Excitotoxic insults such as cerebral ischemia are thought to enhance neuronal autophagy, which is then thought to promote neuronal cell death. Excitotoxic insults indeed increase autophagy markers. Notably, however, autophagy markers can be increased either by autophagy induction (as this enhances their production) or by late-stage autophagy inhibition (as this prevents their degradation during autophagic flux). By comparing each condition with and without protease inhibitors that prevent autophagic degradation of the autophagy marker, the results of this study show that excitotoxic glutamate increases autophagy markers by a late-stage block of autophagy. Initially, this study set out to test if the CaMKII inhibitor tatCN21 mediates its post-insult neuroprotection by regulating autophagy. While tatCN21 partially inhibited basal autophagy in hippocampal neurons, it had no effects on the already blocked autophagy after excitotoxic glutamate insults, indicating that autophagy inhibition is not its neuroprotective mechanism. Additionally, while the autophagy inhibitor chloroquine had no effect, significant neuroprotection was seen instead with two drugs that enhance autophagy induction by different mechanisms, rapamycin (mTOR dependent) and trehalose (mTOR-independent). This suggests that therapeutic approaches should seek to enhance rather than inhibit autophagy, not only in neurodegenerative diseases (where such approach is widely accepted) but also after acute excitotoxic insults. Together, these findings significantly reshape the current view on the mutual cross-regulation of autophagy and excitotoxicity.
CaMKII; glutamate; excitotoxicity; autophagy; neuronal cell death
Autophagy inhibition is a potential therapeutic strategy in cancer, but it is unknown which tumors will benefit. The BRAFV600E mutation has been identified as important in pediatric CNS tumors and is known to affect autophagy in other tumor types. We evaluated CNS tumor cells with BRAFV600E and found that mutant cells (but not wild type) display high rates of induced autophagy, are sensitive to pharmacologic and genetic autophagy inhibition, and display synergy when the clinically used autophagy inhibitor chloroquine was combined with the Raf inhibitor vemurafenib or standard chemotherapeutics. Importantly we also demonstrate chloroquine can improve vemurafenib sensitivity in a resistant ex vivo primary culture and provide the first demonstration in a patient harboring the V600E mutation treated with vemurafenib that addition of chloroquine can improve clinical outcomes. These findings suggest CNS tumors with BRAFV600E are autophagy-dependent and should be targeted with autophagy inhibition in combination with other therapeutic strategies.
Brain tumors; pediatric; autophagy; BRAF; chloroquine
Autophagy delivers cellular material to lysosomes for recycling. This article discusses why both good and bad effects arise because of autophagy and argues that exploiting this knowledge can treat disease.
A glance through Autophagy or any other journal in this field shows that it is very common to block autophagy by RNA interference-based knockdown of ATG mRNAs in mammalian cell lines. Our lab’s experience is that this approach can easily make for failed experiments because good knockdown of even essential autophagy regulators does not necessarily mean you will get good inhibition of autophagy, and, over time, cells can find ways to circumvent the inhibitory effects of the knockdown.
autophagy; shRNA; ATG5; knockdown; RNA interference
Macroautophagy (referred to as autophagy here) is an intracellular degradation pathway enhanced in response to a variety of stresses and in response to nutrient deprivation. This process provides the cell with nutrients and energy by degrading aggregated and damaged proteins as well as compromised organelles. Since autophagy has been linked to diverse diseases including cancer, it has recently become a very interesting target in breast cancer treatment. Indeed, current clinical trials are trying to use chloroquine or hydroxychloroquine, alone or in combination with other drugs to inhibit autophagy during breast cancer therapy since chemotherapy and radiation, regimens that are used to treat breast cancer, are known to induce autophagy in cancer cells. Importantly, in breast cancer, autophagy has been involved in the development of resistance to chemotherapy and to anti-estrogens. Moreover, a close relationship has recently been described between autophagy and the HER2 receptor. Here, we discuss some of the recent findings relating autophagy and cancer with a particular focus on breast cancer therapy.
Autophagy; Cancer; Breast cancer; Chemotherapy; Radiation; Estrogen receptor; HER2; Triple negative breast cancer
Autophagy regulates cell death both positively and negatively, but the molecular basis for this paradox remains inadequately characterized. We demonstrate here that transient cell-to-cell variations in autophagy can either promote cell death or survival depending on the stimulus and cell type. By separating cells with high and low basal autophagy by flow cytometry, we demonstrate that autophagy determines which cells live or die in response to death receptor activation. We have determined that selective autophagic degradation of the phosphatase Fap-1 promotes Fas apoptosis in Type I cells. Conversely, autophagy inhibits apoptosis in Type II cells or upon treatment with TRAIL in either Type I or II cells. These data illustrate that differences in autophagy in a cell population determine cell fate in a stimulus- and cell type-specific manner. This example of selective autophagy of an apoptosis regulator may represent a general mechanism for context-specific regulation of cell fate by autophagy.
The epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of head and neck squamous cell carcinoma (HNSCC), but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in Vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacological or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four out of six HNSCC patient-derived xenografts; however they eventually re-grew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed re-growth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state delaying or completely blocking tumor recurrence.
Head and neck squamous cell cancer; hedgehog pathway; epidermal growth factor receptor pathway; epithelial to mesenchymal transition
Autophagy is intimately associated with eukaryotic cell death and apoptosis. Indeed in some cases the same proteins control both autophagy and apoptosis. Apoptotic signaling can regulate autophagy and conversely autophagy can regulate apoptosis (and most likely other cell death mechanisms). However the molecular connections between autophagy and cell death are complicated and, in different contexts, autophagy may promote or inhibit cell death. Surprisingly, although we know that, at its core, autophagy involves degradation of sequestered cytoplasmic material, and therefore presumably must be mediating its effects on cell death by degrading something, in most cases we have little idea what is being degraded to promote autophagy’s pro- or anti-death activities. Because autophagy is known to play important roles in health and many diseases, it is critical to understand the mechanisms by which autophagy interacts with and affects the cell death machinery since this will perhaps allow new ways to prevent or treat disease. In this chapter we discuss the current state of understanding of these processes.
Apoptosis; caspase; autophagic cell death; TRAIL; BCL proteins; autophagy-mediated protection; ATG protein function
The novel fusion protein, DAB389EGF, is comprised of both the catalytic and translocation domains of diphtheria toxin that are fused to the human epidermal growth factor, providing a targeting and a toxicity component. We tested DAB389EGF for anti-tumor activity in both in vitro and in vivo urinary bladder cancer models.
Human bladder cancer lines were treated with DAB389EGF and assessed for growth inhibition and clonogenic suppression. Using 6–8 week old female athymic nude mice implanted orthotopically with HTB9 cells, DAB389EGF was administered intravesically twice weekly for two weeks. The response of the luciferase expressing HTB9 cells was monitored via bioluminescence as the primary endpoint..
Treatment response with DAB389EGF was specific and robust, with an IC50 ranging from 0.5 to 15ng/ml in 8 tested bladder cancer cell lines, but greater than 50ng/ml in the EGFR-negative H520 control cell line. Simulating short duration intravesical therapy used clinically, a 2 hour treatment exposure of DAB389EGF (10ng/ml) produced clonogenic suppression in three selected bladder cancer cell lines. In vivo, luciferase activity was suppressed in 5 of 6 mice treated with DAB389EGF (70 μl (1ng/μL) per mouse), as compared to only 1 of 6 mice treated with a control DT fusion protein. Histologic assessment of tumor clearance correlated with the bioluminescent changes observed with DAB389EGF treatment. Immunocompetent mice treated with intravesical DAB389EGF did not demonstrate any non-specific systemic toxicity.
The intravesical delivery of targeted-toxin fusion proteins is a novel treatment approach for non-muscle-invasive urinary bladder cancer. With appropriate targeting, the treatments are effective and well tolerated in vivo.
Current treatments for Glioblastoma multiforme (GBM) involve surgery, radiotherapy, and cytotoxic chemotherapy; however, these treatments are not effective and there is an urgent need for better treatments. We investigated GBM cell killing by a novel drug combination involving DT-EGF, an Epidermal Growth Factor Receptor-targeted bacterial toxin, and Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) or antibodies that activate the TRAIL receptors DR4 and DR5. DT-EGF kills GBM cells by a non apoptotic mechanism whereas TRAIL kills by inducing apoptosis. GBM cells treated with DT-EGF and TRAIL were killed in a synergistic fashion in vitro and the combination was more effective than either treatment alone in vivo. Tumor cell death with the combination occurred by caspase activation and apoptosis due to DT-EGF positively regulating TRAIL killing by depleting FLIP, a selective inhibitor of TRAIL receptor-induced apoptosis. These data provide a mechanism-based rationale for combining targeted toxins and TRAIL receptor agonists to treat GBM.
