Phosphodiesterase function and endocrine cells: links to human disease and roles in tumor development and treatment

Abstract

Phosphodiesterases (PDEs) are enzymes that regulate the intracellular levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate, and, consequently, exhibit a central role in multiple cellular functions. The pharmacological exploitation of the ability of PDEs to regulate specific pathways has led to the discovery of drugs with selective action against specific PDE isoforms. Considerable attention has been given to the development of selective PDE inhibitors, especially after the therapeutic success of PDE5 inhibitors in the treatment of erectile dysfunction. Several associations between PDE genes and genetic diseases have been described, and more recently PDE11A and PDE8B have been implicated in predisposition to tumor formation. This review focuses on the possible function of PDEs in a variety of tumors, primarily in endocrine glands, both in tumor predisposition and as potential therapeutic targets.

Introduction

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are important second messengers in signaling, involved in cell proliferation, cell-cycle regulation, and metabolic function. Intracellular cAMP and cGMP levels are controlled both at their production, by activated adenylyl-cyclase and guanylyl-cyclase, which catalyze conversion of ATP and GTP to cAMP and cGMP, respectively, and at their destruction, by cyclic nucleotide phosphodiesterases (PDEs) [1] (Figure 1).

Figure 1.

Summary of cyclic nucleotide signaling pathways: cyclic nucleotides are generated by adenylyl-cyclase and guanylyl-cyclase; the former, activated by G-protein-coupled receptors, and the latter, by molecules such as natriuretic peptide or nitric oxide. In turn, cAMP activates PKA and EPAC. EPAC is involved in the regulation of several cellular processes, including integrin-mediated cell adhesion and cell–cell junction formation [74], exocytosis [75,76,77], and insulin secretion, while PKA is involved in metabolic processes, cell growth, differentiation, and proliferation. cGMP activates PKG which in turn mediates the phosphorylation of proteins involved in apoptosis, inflammation, and other physiologic processes, including smooth muscle contractility [78], the visual transduction cascade, and platelet aggregation. By catalyzing hydrolysis of cAMP and cGMP, PDEs regulate their intracellular concentrations and, consequently, their myriad biological effects.

Phosphodiesterases are enzymes that catalyze the hydrolysis of the 3′ cyclic phosphate bond of cyclic nucleotides. To date, 11 PDE gene families have been identified, based on their amino acid sequences, biochemical properties, and inhibitor profiles. Different PDEs can share the same catalytic function, but may differ in tissue expression and intracellular localization (Table 1) [2].

Table 1.

Summary of human phosphodiesterases: their substrate, tissue expression, subcellular location and inhibitors.

Family	Substrate	Tissue/cellular expression and function	Subcellular localization	Commonly used inhibitors
PDE1A PDE1B PDE1B1–2 PDE1C1–2	cAMP/cGMP	PDE1A: brain and spermatozoa, kidney, liver, pancreas and thyroid gland [79–81]. Heart [82]. Immune cells [83]. Olfactory epithelium [84].	Cytosol	Vinpocetine, IC224, SCH51866, 8-MeoM-IBMX. Zaprinast Sildenafil
PDE2A1–3 PDE3B	cAMP/cGMP	Adrenal glomerulosa [85]. Heart muscle [86]. Immune System [87]. Endothelial permeability and proliferation [88]. Brain [39]. Liver [89].	Membrane: PDE2A3, and PDE2A2 Cytosol: PDE2A1	EHNA, BAY60-7550, PDP, IC933
PDE3A1–3	cAMP/cGMP	Heart [90]. Adiposyte, oosyte, cardiac and vascular smooth muscle, myocardium, platelet [91].	Membrane Cytosol	Milrinone, Tolafentrine, Cilostazol, Cilostamide, Trequinsin, OPC-33540, Dihydropyridazinone, Lixazinone Zardaverine
PDE4A PDE4B PDE4C PDE4D	cAMP	Heart and small intestine [92]. Immune cells [93]. Brain [94].	Membrane	Cilomilast, Rolipram, Ro20-1724, Roflumilast, AWD12281, V11294A, SCH35159, Denbufylline, Arofylline
PDE5A1–3	cGMP	Lung, penis, smooth muscle [15•]. Platelets [95]. Brain [96]. Cardiac muscle [97].	Cytosol	Sildenafil, Tadalafil, DA8159, E402, Vardenafil, Zaprinast, DMPPO, Dipyridamole
PDE6A PDE6B PDE6C	cGMP	Photoreceptors [98]. Pineal gland [99].	Membrane and Cytosol	Zaprinast, Dypyridamole, Sildenafil, Verdenafil, Tadalafil
PDE7A1–2 PDE7B1–3	cAMP	Immune cells [100]. Skeletal and cardiac muscle [101]. Brain [102].	Cytosol	BRL 50481, IC242, Dipyridamole, BMS-586353, Thiadiazoles
PDE8A1–5 PDE8B1–3	cAMP	Immune cells [103]. Heart [104]. Ovary and testes [105]. Thyroid gland [31]. Placenta, Brain [106]. Adrenal gland [28•].	Cytosol and particulate fractions	Dipyridamole
PDE9 A1–6	cGMP	Kidney, spleen, gut, prostate [107]. Brain (Rat) [108].	Cytosol and nucleus	BAY 73–669, SCH51866, Zaprinast
PDE10A1–2	cAMP/cGMP	Brain, testis, thyroid [109].	Cytosol and particulate fractions	Papaverine, Zaprinast Dipyridamole, PQ-10
PDE11A1–4	cAMP/cGMP	Testis, pituitary gland, heart. Kidney, liver [57,110]. Prostate, adrenal, colon [58]	Cytosol	Dipyridamole, Zaprinast

