Original ArticlesSynergistic antitumor effects of radiation and proteasome inhibitor treatment in pancreatic cancer through the induction of autophagy and the downregulation of TRAF6
Introduction
Approximately 30–40% pancreatic cancer patients are diagnosed with locally advanced and not curatively resectable cancer; therefore, therapeutic strategy focuses on the use of different chemotherapeutic combinations or chemoradiation (CRT) [1], [2]. However, pancreatic cancers are among the most intrinsically resistant tumors regarding both ionizing radiation (IR) and chemotherapeutic drugs [3]. Within the context of CRT, the optimal technique for pancreatic irradiation is not clearly defined. Delivery of an adequate IR dose to the pancreas is limited by the radiosensitivity of normal organs in the upper abdomen [1]. Therefore, treatment with chemical modifiers as radiosensitizers in combination with low-dose IR may augment overall therapeutic efficacy. The ubiquitin–proteasome system (UPS) plays a major role in cell homeostasis. UPS is a multistep enzymatic cascade in eukaryotes whereby ubiquitin is conjugated via a lysine residue at position 48 to target proteins for degradation [4]. In the first step of this cascade, ubiquitin-activating enzyme (E1) binds ubiquitin. Ubiquitin is then transferred to ubiquitin-conjugating enzyme (E2). In the final step, ubiquitin ligase (E3) helps transfer ubiquitin to the protein substrate [5]. It is worth mentioning that proteins tagged with lysine 63-linked chains of ubiquitin are not marked for proteasomal degradation. These forms of ubiquitination alter protein function or localization and thereby regulate signaling activation including receptor endocytosis, protein trafficking, and DNA repair [4], [6]. Furthermore, as the proteasome regulates the growth and survival of tumor cells, it represents a potential therapeutic target for the treatment of cancer [7]. Proteasome inhibition leads to the accumulation of misfolded proteins, which results in endoplasmic reticulum (ER) stress, unfolded protein response (UPR) and apoptosis [7], [8]. There are 3 main pathways of ER stress-induced UPR: the inositol-requiring enzyme 1 (IRE1) pathway, the PKR-like ER-resistant kinase (PERK) pathway, and the activating transcription factor 6 (ATF6) pathway [9]. A recent study has shown that mitogen-activated protein kinase (MAPK) signaling pathways play an important role in both ER stress and UPR [10]. ER stress induces JNK and p38 activation, which suggests that these MAPKs play a role in the ER stress response [11], [12].
There are 2 intracellular protein degradation pathways in eukaryotic cells: UPS and autophagy [13]. Inhibition of proteasomal activity or treatment with IR induces autophagy but not apoptosis in cancer cells [14], [15], [16]. Autophagy is a ubiquitous process for degrading misfolded proteins and damaged or old organelles that involves membrane formation followed by fusion of the vesicle with lysosomes [17]. It has been reported that an excess of autophagy induces cell death and may act as a tumor-suppressing mechanism [18]. Contrastingly, autophagy serves as an important mechanism to generate nutrients under cellular stress and may directly contribute to the cell survival following treatment with anti-cancer drugs. Autophagy may ultimately decrease the effect of anticancer therapies [13]. Therefore, autophagy appears to have a dual role in cancer therapy. Whether autophagy has pro-survival or pro-death effects depends on a variety of factors, including cancer cell type, phase and microenvironment. Therefore, live or dead is context dependent. However, in pancreatic cancer, the role of autophagy induced by combined proteasome inhibitor and IR treatment has not yet been fully determined.
It has been reported that K-RAS is mutated in nearly all human pancreatic cancer specimens [19], [20]. The most frequent among K-RAS mutations is glycine to aspartic acid at codon 12 (G12D), followed by glycine to valine at codon 12. Mutant K-RASG12D plays a critical role in the development of chemotherapy resistance and cancer progression for several types of cancer, including pancreatic cancer [21], [22]. Additionally, recent evidence has shown that K-RAS upregulates bone morphogenetic protein-7 (BMP-7) expression/secretion. Subsequently, BMP receptor activation results in the activation of tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β activated kinase-1 (TAK1) in colon cancer [23]. More recently, it was demonstrated that bortezomib (a proteasome inhibitor) induces proteasome-independent degradation of the TRAF6 protein in myelodysplastic syndrome; such as acute myeloid leukemia cell lines and primary cells. Notably, TRAF6 is necessary for maintaining the survival of cells, and its degradation by bortezomib-induced autophagy contributes to cell death [24]. However, whether TRAF6 plays a critical role in the control of autophagy in pancreatic cancer cells remains undetermined.
In the present study, we investigated the anticancer effect of combined IR and MG132 proteasome inhibitor treatment on human pancreatic cancer cells both in vitro and in an orthotopic mouse model. Following combined treatment, the mechanisms of cell death induced by combined treatment were analyzed in human pancreatic cancer cells. Furthermore, the interplay between autophagy and TRAF6 was also examined.
