The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells.

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM, a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells, GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However, the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature, which defines aggressiveness and invasion, and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1, which encode a key regulator of selective autophagy, p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs, DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation, thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast, autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally, DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism, in particular reduced ATP and lactate levels. Taken together, these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells.

Role of the promyelocytic leukaemia protein in cell death regulation.

The promyelocytic leukaemia gene PML was originally identified at the t(15;17) translocation of acute promyelocytic leukaemia, which generates the oncogene PML-retinoic acid receptor α. PML epitomises a subnuclear structure called PML nuclear body. Current models propose that PML through its scaffold properties is able to control cell growth and survival at many different levels. Here we discuss the current literature and propose new avenues for investigation.

The role of autophagy in clinical practice.

Resisting cell death is one of the six hallmarks of cancer. Autophagy is a highly adaptable metabolic process that plays an important role in stressful conditions, such as nutrient deprivation and hypoxia. In these conditions, it is becoming evident that autophagy protects cells, by providing an alternative energy source and by eliminating dysfunctional organelles or proteins. In tumourigenesis, autophagy plays a dual role, which may be related to the different stages in cancer development. The autophagy-mediated removal of damaged proteins and organelles may prevent cancer initiation by limiting tissue inflammation. In contrast, autophagy has been shown to allow established tumours to survive in nutrient-deprived or hypoxic conditions during cancer progression. Key regulators of the autophagy pathway are modulated or aberrantly expressed in cancer and modulating autophagy is an attractive concept for cancer therapy. The difficulties, however, lie in the complexity of the crosstalk between apoptosis and autophagy and the lack of robust tissue biomarkers and in vivo assessment of autophagic flux. Currently there are 19 clinical trials in both solid and haematogenous cancers investigating the efficacy and toxicity of adding an autophagy inhibitor to standard treatment. Hydroxychloroquine, a drug routinely used in the treatment of malaria and autoimmune disorders, is the most common autophagy inhibitor under investigation due to its more favourable toxicity profile. This overview summarises the role of autophagy in cancer initiation, progression and resistance to treatment and thereby the therapeutic benefit that may be gained by modulating its effects.

PML involvement in the p73-mediated E1A-induced suppression of EGFR and induction of apoptosis in head and neck cancers.

Epidermal growth factor receptor (EGFR) tyrosine kinase is commonly overexpressed in human cancers; however, the cellular mechanisms regulating EGFR expression remain unclear. p53, p63 and p73 are transcription factors regulating many cellular targets involved in controlling the cell cycle and apoptosis. p53 activates EGFR expression, whereas TAp63 represses EGFR transcription. The involvement of p73 in the regulation of EGFR has not been reported. Here, a strong correlation between EGFR overexpression and increased levels of the oncogenic DeltaNp73 isoform in head and neck squamous cell carcinoma (HNSCC) cell lines was observed. Ectopic expression of TAp73, particularly TAp73beta, resulted in suppression of the EGFR promoter, significant downregulation of EGFR protein and efficient induction of cell death in all six EGFR-overexpressing HNSCC cell lines. EGFR overexpression from a heterologous LTR promoter protected lung cancer cells from TAp73beta-induced EGFR suppression and apoptosis. Expression of TAp73beta efficiently induced promyelocytic leukaemia (PML) protein expression and PML knockdown by shRNA attenuated the downregulation of EGFR and induction of apoptosis by p73 in HNSCC cells. Furthermore, PML was found to be important for E1A-induced suppression of EGFR and subsequent killing of HNSCC cells. Our data therefore suggest a novel pathway involving PML and p73 in the regulation of EGFR expression.

Stemming out of a new PML era?

The promyelocytic leukaemia protein PML is a growth and tumour suppressor inactivated in acute promyelocytic leukaemia (APL). Recent evidence indicates that PML plays a tumour-suppressive role in cancer of multiple histological origins. However, it is only very recently that PML growth-suppressive functions have been implicated in regulating physiological processes and tissue homoeostasis. In particular, it has been shown that PML is one of the key cell-cycle regulators controlling stem cell function in multiple tissues, from the blood to the brain. As a consequence, PML loss has an impact on tissue development and maintenance of stem cell pools. In addition, new data suggest that PML regulates self-renewal in cancer stem cells. Finally, the oncogenic fusion protein PML/RARalpha, contrary to the conventional view, appears to hijack growth-suppressive pathways to promote transformation of haematopoietic stem cells and to maintain the APL stem cell niche. Overall, these findings not only represent a change in paradigm in the field of PML/APL research, but also contribute to the understanding of fundamental mechanisms underlying stem cell function in vivo. The main objective of this review is to critically discuss the very recent literature on the role of PML in stem cells and tumour-initiating cells. Ultimately, it aims to propose new avenues of investigation.

New insights into the role of PML in tumour suppression.

