Dr Rick Thorne

Extracellular glutamine represents an important energy source for many cancer cells and its metabolism is intimately involved in maintaining redox homeostasis. The heightened metabolic activity within tumor tissues can result in glutamine deficiency, necessitating metabolic reprogramming responses. Here, dual mechanisms involving the stress-responsive transcription factor DDIT3 (DNA damage induced transcript 3) that establishes an interrelationship between glycolysis and mitochondrial respiration are revealed. DDIT3 is induced during glutamine deprivation to promote glycolysis and adenosine triphosphate production via suppression of the negative glycolytic regulator TIGAR. In concert, a proportion of the DDIT3 pool translocates to the mitochondria and suppresses oxidative phosphorylation through LONP1-mediated down-regulation of COQ9 and COX4. This in turn dampens the sustained levels of reactive oxygen species that follow glutamine withdrawal. Together these mechanisms constitute an adaptive survival mechanism permitting tumor cells to survive metabolic stress induced by glutamine starvation.

The TP53 gene is arguably the most important tumor suppressor gene known, contributing multifaceted roles to the process of tumor development. Its protein product p53, is a crucial sequence-specific transcription factor which regulates the expression of a large network of protein-coding genes, as well as thousands of noncoding RNAs (ncRNAs), notably microRNAs and long ncRNAs (lncRNAs). Through a variety of direct and indirect mechanisms, ncRNAs in turn modulate p53 levels and activity. Here the numbers of studies are steadily building which link the contributions of dysregulated ncRNAs to tumorigenesis via their participation throughout the p53 regulatory network. In this review, we will examine how the principal forms of ncRNAs, namely microRNAs, lncRNAs and circular RNAs (circRNAs) function as either effectors or regulators amongst the diversity of p53's cellular responses. We first discuss the more recently discovered connections between miRNAs and p53 signaling before focusing on the remarkable diversity of crosstalk evident between lncRNAs and p53, and subsequently, developing reports linking circRNAs to p53. Highlighted throughout the review are the mechanistic impacts of dysregulated ncRNAs on p53 functions as well as the possible prognostic implications of these interactions. We also describe the emerging connections between ncRNAs and the often-perplexing functions of mutant p53. Finally, in the context of p53 therapeutic approaches, we describe some of the challenges in ncRNA research and their potential for translation.

Long noncoding RNAs (lncRNAs) function through a diverse array of mechanisms that are not presently fully understood. Here, we sought to find lncRNAs differentially regulated in cancer cells resistant to either TNF-related apoptosis-inducing ligand (TRAIL) or the Mcl-1 inhibitor UMI-77, agents that act through the extrinsic and intrinsic apoptotic pathways, respectively. This work identified a commonly up-regulated lncRNA, ovarian adenocarcinoma-amplified lncRNA (OVAAL), that conferred apoptotic resistance in multiple cancer types. Analysis of clinical samples revealed OVAAL expression was significantly increased in colorectal cancers and melanoma in comparison to the corresponding normal tissues. Functional investigations showed that OVAAL depletion significantly inhibited cancer cell proliferation and retarded tumor xenograft growth. Mechanically, OVAAL physically interacted with serine/threonine-protein kinase 3 (STK3), which, in turn, enhanced the binding between STK3 and Raf-1. The ternary complex OVAAL/STK3/Raf-1 enhanced the activation of the RAF protooncogene serine/threonine-protein kinase (RAF)/mito-gen-activated protein kinase kinase 1 (MEK)/ERK signaling cascade, thus promoting c-Myc¿mediated cell proliferation and Mcl-1¿mediated cell survival. On the other hand, depletion of OVAAL triggered cellular senescence through polypyrimidine tract-binding protein 1 (PTBP1)¿mediated p27 expression, which was regulated by competitive binding between OVAAL and p27 mRNA to PTBP1. Additionally, c-Myc was demonstrated to drive OVAAL transcription, indicating a positive feedback loop between c-Myc and OVAAL in controlling tumor growth. Taken together, these results reveal that OVAAL contributes to the survival of cancer cells through dual mechanisms controlling RAF/MEK/ERK signaling and p27-mediated cell senescence.

