Exploration of Shh and BMP paracrine signaling in a prostate cancer xenograft

Stromal–epithelial signaling is a critical regulator of normal prostate development and has been speculated to play an equally important role in the development and progression of prostate cancer. Sonic hedgehog (Shh) and bone morphogenetic proteins (BMP-4, BMP-7), expressed by the urogenital sinus epithelium and mesenchyme, exert reciprocal and coordinate effects on outgrowth of nascent prostate ducts. Over-expression of Shh in the LNCaP xenograft was shown previously to accelerate tumor growth by a paracrine mechanism. A survey of BMP regulators expressed in the developing prostate revealed increased Noggin and BMP-7 mRNA in the stromal component of Shh over-expressing xenografts. In vitro studies demonstrated that treatment of LNCaP cells with BMP-4 and BMP-s7 induced Id-1 expression and inhibited tumor cell proliferation. The activity of BMP-4 was abrogated by co-addition of Noggin; the activity of BMP-7 was not. Quantitative analysis of BMP signaling revealed ambivalent results: decreased tumor cell expression of the BMP response gene Id-1 but increased staining for phospho-SMAD 1,5, 8. To directly test whether increased xenograft tumor growth could be explained by Noggin-mediated blockade of BMP-2/4 effects on tumor cell proliferation, we generated LNCaP xenografts containing stromal cells over-expressing Noggin. Tumor cells in these xenografts exhibited decreased Id-1 and reduced SMAD phosphorylation, but tumor growth was not altered. We conclude that tumor cell Shh expression can induce significant changes in expression of BMP ligands and inhibitors in the stromal microenvironment but that acceleration of LNCaP xenograft tumor growth by Shh over-expression cannot be attributed solely to increased Noggin expression in the tumor stroma.     

Downregulation of PTEN/MMAC/TEP1 expression in human prostate cancer cell line DU145 by growth stimuli

Genetic alterations and/or deletion of the tumor suppressor gene PTEN/MMAC/TEP1 occur in many types of human cancer including prostate cancer. We describe the production of monoclonal antibody against recombinant human PTEN and the study of PTEN gene and protein expression in three commercially available human prostate cancer cell lines, PC-3, LNCaP, and DU 145. Northern blotting analyses showed that LNCaP and DU145 but not PC-3 cells expressed PTEN mRNA. However, Western blotting analyses using a monoclonal antibody against PTEN demonstrated the expression of PTEN protein in DU145 but not LNCaP cells. In DU145 cells, PTEN expression at both the mRNA and protein levels inversely correlated with serum concentrations and levels of PKB/Akt phosphorylation. In addition, the basal activity of PKB/Akt as indicated by level of phosphorylation was higher in prostate cancer cells which do not express PTEN than that in the cells expressing wild type PTEN. Thus, PTEN may play a critical role in regulating cellular signaling in prostate cancer cells.     

Loss of PTEN permits CXCR4-mediated tumorigenesis through ERK1/2 in prostate cancer cells

Loss of PTEN is frequently observed in androgen-independent prostate cancer, resulting in the deregulation of metastatic events. SDF1α activation of CXCR4 induces signaling pathways that have been implicated in prostate metastasis and progression to an advanced disease. The pathways of CXCR4 and PTEN converge, leading to the promotion and regulation of tumorigenesis, respectively. However, loss of PTEN may permit CXCR4 to progress prostate cancer to an advanced disease. In the present study, we investigated the involvement of PTEN in CXCR4-mediated tumorigenesis. When screening advanced metastatic prostate cancer cell lines for PTEN, we observed a loss of expression in PC3 and LNCaP cells whereas Du145 expressed wild-type PTEN. All three cell lines were positive for surface expression of CXCR4. Reconsitution of PTEN induced a mesenchymal to epithelial like morphologic change and inhibited CXCR4-mediated migration and proliferation in PC3 cells. Downregulation of PTEN by siRNA enhanced the CXCR4-mediated migratory behavior of Du145 cells. By Western blot analysis, we observed that PTEN inhibited basal AKT phosphorylation but not ERK1/2 phosphorylation in PTEN-expressing cells. Upon CXCR4 stimulation, PTEN inhibited ERK1/2 phosphorylation but not phosphorylation of AKT. The CXCR4-mediated migration of PC3 cells was through the ERK1/2 pathway, as confirmed by chemical inhibitors. On the basis of these studies, we suggest that loss of PTEN permits CXCR4-mediated functions in prostate cancer cells through the ERK1/2 pathway. Antagonizing CXCR4 and downstream signaling cascades may provide an efficient approach for treating patients with advanced prostate cancer when hormone therapy fails to the stop the growth and containment of tumors.     

Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization

In many human cancers, the cyclin-dependent kinase inhibitor p27(Kip1) is expressed at low or undetectable levels. The decreased p27(Kip1) expression allows cyclin-dependent kinase activity to cause cells to enter into S phase and correlates with poor patient survival. Inhibition of serine/threonine kinase Akt signaling by some pharmacological agents or by PTEN induces G(1) arrest, in part by up-regulating p27(Kip1). However, the role of Akt-dependent phosphorylation in p27(Kip1) regulation is not clear. Here, we show that Akt bound directly to and phosphorylated p27(Kip1). Screening p27(Kip1) phosphorylation sites identified the COOH-terminal Thr(198) residue as a novel site. Further analysis revealed that 14-3-3 proteins bound to p27(Kip1) through Thr(198) only when it was phosphorylated by Akt. Although Akt also phosphorylated p27(Kip1) at Ser(10) and Thr(187), these two sites were not involved in the binding to 14-3-3 proteins. p27(Kip1) phosphorylated at Thr(198) exists only in the cytoplasm. Therefore, Akt promotes cell-cycle progression through the mechanisms of phosphorylation-dependent 14-3-3 binding to p27(Kip1) and cytoplasmic localization.     

The F-box protein SKP2 mediates androgen control of p27 stability in LNCaP human prostate cancer cells

Background

The cyclin-dependent kinase inhibitor p27 is a putative tumor suppressor that is downregulated in the majority of human prostate cancers. The mechanism of p27 down-regulation in prostate cancers in unknown, but presumably involves increased proteolysis mediated by the SCF^SKP2 ubiquitin ligase complex. Here we used the human prostate cancer cell line LNCaP, which undergoes G1 cell cycle arrest in response to androgen, to examine the role of the SKP2 F-box protein in p27 regulation in prostate cancer.

Results

We show that androgen-induced G1 cell cycle arrest of LNCaP cells coincides with inhibition of cyclin-dependent kinase 2 activity and p27 accumulation caused by reduced p27 ubiquitylation activity. At the same time, androgen decreased expression of SKP2, but did not affect other components of SCF^SKP2. Adenovirus-mediated overexpression of SKP2 led to ectopic down-regulation of p27 in asynchronous cells. Furthermore, SKP2 overexpression was sufficient to overcome p27 accumulation in androgen arrested cells by stimulating cellular p27 ubiquitylation activity. This resulted in transient activation of CDK2 activity, but was insufficient to override the androgen-induced G1 block.

Conclusions

Our studies suggest that SKP2 is a major determinant of p27 levels in human prostate cancer cells. Based on our in vitro studies, we suggest that overexpression of SKP2 may be one of the mechanisms that allow prostate cancer cells to escape growth control mediated by p27. Consequently, the SKP2 pathway may be a suitable target for novel prostate cancer therapies.     

