Rate-limiting Event Research Articles

BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of Tcell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.

Parameter estimation for multistage clonal expansion models from cancer incidence data: A practical identifiability analysis.

Many cancers are understood to be the product of multiple somatic mutations or other rate-limiting events. Multistage clonal expansion (MSCE) models are a class of continuous-time Markov chain models that capture the multi-hit initiation–promotion–malignant-conversion hypothesis of carcinogenesis. These models have been used broadly to investigate the epidemiology of many cancers, assess the impact of carcinogen exposures on cancer risk, and evaluate the potential impact of cancer prevention and control strategies on cancer rates. Structural identifiability (the analysis of the maximum parametric information available for a model given perfectly measured data) of certain MSCE models has been previously investigated. However, structural identifiability is a theoretical property and does not address the limitations of real data. In this study, we use pancreatic cancer as a case study to examine the practical identifiability of the two-, three-, and four-stage clonal expansion models given age-specific cancer incidence data using a numerical profile-likelihood approach. We demonstrate that, in the case of the three- and four-stage models, several parameters that are theoretically structurally identifiable, are, in practice, unidentifiable. This result means that key parameters such as the intermediate cell mutation rates are not individually identifiable from the data and that estimation of those parameters, even if structurally identifiable, will not be stable. We also show that products of these practically unidentifiable parameters are practically identifiable, and, based on this, we propose new reparameterizations of the model hazards that resolve the parameter estimation problems. Our results highlight the importance of identifiability to the interpretation of model parameter estimates.

Abstract P6-07-02: CDK4 phosphorylation status and corresponding gene expression profile predict sensitivity to Palbociclib

Abstract P6-07-02: CDK4 phosphorylation status and corresponding gene expression profile predict sensitivity to Palbociclib

Integration of a radiation biomarker into modeling of thyroid carcinogenesis and post-Chernobyl risk assessment.

Strong evidence for the statistical association between radiation exposure and disease has been produced for thyroid cancer by epidemiological studies after the Chernobyl accident. However, limitations of the epidemiological approach in order to explore health risks especially at low doses of radiation appear obvious. Statistical fluctuations due to small case numbers dominate the uncertainty of risk estimates. Molecular radiation markers have been searched extensively to separate radiation-induced cancer cases from sporadic cases. The overexpression of the CLIP2 gene is the most promising of these markers. It was found in the majority of papillary thyroid cancers (PTCs) from young patients included in the Chernobyl tissue bank. Motivated by the CLIP2 findings we propose a mechanistic model which describes PTC development as a sequence of rate-limiting events in two distinct paths of CLIP2-associated and multistage carcinogenesis. It integrates molecular measurements of the dichotomous CLIP2 marker from 141 patients into the epidemiological risk analysis for about 13 000 subjects from the Ukrainian-American cohort which were exposed below age 19 years and were put under enhanced medical surveillance since 1998. For the first time, a radiation risk has been estimated solely from marker measurements. Cross checking with epidemiological estimates and model validation suggests that CLIP2 is a marker of high precision. CLIP2 leaves an imprint in the epidemiological incidence data which is typical for a driver gene. With the mechanistic model, we explore the impact of radiation on the molecular landscape of PTC. The model constitutes a unique interface between molecular biology and radiation epidemiology.

How the Proximal Pocket May Influence the Enantiospecificities of Chloroperoxidase-Catalyzed Epoxidations of Olefins.

Chloroperoxidase-catalyzed enantiospecific epoxidations of olefins are of significant biotechnological interest. Typical enantiomeric excesses are in the range of 66%–97% and translate into free energy differences on the order of 1 kcal/mol. These differences are generally attributed to the effect of the distal pocket. In this paper, we show that the influence of the proximal pocket on the electron transfer mechanism in the rate-limiting event may be just as significant for a quantitatively accurate account of the experimentally-measured enantiospecificities.

Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

Abstract PR06: The reemergence of neural crest progenitor identity is a key event in the initiation of melanoma from a field of cancer-prone melanocytes

Abstract Within a group of cancer-prone cells that harbor a shared oncogenic mutation, only rare clones transition to a malignant state. These rare and transient events occurring during cancer initiation remain incompletely understood, and we thus sought to visualize and molecularly characterize this crucial early step in oncogenesis of melanoma. We previously found that the zebrafish crestin gene, which specifically marks embryonic neural crest and normally turns off at three days of life, is specifically re-expressed in BRAFV600E/p53 mutant melanoma tumors in adult zebrafish. We cloned the crestin promoter/enhancer and developed an EGFP reporter that recapitulates the embryonic expression pattern of crestin mRNA and, crucially, marks de novo melanomas in living BRAFV600E/p53 mutant zebrafish. Remarkably, we also found that crestin:EGFP becomes active when lesions are only a few cells in number, potentially in the first cell of the melanoma. These crestin:EGFP positive patches are transplantable, and these precursor lesions are enriched for expression of neural crest progenitor (NCP) genes including the transcription factors sox10 and dlx2a, among other melanoma- and NCP-associated genes. In order to favor the readoption of an NCP-state, we forced misexpression of sox10 in melanocytes of BRAFV600E/p53 melanoma-prone zebrafish, and this led to enhanced melanoma formation. We then analyzed the chromatin landscape of both human and zebrafish melanoma cells using ChIP-Seq and ATAC-Seq and identified super-enhancers at crestin (in zebrafish), sox10, dlx2a, and other NCP/melanoma loci, which together described an overall chromatin signature consistent with features of the NCP state. These data support a model in which 1) an NCP program stochastically reactivates, as read out by crestin expression, in rare BRAFV600E/p53 mutated melanocytes at the initiation of melanoma formation and 2) reemergence of this NCP state is an important and potentially rate-limiting event in melanoma initiation. We anticipate that progenitor identity reemergence will prove to be a general feature of cancer initiation. This abstract is also presented as Poster B19. Citation Format: Charles K. Kaufman, Christian Mosimann, Andrew Thomas, Zi Peng Fan, Song Yang, Justin Tan, Rachel D. Fogley, Ellen van Rooijen, Elliott Hagedorn, Christie Ciarlo, Richard White, Dominick Matos, Ann-Christin Puller, Cristina Santoriello, Eric Liao, Richard A. Young, Leonard I. Zon. The reemergence of neural crest progenitor identity is a key event in the initiation of melanoma from a field of cancer-prone melanocytes. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR06.

Computational Cancer Biology: An Evolutionary Perspective.

Cancer is a leading cause of death worldwide and represents one of the biggest biomedical research challenges of our time. Tumor progression is caused by somatic evolution of cell populations. Cancer cells expand because of the accumulation of selectively advantageous mutations, and expanding clones give rise to new cell subpopulations with increasingly higher somatic fitness (Fig 1). In the 1970s, Nowell and others established this somatic evolutionary view of cancer [1]. Today, computational biologists have the opportunity to take advantage of large-scale molecular profiling data in order to carve out the principles of tumor evolution and to elucidate how it manifests across cancer types. Analogous to other evolutionary studies, mathematical modeling will be key to the success of understanding the somatic evolution of cancer [2]. Fig 1 Schematic representation of neoplastic transformation. In general, cancer research involves a range of clinical, epidemiological, and molecular approaches, as well as mathematical and computational modeling. An early and very successful example of mathematical modeling was the work of Nordling [3] and of Armitage and Doll [4]. In the 1950s, long before cancer genome data was available, they analyzed cancer incidence data and postulated, based on the observed age-incidence curves, that cancer is a multistep process. In search of these rate-limiting events, cancer progression was then linked to the accumulation of genomic alterations. Since then, the evolutionary perspective on cancer has proven useful in many instances, and the mathematical theory of cancer evolution has been developed much further. However, little clinical benefit could be gained from this approach so far. Much of evolutionary modeling in general, and of cancer in particular, has remained conceptual or qualitative, either because of strong simplifications in the interest of mathematical tractability or lack of informative data. Next-generation sequencing (NGS) technologies and their various applications have changed this situation fundamentally [5]. Today, cancer cells can be analyzed in great detail at the molecular level, and tumor cell populations can be sampled extensively. Driven by this technological revolution, large numbers of high-dimensional molecular profiles of tumors, and even of individual cancer cells, are collected by cancer genome consortia, as well as by many individual labs. Large catalogs of cancer genomes, epigenomes, transcriptomes, proteomes, and other molecular profiles are generated to assess variation among tumors from different patients (intertumor heterogeneity) as well as among individual cells of single tumors (intratumor heterogeneity). These data hold the promise not only of new cancer biology discoveries but also of progress in cancer diagnostics and treatment. Analyzing these complex data and interpreting them in the context of ongoing somatic evolution, disease progression, and treatment response is a major challenge, and the prospects to improve cancer treatment depend critically on progress with these computational and statistical tasks. In the following, we briefly summarize the current state of the art in the field and highlight major challenges that lie ahead, including (i) reconstruction of evolutionary history based on different types of genomic alterations, (ii) functional interpretation of mutations, and (iii) predictive modeling of the evolutionary dynamics of cancer. We argue that an interdisciplinary approach, including statistical and computational data analysis as well as evolutionary modeling of cancer, will be essential for translating technological advances into clinical benefits.

Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment.

Neutrophil recruitment is a hallmark of rapid innate immune responses. Exposure of airways of naive mice to pollens rapidly induces neutrophil recruitment. The innate mechanisms that regulate pollen-induced neutrophil recruitment and the contribution of this neutrophilic response to subsequent induction of allergic sensitization and inflammation need to be elucidated. Here we show that ragweed pollen extract (RWPE) challenge in naive mice induces C-X-C motif ligand (CXCL) chemokine synthesis, which stimulates chemokine (C-X-C motif) receptor 2 (CXCR2)-dependent recruitment of neutrophils into the airways. Deletion of Toll-like receptor 4 (TLR4) abolishes CXCL chemokine secretion and neutrophil recruitment induced by a single RWPE challenge and inhibits induction of allergic sensitization and airway inflammation after repeated exposures to RWPE. Forced induction of CXCL chemokine secretion and neutrophil recruitment in mice lacking TLR4 also reconstitutes the ability of multiple challenges of RWPE to induce allergic airway inflammation. Blocking RWPE-induced neutrophil recruitment in wild-type mice by administration of a CXCR2 inhibitor inhibits the ability of repeated exposures to RWPE to stimulate allergic sensitization and airway inflammation. Administration of neutrophils derived from naive donor mice into the airways of Tlr4 knockout recipient mice after each repeated RWPE challenge reconstitutes allergic sensitization and inflammation in these mice. Together these observations indicate that pollen-induced recruitment of neutrophils is TLR4 and CXCR2 dependent and that recruitment of neutrophils is a critical rate-limiting event that stimulates induction of allergic sensitization and airway inflammation. Inhibiting pollen-induced recruitment of neutrophils, such as by administration of CXCR2 antagonists, may be a novel strategy to prevent initiation of pollen-induced allergic airway inflammation.

Cytokines and Blastocyst Hatching.

Blastocyst implantation into the uterine endometrium establishes early pregnancy. This event is regulated by blastocyst- and/or endometrium-derived molecular factors which include hormones, growth factors, cell adhesion molecules, cytokines and proteases. Their coordinated expression and function are critical for a viable pregnancy. A rate-limiting event that immediately precedes implantation is the hatching of blastocyst. Ironically, blastocyst hatching is tacitly linked to peri-implantation events, although it is a distinct developmental phenomenon. The exact molecular network regulating hatching is still unclear. A number of implantation-associated molecular factors are expressed in the pre-implanting blastocyst. Among others, cytokines, expressed by peri-implantation blastocysts, are thought to be important for hatching, making blastocysts implantation competent. Pro-inflammatory (IL-6, LIF, GM-CSF) and anti-inflammatory (IL-11, CSF-1) cytokines improve hatching rates; they modulate proteases (MMPs, tPAs, cathepsins and ISP1). However, functional involvement of cytokines and their specific mediation of hatching-associated proteases are unclear. There is a need to understand mechanistic roles of cytokines and proteases in blastocyst hatching. This review will assess the available knowledge on blastocyst-derived pro-inflammatory and anti-inflammatory cytokines and their role in potentially regulating blastocyst hatching. They have implications in our understanding of early embryonic loss and infertility in mammals, including humans.

STING Activation by Translocation from the ER Is Associated with Infection and Autoinflammatory Disease.

STING is an ER-associated membrane protein that is critical for innate immune sensing of pathogens. STING-mediated activation of the IFN-I pathway through the TBK1/IRF3 signaling axis involves both cyclic-dinucleotide binding and its translocation from the ER to vesicles. However, how these events are coordinated, and the exact mechanism of STING activation, remain poorly understood. Here, we found that the Shigella effector protein IpaJ potently inhibits STING signaling by blocking its translocation from the ER to ERGIC, even in the context of dinucleotide binding. Reconstitution using purified components revealed STING translocation as the rate-limiting event in maximal signal transduction. Furthermore, STING mutations associated with autoimmunity in humans were found to cause constitutive ER exit and to activate STING independent of cGAMP binding. Together, these data provide compelling evidence for an ER retention and ERGIC/Golgi-trafficking mechanism of STING regulation that is subverted by bacterial pathogens and is deregulated in human genetic disease.

