Functional and mechanistic studies reveal MAGEA3 as a pro-survival factor in pancreatic cancer cells
In the era of personalized therapy, functional annotation of less frequent genetic aberrations will be instrumental in adapting effective therapeutic in clinic. Overexpression of Melanoma associated antigen A3 (MAGEA3) is reported in certain pancreatic cancer (PCA) patients. The major objective of the current study was to investigate the functional role of MAGEA3 in pancreatic cancer cells (PCCs) growth and survival.
Using overexpression (tet-on regulated system and constitutive expression system) and knockdown (by siRNA and shRNA) approach, we dissected the mechanistic role of MAGEA3 in pancreatic cancer pathogenesis. We generated MAGEA3 expressing stable PCA cell lines and mouse primary pancreatic epithelial cells. MAGEA3 was also depleted in certain MAGEA3 positive PCCs by siRNA or shRNA. The stable cells were subjected to in vitro assays like proliferation and survival assays under growth factor deprivation or in the presence of cytotoxic drugs. The MAGEA3 overexpressing or depleted stable PCCs were evaluated in vivo using xenograft model to check the role of MAGEA3 in tumor progression. We also dissected the mechanism behind the MAGEA3 role in tumor progression using western blot analysis and CCL2 neutralization.
MAGEA3 overexpression in PCA cells did not alter the cell proliferation but protected the cells during growth factor deprivation and also in the presence of cytotoxic drugs. However, depletion of MAGEA3 in MAGEA3 positive cells resulted in reduced cell proliferation and increased apoptosis upon growth factor deprivation and also in response to cytotoxic drugs. The in vivo xenograft study revealed that overexpression of MAGEA3 promoted tumor growth however depleting the same hindered the tumor progression. Mechanistically, our in vitro and in vivo study revealed that MAGEA3 has tumor-promoting role by reducing macro-autophagy and overexpressing pro-survival molecules like CCL2 and survivin.
Our data proves tumor-promoting role of MAGEA3 and provides the rationale to target MAGEA3 and/or its functional mediators like CCL2 for PCA, which may have a better impact in PCA therapy.
KeywordsMAGEA3 Pancreatic cancer CCL2 Survivin Autophagy Cancer testis antigen
Bovine serum albumin
Biological safety levels
Dulbecco’s modified eagle’s medium
European collection of authenticated cell cultures
Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
Foetal bovine serum
Hank’s balanced salt solution
Melanoma associated antigen A3
Disodium ethylene diamine tetraacetic acid
National center for biotechnology information
Pancreatic cancer cells
Polymerase chain reaction
Roswell park memorial institute medium
Sodium dodecyl sulfate
Pancreatic cancer (PCA) is of great concern for having six months of median survival period and five year survival of less than 5% . In various cancers including PCA, the known common genetic alterations are still unable to completely explain the oncogenic process, which leads to redefining the genetic basis of the cancer cells [1, 2, 3]. The common genetic alterations involved in pancreatic cancer include activating mutation in KRAS and inactivating mutations in TP53, CDKN2A and SMAD4. In addition to the above common genetic changes, recently different set of genes are also reported to be associated with PCA pathogenesis [1, 2].
Expression of a group of germ cell genes, otherwise known as cancer-testis (CT) antigens are reported in different cancers. Their ectopic expression, in cancer cells is believed to be due to global demethylation during the course of cancer initiation and/or progression and also associated with the disease pathogenesis [3, 4, 5, 6, 7]. Identification of unique cancer-associated genes, which do not express in noncancerous somatic cells, may help to develop immunotherapy against pancreatic cancer . Recent studies have also shown the functional significance of certain CT-antigens in different cancers [9, 10]. The less frequent but cancer-specific genes are now of great importance in designing personalized therapy [11, 12]. One of the most frequently reported germ cell genes across various cancer types (including PCA) is melanoma associated antigen A3 (MAGEA3) . In the past, various studies have shed importance on the functional characterization of the cancer-specific gene MAGEA3; however, its functional role in PCA is yet to be elucidated [14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28].
Cancer cells possess the ability to reprogram their metabolism, which helps them to survive and proliferate under hostile condition . During the transformation process, oncogenes help cancer cells to alter metabolism, and also make the cancer cells dependent on the altered metabolism provided by them (oncogenic dependency) [30, 31]. The oncogenes, involved in metabolic reprogramming in cancer are of great importance in developing targeted therapy [32, 33]. Multiple anti-cancer drugs targeting metabolism are in use and some are under trials . Previous studies have shown the role of MAGEA3 in cancer cells metabolism [16, 17].
