Current status and recommendations for the future of research, teaching, and testing in the biological sciences of radiation oncology: report of the American Society for Radiation Oncology Cancer Biology/Radiation Biology Task Force, executive summary.

In early 2011, a dialogue was initiated within the Board of Directors (BOD) of the American Society for Radiation Oncology (ASTRO) regarding the future of the basic sciences of the specialty, primarily focused on the current state and potential future direction of basic research within radiation oncology. After consideration of the complexity of the issues involved and the precise nature of the undertaking, in August 2011, the BOD empanelled a Cancer Biology/Radiation Biology Task Force (TF). The TF was charged with developing an accurate snapshot of the current state of basic (preclinical) research in radiation oncology from the perspective of relevance to the modern clinical practice of radiation oncology as well as the education of our trainees and attending physicians in the biological sciences. The TF was further charged with making suggestions as to critical areas of biological basic research investigation that might be most likely to maintain and build further the scientific foundation and vitality of radiation oncology as an independent and vibrant medical specialty. It was not within the scope of service of the TF to consider the quality of ongoing research efforts within the broader radiation oncology space, to presume to consider their future potential, or to discourage in any way the investigators committed to areas of interest other than those targeted. The TF charge specifically precluded consideration of research issues related to technology, physics, or clinical investigations. This document represents an Executive Summary of the Task Force report.

Emerging role of sirtuins on tumorigenesis: possible link between aging and cancer.

Aging is the strongest risk factor for cancer development, suggesting that molecular crosstalks between aging and tumorigenesis exist in many cellular pathways. Recently, Sirtuins (Sirt1-7), the mammalian homologues of aging-related sir2α in yeast, have been shown to modulate several major cellular pathways, such as DNA repair, inflammation, metabolism, cell death, and proliferation in response to diverse stresses, and may serve as a possible molecular link between aging and tumorignenesis. In addition, growing evidence suggests that sirtuins are directly implicated in the development of cancer, and they can act as either a tumor suppressor or promoter, depending on the cellular context and tumor types. While the functions of Sirt1 in tumorigenesis have been reported and reviewed in many studies, the connection between sirtuins 2-7 and the development of cancer is less established. Thus, this review will present the recent updates on the emerging roles of Sirt2-7 members in carcinogenesis.

Sirt3, mitochondrial ROS, ageing, and carcinogenesis.

One fundamental observation in cancer etiology is that the rate of malignancies in any mammalian population increases exponentially as a function of age, suggesting a mechanistic link between the cellular processes governing longevity and carcinogenesis. In addition, it is well established that aberrations in mitochondrial metabolism, as measured by increased reactive oxygen species (ROS), are observed in both aging and cancer. In this regard, genes that impact upon longevity have recently been characterized in S. cerevisiae and C. elegans, and the human homologs include the Sirtuin family of protein deacetylases. Interestingly, three of the seven sirtuin proteins are localized into the mitochondria suggesting a connection between the mitochondrial sirtuins, the free radical theory of aging, and carcinogenesis. Based on these results it has been hypothesized that Sirt3 functions as a mitochondrial fidelity protein whose function governs both aging and carcinogenesis by modulating ROS metabolism. Sirt3 has also now been identified as a genomically expressed, mitochondrial localized tumor suppressor and this review will outline potential relationships between mitochondrial ROS/superoxide levels, aging, and cell phenotypes permissive for estrogen and progesterone receptor positive breast carcinogenesis.

SIRT2 maintains genome integrity and suppresses tumorigenesis through regulating APC/C activity.

Members of sirtuin family regulate multiple critical biological processes, yet their role in carcinogenesis remains controversial. To investigate the physiological functions of SIRT2 in development and tumorigenesis, we disrupted Sirt2 in mice. We demonstrated that SIRT2 regulates the anaphase-promoting complex/cyclosome activity through deacetylation of its coactivators, APC(CDH1) and CDC20. SIRT2 deficiency caused increased levels of mitotic regulators, including Aurora-A and -B that direct centrosome amplification, aneuploidy, and mitotic cell death. Sirt2-deficient mice develop gender-specific tumorigenesis, with females primarily developing mammary tumors, and males developing more hepatocellular carcinoma (HCC). Human breast cancers and HCC samples exhibited reduced SIRT2 levels compared with normal tissues. These data demonstrate that SIRT2 is a tumor suppressor through its role in regulating mitosis and genome integrity.

