Pancreatic cancer has been a lethal malignancy worldwide and over 90% is pancreatic ductal adenocarcinoma (PAAD). The overall 5-year survival rate was 7%~9% and 1-year rate less than 20%, to make it worse, the cancer is insensitive to neither chemotherapy nor radiotherapy, even the emerging immunotherapy which has been showing promising clinical effect in other tumors receives limited response in pancreatic cancer, making it urgent to explore the potential gene targets and develop drug-targeting therapies. In the study, multiple GEO profiles data and bioinformatic analysis tools were combine used to explore the genetic information of PAAD and select potential responsible genes during cancer development.
Based on five different GEO cDNA expression profiles GSE15471, GSE16515, GSE41368, GSE43795 and GSE71989 which contain a total of 98 PAAD and 71 normal pancreatic samples, we identified 22491 genes that were differently expressed in cancer vs. normal tissues and then classified them into 4 different groups according to the difference level considering the potential unique functions of each group. Interestingly, further interpretation of the 4 groups of genes indicated that the greater the genes expression difference are, their cellular location were more tend to be far away from cell nuclear. More specifically, the expression difference < 2 fold genes were mainly located in nuclear, and 4 ~ 8 fold genes were mostly in cytoplasm, meanwhile the > 8 fold genes were tend to locate on the cell membrane or in extracellular region. The trend makes reasonable sense considering the fact that except for the certain percent of genes that were synthesized in cell mitochondrion, most human proteins were produced in nuclear abiding by the biology “central dogma” that the direction of genetic information flow is from DNA-RNA-protein, the slight change in the nuclear protein might result in massive proteins change extracellular.
Given the convenience of further IHC experiment validation, which is the most common method for clinical medical diagnosis, and the genes shall harbor more chance to be translated into clinical use if they are suitable to be tested by IHC, we mainly focused on the > 8 fold genes. Interestingly, detailed analysis of the > 8 fold group of extracellular genes revealed that they were mostly extracellular matrix structural constituent regulating related genes, for instance, a certain percent of them were collagen related regulators, which were proven to be associating with PAAD cancer related fibroblasts infiltration, although none direct relation were found between these genes expression and patients overall survival, they certainly affect patients recurrent free survival, stressing the importance of microenvironment construction in cancer development, which is certainly an inspiring direction for further research.
Actually, to identify the potential “unique key genes” during PAAD development, we then combine used KM survival, UALCAN survival and multivariate Cox Regression analysis to successively explore the association between 55 over 8-fold genes and PAAD patient survival, and the results highlighted two genes: GPRC5A and IMUP, which were supported by all three analysis to be associated with both patients overall and recurrence free survival and worked as independent prognostic indicators in PAAD development.
GPRC5A, which is short for G Protein-Coupled Receptor Class C Group 5 Member A, is a member of the GPCR family, locating in 12p13.1 and encoding a protein that is characterized by the signature 7-transmembrane domain motif. And based on the computed physicochemical parameters of the protein, GPRC5A is a hydrophobic protein weighting 40.3KD and mainly locating in cellular membrane and extracellular space, the estimated half-time is 30h and tend to be stable in human cells. Actually, the gene has been reported to play critical roles in embryonic development and epithelial cell differentiation, the dysregulation of GPRC5A was known to be involved in multiple cancers including lung, breast, colon and other types of cancers[5, 38]. In the study, we mainly focused on its potential regulation on PAAD development.
Meanwhile, IMUP, which is more commonly known as C19orf33 being short for chromosome 19 open reading frame 33 and locating on 19q13.2, encodes a hydrophilic protein weighing 10.9KD. And the encoded protein probably locates in nucleoplasm, cytosol and plasma membrane, with the estimated half-time as 30h in human cells and potentially relates with human placental development. The aberrant dysregulation of IMUP has been reported in endometrial carcinoma and pre-eclampsia[39–41].
Interestingly, besides the strong correlation between GPRC5A and IMUP genes expression indicating their potentially similar biological functions and close involving signaling pathways in PAAD, the aberrant higher expression of both genes were indicated to be relating with worse patients both overall survival and recurrence free survival, supported not only by previous online analysis, but also IHC experiments using local hospital PAAD tissue array which was produced using 62 local PAAD patients samples and matched normal pancreatic tissues. A more inspiring discovery is that based on the expression of these two genes, pancreatic cancer patients could be divided into 4 groups, the patients with neither GRPC5A nor IMUP expression shows much better prognosis than the patients with either gene expression, and the survival of patients with both genes expression was the worst in 4 groups, indicating the drug developing potentiality of the two genes.
Besides the mRNA expression, other types of variation of GPRC5A and IMUP were also explored which results revealed basically a similar result that the gene amplification works as the main variation type for both GPRC5A and IMUP in PAAD, only occasional gene mutation or deletion occurs. Moreover, none specific relation was found between neither gene expression nor immune cells infiltration.
Additionally, to evaluate the potential correlation between GPRC5A and IMUP expression with PAAD clinical parameters, both UALCAN online analysis and TCGA original clinical data were used, both results indicated the genes were more higher expressed in patients with more advance cancer stage and grade, supporting the potential clinical value of the genes for indicating cancer developing. Recent reports highlighted that GPRC5A and IMUP were involved in several human cancers via participated in various tumor-associated signaling pathways, for instance the nuclear factor (NF)-κB[42–44], signal transducer and activator of transcription (STAT) 3[45, 46], and focal adhesion kinase (FAK)/Src signaling[47–51], however, they are still on the way to be validated in PAAD.
Although the current result is not yet enough to classify GPRC5A or IMUP as new useful clinical drug targets, comprehensive studies and clinical trials are needed to confirm the findings before promoting the clinical utility of the genes in PAAD clinical treatment, the results shall provide meaningful insight into better understanding of the disease. We sincerely hope the study will provoke worldwide researchers’ interest to further explore the pancreatic cancer deeply and benefit the suffering patients in the near future.