Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.

The therapeutic potential of autophagy for the treatment cancer and other diseases is beset by paradoxes stemming from the complexity of the interactions between the apoptotic and autophagic machinery. The simplest question of how autophagy acts as both a protector and executioner of cell death remains the subject of substantial controversy. Elucidating the molecular interactions between the processes will help us understand how autophagy can modulate cell death, whether autophagy is truly a cell death mechanism and how these functions are regulated. We suggest that despite many connections between autophagy and apoptosis, a strong causal relationship wherein one process controls the other, has not been adequately demonstrated. Knowing when and how to modulate autophagy therapeutically depends on understanding these connections.

Autophagy is a catabolic process that turns over long-lived proteins and organelles and contributes to cell and organism survival in times of stress. Current cancer therapies including chemotherapy and radiation are known to induce autophagy within tumor cells. This is therefore an attractive process to target during cancer therapy as there are safe, clinically available drugs known to both inhibit and stimulate autophagy. However, there are conflicting positive and negative effects of autophagy and no current consensus on how to manipulate autophagy to improve clinical outcomes. Careful and rigorous evaluation of autophagy with a focus on how to translate laboratory findings into relevant clinical therapies remains an important aspect of improving clinical outcomes in patients with malignant disease.

The study of autophagy is rapidly expanding, and our knowledge of the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. The vocabulary associated with autophagy has grown concomitantly. In fact, it is difficult for readers—even those who work in the field—to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors and chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, and the roles of accessory components and structures that are associated with autophagy.

Vitamin D3 is a promising preventative and therapeutic agent for prostate cancer, but its implementation is hampered by a lack of understanding about its mechanism of action. Upon treatment with 1α,25 dihydroxyvitamin D3 (vitamin D3), the metabolically active form of vitamin D3, adult prostate progenitor/stem cells (PrP/SC) undergo cell-cycle arrest, senescence, and differentiation to an androgen receptor-positive luminal epithelial cell fate. Microarray analyses of control- and vitamin D3-treated PrP/SC revealed global gene expression signatures consistent with induction of differentiation. Interestingly, one of the most highly-upregulated genes by vitamin D3 was the pro-inflammatory cytokine interleukin-1 alpha (IL1α). Systems biology analyses supported a central role for IL1α in the vitamin D3 response in PrP/SC. siRNA-mediated knockdown of IL1α abrogated vitamin D3-induced growth suppression, establishing a requirement for IL1α in the anti-proliferative effects of vitamin D3 in PrP/SC. These studies establish a system to study the molecular profile of PrP/SC differentiation, proliferation, and senescence, and they point to an important new role for IL1α in vitamin D3 signaling in prostate progenitor/stem cells.

Autophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. It contributes to energy and organelle homeostasis and the preservation of proteome and genome integrity. Although a role in cancer is unquestionable, there are conflicting reports that autophagy can be both oncogenic and tumor suppressive, perhaps indicating that autophagy has different roles at different stages of tumor development. In this report, we address the role of autophagy in a critical stage of cancer progression—tumor cell invasion. Using a glioma cell line containing an inducible shRNA that targets the essential autophagy gene Atg12, we show that autophagy inhibition does not affect cell viability, proliferation or migration but significantly reduces cellular invasion in a 3D organotypic model. These data indicate that autophagy may play a critical role in the benign to malignant transition that is also central to the initiation of metastasis.

One impediment to the use of TRAIL receptor targeted agents as anti-tumor drugs is the evolution of resistance, a common problem in cancer. On the other hand, many different kinds of drugs synergize with TRAIL in TRAIL-sensitive tumor cells, raising the question whether one can overcome resistance with the same drugs producing synergy. This is an important question, because recent clinical trials suggest that combination treatments with cytotoxic drugs and TRAIL receptor-targeted agents do not provide additional benefit compared with cytotoxic agents on their own. Such results might be expected if drug combinations that synergize in sensitive tumor cells but cannot overcome TRAIL resistance are used in patients whose tumors were not selected for retention of TRAIL sensitivity. We tested this idea by creating isogenic tumor cells with acquired TRAIL resistance or defined mechanisms of resistance that occur in human tumors then compared them to the TRAIL-sensitive parental cell line. Although diverse classes of anti-cancer drug were all able to synergize with TRAIL in sensitive cells, most agents were unable to overcome resistance and there was no relationship between the amount of synergy seen with a particular agent and its ability to overcome acquired resistance. An important exception was proteasome inhibitors, which were however able to overcome diverse resistance mechanisms. Our findings suggest that one should select drugs for TRAIL receptor agonist combination therapy based not just on their ability to synergize but rather on their ability to both overcome resistance as well as synergize.

