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1.  Immunohistochemical detection of cytoplasmic LC3 puncta in human cancer specimens 
Autophagy  2012;8(8):1175-1184.
Autophagy is an evolutionarily conserved catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Alterations in autophagic signaling are found in several human diseases including cancer. Here, we describe a validated immunohistochemical protocol for the detection of LC3 puncta in human formalin-fixed, paraffin-embedded cancer specimens that can also be applied to mouse tissues. In response to systemic chemotherapy, autophagy-competent mouse tumors exhibited LC3 puncta, which did not appear in mouse cancers that had been rendered autophagy-deficient by the knockdown of Atg5 or Atg7. As compared with normal tissues, LC3 staining was moderately to highly elevated in the large majority of human cancers studied, albeit tumors of the same histological type tended to be highly heterogeneous in the number and intensity of LC3 puncta per cell. Moreover, tumor-infiltrating immune cells often were highly positive for LC3. Altogether, this protocol for LC3 staining appears suitable for the specific detection of LC3 puncta in human specimens, including tissue microarrays. We surmise that this technique can be employed for retrospective or prospective studies involving large series of human tumor samples.
PMCID: PMC3973657  PMID: 22647537
autophagosomes; CT26; immunohistochemistry; lysosomes; macroautophagy; MCA205
2.  Why Sick Cells Produce Tumors 
Autophagy  2007;3(5):502-505.
Cells exploit autophagy for survival to metabolic stress in vitro as well as in tumors where it localizes to regions of metabolic stress suggesting its role as a survival pathway. Consistent with this survival function, deficiency in autophagy impairs cell survival, but also promotes tumor growth, creating a paradox that the loss of a survival pathway leads to tumorigenesis. There is evidence that autophagy is a homeostatic process functioning to limit the accumulation of poly-ubiquitinated proteins and mutant protein aggregates associated with neuronal degeneration.2,3 Interestingly, we found that deficiency in autophagy caused by monoallelic loss of beclin1 or deletion of atg5 leads to accelerated DNA damage and chromosomal instability demonstrating a mutator phenotype.4 These cells also exhibit enhanced chromosomal gains or losses suggesting that autophagy functions as a tumor suppressor by limiting chromosomal instability. Thus the impairment of survival to metabolic stress due to deficiency in autophagy may be compensated by an enhanced mutation rate thereby promoting tumorigenesis. The protective role of autophagy may be exploited in developing novel autophagy modulators as rational chemotherapeutic as well as chemopreventive agents.
PMCID: PMC2866178  PMID: 17611387
autophagy; metabolism; beclin1; atg5; DNA damage; chromosomal instability; cancer
3.  Role of Autophagy in Breast Cancer 
Autophagy  2007;3(6):610-613.
Autophagy is an evolutionarily conserved process of cytoplasm and cellular organelle degradation in lysosomes. Autophagy is a survival pathway required for cellular viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. In neurons, constitutive autophagy limits accumulation of polyubiquitinated proteins and prevents neuronal degeneration. Therefore, autophagy has emerged as a homeostatic mechanism regulating the turnover of long-lived or damaged proteins and organelles, and buffering metabolic stress under conditions of nutrient deprivation by recycling intracellular constituents. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin1+/− mice are tumor-prone. We found that allelic loss of beclin1 renders immortalized mouse mammary epithelial cells susceptible to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. Thus, loss of the prosurvival role of autophagy likely contributes to breast cancer progression by promoting genome damage and instability. Exploring the yet unknown relationship between defective autophagy and other breast cancer-promoting functions may provide valuable insight into the pathogenesis of breast cancer and may have significant prognostic and therapeutic implications for breast cancer patients.
PMCID: PMC2859167  PMID: 17786023
autophagy; breast cancer; beclin1; DNA damage; genomic instability
4.  Tumor suppression by autophagy through the management of metabolic stress 
Autophagy  2008;4(5):563-566.