Tumor suppressor Lzap regulates cell cycle progression, doming, and zebrafish epiboly.

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here, we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract, and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly--the earliest morphogenetic movement in animal development--which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation.

Prostaglandin Gbetagamma signaling stimulates gastrulation movements by limiting cell adhesion through Snai1a stabilization.

Gastrulation movements form the germ layers and shape them into the vertebrate body. Gastrulation entails a variety of cell behaviors, including directed cell migration and cell delamination, which are also involved in other physiological and pathological processes, such as cancer metastasis. Decreased Prostaglandin E(2) (PGE(2)) synthesis due to interference with the Cyclooxygenase (Cox) and Prostaglandin E synthase (Ptges) enzymes halts gastrulation and limits cancer cell invasiveness, but how PGE(2) regulates cell motility remains unclear. Here we show that PGE(2)-deficient zebrafish embryos, impaired in the epiboly, internalization, convergence and extension gastrulation movements, exhibit markedly increased cell-cell adhesion, which contributes to defective cell movements in the gastrula. Our analyses reveal that PGE(2) promotes cell protrusive activity and limits cell adhesion by modulating E-cadherin transcript and protein, in part through stabilization of the Snai1a (also known as Snail1) transcriptional repressor, an evolutionarily conserved regulator of cell delamination and directed migration. We delineate a pathway whereby PGE(2) potentiates interaction between the receptor-coupled G protein betagamma subunits and Gsk3beta to inhibit proteasomal degradation of Snai1a. However, overexpression of beta-catenin cannot stabilize Snai1a in PGE(2)-deficient gastrulae. Thus, the Gsk3beta-mediated and beta-catenin-independent inhibition of cell adhesion by Prostaglandins provides an additional mechanism for the functional interactions between the PGE(2) and Wnt signaling pathways during development and disease. We propose that ubiquitously expressed PGE(2) synthesizing enzymes, by promoting the stability of Snai1a, enable the precise and rapid regulation of cell adhesion that is required for the dynamic cell behaviors that drive various gastrulation movements.

Frizzled 8a function is required for oligodendrocyte development in the zebrafish spinal cord.

Oligodendrocytes are the myelinating cells in the central nervous system. The development of oligodendrocytes is mediated by complex signaling networks, including Wnt signaling. Although Wnt signaling has been studied in various aspects of neurogenesis, the distinct roles of various Frizzled receptors that mediate the Wnt signaling in the CNS remain virtually unknown. In order to understand the specific function of Wnt signaling in oligodendrocyte development, we focused on the Frizzled 8a (Fz8a) receptor. Here we show that Fz8a plays a critical role in the specification and maturation of oligodendrocyte progenitor cells (OPCs) in the ventral spinal cord. Loss of Fz8a function perturbed the proliferation and organization of radial glial cells that give rise to OPCs in the ventral precursor region of spinal cord. In addition, we demonstrate that Wnt signaling activation after the specification of OPCs blocks the formation of mature oligodendrocytes and results in the elimination of OPCs.

Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer.

Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition could enhance radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining) and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.

Switch between apoptosis and autophagy: radiation-induced endoplasmic reticulum stress?

The induction of cell death by radiation has largely been attributed to pro-apoptotic mechanisms. Autophagy, an alternative form of programmed cell death, has recently been shown to contribute significantly to anti-neoplastic effects of radiation therapy. In light of this, ER stress has been shown to trigger both apoptosis and autophagy, and act as an important mediator linking the two programmed cell death pathways. Recent data reveal that ER stress leads to activation of autophagosome formation with LC3 conversion via either PERK-eIF2a pathway or IRE1-JNK pathway. In this focused review, we summarize the main molecular mediators that control cellular "switches" between apoptosis and autophagy pathways by utilizing radiation therapy as a model.

NSAIDs and cancer prevention: targets downstream of COX-2.

Preclinical and clinical studies have clearly shown a benefit of nonsteroidal anti-inflammatory drug (NSAID) use in reducing cancer risk. However, the adverse gastrointestinal and cardiovascular side effects associated with NSAIDs and COX-2 selective inhibitors (coxibs) have provoked more scrutiny of the precise role of specific downstream mediators in the prostaglandin (PG) signaling cascade. NSAIDs and coxibs inhibit PG biosynthesis. One of the PGs produced at high levels in the tumor microenvironment is PGE(2), which is thought to play a major role in cancer progression. Thus, a better understanding of PGE(2) signaling could enable identification of novel and safer therapeutic targets downstream of the cyclooxygenase enzymes. We review the emerging molecular mechanisms by which COX-2-derived PGE(2) is involved in cancer progression and delineate potential opportunities for development of novel pharmacologic approaches utilizing this pathway.