Although autophagy has been shown to have a clear role as a tumor suppressor mechanism, its role in cancer treatment is still controversial. Because autophagy is a survival pathway activated during nutrient deprivation and other stresses, it is reasonable to think that autophagy can function as a tumor cell survival mechanism activated after cancer treatment. Such a mechanism could be widely important because most cancer treatments induce autophagy in tumor cells. Indeed, many papers have presented data suggesting that tumor cell autophagy induced by anticancer treatment inhibits tumor cell killing. However, it has also been proposed that autophagy is a cell death mechanism that could function as a backup when apoptosis is disabled. The fact that there are active clinical trials in patients both using autophagy inhibitors or inducers together with other cancer treatments underscores the importance of understanding and distinguishing between these opposing ideas. Here we discuss some of the recent work studying the role of autophagy with different cancer therapies.

Summary

In cancer treatment, apoptosis is a well-recognized cell death mechanism through which cytotoxic agents kill tumor cells. Here we report that dying tumor cells use the apoptotic process to generate potent growth-stimulating signals to stimulate the repopulation of tumors undergoing radiotherapy. Surprisingly, activated caspase 3, a key executioner of apoptosis, plays key roles in the growth stimulation. One downstream effector that caspase 3 regulates is prostaglandin E2, which can potently stimulates growth of surviving tumor cells. Deficiency of caspase 3 either in tumor cells or in tumor stroma caused significant tumor sensitivity to radiotherapy in xenograft or mouse tumors. In human cancer patients, higher levels of activated caspase 3 in tumor tissues are correlated with significantly increased rate of recurrence and deaths. We propose the existence of a “Phoenix Rising” pathway of cell death-induced tumor repopulation in which caspase 3 plays key roles.

Purpose

There is growing evidence implicating the importance of the insulin-like growth factor (IGF) pathway in colorectal cancer (CRC) based upon the results of population studies, and preclinical experiments. However, the combination of an IGF-1 receptor (IGF-1R) inhibitor with standard CRC chemotherapies has not yet been evaluated. In this study, we investigated the interaction between the dual IGF-1R/IR tyrosine kinase inhibitor (TKI), PQIP, and standard chemotherapies in CRC cell line models.

Experimental Design

The antiproliferative effects of PQIP, as a single agent and in combination with 5-fluorouracil, oxaliplatin, or SN38 were analyzed against four CRC cell lines. Downstream effector proteins, apoptosis and cell cycle were also assessed in the combination of PQIP and SN-38. Lastly, the efficacy of OSI-906 (a derivative of PQIP) combined with irinotecan was further tested using a human CRC xenograft model.

Results

Treatment with the combination of PQIP and each of three chemotherapies resulted in an enhanced decrease in proliferation of all four colorectal cancer cell lines compared to single agent treatment. This inhibition was not associated with a significant induction of apoptosis, but was accompanied by cell cycle arrest and changes in phosphorylation of Akt. Interestingly, antitumor activity between PQIP and SN-38 in vitro was also reflected in the human CRC xenograft model.

Conclusions

Combination treatment with the dual IGF-1R/IR TKI, PQIP, and standard CRC chemotherapy resulted in enhanced antiproliferative effects against CRC cell line models providing a scientific rationale for the testing of OSI-906 and standard CRC treatment regimens.

Introduction

Despite modern targeted therapy, metastatic renal cell carcinoma (RCC) remains a deadly disease. Interferon alpha (IFNα) is currently used to treat this condition, mainly in combination with the targeted anti-VEGF antibody, bevacizumab. Apo2 ligand/Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a novel anti-neoplastic agent now in early phase clinical trials. IFNα and TRAIL can act synergistically to kill cancer cells, but this has never been tested in the context of RCC. We hypothesized that TRAIL and IFNα can synergistically induce apoptosis in RCC cells.

