Oncogenic Ras and ΔNp63α cooperate to recruit immunosuppressive polymorphonuclear myeloid-derived suppressor cells in a mouse model of squamous cancer pathogenesis.

Introduction: Amplification of human chromosome 3q26-29, which encodes oncoprotein ΔNp63 among other isoforms of the p63 family, is a feature common to squamous cell carcinomas (SCCs) of multiple tissue origins. Along with overexpression of ΔNp63, activation of the protooncogene, RAS, whether by overexpression or oncogenic mutation, is frequently observed in many cancers. In this study, analysis of transcriptome data from The Cancer Genome Atlas (TCGA) demonstrated that expression of TP63 mRNA, particularly ΔNp63 isoforms, and HRAS are significantly elevated in advanced squamous cell carcinomas of the head and neck (HNSCCs), suggesting pathological significance. However, how co-overexpressed ΔNp63 and HRAS affect the immunosuppressive tumor microenvironment (TME) is incompletely understood. Methods: Here, we established and characterized an immune competent mouse model using primary keratinocytes with retroviral-mediated overexpression of ΔNp63α and constitutively activated HRAS (v-rasHa G12R) to evaluate the role of these oncogenes in the immune TME. Results: In this model, orthotopic grafting of wildtype syngeneic keratinocytes expressing both v-rasHa and elevated levels of ΔNp63α consistently yield carcinomas in syngeneic hosts, while cells expressing v-rasHa alone yield predominantly papillomas. We found that polymorphonuclear (PMN) myeloid cells, experimentally validated to be immunosuppressive and thus representing myeloid-derived suppressor cells (PMN-MDSCs), were significantly recruited into the TME of carcinomas arising early following orthotopic grafting of ΔNp63α/v-rasHa-expressing keratinocytes. ΔNp63α/v-rasHa-driven carcinomas expressed higher levels of chemokines implicated in recruitment of MDSCs compared to v-rasHa-initiated tumors, providing a heretofore undescribed link between ΔNp63α/HRAS-driven carcinomas and the development of an immunosuppressive TME. Conclusion: These results support the utilization of a genetic carcinogenesis model harboring specific genomic drivers of malignancy to study mechanisms underlying the development of local immunosuppression.

Delineating functional mechanisms of the p53/p63/p73 family of transcription factors through identification of protein-protein interactions using interface mimicry.

Members of the p53 family of transcription factors-p53, p63, and p73-share a high degree of homology; however, members can be activated in response to different stimuli, perform distinct (sometimes opposing) roles and are expressed in different tissues. The level of complexity is increased further by the transcription of multiple isoforms of each homolog, which may interact or interfere with each other and can impact cellular outcome. Proteins perform their functions through interacting with other proteins (and/or with nucleic acids). Therefore, identification of the interactors of a protein and how they interact in 3D is essential to fully comprehend their roles. By utilizing an in silico protein-protein interaction prediction method-HMI-PRED-we predicted interaction partners of p53 family members and modeled 3D structures of these protein interaction complexes. This method recovered experimentally known interactions while identifying many novel candidate partners. We analyzed the similarities and differences observed among the interaction partners to elucidate distinct functions of p53 family members and provide examples of how this information may yield mechanistic insight to explain their overlapping versus distinct/opposing outcomes in certain contexts. While some interaction partners are common to p53, p63, and p73, the majority are unique to each member. Nevertheless, most of the enriched pathways associated with these partners are common to all members, indicating that the members target the same biological pathways but through unique mediators. p63 and p73 have more common enriched pathways compared to p53, supporting their similar developmental roles in different tissues.

Intersection of the p63 and NF-κB pathways in epithelial homeostasis and disease.

