Autophagy in pancreatic cancer: An emerging mechanism of cell death

Pancreatic cancer, the fourth leading cause of cancer-related death in the United States, is resistant to current chemotherapies. Therefore, identification of different pathways of cell death is important to develop novel therapeutics. Our previous study has shown that triptolide, a diterpene triepoxide, inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. However, the mechanism by which triptolide kills pancreatic cancer cells was not known, hence, this study aimed at elucidating it. Our study reveals that triptolide kills diverse types of pancreatic cancer cells by two different pathways; it induces caspase-dependent apoptotic death in some cell lines and death via a caspase-independent autophagic pathway in the other cell lines tested. Triptolide-induced autophagy requires autophagy-specific genes, atg5 or beclin 1 and its inhibition results in cell death via the apoptotic pathway, whereas inhibition of both autophagy and apoptosis rescues triptolide-mediated cell death. Our study shows for the first time that induction of autophagy by triptolide has a pro-death role in pancreatic cancer cells. Since triptolide kills diverse pancreatic cancer cells by different mechanisms, it makes an attractive chemotherapeutic agent for future use against a broad spectrum of pancreatic cancers.Key words: pancreatic cancer, triptolide, apoptosis, caspase-3Pancreatic adenocarcinoma is one of the most lethal human malignancies. It is the fourth leading cause of cancer-related death in the United States. The five-year survival rate for pancreatic cancer is estimated to be <5% due to its aggressive growth, metastasis and resistance to radiation and most systemic chemotherapies. Hence, efforts are ongoing to understand the pathobiology of pancreatic cancer to develop innovative and effective therapies against it. A promising candidate for future therapeutic use against pancreatic cancer is a diterpene triepoxide, triptolide. Our previous studies show that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. Since the mechanism by which triptolide kills pancreatic cancer cells was not known, we decided to elucidate it.The K-ras, p53, p16 and DPC4 genes are the most frequently altered genes in pancreatic adenocarcinoma. In this study we have used diverse pancreatic cancer cell lines, MiaPaCa-2, Capan-1, S2-013 and S2-VP10 cells, which have mutations in all the above-mentioned genes and BxPC-3 and Hs766T cells, which have mutations in the p53, p16 and DPC4 genes, but have a wild-type K-ras gene. The treatment of all the cell lines with triptolide results in a significant time- and dose-dependent decrease in cell viability, independent of cell cycle arrest. After treatment with triptolide, only MiaPaCa-2, Capan-1 and BxPC-3 cells show an increase in the apoptosis parameters: cytochrome c release from mitochondria into the cytosol, caspase-3 activation and phosphatidylserine externalization. In contrast to this, S2-013, S2-VP10 and Hs766T cells show an induction of autophagy: an increase in LC3-II levels (by immunoblotting and immufluorescence), increase in acridine orange-positive cells, inhibition of the PtdIns3K/Akt/mTOR pathway and induction of the ERK1/2 pathway. Also, none of the cell lines tested show necrosis as evidenced by the absence of the release of lactate dehydrogenase. These results indicate that triptolide induces apoptosis in MiaPaCa-2, Capan-1 and BxPC-3 cells, whereas it induces autophagy in S2-013, S2-VP10 and Hs766T cells.Since the role of autophagy in cancer was controversial we investigated whether triptolide-induced autophagy has a prosurvival or a pro-death role. As autophagy-associated cell death is independent of caspase-3, we tested the effect of triptolide on pancreatic cancer cells in the absence of caspase-3. Treatment of cells with triptolide post-caspase-3 knockdown shows a significant rescue of cell viability only in MiaPaCa-2, but not S2-013 or S2-VP10 cells. This indicates that in contrast to MiaPaCa-2, triptolide-mediated cell death in S2-013 and S2-VP10 cells is independent of caspase-3. Next, we tested the role of autophagy in triptolide-mediated cell death in pancreatic cancer cells. In spite of a knockdown of autophagy-specific genes (atg5 and beclin 1), treatment of S2-013 and S2-VP10 cells with triptolide show a significant decline in cell viability, which is comparable to the cells treated with triptolide in the presence of autophagy genes. Subsequently we show that death in the absence of autophagy-specific genes is due to the utilization of an alternate cell death pathway, apoptosis. Furthermore, in the absence of both autophagy-specific and apoptosis-specific genes, triptolide-mediated cell death is rescued in S2-013 and S2-VP10 cells. Thus, these results confirm that triptolide-induced autophagy has a pro-death role in S2-013 and S2-VP10 cells and that these cells do not have a defect in the apoptotic machinery; however, they respond to triptolide by activating the autophagic pathway instead of the apoptotic pathway. Our studies also reveal the presence of a crosstalk between the two cell death pathways, apoptosis and autophagy, in pancreatic cancer cells.In conclusion, our study shows for the first time that triptolide induces autophagy in pancreatic cancer cells. It sheds light on the fundamental question as to whether autophagy is protective or causes cell death, proving convincingly that induction of autophagy causes cell death of some pancreatic cancer cells. Although a basal level of autophagy is necessary to maintain cellular homeostasis, its prosurvival role can be switched into a cell death mechanism if the amplitude of autophagy increases above a threshold level which is incompatible with viability, as seen in S2-013, S2-VP10 and Hs766T cells after triptolide treatment. Furthermore, there exists a crosstalk between apoptosis and autophagy in S2-013 and S2-VP10 cells; either both pathways function independently to kill the cells, with autophagy being the preferred pathway or autophagy antagonizes apoptosis and hence apoptosis is seen only after inhibiting autophagy. Although there is no direct correlation between the selection of cell death pathway in response to triptolide and the genotype of the cell lines, the choice of autophagic cell death pathway could depend on the metastatic potential of the cells; S2-013, S2-VP10 and Hs766T cell lines being more metastatic than the others, which merits further investigation. In conclusion, the ability of triptolide to induce cell death in diverse pancreatic cancer cells by either mechanism makes it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.     