Cancer cells in quiescence (G phase) are resistant to death, and re-entry of quiescent cancer cells into the cell-cycle plays an important role in cancer recurrence. Here we show that two p53-responsive miRNAs utilize distinct but complementary mechanisms to promote cancer cell quiescence by facilitating stabilization of p27. Purified quiescent B16 mouse melanoma cells expressed higher levels of miRNA-27b-3p and miRNA-455-3p relative to their proliferating counterparts. Induction of quiescence resulted in increased levels of these miRNAs in diverse types of human cancer cell lines. Inhibition of miRNA-27b-3p or miRNA-455-3p reduced, whereas its overexpression increased, the proportion of quiescent cells in the population, indicating that these miRNAs promote cancer cell quiescence. Accordingly, cancer xenografts bearing miRNA-27b-3p or miRNA-455-3p mimics were retarded in growth. miRNA-27b-3p targeted cyclin-dependent kinase regulatory subunit 1 (CKS1B), leading to reduction in p27 polyubiquitination mediated by S-phase kinase-associated protein 2 (Skp2). miRNA-455-3p targeted CDK2-associated cullin domain 1 (CAC1), which enhanced CDK2-mediated phosphorylation of p27 necessary for its polyubiquitination. Of note, the gene encoding miRNA-27b-3p was embedded in the intron of the chromosome 9 open reading frame 3 gene that was transcriptionally activated by p53. Similarly, the host gene of miRNA-455-3p, collagen alpha-1 (XXVII) chain, was also a p53 transcriptional target. Collectively, our results identify miRNA-27b-3p and miRNA-455-3p as important regulators of cancer cell quiescence in response to p53 and suggest that manipulating miRNA-27b-3p and miRNA-455-3p may constitute novel therapeutic avenues for improving outcomes of cancer treatment. Significance: Two novel p53-responsive microRNAs whose distinct mechanisms of action both stabilize p27 to promote cell quiescence and may serve as therapeutic avenues for improving outcomes of cancer treatment. 0

The oncoprotein c-Myc plays an important role in regulating glycolysis under normoxia; yet, in cancer cells, HIF1a, which is essential for driving glycolysis under hypoxia, is often up-regulated even in the presence of oxygen. The relationship between these two major regulators of the Warburg effect remains to be fully defined. Here we demonstrate that regulation of a long noncoding RNA (lncRNA), named IDH1-AS1, enables c-Myc to collaborate with HIF1a in activating the Warburg effect under normoxia. c-Myc transcriptionally repressed IDH1-AS1, which, upon expression, promoted homodimerization of IDH1 and thus enhanced its enzymatic activity. This resulted in increased a-KG and decreased ROS production and subsequent HIF1a down-regulation, leading to attenuation of glycolysis. Hence, c-Myc repression of IDH1-AS1 promotes activation of the Warburg effect by HIF1a. As such, IDH1-AS1 overexpression inhibited cell proliferation, whereas silencing of IDH1-AS1 promoted cell proliferation and cancer xenograft growth. Restoring IDH1-AS1 expression may therefore represent a potential metabolic approach for cancer treatment.

MTH1 helps prevent misincorporation of ROS-damaged dNTPs into genomic DNA; however, there is little understanding of how MTH1 itself is regulated. Here, we report that MTH1 is regulated by polyubiquitination mediated by the E3 ligase Skp2. In melanoma cells, MTH1 was upregulated commonly mainly due to its improved stability caused by K63-linked polyubiquitination. Although Skp2 along with other components of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex was physically associated with MTH1, blocking the SCF function ablated MTH1 ubiquitination and expression. Conversely, overexpressing Skp2-elevated levels of MTH1 associated with an increase in its K63-linked ubiquitination. In melanoma cell lines and patient specimens, we observed a positive correlation of Skp2 and MTH1 expression. Mechanistic investigations showed that Skp2 limited DNA damage and apoptosis triggered by oxidative stress and that MAPK upregulated Skp2 and MTH1 to render cells more resistant to such stress. Collectively, our findings identify Skp2-mediated K63-linked polyubiquitination as a critical regulatory mechanism responsible for MTH1 upregulation in melanoma, with potential implications to target the MAPK/Skp2/MTH1 pathway to improve its treatment.

Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates phosphatidylinositol 3-kinase signaling and is a tumor suppressor in some types of cancers. However, we have found that it is frequently upregulated in human colon cancer cells. Here we show that silencing of INPP4B blocks activation of Akt and serum-and glucocorticoid-regulated kinase 3 (SGK3), inhibits colon cancer cell proliferation and retards colon cancer xenograft growth. Conversely, overexpression of INPP4B increases proliferation and triggers anchorage-independent growth of normal colon epithelial cells. Moreover, we demonstrate that the effect of INPP4B on Akt and SGK3 is associated with inactivation of phosphate and tensin homolog through its protein phosphatase activity and that the increase in INPP4B is due to Ets-1-mediated transcriptional upregulation in colon cancer cells. Collectively, these results suggest that INPP4B may function as an oncogenic driver in colon cancer, with potential implications for targeting INPP4B as a novel approach to treat this disease.

The immune and nociceptive systems are shaped during the neonatal period where they undergo fine-tuning and maturation. Painful experiences during this sensitive period of development are known to produce long-lasting effects on the immune and nociceptive responses. It is less clear, however, whether inflammatory pain responses are primed by neonatal exposure to mild immunological stimuli, such as with lipopolysaccharide (LPS). Here, we examine the impact of neonatal LPS exposure on inflammatory pain responses, peripheral and hippocampal interleukin-1ß (IL-1ß), as well as mast cell number and degranulation in preadolescent and adult rats. Wistar rats were injected with LPS (0.05 mg/kg IP, Salmonella enteritidis) or saline on postnatal days (PNDs) 3 and 5 and later subjected to the formalin test at PNDs 22 and 80-97. At both time-points, and one-hour after formalin injection, blood and hippocampus were collected for measuring circulating and central IL-1ß levels using ELISA and Western blot, respectively. Paw tissue was also isolated to assess mast cell number and degree of degranulation using Toluidine Blue staining. Behavioural analyses indicate that at PND 22, LPS-challenged rats displayed enhanced flinching (p<.01) and licking (p<.01) in response to formalin injection. At PNDs 80-97, LPS-challenged rats exhibited increased flinching (p<.05), an effect observed in males only. Furthermore, neonatal LPS exposure enhanced circulating IL-1ß and mast cell degranulation in preadolescent but not adult rats following formalin injection. Hippocampal IL-1ß levels were increased in LPS-treated adult but not preadolescent rats in response to formalin injection. These data suggest neonatal LPS exposure produces developmentally regulated changes in formalin-induced behavioural responses, peripheral and central IL-1ß levels, as well as mast cell degranulation following noxious stimulation later in life. These findings highlight the importance of immune activation during the neonatal period in shaping immune response and pain sensitivity later in life. This is of clinical relevance given the high prevalence of bacterial infection during the neonatal period, particularly in the vulnerable population of preterm infants admitted to neonatal intensive care units.

The Hippo pathway is emerging as a critical nexus that balances self-renewal of progenitors against differentiation; however, upstream elements in vertebrate Hippo signalling are poorly understood. High expression of Fat1 cadherin within the developing neuroepithelium and the manifestation of severe neurological phenotypes in Fat1-knockout mice suggest roles in neurogenesis. Using the SH-SY5Y model of neuronal differentiation and employing gene silencing techniques, we show that FAT1 acts to control neurite outgrowth, also driving cells towards terminal differentiation via inhibitory effects on proliferation. FAT1 actions were shown to be mediated through Hippo signalling where it activated core Hippo kinase components and antagonised functions of the Hippo effector TAZ. Suppression of FAT1 promoted the nucleocytoplasmic shuttling of TAZ leading to enhanced transcription of the Hippo target gene CTGF together with accompanying increases in nuclear levels of Smad3. Silencing of TAZ reversed the effects of FAT1 depletion thus connecting inactivation of TAZ-TGFbeta signalling with Hippo signalling mediated through FAT1. These findings establish FAT1 as a new upstream Hippo element regulating early stages of differentiation in neuronal cells.

