Kinase inhibitors are effective cancer therapies, but tumors frequently develop resistance. Current strategies to circumvent resistance target the same or parallel pathways. We report here that targeting a completely different process, autophagy, can overcome multiple BRAF inhibitor resistance mechanisms in brain tumors. BRAFV600Emutations occur in many pediatric brain tumors. We previously reported that these tumors are autophagy-dependent and a patient was successfully treated with the autophagy inhibitor chloroquine after failure of the BRAFV600E inhibitor vemurafenib, suggesting autophagy inhibition overcame the kinase inhibitor resistance. We tested this hypothesis in vemurafenib-resistant brain tumors. Genetic and pharmacological autophagy inhibition overcame molecularly distinct resistance mechanisms, inhibited tumor cell growth, and increased cell death. Patients with resistance had favorable clinical responses when chloroquine was added to vemurafenib. This provides a fundamentally different strategy to circumvent multiple mechanisms of kinase inhibitor resistance that could be rapidly tested in clinical trials in patients with BRAFV600E brain tumors.
Cancers of the brain and spine are the second most common kind of tumor in children, after cancers of the blood and bone marrow. Yet brain and spine tumors kill more children than any other cancer, in part because many become resistant to treatment.
Like in other cancers, cells in brain and spine tumors often use a process called autophagy to survive the treatments that are used to try and kill them. This process allows cells to recycle proteins and other things inside the cell and use them for energy when the cell is under stress. In 2014, researchers reported that brain tumors carrying a mutation called BRAFV600E rely on autophagy to survive treatment with medications that target this mutation. These findings suggested that blocking autophagy might make the medications more effective against BRAFV600Emutant tumors and overcome the resistance.
Now, Mulcahy Levy et al. – who include most of the researchers involved in the 2014 study – report that blocking autophagy does indeed overcome this kind of resistance in multiple types of tumor. The experiments made use of human brain tumor cells that can be grown in the laboratory and have been widely studied, as well as samples collected from patients.
Mulcahy Levy et al. were able to block autophagy in the tumor cells by using genetic methods and, importantly, by using an approved and inexpensive drug that could be rapidly translated into clinical trials. Together these findings suggest that blocking autophagy in patients might be a safe and effective strategy to improve their response to existing therapies that target the BRAFV600E mutation. Future clinical trials are now needed to test more patients and verify if this treatment plan can be broadly effective in patients with these types of brain cancers.
autophagy; BRAF V600E; brain tumor; chloroquine; pediatric; Human
Macroautophagy (hereafter autophagy) is an evolutionarily-ancient mechanism by which cellular material is delivered to lysosomes for degradation. Autophagy and cell death are intimately linked. For example, both processes often use the same molecular machinery and recent work suggests that autophagy has great influence over a cell’s decision to live or die. However, this decision making is complicated by the fact that autophagy’s role in determining whether a cell should live or die goes both ways—autophagy inhibition can result in more or less cell death depending on the death stimulus, cell type, or context. Autophagy may also differentially affect different types of cell death. Here we discuss recent literature that helps make sense of this seemingly inconsistent role of autophagy in influencing a cell to live or die.
Macroautophagy; autophagy; apoptosis; necroptosis; necrosis; autosis; MOMP; caspases; BH3 only proteins; PUMA; FAP-1
Autophagy is a catabolic process that facilitates nutrient recycling via degradation of damaged organelles and proteins through lysosomal mediated degradation. Alterations in this complex, and tightly regulated process, lead to disease. Autophagy is widely accepted as cytoprotective against neurodegenerative diseases and a variety of clinical interventions are moving forward to increase autophagy as a therapeutic intervention. Autophagy has both positive and negative roles in cancer and this has led to controversy over whether or how autophagy manipulation should be attempted in cancer therapy. Nevertheless, cancer is the disease where most current activity in trying to manipulate autophagy for therapy is taking place and dozens of clinical trials are using autophagy inhibition with Chloroquine or Hydroxychloroquine in combination with other drugs for the treatment of multiple neoplasms. Here, we review recent literature implicating autophagy in neurodegenerative diseases and cancer and highlight some of the opportunities, controversies and potential pitfalls of therapeutically targeting autophagy.