A polyubiquitin chain reaction: parkin recruitment to damaged mitochondria.

Mutations in the E3 ubiquitin ligase Parkin or the mitochondrial kinase PINK1 cause autosomal recessive forms of Parkinson’s disease [1, 2]. Genetic and cell biological studies have implicated PINK1 and Parkin as critical elements in mitophagy, a mitochondrial quality control pathway that involves the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system [1, 2]. Under basal conditions, PINK1 is processed by mitochondrial proteases and targeted for degradation by the UPS [1, 2]. Following persistent mitochondrial damage (e.g., treatment with the mitochondrial uncoupling agent CCCP) PINK1 is stabilized and accumulates in an active form on the outer mitochondrial membrane [1, 2]. Although PINK1 activity is essential for the mitochondrial recruitment of cytoplasmic Parkin and for the subsequent ubiquitin-dependent clearance of damaged mitochondria, the mode of Parkin activation and recruitment has been elusive [1, 2].

Abstract A06: The role of the epidermal growth factor receptor (EGFR) in primary tumor cell escape and development of an intravasation-sustaining vasculature

Abstract Metastasis of malignant cells is regarded as the leading cause of cancer-related deaths. Metastasis is a complex process whereby a subset or individual cancer cells disseminate from the primary tumor to distant/secondary sites. Intravasation, which is the entry of escaping tumor cells into the vasculature, is a critical and rate-limiting early event in the metastatic cascade. The complex process of intravasation is difficult to model and quantify in vivo, and therefore it remains the least studied step of the metastatic cascade. To study intravasation, we employ the chick embryo model involving grafting of human cancer cells on the chorioallantoic membrane (CAM), the highly vascularized tissue capable of supporting primary tumor growth and development of angiogenic vasculature. The epidermal growth factor receptor (EGFR) is known to play an important role in cancer progression and EGFR-induced signaling has been associated with tumor invasion and metastasis. In this study, we investigated the specific roles of EGFR in the early steps of tumor metastasis such as escape of cancer cells from the primary tumor and development of angiogenic vasculature, the two critical events required for tumor cell intravasation. For this purpose we employed the EGFR-overexpressing highly disseminating variant of human fibrosarcoma HT-1080 cell line, HT-hi/diss, in several independent in vivo CAM models. In the spontaneous metastasis model, siRNA silencing of EGFR significantly reduced the levels of HT-hi/diss intravasation, suggesting a role of EGFR overexpression in tumor cell escape and/or development of intravasation-sustaining vasculature. We have demonstrated that EGFR silencing prevented HT-hi/diss cell escape from the primary microtumors in the intramesodermal model, linking EGFR-regulated cell motility to tumor cell intravasation. We further investigated the role of EGFR in development of tumor-associated vasculature. By using our newly developed topical microtumor model, we have demonstrated that both control and EGFR-silenced cells attract comparable numbers of angiogenic vessels converging to developing primary microtumors. However, epifluorescence microscopy revealed dramatic difference in the microarchitecture of blood vessels within the two types of microtumors. Whereas control microtumors exhibited well-developed networks of intratumoral blood vessels with lumen diameter of 15-30 µm, EGFR-silenced microtumors contained only thin blood vessels with lumen diameter less than 15 µm. This lack of appropriately dilated intratumoral vasculature in EGFR-silenced tumors directly correlated with a significant decrease in vascular permeability concomitant with the suppression of tumor cell intravasation. These data highlight a specific aspect of EGFR functionality that is different from its well-established role in transcriptional regulation of genes required for tumor growth and suggest a novel mechanism whereby tumor cell EGFR regulates microenvironmental cues for induction, development, and functionality of an intravasation-sustaining vasculature. Citation Format: Petra Minder, Elena I. Deryugina, James P. Quigley. The role of the epidermal growth factor receptor (EGFR) in primary tumor cell escape and development of an intravasation-sustaining vasculature. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A06. doi:10.1158/1538-7445.CHTME14-A06

A functional SNP in the MDM2 promoter mediates E2F1 affinity to modulate cyclin D1 expression in tumor cell proliferation.

