Prostate cancer is the most common visceral malignancy in US men and the second leading cause of cancer deaths. The clinical behavior of prostate cancer is extremely heterogeneous, ranging from indolent disease to aggressive, metastatic cancer with rapid mortality. While many advances have been made in our understanding of prostate cancer biology, there are still significant gaps in our knowledge regarding the regulation of critical pathways regulating prostate cancer progression.
Activation of phosphatidyl-inositol-3 kinase (PI3K) pathway and Akt serine-threonine kinases play a central role in prostate cancer initiation and progression (for review see (1
)). Following the activation of PI3K by tyrosine kinase receptors or other cell surface receptors, the resulting lipid second messenger product phospholipids phosphatidylinositol 3, 4, 5-trisphosphate (PI-3,4,5-P3
) or phosphatidylinositol 3, 4-bisphosphate (PI-3,4-P2
), recruit Akt to the plasma membrane and bind to its pleckstrin homology (PH) domain. This binding leads to the conformational change in Akt, resulting in phosphorylation at Ser-473 in the regulatory domain. Phosphorylated Akt can then phosphorylate and regulate the function of many cellular proteins involved in cell proliferation, survival and mobility- processes that are critical for tumorigenesis and metastasis (1
). The net result of Akt activation include enhanced cell proliferation (1
), decreased apoptosis (1
) and increased tumor angiogenesis (1
), all of which can promote prostate cancer progression.
The NF-κB pathway also plays a critical role in prostate cancer progression (6
). NF-κB is present in cells as a heterodimer of two subunits, p50 and p65. This complex is retained in the cytoplasm of unstimulated cells by its interaction with IκBα. After stimulation of cells by cytokines and/or growth factors, IκBα is phosphorylated by the IKK complex, leading to degradation of IκBα by the 26S proteosome. This allows translocation of the NF-κB complex to the nucleus where it can activate transcription of a number of genes that can promote neoplastic progression in prostate cancer (6
). These include Bcl-2, c-myc, IL-6, IL-8, VEGF, MMP9, μPA and μPAR, all of them may play a role in prostate cancer initiation and progression. Consistent with these biological activities, immunohistochemical studies have shown that increased nuclear NF-kB staining is a strong independent predictor of biochemical recurrence following radical prostatectomy (10
Given the critical role of the PI3-K pathway in many cellular processes, it is not surprising that it is regulated by both positive and negative regulators. The PTEN tumor suppressor gene encodes a lipid phosphatase and is inactivated in a wide variety of malignant neoplasms, including prostate carcinoma (1
). The tumor suppressor activity of the PTEN tumor suppressor gene is primarily due to its ability to dephosphorylate phosphatidylinositol (3
) phosphate at the 3-position and negatively regulate the activity of the PI3-K pathway (1
). A novel group of positive regulators of the PI3-K pathway are the PIKE/GGAP2 proteins (19
). These proteins are all encoded by a single gene on chromosome 12q13.3 (21
). PIKE-L and PIKE-S are alternatively spliced variants of the same transcript (21
). The PIKE-A/GGAP2 transcript arises from an alternative promoter within the PIKE gene (22
), which was first identified as PIKE-S. It contains N-terminal proline-rich domains, a Ras homology domain (G domain), and a pleckstrin homology domain. PIKE-L is an alternatively spliced isoform which also contains a C-terminal Arf-GAP domain and two ankyrin repeats. Both of these proteins can bind PI3-K via their proline-rich domains and modulate its activity in the central nervous system and may play an important role in central nervous system biology (19
). The third form, known as GGAP2 or PIKE-A (we will use the designation GGAP2), is expressed in cancer (27
The GGAP2 protein is similar to PIKE-L except the N-terminal proline-rich domains due to an alternative promoter. This protein was originally characterized in 2003 by Xia et al as GGAP2 (23
). It was shown that GGAP2 has a GTPase activity, as expected from its RAS homology domain, and could play critical roles in modulating many normal cellular processes as well as in neoplastic transformation and tumor progression. The GAP domain can activate the GTPase activity via either intramolecular or intermolecular interaction. Further studies of the PIKE-A/GGAP2 protein (22
) demonstrated that the protein binds to activated Akt and this binding is promoted by GTP binding. In addition, the GTPase domain of GGAP2 is responsible for binding activated Akt. This binding enhances Akt activation while a dominant negative form of GGAP2 decreases Akt activity. Increased GGAP2 protein results in increased invasion and resistance to apoptosis in glioblastoma cell lines and Akt is necessary for these changes in the phenotype (22
). In summary, GGAP2 can promote Akt activity via direct binding with the protein, which can be modulated by GTP binding.
There is evidence linking alterations of GGAP2 activity to neoplastic transformation. The GGAP2 locus at 12q13.3 is amplified in glioblastoma cell lines, primary glioma cultures, and in glioblastoma tumors from patients (22
). More than 90% of glioblastomas overexpress GGAP2 (26
), indicating that it is probably a key target in this amplicon. GGAP2 is also amplified in sarcoma and neuroblastoma cell lines (22
). Dot blot hybridization by Liu et al (27
) showed the increased expression of GGAP2/PIKE-A mRNA in a wide variety of human malignancies, including 2 of 4 prostate cancers. Thus, increased GGAP2 activity is seen in a number of human malignancies secondary to amplification and/or overexpression. A number of comparative genomic hybridization studies of prostate cancer have shown gain of the 12q13.3 region where the GGAP2 gene is located (28
). We therefore undertook studies to examine the potential role of GGAP2 in human prostate cancer.