Sweet DREAMs for hippo

The Hippo pathway coordinates organ size and cell proliferation. The retinoblastoma family of proteins regulates progression through the G0/G1 phase of the cell cycle. Disruption of either pathway contributes to cancer formation. Three recent studies in Genes & Development reveal how cellular proliferation is coordinated between these pathways. Here we discuss the implications of these studies and the new questions that they raise. © 2011 by Cold Spring Harbor Laboratory Press

Requirements for E1A dependent transcription in the yeast Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>The human adenovirus type 5 early region 1A (E1A) gene encodes proteins that are potent regulators of transcription. E1A does not bind DNA directly, but is recruited to target promoters by the interaction with sequence specific DNA binding proteins. In mammalian systems, E1A has been shown to contain two regions that can independently induce transcription when fused to a heterologous DNA binding domain. When expressed in <it>Saccharomyces cerevisiae</it>, each of these regions of E1A also acts as a strong transcriptional activator. This allows yeast to be used as a model system to study mechanisms by which E1A stimulates transcription.</p> <p>Results</p> <p>Using 81 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, CSR, INO80, ISW1, NuA3, NuA4, Mediator, PAF, RSC, SAGA, SAS, SLIK, SWI/SNF and SWR1 transcriptional regulatory complexes on E1A dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on transcriptional activation.</p> <p>Conclusion</p> <p>Our analysis indicates that the two activation domains of E1A function via distinct mechanisms, identify new factors regulating E1A dependent transcription and suggest that yeast can serve as a valid model system for at least some aspects of E1A function.</p

Conserved region 3 of human papillomavirus 16 E7 contributes to deregulation of the retinoblastoma tumor suppressor

The human papillomavirus (HPV) E7 oncoprotein binds cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. A key target of E7 is the product of the retinoblastoma susceptibility locus (pRb). This interaction results in the release of E2F transcription factors and drives the host cell into the S phase of the cell cycle. E7 binds pRb via a high-affinity binding site in conserved region 2 (CR2) and also targets a portion of cellular pRb for degradation via the proteasome. Evidence suggests that a secondary binding site exists in CR3, and that this interaction influences pRb deregulation. Additionally, evidence suggests that CR3 also participates in the degradation of pRb. We have systematically analyzed the molecular mechanisms by which CR3 contributes to deregulation of the pRb pathway by utilizing a comprehensive series of mutations in residues predicted to be exposed on the surface of HPV16 E7 CR3. Despite differences in the ability to interact with cullin 2, all CR3 mutants degrade pRb comparably to wild-type E7. We identified two specific patches of residues on the surface of CR3 that contribute to pRb binding independently of the high-affinity CR2 binding site. Mutants within CR3 that affect pRb binding are less effective than the wild-type E7 in overcoming pRb-induced cell cycle arrest. This demonstrates that the interaction between HPV16 E7 CR3 and pRb is functionally important for alteration of the cell cycle. © 2012, American Society for Microbiology

Adenovirus E1A directly targets the E2F/DP-1 complex

Deregulation of the cell cycle is of paramount importance during adenovirus infection. Adenovirus normally infects quiescent cells and must initiate the cell cycle in order to propagate itself. The pRb family of proteins controls entry into the cell cycle by interacting with and repressing transcriptional activation by the E2F transcription factors. The viral E1A proteins indirectly activate E2F-dependent transcription and cell cycle entry, in part, by interacting with pRb and family members to free the E2Fs. We report here that an E1A 13S isoform can unexpectedly activate E2F-responsive gene expression independently of binding to the pRb family of proteins. We demonstrate that E1A binds to E2F/DP-1 complexes through a direct interaction with DP-1. E1A appears to utilize this binding to recruit itself to E2F-regulated promoters, and this allows the E1A 13S protein, but not the E1A 12S protein, to activate transcription independently of interaction with pRb. Importantly, expression of E1A 13S, but not E1A 12S, led to significant enhancement of E2F4 occupancy of E2F sites of two E2F-regulated promoters. These observations identify a novel mechanism by which adenovirus deregulates the cell cycle and suggest that E1A 13S may selectively activate a subset of E2F-regulated cellular genes during infection. © 2011, American Society for Microbiology

E1A Activates Transcription of p73 and Noxa to Induce Apoptosis

p73, a member of the p53 family of proteins, transcriptionally activates a number of genes involved in the control of cell cycle and apoptosis. Overexpression of p73 was detected in a large number of primary head and neck cancers, and in the established cell lines examined, these all contained inactivating p53 mutations. The significance of p73 overexpression in the pathogenesis of head and neck cancer is currently unclear. We have shown that the expression of adenovirus 5 E1A in a panel of head and neck cancer cell lines induces apoptosis independently of their p53 status. In this study we examined the role of p73 and its transcriptional targets in E1A-mediated induction of apoptosis. E1A expression resulted in significant activation of the TAp73 promoter but had no effect on the alternative, DeltaNp73 promoter. E1A also increased expression of endogenous TAp73 mRNA and protein. E1A mutants lacking the p300- and/or pRB-binding sites showed reduced ability to activate the TAp73 promoter. Additionally, mutations in the E2F1-binding sites in the TAp73 promoter impaired activation by E1A. Importantly, expression of the 13S isoform of E1A substantially induced the p53 apoptotic target Noxa in several p53-deficient cancer cell lines. Our results indicate that E1A activation of p73 and the p53 apoptotic target Noxa can occur in the absence of a functional p53. This activation is likely to play a key role in the mechanism of p53-independent apoptosis induced by E1A in some cancers and may provide an avenue for future cancer therapies