Cyclooxygenase-1-derived PGE2 promotes cell motility via the G-protein-coupled EP4 receptor during vertebrate gastrulation.

Gastrulation is a fundamental process during embryogenesis that shapes proper body architecture and establishes three germ layers through coordinated cellular actions of proliferation, fate specification, and movement. Although many molecular pathways involved in the specification of cell fate and polarity during vertebrate gastrulation have been identified, little is known of the signaling that imparts cell motility. Here we show that prostaglandin E(2) (PGE(2)) production by microsomal PGE(2) synthase (Ptges) is essential for gastrulation movements in zebrafish. Furthermore, PGE(2) signaling regulates morphogenetic movements of convergence and extension as well as epiboly through the G-protein-coupled PGE(2) receptor (EP4) via phosphatidylinositol 3-kinase (PI3K)/Akt. EP4 signaling is not required for proper cell shape or persistence of migration, but rather it promotes optimal cell migration speed during gastrulation. This work demonstrates a critical requirement of PGE(2) signaling in promoting cell motility through the COX-1-Ptges-EP4 pathway, a previously unrecognized role for this biologically active lipid in early animal development.

Fishing for prostanoids: deciphering the developmental functions of cyclooxygenase-derived prostaglandins.

Prostaglandin G/H synthases (PGHS), commonly referred to as cyclooxygenases (COX-1 and COX-2), catalyze a key step in the synthesis of biologically active prostaglandins (PGs), the conversion of arachidonic acid (AA) into prostaglandin H(2) (PGH(2)). PGs have important functions in a variety of physiologic and pathologic settings, including inflammation, cardiovascular homeostasis, reproduction, and carcinogenesis. However, an evaluation of prostaglandin function in early development has been difficult due to the maternal contribution of prostaglandins from the uterus. The emergence of zebrafish as a model system has begun to provide some insights into the roles of this signaling cascade during vertebrate development. In zebrafish, COX-1 derived prostaglandins are required for two distinct stages of development, namely during gastrulation and segmentation. During gastrulation, PGE(2) signaling promotes cell motility, without altering the cell shape or directional migration of gastrulating cells. During segmentation, COX-1 signaling is also required for posterior mesoderm development, including the formation of vascular tube structures, angiogenesis of intersomitic vessels, and pronephros morphogenesis. We propose that deciphering the role for prostaglandin signaling in zebrafish development could yield insight and ultimately address the mechanistic details underlying various disease processes that result from perturbation of this pathway.

Cyclooxygenase-1 signaling is required for vascular tube formation during development.

Prostaglandin endoperoxide synthases (PTGS), commonly referred to as cyclooxygenases (COX-1 and COX-2), catalyze the key step in the synthesis of biologically active prostaglandins (PGs), the conversion of arachidonic acid (AA) into prostaglandin H2 (PGH2). Although COX and prostaglandins have been implicated in a wide variety of physiologic processes, an evaluation of the role of prostaglandins in early mammalian development has been difficult due to the maternal contribution of prostaglandins from the uterus: COX null mouse embryos develop normally during embryogenesis. Here, we verify that inhibition of COX-1 results in zebrafish gastrulation arrest and shows that COX-1 expression becomes restricted to the posterior mesoderm during somitogenesis and to posterior mesoderm organs at pharyngula stage. Inhibition of COX-1 signaling after gastrulation results in defective vascular tube formation and shortened intersomitic vessels in the posterior body region. These defects are rescued completely by PGE(2) treatment or, to a lesser extent, by PGF(2alpha), but not by other prostaglandins, such as PGI(2), TxB(2), or PGD(2). Functional knockdown of COX-1 using antisense morpholino oligonucleotide translation interference also results in posterior vessel defect in addition to enlarged posterior nephric duct, phenocopying the defects caused by inhibition of COX-1 activity. Together, we provide the first evidence that COX-1 signaling is required for development of posterior mesoderm organs, specifically in the vascular tube formation and posterior nephric duct development.