Overexpression of ΔNp63α, a member of the p53/p63/p73 family of transcription factors, is a molecular attribute of human squamous cancers of the head and neck, lung and skin. The TP63 gene plays important roles in epidermal morphogenesis and homeostasis, regulating diverse biological processes including epidermal fate decisions and keratinocyte proliferation and survival. When overexpressed experimentally in primary mouse keratinocytes, ΔNp63α maintains a basal cell phenotype including the loss of normal calcium-mediated growth arrest, at least in part through the activation and enhanced nuclear accumulation of the c-rel subunit of NF-κB (Nuclear Factor-kappa B). Initially identified for its role in the immune system and hematopoietic cancers, c-Rel has increasingly been associated with solid tumors and other pathologies. ΔNp63α and c-Rel have been shown to be associated in the nuclei of ΔNp63α overexpressing human squamous carcinoma cells. Together, these transcription factors cooperate in the transcription of genes regulating intrinsic keratinocyte functions, as well as the elaboration of factors that influence the tumor microenvironment (TME). This review provides an overview of the roles of ΔNp63α and c-Rel in normal epidermal homeostasis and elaborates on how these pathways may intersect in pathological conditions such as cancer and the associated TME.

Molecular Mechanisms of p63-Mediated Squamous Cancer Pathogenesis.

The p63 gene is a member of the p53/p63/p73 family of transcription factors and plays a critical role in development and homeostasis of squamous epithelium. p63 is transcribed as multiple isoforms; ΔNp63α, the predominant p63 isoform in stratified squamous epithelium, is localized to the basal cells and is overexpressed in squamous cell cancers of multiple organ sites, including skin, head and neck, and lung. Further, p63 is considered a stem cell marker, and within the epidermis, ΔNp63α directs lineage commitment. ΔNp63α has been implicated in numerous processes of skin biology that impact normal epidermal homeostasis and can contribute to squamous cancer pathogenesis by supporting proliferation and survival with roles in blocking terminal differentiation, apoptosis, and senescence, and influencing adhesion and migration. ΔNp63α overexpression may also influence the tissue microenvironment through remodeling of the extracellular matrix and vasculature, as well as by enhancing cytokine and chemokine secretion to recruit pro-inflammatory infiltrate. This review focuses on the role of ΔNp63α in normal epidermal biology and how dysregulation can contribute to cutaneous squamous cancer development, drawing from knowledge also gained by squamous cancers from other organ sites that share p63 overexpression as a defining feature.

Brd4 is displaced from HPV replication factories as they expand and amplify viral DNA.

Replication foci are generated by many viruses to concentrate and localize viral DNA synthesis to specific regions of the cell. Expression of the HPV16 E1 and E2 replication proteins in keratinocytes results in nuclear foci that recruit proteins associated with the host DNA damage response. We show that the Brd4 protein localizes to these foci and is essential for their formation. However, when E1 and E2 begin amplifying viral DNA, Brd4 is displaced from the foci and cellular factors associated with DNA synthesis and homologous recombination are recruited. Differentiated HPV-infected keratinocytes form similar nuclear foci that contain amplifying viral DNA. We compare the different foci and show that, while they have many characteristics in common, there is a switch between early Brd4-dependent foci and mature Brd4-independent replication foci. However, HPV genomes encoding mutated E2 proteins that are unable to bind Brd4 can replicate and amplify the viral genome. We propose that, while E1, E2 and Brd4 might bind host chromatin at early stages of infection, there is a temporal and functional switch at later stages and increased E1 and E2 levels promote viral DNA amplification, displacement of Brd4 and growth of a replication factory. The concomitant DNA damage response recruits proteins required for DNA synthesis and repair, which could then be utilized for viral DNA replication. Hence, while Brd4 can enhance replication by concentrating viral processes in specific regions of the host nucleus, this interaction is not absolutely essential for HPV replication.

Hitchhiking on host chromatin: how papillomaviruses persist.

