PC is the fourth leading cause of cancer related deaths in the United States with only about 20% of the patients surviving for 2 years and only 6% for five years. Its incidence to mortality ratio has remained virtually unchanged for the past several decades despite a considerable understanding of its biology 
. This high mortality rate among PC patients is due to the tendency of the cancer cells to metastasize early. This, together with the poor response to therapy and high-rate of recurrence makes it one of the most lethal cancers known to man 
. PC is often diagnosed at an advanced stage, chiefly due to the lack of reliable early diagnostic markers 
. Therefore, there is an urgent need to identify specific early detection marker(s) and suitable molecular targets to combat PC. Mucins are one of the major biomarkers that have emerged in recent years as highly specific diagnostic markers in several malignancies including pancreatic, gynecologic and aerodigestive tract malignancies 
. Further, their aberrant expression has been demonstrated to modulate cell growth, differentiation, transformation, adhesion, invasion and immune surveillance 
CA125/MUC16 is a tumor biomarker that is currently used for the follow-up of patients with ovarian cancer 
. However, its role in PC pathogenesis remains unexplored. In the present study, we examined the expression pattern of MUC16 in PC tissues and compared it with that in the normal pancreas. Further, we also studied its association with PC development and with tumor stage, grade and metastasis. Interestingly, our results showed that the normal pancreas does not express MUC16 but its expression is significantly upregulated in 12/17 PC and 1/6 pancreatitis tissue. It has been previously shown that there is an association between the carcinoma of the pancreas and chronic pancreatitis (sporadic and familial) and the standardized incident ratio of developing PC in chronic pancreatitis patients is 14–18 
. This observation of MUC16 being significantly upregulated in PC is coherent with the expression of other membrane bound mucins MUC4 and MUC1 which are also aberrantly expressed in PC and have been identified as potential diagnostic markers for this malignancy 
We also observed that MUC16 expression increased progressively with loss of differentiation of the PC tumor. It has been previously observed that PC patients who have been diagnosed with distant metastasis had the tendency to have poorly differentiated pancreatic adenocarcinoma 
. Thus, our findings suggest that MUC16 may have an important role to play in the progression and metastasis of PC.
According to the well-known progression model for PC, ductal carcinoma develops from non-neoplastic ducts though a series of pre-malignant lesions termed as PanINs 
. We observed that MUC16 expression increases progressively from PanIN I to PanIN III. Particularly, the percentage of MUC16 positivity in high-grade PanIN lesions was significantly higher than that of low-grade PanINs. This suggests that MUC16 expression is altered at the stage of pancreatic dysplasia and may play a critical role in the progression of PC. MUC4, another membrane bound mucin has been previously shown to be differentially upregulated with progressively increasing dysplasia, suggesting the possibility that there may be certain common regulatory pathways that modulate the expression of both these mucins during PC development 
The high mortality rate in PC patients is due to the frequent occurrence of distant metastasis. In an analysis of 4,012 autopsies performed on PC patients between 1914 and 1943 it was reported that the most common site of distant metastasis was the liver, followed by the peritoneum, lung and pleura, bones and the adrenal glands 
. But PC is not limited to these organs. Even small PCs (<2 cm in diameter) exhibit metastasis, supporting the premise that PC is a malignancy that metastasizes very early during its progression 
Among the several molecules observed to play a role in the metastasis of PC cells, mucins have emerged as one of the key determinants. We have previously shown that MUC4, another transmembrane mucin when expressed promotes metastasis and invasiveness in PC cells 
. In the present study, we observed that those primary PCs that express MUC16 also express MUC16 with nearly equal intensity in the metastatic sites. This suggests that PC cells may maintain their expression of MUC16 during the metastatic process. MUC16 has been previously demonstrated to be important in the metastasis of solid tumors to the central nervous system via its interaction with mesothelin, a protein differentially expressed in normal mesothelial cells, mesotheliomas and some other mesenchymal malignancies 
. Hence it is possible that MUC16 might interact with mesothelin and facilitates metastasis in PC.
The molecular mechanisms driving metastasis in PC requires a better understanding of proteins that modulate epithelial-mesenchymal transition (EMT) and the reverse process (MET), which are necessary for the detachment and re-attachment of tumor cells at the site of metastasis respectively. The results of our study suggest that MUC16 might have a role for the development and progression of PC and studying its specific role in the progression of PC will be the basis of our next study. Its upregulation, which was particularly strong during the late stages of PC dysplasia, suggests that the mechanisms that turn on its expression are possibly turned on late during PC development. Studies on MUC4 mucin have revealed that its expression is silenced in normal ducts by virtue of hypermethylation of its promoter 
. Whether similar epigenetic changes also regulate MUC16 expression remains to be examined in future studies. The detection of MUC16 in high-grade PanINs (considered to be the true dysplastic lesions with a high risk for invasive cancer) suggests its potential use in the early detection of potentially malignant lesions in the pancreas. MUC16 is also shed in the bloodstream (known as CA125), making it an attractive molecule for investigation as a potential secreted biomarker for PC 
Further to identify a suitable in vitro
model to investigate the functional role of MUC16, a panel of PC cell lines was screened for MUC16 expression both at the protein and the mRNA level. On performing western blot analysis, it was observed that MUC16 expression either appeared as a single band or as a streaky band. It has been previously shown that when mucins are treated with 2-mercaptoethanol and analyzed on a SDS-PAGE gel, a streaky band is obtained as the mucins have been reduced from its oligomeric structure to its monomeric form. This monomeric form enables mucins to migrate faster on the gel and the intact oligomers remain in the well 
. This thus explains the differential expression pattern of MUC16 obtained across the various PC cell lines screened. In addition, we also observed that MUC16 expressing cell lines, such as Capan 1 (liver met), Colo 357 (lymph node met) and T3M4 (lymph node met) were derived from metastatic sites while the MUC16 non expressing cell lines such as Panc1, AsPC1 and BxPC3 were isolated from the primary tumor site (pancreas). Further, from the RT-PCR studies we observed that the mRNA levels of some PC cell lines did not corroborate with their corresponding protein levels. We speculate that MUC16 mRNA undergoes post transcriptional processing in certain cell lines. We are currently performing preliminary studies to further investigate this hypothesis.
In conclusion, our study shows that MUC16 is expressed only in pancreatic adenocarcinomas when compared to undetectable levels in the normal pancreas. The expression of MUC16 is stronger with progressive worsening pancreatic dysplasia (from PanIN I lesion to PanIN III). A strong expression pattern of MUC16 was observed in matched primary tumors and metastatic tumors at all the sites examined (liver, lung, lymph nodes and omentum/diaphragm) suggesting that MUC16 could be playing an important role in the progression and metastasis of PC. Further, MUC16 expression was observed in several PC cell lines at both the protein and the mRNA level. Overall, these results suggest a potential implication of MUC16 in PC pathogenesis and provide a basis for future studies aimed at unraveling the functions of this large membrane bound glycoprotein in PC.