Pancreatic cancer accounts for 3% of all cancers and approximately 7% of all cancer-related deaths in the USA. The survival rates of pancreatic cancer patients are low relative to those of other cancers that are considered incurable (Cameron
It is well documented that the KRAS gene is mutated in approximately 90% of pancreatic cancer patients and works in an early and initiating event, which is sufficient to drive premalignant lesion formation into pancreatic cancer (Biankin
Oncogenic KRAS drives the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways, which are important for proliferation, survival, and tumorigenesis in pancreatic cancer (Fresno Vara
Therefore, in the present study, we developed inRas37 KRAS-targeting antibody that demonstrated a strong binding affinity to the KRAS-GTP form and investigated whether inRas37 could enhance the antitumor effect of BEZ-235, a PI3K inhibitor, by overcoming MAPK pathway reactivation in pancreatic cancer.
Human MIAPaCa-2 and PANC-1 pancreatic cancer cells were purchased from the American Type Culture Collection (ATCC, VA, USA). MIA PaCa-2 and PANC-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Gyeongsan, Korea) with 1% antibiotic-antimycotic and 10% fetal bovine serum (FBS). Cultures were maintained in an incubator with 95% air and 5% CO2 at 37°C.
Based on our previous study, plasmids generating heavy chain (pciw3.4-inras37-HC) and light chain (pciw3.4-inRas37-LC) were produced.
The 96-well Nunc MaxisorpTM ELISA plates (Nalgene Nunc, NY, USA) were coated for 1 h at 37°C with inRas37 and inCT37 (1, 10, and 100 nM), washed with washing buffer (Tris-buffered saline with 0.1% Tween 20 [TBST] and 10 mM MgCl2, pH 7.4), and then blocked with blocking buffer (TBST, 10 mM MgCl2, 4% BSA, pH 7.4) for 1 h at room temperature (RT). After washing, His-fused KRASG12D-GppNHp (1, 10, and 100 nM) and His-fused KRASG12D-GDP were incubated in each wells for 1 h at 37°C. After washing, bound proteins were detected by labeling with horseradish peroxidase (HRP)-conjugated goat anti-His antibody (Sigma Aldrich, MO, USA) and washed. Subsequent incubation with ultra TMB-ELISA solution (Thermo Fisher Scientific) was performed for 1 min, and then stopped with stop buffer (1 M H2SO4). The plate absorbance was read at 450 nm using a microplate reader (BioTek Instruments, VT, USA).
MIA PaCa-2 and PANC-1 cells were seeded at 8×102 cells/well in 94-well ultra-low attachment plates (Falcon, NY, USA) and were treated with inRas37 (0, 2, and 5 μM) and/or BEZ-235 (50 nM) every 2 days for 1 week. Subsequently, 13.5 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution (Promega, Madison, WI, USA) was added to each well and incubated for 3 h at 37°C. Absorbances were read at 490 nm using a microplate reader (BioTek Instruments). The MTS assay was performed in triplicate.
Human pancreatic cancer cells were seeded at 1×103 cells/well in ultra-low attachment round 96-well plates (Falcon) and were treated with inRAS37 and BEZ235 every 2 days for 1 week, followed by MTS solution at a 1:10 dilution in total volume for 4 h at 37°C. The absorbance was measured at 490 nm using a microplate reader (BioTek Instruments).
MIAPaCa-2 and PANC-1 cells were washed with Dulbecco’s phosphate buffered saline (DPBS) and lysed with RIPA buffer (Biosesang, Korea) containing 1% Triton X-100, Xpert protease inhibitor, and phosphatase inhibitor Cocktail (GenDEPOT, TX, USA). Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, MA, USA). Protein transfer was verified using the Ponceau S staining solution (Amresco, OH, USA), and the blots were then incubated with the appropriate primary (1:500, except for β-actin [1:10,000]) and the secondary antibodies (1:1,000, except for β-actin [1:20,000]) conjugated to HRP. Antibody binding was detected using an enhanced chemiluminescence reagent (Bio-Rad, CA, USA) using primary antibodies specific to the proteins of interest, and the proteins were detected using X-ray film and enhanced chemiluminescence reagent. Primary antibodies were used against the following: p-ERK, ERK, p-AKT, AKT, and β-actin (Cell Signaling Technologies, MA, USA) and p-BRAF (Santa Cruz Biotechnology, TX, USA), and the secondary antibodies were purchased from Cell Signaling Technologies.
MIA PaCa-2 and PANC-1 cells were seeded in 6-well plates at a density of 0.8×106 and 1.5×106 cells/well, respectively. After 24 h of incubation at 37°C, a straight scratch was made on the wells using a cell scratcher for the 6-well plate (SPL, Gyeonggi, Korea). The cells were then washed three times with PBS and further treated with inRas37 (1 µM) and/or BEZ-235 (30 nM) in DMEM with 10% FBS and 1% antibiotic-antimycotic. After incubating for 72 h, the gap size on the wells was measured and recorded, and then compared with the initial gap size at 0 h. Using the Image J software (LOCI, WI, USA), the cells that moved over the scratched line were counted.
