Pituitary adenomas account for ~15% of primary intracranial neoplasms and are discovered in up to 25% of unselected autopsy specimens (2
). PRL-secreting adenomas account for ~40% of all pituitary tumors (1
). Indications for prolactinoma therapy include hyperprolactinemia-associated hypogonadism, infertility and osteoporosis, as well as central compressive effects (1
). Drug intolerance or development of dopamine agonist resistance may constrain medical therapy of benign prolactinomas (3
). Effective drug therapies are also required for recurrent invasive macroprolactinomas or those that undergo rare malignant transformation and are resistant to attempts at surgery and radiation (4
We show here that human prolactinomas express ErbB3 receptors, and that heregulin, an ErbB3 ligand, induces PRL gene expression. Expression of other ErbB receptor family members EGFR (22
) and p185her2/neu
) have been reported in the normal anterior pituitary. EGFR (9
) and p185her2/neu
) expression in pituitary adenomas have been associated with more invasive tumor phenotypes (9
), suggesting a role for these receptors in aggressive tumor behavior. Estrogen-mediated rat lactotroph hyperplasia is also associated with upregulation of pituitary TGFα, an EGFR ligand (30
), and lactotroph TGFα overexpression in transgenic mice results in hyperplasia and pituitary adenoma formation (31
). EGFR tyrosine kinase inhibition was shown to inhibit EGF-induced rat lactotroph differentiation and tumor growth (14
), indicating a role for EGFR signaling in prolactinoma regulation.
ErbB3 activation and subsequent PI3K / Akt signaling may contribute to TKI resistance in breast and lung cancer (15
and ErbB3 are functionally incomplete receptors. The extracellular domain of p185her2/neu
appears to be devoid of ligand-binding activity, while ErbB3 contains a non-functional kinase domain devoid of catalytic kinase activity. However, the p185her2/neu
/ ErbB3 heterodimer is the most active signaling dimer in the ErbB family (8
). Ligand induction leads to ErbB3 trans-phosphorylation (32
), the only ErbB receptor which couples directly to PI3K / Akt (33
). Although pituitary heregulin (glial growth factor) expression has been reported (34
), ErbB3 expression and the role of p185her2/neu
/ ErbB3 signaling in pituitary tumorigenesis were not heretofore known.
Rat lactosomatotroph GH4C1 cells are shown here to express functional ErbB receptor members (), and p185c-neu
tyrosine phosphorylation was observed in response to both EGFR and ErbB3 activation, suggesting p185c-neu
as the preferred dimerization partner in these cells. p185c-neu
tyrosine phosphorylation was detected only in response to ligand induction, suggesting expression of the non-mutated proto-oncogenic p185c-neu
, similar to the observed absence of p185her2/neu
activating mutations in human pituitary adenomas (28
). Heregulin induction of PRL (), suggests a functional role for p185c-neu
/ ErbB3 signaling in PRL but not GH control, consistent with known GH regulation (35
). In contrast to EGFR and p185her2/neu
), non-tumorous pituitary lactotrophs likely do not express ErbB3, as we did not observe PRL regulation by heregulin in primary rat pituitary cell cultures. ErbB receptor-mediated PRL regulation may be selective for transformed lactotrophs with altered intracellular signaling and loss of dopaminergic control. Indeed, GH3 / GH4C1 cells do not express functional D2 dopamine receptors (36
) and are resistant to dopamine agonists, similar to drug-resistant aggressive prolactinomas.
Aberrant Akt (37
) and MAPK (38
) activity in human pituitary tumors, and heregulinmediated ErbB3 and p185c-neu
tyrosine phosphorylation and receptor heterodimerization we observed, was associated with ERK and Akt activation. EGF, the EGFR ligand, also activated ERK but not PI3K / Akt signaling, as previously shown for GH3 cells (14
), demonstrating the requirement of pituitary ErbB3 activation for PI3K / Akt signaling induction. As targeted ErbB3-specific therapies are not currently available, we employed pharmacological inhibition of the receptor with gefitinib, an EGFR tyrosine kinase inhibitor, which induces formation of inactive heterodimers between ErbB3 and other ErbB receptor members, thereby blocking heregulin signaling (19
). Indeed, gefitinib treatment suppressed ErbB3 and p185c-neu
tyrosine phosphorylation and downstream signaling. Dose-dependent differences of gefitinib inhibition on receptor (higher doses) vs. ERK and Akt activation (lower doses) are likely due to differences in assay sensitivities, as the blots for immunoprecipitated ErbB3 and p185c-neu
show total tyrosine phosphorylation load, and are not reflective of specific tyrosine residues which signal for ERK and Akt. Although low doses of gefitinib could inhibit trans-activated EGFR on ERK signaling by heregulin, the ligand did not appear to induce EGFR trans-activation ().
and ErbB3 downregulation suppressed heregulin effects, indicating the requirement of functional pituitary p185c-neu
/ ErbB3 signaling for heregulin action on PRL. In the presence of heregulin, specific pharmacologic and siRNA-mediated inhibition of ERK but not Akt signaling mediated PRL regulation, consistent with known Ras/Raf/MAPK/Ets-1-mediated PRL regulation in GH3/GH4 cells (39
). However, downstream involvement of additional Ras/MAPK-independent mechanisms (as has been shown for EGF-mediated PRL regulation (41
) cannot be excluded. In contrast to PRL-releasing peptide (PrRP) and insulin, however, which signal to the proximal rat PRL promoter in a pathway that involves PI3K/Akt (43
/ ErbB3-mediated PI3K/Akt activation does not appear to induce lactotroph differentiation but could reflect changes leading to a more aggressive cell phenotype.
The unavailability of functional human pituitary cell lines and scarcity of surgically obtained human prolactinoma specimens renders mechanistic analysis of heregulin signaling in human prolactinomas an experimental challenge. We examined p185her2/neu
and ErbB3 expression in 7 benign prolactinomas and one carcinoma (Hardy class 4) by immunofluorescence and demonstrate p185her2/neu
expression in the majority of tumors (7/8). In addition, we now also show ErbB3 expression in a subset of these tumors (4/8), a novel finding which may indicate altered patterns of tumor differentiation. Tumors which were immunoreactive for ErbB3 expressed relatively high levels of Ki67, a marker of increased pituitary cell proliferation (44
). ErbB3 immunoreactivity was also detected in an aggressive carcinoma specimen. Quantitative PCR analysis of available RNA samples derived from a single individual who initially presented with a benign prolactinoma which recurred as a malignant prolactinoma with dopamine resistance and failed surgery and radiation, revealed dramatic upregulation of ErbB3 mRNA expression in the second tumor sample.
In addition to potential efficacy of temozolomide therapy (45
) and the development of new generation PRL receptor antagonists (47
), these results highlight the importance of examining ErbB receptor expression in surgical prolactinoma specimens. Benign prolactinomas usually respond effectively to medical therapy with dopamine agonists, and drug resistance may be an early sign of aggressive tumor behavior. In very rare cases of malignant tumor transformation during the observed course of the disease, and resistance to conventional therapy, information on progressive ErbB receptor status would provide an additional tool for determining therapy options for controlling tumor cell growth and excess PRL secretion.