Pancreatic cancer is considered to be the fourth leading cause of cancer related death due to its drug resistance and rapid metastasis.23
The aim of this study was to evaluate the role of Cav-1 in the regulation of epithelial to mesenchymal transition (EMT) in pancreatic cancer. Importantly, EMT has been shown to play an important role in aggressive pancreatic cancer progression.23
EMT is a biological and molecular process in which epithelial cells lose cell polarity and gain a fibroblastic spindle-shape morphology allowing them to infiltrate tissues and invade organs.24
Interestingly, here we show that enforcing Cav-1 expression induced profound alterations in the morphology of Panc 10.05 pancreatic cancer cells. Panc 10.05 cells expressing Cav-1 displayed cell-cell adherens, which were absent in the spindle-shape control cells. To better characterize the phenotype, we then examined the levels of E-cadherin, which is responsible for cell adherence and tight junctions. Protein gel blot and immunofluorescence analysis indicated that E-cadherin expression was undetectable in Panc10/pBabe cells but was readily restored in Panc10/Cav-1 cells. Of great interest, E-cadherin was localized at the cell membrane in Panc10/Cav-1 cells. Restoration of E-cadherin expression is critical, as loss of E-cadherin correlates with undifferentiated and anaplastic pancreatic tumors leading to worse prognosis as compared with differentiated pancreatic tumors that express E-cadherin.25
It is well known that β-catenin-dependent transcription plays an important role in the initiation of EMT and invasion of cancer cells.26
Interestingly, we show here that Cav-1 expression was sufficient to restore the expression of β-catenin at the plasma membrane. Consistent with these findings, we have previously reported that Cav-1 stabilizes β-catenin at the cell membrane, preventing the formation of the complex of β-catenin with the transcription factor Lef-1, β-catenin nuclear translocation and activation of gene transcription.27
β-catenin-dependent transcription is also promoted by Snail, that has a reciprocal correlation with E-cadherin expression.2
Snail transcriptionally represses E-cadherin by recruiting histone deacetylases (HDAC) to the E-boxes of the E-cadherin promoter.30
Interestingly, Yin et al. showed that Snail overexpression correlated with lymph node invasion and distant metastasis in human pancreatic cancer.31
Also, Snail overexpression in Panc-1 pancreatic cancer cells enhanced metastatic potential after orthotopic injection in the pancreas of nude mice and promoted chemoresistance to 5-fluorouracil or gemcitabine. Here, we present compelling evidence showing that Cav-1 is sufficient to downregulate Snail expression, and to sensitize pancreatic cancer cells to chemotherapy.
To mechanistically understand how Cav-1 affects Snail expression, we investigated the three main molecules responsible for Snail activation, namely ERK, Smad2 and AKT, which are involved in the mitogenactivated protein kinase (MAPK), TGFβ and phosphatidylinositol-3-kinase (PI3K)/AKT pathways, respectively.18,32–34
All three pathways are key for Snail activation and EMT initiation and maintenance. Li et al. demonstrated that E-cadherin re-expression in breast cancer cells was not sufficient to revert the EMT process unless ERK was suppressed.35
Moreover, previous studies have shown that MAPK modulates the role of TGFβ to act as a tumor promoter in human pancreatic cancer cells.36
TGFβ pathway phosphorylates and activates Smad2, inducing Smad2 nuclear translocation and binding to transcriptional factors, such ZEB1 or ZEB2, that control EMT.37
AKT pathway is not only an EMT inducer, but also it is a pro-survival and metastasis promoter molecule.18
Remarkably, our results show that Cav-1 greatly decreases the activation of ERK and Smad2 and AKT, restoring epithelial cell morphology.
We also found that Cav-1 expression altered certain key features associated with EMT, such as migration, invasion and chemoresistance. Consistent with our current results, a previous study has demonstrated that Cav-1 suppresses migration and invasion in pancreatic cancer cells by inhibition of MAPK signaling pathway.38
Chemoresistance is a serious concern in the clinical management of pancreatic cancer patients. Many previous studies indicated that the pro-survival AKT pathway is responsible for doxorubicin resistance in breast, lung, gastric and uterine cancer.22,39–42
Moreover, recent studies implied that drug resistance in pancreatic cancer inversely correlates with E-cadherin expression and re-expression of E-cadherin sensitizes pancreatic cancer cells to cytotoxic drugs.16
Importantly, we show here that Cav-1 downregulated AKT expression and activation, and sensitized pancreatic cancer cells to doxorubicin-induced cell death. Also, Arumugam et al. indicated that EMT induces chemoresistant toward gemcitabine and 5-flurouracil in pancreatic cancer cells.16
Consistent with these data, we show here that Cav-1 expression inhibits EMT process and leads to cancer cell chemosensitization.
Previous studies demonstrated that Cav-1 inhibits tumor growth.5,38
Our current results show that Cav-1 expression greatly blocks tumor formation in an in vivo xenograft model. More interestingly, Cav-1 expressing tumors displayed nests of differentiated cells, which were completely absent in control tumors. These nests of differentiated cells expressed high levels of E-cadherin and β-catenin at the plasma membrane. These remarkable findings suggest a critical role of Cav-1 in cell differentiation and epithelial cell plasticity.
Although our results describe Cav-1 as a tumor suppressor in pancreatic cancer, clinical data portrays Cav-1 as a tumor promoter and high Cav-1 expression correlates with high tumor grade.43
However, Cav-1 is not an independent prognostic factor in pancreatic cancer and predicts survival only when combined with other biomarkers, such as FASN.43
This discrepancy may be explained by the fact that in higher tumor grades Cav-1 loses its tumor suppression role or gains an oncogenic function, potentially by genetic mutations.44
A biphasic differential expression of Cav-1 was previously suggested in other types of human cancers, including oral cancers, where Cav-1 is highly expressed in early stage disease, but lost in metastatic and advanced lesions.45
Another possible explanation is that E-cadherin expression is necessary for Cav-1 to act as a tumor suppressor, as E-cadherin inhibits the β-catenin-TCF oncogenic pathway.46
Loss of E-cadherin in high grade tumors may promote Cav-1 interaction with other partners, such as FASN, and induce an oncogenic switch in Cav-1 function. However, the relation of Cav-1 and E-cadherin appears to be important to determine the behavior of Cav-1, which in turn deeply affects the behavior of pancreatic cancer cells.
In conclusion, our data demonstrate that Cav-1 plays a critical role in promoting pancreatic cancer cells differentiation, and implicate that Cav-1 may be a promising therapy for pancreatic cancer. We showed that Cav-1 restored the epithelial status of pancreatic cancer cells, cell differentiation and maintained E-cadherin at plasma membrane. Delivery of Cav-1 by gene therapy or by peptide administration may hold the promise to effectively treat or retard pancreatic cancer progression. For example, systemic administration of a cell-permeable Cav-1 peptide has been employed to ameliorate signs of lung fibrosis in a pre-clinical model of scleroderma, in whose pathogenesis a loss of Cav-1 plays a crucial role. Thus, restoration of Cav-1 function by treatment with a Cav-1 peptide may be a novel therapeutic approach for pancreatic cancer.