Nerve infiltration is essential to prostate cancer progression, but the mechanism by which nerves are attracted to prostate tumors remains unknown. We report that the precursor of nerve growth factor (proNGF) is overexpressed in prostate cancer and involved in the ability of prostate cancer cells to induce axonogenesis. A series of 120 prostate cancer and benign prostate hyperplasia (BPH) samples were analyzed by IHC for proNGF. ProNGF was mainly localized in the cytoplasm of epithelial cells, with marked expression in cancer compared with BPH. Importantly, the proNGF level positively correlated with the Gleason score (n = 104, t<inf>B</inf> = 0.51). A higher level of proNGF was observed in tumors with a Gleason score of =8 compared with a Gleason score of 7 and 6 (P < 0.001). In vitro, proNGF was detected in LNCaP, DU145, and PC-3 prostate cancer cells and BPH-1 cells but not in RWPE-1 immortalized nontumorigenic prostate epithelial cells or primary normal prostate epithelial cells. Co-culture of PC12 neuronal-like cells or 50B11 neurons with PC-3 cells resulted in neurite outgrowth in neuronal cells that was inhibited by blocking antibodies against proNGF, indicating that prostate cancer cells can induce axonogenesis via secretion of proNGF. These data reveal that ProNGF is a biomarker associated with high-risk prostate cancers and a potential driver of infiltration by nerves.

Fat1 is a single pass transmembrane protein and the largest member of the cadherin superfamily. Mouse knockout models and in vitro studies have suggested that Fat1 influences cell polarity and motility. Fat1 is also an upstream regulator of the Hippo pathway, at least in lower vertebrates, and hence may play a role in growth control. In previous work we have established that FAT1 cadherin is initially cleaved by proprotein convertases to form a noncovalently linked heterodimer prior to expression on the cell surface. Such processing was not a requirement for cell surface expression, since melanoma cells expressed both unprocessed FAT1 and the heterodimer on the cell surface. Here we further establish that the site 1 (S1) cleavage step to promote FAT1 heterodimerisation is catalysed by furin and we identify the cleavage site utilised. For a number of other transmembrane receptors that undergo heterodimerisation the S1 processing step is thought to occur constitutively but the functional significance of heterodimerisation has been controversial. It has also been generally unclear as to the significance of receptor heterodimerisation with respect to subsequent post-translational proteolysis that often occurs in transmembrane proteins. Exploiting the partial deficiency of FAT1 processing in melanoma cells together with furin-deficient LoVo cells, we manipulated furin expression to demonstrate that only the heterodimer form of FAT1 is subject to cleavage and subsequent release of the extracellular domain. This work establishes S1-processing as a clear functional prerequisite for ectodomain shedding of FAT1 with general implications for the shedding of other transmembrane receptors. © 2014.

The vertebrate Fat cadherins comprise a small gene family of four members, Fat1-Fat4, all closely related in structure to Drosophila ft and ft2. Over the past decade, knock-out mouse studies, genetic manipulation, and large sequencing projects has aided our understanding of the function of vertebrate Fat cadherins in tissue development and disease. The majority of studies of this family have focused on Fat1, with evidence now showing it can bind enable (ENA)/Vasodilator-stimulated phosphoprotein (VASP), ß-catenin and Atrophin proteins to influence cell polarity and motility; HOMER-1 and HOMER-3 proteins to regulate actin accumulation in neuronal synapses; and scribble to influence the Hippo signaling pathway. Fat2 and Fat3 can regulate cell migration in a tissue specific manner and Fat4 appears to influence both planar cell polarity and Hippo signaling recapitulating the activity of Drosophila ft. Knowledge about the exact downstream signaling pathways activated by each family member remains in its infancy, but it is becoming clearer that they have tissue specific and redundant roles in development and may be lost or gained in cancer. In this review, we summarize the recent progress on understanding the role of the Fat cadherin family, integrating the current knowledge of molecular interactions and tissue distributions, together with the accumulating evidence of their changed expression in human disease. The latter is now beginning to promote interest in these molecules as both biomarkers and new targets for therapeutic intervention. © 2013 Wiley Periodicals, Inc.

Elucidation of the molecular mechanisms by which 5-fluorouracil (5-FU) induces apoptosis is required in order to understand the resistance of colorectal cancer (CRC) cells to 5-FU. In the current study, 5-FU-induced apoptosis was assessed using the propidium iodide method. Involvement of protein kinase C (PKC) was assessed by evaluating the extent of their activation in CRC, following treatment with 5-FU, using biochemical inhibitors and western blot analysis. The results revealed that 5-FU induces varying degrees of apoptosis in CRC cells; HCT116 cells were identified to be the most sensitive cells and SW480 were the least sensitive. In addition, 5-FU-induced apoptosis was caspase-dependent as it appeared to be initiated by caspase-9. Furthermore, PKCe was marginally expressed in CRC cells and no changes were observed in the levels of cleavage or phosphorylation following treatment with 5-FU. The treatment of HCT116 cells with 5-FU increased the expression, phosphorylation and cleavage of PKCd. The inhibition of PKCd was found to significantly inhibit 5-FU-induced apoptosis. These results indicated that 5-FU induces apoptosis in CRC by the activation of PKCd and caspase-9. In addition, the levels of PKCd activation may determine the sensitivity of CRC to 5-FU.