Autophagy and cancer

Autophagy is a homeostatic and evolutionarily conserved mechanism of self-digestion by which the cells degrade and recycle long-lived proteins and excess or damaged organelles.Autophagy is activated in response to both physiological and pathological stimuli including growth factor depletion,energy deficiency or the upregulation of Bcl-2 protein expression.A novel role of autophagy in various cancers has been proposed.Interestingly,evidence that supports both a positive and negative role of autophagy in the pathogenesis of cancer has been reported.As a tumor suppression mechanism,autophagy maintains genome stability,induces senescence and possibly autophagic cell death.On the other hand,autophagy participates in tumor growth and maintenance by supplying metabolic substrate,limiting oxidative stress,and maintaining cancer stem cell population.It has been proposed that the differential roles of autophagy in cancer are disease type and stage specific.In addition,substrate selectivity might be involved in carrying out the specific effect of autophagy in cancer,and represents one of the potential directions for future studies.     

Autophagy and cancer

(Macro)autophagy is a cellular membrane trafficking process that serves to deliver cytoplasmic constituents to lysosomes for degradation. At basal levels, it is critical for maintaining cytoplasmic as well as genomic integrity and is therefore key to maintaining cellular homeostasis. Autophagy is also highly adaptable and can be modified to digest specific cargoes to bring about selective effects in response to numerous forms of intracellular and extracellular stress. It is not a surprise, therefore, that autophagy has a fundamental role in cancer and that perturbations in autophagy can contribute to malignant disease. We review here the roles of autophagy in various aspects of tumor suppression including the response of cells to nutrient and hypoxic stress, the control of programmed cell death, and the connection to tumor-associated immune responses.     

