In this study, we demonstrated that the 14-3-3σ protein level was increased in a majority of pancreatic cancer tissues studied (~71%) compared to their corresponding normal tissues and that the 14-3-3σ protein level correlated with lymph node metastasis and patient survival. We also showed that the increased expression of 14-3-3σ correlated with poor cellular response to γ-irradiation and anticancer drugs and that stable over-expression of ectopic 14-3-3σ in a human pancreatic cancer cell line, MiaPaCa-2, significantly increased its resistance level to γ-irradiation as well as anticancer drugs in part by causing resistance to the apoptosis induced by the treatments and by arresting cells in G2/M phase.
It has been reported that 14-3-3σ expression is lost or decreased in human cancers of breast [19
], liver [21
], vulva [22
], mouth [23
], as well as neuroendocrine tumors [24
], and small and non-small cell lung [25
] cancers. This decrease is thought to be due to hypermethylation of the CpG islands of the 14-3-3σ gene [19
]. However, recent studies revealed that the loss of 14-3-3σ expression in breast cancers is a sporadic event and that its expression is up-regulated in some breast tumors [29
]. From these later studies, it appears that majority of breast cancer cells with basal/myoepithelial phenotype show 14-3-3σ expression whereas some of the breast cancer cells with luminal epithelial cell differentiation show decreased or loss of 14-3-3σ expression. Furthermore, it has also been reported that 14-3-3σ expression is increased in lung cancers [31
], and head and neck squamous cell carcinomas [32
In this study, we demonstrated that 14-3-3σ protein was increased significantly in the majority (17 of 24) of paired fresh-frozen pancreatic cancer tissues examined. Previously, it has also been observed that 14-3-3σ is one of the genes with increased expression at the mRNA level in pancreatic cancer tissues using microarray profiling or real time PCR [11
]. In the study by Friess et al. [11
], a microarray analysis of 5600 human genes in 8 human pancreatic ductal adenocarcinomas and 8 normal tissues showed that 14-3-3σ is one of 120 genes that had increased expression at the mRNA level in cancer compared to normal tissues. Similarly, Iacobuzio-Donahue et al. [13
] reported that 14-3-3σ is one of the genes with increased expression of mRNA in a cDNA microarray profiling study of 17 infiltrating pancreatic cancer tissues compared with 5 normal pancreatic tissues. Using cell lines and methylation specific PCR analysis it was found that the increased expression of 14-3-3σ mRNA in pancreatic cancer cells may be due to hypomethylation of its promoter region. This later observation is interesting since 14-3-3σ in other cancers such as breast cancer is likely to be hypermethylated, resulting in silencing of its expression. Again in a third study, Logsdon et al. [12
] used microarray analysis of 10 pancreatic cancer and 5 normal tissues and found 14-3-3σ among 188 genes that had increased mRNA expression in pancreatic cancer tissues. Using laser capture dissection and real time PCR, Neupane and Korc [33
] compared the mRNA levels of all seven 14-3-3 genes in 3 normal and 5 pancreatic ductal adenocarcinoma tissues and found that 14-3-3σ is the only gene that has significantly higher expression in cancer tissues. However, all the above studies evaluated the expression of 14-3-3σ at its mRNA level. Since the mRNA level does not always predict the protein level, we performed the current study to determine 14-3-3σ protein expression in pancreatic cancers and found that the 14-3-3σ protein level is indeed significantly increased in pancreatic cancers. This observation is consistent with a previous study by Hustinx et al. [34
]. Taken together, these studies clearly show that 14-3-3σ expression is increased at both its mRNA and protein levels in pancreatic cancer tissues and the increase may be due to decreased methylation of its promoter.