Regulation of androgen receptor signaling by PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor through distinct mechanisms in prostate cancer cells

Defects in the PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor gene have been found in many human cancers including breast and prostate. Here we show that PTEN suppresses androgen receptor (AR) activity via a phosphatidylinositol-3-OH kinase/Akt-independent pathway in the early passage numbers prostate cancer LNCaP cells. We provide the direct links between PTEN and androgen/AR signaling by demonstrating that AR directly interacts with PTEN. The interaction between PTEN and AR inhibits the AR nuclear translocation and promotes the AR protein degradation that result in the suppression of AR transactivation and induction of apoptosis. The minimum interaction peptide within AR (amino acids 483-651) disrupts the interaction of PTEN with AR and reduces the PTEN effect on AR transactivation and apoptosis. Genetic approaches using PTEN-null mouse embryonic fibroblasts (MEFs) further demonstrate that both AR expression and AR activity were much higher in PTEN-null MEFs than wild-type MEFs, and reintroducing PTEN into PTEN-null MEFs dramatically reduced AR protein levels and AR activity. Interestingly, we also found that PTEN could suppress AR activity via the phosphatidylinositol-3-OH kinase/Akt-dependent pathway in the higher passage number LNCaP cells, because restoration of Akt activity blocks the effect of PTEN on AR activity. Together, these contrasting PTEN effects on AR activity in the same prostate cancer cell line with different passage numbers suggest that PTEN, via distinct mechanisms, differentially regulates AR in various stages of prostate cancers.     

Androgens as therapy for androgen receptor-positive castration-resistant prostate cancer

Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in Western countries. While surgery is often successful for organ-confined prostate cancer, androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. Shortening the period of androgen ablation therapy may benefit prostate cancer patients. Intermittent Androgen Deprivation therapy improves quality of life, reduces toxicity and medical costs, and delays disease progression in some patients. Cell culture and xenograft studies using androgen receptor (AR)-positive castration-resistant human prostate cancers cells (LNCaP, ARCaP, and PC-3 cells over-expressing AR) suggest that androgens may suppress the growth of AR-rich prostate cancer cells. Androgens cause growth inhibition and G1 cell cycle arrest in these cells by regulating c-Myc, Skp2, and p27^Kip via AR. Higher dosages of testosterone cause greater growth inhibition of relapsed tumors. Manipulating androgen/AR signaling may therefore be a potential therapy for AR-positive advanced prostate cancer.     

Cell-permeable carboxyl-terminal p27(Kip1) peptide exhibits anti-tumor activity by inhibiting Pim-1 kinase

The incidence and death rate of prostate cancer is increasing rapidly. In addition, the low sensitivity of prostate cancer to chemotherapy makes it difficult to treat this condition. The serine/threonine kinase Pim-1 plays an important role in cell cycle progression and apoptosis inhibition, resulting in prostate tumorigenesis. Therefore, Pim-1 inhibition has been expected to be an attractive target for developing new anti-cancer drugs. However, no small compounds targeting Pim-1 have progressed to clinical use because of their lack of specificity. Here, we have reported a new cell-permeable Pim-1 inhibitory p27(Kip1) peptide that could interfere with the binding of Pim-1 to its substrates and act as an anti-cancer drug. The peptide could bind to Pim-1 and inhibit phosphorylation of endogenous p27(Kip1) and Bad by Pim-1. Treatment of prostate cancer with the peptide induces G(1) arrest and subsequently apoptosis in vitro. However, the peptide showed almost no growth inhibitory or apoptosis-inducing effects in normal cells. The peptide could inhibit tumor growth in in vivo prostate cancer xenograft models. Moreover, the peptide treatment could overcome resistance to taxol, one of the first line chemotherapeutic agents for prostate cancer, and a combination of the peptide with taxol synergistically inhibited prostate cancer growth in vivo. These results indicate that a Pim-1 inhibitory p27(Kip1) peptide could be developed as an anti-cancer drug against prostate cancer.     

Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.     