Overexpression of MAGEA3 has been reported in multiple malignancies including PCA [34, 35, 36, 37, 38, 39, 40]. Unlike malignancies such as melanoma, the frequency of MAGEA3 overexpression in PCA has been reported as moderate, which supports the known low immunogenic properties of PCA cells [41, 42]. In the recent past studies have shown potential pro-tumorigenic role of MAGEA3 in different other malignancies [15, 16, 23, 43, 44]. The poor prognosis of patients is also reported to be associated with MAGEA3 expression in different cancers including pancreatic cancer [5, 45, 46]. In the current study, we report the mechanistic role of MAGEA3 in pancreatic cancer cells (PCCs) growth and survival in the presence and absence of growth factor (GF). Moreover, the study provided experimental evidence that suggests the role of MAGEA3 in conferring chemo-resistance to PCCs.
Materials and methods
The cell lines used in this study are LNCap, BxPC3, AsPC1, MiaPaCa2, PANC1, SW1990, and HEK293T. Pancreatic cancer cell lines were procured through Sigma-Aldrich (ECACC authenticated cell lines) and were confirmed through STR profiling. Cells were routinely screened for mycoplasma contamination and were grown in a sterile humidified chamber with 5% CO2 and at 37 °C. LNCap and BxPC3 cells were maintained in RPMI media containing 10% FBS (Invitrogen), 100 units/mL penicillin and 100 mg/mL streptomycin; however AsPC1, MiaPaCa2, PANC1, SW1990 and HEK293T were grown in DMEM supplemented with above-mentioned concentration of FBS, penicillin and streptomycin.
Knockdown studies were conducted using siRNA transfection (Additional file 1: Table S2) and shRNA-stable cells. siRNA was transfected using RNAiMax (Additional file 1: Table S4) following manufacturers instruction. shRNA stable cells were generated using lentiviral particles of Lenti-sh-MAGEA3si1 or Lenti-sh-Contol.
Isolation, characterization and genetic manipulation of mouse pancreatic epithelial cells
Untransformed cell lines like HEK293, NIH3T3 and immortalized primary epithelial cells are of instrumental in evaluating the function of a gene, especially in oncogenic context. In our study, we isolated and cultured mouse pancreatic epithelial cells using an established protocol . Briefly, after harvesting the mouse pancreas in aseptic condition, the same was digested with collagenase type V enzyme solution (0.1 mg/mL) at 37 °C for 20 min. The enzymatic reaction was stopped; the cell pellet was washed and resuspended in culture media. The cells were seeded onto collagen-coated plates to favor the epithelial cells and grown in the presence of specific growth factors required for epithelial cells (Additional file 2: Figure S1a and S1b). The homogenous population of epithelial cells was obtained after third passage (Additional file 2: Figure S1c). The isolated cells, were confirmed for their epithelial origin by using E-cadherin as a marker (Additional file 2: Figure S1d) and manipulated with an expression construct of GFP or mouse KRASG12D-HA or humanMAGEA3-HA or humanMAGEA3. The stable mouse primary pancreatic epithelial cells expressing above proteins (Additional file 2: Figure S1e and S1f) were established using puromycin (3 μg/mL) selection.
Spheroids of control cells or MAGEA3 overexpressing stable cells were generated by hanging drop method in the presence or absence of doxycycline. After 72 h, the formed spheroids were transferred to 0.6% low melting agar-coated plates (to prevent attachment) and incubated at 37 °C, 5% CO2 with desired experimental conditions followed by further analysis.
RNA isolation and cDNA synthesis
Total RNA was isolated from different cell lines after completing the appropriate duration of experiments using RNA easy kit following manufacturers instruction. DNA contamination was eliminated during RNA isolation by on-column DNA digestion using RNase-free-DNase set. Total RNA from different samples were quantified using UV-visible spectrophotometer (Eppendorf). Two microgram of RNA was used for cDNA synthesis using High capacity cDNA synthesis kit (Life Technologies).
Quantitative PCR analysis
Gene expression analysis was done in LC-480 (Roche) platform. The used gene-specific primers (Additional file 1: Table S1) were procured from Euorofins Genomics.