Autophagy and cancer therapy

Autophagy is a dynamic process of protein degradation, which is typically observed during nutrient deprivation. Recently, interest in autophagy has been renewed among oncologists, because different types of cancer cells undergo autophagy after various anticancer therapies. This type of nonapoptotic cell death has been documented mainly by observing morphological changes, e.g., numerous autophagic vacuoles in the cytoplasm of dying cells. Thus, autophagic cell death is considered programmed cell death type II, whereas apoptosis is programmed cell death type I. These two types of cell death are predominantly distinctive, but many studies demonstrate cross-talk between them. Whether autophagy in cancer cells causes death or protects cells is controversial. In multiple studies, autophagy has been inhibited pharmacologically or genetically, resulting in contrasting outcomes--survival or death--depending on the specific context. Interestingly, the regulatory pathways of autophagy share several molecules with the oncogenic pathways activated by tyrosine kinase receptors. Tumor suppressors such as Beclin 1, PTEN and p53 also play an important role in autophagy induction. Taken together, these accumulating data may lead to development of new cancer therapies that manipulate autophagy.     

Autophagy in cancer biology and therapy

The role of macroautophagy （hereafter autophagy） in cancer biology and response to clinical intervention is complex. It is clear that autophagy is dysregulated in a wide variety of tumor settings, both during tumor initiation and progression, and in response to therapy. However, the pleiotropic mechanistic roles of autophagy in controlling cell behavior make it difficult to predict in a given tumor setting what the role of autophagy, and, by extension, the therapeutic outcome of targeting autophagy, might be. In this review we summarize the evidence in the literature supporting pro- and anti-tumorigenic and -therapeutic roles of autophagy in cancer. This overview encompasses roles of autophagy in nutrient management, cell death, cell senescence, regulation of proteotoxic stress and cellular homeostasis, regulation of tumor-host interactions and participation in changes in metabolism. We also try to understand, where possible, the mechanistic bases of these roles for autophagy. We specifically expand on the emerging role of genetically- engineered mouse models of cancer in shedding light on these issues in vivo. We also consider how any or all of the above functions of autophagy proteins might be targetable by extant or future classes of pharmacologic agents. We conclude by briefly exploring non-canonical roles for subsets of the key autophagy proteins in cellular processes, and how these might impact upon cancer.     

Autophagy and the pancreatic beta-cell in human type 2 diabetes

Pancreatic beta-cell dysfunction is central to the development and worsening of type 2 diabetes. Whereas beta-cell apoptosis plays a major role in reducing beta-cell mass in diabetes, alterations of autophagy can also lead to beta-cell death, as recently demonstrated in type 2 diabetic subjects. In addition, several studies with cell lines and rodent models have shown the importance of autophagy in regulating beta-cell survival and function. Although most of the underlying molecular mechanisms remain to be elucidated, this growing evidence raises interest in the role of autophagy in beta-cell pathophysiology and suggests the possibility of exploring autophagic processes to develop tools for protection of the pancreatic beta-cell in type 2 diabetes.     

Autophagy,citrullination and cancer

A cell needs to maintain a balance between biosynthesis and degradation of cellular components to maintain homeostasis. There are 2 pathways, the proteasome, which degrades short-lived proteins, and the autophagy/lysosomal pathway, which degrades long-lived proteins and organelles. Both of these pathways are also involved in antigen presentation or the effective delivery of peptides to MHC molecules for presentation to T cells. Autophagy (macroautophagy) is a key player in providing substantial sources of citrullinated peptides for loading onto MHC-II molecules to stimulate CD4⁺ T cell responses. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. We therefore investigated if citrullinated peptides could stimulate CD4⁺ T cell responses that would recognize these modifications produced during autophagy within tumor cells. Focusing on the intermediate filament protein VIM (vimentin), we generated citrullinated VIM peptides for immunization experiments in mice. Immunization with these peptides induced CD4⁺ T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 d after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. These results show how CD4⁺ cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides produced during autophagy may offer especially attractive vaccine targets for cancer therapy.     