The PML gene is involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), which generates the oncogenic fusion protein PML (promyelocytic leukaemia protein)-retinoic acid receptor alpha. The PML protein localises to a subnuclear structure called the PML nuclear domain (PML-ND), of which PML is the essential structural component. In APL, PML-NDs are disrupted, thus implicating these structures in the pathogenesis of this leukaemia. Unexpectedly, recent studies indicate that PML and the PML-ND play a tumour suppressive role in several different types of human neoplasms in addition to APL. Because of PML's extreme versatility and involvement in multiple cellular pathways, understanding the mechanisms underlying its function, and therefore role in tumour suppression, has been a challenging task. In this review, we attempt to critically appraise the more recent advances in this field and propose new avenues of investigation.

New insights into the cytoplasmic function of PML.

PML is a tumour suppressor inactivated in Acute Promyelocytic Leukaemia (APL). PML is the essential component of a subnuclear structure called the PML nuclear body (PML-NB), which is disrupted in APL. By targeting different cellular proteins to this structure, PML can either hamper or potentiate their functions. The PML transcript undergoes alternative splicing to generate both nuclear and cytoplasmic isoforms. Most of the research in this field has focused its attention on studying nuclear PML. Nevertheless, new exciting studies show that cytoplasmic PML may control essential cellular functions, thus opening new avenues for investigation.

Daxx is required for stress-induced cell death and JNK activation.

Daxx has been implicated in the modulation of apoptosis in response to various stimuli. In the nucleus, Daxx interacts and colocalizes with the promyelocytic leukemia protein (PML) into the PML-nuclear body. Moreover, overexpressed Daxx positively modulates FAS-ligand and TGFbeta-induced apoptosis. However, recent reports indicate that Daxx can also act as an antiapoptotic factor. As most studies on the role of Daxx in cell death have been conducted using tumour cell lines, we analysed the function of Daxx in physiological settings. We found that Daxx is induced upon exposure to ultraviolet (UV) irradiation and hydrogen peroxide treatment. We employed RNA interference to downregulate Daxx in primary fibroblasts. Remarkably, Daxx-depleted cells are resistant to cell death induced by both UV irradiation and oxidative stress. Furthermore, the downregulation of Daxx results in impaired MKK/c-Jun-N-terminal kinase (JNK) activation. This is the first evidence that Daxx promotes cell death and JNK activation in physiological conditions.

Caspase cleavage enhances the apoptosis-inducing effects of BAD.

The function of BAD, a proapoptotic member of the Bcl-2 family, is regulated primarily by rapid changes in phosphorylation that modulate its protein-protein interactions and subcellular localization. We show here that, during interleukin-3 (IL-3) deprivation-induced apoptosis of 32Dcl3 murine myeloid precursor cells, BAD is cleaved by a caspase(s) at its N terminus to generate a 15-kDa truncated protein. The 15-kDa truncated BAD is a more potent inducer of apoptosis than the wild-type protein, whereas a mutant BAD resistant to caspase 3 cleavage is a weak apoptosis inducer. Truncated BAD is detectable only in the mitochondrial fraction, interacts with BCL-X(L) at least as effectively as the wild-type protein, and is more potent than wild-type BAD in inducing cytochrome c release. Human BAD, which is 43 amino acids shorter than its mouse counterpart, is also cleaved by a caspase(s) upon exposure of Jurkat T cells to anti-FAS antibody, tumor necrosis factor alpha (TNF-alpha), or TRAIL. Moreover, a truncated form of human BAD lacking the N-terminal 28 amino acids is more potent than wild-type BAD in inducing apoptosis. The generation of truncated BAD was blocked by Bcl-2 in IL-3-deprived 32Dcl3 cells but not in Jurkat T cells exposed to anti-FAS antibody, TNF-alpha, or TRAIL. Together, these findings point to a novel and important role for BAD in maintaining the apoptotic phenotype in response to various apoptosis inducers.

Cytokeratin immunoreactivity in mouse tissues: study of different antibodies with a new detection system.

The cross-reactivity of a group of monoclonal antibodies (MABs) generated against human cytokeratins (CKs) was investigated in mouse tissues. Formalin-fixed and paraffin-embedded sections of lung, stomach, small and large intestine, liver, and kidney were immunostained with MABs after epitope retrieval with enzyme digestion. AE1/AE3, a "cocktail" of two MABs that recognizes basic and acidic CKs, 5D3 MAB to low molecular weight CKs (8, 18, and 19), and monospecific MABs to CK 7 and 20 were tested. Additionally, CK 17 and 34betaE12 MABs to high molecular weight CKs were evaluated in the same organs and in sections from skin and preputial glands. We employed the new universal animal system (ARK) as the detection system. The results showed intense reactivity for the first group of antibodies used, with topographic distribution similar to that in human tissues, with the exception of CK 7 in lung parenchyma, which displayed reactivity only in type II pneumocytes, with negativity of adjacent bronchial epithelium. Also of note was the lack of reaction of liver hepatocytes and renal tubular cells to AE1/AE3 and 5D3 MABs. Regarding the second group of antibodies, no reaction was obtained for CK 17 in the tissues tested. On the contrary, 34betaE12 MAB yielded intense reactivity in cells of epidermis and hair follicles. Compared to other detection systems used previously in this animal, ARK produced a well-defined reactivity at the cellular level without any background. We conclude that a useful panel of anti-CK antibodies commonly used in human pathology can be applied successfully to mouse tissues after enzyme digestion, leading to a more accurate definition of cellular populations in this laboratory animal.