Generation of MAGEA3 overexpression construct
Non-Viral Constitutive expression system
The human (hu) MAGEA3 complete coding sequence (gi|16,877,053|gb|BC016803.1) was obtained from the NCBI database and primers with proper restriction sites were designed. To amplify MAGEA3, mRNA was isolated from LNCaP cells and cDNA was prepared. Then using the following primers the MAGEA3 whole coding sequence was amplified.
EcoRV-huMA3F- 5′ GCGGATATCCATCATGCCTCTTGAGCAG and XhoI-huMA3-ST-R- 5′ GCGCTCGAGTCATCACTCTTCCCCCTCT or XhoI-huMA3-NST-R- 5′ GCGCTCGAGCTCTTCCCCCTCT).
The amplified product was gel-purified, ligated in pCR-Blunt II-TOPO (AmpR) vector and transformed. The positive clones were screened through colony PCR and restriction digestion and then sequenced before sub-cloning into the pCMV-3tag-3A expression vector. The gene was sub-cloned into pCMV-3tag-3A expression vector (NeoR) between the EcoRV and XhoI restriction enzyme sites. The gene was cloned into pCMV-3tag-3A vector with or without Flag-tag. The clones were screened through restriction digestion and positive clones were again sequenced to confirm the right orientation and ORF of the target gene.
Constitutive and Tetracycline regulated (Tet-On) lentiviral expression system
Human MAGEA3 cds was cloned into pLenti-CMV-Puro-Dest vector (constitutive promoter) or pSIN-TRE-Lenti (TRE: tetracycline response element; tetracycline-regulated promoter) and muKRASG12D cds was cloned into pLenti-CMV-Puro-Dest vector (constitutive promoter) using gateway cloning strategy. Primers were designed along with the recombination sites (attB1 or attB2) flanking the gene-specific sequences. The primers used were listed (Additional file 1: Table S1). The PCR products attB1-MAGEA3-ST-attB2, attB1-MAGEA3-HA-attB2 and attB1-muKRASG12D-HA-attB2 were amplified using Platinum Pfx proofreading enzyme PCR kit (Invitrogen); the PCR products were analyzed on 1% agarose gel, specific desired bands were excised and purified using gel purification kit (GE). 150 ng of purified PCR products were recombined to pDONR221 using BP clonase reaction mix (Invitrogen) at 25 °C for overnight. The reaction was stopped by incubating with proteinase K at 37 °C for 10 min. The products were transformed into Stbl3 chemically competent E.coli cells and plated on agar plates containing 50 μg/mL of kanamycin. After overnight incubation at 37 °C, the single colonies on agar plates were picked and inoculated (LB broth media, 50 μg/mL kanamycin) for plasmid isolation. The isolated donor plasmids were quantified and sequenced. The donor plasmids containing attL1-MAGEA3-ST-attL2, attL1-MAGEA3-HA-attL2 or attL1- muKRASG12D-HA-attL2 were recombined with the destination vector pLenti-CMV-Puor-Dest  or pSIN-TRE-Lenti (having attR1---attR2) using LR clonase reaction mix (Invitrogen). 150 ng of each plasmid was taken for recombination reaction and the reaction was set for overnight at 25 °C. After stopping the reaction with proteinase K as described above, the products were transformed into Stbl3 competent cells, plated on agar plates containing 100 μg/mL of ampicillin and incubated at 37 °C for overnight. The single colonies were inoculated into LB broth (100 μg/mL, ampicillin) media and incubated at 37 °C, 200 rpm for overnight followed by plasmid isolation and sequencing. From the sequencing results, we confirmed the cloning of human MAGEA3 cds with or without HA-tag into the pLenit-CMV-Puro-Dest constitutive lentiviral expression vector and tetracycline regulated expression vector pSIN-TRE-Lenti. The proper cloning of muKRASG12D-HA into pLenit-CMV-Puro-Dest constitutive lentiviral expression vector was also confirmed through sequencing. The sequence of siRNA and shRNA used in this study are mentioned (Additional file 1: Table S2).