Elevation of cAMP induces activation of cAMP-dependent protein kinase A (PKA). PKA is a heterotetramer formed by two catalytic subunits (C) and two regulatory subunits (R) [3]. In the absence of cAMP, PKA is inactive. Upon cAMP binding to the R-PKA, the catalytic subunits are released and phosphorylate different targets, including the cAMP response element binding (CREB) protein, a transcription factor that regulates genes involved in metabolism and proliferation [4,5]. Similarly, cGMP activates protein kinase G (PKG) which catalyzes the phosphorylation of downstream proteins involved in several physiologic functions, such as glycogenolysis, ion channel conductance, and apoptosis [6].

Dysregulation of cAMP homeostasis can be linked to tumorigenesis, both directly and indirectly [7]. Some tumor cells overexpress phosphodiesterases and exhibit lower cAMP levels [8], whereas other tumor types have increased cAMP levels as a protective mechanism against malignancy [9]. Thus, understanding the molecular basis of cAMP signaling can provide new insights for improved pharmaceutical targeting of cancer cells [10,11].

PDEs and endocrine glands: tumors and other phenotypes

Alterations in cAMP signaling pathways have been linked to tumorigenesis at different levels. First, activating mutations of the stimulatory G protein of adenylyl-cyclase, which induces high cAMP levels, leads to endocrine and nonendocrine manifestations in McCune Albright syndrome (MAS) [12]. Second, inactivating germline mutations in the alpha regulatory subunit gene of the PKA gene (PRKARIA) lead to the Carney complex (CNC) [13]. CNC is an autosomal dominant disease characterized by skin pigmentary abnormalities, cardiac myxomas, schwannomas, and endocrine tumors, the most frequent being a type of adrenocortical hyperplasia named primary pigmented nodular adrenocortical disease (PPNAD) [14]. PRKAR1A is located on chromosome 17q22–24, and more than a hundred different mutations of this gene have been described [13,15•,16–19].

Altered cAMP signaling, somatic PRKAR1A mutations, and somatic losses in the 17q22–24 locus have all been reported in adrenocortical adenomas and adrenocortical cancer. Specifically, 17q22–24 losses were found in 23% and 53% of adrenocortical adenomas and adrenocortical cancer samples, respectively. Both cancers and adenomas with 17q losses had higher PKA activity in response to cAMP when compared to similar tumors without 17q losses [20•].

A third link between cAMP and tumorigenesis is through altered PDEs. Inactivating molecular defects in PDEs lead to high cAMP or cGMP levels that in turn generate a continuous activation of the cAMP/PKA cascade. In 2006, our laboratory identified five PDE11A mutations in a group of 16 patients with adrenocortical hyperplasia. Three of these mutations led to premature terminations with truncated proteins, and the other two were missense mutations (R804H and R867G), leading to defective proteins [21••].

Although germline PDE11A truncating-protein mutations are seen in the general population, they are significantly more common among patients with adrenal hyperplasia [22]. Somatic missense mutations are frequently found in adrenocortical tumors: adrenocortical cancer (ACA), adrenocortical adenomas, and corticotrophin (ACTH)-independent macronodular adrenal hyperplasia or AIMAH. In line with the above, higher cAMP levels and lower PDE11A expression were observed in AIMAH and ACA tissues studied by immunohistochemistry [23•]. Interestingly, a higher frequency of PDE11A variants has been found in patients with PRKAR1A mutations, suggesting a contribution of PDE11A to adrenal and testicular tumor formation in CNC [24•]. More recently, PDE11A genetic defects were found to be significantly increased in prostatic cancer patients, compared with healthy controls, suggesting that PDE11A genetic variants may play a role in susceptibility to prostatic cancer, as well [25••].

A second PDE found to be involved in adrenocortical tumor predisposition was PDE8B; its locus on chromosome 5 was the second most highly linked to adrenal hyperplasias in a genome-wide study [21••]. A PDE8B missense mutation (p.H305P) was then described in a young girl with isolated micronodular adrenocortical disease. Functional studies showed high levels of cAMP in HEK293 cells transfected with the mutant gene [26]. Subsequently, additional three novel mutations in PDE8B were described in patients with adrenal tumors [27]. PDE8 is highly expressed in adrenal tissues [28•], and has an important role in steroidogenesis in adrenals, as recently demonstrated [29]. AIMAH and cortisol-producing adenomas specimens were found to have high cAMP levels and, interestingly, decreased PDE activity was shown in cortisol-producing adenomas [30•].

PDE8 is highly expressed in the pituitary gland [31]. A strong association between high TSH levels and polymorphisms in the PDE8B gene was described in a genome-wide association study [32]. The segregation of those polymorphic variants in a family with micronodular adrenal disease, with a PDE8 defect leading to Cushing syndrome was also studied [26]. The analysis revealed separate segregation of an inactivating PDE8B allele from the high TSH-predisposing allele, and showed low TSH levels in individuals who carry an inactivating PDE8B allele [28•].

An association between PDE10A and hypothyroidism was found in a study comprising 1258 individuals from three Alpine villages. In this study, a combination of linkage and association in families with hyperthyrotropinemia pointed to PDE10A and DACT2 as candidate genes. Genome association of the TSH values in a different population set supported the involvement of the PDE10A locus [33].

An association between upregulation of PDEs in a growth hormone (GH)-producing pituitary adenoma carrying a GNAS mutation has been investigated; increased PDE4C and PDE4D expression and activity were discussed as a possible protective mechanism against GNAS-dependent activation of the cAMP pathway [34].

PDE inhibitors and cancer

Vinpocetine, a PDE1 inhibitor, is used for the prevention of cerebrovascular disease and cognitive impairment, and to date no significant side effects or toxicity have been reported. Although the use of PDE1 inhibitors has not been associated with effects on tumorigenesis, in vitro cell studies have suggested a role for PDE1 inhibitors in controlling cell malignancy. For example, inhibition of PDE1B stimulates apoptosis in human leukemia cells [35]. Likewise, PDE1C is overexpressed in human malignant melanoma-associated cells, and growth is inhibited by vinpocetine [1,36,37].

Different PDE2 inhibitors have been experimentally tested but have not been used in humans. PDE2 inhibitors have been mainly tested for effects on endothelial permeability, and to treat learning and memory disorders in animal studies [38,39]. One of them, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), has been reported to increase intracellular cAMP levels in human umbilical vein endothelial cells (HUVEC) and inhibit angiogenesis, which can be associated with tumor development and other proliferative pathologies [40].

Cilostazol (Pletal^®), a dual inhibitor of PDE3 and adenosine uptake, is used for the treatment of intermittent claudication, due to its anti-aggregant and vasodilator properties [36]. Cilostazol has been tested as a tool for the inhibition of breast cancer metastasis in mice, due to its ability to restrict the aggregability of mouse platelets [41]. Also, cilostazol was reported to block human colon cancer cell motility, and might be effective as an anti-metastasis drug [42].

Rolipram is a PDE4 inhibitor marketed as antidepressant in several countries [43]. Rolipram enhanced the survival of mice bearing xenografts of U87 glioblastoma cells, and augmented the antitumor effect of chemotherapy and radiotherapy [44]. Incubation of a human alveolar epithelial type II cell line with rolipram resulted in inhibition of epithelial-mesenchymal transition (EMT), which is a critical event in the pathogenesis of organ fibrosis and cancer, suggesting that this drug can be used to depress EMT in lung cancer [45]. In CLL cells, rolipram, in a dose-dependent manner, increased intracellular cAMP levels and induced apoptosis [46,47].

Exisulind, a dual inhibitor for PDE4 and PDE5, is a novel drug with proapoptotic properties. In colon cancer cells and in rat bladder tumors; exisulind reduced multiplicity and incidence of the tumorigenic events [48,49]. Zaprinast was the first PDE5 inhibitor used in humans as a mast cell-stabilizer in allergy treatment. Other specific PDE5 inhibitors are sildenafil, vardenafil, and tadalafil which are used for treatment of erectile dysfunction [50]. An interesting connection between PDE5 and melanoma cell invasion has been described by Arozarena et al. [51•]. This study showed that downregulation of PDE5A in melanoma cells led to increased cGMP levels, which in turn caused a mild deceleration in cellular proliferation, but a larger effect on cell contractility. These events culminated in an increased invasion of melanoma cells [51•].

All PDE5 inhibitors weakly inhibit PDE6, which is expressed in rod and cone photoreceptors. This inhibition results in mild and transient visual symptoms that correlate with the inhibitor plasma concentrations [52,53]. The possible effect of therapeutic levels of tadalafil in the physiology of testis and spermatozoids has been a topic of studies that remain inconclusive [54,55,56]. In human tissues that express PDE11A (prostate, pituitary, heart, liver, skeletal muscle testis, bladder, and adrenal gland), no adverse effects related to the use of tadalafil or other PDE5 inhibitors that inhibit PDE11A have been reported to date [21••,55,57–59].

The use of PDE5 inhibitors as possible modulators of cell growth, division, and death has been reported. Sildefanil has been shown to induce an augmented endogenous antitumor immunity in several mouse tumor models [60]. Similar to the aforementioned activity of PDE4 inhibitors, vardenafil and sildenafil can induce apoptosis of chronic lymphocytic leukemia cells in vitro in an induced caspase-dependent mechanism [61].

Another inhibitor of PDE5, sulindac, can inhibit growth and induce apoptosis in human breast tumor cells, through elevation of cGMP and subsequent activation of PKG [62]. Sulindac also induces apoptosis in a non-small cell lung cancer orthotopic lung tumor model via a mechanism involving PDE inhibition — a finding consistent with a cGMP-regulated apoptosis pathway [63]. Furthermore, the use of antisense RNAi that suppresses PDE5 activity in human colon tumor cells inhibited cell growth by inducing cell apoptosis and delaying cell-cycle progression [64].

The effects of sildenafil, tadalafil, and vardenafil were also investigated in human stromal cells involved in bilateral prostatic hypertrophy (BPH). Vardenafil significantly inhibited human stromal cell proliferation in a dose-dependent manner [65]. A possible underlying mechanism involved blocking the degradation of cGMP, thereby augmenting the bioactivity of nitric oxide (NO), which, in turn, inhibited NADPH oxidase (NOX) and contributed to the reduction of superoxide (O₂⁻), a free radical thought to be involved in the genesis of BPH [66]. However, another study showed that inhibition of PDE5 can induce cell proliferation, and enhance new vessel growth and cell migration through activation of MAPKs [67,68]. In addition, an intracellular NO-induced apoptosis mechanism, which was enhanced by Ca²⁺-dependent NOX activation, was inhibited by downregulation of calcium transport exerted by PDE5 inhibitors [69,70,71].

Regarding the role of PDE7 in cell apoptosis, as previously mentioned for PDE4, the high expression of PDE7B in chronic lymphoid leukemia (CLL) cells, and PDE7 inhibitor-induced apoptosis can imply a new therapeutic target for this entity [72].

A very selective PDE8 inhibitor, PF-04957325 (Pfizer Inc., Groton Laboratories, Groton, CT), has been used in the characterization of T-cell adhesion and proliferation [73]. The association between PDE8B genetic defects and adrenal hyperplasia was described above, and although the use of PF-04957325 is known to potentiate steroidogenesis in Y-1 adrenal cell line and in mouse primary adrenocortical cells, no other reference of effects of PDE8 inhibitors on adrenal hyperplasia or tumorigenesis has been reported [29].

As mentioned, PDE11A defects have been described in different endocrine tumors, and high PDE11A immunoreactivity was detected by immunohistochemistry in renal, prostate, colon, lung, and breast carcinoma tissues [58]. However, PDE11A-specific inhibitors are not available to characterize the role for PDE11A in these tumors.

Conclusions

The continuous interest in PDE research since their discovery goes hand in hand with the development of their inhibitors which are used, first, to biologically characterize PDEs in different tissues and understand their involvement in various physiological and pathological settings, and, second, to selectively target PDEs in the treatment of diseases, avoiding adverse effects. Growing evidence supports a role for cyclic nucleotide signaling pathways in endocrine cell growth and proliferation, and possible tumor development. Additional studies are needed for more conclusive evidence and for the investigation of the role of PDE-modulating drugs in fighting tumor development.