•Autophagy is a complex process that can be pharmacologically targeted at multiple steps.•Autophagy protects against neurodegeneration and enhanced autophagy decreases pathogenesis of neurodegenerative diseases.•Autophagy is both pro and anti-tumorigenic depending on the stage of tumorigenesis and mutational and oncogenic background.•Clinical trials are ongoing to increase autophagy in neurodegenerative diseases and inhibit autophagy to treat cancer.
Autophagy; Neurodegenerative disease; Cancer; Clinical trials
Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is an essential part of the cellular machinery that executes “programmed” or “regulated” necrosis. Here we show that programmed necrosis is activated in response to many chemotherapeutic agents and contributes to chemotherapy-induced cell death. However, we show that RIP3 expression is often silenced in cancer cells due to genomic methylation near its transcriptional start site, thus RIP3-dependent activation of MLKL and downstream programmed necrosis during chemotherapeutic death is largely repressed. Nevertheless, treatment with hypomethylating agents restores RIP3 expression, and thereby promotes sensitivity to chemotherapeutics in a RIP3-dependent manner. RIP3 expression is reduced in tumors compared to normal tissue in 85% of breast cancer patients, suggesting that RIP3 deficiency is positively selected during tumor growth/development. Since hypomethylating agents are reasonably well-tolerated in patients, we propose that RIP3-deficient cancer patients may benefit from receiving hypomethylating agents to induce RIP3 expression prior to treatment with conventional chemotherapeutics.
RIP3 (RIPK3); MLKL; programmed necrosis; chemotherapy; hypomethylating agents
Autophagy is a mechanism by which cells degrade cellular material to provide nutrients and energy for survival during stress. The autophagy is thought to be a critical process for cancer stem (CSC) or tumor initiating cell maintenance but the mechanisms by which autophagy supports survival of CSCs remain poorly understood. In this study, inhibition of autophagy by knockdown of ATG7 or BECN1 modified the CD44+/CD24low/− population in breast cancer cells by regulating CD24 and IL6 secretion. In a breast cancer cell line that is independent of autophagy for survival, autophagy inhibition increased IL6 secretion to the media. On the other hand, in an autophagy-dependent cell line, autophagy inhibition decreased IL6 secretion, cell survival and mammosphere formation. In these cells, IL6 treatment or conditioned media from autophagy-competent cells rescued the deficiency in mammosphere formation induced by autophagy inhibition. These results reveal that autophagy regulates breast CSC maintenance in autophagy-dependent breast cancer cells by modulating IL6 secretion implicating autophagy as a potential therapeutic target in breast cancer.
autophagy; breast cancer; cancer stem cells; IL6; mammosphere
Autophagy inhibition is a potential therapeutic strategy in central nervous system (CNS) tumors. The BRAFV600E mutation is known to affect autophagy. Our studies indicate CNS tumor cells with BRAFV600E mutant cells (but not wild type) display high rates of induced autophagy, are sensitive to autophagy inhibition, and display synergy when chloroquine is combined with the RAF kinase inhibitor vemurafenib or standard chemotherapeutics. Our studies also indicate chloroquine can improve vemurafenib sensitivity in intrinsically resistant cells and in a patient with induced-vemurafenib resistance. These findings suggest CNS tumors with BRAFV600E are autophagy-dependent and that identification of BRAFV600E may be a marker to identify pediatric patients with the best potential response to autophagy inhibition.
brain tumors; pediatric; autophagy; BRAF; chloroquine; BRAF, B-Raf proto-oncogene; serine/threonine kinase; CNS, central nervous system; WT, wild type
Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.
