c-MYC: more than just a matter of life and death.

Deregulated expression of c-MYC occurs in a broad range of human cancers and is often associated with poor prognosis, indicating a key role for this oncogene in tumour progression. However, as established human tumours often bear multiple genetic lesions, it is difficult to determine whether c-MYC is instrumental in the initiation/progression of the tumour, or indeed whether inactivating c-MYC would lead to tumour regression. Regulatable transgenic mouse models of oncogenesis have shed light on these issues and provide hope for effective cancer therapies.

Deciphering c-MYC-regulated genes in two distinct tissues.

BACKGROUND: The transcription factor MYC is a critical regulator of diverse cellular processes, including both replication and apoptosis. Differences in MYC-regulated gene expression responsible for such opposing outcomes in vivo remain obscure. To address this we have examined time-dependent changes in global gene expression in two transgenic mouse models in which MYC activation, in either skin suprabasal keratinocytes or pancreatic islet ß-cells, promotes tissue expansion or involution, respectively. RESULTS: Consistent with observed phenotypes, expression of cell cycle genes is increased in both models (albeit enriched in ß-cells), as are those involved in cell growth and metabolism, while expression of genes involved in cell differentiation is down-regulated. However, in ß-cells, which unlike suprabasal keratinocytes undergo prominent apoptosis from 24 hours, there is up-regulation of genes associated with DNA-damage response and intrinsic apoptotic pathways, including Atr, Arf, Bax and Cycs. In striking contrast, this is not the case for suprabasal keratinocytes, where pro-apoptotic genes such as Noxa are down-regulated and key anti-apoptotic pathways (such as Igf1-Akt) and those promoting angiogenesis are up-regulated. Moreover, dramatic up-regulation of steroid hormone-regulated Kallikrein serine protease family members in suprabasal keratinocytes alone could further enhance local Igf1 actions, such as through proteolysis of Igf1 binding proteins. CONCLUSIONS: Activation of MYC causes cell growth, loss of differentiation and cell cycle entry in both ß-cells and suprabasal keratinocytes in vivo. Apoptosis, which is confined to ß-cells, may involve a combination of a DNA-damage response and downstream activation of pro-apoptotic signalling pathways, including Cdc2a and p19(Arf)/p53, and downstream targets. Conversely, avoidance of apoptosis in suprabasal keratinocytes may result primarily from the activation of key anti-apoptotic signalling pathways, particularly Igf1-Akt, and induction of an angiogenic response, though intrinsic resistance to induction of p19(Arf) by MYC in suprabasal keratinocytes may contribute.

Micro-Net: A unified model for segmentation of various objects in microscopy images.

Object segmentation and structure localization are important steps in automated image analysis pipelines for microscopy images. We present a convolution neural network (CNN) based deep learning architecture for segmentation of objects in microscopy images. The proposed network can be used to segment cells, nuclei and glands in fluorescence microscopy and histology images after slight tuning of input parameters. The network trains at multiple resolutions of the input image, connects the intermediate layers for better localization and context and generates the output using multi-resolution deconvolution filters. The extra convolutional layers which bypass the max-pooling operation allow the network to train for variable input intensities and object size and make it robust to noisy data. We compare our results on publicly available data sets and show that the proposed network outperforms recent deep learning algorithms.

Brief inactivation of c-Myc is not sufficient for sustained regression of c-Myc-induced tumours of pancreatic islets and skin epidermis.