Autophagy plays a critical protective role maintaining energy homeostasis and protein and organelle quality control. These functions are particularly important in times of metabolic stress and in cells with high energy demand such as cancer cells. In emerging cancer cells, autophagy defect may cause failure of energy homeostasis and protein and organelle quality control, leading to the accumulation of cellular damage in metabolic stress. Some manifestations of this damage, such as activation of the DNA damage response and generation of genome instability may promote tumor initiation and drive cell-autonomous tumor progression. In addition, in solid tumors, autophagy localizes to regions that are metabolically stressed. Defects in autophagy impair the survival of tumor cells in these areas, which is associated with increased cell death and inflammation. The cytokine response from inflammation may promote tumor growth and accelerate cell non-autonomous tumor progression. The overreaching theme is that autophagy protects cells from damage accumulation under conditions of metabolic stress allowing efficient tolerance and recovery from stress, and that this is a critical and novel tumor suppression mechanism. The challenge now is to define the precise aspects of autophagy, including energy homeostasis and protein and organelle turnover, that are required for the proper management of metabolic stress that suppress tumorigenesis. Furthermore, we need to be able to identify human tumors with deficient autophagy, and to develop rational cancer therapies that take advantage of the altered metabolic state and stress responses inherent to this autophagy defect.
PMCID: PMC2857579  PMID: 18326941
autophagy; beclin1; cancer
5.  Role of Autophagy in Cancer 
Autophagy  2007;3(1):28-31.
Human breast, ovarian, and prostate tumors display allelic loss of the essential autophagy gene beclin1 with high frequency, and an increase in the incidence of tumor formation is observed in beclin1+/− mutant mice. These findings suggest a role for beclin1 and autophagy in tumor suppression; however, the mechanism by which this occurs has been unclear. Autophagy is a bulk degradation process whereby organelles and cytoplasm are engulfed and targeted to lysosomes for proteolysis,1,2 There is evidence that autophagy sustains cell survival during nutrient deprivation through catabolism, but also that autophagy is a means of achieving cell death when executed to completion. If or how either of these diametrically opposing functions proposed for autophagy may be related to tumor suppression is unknown. We found that metabolic stress is a potent trigger of apoptotic cell death, defects in which enable long-term survival that is dependent on autophagy both in vitro and in tumors in vivo.3 These findings raise the conundrum whereby inactivation of a survival pathway (autophagy) promotes tumorigenesis. Interestingly, when cells with defects in apoptosis are denied autophagy, this creates the inability to tolerate metabolic stress, reduces cellular fitness, and activates a necrotic pathway to cell death. This necrosis in tumors is associated with inflammation and enhancement of tumor growth, due to the survival of a small population of surviving, but injured, cells in a microenvironment that favors oncogenesis. Thus, by sustaining metabolism through autophagy during periods of metabolic stress, cells can limit energy depletion, cellular damage, and cell death by necrosis, which may explain how autophagy can prevent cancer, and how loss of a survival function can be tumorigenic.
PMCID: PMC2770734  PMID: 16969128
autophagy; apoptosis; necrosis; Beclin1; cancer
6.  Autophagic cell death unraveled: Pharmacological inhibition of apoptosis and autophagy enables necrosis 
Autophagy  2008;4(4):399-401.
Apoptosis is a well-characterized pathway to cell death, yet how it is related to other forms of cell death such as necrosis, and possibly also autophagic cell death has not been entirely clear. Difficulties arise because necrotic cell death is poorly characterized at the molecular level, and also because autophagy is primarily a survival pathway that has been associated with cell death induction in some circumstances. A common theme appears to be now emerging where autophagy promotes survival of apoptosis-defective cells, and inhibition of the autophagy survival function in this setting represents a means to divert cells into a necrotic cell fate. In cells denied the ability to commit suicide by apoptosis, and that are also unable to access the autophagy survival mechanism to sustain homeostasis, necrosis is the default activity. This was most recently illustrated with the discovery that the caspase and apoptosis inhibitor, zVAD, also inhibits a lysosomal protease, and thereby autophagy, and it is this dual inhibition that is responsible for induction of necrotic cell death.1 This radically alters the interpretation of earlier findings reporting induction of autophagic cell death by zVAD,2 instead, suggests that autophagy functions to promote cell survival.
PMCID: PMC2696931  PMID: 18367872
autophagy; autophagic cell death; apoptosis; necrosis; mTOR; zVAD; Cathepsin B

Results 1-6 (6)