Methods

RCC cell lines were treated with recombinant TRAIL and/or IFNα. Viability and apoptosis were assessed by MTS assay, flow cytometery, and western blot. Synergy was confirmed by isobologram analysis. IFNα induced changes in signaling by RCC cells were assessed by western blot, flow cytometry, and ELISA.

Results

TRAIL and IFNα act synergistically to increase apoptotic cell death in RCC cells. Treatment with IFNα alters these cells ability to activate ERK and inhibiting ERK with UO126 abrogates the apoptotic synergy between TRAIL and IFNα. IFNα does not induce changes in TRAIL or death receptor expression, nor does it change other known mediators of the intrinsic and extrinsic apoptotic cascade in RCC cells.

Conclusion

TRAIL plus IFNα synergistically induces apoptosis in RCC cells. The mechanism is due at least in part to IFNα mediated changes in ERK activation. Combination therapy with TRAIL and IFNα may be a novel approach to systemically treat advanced RCC and warrants further testing in vivo.

Astrocytomas account for the majority of malignant brain tumors diagnosed in both adult and pediatric patients. The therapies available to treat these neoplasms are limited, and the prognosis associated with high-grade lesions is extremely poor. Mer (MerTK) and Axl receptor tyrosine kinases (RTK) are expressed at abnormally high levels in a variety of malignancies, and these receptors are known to activate strong antiapoptotic signaling pathways that promote oncogenesis. In this study, we found that Mer and Axl mRNA transcript and protein expression were elevated in astrocytic patient samples and cell lines. shRNA-mediated knockdown of Mer and Axl RTK expression led to an increase in apoptosis in astrocytoma cells. Apoptotic signaling pathways including Akt and extracellular signal–regulated kinase 1/2, which have been shown to be activated in resistant astrocytomas, were downregulated with Mer and Axl inhibition whereas poly(ADP-ribose) poly-merase cleavage was increased. Furthermore, Mer and Axl shRNA knockdown led to a profound decrease of astrocytoma cell proliferation in soft agar and a significant increase in chemosensitivity in response to temozolomide, carboplatin, and vincristine treatment. Our results suggest Mer and Axl RTK inhibition as a novel method to improve apoptotic response and chemosensitivity in astrocytoma and provide support for these oncogenes as attractive biological targets for astrocytoma drug development.

In order to metastasize, tumor cells must adapt to untoward, stressful microenvironments as they disseminate into the systemic circulation and colonize distant organ sites. Autophagy, a tightly regulated lysosomal self-digestion process that is upregulated during cellular stress, has been demonstrated to suppress primary tumor formation, but how autophagy influences metastasis remains unknown. Autophagy may inhibit metastasis by promoting anti-tumor inflammatory responses or by restricting the expansion of dormant tumor cells into macrometastases. Conversely, self-eating may promote metastasis by enhancing tumor cell fitness in response to environmental stresses, such as anoikis, during metastatic progression. Because autophagy is titratable, it may serve both pro- and anti-metastatic functions depending on the contextual demands placed on tumor cells throughout the metastatic process.

Background

Signaling by Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) and Fas ligand (FasL) has been proposed to contribute to the chemosensitivity of tumor cells treated with various other anti-cancer agents. However, the importance of these effects and whether there are differences in vitro and in vivo is unclear.

Methodology/Principal Findings

To assess the relative contribution of death receptor pathways to this sensitivity and to determine whether these effects are intrinsic to the tumor cells, we compared the chemosensitivity of isogenic BJAB human lymphoma cells where Fas and TRAIL receptors or just TRAIL receptors were inhibited using mutants of the adaptor protein FADD or by altering the expression of the homeobox transcription factor Six1. Inhibition of TRAIL receptors did not affect in vitro tumor cell killing by various anti-cancer agents indicating that chemosensitivity is not significantly affected by the tumor cell-intrinsic activation of death receptor signaling. However, selective inhibition of TRAIL receptor signaling caused reduced tumor regression and clearance in vivo when tested in a NOD/SCID mouse model.