BACKGROUND: Tumour regression observed in many conditional mouse models following oncogene inactivation provides the impetus to develop, and a platform to preclinically evaluate, novel therapeutics to inactivate specific oncogenes. Inactivating single oncogenes, such as c-Myc, can reverse even advanced tumours. Intriguingly, transient c-Myc inactivation proved sufficient for sustained osteosarcoma regression; the resulting osteocyte differentiation potentially explaining loss of c-Myc's oncogenic properties. But would this apply to other tumours? RESULTS: We show that brief inactivation of c-Myc does not sustain tumour regression in two distinct tissue types; tumour cells in pancreatic islets and skin epidermis continue to avoid apoptosis after c-Myc reactivation, by virtue of Bcl-xL over-expression or a favourable microenvironment, respectively. Moreover, tumours progress despite reacquiring a differentiated phenotype and partial loss of vasculature during c-Myc inactivation. Interestingly, reactivating c-Myc in beta-cell tumours appears to result not only in further growth of the tumour, but also re-expansion of the accompanying angiogenesis and more pronounced beta-cell invasion (adenocarcinoma). CONCLUSIONS: Given that transient c-Myc inactivation could under some circumstances produce sustained tumour regression, the possible application of this potentially less toxic strategy in treating other tumours has been suggested. We show that brief inactivation of c-Myc fails to sustain tumour regression in two distinct models of tumourigenesis: pancreatic islets and skin epidermis. These findings challenge the potential for cancer therapies aimed at transient oncogene inactivation, at least under those circumstances where tumour cell differentiation and alteration of epigenetic context fail to reinstate apoptosis. Together, these results suggest that treatment schedules will need to be informed by knowledge of the molecular basis and environmental context of any given cancer.

Oxidised lipoproteins may promote inflammation through the selective delay of engulfment but not binding of apoptotic cells by macrophages.

During normal tissue homeostasis apoptotic cells (AC) are rapidly recognised and engulfed by neighbouring cells or macrophages (Mphi), thus preventing an inflammatory response. Conversely, in chronically inflamed tissues, including the atherosclerotic artery and rheumatoid joint, removal of AC is defective despite the co-localisation of seemingly adequate numbers of Mphi. Mechanisms preventing removal of AC in vivo remain obscure, but might include oxidised low-density lipoprotein (ox-LDL), which is abundant in chronic inflammatory lesions. Although implicated in their pathogenesis, defining the role of ox-LDL on inflammatory processes has proved complicated. In fact, seemingly contradictory results have previously been described, though these may in part reflect the heterogeneous nature of ox-LDL applied in these studies. We wished to investigate the effect of physiologically representative ox-LDL on the binding and engulfment of apoptotic vascular smooth muscle cells (VSMC) and fibroblasts, as these have previously been shown to co-localise with Mphi in chronically inflamed tissues in vivo. We show that Mphi recognition of AC in vitro is not affected at physiological levels of ox-LDL. However, engulfment of intact AC is dramatically reduced/delayed. Importantly, in the absence of ox-LDL rapid phagocytosis of intact AC suppresses Mphi inflammatory cytokine release. In striking contrast, in the presence of ox-LDL, despite binding of AC to Mphi, release of IL-6 and MCP-1 is no longer suppressed. We propose that ox-LDL could maintain an inflammatory response by inhibiting the engulfment of AC, required for Mphi de-activation. This mechanism may contribute to chronic persisting inflammation in the atherosclerotic artery and rheumatoid joint.

A simple matter of life and death-the trials of postnatal Beta-cell mass regulation.

Pancreatic beta-cells, which secrete the hormone insulin, are the key arbiters of glucose homeostasis. Defective beta-cell numbers and/or function underlie essentially all major forms of diabetes and must be restored if diabetes is to be cured. Thus, the identification of the molecular regulators of beta-cell mass and a better understanding of the processes of beta-cell differentiation and proliferation may provide further insight for the development of new therapeutic targets for diabetes. This review will focus on the principal hormones and nutrients, as well as downstream signalling pathways regulating beta-cell mass in the adult. Furthermore, we will also address more recently appreciated regulators of beta-cell mass, such as microRNAs.

Re-expression of IGF-II is important for beta cell regeneration in adult mice.