Autophagy and cancer cell metabolism

Autophagy is a catabolic process involving lysosomal turnover of proteins and organelles for maintenance of cellular homeostasis and mitigation of metabolic stress. Autophagy defects are linked to diseases, such as liver failure, neurodegeneration, inflammatory bowel disease, aging and cancer. The role of autophagy in tumorigenesis is complex and likely context-dependent. Human breast, ovarian and prostate cancers have allelic deletions of the essential autophagy regulator BECN1 and Becn1(+/-) and other autophagy-deficient transgenic mice are tumor-prone, whereas tumors with constitutive Ras activation, including human pancreatic cancers, upregulate basal autophagy and are commonly addicted to this pathway for survival and growth; furthermore, autophagy suppression by Fip200 deletion compromises PyMT-induced mammary tumorigenesis. The double-edged sword function of autophagy in cancer has been attributed to both cell- and non-cell-autonomous mechanisms, as autophagy defects promote cancer progression in association with oxidative and ER stress, DNA damage accumulation, genomic instability and persistence of inflammation, while functional autophagy enables cancer cell survival under stress and likely contributes to treatment resistance. In this review, we will focus on the intimate link between autophagy and cancer cell metabolism, a topic of growing interest in recent years, which has been recognized as highly clinically relevant and has become the focus of intense investigation in translational cancer research. Many tumor-associated conditions, including intermittent oxygen and nutrient deprivation, oxidative stress, fast growth and cell death suppression, modulate, in parallel and in interconnected ways, both cellular metabolism and autophagy to enable cancer cells to rapidly adapt to environmental stressors, maintain uncontrolled proliferation and evade the toxic effects of radiation and/or chemotherapy. Elucidating the interplay between autophagy and tumor cell metabolism will provide unique opportunities to identify new therapeutic targets and develop synthetically lethal treatment strategies that preferentially target cancer cells, while sparing normal tissues.     

Autophagy in malignant transformation and cancer progression

Autophagy plays a key role in the maintenance of cellular homeostasis. In healthy cells, such a homeostatic activity constitutes a robust barrier against malignant transformation. Accordingly, many oncoproteins inhibit, and several oncosuppressor proteins promote, autophagy. Moreover, autophagy is required for optimal anticancer immunosurveillance. In neoplastic cells, however, autophagic responses constitute a means to cope with intracellular and environmental stress, thus favoring tumor progression. This implies that at least in some cases, oncogenesis proceeds along with a temporary inhibition of autophagy or a gain of molecular functions that antagonize its oncosuppressive activity. Here, we discuss the differential impact of autophagy on distinct phases of tumorigenesis and the implications of this concept for the use of autophagy modulators in cancer therapy.     

Autophagy signaling and the cogwheels of cancer

The downregulation of macroautophagy observed in cancer cells is associated with tumor progression. The regulation of macroautophagy by signaling pathways overlaps with the control of cell growth, proliferation, cell survival and death. Several tumor suppressor genes (PTEN, TSC2 and p53) involved in the mTOR signaling network have been shown to stimulate autophagy. In contrast, the oncoproteins involved in this network have the opposite effect. These findings, together with the discovery that haploinsufficiency of the tumor suppressor beclin 1 promotes tumorigenesis in various tissues in transgenic mice, give credibility to the idea that autophagy is a tumor suppressor mechanism. The induction of macroautophagy by cancer treatments may also contribute to cell eradication. However, cancer cells sometimes mobilize autophagic capacities in response to various stimuli without a fatal outcome, suggesting that they can also exploit macroautophagy for their own benefit.     

Autophagy induction for the treatment of cancer

Cancer can be viewed in 2 rather distinct ways, namely (i) as a cell-autonomous disease in which malignant cells have escaped control from cell-intrinsic barriers against proliferation and dissemination or (ii) as a systemic disease that involves failing immune control of aberrant cells. Since macroautophagy/autophagy generally increases the fitness of cells as well as their resistance against endogenous or iatrogenic (i.e., relating to illness due to medical intervention) stress, it has been widely proposed that inhibition of autophagy would constitute a valid strategy for sensitizing cancer cells to chemotherapy or radiotherapy. Colliding with this cell-autonomous vision, however, we found that immunosurveillance against transplantable, carcinogen-induced or genetically engineered cancers can be improved by pharmacologically inducing autophagy with caloric restriction mimetics. This positive effect depends on autophagy induction in cancer cells and is mediated by alterations in extracellular ATP metabolism, namely increased release of immunostimulatory ATP and reduced adenosine-dependent recruitment of immunosuppressive regulatory T cells into the tumor bed. The combination of autophagy inducers and chemotherapeutic agents is particularly efficient in reducing cancer growth through the stimulation of CD8⁺ T lymphocyte-dependent anticancer immune responses.     