The MDM2 oncogene, a negative regulator of p53, has a functional polymorphism in the promoter region (SNP309) that is associated with multiple kinds of cancers including non-melanoma skin cancer. SNP309 has been shown to associate with accelerated tumor formation by increasing the affinity of the transcriptional activator Sp1. It remains unknown whether there are other factors involved in the regulation of MDM2 transcription through a trans-regulatory mechanism. In this study, SNP309 was verified to be associated with overexpression of MDM2 in tumor cells. Bioinformatics predicts that the T to G substitution at SNP309 generates a stronger E2F1 binding site, which was confirmed by ChIP and luciferase assays. E2F1 knockdown downregulates the expression of MDM2, which confirms that E2F1 is a functional upstream regulator. Furthermore, tumor cells with the GG genotype exhibited a higher proliferation rate than TT, correlating with cyclin D1 expression. E2F1 depletion significantly inhibits the proliferation capacity and downregulates cyclin D1 expression, especially in GG genotype skin fibroblasts. Notably, E2F1 siRNA effects could be rescued by cyclin D1 overexpression. Taken together, a novel modulator E2F1 was identified as regulating MDM2 expression dependent on SNP309 and further mediates cyclin D1 expression and tumor cell proliferation. E2F1 might act as an important factor for SNP309 serving as a rate-limiting event in carcinogenesis.

Targeted deletion of the C-terminus of the mouse adenomatous polyposis coli tumor suppressor results in neurologic phenotypes related to schizophrenia

BackgroundLoss of adenomatous polyposis coli (APC) gene function results in constitutive activation of the canonical Wnt pathway and represents the main initiating and rate-limiting event in colorectal tumorigenesis. APC is likely to participate in a wide spectrum of biological functions via its different functional domains and is abundantly expressed in the brain as well as in peripheral tissues. However, the neuronal function of APC is poorly understood. To investigate the functional role of Apc in the central nervous system, we analyzed the neurological phenotypes of Apc1638T/1638T mice, which carry a targeted deletion of the 3′ terminal third of Apc that does not affect Wnt signaling.ResultsA series of behavioral tests revealed a working memory deficit, increased locomotor activity, reduced anxiety-related behavior, and mildly decreased social interaction in Apc1638T/1638T mice. Apc1638T/1638T mice showed abnormal morphology of the dendritic spines and impaired long-term potentiation of synaptic transmission in the hippocampal CA1 region. Moreover, Apc1638T/1638T mice showed abnormal dopamine and serotonin distribution in the brain. Some of these behavioral and neuronal phenotypes are related to symptoms and endophenotypes of schizophrenia.ConclusionsOur results demonstrate that the C-terminus of the Apc tumor suppressor plays a critical role in cognitive and neuropsychiatric functioning. This finding suggests a potential functional link between the C-terminus of APC and pathologies of the central nervous system.

Identification of Pauses during Formation of HIV-1 Virus Like Particles

HIV Gag polymerizes on the plasma membrane to form virus like particles (VLPs) that have similar diameters to wild-type viruses. We use multicolor, dual-penetration depth, total internal reflection fluorescence microscopy, which corrects for azimuthal movement, to image the assembly of individual VLPs from the time of nucleation to the recruitment of VPS4 (a component of the endosomal sorting complexes required for transport, and which marks the final stage of VLP assembly). Using a mathematical model for assembly and maximum-likelihood comparison of fits both with and without pauses, we detect pauses during Gag polymerization in 60% of VLPs. Pauses range from 2 to 20 min, with an exponentially distributed duration that is independent of cytosolic Gag concentration. VLPs assembled with late domain mutants of Gag (which do not recruit the key endosomal sorting complexes required for transport proteins Alix or TSG101) exhibit similar pause distributions. These pauses indicate that a single rate-limiting event is required for continuation of assembly. We suggest that pauses are either related to incorporation of defects in the hexagonal Gag lattice during VLP assembly or are caused by shortcomings in interactions of Gag with essential and still undefined cellular components during formation of curvature on the plasma membrane.

Abstract 839: An uncharacterized mechanism of cell death independent of p53 mutation status.