The invasion assay was performed in a 24-well plate with 8.0-μm pore size transwell (Corning). Inserts were coated with 10% Matrigel for 24 h at 37°C before cell seeding. Then, 5×104 MIA PaCa-2 and PANC-1 cells were seeded on the insert with serum-free medium treated with inRas37 (1 µM) and/or BEZ-235 (30 µM). The lower chambers were filled with DMEM complete medium (10% FBS and 1% antibiotics). After 72 h incubation at 37°C, the cells that passed through the 0.8-µm pore were stained with 0.5% crystal violet. The invaded cells were photographed at 100× and counted (per microscopic field).
MIA PaCa-2 cells (7×106 cells/mouse) were implanted in the right flanks of 4-weeks-old male BALB/c nude mice (Orient Bio, Seoul, Korea) weighing 20 g. All animal experiments were approved by the Animal and Ethics Review Committee of Inha University (Incheon, Korea) and were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee. When tumor size reached approximately 50-100 mm3, the mice were divided into four groups. Mice were administered 30 mg/kg BEZ-235 and/or 30 mg/kg inRas37 intravenously three times a week for 23 days. Tumor volumes and weights were measured three times a week. Tumor volumes were calculated as (short axis)2×(long axis)/2. At the end of the experiment, the mice were sacrificed, and the primary tumors were harvested.
TUNEL staining was performed in formalin-fixed, deparaffinized tumor tissue sections using an ApopTag peroxidase
Xenograft tumor tissue samples were fixed in 10% buffered formaldehyde at 4°C overnight, embedded in paraffin, and sectioned to obtain 8-µm-thick slices. Hematoxylin and eosin (H&E) staining were performed after deparaffinization. Finally, the pathological morphology of the tissue was examined in at least 3 randomly selected fields at 100× magnification.
Xenograft tumor tissue Immunostaining was performed after deparaffinization. Antigen retrieval was performed by heating in citrate buffer (pH 6.0) for 20 min. Next, the sections were permeabilized with Triton X-100 for 15 min and endogenous peroxidase activity was quenched using 0.3% H2O2 for 10 min at RT. Sections were gently washed with distilled water, blocked with CAS block solution (Zymed Laboratories, CA, USA) for 1 h at RT, and incubated with 1:50 dilutions of primary antibodies (α-SMA (Sigma Aldrich) and p-AKT, fibronectin, and collagen I (Abcam, Cambridge, UK)) at 4°C overnight. After gently washing twice with PBS, tumor sections were incubated with 1:100 dilutions of secondary antibody conjugated to FITC or TEXAS RED for 1 h at RT and stained with 4,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich) to visualize the nuclei. After gently washing twice with PBS, the slides were covered with mounting solution (Vectashield, CA, USA) before being viewed under a confocal laser scanning microscope (Olympus).
Statistical significance was determined using analysis of variance (ANOVA) or unpaired Student’s t-test. Results are presented as the mean ± standard deviation (SD), and
We previously reported the KRAS-targeting antibody RT11-i which recognizes integrin αvβ3/5 on the cancer cell surface (Shin
To investigate the inRas37 targeting efficiency for GTP-bound KRAS active form, we used ELISA using non-hydrolyzable GTP analogs (KRASG12D∙GppNHp) that bind to the active form of human KRASG12D mutants and the inactive form of GDP (KRASG12D∙GDP). inRas37 strongly bound to the active form of KRAS (KRASG12D∙GppNHp); however, it did not bind to the inactive form of KRAS (KRASG12D∙GDP; Fig. 1B). Moreover, as a control antibody, inCT37 containing the same components as inRas37 without KRAS-binding activity did not bind to active form of KRAS. These results indicated that inRas37 selectively bound to the active form of KRAS∙GTP.
Next, we investigated whether inRas37 has anticancer effects on pancreatic cancer cells with KRAS mutations. Previously, we found ανβ3 and ανβ5 to be highly expressed in various pancreatic cancer cells (Kang
In previous study, a dual PI3K/mTOR inhibitor, BEZ-235, was reported to inhibit cell growth and proliferation in various cancer cells (Serra
KRAS drives tumor progression by regulating the MAPK and AKT signaling pathways, which are highly activated in human pancreatic cancer cells with KRAS mutation, which involve cell proliferation, survival, and metastasis (Hancock, 2003; Keleg
Pancreatic cancer is characterized by metastasis via migration and local invasion into surrounding tissues, and KRAS, in particular, drives invasion and maintains metastasis (Keleg
To evaluate the therapeutic potential of inRas37 and BEZ-235 co-treatment
Pancreatic cancer patients have few viable therapeutic options. It is clear that KRAS mutations frequently occur and are early drivers of pancreatic cancer progression. KRAS accelerates the activity of the RAF/MEK/ERK and PI3K/AKT pathways, which are the drivers of tumor growth in the majority of pancreatic cancer (Jones
Antibody drugs have the advantages of long half-life in human blood and less toxicity to normal cells, which offer targeted therapy in pancreatic cancer compared with cytotoxic drugs or small molecule inhibitors (Scott
BEZ235 inhibits multiple class I PI3K isoforms and mTORC1/2 kinase activity, exerts potent anticancer activity, and attenuates PI3K reactivation and mTORC2-mediated AKT reactivation (Chiarini
In conclusion, MEK reactivation by BEZ-235 was inhibited by a combination treatment with inRAS37, a KRAS-targeting antibody. In addition, this combination synergistically suppressed pancreatic cancer cell growth
This research was supported by the National Research Foundation (NRF) Grant (2021R1A2B5B03086410, 2021R1A5A2031612, 2019M3E5D1A02069621), Republic of Korea.
The authors declare that they have no conflict of interest.