Conclusions

These data show that TRAIL receptor signaling in tumor cells can determine chemosensitivity in vivo but not in vitro and thus imply that TRAIL resistance makes tumors less susceptible to conventional cytotoxic anti-cancer drugs as well as drugs that directly target the TRAIL receptors.

The characteristics of tumor cell killing by an anti-cancer agent can determine the long-term effectiveness of the treatment. For example, if dying tumor cells release the immune modulator HMGB1 after treatment with anti-cancer drugs, they can activate a tumor-specific immune response that boosts the effectiveness of the initial treatment. Recent work from our group examined the mechanism of action of a targeted toxin called DT-EGF that selectively kills Epidermal Growth Factor Receptor-expressing tumor cells. We found that DT-EGF kills glioblastoma cells by a caspase-independent mechanism that involves high levels of autophagy, which inhibits cell death by blocking apoptosis. In contrast, DT-EGF kills epithelial tumor cells by caspase-dependent apoptosis and in these cells autophagy is not induced. These differences allowed us to discover that the different death mechanisms were associated with differences in the release of HMGB1 and that autophagy induction is required and sufficient to cause release of HMGB1 from the dying cells. These data identify a new function for autophagy during cell death and open up the possibility of manipulating autophagy during cancer treatment as a way to influence the immunogenicity of dying tumor cells.

Macroautophagy (hereafter referred to as autophagy) can increase or decrease the amount of cell death in response to various stimuli. To test if autophagy also controls the characteristics associated with dying cells, we studied tumor cell killing by Epidermal Growth Factor Receptor (EGFR)-targeted diphtheria toxin (DT-EGF). DT-EGF kills epithelial and glioblastoma tumor cells with similar efficiency but by different mechanisms that depend on whether the cells activate autophagy when treated with the drug. Dying cells in which autophagy is induced selectively release the immune modulator HMGB1 without causing lysis of the cell membrane and classical necrosis. Conversely, cells that are killed by DT-EGF where autophagy is blocked, activate caspases but retain HMGB1. These data suggest that it may be feasible to manipulate the immunogenicity of dying cells by increasing or decreasing autophagy.

Depending on the circumstances, autophagy can prevent, cause or regulate the kinetics of cell death. Consequently, it is particularly important that experimental strategies to study these responses are designed carefully. Here, I discuss some of the issues to be considered when designing such experiments.

Tumor Necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) receptors are attractive therapeutic targets in cancer because agents that activate these receptors directly induce tumor cell apoptosis and have low toxicity to normal tissues. Consequently, several different drugs that target these receptors (recombinant TRAIL and various agonistic antibodies that activate one of the two TRAIL receptors) have been developed and are being tested in human clinical trials. However, in vitro and in vivo data suggest that resistance to these agents may limit their clinical effectiveness. In this review, we discuss recent findings about some of the ways these resistance mechanisms arise, potential biomarkers to identify TRAIL resistance in patients (Six1, GALNT14, XIAP, certain microRNAs) and potential ways to circumvent resistance and resensitize tumors.

Apoptosis and autophagy are genetically-regulated, evolutionarily-conserved processes that regulate cell fate. Both apoptosis and autophagy are important in development and normal physiology and in a wide range of diseases. Recent studies show that despite the marked differences between these two processes, their regulation is intimately connected and the same regulators can sometimes control both apoptosis and autophagy. In this review, I discuss some of these findings, which provide possible molecular mechanisms for crosstalk between apoptosis and autophagy and suggest that it may be useful to think of these processes as different facets of the same cell death continuum rather than completely separate processes.

Background

Acute myelogenous leukemia (AML) is the second most common leukemia with approximately 13,410 new cases and 8,990 deaths annually in the United States. A novel fusion toxin treatment, diphtheria toxin GM-CSF (DT-GMCSF) has been shown to selectively eliminate leukemic repopulating cells that are critical for the formation of AML. We previously showed that DT-GMCSF treatment of U937 cells, an AML cell line, causes activation of caspases and the induction of apoptosis.

Methods and Findings

In this study we further investigate the mechanisms of cell death induced by DT-GMCSF and show that, in addition to the activation of caspase-dependent apoptosis, DT-GMCSF also kills AML cells by simultaneously activating caspase-independent necroptosis. These mechanisms depend on the ability of the targeted toxin to inhibit protein synthesis, and are not affected by the receptor that is targeted or the mechanism through which protein synthesis is blocked.