Persistent viruses need mechanisms to protect their genomes from cellular defenses and to ensure that they are efficiently propagated to daughter host cells. One mechanism by which papillomaviruses achieve this is through the association of viral genomes with host chromatin, mediated by the viral E2 tethering protein. Association of viral DNA with regions of active host chromatin ensures that the virus remains transcriptionally active and is not relegated to repressed heterochromatin. In addition, viral genomes are tethered to specific regions of host mitotic chromosomes to efficiently partition their DNA to daughter cells. Vegetative viral DNA replication also initiates at specific regions of host chromatin, where the viral E1 and E2 proteins initiate a DNA damage response that recruits cellular DNA damage and repair proteins to viral replication foci for efficient viral DNA synthesis. Thus, these small viruses have capitalized on interactions with chromatin to efficiently target their genomes to beneficial regions of the host nucleus. This article is part of a Special Issue entitled: Chromatin in time and space.

The papillomavirus E1 helicase activates a cellular DNA damage response in viral replication foci.

The papillomavirus E1 and E2 proteins are essential for viral genome replication. E1 is a helicase that unwinds the viral origin and recruits host cellular factors to replicate the viral genome. E2 is a transcriptional regulator that helps recruit the E1 helicase to the origin and also plays a role in genome partitioning. We find that when coexpressed, the E1 and E2 proteins from several papillomavirus types localize to defined nuclear foci and result in growth suppression of the host cells. Growth suppression was due primarily to E1 protein function, and nuclear expression of E1 was accompanied by activation of a DNA damage response, resulting in phosphorylation of ATM, Chk2, and H2AX. Growth suppression and ATM activation required the ATPase and origin-specific binding functions of the E1 protein and resulted in active DNA repair, as evidenced by incorporation of nucleotide analogs and detection of free DNA ends. In the presence of the E2 protein, these activities became localized to nuclear foci. We postulate that these foci represent viral replication factories and that a cellular DNA damage response is activated to facilitate replication of viral DNA.

Mutational analysis of conserved aspartic acid residues in the Methanothermobacter thermautotrophicus MCM helicase.

Minichromosome maintenance (MCM) helicases are thought to function as the replicative helicases in archaea and eukarya, unwinding the duplex DNA in the front of the replication fork. The archaeal MCM helicase can be divided into three parts, the N-terminal, catalytic, and C-terminal regions. The N-terminal part of the protein is divided into three domains, A, B, and C, and was shown to be involved in protein multimerization and binding to single- and double-stranded DNA. Two Asp residues found in domain C are conserved among MCM proteins from different archaea. These residues are located in a loop at the interface with domain A. Mutations of these residues in the Methanothermobacter thermautotrophicus MCM protein, Asp202 and Asp203, to Asn result in a significant reduction in the ability of the enzyme to bind DNA and in lower thermal stability. However, the mutant proteins retained helicase and ATPase activities. Further investigation of the DNA binding revealed that the presence of ATP rescues the DNA binding deficiencies by these mutant proteins. Possible roles of these conserved residues in MCM function are discussed.

Different residues on the surface of the Methanothermobacter thermautotrophicus MCM helicase interact with single- and double-stranded DNA.

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea, separating the two strands of chromosomal DNA during replication. The catalytic activity resides within the C-terminal region of the MCM protein, while the N-terminal portion plays an important role in DNA binding and protein multimerization. An alignment of MCM homologues from several archaeal species revealed a number of conserved amino acids. Here several of the conserved residues located on the surface of the helicase have been mutated and their roles in MCM functions determined. It was found that some mutations result in increased affinity for ssDNA while the affinity for dsDNA is decreased. Other mutants exhibit the opposite effect. Thus, the data suggest that these conserved surface residues may participate in MCM-DNA interactions.

Unwinding the structure and function of the archaeal MCM helicase.

During chromosomal DNA replication, the replicative helicase unwinds the duplex DNA to provide the single-stranded DNA substrate for the polymerase. In archaea, the replicative helicase is the minichromosome maintenance (MCM) complex. The enzyme utilizes the energy of ATP hydrolysis to translocate along one strand of the duplex and unwind the complementary strand. Much progress has been made in elucidating structure and function since the first report on the biochemical properties of an archaeal MCM protein in 1999. We now know the biochemical and structural properties of the enzyme from several archaeal species and some of the mechanisms by which the enzyme is regulated. This review summarizes recent studies on the archaeal MCM protein and discusses the implications for helicase function and DNA replication in archaea.