Diphtheria toxin; TRAIL; apoptosis; autophagy
Brain and spinal tumors are the second most common malignancies in childhood after leukemia, and they remain the leading cause of death from childhood cancer. The role of autophagy, a catabolic cellular process used to provide energy during times of stress, in pediatric tumors is unknown. Here we present studies done in pediatric medulloblastoma cell lines (DAOY, ONS76) and atypical teratoid/rhabdoid tumor cell lines (BT-16, BT-12) to test this role. Autophagy was inhibited using siRNA against autophagy related genes ATG12 and ATG7 or pharmacologically induced using rapamyacin or inhibited using chloroquine to test the effect of autophagy on chemosensitivity. We found that when pediatric brain tumor cells are under starvation stress or exposed to known autophagy inducers, they show increased levels of autophagy. We also found that current chemotherapeutics (CCNU, cisplatin) stimulate autophagy in pediatric brain cancer cells. Silencing of ATG12 and ATG7 prevents this increase and autophagy can be pharmacologically stimulated and inhibited. Although autophagy can be induced and inhibited in these cell lines, the effect of autophagy on tumor cell killing is small. This may have significant clinical relevance in the future planning of therapeutic regimens for pediatric brain tumors.
The semi-synthetic vitamin E derivative alpha-tocopheryloxyacetic acid (α-TEA) induces tumor cell apoptosis and may offer a simple adjuvant supplement for cancer therapy if its mechanisms can be better understood. Here we report that α-TEA also triggers tumor cell autophagy and that it improves cross-presentation of tumor antigens to the immune system. α-TEA stimulated both apoptosis and autophagy in murine mammary and lung cancer cells and inhibition of caspase-dependent apoptosis enhanced α-TEA-induced autophagy. Cell exposure to α-TEA generated double membrane-bound vesicles indicative of autophagosomes, which efficiently cross-primed antigen-specific CD8+ T cells. Notably, vaccination with dendritic cells pulsed with α-TEA-generated autophagosomes reduced lung metastases and increased the survival of tumor-bearing mice. Taken together, our findings suggest that both autophagy and apoptosis signaling programs are activated during α-TEA-induced tumor cell killing. We suggest that the ability of α-TEA to stimulate autophagy and enhance cross-priming of CD8+ T cells might be exploited as an adjuvant strategy to improve stimulation of anti-tumor immune responses.
α-TEA; autophagy; cross-presentation; tumor immunity
The presence of Six1 mRNA gene portends a poor prognosis in ovarian cancer. We describe validation of a Six1 specific antibody and evaluate its association with tumorigenicity and prognosis in ovarian cancer.
A Six1 antibody (Six1cTerm) was raised to residues downstream of the Six1 homeodomain, representing its unique C-terminus as compared to other Six family members. Cells were transfected with Six1–Six6 and Western blot was performed to demonstrate Six1 specificity. Ovarian cancer cell lines were analyzed for Six1 mRNA and Six1cTerm and tumorigenicity was evaluated. Ovarian cancer tissue microarrays (OTMA) were analyzed for Six1cTerm by immunohistochemistry and scored by two blinded observers. The metastatic tumors of 15 stage IIIC high grade serous ovarian cancers were analyzed with Six1 mRNA and Six1cTerm and expression was compared to clinical factors and survival.
The Six1cTerm antibody is specific for Six1. Cell line tumorigenicity in SCID mice correlates with Six1 levels both by mRNA(p=0.001, Mann–Whitney U test) and by protein (presence vs. absence, p=0.05 Fischer's Exact test). Six1 protein was present in up to 54% of OTMA specimens. Six1 protein expression in omental/peritoneal metastases correlated with worsened survival in a sample (n=15) of high grade serous stage IIIC ovarian cancers (p=0.001).
The Six1cTerm antibody is specific and able to detect Six1 in cell lines and tumor tissue. Six1 protein detection is common in ovarian cancer and is associated with tumorigenicity and poor prognosis in this group of patient samples. Six1cTerm antibody should be further validated as prognostic tool.
Ovarian cancer; Homeoprotein; Homeobox gene; Six1; Prognosis
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.
chloroquine; cisplatin; PtdIns3K; LY294002; mTOR; rapamycin; autophagy; breast cancer
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.
Autophagy; Cancer; Cancer therapy; Cell death; Clinical trial; Chloroquine
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.
autophagy; lysosome; mitophagy; pexophagy; stress; vacuole
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.
vitamin D; interleukin-1 alpha; prostate stem cell; differentiation; senescence
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.
autophagy; cancer; invasion; migration; organotypic model
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.
TRAIL; Six1; chemoresistance; lymphoma
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.
autophagy; cancer; cancer therapy; cell death; apoptosis
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.
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.
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.
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.
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.
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.
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.
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.
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.
Apo2 ligand/Tumor necrosis factor related apoptosis inducing ligand (TRAIL); kidney cancer; immunotherapy; apoptosis; ERK
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.