical circuitry that guides the life cycle of these biomolecules, from transcription to degradation. Finally, we contrast the regulatory properties of protein motifs and the regulatory elements of DNA and (pre-)mRNAs, advocating that co-regulation, co-operativity, and motif-driven regulatory programs are common mechanisms that emerge from the use of simple, evolutionarily plastic regulatory modules

C-elegans ced-13 can promote apoptosis and is induced in response to DNA damage

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein. We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes