In this study, we show that PDE4A1 overexpression stimulates brain tumor growth in vivo
, while inhibition of PDE4 suppresses tumor growth and augments the anti-tumor effects of chemo- and radiation therapies. PDE4 has previously been shown to be widely expressed in a number of different human tumor cell lines (4
). In addition, we now show that PDE4A is also expressed in astrocytomas, medulloblastomas, oligodendrogliomas, ependymomas and meningiomas, indicating that PDE4A activity may be essential to the growth of all brain tumors.
To better define the role of PDE4 in brain tumor biology, we overexpressed PDE4A1, a brain specific isoform, in U87 glioblastoma and Daoy medulloblastoma cells (26
). Stimulation of growth occurred in both brain tumor models. U87 cells were derived from a malignant astrocytoma. These tumors typically carry mutations in the MAPK, PI3K, Rb and MDM2/p53 pathways (28
) and, consistent with this, U87 cells possess deletion of p14/p16
). Daoy cells were derived from a desmoplastic medulloblastoma. Medulloblastomas are characterized by mutational activation of the sonic hedgehog, Wnt, and Myc pathways (30
). Daoy cells also carry a mutation of TP53
). Despite these differences in molecular profile, PDE4A1 overexpression stimulated both U87 and Daoy growth in vivo
. These data suggest that PDE4A1 effects were not dependent upon the locus into which the transgene inserted and were not specific to pathways activated by mutation in these tumors. Instead, these results indicate that PDE4A1 is a positive regulator of brain tumor growth.
It will be important to determine whether the cAMP-hydrolyzing activity of PDE4A1 is essential to its growth promoting effects. Previously, we and others have suggested that low levels of cAMP stimulate brain tumor growth (3
). The current study lends additional support to this model as the effects of PDE4A1 overexpression and Rolipram are most likely the result of changes in cAMP levels. The work of Houslay and others has shown that PDE4s play multiple roles in intracellular signaling. These roles include the regulation of receptor desensitization and G protein switching, as well as PKA and Erk activation (5
). There are at least 20 different isoforms of PDE4 that can be inhibited with Rolipram. Each contains a unique N-terminal region that appears to regulate specific compartmentalized functions (6
). For example, the N-terminal regions of PDE4A4 and PDE4A5 interact with the SH3 homology domains of Src family kinases, while the N terminal region of PDE4A1 facilitates membrane insertion, particularly into the trans-Golgi membrane (35
). Thus overexpression of even catalytically inactive PDE4 isoforms can alter signaling functions by displacing active endogenous PDE4 from necessary binding sites and functioning as dominant negative constructs (8
). The PDE4A1 isoform lacks the regulatory and protein-protein interacting domains that are found in longer PDE4 isoforms (5
). Thus, the growth-promoting effects of PDE4A1 overexpression are most readily attributed to increased cAMP hydrolysis as evidenced by the observed decrease in levels of cAMP.
In this regard, the localization of PDE4A1 to the membranes of the Golgi apparatus and Golgi-derived vesicles is of interest. While global changes in cAMP were detected in our cells and xenografts overexpressing PDE4A1, PDE4 effects can also be limited to regulating local cAMP dependent functions. For instance, targeted deletion of PDE4B revealed that it, and not PDE4A or PDE4D was necessary for regulating TLR signaling in macrophages (9
). The effect of PDE4B loss on macrophage responses to LPS stimulation was dependent upon protein kinase A activation but was not associated with global changes in cAMP levels. These data suggest that localization of PDE4B is necessary to regulate cAMP levels in a critical subcompartment for TLR signaling. Thus it will be essential to identify Golgi-localized mediators of PDE4A1 effects on tumor growth and to determine whether elevation of cAMP levels antagonizes their growth promoting functions.
Elevation of cAMP as a therapeutic strategy for cancer has previously been examined, but was limited by the excessive toxicity of the early pleiotropic phosphodiesterase inhibitors (36
). Specific phosphodiesterase inhibitors such as Rolipram are better tolerated at therapeutic doses (38
) and exhibit strong anti-tumor effects. (10
). Prior studies suggested a therapeutic role for Rolipram in the treatment of brain tumors and colon cancer (3
). To our knowledge, this is the first report to demonstrate a survival advantage in response to Rolipram treatment over an extended preclinical trial.
The potential importance of Rolipram therapy was most evident upon analysis of bioluminescence data pertaining to the highly effective therapies of radiation and temozolomide, Rolipram and temozolomide, and Rolipram, radiation, and temozolomide. Though the initial pattern of tumor growth was similar among these three groups, the value of Rolipram treatment became most evident at 12 weeks of therapy. Therapy with either Rolipram and temozolomide or radiation and temozolomide resulted in cessation of further tumor growth, but tumors remained viable as evidenced by their persistent bioluminescent signal. In contrast, treatment with Rolipram in conjunction with radiation and temozolomide resulted in nearly complete loss of bioluminescence, indicating tumor regression.
Human studies have shown that a major determinant of response to alkylator therapy like temozolomide is the expression and activity of the DNA repair enzyme methyl glutamyl methyl transferase (MGMT) (39
). In patients with reduced MGMT expression through promoter methylation, there is a greater response to temozolomide therapy. Thus one possible mechanism whereby Rolipram enhances the effects of temozolomide and radiation might be the inhibition of MGMT. Consistent with published studies we found that Daoy and U87 cells express MGMT (40
) but this was unaffected by Rolipram treatment in vitro
(data not shown). Thus the mechanism by which Rolipram augments the activity of radiation and temozolomide may be distinct from modulating DNA alkylation.
The present study strongly indicates that PDE4 inhibitors should be evaluated in clinical trials for brain tumors. Our data suggest that PDE4 inhibition may be the key adjunct to standard chemotherapy and radiation therapy to promote brain tumor regression. Furthermore, the equivalence of combined radiation and temozolomide to Rolipram and temozolomide suggests that PDE inhibitors might prove to be suitable alternatives for radiation when combined with chemotherapy. This may be especially important for young patients for whom irradiation is not an option due to the detrimental effects radiation has on their developing brains.
The potential for Rolipram to be a useful agent in brain tumor therapy is emphasized by its ability to penetrate into and treat diseases of the central nervous system. However, clinical application of Rolipram might also be limited by its toxicities. In multiple clinical trials for depression and multiple sclerosis, Rolipram was relatively well tolerated with nausea and vomiting the most commonly reported side effect (38
). However, in long-term toxicity studies in rats, high dose Rolipram therapy was also associated with prolactinomas and mammary adenocarcinoma (41
). While serious, these side effects are not substantially different from the nausea, vomiting and secondary cancer risks of commonly used treatments for brain tumors like radiation therapy and temozolomide. Therefore, if Rolipram demonstrated a unique therapeutic effect its known toxicities would not preclude its use in the treatment of malignant brain tumors. However, newer PDE4 subfamily-specific inhibitors may prove equal or more efficacious and less toxic (6
). Thus, additional investigation into whether other PDE4 isoforms can also regulate brain tumor growth, and whether subfamily specific inhibitors exhibit equal efficacy to Rolipram is essential.