Production of lentiviral particle of expression constructs
All lentiviral works were done in BSL2 facility after institutional biosafety committee approval (Institute of Life Sciences, Bhubaneswar). The generated tetracycline-regulated MAGEA3/MAGEA3-HA or constitutive MAGEA3/MAGEA3-HA/GFP/muKRASG12D-HA expression constructs were packaged into lentiviral particles using packaging plasmids PMD2.G and pCMVR8.74. The detailed procedure for generation of lentiviral particles of the above said constructs is as follows. About 1.5 × 106 number of HEK293T cells were seeded in 10 mL media (DMEM containing 10% FBS and 1% penicillin/streptomycin) into 10 cm tissue culture plate and incubated in a humidified incubator at 37 °C and 5% CO2. After 24 h of seeding, the media was changed; further, after 6 h, the cells were transfected with the expression constructs of MAGEA3 and packaging plasmids (PMD2.G and pCMVR8.74) using CalPhos™ Mammalian Transfection Kit (TaKaRa) as per the manufacturer’s instruction. Briefly for a single transfection reaction, 10 μg of target gene expression construct, 4 μg of PMD2.G and 6 μg of pCMVR8.74 were taken in an eppendorf tube, to which 293 μL of 0.1X TE buffer (pH -8) was added following the addition of 155 μL of dH2O into the cocktail mix. The above mix was vortexed and 50.2 μL of 2.5 M CaCl2 was added and again under vortexing condition 506 μL of 2xHBSS was added dropwise. The cocktail mix was vortexed well and kept at RT undisturbed for 5 min. After 5 min the mixture was added to the HEK293T cells dropwise and the cells were incubated for 18 h at 37 °C in a humidified incubator supplied with 5% CO2. After 18 h, 10 mL of fresh complete media was added to the transfected cells and incubated at 37 °C in a humidified incubator supplied with 5% CO2 for 24 h. After 24 h the culture media was collected and filtered through 0.45 μm syringe filter. 3 mL (1/3rd of the filtrate) of Lenti-X concentrator (TaKaRa) was added to the filtrate, mixed by gentle inversion and incubated overnight at 4 °C, followed by centrifugation at 1500 x g for 45 min at 4 °C to obtain an off white pellet. The pellet was reconstituted in 200 μL of incomplete DMEM media, aliquoted into 20 μL in eppendorf tubes and stored at − 80 °C till use.
Proliferation assay and cell viability assay
To study the growth pattern/proliferation rate of the cancer cells MTT assay was done. For MTT assay cells were seeded at a density of 800 cells/well in 96 well tissue culture plates, grown for indicated days in presence or absence of doxycycline. MTT was added every day to three wells of each condition and incubated at 37 °C for 4 h, the crystal was dissolved in DMSO and the absorbance was taken at 570 nm. The graph represents the growth rate.
In case of spheroids, the number of viable cells present per spheroid was determined by dissociating the spheroids followed by counting on haemocytometer after trypan blue staining.
Crystal violet assay
For crystal violet assay, 50 thousand cells were seeded per well of 6 well plate or 35 mm culture dish. After the appropriate experimental duration, cells were stained with 0.5% crystal violet solution, dried, imaged; crystal violet was dissolved in 10% acetic acid and quantified by taking absorbance at 540 nm.
Annexin-V and PI were used to detect apoptotic cell death. Cells, after the appropriate duration of treatment or condition were collected and washed with PBS. The cells were stained with annexin-V-FITC only, PI only or annexin-V-FITC + PI solution and analyzed in flowcytometer (BD FACS Calibur).
Protein lysate was prepared in RIPA buffer (Radio-Immuno-Precipitation Assay buffer; 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin) containing protease inhibitor and phosphatase inhibitor and stored at − 80 °C until use. Proteins were first separated in SDS-polyacrylamide gel and then transferred to 0.45 μm PVDF membrane. The blots were incubated with desired primary antibodies (Additional file 1: Table S3) at 4 °C for overnight in gentle shaking followed by one hour incubation with peroxidise-conjugated secondary antibody at room temperature and then the bands were detected in chemidoc or X-ray film using ECL substrate.
Cells were seeded on poly-L-lysine coated coverslips placed in 35 mm tissue culture plates and after the end of the experimental condition, the cells were washed with PBS followed by fixation and permeabilization using ice-cold methanol:acetone (1:1) at − 20 °C for 10 min. For E-cadherin, the cells were fixed with 4% formalin but not permeabilized. Then, after washing with PBS the cells were blocked with 2% BSA (in PBS) for 30 min at room temperature with gentle shaking. After blocking, the cells were incubated with primary antibody at 4 °C overnight. Then the coverslips were washed with PBS and incubated with secondary antibody conjugated with alexafluor 594 (red) or alexafluor 488 at RT for 45 min in dark. Finally, the coverslips were washed two times with PBS and incubated with Phalloidin-555 to stain the cytoskeleton and/or mounted with ProLong® Gold Antifade Mountant with DAPI (nuclear stain). The slides were visualized under fluorescence microscope (ApoTome.2, ZEISS) and images were captured.
