The Molecular Mechanism Of K-ras Mutation Dependence Of The Crosstalk Between NF-κB And Hedgehog Pathways In PDAC

Background and aimsPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancy. Although many improvements have been made in anti-cancer therapy, the overall 5-year survival rate is still below 5%. Now, PDAC was still one of the poorest prognoses among all cancers, because of lacking understanding of its molecular mechanisms.As reported, many elements involved in PDAC formation, such as abnormal activity of Hedgehog (HH) signaling pathway, inflamation and K-ras mutation, and so on.Abnormal HH overactivation is an early contributor of PDAC. This signaling pathway involved in morphogenesis, and its aberrant activation is common in PDAC. HH signaling can be activated by two ways:ligands associated activation and mutation of its members caused activation. And in most cases, it is triggered by ligands. HH pathway has three ligands:Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh), among them, Shh is the dominant one in human. The nuclear translocation of the glioma-associated oncogene (Gli) family, transcription factors of HH signaling pathway, means HH pathway activation. In Gli family, Gli1 is the most important activator transcription factor, and the marker of HH activation.Inflammation has drawn more and more attention as central factors in the tumorigenesis of many cancers, PDAC included. Transcription nuclear factor-kappa B (NF-κB) is a critical member of inflammation. A variety of stimuli, such as interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), could realize NF-κB activation.K-ras mutations occurs very early and frequently in PDAC. Magliano and Logsdon pointed out, individual K-ras proteins function as binary molecular switches. Only when they bound to guanosine triphosphate (GTP), they can regulate cell activities. Normal Kras is rapidly inactivated due to the effect of the GTPase-activating proteins (GAPs), which help hydrolyze GDP to GTP. Mutations of the K-ras gene, produce a single amino acid change, which resulting in mutant Ras proteins that are insensitive to the function of GAP and stay constitutively active.The three factors above may interact with each other to promote the formation of PDAC. Although, some studies discussed the crosstalk between NF-κB and HH pathway, K-ras mutation was not included as variable. So in our researches, we focused on K-ras mutation’s role in the crosstalk between NF-κB and HH pathway. To further unveil the mechanism of PDAC process.We do the studies as follows:1. Testing the expression of HH pathway(Shh and Glil) and NF-κB pathway, K-ras mutation in PDAC tissues and cells, analyzing the correlation between the three factors.Methods and materials:We collected 32 PDAC tissues and 3 PDAC cell lines, testing Shh, nuclear Gli1 and NF-κB protein expression by IHC, qRT-PCR and western-blot; checking K-ras gene expression(codon 12 and 13) by DNA sequencing.Results:We found the positive correlation between Shh, Glil and NF-kB were exsited in PDAC tissues and cells, and meanwhile, this kind of connection was dependent on K-ras mutation to some extent.2. Observing regulating effect of NF-kB activation on HH pathway activation in PDAC cells, and analyzing their K-ras mutation dependence.Methods and materials:We choosing wild-type K-ras cell line BxPC-3 and mutant K-ras cell lines Panc-1 (codon 12 mutation) and SW1990 (codon 13 mutation) for further studies. We stimulated the cells by IL-1β and TNF-a (using pure culture medium as control), testing the expression of Shh, Glil and NF-κB by qRT-PCR and western-blot, meanwile testing the proliferation and apoptosis changes, and at last analysis the relationship with K-ras mutation.Results:Our data showed, in mutant K-ras cell lines, inflammatory factor stimulation resulted in NF-κB activation, and NF-κB activation promoted HH pathway activation, enhanced malignant biological behavior; but in wild-type K-ras cells, this intervening couldn’t get significant effect. Which means, in PDAC, there existed crosstalk between NF-κB and HH pathway, and it was somehow dependent in K-ras mutation.3. Observing regulating effect of HH activation on NF-κB pathway activation in PDAC cells, and analyzing their K-ras mutation dependence.Methods and materials:The same as Part 2, we transfected mutant K-ras cell line (SW1990) with Gli1 cDNA and stimulated all 3 cell lines with Shh, then we testd the expression of Shh, Glil and NF-κB by qRT-PCR and western-blot, meanwhile examined the proliferation and apoptosis changes, and at last analyzed the relationship with K-ras muation.Results:In mutant K-ras cell lines, intervening resulted in HH pathway activation, and HH pathway activation promoted NF-κB pathway activation, enhanced malignant biological behavior; but in wild-type K-ras cells, this intervening couldn’t get significant effect. Which means, in PDAC, there existed crosstalk between HH pathway and NF-κB, and it was somehow dependent in K-ras mutation.4. The molecular mechanism of K-ras mutation dependence of the crosstalk between HH pathway and NF-κB.Methods and materials:Based on the results of Part 2 and 3, we further discussed the molecular mechanism. We estimated K-ras protein expression and K-ras activities by detecting p-/t-ERK1/2 and p-/t-AKT1 expression, as well as Ras activity. Then we knocked-down K-ras expression of 3 cell lines, with si-Kras, and up-regulated mutant K-ras expression in BxPC-3 cells by mutant-K-ras cDNA (G12D) transfection to observe these effect on K-ras activities and the crosstalk between HH pathway and NF-κB, to further confirm K-ras mutation dependence of the crosstalk between HH pathway and NF-κB.Results:It turned out, changes in K-ras activities is the key molecular mechanism of K-ras mutation dependence of the crosstalk between HH pathway and NF-κB.5. K-ras mutation dependence of the crosstalk between HH pathway and NF-κB in animal models.Methods and materials:In view of the results of cell researches,we did animal studies to explore K-ras mutation dependence of the crosstalk between HH pathway and NF-κB. We got xenograft tumor from ①both normal SW1990 and Gli1 cDNA stable transfected SW1990,②SW1990, Panc-1 and BxPC-3, which were treated by PBS, Shh, IL-1β and TNF-a. Then we tested Shh, and nuclear Gli1 and NF-κB protein expression, disccussed the crosstalk between HH pathway and NF-κB, and its K-ras dependence. As well as their effect on tumor growth.Results:Animal experiments showed that HH pathway activation could active NF-κB and promoted tumor gowth; K-ras mutation dependence of the crosstalk between HH pathway and NF-κB were also existed in animals.Conclusions1. K-ras mutation, HH pathway activation and NF-κB activation were existed in PDAC; The positive correlation between HH pathway acitvation and NF-kB activation was based on mutant K-ras gene expression.2. NF-κB activation, and NF-κB induced HH pathway activation were based on mut-K-ras gene expression, in PDAC.3. Classic HH pathway activation, and HH pathway induced NF-κB activation were based on mut-K-ras gene expression, in PDAC.4. K-ras activity played an important role in classic HH pathway activation, NF-κB activation,and their interaction were based on K-ras mutant gene expression.5. In animal models, we proved that classic HH pathway activation, NF-κB activation,and their interaction were based on K-ras mutant gene expression.