MEK1 And MEK2 Isoforms Regulate Distinct Functions In Pancreatic Cancer Cells

IntroductionBy the time it is diagnosed, pancreatic cancer has often undergone extensive local invasion and/or metastasis, precluding a therapeutic surgical solution. Development of new molecular therapeutic methods targeting the invasion/metastasis-related factors is a promising avenue for improving the prognosis of patients with pancreatic cancer. However, the cellular and molecular mechanisms of invasion/metastasis of pancreatic cancer have not been fully elucidated.Two hamster pancreatic cancer cell lines with different potentials for invasion and metastasis after intra-pancreatic transplantation, PC-1 (low potential) and PC-1.0 (high potential), were established from a pancreatic ductal carcinoma induced by N-nitrosobis (2-oxopropyl) amine (BOP) in a Syrian golden hamster. In our previous study, gene expression differences between PC-1.0 and PC-1 cells were examined using the Representational Difference Analysis (RDA) method. We identified mitogen-activated protein kinase kinase 2 (MEK2) as a factor that was correlated with the invasion and metastasis potential of these cell lines. Further investigation confirmed that MEK2 was involved in invasion/metastasis of both hamster and human pancreatic cancer cells.MEK2 is a key kinase in the mitogen-activated protein kinase (MAPK) signal transduction pathway, which is involved in many cellular processes, including cell proliferation, differentiation, cell division, stress, and apoptosis. The MAPK pathways are evolutionarily conserved in all eukaryotes and can be organized into a three-kinase hierarchy:a MAPK, a MAPK activator (MAP kinase kinase, MKK or MEK), and a MAP kinase kinase activator (MAP kinase kinase kinase, MAPKKK) (10). MEK1 and MEK2 are the only two isoforms of the MEK family and are commonly reffered to as MEK 1/2. Their amino acid sequences are～85% identical and they are expressed ubiquitously in cell lines and tissues. Although it is commonly assumed that the two isoforms are functionally equivalent, several lines of evidence, however, indicate that they are regulated differentially and may exert non-redundant functions (11-13). Thus far, the individual roles of MEK1 and MEK2 in pancreatic cancer cells remain to be explored.ObjectiveIn this study, short-hairpin RNAs (shRNAs) expressed in a retroviral vector were used to specifically silence the expressions of MEK1 and MEK2 in the highly invasive and metastatic pancreatic cancer cell line PC-1.0 to establish the stable subclones of the PC-1.0 cells and to demonstrate the distinct functions regulation of MEK1 and MEK2 in pancreatic cancer cells.Materials and Methods1. MaterialsWe used two hamster pancreatic cancer cell lines:the weakly invasive and rarely metastatic cell line, PC-1, and the highly invasive and metastatic cell line, PC-1.0. The PC-1 cell line was established from pancreatic ductal/ductular adenocarcinomas induced by BOP in a Syrian golden hamster.We used RNAi (Clontech Laboratories, Inc., CA, USA) tool to design the shRNA sequences according to the sequences of the MEK1 and MEK2. We used RNAi-Ready pSIREN-RetroQ-Zsgreen plasmid to establish the MEK1 and MEK2 shRNA. To establish the stable subclones of the PC-1.0 cells expressed the MEK1 or MEK2 shRNA, We used GP2-293 packaging cell to infect them.We used fluorescence microscope to optimize the cells. These cell lines were incubated in RPMI-1640 (Gibco-BRL, Grand Island, NY), supplemented with 10% foetal bovine serum (Bioserum, Victoria, Australia),100 units/ml penicillin G, and 100μg/ml streptomycin at 37℃in a humidified atmosphere of 5% CO2 and 95% air. The cells were serum starved overnight before experiments.2. MethodsWe utilised shRNA-mediated knockdown of their two mRNAs individually. We used PT-PCR, Western blot to optimize the right cells within several ones.We studied the effects of MEK1 and MEK2 knockdown on proliferation, mitotic arrest, in vitro invasion capability and cell morphology in PC-1.0 cells.Results1. Construction of MEKl-shRNA and MEK2-shRNA plasmidsTo block endogenous MEK1 or MEK2, we tested three shRNA sequences against each gene in the RNAi-Ready pSIREN-RetroQ-Zsgreen vector. We found that all three shRNAs targeting either gene lowered endogenous MEK1 or MEK2 mRNA levels, respectively, with maximum reductions of 98.4%(MEKl-shRNA 3) and 77.5% (MEK2-shRNA 3). The MEK1-shRNA 3 and MEK2-shRNA 3 vectors also reduced MEK1 and MEK2 protein levels respectively, compared with the control vectors. Thus, these two vectors that achieved the greatest degree of knockdown were selected for the following experiments.2. Cellular morphology and growth patterns in MEK1-and MEK2-knockdown PC-1.0 cellsWe found that the MEK2-knockdown PC-1.0 cells grew in an aggregated or clumped pattern in culture. However, MEK1-knockdown cells showed no change in growth pattern compared with that of empty vector-infected PC-1 cells.3. Effects of MEK1 and MEK2 knockdown on proliferation of PC-1.0 cellsWe found that down-regulation of MEK1 inhibited proliferation and growth of PC-1.0 cells. In particular,48 h and 72 h after incubation, PC-1.0 cells showed markedly decreased proliferative activity, with inhibition rates of 62.0% and 60.5%, respectively, compared with the control cells (P<0.05). On the other hand, there was no statistically significant difference in the growth of PC-1.0 cells after down-regulation of MEK2 gene expression compared with the control cells (P> 0.05).4. Effects of MEK1 and MEK2 knockdown on cell-cycle phase in PC-1.0 cellsThe cell-cycle distributions of MEK1-knockdown and MEK2-knockdown PC-1.0 cells were analysed using a FACScan flow cytometer. In MEK1-knockdown cells, we found only 8.1±0.7% of cells in the S phase of mitosis. In contrast,26.6%±5.4% of control PC-1.0 cells were in S phase (P＜0.05), and there was no significant difference between the MEK2-knockdown cells and the control cells (P> 0.05).5. Effects of MEK1 and MEK2 knockdown on in vitro invasion in PC-1.0 cellsThe control cells exhibited a strong invasive capability (invasive cell number=52.6±5.8). On the contrary, MEK2 knockdown markedly inhibited the invasive capability of PC-1.0 cells compared with the control cells (invasive cell number=20.1±3.1, P＜0.05). However, down-regulation of MEKl showed no significant inhibitory effect on the invasive capability of PC-1.0 cells (invasive cell number=48.4±4.3, P> 0.05).Conclusions1. MEKl and MEK2 mediate different biological functions in pancreatic cancer cells. MEK1 was an effective and specific method of inhibiting cell proliferation and inducing G0/G1 arrest. Moreover, targeting MEK2 mRNA specifically disrupted cell morphology and reduced the invasive ability of cultured pancreatic cancer cells.2. MEK1 and MEK2 have the potential to serve as therapeutic agents in the treatment of pancreatic cancer.