Background: Macrophage migration inhibitory factor (MIF) is a widely expressed cytokine involved in a variety of cellular processes including cell cycle regulation and the control of proliferation. Overexpression of MIF has been reported in a number of cancer types and it has previously been shown that MIF is upregulated in melanocytic tumours with the highest expression levels occurring in malignant melanoma. However, the clinical significance of high MIF expression in melanoma has not been reported. Methods: MIF expression was depleted in human melanoma cell lines using siRNA-mediated gene knockdown and effects monitored using in vitro assays of proliferation, cell cycle, apoptosis, clonogenicity and Akt signalling. In silico analyses of expression microarray data were used to correlate MIF expression levels in melanoma tumours with overall patient survival using a univariate Cox regression model. Results: Knockdown of MIF significantly decreased proliferation, increased apoptosis and decreased anchorage-independent growth. Effects were associated with reduced numbers of cells entering S phase concomitant with decreased cyclin D1 and CDK4 expression, increased p27 expression and decreased Akt phosphorylation. Analysis of clinical outcome data showed that MIF expression levels in primary melanoma were not associated with outcome (HR = 1.091, p = 0.892) whereas higher levels of MIF in metastatic lesions were significantly associated with faster disease progression (HR = 2.946, p = 0.003 and HR = 4.600, p = 0.004, respectively in two independent studies). Conclusions: Our in vitro analyses show that MIF functions upstream of the PI3K/Akt pathway in human melanoma cell lines. Moreover, depletion of MIF inhibited melanoma proliferation, viability and clonogenic capacity. Clinically, high MIF levels in metastatic melanoma were found to be associated with faster disease recurrence. These findings support the clinical significance of MIF signalling in melanoma and provide a strong rationale for both targeting and monitoring MIF expression in clinical melanoma.

Background: A useful application of flow cytometry is the investigation of cell receptor-ligand interactions. However such analyses are often compromised due to problems interpreting changes in ligand binding where the receptor expression is not constant. Commonly, problems are encountered due to cell treatments resulting in altered receptor expression levels, or when cell lines expressing a transfected receptor with variable expression are being compared. To overcome this limitation we have developed a Microsoft Excel spreadsheet that aims to automatically and effectively simplify flow cytometric data and perform statistical tests in order to provide a clearer graphical representation of results. Results: To demonstrate the use and advantages of this new spreadsheet method we have investigated the binding of the transmembrane adhesion receptor CD44 to its ligand hyaluronan. In the first example, phorbol ester treatment of cells results in both increased CD44 expression and increased hyaluronan binding. By applying the spreadsheet method we effectively demonstrate that this increased ligand binding results from receptor activation. In the second example we have compared AKR1 cells transfected either with wild type CD44 (WT CD44) or a mutant with a truncated cytoplasmic domain (CD44-T). These two populations do not have equivalent receptor expression levels but by using the spreadsheet method hyaluronan binding could be compared without the need to generate single cell clones or FACS sorting the cells for matching CD44 expression. By this method it was demonstrated that hyaluronan binding requires a threshold expression of CD44 and that this threshold is higher for CD44-T. However, at high CD44-T expression, binding was equivalent to WT CD44 indicating that the cytoplasmic domain has a role in presenting the receptor at the cell surface in a form required for efficient hyaluronan binding rather than modulating receptor activity. Conclusion: Using the attached spreadsheets and instructions, a simple post-acquisition method for analysing bivariate flow cytometry data is provided. This method constitutes a straightforward improvement over the standard graphical output of flow cytometric data and has the significant advantage that ligand binding can be compared between cell populations irrespective of receptor expression levels. © 2004 Tzircotis et al; licensee BioMed Central Ltd.