Peter D. Adams

p53 status determines the role of autophagy in pancreatic tumour development

Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Autophagy has a major role in cellular homeostasis and has been implicated in various forms of human disease. The role of autophagy in cancer seems to be complex, with reports indicating both pro-tumorigenic and tumour-suppressive roles. Here we show, in a humanized genetically-modified mouse model of pancreatic ductal adenocarcinoma (PDAC), that autophagy’s role in tumour development is intrinsically connected to the status of the tumour suppressor p53. Mice with pancreases containing an activated oncogenic allele of Kras (also called Ki-Ras)—the most common mutational event in PDAC—develop a small number of pre-cancerous lesions that stochastically develop into PDAC over time. However, mice also lacking the essential autophagy genes Atg5 or Atg7 accumulate low-grade, pre-malignant pancreatic intraepithelial neoplasia lesions, but progression to high-grade pancreatic intraepithelial neoplasias and PDAC is blocked. In marked contrast, in mice containing oncogenic Kras and lacking p53, loss of autophagy no longer blocks tumour progression, but actually accelerates tumour onset, with metabolic analysis revealing enhanced glucose uptake and enrichment of anabolic pathways, which can fuel tumour growth. These findings provide considerable insight into the role of autophagy in cancer and have important implications for autophagy inhibition in cancer therapy. In this regard, we also show that treatment of mice with the autophagy inhibitor hydroxychloroquine, which is currently being used in several clinical trials, significantly accelerates tumour formation in mice containing oncogenic Kras but lacking p53.

Molecular Dissection of Formation of Senescence-Associated Heterochromatin Foci

ABSTRACT Senescence is characterized by an irreversible cell proliferation arrest. Specialized domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), are thought to contribute to the irreversible cell cycle exit in many senescent cells by repressing the expression of proliferation-promoting genes such as cyclin A. SAHF contain known heterochromatin-forming proteins, such as heterochromatin protein 1 (HP1) and the histone H2A variant macroH2A, and other specialized chromatin proteins, such as HMGA proteins. Previously, we showed that a complex of histone chaperones, histone repressor A (HIRA) and antisilencing function 1a (ASF1a), plays a key role in the formation of SAHF. Here we have further dissected the series of events that contribute to SAHF formation. We show that each chromosome condenses into a single SAHF focus. Chromosome condensation depends on the ability of ASF1a to physically interact with its deposition substrate, histone H3, in addition to its cochaperone, HIRA. In cells entering senescence, HP1γ, but not the related proteins HP1α and HP1β, becomes phosphorylated on serine 93. This phosphorylation is required for efficient incorporation of HP1γ into SAHF. Remarkably, however, a dramatic reduction in the amount of chromatin-bound HP1 proteins does not detectably affect chromosome condensation into SAHF. Moreover, abundant HP1 proteins are not required for the accumulation in SAHF of histone H3 methylated on lysine 9, the recruitment of macroH2A proteins, nor other hallmarks of senescence, such as the expression of senescence-associated β-galactosidase activity and senescence-associated cell cycle exit. Based on our results, we propose a stepwise model for the formation of SAHF.

Defective S Phase Chromatin Assembly Causes DNA Damage, Activation of the S Phase Checkpoint, and S Phase Arrest

The S phase checkpoint protects the genome from spontaneous damage during DNA replication, although the cause of damage has been unknown. We used a dominant-negative mutant of a subunit of CAF-I, a complex that assembles newly synthesized DNA into nucleosomes, to inhibit S phase chromatin assembly and found that this induced S phase arrest. Arrest was accompanied by DNA damage and S phase checkpoint activation and required ATR or ATM kinase activity. These results show that in human cells CAF-I activity is required for completion of S phase and that a defect in chromatin assembly can itself induce DNA damage. We propose that errors in chromatin assembly, occurring spontaneously or caused by genetic mutations or environmental agents, contribute to genome instability.

Dynamics of Histone H3 Deposition In Vivo Reveal a Nucleosome Gap-Filling Mechanism for H3.3 to Maintain Chromatin Integrity

Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity.

Retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin-cdk complexes.

ABSTRACT Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.

Structural organization of the pentameric transmembrane alpha-helices of phospholamban, a cardiac ion channel.

Phospholamban is a 52 amino acid calcium regulatory protein found as pentamers in cardiac SR membranes. The pentamers form through interactions between its transmembrane domains, and are stable in SDS. We have employed a saturation mutagenesis approach to study the detailed interactions between the transmembrane segments, using a chimeric protein construct in which staphylococcal nuclease (a monomeric soluble protein) is fused to the N‐terminus of phospholamban. The chimera forms pentamers observable in SDS‐PAGE, allowing the effects of mutations upon the oligomeric association to be determined by electrophoresis. The disruptive effects of amino acid substitutions in the transmembrane domain were classified as sensitive, moderately sensitive or insensitive. Residues of the same class lined up on faces of a 3.5 amino acids/turn helical projection, allowing the construction of a model of the interacting surfaces in which the helices are associated in a left‐handed pentameric coiled‐coil configuration. Molecular modeling simulations (to be described elsewhere in detail) confirm that the helices readily form a left‐handed coiled‐coil helical bundle and have yielded molecular models for the interacting surfaces, the best of which is identical to that predicted by the mutagenesis. Residues lining the pore show considerable structural sensitivity to mutation, indicating that care must be taken in interpreting the results of mutagenesis studies of channels. The cylindrical ion pore (minimal diameter of 2 A) appears to be defined largely by hydrophobic residues (I40, L43 and I47) with only two mildly polar elements contributed by sulfurs in residues C36 and M50.

Regulation of the retinoblastoma tumor suppressor protein by cyclin/cdks

The retinoblastoma tumor suppressor protein (pRB) is a paradigm for understanding cell cycle- and proliferation-dependent transcription and how deregulation of this process contributes to the neoplastic process in humans. The ability of pRB to regulate transcription, and consequently cell proliferation and differentiation, is regulated by the activity of cyclin/cdks. In general, phosphorylation of pRB by cyclin/cdks inactivates pRB-mediated transcriptional inhibition and growth suppression. However, it is apparent that pRB is a multi-functional protein that can inhibit transcription through various mechanisms. This review focuses on recent data to suggest that different pRB functions are progressively and cooperatively inactivated by multiple cyclin/cdk complexes during G1- and S-phase. The implications of such a model for pRB-mediated tumor suppression are discussed.

Lysosome-mediated processing of chromatin in senescence

Senescent cells extrude fragments of chromatin from the nucleus into the cytoplasm, where they are processed by an autophagic/lysosomal pathway.

Autophagy mediates degradation of nuclear lamina

Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3–lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3–lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.

Definition of pRB- and p53-dependent and -independent steps in HIRA/ASF1a-mediated formation of senescence-associated heterochromatin foci.

ABSTRACT Cellular senescence is an irreversible proliferation arrest triggered by short chromosome telomeres, activated oncogenes, and cell stress and mediated by the pRB and p53 tumor suppressor pathways. One of the earliest steps in the senescence program is translocation of a histone chaperone, HIRA, into promyelocytic leukemia (PML) nuclear bodies. This relocalization precedes other markers of senescence, including the appearance of specialized domains of facultative heterochromatin called senescence-associated heterochromatin foci (SAHF) and cell cycle exit. SAHF represses expression of proliferation-promoting genes, thereby driving exit from the cell cycle. HIRA bound to another histone chaperone, ASF1a, drives formation of SAHF. Here, we show that HIRAs translocation to PML bodies occurs in response to all senescence triggers tested. Dominant negative HIRA mutants that block HIRAs localization to PML bodies prevent formation of SAHF, as does a PML-RARα fusion protein which disrupts PML bodies, directly supporting the idea that localization of HIRA to PML bodies is required for formation of SAHF. Significantly, translocation of HIRA to PML bodies occurs in the absence of functional pRB and p53 tumor suppressor pathways. However, our evidence indicates that downstream of HIRAs localization to PML bodies, the HIRA/ASF1a pathway cooperates with pRB and p53 to make SAHF, with the HIRA/ASF1a and pRB pathways acting in parallel. We present evidence that convergence of the HIRA/ASF1a and pRB pathways occurs through a DNAJ-domain protein, DNAJA2.