Xenograft mouse model
The animal experiment reported in this study was approved from the institutional animal ethical committee (IAEC), Institute of Life Sciences, Bhubaneswar, India. The female nude mice were purchased from Vivo Bio Tech Ltd., Telangana, India and maintained in animal house facility at ILS. Two million cancer cells were injected subcutaneously on the rear flanks and grouped as indicated below. AsPC1-GFP D−/AsPC1-TRE-MAGEA3-HA D- (Left flank/Right flank), n = 5; AsPC1-GFP D+/AsPC1-TRE-MAGEA3-HA D+ (Left flank/Right flank), received 50 ng/mL doxycycline in drinking water, n = 5; AsPC1-pLentiMAGEA3-HA/AsPC1-pLentiMAGEA3 (Left flank/Right flank), n = 5. BxPC3-GFP/ BxPC3-MAGEA3 (Left flank/Right flank), n = 5; BxPC3-sh-ve/BxPC3-sh1 (Left flank/Right flank), n = 3. Tumor volume and body weight were recorded on every alternate day. Tumor volume was calculated using the formulae Volume (mm3) = (Length x Width2)/2. Part of each tumor was snap frozen and remaining were fixed in 10% buffered formalin solution for histological analysis. BrdU was injected into the peritoneum at 2 mg/20 g mice (100 mg/kg of body weight) one hour prior to the sacrifice of animals.
Tissue or spheroid processing and immunohistochemistry
After the end of experimental condition time point, spheroids were fixed in 10% buffered formalin at 4 °C for 48 h and then processed and embedded in paraffin and block was made. Sections of 4 μm thickness were made and processed for immunohistochemical staining. The tumors isolated from animals were kept in formalin for 7 days and then processed for embedding into paraffin blocks followed by sectioning and immuunohistochemical staining procedures as reported earlier . For BrdU staining, the tissue sections were deparaffinized, rehydrated followed by washing with PBST (PBS + 0.1% TritonX-100). After washing the sections were treated with proteinase K (20 μg/mL in PBS) followed by washing with PBS. Then the sections were incubated with 2 N HCl and then with sodium borate solution (0.1 M). Further, the sections were washed with PBS and treated with 3% H2O2. Again, the sections were washed with PBS and blocked with the mouse on mouse blocking solution. Then, the primary antibody against BrdU was applied and incubated overnight at 4 °C. Afterward, the sections were washed with PBS and incubated with biotinylated secondary antibody and processed with DAB based immunohistochemical detection methodology, which includes incubation with ABC reagent and developed with DAB. After staining and mounting with cover-slip, the slides were visualized under a microscope (Leica ICC500) and images were captured. The number of cells positive for staining were quantified using ImageJ software.
An equal number (2 × 105) of control and MAGEA3 stable cells were seeded onto 6 well culture plates and cultured with equal volume (3 mL) of culture media. After completion of the required experimental time period, the culture media (conditioned media, CM) were collected and assayed for the presence of human CCL2 using sandwich ELISA (R&D systems) following manufacturers instruction.
All the in vitro experiments were performed thrice and the data presented here were from a representative experiment as the mean ± standard error of the mean (SEM). The data were analysed statistically by Student’s t-test or two-way ANOVA using GraphPad prism 5.00 (GraphPad Software, Inc., San Diego, USA).
MAGEA3 expression in different parental pancreatic cancer cell lines and their derivatives
It has been reported that MAGEA3 is expressed in a sporadic pattern in tumor tissues, which limits the strategy of immunotherapy using MAGEA3 as a candidate [46, 50, 51]. We sought to check the expression status of MAGEA3 in different PCA cell lines. We observed almost undetectable level of MAGEA3 in AsPC1, MiaPaCa2, SW1990 and PANC1 pancreatic cancer cells both at mRNA (Additional file 3: Figure S2a) and protein level (Additional file 3: Figure S2b). However, we observed a high level of MAGEA3 in BxPC3 pancreatic cancer cells (Additional file 3: Figure S2a and S2b). The data shows cell line dependent expression pattern of MAGEA3 in PCA, and further supports the known sporadic nature of its expression in different other cancers.