ATP release; autophagy; calreticulin; endoplasmic reticulum stress; HMGB1; immunotherapy; APC, antigen-presenting cell; ATF6, activating transcription factor 6; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BCL2, B-cell CLL/lymphoma 2 protein; CALR, calreticulin; CTL, cytotoxic T lymphocyte; Δψm, mitochondrial transmembrane potential; DAMP, damage-associated molecular pattern; DAPI, 4′,6-diamidino-2-phenylindole; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide; EIF2A, eukaryotic translation initiation factor 2A; ER, endoplasmic reticulum; FLT3LG, fms-related tyrosine kinase 3 ligand; G3BP1, GTPase activating protein (SH3 domain) binding protein 1; GFP, green fluorescent protein; H2B, histone 2B; HMGB1, high mobility group box 1; HSP, heat shock protein; HSV-1, herpes simplex virus type I; ICD, immunogenic cell death; IFN, interferon; IL, interleukin; MOMP, mitochondrial outer membrane permeabilization; PDIA3, protein disulfide isomerase family A; member 3; PI, propidium iodide; RFP, red fluorescent protein; TLR, Toll-like receptor; XBP1, X-box binding protein 1
Chloroquine (CQ) is an antimalarial drug and late-stage inhibitor of autophagy currently FDA-approved for use in the treatment of rheumatoid arthritis and other autoimmune diseases. Based primarily on its ability to inhibit autophagy, CQ and its derivative, hydroxychloroquine, are currently being investigated as primary or adjuvant therapy in multiple clinical trials for cancer treatment. Oncogenic RAS has previously been shown to regulate autophagic flux, and cancers with high incidence of RAS mutations, such as pancreatic cancer, have been described in the literature as being particularly susceptible to CQ treatment, leading to the hypothesis that oncogenic RAS makes cancer cells dependent on autophagy. This autophagy “addiction” suggests that the mutation status of RAS in tumors could identify patients who would be more likely to benefit from CQ therapy. Here we show that RAS mutation status itself is unlikely to be beneficial in such a patient selection because oncogenic RAS does not always promote autophagy addiction. Moreover, oncogenic RAS can have opposite effects on both autophagic flux and CQ sensitivity in different cells. Finally, for any given cell type, the positive or negative effect of oncogenic RAS on autophagy does not necessarily predict whether RAS will promote or inhibit CQ-mediated toxicity. Thus, although our results confirm that different tumor cell lines display marked differences in how they respond to autophagy inhibition, these differences can occur irrespective of RAS mutation status and, in different contexts, can either promote or reduce chloroquine sensitivity of tumor cells.
autophagy; autophagy dependence; cell death; chloroquine; RAS
Recent studies have highlighted the opportunity to treat cancer by
inhibiting autophagy but have also raised important caveats with this idea. A
paper in this issue of Cancer Discovery adds to accumulating
evidence suggesting that we should focus our efforts (at least initially) on
specific tumors where we are most likely to see beneficial effects.
autophagy; cancer; phase I trial; translational; treatment
Autophagy is a lysosomal degradation process that may act as a mechanism of survival in a variety of cancers. While pharmacologic inhibition of autophagy with hydroxychloroquine (HCQ) is currently being explored in human clinical trials, it has never been evaluated in canine cancers. Non-Hodgkin lymphoma (NHL) is one of the most prevalent tumor types in dogs and has similar pathogenesis and response to treatment as human NHL. Clinical trials in canine patients are conducted in the same way as in human patients, thus, to determine a maximum dose of HCQ that can be combined with a standard chemotherapy, a Phase I, single arm, dose escalation trial was conducted in dogs with spontaneous NHL presenting as patients to an academic, tertiary-care veterinary teaching hospital. HCQ was administered daily by mouth throughout the trial, beginning 72 h prior to doxorubicin (DOX), which was given intravenously on a 21-d cycle. Peripheral blood mononuclear cells and biopsies were collected before and 3 d after HCQ treatment and assessed for autophagy inhibition and HCQ concentration. A total of 30 patients were enrolled in the trial. HCQ alone was well tolerated with only mild lethargy and gastrointestinal-related adverse events. The overall response rate (ORR) for dogs with lymphoma was 93.3%, with median progression-free interval (PFI) of 5 mo. Pharmacokinetic analysis revealed a 100-fold increase in HCQ in tumors compared with plasma. There was a trend that supported therapy-induced increase in LC3-II (the cleaved and lipidated form of microtubule-associated protein 1 light chain 3/LC3, which serves as a maker for autophagosomes) and SQSTM1/p62 (sequestosome 1) after treatment. The superior ORR and comparable PFI to single-agent DOX provide strong support for further evaluation via randomized, placebo-controlled trials in canine and human NHL.
autophagy; lymphoma; canine model; hydroxychloroquine; doxorubicin
TRAIL and agonistic antibodies against TRAIL death receptors kill tumor cells while causing virtually no damage to normal cells. Several novel drugs targeting TRAIL receptors are currently in clinical trials. However, TRAIL resistance is a common obstacle in TRAIL based therapy and limits the efficiency of these drugs. In this review article we discuss different mechanisms of TRAIL resistance and how they can be predicted and therapeutically circumvented. In addition, we provide a brief overview of all TRAIL based clinical trials conducted so far. It is apparent that although the effects of TRAIL therapy are disappointingly modest overall, a small subset of patients responds very well to TRAIL. We argue that the true potential of targeting TRAIL death receptors in cancer can only be reached when we find efficient ways to select for those patients that are most likely to benefit from the treatment. To achieve this, it is crucial to identify biomarkers that can help us predict TRAIL sensitivity.