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Cell culture
Human pancreatic cancer cell lines MIA PaCa-2 (ATCC CRL-1420), PANC-1 (ATCC CRL-1469) and BxPC-3 (ATCC CRL-1687) were obtained from the American Type Culture Collection (ATCC). Human pancreatic duct epithelial (HPDE) cells were obtained from Dr. Y.S. Shan (Institute of Clinical Medicine and Department of Surgery, College of Medicine, National Cheng Kung University, Tainan, Taiwan) [25]. Details of cell culture are described in Supplementary Appendix S1: Materials and Methods.
Irradiation treatment and cell viability assay
IR was performed
Synergistic cytotoxicity and survival curves following treatment of pancreatic cancer cells with MG132 and IR either alone or in combination
We investigated the cytotoxic effect of MG132 or IR treatment on 2 different pancreatic cancer cell lines (Fig. 1A and B). MG132 and IR inhibited cell viability of both human pancreatic cancer cell lines (MIA PaCa-2 and PANC-1) in a dose- and time-dependent manner. Fig. 1C and E highlights the cell viability of MIA PaCa-2 and PANC-1 cells following treatment with MG132 and IR in combination. Significant enhancement of toxicity was observed in the combined treatment compared with MG132 and IR
Discussion
The role of autophagy in cancer cell death and cell survival remains highly controversial [36]. Accumulated evidence suggests that a low or moderate level of autophagy is protective, whereas a high level of autophagy promotes cell death via excessive degradation of molecules and organelles, such as catalase and mitochondria [38], [39]. Autophagic flux is defined as the quantity of degradable material transported from the autophagosome to the lysosome. Increased levels of autophagosomes could
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This study was supported by the Ministry of Science and Technology, Taiwan (MOST 103-2314-B-006-060-MY2), the Chi Mei Medical Center, Tainan, Taiwan (CMNCKU10316) and the Medical Science and Technology Research Grant, National Cheng Kung University Hospital (NCKUH-10307004 and NCKUH-10408011).
References (53)
- et al.
Radiotherapy technical considerations in the management of locally advanced pancreatic cancer: American–French consensus recommendations
Int. J. Radiat. Oncol. Biol. Phys
(2012) - et al.
Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-kappaB and pp38 mitogen-activated protein kinase
J. Biol. Chem
(2004) - et al.
Elevated gadd153/chop expression and enhanced c-Jun N-terminal protein kinase activation sensitizes aged cells to ER stress
Exp. Gerontol
(2004) - et al.
Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability
Am. J. Pathol
(2007) - et al.
Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes
Cell
(1988) - et al.
Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse
Cancer Cell
(2003) - et al.
TAK1 inhibition promotes apoptosis in KRAS-dependent colon cancers
Cell
(2012) - et al.
Cytotoxic effects of bortezomib in myelodysplastic syndrome/acute myeloid leukemia depend on autophagy-mediated lysosomal degradation of TRAF6 and repression of PSMA1
Blood
(2012) - et al.
A gene expression signature of epithelial tubulogenesis and a role for ASPM in pancreatic tumor progression
Gastroenterology
(2013) - et al.
Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors
Adv. Enzyme Regul
(1984)
ATM blocks tunicamycin-induced endoplasmic reticulum stress
FEBS Lett
Progress in the unraveling of the endoplasmic reticulum stress/autophagy pathway and cancer: implications for future therapeutic approaches
Drug Resist. Updat
Autophagic clearance of aggregate-prone proteins associated with neurodegeneration
Methods Enzymol
Methods in mammalian autophagy research
Cell
Modulation of endoplasmic reticulum (ER) stress-induced autophagy by C/EBP homologous protein (CHOP) and inositol-requiring enzyme 1alpha (IRE1alpha) in human colon cancer cells
Biochem. Biophys. Res. Commun
Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages
Eur. J. Cancer
The implications of angiogenesis for the biology and therapy of cancer metastasis
Cell
Development and validation of automatic tools for interactive recurrence analysis in radiation therapy: optimization of treatment algorithms for locally advanced pancreatic cancer
Radiat. Oncol
AACR highlights: promise for treating pancreatic cancer
J. Natl. Cancer Inst
Novel proteasome inhibitors to overcome bortezomib resistance
J. Natl. Cancer Inst
The ubiquitin-proteasome pathway and its role in cancer
J. Clin. Oncol
Regulation of DNA repair by ubiquitylation
Nat. Rev. Mol. Cell Biol
Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis
Cancer Res
Bortezomib inhibits PKR-like endoplasmic reticulum (ER) kinase and induces apoptosis via ER stress in human pancreatic cancer cells
Cancer Res
Signal integration in the endoplasmic reticulum unfolded protein response
Nat. Rev. Mol. Cell Biol
The role of MAPK signalling pathways in the response to endoplasmic reticulum stress
Biochim. Biophys. Acta
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These authors contributed equally to this work.