Abstract Resistance to chemotherapeutic agents is often a rate-limiting event in the treatment of metastatic cancers. Mutations of TP53 or dysfunctions in the p53 pathway are known to play primary roles in the resistance to apoptosis-inducing agents. A stapled alpha-helical p53 peptide (SAH-p53) capable of disrupting the p53-HDM2 and p53-HDMX complexes and restoring p53 function has been found to induce a death response in breast, melanoma and choriocarcinoma cancer cells independent of p53 mutational status. We have recently discovered that the mechanism of death induced by SAH-p53 does not exhibit all of the characteristics of classical apoptosis, necrosis or autophagy independently. The binding of SAH-p53 to HDM2, HDMX, or a yet unidentified target results in a mechanism that leads to the intracellular aggregation of proteins and lipids that are extruded from the cell, resulting in catastrophic damage to the integrity of the cell membrane and culminating in cell death. This type of cell death involves the loss of cytoplasm, generation of phase-clear vacuole-like structures, apparent degradation of cellular membrane components, and the significant release of uniform non-vesicular cellular components. The cellular debris, while yet undefined, displays orange staining with acridine orange as well as accumulation of MitoTracker, suggesting that the debris contains hydrophobic regions. Thus far it has been established that there is a lack of any membrane blebbing, no chromatin condensation, no apoptosis signaling events or typical necrotic bubble formation after treatment with SAH-p53 in cells harboring wild-type p53, mutant p53, or null p53, suggesting an alternative mechanism of cell death. Data suggest the manifestation of early autophagy within 6 hours of SAH-p53 treatment as evidenced by LC3 accumulation, yet this phenotype is lost within 24 hours post-treatment. In addition, the pretreatment of the cells with chloroquine does not inhibit the release of debris and changes in morphology characteristic of SAH-p53 treatment, suggesting that the mechanism of death is independent of classical autophagy. We have begun to uncover the precise mechanism of cell death through the identification and validation of the transcriptional program regulated by SAH-p53 in both wild-type and mutant p53-containing cells via microarray analysis. The complete understanding and characterization of this novel mechanism may be exploited to overcome resistance to apoptosis and thus lead to a more effective way of treating chemotherapy-resistant metastatic cancers. Citation Format: Bethanie L. Morrison, Elisabeth Russell, Joseph Mitala, Federico Bernal. An uncharacterized mechanism of cell death independent of p53 mutation status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 839. doi:10.1158/1538-7445.AM2013-839

Critical Role of S1PR1 and Integrin β4 in HGF/c-Met-mediated Increases in Vascular Integrity

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met. We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.

Management of Recurrent Hepatocellular Carcinoma in Liver Transplant Recipients: A Systematic Review

Hepatocellular carcinoma is the most common form of liver cancer, representing 70% to 85% of primary hepatic malignancies in adults. Liver transplant is an optimal treatment for patients with hepatocellular carcinoma because it eliminates the malignancy as well as the often-underlying liver cirrhosis and restores normal liver function. Since the development of strict selection criteria in hepatocellular carcinoma patients undergoing liver transplant with the implementation of the Milan criteria, patient survival and recurrence rates after liver transplant have dramatically improved. However, several research groups are now seeking to expand this criteria to include more patients with larger tumors who may achieve similar postliver transplant survival rates as those patients meeting current eligibility requirements. Currently, in approximately 20% of patients, hepatocellular carcinoma recurrence is still the rate-limiting event that clearly affects patient survival. Given the limited number of grafts available for transplant, the poor prognosis of untreated hepatocellular carcinoma, and the recent notion of expanding selection criteria, strategies for reducing the rate of, monitoring and treating hepatocellular carcinoma recurrence, in both pretransplants and posttransplants, are explored in this review. We review the available literature to better understand current strategies available to optimize long-term clinical outcomes.

Smad4 disruption accelerates keratinocyte reepithelialization in murine cutaneous wound repair

Keratinocyte reepithelialization is a rate-limiting event in cutaneous wound repair, which involves the migration and proliferation of keratinocytes to cover the denuded dermal surface. Transforming growth factor-β1 (TGF-β1) has the ability to induce epithelial cell migration while inhibiting proliferation, and controversial results have been generated regarding the effect of TGF-β signaling on reepithelialization. In this study, full-thickness skin wounds were made in keratinocyte-specific Smad4 knockout and the control mice. The wound closure, reepithelialization, keratinocyte proliferation, myofibroblast numbers and collagen deposition of were assessed. The results showed that the proliferation of keratinocytes increased, which accelerated the reepithelialization, and led to faster wound repair in the epidermis of Smad4 mutant mice. Upregulation of keratin 17, 14-3-3 sigma and phosphorylated AKT in the hyperproliferative epidermis may be correlated with the accelerated reepithelialization. We conclude that Smad4 plays an inhibitory role in the keratinocyte-mediated reepithelialization of wound healing.