To investigate the functional role of MAGEA3 in PCA, we adopted ectopic gene expression and silencing approaches. Previously, constitutive overexpression of MAGEA3 in different cancer cell lines has been used to investigate its functional role. Experimentally, constitutive or regulated overexpression of genes has their intrinsic strength and weakness. Hence, to have a convincing conclusion about the function of MAGEA3 in PCCs, we generated MAGEA3 overexpressing cell lines that express MAGEA3 in a constitutive or regulated fashion. Further to distinguish the ectopically expressed MAGEA3 from endogenous MAGEA3, we incorporated HA-tag at the C-terminal end of the MAGEA3 open reading frame. The stable cell line with inducible MAGEA3 construct (cell line name-TRE-MAGEA3/MAGEA3-HA) expressed MAGEA3 at basal level (Additional file 3: Figure S2c-f) and the level increased in response to doxycycline with a minimum dose of 50 ng/mL (Additional file 3: Figure S2c). All the experiments were carried out with 100 ng/mL of doxycycline for optimal MAGEA3 expression wherever required. Doxycycline of 100 ng/mL induced the MAGEA3 expression within 4 h of treatment and continues to induce the protein expression at least till 10 days if treated once (Additional file 3: Figure S2d). In generated stable cells, MAGEA3 was expressed in native form (Additional file 3: Figure S2e and S2g) or with a HA-tag at its C-terminal (Additional file 3: Figure S2c, S2d and S2g) and importantly both the forms were detected by anti-MAGEA3 antibody with a little shift of HA-tag form (Additional file 3: Figure S2g). Thus, we confirmed that the generated constructs effectively express MAGEA3 and is detectable through anti-MAGEA3 antibody. At the same time, to rule out the non-specific side effect of overexpression system and confirm the findings of the overexpression studies, we used siRNA/shRNA mediated knockdown of MAGEA3 in BxPC3 cells. We tried two siRNA sequences (Additional file 1: Table S2) targeting MAGEA3  and both the siRNA were equally efficient to reduce MAGEA3 expression in BxPC3 pancreatic cancer cells (Additional file 3: Figure S2h and S2i).
Ectopic MAGEA3 expression does not affect the pancreatic cancer cells’ growth but knockdown affects the viability of pancreatic cancer cells in vitro
MAGEA3 provides survival advantage to pancreatic cancer cells in growth factor deprived condition
MAGEA3 inhibits autophagy in growth factor deprived pancreatic cancer cells
MAGEA3 activates alternative survival pathway in growth factor deprived condition
Role of MAGEA3 in tumor progression in vivo
Targeting MAGEA3 enhanced cell death to therapeutics in vitro
In addition to the existing list of oncogenes/proto-oncogenes, recent reports on the identification of cancer-specific genes and their potential in cancer pathogenesis lead to the identification of new molecular targets [1, 2, 3, 4, 11]. For a long time, MAGEA3 has been on light for hope as cancer immunotherapeutic [63, 64]; however, recent findings emphasizes on its functional role in the pathogenesis of different malignancies [16, 20, 22, 27]. In the current study, we report the cancer-promoting/favoring role of MAGEA3 in PCA. Our findings suggest that the PCCs expressing MAGEA3 are dependent on it to survive and thrive both in growth factor enriched and growth factor depleted condition (Figs. 1 and 2). However the cells, those are negative for MAGEA3 expression, don’t depend on it when desired growth factors are available (Fig. 1), but in growth factor limiting condition, ectopic expression of MAGEA3 is able to help the cancer cells to survive (Fig. 2 and Additional file 4: Figure S3). The cell lines used in this study have different oncogenic KRAS and P53 status (BxPC3 has WT-KRAS and Mut-TP53; AsPC1, MiaPca2 and PANC1 have Mut-KRAS and Mut-TP53) ; however, in the current study, we have not experimentally explored the correlation between KRAS and P53 status with MAGEA3 function. Hence, future studies in these aspects might elucidate the influence of common genetic aberrations on MAGEA3 function in pancreatic cancer cells.