BACKGROUND: The key factors which support re-expansion of beta cell numbers after injury are largely unknown. Insulin-like growth factor II (IGF-II) plays a critical role in supporting cell division and differentiation during ontogeny but its role in the adult is not known. In this study we investigated the effect of IGF-II on beta cell regeneration. METHODOLOGY/PRINCIPAL FINDINGS: We employed an in vivo model of 'switchable' c-Myc-induced beta cell ablation, pIns-c-MycER(TAM), in which 90% of beta cells are lost following 11 days of c-Myc (Myc) activation in vivo. Importantly, such ablation is normally followed by beta cell regeneration once Myc is deactivated, enabling functional studies of beta cell regeneration in vivo. IGF-II was shown to be re-expressed in the adult pancreas of pIns-c-MycER(TAM)/IGF-II(+/+) (MIG) mice, following beta cell injury. As expected in the presence of IGF-II beta cell mass and numbers recover rapidly after ablation. In contrast, in pIns-c-MycER(TAM)/IGF-II(+/-) (MIGKO) mice, which express no IGF-II, recovery of beta cell mass and numbers were delayed and impaired. Despite failure of beta cell number increase, MIGKO mice recovered from hyperglycaemia, although this was delayed. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that beta cell regeneration in adult mice depends on re-expression of IGF-II, and supports the utility of using such ablation-recovery models for identifying other potential factors critical for underpinning successful beta cell regeneration in vivo. The potential therapeutic benefits of manipulating the IGF-II signaling systems merit further exploration.

c-Myc directly induces both impaired insulin secretion and loss of ß-cell mass, independently of hyperglycemia in vivo.

c-Myc (Myc) is a mediator of glucotoxicity but could also independently compromise ß-cell survival and function. We have shown that after Myc activation in adult ß-cells in vivo, apoptosis is preceded by hyperglycemia, suggesting glucotoxicity might contribute to Myc-induced apoptosis. To address this question conditional Myc was activated in ß-cells of adult pIns-c-MycER(TAM) mice in vivo in the presence or absence of various glucose-lowering treatments, including exogenous insulin and prior to transplantation with wild-type islets. Changes in blood glucose levels were subsequently correlated with changes in ß-cell mass and markers of function/differentiation. Activation of c-Myc resulted in reduced insulin secretion, hyperglycemia and loss of ß-cell differentiation, followed by reduction in mass. Glucose-lowering interventions did not prevent loss of ß-cells. Therefore, Myc can cause diabetes by direct effects on ß-cell apoptosis even in the absence of potentially confounding secondary hyperglycemia. Moreover, as loss of ß-cell differentiation/function and hyperglycemia are not prevented by preventing ß-cell apoptosis, we conclude that Myc might contribute to the pathogenesis of diabetes by directly coupling cell cycle entry and ß-cell failure through two distinct pathways.

Non-ß-cell progenitors of ß-cells in pregnant mice.

Pregnancy is a normal physiological condition in which the maternal ß-cell mass increases rapidly about two-fold to adapt to new metabolic challenges. We have used a lineage tracing of ß-cells to analyse the origin of new ß-cells during this rapid expansion in pregnancy. Double transgenic mice bearing a tamoxifen-dependent Cre-recombinase construct under the control of a rat insulin promoter, together with a reporter Z/AP gene, were generated. Then, in response to a pulse of tamoxifen before pregnancy, ß-cells in these animals were marked irreversibly and heritably with the human placental alkaline phosphatase (HP AP). First, we conclude that the lineage tracing system was highly specific for ß-cells. Secondly, we scored the proportion of the ß-cells marked with HP AP during a subsequent chase period in pregnant and non-pregnant females. We observed a dilution in this labeling index in pregnant animal pancreata, compared to nonpregnant controls, during a single pregnancy in the chase period. To extend these observations we also analysed the labeling index in pancreata of animals during the second of two pregnancies in the chase period. The combined data revealed statistically-significant dilution during pregnancy, indicating a contribution to new beta cells from a non-ß-cell source. Thus for the first time in a normal physiological condition, we have demonstrated not only ß-cell duplication, but also the activation of a non-ß-cell progenitor population. Further, there was no transdifferentiation of ß-cells to other cell types in a two and half month period following labeling, including the period of pregnancy.

