PI3K/PTEN/AKT signaling regulates prostate tumor angiogenesis

Abstract

PI3K pathway exerts its function through its downstream molecule AKT in regulating various cell functions including cell proliferation, cell transformation, cell apoptosis, tumor growth and angiogenesis. PTEN is an inhibitor of PI3K, and its loss or mutation is common in human prostate cancer. But the direct role and mechanism of PI3K/PTEN signaling in regulating angiogenesis and tumor growth in vivo remain to be elucidated. In this study, by using chicken chorioallantoic membrane (CAM) and in nude mice models, we demonstrated that inhibition of PI3K activity by LY294002 decreased PC-3 cells-induced angiogenesis. Reconstitution of PTEN, the molecular inhibitor of PI3K in PC-3 cells inhibited angiogenesis and tumor growth. Immunohistochemical staining indicated that PTEN expression suppressed HIF-1α, VEGF and PCNA expression in the tumor xenographs. Similarly, expression of AKT dominant negative mutant also inhibited angiogenesis and tumor growth, and decreased the expression of HIF-1α and VEGF in the tumor xenographs. These results suggest that inhibition of PI3K signaling pathway by PTEN inhibits tumor angiogenesis and tumor growth. In addition, we found that AKT is the downstream target of PI3K in controlling angiogenesis and tumor growth, and PTEN could inhibit angiogenesis by regulating the expression of HIF-1 and VEGF expression through AKT activation in PC-3 cells.

Introduction

The phosphatidylinotidol 3-kinase (PI3K) is a heterodimeric enzyme composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit [1]. PI3K catalyzes the production of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 helping to recruit AKT (protein kinase B) and PDK1 to the membrane [1], [2], [3], [4], [5]. When activated, AKT transmits survival signals from growth factors and inactivates the apoptotic pathways [6], [7].

PTEN (phosphatase and tensin homolog deleted on chromosome ten)/MMAC (mutated in multiple advanced cancers) is a tumor suppressor gene [3], [8]. The PTEN tumor suppressor gene is lost or mutated in a large number of human cancers including prostate cancer [8], [9], [10]. Deregulation of PI3K pathway is commonly observed in many human cancers. This deregulation can be caused either by loss of PTEN, or by constitutive activation of PI3K or its target AKT. Activation of PI3K/AKT may potentiate cell survival, cell migration, proliferation, and cytoskeletal rearrangement. In addition, PI3K pathway is implicated in upregulating vascular endothelial growth factor (VEGF). Studies show that overexpression of PI3K or AKT correlates with increased VEGF levels [11], [12], [13], [14], and overexpression of PTEN in chicken embryo inhibited embryonic angiogenesis [15].

Angiogenesis is the formation of new blood vessels from preexisted ones. It is critical for tumor growth. The tumor cannot grow more than 1–2 mm in diameter without angiogenesis [16]. Tumor angiogenesis is stimulated by a few angiogenic factors, among which VEGF is a fundamental regulator [17]. Hypoxia inducible factor 1 (HIF-1) is a transcription factor that activates the transcription of many genes, including VEGF [18]. HIF-1 is composed of HIF-1α and β subunits [19], [20]. HIF-1α expression can be stimulated by a low concentration of oxygen or loss of function of VHL and PTEN [21], [22]. HIF-1α expression can be induced by growth factors through activation of PI3K/AKT signaling [23], [24]. PI3K/AKT signaling regulates tumor angiogenesis via HIF-1α/VEGF.

Prostate cancer is one of the most commonly diagnosed male malignancy in the United States, affecting 1 in 9 men over 65 years of age [25]. Several lines of evidence implicate that loss of PTEN is a major factor causing human prostate carcinogenesis. PTEN was found to be mutated or deleted in many prostate cancer cell lines including LNCaP, PC-3, NCI H660 [8], [10]. It was shown that PTEN is frequently lost in prostate cancer cells [26], that it undergoes homozygous deletions in ∼ 10% of primary prostate tumors [27], and that alterations are frequently occurred in metastatic prostate cancer [28].

Elevated levels of VEGF are found in the serum and urine of prostate cancer patients, suggesting that VEGF plays an important role in prostate cancer. PI3K/PTEN pathway regulated HIF-1α and VEGF expression in prostate cancer cell lines [11], [14], [28], indicating that this pathway may play an important role in regulating prostate tumor angiogenesis. However, there is a lack of direct evidence to show that PI3K/PTEN pathway regulates prostate tumor angiogenesis in vivo. We have successfully established tumor cells-induced angiogenesis and tumor growth models on chicken chorioallantoic membrane (CAM) and in nude mice [12], [15], [29]. The goal of this study is to investigate: 1) whether PI3K activity is required for angiogenesis of prostate tumor; 2) whether restoration of PTEN expression in prostate cancer cells inhibits tumor angiogenesis and growth on CAM in vivo; 3) whether the level and activity of PI3K and PTEN affect HIF-1 and VEGF expression in tumors; and 4) whether AKT is downstream target of PI3K for regulating angiogenesis.