In cancer cells, impaired autophagy was reported to be an important protumorigenic mechanism that supports the metabolic switching of cancer cells and favors Warburg’s effect [15, 16, 20, 56, 57]. Though early autophagy inhibition during GF-deprivation may provide pro-survival gain; prolonged autophagy inhibition can be lethal during continuous energy stress [56, 57]. Our study demonstrates that MAGEA3 reduces the autophagy level in PCCs, which agrees with earlier reports [15, 16, 17, 20]. Importantly, the reduced autophagy level during growth factor limitation provided a pro-survival advantage to the MAGEA3 over-expressing PCCs. Cancer cells have been reported to produce and use important chemokines in survival signalling pathways, which can be a potential factor responsible for the survival of cancer cells in prolonged GF-deprivation [58, 59, 66, 67]. Upon ectopic expression of MAGEA3, CCL2 and survivin were found to be up-regulated; conversely, upon MAGEA3 depletion both CCL2 and survivin level was reduced. A previous report had already shown the integrated role of CCL2 and survivin in reducing autophagy and providing survival advantage [60, 61]. Acquisition of intrinsic properties to overcome various metabolic stresses including GF-deprivation is one of the essential hallmarks of cancer cells [68, 69]. In cell line based in vivo tumor models, although the cells that are used to generate tumors are already transformed, but in absence of proper angiogenesis (during the initial establishment of tumors and at the core region of the fast-growing tumors) many cancer cells encounter sustained GF-deprivation and succumb to death. In our study, the presence of more number of cancer cells in MAGEA3 overexpressing tumors might be due to better survival and/or proliferation of cancer cells in tumor tissues (Fig. 6d and e). Our in vitro studies have provided convincing evidence that shows the role of MAGEA3-mediated autophagy inhibition as a potential mechanism through which MAGEA3 directly promotes survival of GF-deprived PCCs in vitro. However, in the in vivo context, in addition to this direct effect, MAGEA3 might have also indirectly affected the overall tumor growth by modulating different stromal events like angiogenesis. CCL2 is known to promote angiogenesis in different cancers [70, 71, 72]; hence, MAGEA3-mediated CCL2 overexpression in pancreatic cancer cells (Fig. 4c-e and g) might have also indirectly contributed to the overall tumor growth in vivo, which warrants further investigation. We also found that MAGEA3 is able to upregulate survivin in mouse primary pancreatic epithelial cells (Fig. 4i), which indicates the possible oncogenic role of MAGEA3 and needs further investigation. Our study involves the molecule MAGEA3 which is highly similar to MAGEA6. Although MAGEA3 and MAGEA6 are two different gene products, but due to their high similarity at protein level (> 90%), in different literatures both the genes have been reported together (MAGEA3/6) . In the current study, we have used MAGEA3 coding sequence for its overexpression; however, the siRNA used by us might act on both the genes. Hence, we believe that the findings of the current study might also be true for MAGEA6 function in PCA.
Together, our study confirms the survival advantage provided by MAGEA3 during the hostile condition to PCCs. Moreover, the results of this study provide a rationale to target MAGEA3 and/or associated molecules like CCL2 for personalized PCA therapy. Further, we targeted MAGEA3 in PCCs and observed enhanced cytotoxic effect of various existing molecules upon MAGEA3 depletion but the cytotoxic effect is reduced when MAGEA3 is overexpressed in PCCs. Thus, it cautious the strategy to overexpress molecules like MAGEA3 in pancreatic cancer cells, which will make them more immunogenic like in other cancers [73, 74, 75, 76] but may have a serious negative consequence. In the future, along with existing chemotherapy, MAGEA3-targeted therapy can be explored for a better therapeutic approach against PCA.
Our study provides experimental evidence that suggests MAGEA3 is an important survival molecule for pancreatic cancer cells under metabolic and genotoxic stress conditions. The mechanistic study revealed that CCL2 and/or survivin are two possible functional mediators of MAGEA3. Thus, we propose that targeting MAGEA3 may have a better impact on PCA therapy.
We thank Dr. Sunil K Raghav for providing the tet-on empty vector and Dr. Rajeeb K Swain for providing pLentiCMV-Puro Dest empty vector and pLentiCMV-GFP control vector. We also thank Mr. Madan Mohan Mallick and Mr. Sushanta Kumar Swain for their efficient technical support. We acknowledge the director, Institute of Life sciences for supporting the project.
BD and SS designed and interpreted the experiments; BD performed the experiments; BD and SS analysed the data and wrote the manuscript. Both authors read and approved the final manuscript.
The project is funded by Department of Biotechnology (DBT; BT/PR13502/MED/30/1555/2015), Government of India.
Ethics approval and consent to participate
All animal procedures were approved by the Institutional Animal Ethical Committee, Institute of Life Sciences, Bhubaneswar, India.
Consent for publication
The authors declare that they have no competing interests.
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