We described here that: (a) cystic cholangiocytes are hyper-proliferative and have an abnormal cell cycle; (b) Cdc25A is overexpressed in cystic cholangiocytes of humans and rodents with PKD/PLD; (c) in Cdc25A+/− :Pkhd1del2/del2 crossbred mice, liver weights and cystic and fibrotic areas are decreased compared to Pkhd1del2/del2 counterparts supporting the importance of Cdc25A in hepatic cystogenesis; (d) VK3 decreases proliferation and causes cell cycle arrest of PCK cholangiocytes; (e) VK3 reduces cyst growth in 3-D cultures; (f) the intrahepatic biliary tree volume is significantly less after VK3; (g) in vivo in PCK rats and Pkd2ws25/− mice, liver and kidney weights, hepato-renal cystic and fibrotic areas, and mitotic and apoptotic indices are all decreased after VK3 treatment; (h) PM-20 attenuates hepato-renal cystogenesis in Pkd2ws25/− mice; and (i) VK3 affects the expression of multiple cell cycle proteins. These findings provide new insights into the pathogenesis of PKD/PLD and present a rationale for the use of Cdc25A inhibitors to halt cyst progression.
Cdc25A over-expression in cystic cholangiocytes is likely linked to disturbances in several signaling pathways. First, elevated Cdc25A is associated with reduced expression of its regulator, miR-15a.4
Second, Cdk2 (i.e., a down-stream effector of Cdc25A) phosphorylates Cdc25A in a positive feedback loop increasing its activity.34
We observed (unpublished data) that Ckd2 is up-regulated in cystic cholangiocytes; therefore its overexpression may add to Cdc25A elevation. Importantly, Cdk2 is also a miR-15a target. Third, the levels of many RTK’ (such as EGFR, VGFR, IGFR, HGFR) and several components of the RTK pathway (i.e., Raf, MEK and ERK) are increased in hepato-renal cysts.2, 7
Raf kinase forms a functional complex with Cdc25A while ERK is known to directly phosphorylate Cdk2 further suggesting a connection between mitogenic signaling and the cell cycle machinery.35
Additional studies are underway in our laboratory to directly assess all these possibilities. Be that as it may, accumulating data suggest a major role for Cdc25A in hepatic cystogenesis.
Based on our published evidence that hepatic cystogenesis is linked to elevated Cdc25A in cholangiocytes,4
we hypothesized that its reduction might decrease cyst growth. To this end, we cross-breed the Pkhd1del2/del2
mice (they develop hepatic cysts and have increased Cdc25A expression) with Cdc25A+/−
mice that have reduced Cdc25A levels. We anticipated that in crossbred (i.e., Pkhd1del2/del2
mice), cystogenesis should be less due to Cdc25A diminution. As expected, in Cdc25A+/−
mice, decreased Cdc25A expression was associated with reduced liver weights and less evident cystogenesis. The mechanism/s by which Cdc25A depletion affects hepatic cystogenesis is not yet understood. However, our indirect in vitro
and in vivo
data suggest that cell cycle progression and cell proliferation might be involved since suppression of Cdc25A by VK3 affected the distribution of the cell cycle phases, inhibited rate of cell proliferation and cyst growth.
Various pharmacological inhibitors of Cdc25 phosphatases have been developed.20, 21, 36, 37
Studies testing the effects of VK family members on cancer cell growth have shown that VK3 has the strongest antiproliferative capacity directly affecting Cdc25A.38
Mechanisms of VK3 action might relate to: (i) an oxidative effect produced by redox cycling of VK3; (ii) modulation of transcriptional factors; (iii) perturbation of cell proliferation; and (iv) cell cycle arrest with consequent inhibition of proliferation due to direct binding of inhibitor to Cdc25A.21, 23, 38-42
Consistent with these possibilities, we observed inhibited proliferation of PCK cholangiocytes which was coupled with cell cycle arrest after treatment. In contrast, VK3 has no effect on the cell cycle in normal cholangiocytes suggesting that suppression of their proliferation might involve other mechanisms including those described above. VK3 also slows down the growth of cystic structures in 3-D cultures. Finally, in vivo
in two animal models of PKD/PLD, VK3 halted hepato-renal cystogenesis by decreasing mitotic indices and reducing PCNA expression. Another inhibitor used in this study, PM-20, also attenuated hepato-renal cystogenesis in vivo
mice. PM-20 is known to bind and inhibit the Cdc25A activity subsequently affecting cell proliferation and tumor growth.20, 22
Indeed, mitotic indices were decreased in PM-20 treated mice.
To begin to elucidate the mechanisms by which VK3 affects hepatic cyst growth, we analyzed the expression of the cell cycle proteins. We found that treatment of PCK rats with VK3 decreased levels of Cdc25A and proteins known to be regulated by Cdc25A; reduced the phosphorylated status of Rb, and increased the phosphorylated status of Cdk1 and Cdk2. These observations are in agreement with previous reports.36, 41, 42
Increased phosphorylation of Cdks suggest that VK3 might affect cell proliferation and cell cycle via direct inactivation of Cdc25A which is usually correlated with protein levels.35, 43, 44
Consistent with this view, treatment of PCK rats with VK3 for 8 weeks decreased Cdc25A expression. Finally, we evaluated the phosphorylated status of Erk1/2. We have recently shown the significance of Erk1/2 signaling in hepatic cystogenesis.7
In particular, expansion of PCK-derived hepatic cysts in 3-D cultures as well as enhanced proliferation of cystic cholangiocytes was Mek/Erk1/2-dependent.7
In the present study, VK3 affected Mek/Erk1/2 pathway by decreasing the level of Erk1/2 activation. The mechanism of this inhibition is currently unclear; however, suppressed Erk1/2 phosphorylation was observed in vivo
mice (the animal model of PKD) after 5 weeks of treatment with Cdk inhibitor, roscovitine.45
Taken together, our study provides the first experimental evidence that Cdc25A targeting with VK3 and PM-20 decreases proliferation of cystic cholangiocytes halting the expansion of hepatic cysts. Moreover, VK3 and PM-20 also has positive effects on renal cytogenesis.
In conclusion, we believe that Cdc25A represents a potential therapeutic target and this study provides a rationale for examining the role of VK3, PM-20 or other Cdc25 inhibitors that are now at developmental/testing stage37
in the treatment of PKD/PLD.