Bcl-2 expression restores the leukemogenic potential of a BCR/ABL mutant defective in transformation.

Growth factor-dependent hematopoietic cell lines expressing the BCR/ABL oncoprotein of the Ph chromosome show growth factor-independent proliferation and resistance to apoptosis. Apoptosis resistance of BCR/ABL-expressing cells may depend on enhanced expression of anti-apoptotic proteins as well as reduced expression and/or inactivation of pro-apoptotic proteins. Compared to myeloid precursor 32Dcl3 cells expressing wild type BCR/ABL, cells expressing a BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185 delta BCR) are susceptible to apoptosis induced by interleukin-3 (IL-3) deprivation. These cells exhibited the hypophosphorylated apoptotic BAD and markedly reduced levels of Bcl-2. Upon ectopic expression of Bcl-2, these cells showed no changes in BAD phosphorylation, but they became apoptosis-resistant and proliferated in the absence of IL-3, albeit more slowly than cells expressing wild type BCR/ABL. Moreover, the p185 delta BCR/Bcl-2 double transfectants were leukemogenic when injected into immunodeficient mice, but Bcl-2 expression did not restore the leukemia-inducing effects of p185 delta BCR to the levels of wild type BCR/ABL. Leukemic cells recovered from the spleen of mice injected with p185 delta BCR/Bcl-2 cells did not show rearrangements in the Bcl-2 genomic locus, but they exhibited enhanced proliferation in culture and induced a rapidly fatal disease process when inoculated in secondary recipient mice. Together, these data support the importance of anti-apoptotic pathways for BCR/ABL-dependent leukemogenesis and suggest that Bcl-2 expression promotes secondary changes leading to a more aggressive tumor phenotype. (Blood. 2000;96:3915-3921)

The function of PML in p53-dependent apoptosis.

The PML gene of acute promyelocytic leukaemia (APL) encodes a growth- and tumour-suppresor protein that is essential for several apoptotic signals. The mechanisms by which PML exerts its pro-apoptotic function are still unknown. Here we show that PML acts as a transcriptional co-activator with p53. PML physically interacts with p53 both in vitro and in vivo and co-localizes with p53 in the PML nuclear body (PML-NB). The co-activatory role of PML depends on its ability to localize in the PML-NB. p53-dependent, DNA-damage-induced apoptosis, transcriptional activation by p53, the DNA-binding ability of p53, and the induction of p53 target genes such as Bax and p21 upon gamma-irradiation are all impaired in PML-/- primary cells. These results define a new PML-dependent, p53-regulatory pathway for apoptosis and shed new light on the function of PML in tumour suppression.

Versatility of BCR/ABL-expressing leukemic cells in circumventing proapoptotic BAD effects.

BAD, the proapoptotic member of the "BH3-only" subfamily of BCL-2 proteins, is inactivated by phosphorylation at serines 112 and 136 and by sequestration in the cytoplasm where it interacts with members of the 14-3-3 family. In BCR/ABL-expressing cells, BAD is constitutively phosphorylated and mainly cytoplasmic, whereas in cells expressing BCR/ABL mutants unable to protect from apoptosis, BAD is nonphosphorylated. We show here that both the wild-type (WT) and the S112A/ S136A double mutant (DM) BAD are more potent inducers of apoptosis in parental than in BCR/ABL-expressing 32D myeloid precursor cells. Stable lines of parental cells expressing DM BAD could not be established and most clones from WT BAD retrovirus-infected parental cells lost BAD expression. On IL-3 withdrawal from parental 32D cells, BAD was rapidly dephosphorylated by the serine-threonine phosphatase 1 alpha, and localized in the mitochondria, whereas it remained phosphorylated and did not localize to the mitochondria in the cohort of BCR/ABL-expressing cells escaping apoptosis induced by WT BAD. Moreover, these cells showed high levels of BCL-2 and BCL-X(L) expression. The cohort of BCR/ABL-expressing cells resistant to apoptosis induced by DM BAD showed only high levels of BCL-2 and BCL-X(L). These findings suggest that BCR/ABL-expressing cells are more versatile than normal hematopoietic progenitors in counteracting the apoptotic potential of BAD, and raise the possibility that tumor cells activate multiple antiapoptotic pathways for survival in the face of death-inducing stimuli. (Blood. 2000;96:676-684)

The transcriptional role of PML and the nuclear body.

The PML gene encodes a tumour suppressor protein associated with a distinct subnuclear domain, the nuclear body. Various functions have been attributed to the PML nuclear body, but its main biochemical role is still unclear. Recent findings indicate that PML is essential for the proper formation of the nuclear body and can act as a transcriptional co-factor. Here we summarize the current understanding of the biological functions of PML and the nuclear body, and discuss a role for these intra-nuclear structures in the regulation of transcription.