Integrating semantic annotation and information visualization for the analysis of multichannel fluorescence micrographs from pancreatic tissue.

The challenging problem of computational bioimage analysis receives growing attention from life sciences. Fluorescence microscopy is capable of simultaneously visualizing multiple molecules by staining with different fluorescent dyes. In the analysis of the result multichannel images, segmentation of ROIs resembles only a first step which must be followed by a second step towards the analysis of the ROI's signals in the different channels. In this paper we present a system that combines image segmentation and information visualization principles for an integrated analysis of fluorescence micrographs of tissue samples. The analysis aims at the detection and annotation of cells of the Islets of Langerhans and the whole pancreas, which is of great importance in diabetes studies and in the search for new anti-diabetes treatments. The system operates with two modules. The automatic annotation module applies supervised machine learning for cell detection and segmentation. The second information visualization module can be used for an interactive classification and visualization of cell types following the link-and-brush principle for filtering. We can compare the results obtained with our system with results obtained manually by an expert, who evaluated a set of example images three times to account for his intra-observer variance. The comparison shows that using our system the images can be evaluated with high accuracy which allows a considerable speed up of the time-consuming evaluation process.

Effects of c-MYC activation on glucose stimulus-secretion coupling events in mouse pancreatic islets.

Alteration of pancreatic beta-cell survival and Preproinsulin gene expression by prolonged hyperglycemia may result from increased c-MYC expression. However, it is unclear whether c-MYC effects on beta-cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key beta-cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifen-switchable form of c-MYC in beta-cells (MycER) and their wild-type littermates. Tamoxifen treatment of wild-type islets did not affect their cell survival, Preproinsulin gene expression, and glucose stimulus-secretion coupling. In contrast, tamoxifen-mediated c-MYC activation for 2-3 days triggered cell apoptosis and decreased Preproinsulin gene expression in MycER islets. These effects were accompanied by mitochondrial membrane hyperpolarization at all glucose concentrations, a higher resting intracellular calcium concentration ([Ca(2+)](i)), and lower glucose-induced [Ca(2+)](i) rise and islet insulin content, leading to a strong reduction of glucose-induced insulin secretion. Compared with these effects, 1-wk culture in 30 mmol/l glucose increased the islet sensitivity to glucose stimulation without reducing the maximal glucose effectiveness or the insulin content. In contrast, overnight exposure to a low H(2)O(2) concentration increased the islet resting [Ca(2+)](i) and reduced the amplitude of the maximal glucose response as in tamoxifen-treated MycER islets. In conclusion, c-MYC activation rapidly stimulates apoptosis, reduces Preproinsulin gene expression and insulin content, and triggers functional alterations of beta-cells that are better mimicked by overnight exposure to a low H(2)O(2) concentration than by prolonged culture in high glucose.

Regulated beta-cell regeneration in the adult mouse pancreas.

Several studies have shown that the adult pancreas possesses a limited potential for beta-cell regeneration upon tissue injury. One of the difficulties in studying beta-cell regeneration has been the lack of a robust, synchronized animal model system that would allow controlled regulation of beta-cell loss and subsequent proliferation in adult pancreas. Here we present a transgenic mouse regeneration model in which the c-Myc transcription factor/mutant estrogen receptor (cMycER(TAM)) fusion protein can be specifically activated in mature beta-cells. We have studied these transgenic mice by immunohistochemical and biochemical methods to assess the ablation and posterior regeneration of beta-cells. Activation of the cMycER(TAM) fusion protein results in synchronous and selective beta-cell apoptosis followed by the onset of acute diabetes. Inactivation of c-Myc leads to gradual regeneration of insulin-expressing cells and reversal of diabetes. Our results demonstrate that the mature pancreas has the ability to fully recover from almost complete ablation of all existing beta-cells. Our results also suggest the regeneration of beta-cells is mediated by replication of beta-cells rather than neogenesis from pancreatic ducts.

c-Myc and downstream targets in the pathogenesis and treatment of cancer.