Macroautophagy has been implicated in numerous diseases, yet our understanding of the proteins responsible for the turnover of specific cargo by autophagy is limited. In a recent paper published in Nature, Mancias et al. used quantitative proteomics to identify a cohort of autophagosome-enriched proteins, one of which, nuclear receptor coactivator 4 (NCOA4) was shown to be required for the selective delivery of ferritin to the lysosome, ultimately regulating intracellular iron by autophagic turnover of ferritin, or ferritinophagy.
We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.
autophagy; flow cytometry; autophagic flux; GFP-LC3; cell sorting; quantification
Activating mutations in the RAS oncogene are common in cancer but are difficult to therapeutically target. RAS activation promotes autophagy, a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses. Here we report that casein kinase 1α (CK1α), a ubiquitously expressed serine/threonine kinase, is a key negative regulator of oncogenic RAS–induced autophagy. Depletion or pharmacologic inhibition of CK1α enhanced autophagic flux in oncogenic RAS–driven human fibroblasts and multiple cancer cell lines. FOXO3A, a master longevity mediator that transcriptionally regulates diverse autophagy genes, was a critical target of CK1α, as depletion of CK1α reduced levels of phosphorylated FOXO3A and increased expression of FOXO3A-responsive genes. Oncogenic RAS increased CK1α protein abundance via activation of the PI3K/AKT/mTOR pathway. In turn, elevated levels of CK1α increased phosphorylation of nuclear FOXO3A, thereby inhibiting transactivation of genes critical for RAS-induced autophagy. In both RAS-driven cancer cells and murine xenograft models, pharmacologic CK1α inactivation synergized with lysosomotropic agents to inhibit growth and promote tumor cell death. Together, our results identify a kinase feedback loop that influences RAS-dependent autophagy and suggest that targeting CK1α-regulated autophagy offers a potential therapeutic opportunity to treat oncogenic RAS–driven cancers.
Autophagy is a protein and organelle degradation pathway that is involved in diverse diseases including cancer. Recent evidence suggests that autophagy is a cell survival mechanism in tumor cells and that its inhibition especially in combination with other therapy could be beneficial but it remains unclear if all cancer cells behave the same way when autophagy is inhibited. We inhibited autophagy in a panel of breast cancer cell lines and found that some of them are dependent on autophagy for survival even in nutrient rich conditions without any additional stress while others need autophagy only when stressed. Survival under unstressed conditions is due to cell type specific autophagy regulation of STAT3 activity and this phenotype is enriched in triple negative cell lines. This autophagy-dependency affects response to therapy because autophagy inhibition reduced tumor growth in vivo in autophagy-dependent but not in autophagy-independent breast tumors while combination treatment with autophagy inhibitors and other agent was preferentially synergistic in autophagy-dependent cells. These results imply that autophagy-dependence represents a tumor cell specific characteristic where autophagy inhibition will be more effective. Moreover, our results suggest that autophagy inhibition might be a potential therapeutic strategy for triple negative breast cancers, which currently lack an effective targeted treatment.
autophagy; breast cancer; STAT3
Macroautophagy is thought to protect against apoptosis, however underlying mechanisms are poorly understood. We examined how autophagy affects canonical death receptor-induced mitochondrial outer membrane permeabilization (MOMP) and apoptosis. MOMP occurs at variable times in a population of cells and this is delayed by autophagy. Additionally, autophagy leads to inefficient MOMP after which some cells die through a slower process than typical apoptosis and, surprisingly, can recover and divide afterwards. These effects are associated with p62/SQSTM1-dependent selective autophagy causing PUMA levels to be kept low through an indirect mechanism whereby autophagy affects constitutive levels of PUMA mRNA. PUMA depletion is sufficient to prevent the sensitization to apoptosis that occurs when autophagy is blocked. Autophagy can therefore control apoptosis via a key regulator that makes MOMP faster and more efficient thus ensuring rapid completion of apoptosis. This identifies a molecular mechanism whereby cell fate decisions can be determined by autophagy.
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