ATP hydrolysis and DNA binding confer thermostability on the MCM helicase.

The minichromosome maintenance (MCM) helicase is the replicative helicase in archaea. The enzyme utilizes the energy derived from ATP hydrolysis to translocate along one strand of the DNA and unwind the complementary strand. Here, the effect of DNA and ATP on the thermostability of the Methanothermobacter thermautotrophicus MCM protein was determined by differential scanning calorimetry. The MCM protein shows a single thermal transition at 67 degrees C. The stability is dramatically altered with the appearance of a second thermal transition up to 10 degrees C higher in the presence of DNA and either ATP or ADP-AlF(4)(-), a transition-state analogue of ATP, bound to MCM. In the presence of DNA and ADP or the nonhydrolyzable ATP analogues ATPgammaS and AMP-PNP, however, only a single thermal transition is observed at temperatures slightly higher than the transition temperature of MCM alone. Thus, the results suggest that ATP hydrolysis proceeds through a transition state that decouples an interaction between the N-terminal DNA binding domain and the C-terminal catalytic domain in the presence of DNA.

How is the archaeal MCM helicase assembled at the origin? Possible mechanisms.

In order for any organism to replicate its DNA, a helicase must unwind the duplex DNA in front of the replication fork. In archaea, the replicative helicase is the MCM (minichromosome maintenance) helicase. Although much is known about the biochemical properties of the MCM helicase, the mechanism of assembly at the origin of replication is unknown. In the present paper, several possible mechanisms for the loading process are described.

Cloning, Purification, and Partial Characterization of the Halobacterium sp. NRC-1 Minichromosome Maintenance (MCM) Helicase.

The MCM gene from the archaeon Halobacterium, with and without its intein, was cloned into an Escherichia coli expression vector, overexpressed and the protein was purified and antibodies were generated. The antibodies were used to demonstrate that in vivo only the processed enzyme, without the intein, could be detected.

Thermoplasma acidophilum Cdc6 protein stimulates MCM helicase activity by regulating its ATPase activity.

The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. In most archaeal species studied, the interaction between MCM and the initiator protein, Cdc6, inhibits helicase activity. To date, the only exception is the helicase and Cdc6 proteins from the archaeon Thermoplasma acidophilum. It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity. It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM. Also, through the use of site-specific substitutions, and truncated and chimeric proteins, it was shown that an intact Cdc6 protein is required for this stimulation. ATP binding and hydrolysis by the Cdc6 protein is not needed for the stimulation. The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity.

Cryo-electron microscopy reveals a novel DNA-binding site on the MCM helicase.

The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize the interplay between the MCM helicase and DNA prior to the melting of the double helix, we determined the structure of an archaeal MCM orthologue bound to a 5.6-kb double-stranded DNA segment, using cryo-electron microscopy. DNA wraps around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. Our findings provide novel insights into the role of the MCM complex during the initiation step of DNA replication.

Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein.

Minichromosome maintenance (MCM) helicases are the presumptive replicative helicases, thought to separate the two strands of chromosomal DNA during replication. In archaea, the catalytic activity resides within the C-terminal region of the MCM protein. In Methanothermobacter thermautotrophicus the N-terminal portion of the protein was shown to be involved in protein multimerization and binding to single and double stranded DNA. MCM homologues from many archaeal species have highly conserved predicted amino acid similarity in a loop located between beta7 and beta8 in the N-terminal part of the molecule. This high degree of conservation suggests a functional role for the loop. Mutational analysis and biochemical characterization of the conserved residues suggest that the loop participates in communication between the N-terminal portion of the helicase and the C-terminal catalytic domain. Since similar residues are also conserved in the eukaryotic MCM proteins, the data presented here suggest a similar coupling between the N-terminal and catalytic domain of the eukaryotic enzyme.