The finding that the 14-3-3σ protein level is increased in pancreatic cancers is interesting considering that increased 14-3-3σ expression has been shown to cause drug resistance in breast and prostate cancer cell lines [9
]. Indeed, we showed that the increased 14-3-3σ expression in pancreatic cancers appears to correlate with lymph node metastasis, a marker of poor prognosis, and poor survival in a post hoc analysis. Furthermore, over-expression of ectopic 14-3-3σ in a pancreatic cancer cell line, MiaPaCa-2, caused resistance to anticancer drugs as well as to γ-irradiation while knocking down its expression in BxPc-3 cells reduced the resistance. Thus, the increased 14-3-3σ expression in pancreatic cancer cells may contribute to the failure in the treatment of human pancreatic cancers. Clearly, a more extensive prospective study with more patient samples is needed to further warrant this conclusion.
Recently, it has also been found that patients with breast carcinomas (both luminal or basal/myoepithelial phenotypes) that have higher cytoplasmic staining of 14-3-3σ also have shorter survival compared with patients whose tumors have lower 14-3-3σ staining [30
]. In estrogen receptor positive tumors, the relationship between 14-3-3σ and poor prognosis is even more significant. This later finding is interesting because it has been shown previously that estrogen regulates the stability of 14-3-3σ by affecting its proteosome-mediated degradation [35
]. In another study of cyclin B1 expression in breast carcinoma, Suzuki et al. also showed that the expression of cyclin B1 and 14-3-3σ have a positive correlation and predict a poor prognosis [36
]. In a third study, it was suggested that 14-3-3σ expression is an independent prognostic marker for poor survival of colorectal cancer patients [37
]. In prostate cancers, we previously found an increase in 14-3-3σ expression as tumor progresses [38
]. Adenocarcinomas with high Gleason scores (>7) had significantly higher staining intensities and higher percentages of 14-3-3σ immunoreactive cells than adenocarcinomas with low scores (<7). Adenocarcinomas with lymph node metastases had higher percentages of 14-3-3σ expression compared with adenocarcinomas without lymph node metastases. Thus, over-expression of 14-3-3σ may cause resistance to therapies in various cancers. However, it is noteworthy that opposite observations have also been made in endometrial cancer [39
], head and neck cancer [40
], NSCLC [41
], and nasopharyngeal carcinoma [42
] where it was found that the absence or low expression level of 14-3-3σ predicts poor survival. The reason for the difference between these cancers is currently unknown.
Several popular anticancer drugs used for cancer therapy such as mitoxantrone and Adriamycin are topo II inhibitors which exert their anti-neoplastic effects in susceptible cells by inducing apoptosis, mainly through their ability to induce DNA double-strand breaks. Similarly, γ-irradiation also causes apoptosis via inducing double strand DNA breaks. However, radiation has also been suggested to induce cell death via mitotic catastrophe. In response to a DNA double-strand break, cancer cells undergo apoptosis or go into cell cycle arrest for repair of DNA which prevents replication of damaged DNA or aberrant mitosis which leads to mitotic catastrophe and apoptosis, thereby, attenuating the toxic effect of the treatments. 14-3-3σ has been shown to be essential for maintaining G2, G1, S cell cycle arrest following DNA damage by interacting with and negatively regulating cyclin dependent kinase Cdc2, CDK2 and CDK4, which are responsible for cell cycle progression, and prevent mitotic catastrophe and apoptosis [8
]. Cells without 14-3-3σ are unable to arrest their cell cycle progression and will undergo mitotic catastrophe and apoptosis following DNA damage [43
]. Furthermore, 14-3-3σ interacts and negatively regulates Bax and possibly BAD, two pro-apoptotic proteins that regulate the release of mitochondria apoptogenic factors [44
]. Thus, increased 14-3-3σ expression in pancreatic cancer cells likely causes resistance to DNA damage-induced cell death.
Gemcitabine, a commonly used drug for pancreatic cancer treatment, does not cause DNA double strand breaks. However, incorporation of gemcitabine into DNA also causes damage to DNA by generating premature termination of DNA replication. The observation that over-expression of ectopic 14-3-3σ increases while knocking down its expression reduces resistance to gemcitabine, suggests that 14-3-3σ may also play a role in regulating repair of premature terminations.