Conclusions

We conclude that fusion toxin proteins may be effective for treating AML cells whether or not they are defective in apoptosis.

Although evasion of apoptosis is thought to be required for the development of cancer, it is unclear which cell death pathways are evaded. We previously identified a novel epithelial cell death pathway that works in normal cells but is inactivated in tumor cells, implying that it may be targeted during tumor development. The pathway can be activated by the Fas-associated death domain (FADD) of the adaptor protein but is distinct from the known mechanism of FADD-induced apoptosis through caspase-8. Here, we show that a physiological signal (tumor necrosis factor-related apoptosis-inducing ligand) can kill normal epithelial cells through the endogenous FADD protein by using the novel FADD death domain pathway, which activates both apoptosis and autophagy. We also show that selective resistance to this pathway occurs when primary epithelial cells are immortalized and that this occurs through a mechanism that is independent of known events (telomerase activity, and loss of function of p53, Rb, INK4a, and ARF) that are associated with immortalization. These data identify a novel cell death pathway that combines apoptosis and autophagy and that is selectively inactivated at the earliest stages of epithelial cancer development.

The adapter protein FADD consists of two protein interaction domains: a death domain and a death effector domain. The death domain binds to activated death receptors such as Fas, whereas the death effector domain binds to procaspase 8. An FADD mutant, which consists of only the death domain (FADD-DD), inhibits death receptor–induced apoptosis. FADD-DD can also activate a mechanistically distinct, cell type–specific apoptotic pathway that kills normal but not cancerous prostate epithelial cells. Here, we show that this apoptosis occurs through activation of caspases 9, 3, 6, and 7 and a serine protease. Simultaneous inhibition of caspases and serine proteases prevents FADD-DD–induced death. Inhibition of either pathway alone does not prevent cell death but does affect the morphology of the dying cells. Normal prostate epithelial cells require both the caspase and serine protease inhibitors to efficiently prevent apoptosis in response to TRAIL. In contrast, the serine protease inhibitor does not affect TRAIL-induced death in prostate tumor cells suggesting that the FADD-DD–dependent pathway can be activated by TRAIL. This apoptosis pathway is activated in a cell type–specific manner that is defective in cancer cells, suggesting that this pathway may be targeted during cancer development.

The adapter protein tumor necrosis factor receptor (TNFR)1–associated death domain (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex at the cell membrane. Here we show that TRADD contains a nuclear export and import sequence that allow shuttling between the nucleus and the cytoplasm. In the absence of export, TRADD is found within nuclear structures that are associated with promyelocytic leukemia protein (PML) nuclear bodies. In these structures, the TRADD death domain (TRADD-DD) can activate an apoptosis pathway that is mechanistically distinct from its action at the membrane-bound TNFR1 complex. Apoptosis by nuclear TRADD-DD is promyelocytic leukemia protein dependent, involves p53, and is inhibited by Bcl-xL but not by caspase inhibitors or dominant negative FADD (FADD-DN). Conversely, apoptosis induced by TRADD in the cytoplasm is resistant to Bcl-xL, but sensitive to caspase inhibitors and FADD-DN. These data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.

Hypertrophic growth of cardiac muscle cells is induced by a variety of physiological and pathological stimuli and is associated with a number of changes, including activation of genes such as atrial natriuretic factor. We found that two serum response element (SRE)-like DNA elements, one of which does not meet the consensus sequence and binds serum response factor (SRF) with low affinity, regulate the activity of this promoter. Surprisingly, the ability to induce the promoter by two different physiologic stimuli, as well as various activated transcription factors, including SRF-VP16, was primarily dependent upon the nonconsensus rather than the consensus SRE. This SRE controls the induction of gene expression via an unusual mechanism in that it is required to allow some, but not all, active transcription factors at unrelated sites on the promoter to stimulate gene expression. Thus, in addition to regulation of SRF activity by growth stimuli, regulation of a low-affinity SRE element controls inducible gene expression by modulating the ability of other transcription factors to stimulate the transcription machinery.