Genomic Sequencing Of Key Genes In Pancreatic Cancer And Function Validation

Pancreatic cancer is an extremely malignant disease associated with poor prognosis.The5-year survival rate is less than5%in pancreatic cancer patients. Although moderateprogresses in diagnosis and therapy have been made, the statistics remains unchanged forthe past several years. It was estimated that44,030patients were diagnosed with pancreaticcancer in the U.S. in2010, and among these patients,37,660patients had died. Currently,surgical resection is the only curative treatment. Unfortunately, only about10-15%ofpatients are eligible for surgical resection and cancer recurrences for these patients arecommon. Consequently there is an urgent need for developing novel methods for earlydiagnosis and therapy of pancreatic cancer.Upon completion of the Human Genome Project, whole-genome sequencing hasushered in a new era of molecular medicine, revolutionizing the ideas of personalizedprevention, diagnosis and therapy in the biomedical field. This ground-breakingtechnology has provided scientists the tools to probe gene function in a more efficientmanner. Human Genome Project had sequenced approximately20,000-25,000genes,consisting of3billion base pairs of human DNA, and identified enormous amount ofmutations/Single Nucleotide Polymorphisms (SNPs). However, the application of the massdata, generated by whole-genomic sequencing, to cancer research still remains a challenge.In the past decade, the rapid development as well as refinement of sequencing technologiesnot only significantly reduced the sequencing turnaround time at lower cost, but alsoprovided a detailed analysis of the epigenome and transcriptome on a genome-wide scale.Moreover, a new phase of genomic research that allows decoding the genome beyondsequencing has appeared on the horizon to facilitate the understanding of cancerpathogenesis due to aberrant genetic programming. However, Cancer is a genetic disorder,and the aberrant genetic programming that impacts protein expression ultimately leads toimpaired cell function. Therefore, the whole-genomic sequencing may hold potential inunderstating the molecular mechanism and providing personalized therapy for cancerpatients in the near future.A number of mutations in key genes such as KRAS, SMAD4, CDKN2A (p16) andTP53(p53) have been identified in human pancreatic cancer. Discoveries of these mutatedgenes have provided important insight on the development of pancreatic cancer andsuggested new diagnostic and therapeutic strategies for the patients. Recent studies haveidentified new molecular targets in pancreatic cancer, whose overexpression promote pancreatic cancer progression, such as ZIP4and PDX-1genes. Our previous studiesindicated that ZIP4is upregulated in human pancreatic cancer, and silencing of ZIP4led todecreased pancreatic cancer growth and increased survival rate in a xenograft mouse model.PDX-1has been shown to exert oncogenic properties in pancreatic cancer. Those datastrongly suggest that ZIP4and PDX-1are novel molecular targets in pancreatic cancer andmight serve as cancer master switch genes in pancreatic cancer development andprogression. Therefore, detailed genetic profiling study on those above mentioned keygenes in pancreatic cancer is of great interest in elucidating the molecular mechanisms ofthis deadly disease.Animal models, especially mouse models, are often employed for studying thegenetic variations and the biological relevance in the progression of cancer. Panc02and itsmetastatic derivative Panc02-H7cell lines were established to study the pancreatic cancergenetics and immunology in a syngeneic mouse model. After implantations of these cellsin the pancreas, the highly proliferating Panc02-H7cells disseminated to peritoneum andmetastasized to distal organs, showing more aggressive invasion compared to the parentalPanc02cells. These two mouse pancreatic cancer cells are important tools to study theimmune response against pancreatic cancer in immune competent mice and test newimmunotherapy drugs and vaccines. Therefore, it is critical to examine the genomic profileof key genes in human pancreatic cancer such as KRAS, SMAD4, CDKN2A (p16), TP53(p53), ZIP4, and PDX-1in these mouse pancreatic cancer cell lines and compare theirgenomic sequences in order to pinpoint the driver mutations or other genetic variationswhich give rise to the substantial biological differences of those cells.Based on the above situation,we performed genomic sequencing of key genes ofpancreatic cancer in mouse pancreatic cancer cells and normal C57BL/6mouse pancreastissue,and then further validate the function of the new SMAD4mutation. The aim was topinpoint the driver mutations or other genetic variations which give rise to the substantialbiological differences of those cells. We also performed genomic sequencing of ZIP4inhuman pancreatic cancer paired tissues and serum to deeply study the role of ZIP4in thepathogenesis of pancreatic cancer. So this study contained two parts, as described below.1. Genomic sequencing of key genes of pancreatic cancer in mouse pancreaticcancer cellsAim: to pinpoint the driver mutations or other genetic variations which give rise tothe substantial biological differences of those cells. Methods: we designed multiple primer sets to cover the coding regions of six genesincluding KRAS, SMAD4, CDKN2A (p16), TP53(p53), ZIP4, and PDX-1; then performexome sequencing of these genes in the two cells; Sequences from the normal syngeneicC57BL/6mouse pancreas, the derived tumoral cell lines were aligned and comparedamong themselves, and to the mouse reference genome (NCBI) using the sequencealignment software Sequencher v4.7; Real time PCR and Western Blot analysis test theRNA and protein expression; construct mutation plasmid by Site-Directed Mutagenesismethod; Finally, Dual-Luciferase Reporter Assays validated the mutation function.Results:(1) KRAS gene has a single nucleotide polymorphism(SNP) in two mousepancreatic cancer cell lines. We found a synonymous polymorphism in the normalpancreas of C57BL/6mouse, Panc02, and Panc02-H7cells in codon32which led to a TATto TAC coding sequence change without any amino acid sequence change.(2) A novelnon-synonymous mutation in SMAD4Gene was identified in two mouse pancreatic cancercell lines-GAA to TAA (Glu to stop) at codon174. The novel SMAD4mutation that wediscovered in the mouse Panc02and Panc02-H7cells is located in the SMAD4proteinlinker region, leading to the early termination of the translation and a truncated SMAD4protein, which completely lost the MH2domain.(3)No mutation was found in KRAS,SMAD4, CDKN2A (p16), TP53(p53), ZIP4, and PDX-1genes of these two mousepancreatic cancer cell lines.(4) RNA and protein expression in Panc02, Panc02-H7andPanc-1cells. A specific SMAD4band was clearly seen in Panc-1cells, but no band of fulllength SMAD4was observed in Panc02and Panc02-H7cells. Real-time PCR resultshowed that the endogenous SMAD4mRNA were present in both cell lines.(5) Thefunction of this mutant SMAD4in TGF-β induced SMAD4dependent signaling pathway.Panc-1cells responded to TGF-β, and the transfection of either mutant SMAD4-m174orwild type SMAD4did not impact the response to TGF-β in Panc-1cells Panc02cells aloneor Panc02cells with the overexpression of the mutant SMAD4-m174did not respond toTGF-β stimulation, while the response to TGF-β was rescued when the wild type SMAD4was introduced into the Panc02cells. Panc02-H7cells did not respond to TGF-β, andintroduction of wild type SMAD4did not rescue the response either.Conclusion: A mutation in SMAD4was identified in both cell lines. Thishomozygote G to T mutation in the first position of codon174(GAA) generated a stopcodon resulting in the translation of a truncated protein. Further functional analysisindicates that different TGF-β-SMAD signaling pathways were involved in those two mouse cell lines, which may explain the phonotypic difference between the two cells. Asingle nucleotide polymorphism (SNP) in KRAS gene (TAT to TAC at codon32) was alsoidentified in the normal pancreas DNA of the syngenic mouse and in both derived tumoralPanc02and Panc02-H7cells. No mutation or SNP was found in CDKN2A (p16), TP53(p53), ZIP4, and PDX-1genes in these two cell lines. The absence of mutations in genessuch as KRAS, TP53, and CDKN2A, which are considered as key genes in thedevelopment of human pancreatic cancer suggests that SMAD4might play a central anddecisive role in mouse pancreatic cancer. These results also suggest that other mechanismsare involved in the substantial phenotypic difference between these two mouse pancreaticcancer cell lines.2. Genomic sequencing of ZIP4in pancreatic cancerAim: to illustrate the mutation or SNPs of ZIP4in pancreatic cancerMethods: Genomic sequencing of ZIP4in pancreatic cancer tissues and serum onABI3700DNA Sequencers. Sequence data were assembled and the acquired and inheritedpolymorphisms was identified within and surrounding each exon by parallel comparing ofthe tumor DNA sequence with the patient blood DNA and normal pancreas samples. Thecall of the mutations was performed according to automated analysis techniques in used bythe HGSC such as SNP Detector Version2. All positive mutations were manuallyverified in Consed.Results: Totally17SNPs were identified, and there are7SNPs which were notreported previously.3SNPs located in promoter,4in intron and10in exome. Among the10SNPs,3was synonymous mutation and7was non synonymous mutation (Thr19Met,Ala58Thr, Pro84Leu, Ala114Thr, Glu284Lys, Thr357Ala and Pro484Ser). For rs2280838(Ala58Thr), rs17855765(Ala114Thr) and rs2272662(Thr357Ala) located in exome,belonged to non synonymous mutation and have a high proportion of Hetero and Homominor, maybe they are associated with pancreatic cancer progression.Conclusion: Genomic background of ZIP4in pancreatic cancer patients wasidentified. Further study was needed to validate the SNPs function, especially rs2280838(Ala58Thr), rs17855765(Ala114Thr) and rs2272662(Thr357Ala) and survey theassociation between ZIP4SNPs and pancreatic cancer in a large sample of patients.Based on the above experiments, the final conclusions were as followings:1. By performing exome sequencing of these genes in the two cells, one KRAS SNPand one SMAD4mutation was identified. The SMAD4mutation lost the normal function of MH2domain of SMAD4.2. Function validation conformed that SMAD4mutation resulted to aberrant TGF-βsignal pathway in two mouse pancreatic cells.3. TGF-β signal pathway is different between the two cells. There are other TGF-βsignal pathway elements mutations in Panc02-H7cell.4. There was no ZIP4mutation in pancreatic cancer. Parts of ZIP4SNPs wereassociated with pancreatic cancer. Further studies are needed.All in all, SMAD4and ZIP4play a vital role in the development of pancreatic cancer.Genomic sequencing of the genes can facilitate to illustrate the pathogenesis of pancreaticcancer.