The c-Myc oncoprotein is a master regulator of genes involved in diverse cellular processes. Situated upstream of signalling pathways regulating cellular replication/growth as well as apoptosis/growth arrest, c-Myc may help integrate processes determining cell numbers and tissue size in physiology and disease. In cancer, this 'dual potential' allows c-Myc to act as its own tumour suppressor. Evidently, given that deregulated expression of c-Myc is present in most, if not all, human cancers (Table 1) and is associated with a poor prognosis, by implication these in-built 'failsafe' mechanisms have been overcome. To explore the complex activity of c-Myc and its potential as a therapeutic target 'post-genome era' technologies for determining global gene expression alongside advanced new models for the study of tumourigenesis in vivo have proved invaluable. Thus, many recent studies have provided encouragement for the therapeutic targeting of c-Myc in cancer and have revealed new protein targets for manipulating aspects of c-Myc activity. The remarkable regression of even advanced and genetically unstable tumours, seen following deactivation of c-Myc in various models is particularly exciting. This review will discuss what is known about the role of c-Myc in growth deregulation and cancer and will conclude with a discussion of the most promising recent developments in Myc-targeted therapeutics.

The c-MYC oncoprotein as a treatment target in cancer and other disorders of cell growth.

The c-MYC proto-oncogene is essential for cellular proliferation but, paradoxically, may also promote cell death. Deregulated expression of c-MYC is present in most, if not all, human cancers, and is associated with a poor prognosis. However, given that human tumours at diagnosis generally carry multiple genetic lesions that have accumulated during (although they are not necessarily essential for) tumour progression, it has proved difficult to attribute a specific role to any given single factor or indeed to explore the therapeutic potential of selectively mitigating their biological functions. Regulatable transgenic mouse models of oncogenesis have shed light on these issues, influenced our thinking about cancer and provided encouragement for the future development of cancer therapies based on targeting individual oncogenes such as c-MYC. Although still in its infancy, encouraging results have been reported using antisense oligodeoxynucleotide-based methods, as well as other approaches to interfere with MYC expression both in vitro and in vivo.

The many faces of c-MYC.

The proto-oncogene c-MYC is implicated in various physiological processes-cell growth, proliferation, loss of differentiation, and cell death (apoptosis). Oncogenic c-MYC implies constitutive or deregulated expression of c-MYC and is associated with many human cancers often with poor prognosis. Recently, c-MYC has been implicated in the loss and dysfunction of insulin-producing beta cells in diabetes. Intriguingly, this raises the possibility that c-Myc may be a key contributor to disease, not only by deregulating cell proliferation, which is well established, but also by virtue of its opposing role in engendering apoptosis. However, given the fact that human diseases at diagnosis are generally advanced and pathologically complex, it is generally difficult to attribute a specific pathogenic role to c-MYC, or indeed any given single factor, or to assess the potential of therapies targeting individual such factors. Regulatable transgenic mouse models have shed light on these issues, have influenced our thinking about cancer, and have provided encouragement for the future development of cancer therapies based on targeting individual oncogenes such as c-MYC. Although still in its infancy, encouraging results have been reported for several approaches using gene targeting to interfere with c-MYC expression or activity both in vitro and in vivo.

Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression.

To explore the role of c-Myc in carcinogenesis, we have developed a reversible transgenic model of pancreatic beta cell oncogenesis using a switchable form of the c-Myc protein. Activation of c-Myc in adult, mature beta cells induces uniform beta cell proliferation but is accompanied by overwhelming apoptosis that rapidly erodes beta cell mass. Thus, the oncogenic potential of c-Myc in beta cells is masked by apoptosis. Upon suppression of c-Myc-induced beta cell apoptosis by coexpression of Bcl-x(L), c-Myc triggers rapid and uniform progression into angiogenic, invasive tumors. Subsequent c-Myc deactivation induces rapid regression associated with vascular degeneration and beta cell apoptosis. Our data indicate that highly complex neoplastic lesions can be both induced and maintained in vivo by a simple combination of two interlocking molecular lesions.