Corepressor/coactivator paradox: potential constitutive coactivation by corepressor splice variants

The functional consequences of the interaction of transcriptional coregulators with the human thyroid hormone receptor (TR) in mammalian cells are complex. We have used the yeast, Saccharomyces cerevisiae, which lack endogenous nuclear receptors (NRs) and NR coregulators, as a model to decipher mechanisms regulating transcriptional activation by TR. In effect, this system allows the reconstitution of TR mediated transcription complexes by the expression of specific combinations of mammalian proteins in yeast. In this yeast system, human adenovirus 5 early region 1A (E1A), a natural N-CoR splice variant (N-CoR(I)) or an artificial N-CoR truncation (N-CoR(C)) coactivate unliganded TRs and these effects are inhibited by thyroid hormone (TH). E1A contains a short peptide sequence that resembles known corepressor-NR interaction motifs (CoRNR box motif, CBM), and this motif is required for TR binding and coactivation. N-CoR(I) and N-CoR(C) contain three CBMs, but only the C-terminal CBM1 is critical for coactivation. These observations in a yeast model system suggest that E1A and N-CoR(I) are naturally occurring TR coactivators that bind in the typical corepressor mode. These findings also raise the possibility that alternative splicing events which form corepressor proteins containing only C-terminal CBM motifs could represent a novel mechanism in mammalian cells for regulating constitutive transcriptional activation by TRs

The human papillomavirus E7 proteins associate with p190RhoGAP and alter its function

Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis. © 2014, American Society for Microbiology

High-throughput testing in head and neck squamous cell carcinoma identifies agents with preferential activity in human papillomavirus-positive or negative cell lines.

Head and neck squamous cell carcinoma (HNSCC) is a common cancer diagnosis worldwide. Despite advances in treatment, HNSCC has very poor survival outcomes, emphasizing an ongoing need for development of improved therapeutic options. The distinct tumor characteristics of human papillomavirus (HPV)-positive vs. HPV-negative disease necessitate development of treatment strategies tailored to tumor HPV-status. High-throughput robotic screening of 1,433 biologically and pharmacologically relevant compounds at a single dose (4 μM) was carried out against 6 HPV-positive and 20 HPV-negative HNSCC cell lines for preliminary identification of therapeutically relevant compounds. Statistical analysis was further carried out to differentiate compounds with preferential activity against cell lines stratified by the HPV-status. These analyses yielded 57 compounds with higher activity in HPV-negative cell lines, and 34 with higher-activity in HPV-positive ones. Multi-point dose-response curves were generated for six of these compounds (Ryuvidine, MK-1775, SNS-032, Flavopiridol, AZD-7762 and ARP-101), confirming Ryuvidine to have preferential potency against HPV-negative cell lines, and MK-1775 to have preferential potency against HPV-positive cell lines. These data comprise a valuable resource for further investigation of compounds with therapeutic potential in the HNSCC

A case report and genetic characterization of a massive acinic cell carcinoma of the parotid with delayed distant metastases.

We describe the presentation, management, and clinical outcome of a massive acinic cell carcinoma of the parotid gland. The primary tumor and blood underwent exome sequencing which revealed deletions in CDKN2A as well as PPP1R13B, which induces p53. A damaging nonsynonymous mutation was noted in EP300, a histone acetylase which plays a role in cellular proliferation. This study provides the first insights into the genetic underpinnings of this cancer. Future large-scale efforts will be necessary to define the mutational landscape of salivary gland malignancies to identify therapeutic targets and biomarkers of treatment failure

Repurposing Albendazole: new potential as a chemotherapeutic agent with preferential activity against HPV-negative head and neck squamous cell cancer.

Albendazole is an anti-helminthic drug that has been shown to exhibit anti-cancer properties, however its activity in head and neck squamous cell cancer (HNSCC) was unknown. Using a series of in vitro assays, we assessed the ability of albendazole to inhibit proliferation in 20 HNSCC cell lines across a range of albendazole doses (1 nM-10 μM). Cell lines that responded to treatment were further examined for cell death, inhibition of migration and cell cycle arrest. Thirteen of fourteen human papillomavirus-negative HNSCC cell lines responded to albendazole, with an average IC50 of 152 nM. In contrast, only 3 of 6 human papillomavirus-positive HNSCC cell lines responded. Albendazole treatment resulted in apoptosis, inhibition of cell migration, cell cycle arrest in the G2/M phase and altered tubulin distribution. Normal control cells were not measurably affected by any dose tested. This study indicates that albendazole acts to inhibit the proliferation of human papillomavirus-negative HNSCC cell lines and thus warrants further study as a potential chemotherapeutic agent for patients suffering from head and neck cancer