Autophagy,senescence and tumor dormancy in cancer therapy

The relationships between autophagy and apoptosis have been examined quite extensively and have often been shown to be reciprocally regulated responses to stresses such as exposure of the tumor cells to chemotherapeutic drugs and radiation. However, there is now evidence that autophagy may also play a role in tumor dormancy. Given that tumor dormancy and disease recurrence are poorly understood phenomenon which are nevertheless critical elements of patient morbidity and mortality, this commentary develops the postulate that autophagy and senescence may be related responses that influence the capacity of the tumor cell to maintain a prolonged state of growth arrest that can be succeeded by tumor regrowth and disease recurrence.     

肿瘤细胞的自噬和窖蛋白-1

自噬作用是一种细胞通过溶酶体自我降解的过程,在肿瘤的形成和发展中起着双重作用。窖蛋白-1(Caveolin-1,Cav-1)作为胞膜窖的标志蛋白,介导许多生理和病理过程,包括胞膜窖的形成、膜泡运输、维持细胞胆固醇稳态、信号转导和肿瘤的发生。近年来,许多研究表明肿瘤细胞自噬和Cav-1存在一定关系。文中就近年来肿瘤细胞的自噬与Cav-1的关系及其在肿瘤发生和发展过程中的作用进行综述。     

Epiregulin is Up-regulated in pancreatic cancer and stimulates pancreatic cancer cell growth

Epiregulin belongs to the epidermal growth factor (EGF) family of polypeptides. Previous studies have underscored the important role of the EGF family of ligands and receptors in the pathology of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). It is not known, however, whether epiregulin may also have a role in these diseases. Therefore, in the present study we investigated the expression and function of epiregulin in five pancreatic cancer cell lines and in PDAC and CP tissue samples. Epiregulin mRNA was present at high (MIA-PaCa-2 cells) or moderate levels (ASPC-1, CAPAN-1, and T3M4) in most cells, but was below detection levels in PANC-1 cells. All the cell lines exhibited a dose-dependent increase in growth in response to recombinant human epiregulin. Epiregulin mRNA levels were increased 2.1-fold in PDAC samples (P < 0.01) and 1.7-fold in CP samples (P < 0.01), when compared with the normal controls. There was no correlation between epiregulin mRNA levels and tumor stage or grade. By in situ hybridization, a moderate to intense epiregulin mRNA signal was present in most pancreatic cancer cells in PDAC. In contrast, only a weak (normal pancreas) to moderate (CP) signals were present in the ductal and acinar cells in CP. These findings suggest that epiregulin may contribute to the pathobiology of PDAC, and may also have a role in CP.     

Autophagy regulation in pancreatic acinar cells is independent of epidermal growth factor receptor signaling

Autophagy is an intracellular degradation system in eukaryotic cells that occurs at a basal level. It can also be induced in response to environmental signals including nutrients, hormones, microbial pathogens, and growth factors, although the mechanism is not known in detail. We previously demonstrated that excessive autophagy is induced within pancreatic acinar cells deficient in Spink3, which is a trypsin inhibitor. SPINK1, the human homolog of murine Spink3, has structural similarity to epidermal growth factor (EGF), and can bind and stimulate the EGF receptor (EGFR). To analyze the role of the EGFR in pancreatic development, in the regulation of autophagy in pancreatic acinar cells, and in cerulein-induced pancreatitis, we generated and examined acinar cell-specific Egfr-deficient (Egfr^−/−) mice. Egfr^−/− mice showed no abnormalities in pancreatic development, induction of autophagy, or cerulein-induced pancreatitis, suggesting that Egfr is dispensable for autophagy regulation in pancreatic acinar cells.