mLST8 Promotes mTOR-Mediated Tumor Progression

The activity of the mechanistic target of rapamycin (mTOR) is elevated in various types of human cancers, implicating a role in tumor progression. However, the molecular mechanisms underlying mTOR upregulation remain unclear. In this study, we found that the expression of mLST8, a required subunit of both mTOR complex 1 (mTORC1) and complex 2 (mTORC2), was upregulated in several human colon and prostate cancer cell lines and tissues. Knockdown of mLST8 significantly suppressed mTORC1 and mTORC2 complex formation, and it also inhibited tumor growth and invasiveness in human colon carcinoma (HCT116) and prostate cancer (LNCaP) cells. Overexpression of mLST8 induced anchorage-independent cell growth in normal epithelial cells (HaCaT), although mLST8 knockdown had no effect on normal cell growth. mLST8 knockdown reduced mTORC2-mediated phosphorylation of AKT in both cancer and normal cells, whereas it potently inhibited mTORC1-mediated phosphorylation of 4E-BP1 specifically in cancer cells. These results suggest that mLST8 plays distinct roles in normal and cancer cells, depending upon its expression level, and that mLST8 upregulation may contribute to tumor progression by constitutively activating both the mTORC1 and mTORC2 pathways.     

Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human prostate cancer

Prostate cancer is one of the most common cancers among men. Recent studies demonstrated that PI3K signaling is an important intracellular mediator which is involved in multiple cellular functions including proliferation, differentiation, anti-apoptosis, tumorigenesis, and angiogenesis. In the present study, we demonstrate that the inhibition of PI3K activity by LY294002, inhibited prostate cancer cell proliferation and induced the G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins including cyclin D1, CDK4, and Rb phosphorylation at Ser780, Ser795, and Ser807/811, whereas expression of CDK6 and beta-actin was not affected by LY294002. The expression of cyclin kinase inhibitor, p21(CIP1/WAF1), was induced by LY294002, while levels of p16(INK4) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation and p70(S6K), but not MAPK. PI3K regulates cell cycle through AKT, mTOR to p70(S6K). The mTOR inhibitor rapamycin has similar inhibitory effects on G(1) cell cycle progression and expression of cyclin D1, CDK4, and Rb phosphorylation. These results suggest that PI3K mediates G(1) cell cycle progression and cyclin expression through the activation of AKT/mTOR/p70(S6K) signaling pathway in the prostate cancer cells.     

CXCR4 and PTEN are involved in the anti-metastatic regulation of anethole in DU145 prostate cancer cells

Anethole has been known to have chemopreventive activities as a suppressor of the incidence and multiplicity of both invasive and noninvasive carcinomas. The goal of this study was to understand the anti-metastatic effect of anethole through C-X-C chemokine receptor type 4 (CXCR4)/tumor suppressor phosphatase and tensin homologue (PTEN) axis in DU145 prostate cancer cells. Anethole reduced both of the RNA level and the protein level of CXCR4 in a dose-dependent manner without cytotoxicity. Anethole also reduced the expression of CXCR4 and prolonged the expression of PTEN in DU145 prostate cancers. The phosphorylation of AKT and phosphatidylinositol-3kinase (PI3K) were decreased with anethole. The inhibition metastatic effect of anethole was arisen from down-regulating CXCR4 and up-regulating PTEN. Morphologically, anethole significantly inhibited the invasion of DU145 cell and down-regulated the activities of matrix-metalloproteinase (MMPs) in a dose-dependent manner. However, anethole didnot decrease the phosphorylation of PI3K and AKT while PTEN was silenced. Furthermore, the CXCR4 inhibition of anethole was not caused to proteasomal or lysosomal of CXCR4.     

Interleukin-6 induces G1 arrest through induction of p27(Kip1), a cyclin-dependent kinase inhibitor, and neuron-like morphology in LNCaP prostate tumor cells

Prostate carcinoma cells express high levels of interleukin-6 (IL-6) and IL-6 receptor. In this study, we examined the effect of IL-6 on LNCaP human prostate carcinoma cells. IL-6 induces G1 growth arrest of LNCaP. Following IL-6 treatment of LNCaP, Western blot analysis showed that the protein levels of cyclin-dependent kinase-2 (CDK2), CDK4, and CDK6 were decreased, while accumulation of CDK inhibitor p27(Kip1) was rapidly and markedly induced. In vitro kinase assays revealed that the CDK-associated histone H1 and CDK4- and CDK6-associated pRb kinase activities were significantly inhibited in IL-6-treated LNCaP. Further, a significant amount of p27(Kip1) was co-precipitated with CDK2, CDK4 and CDK6, as detected in immunoprecipitation experiments. Thus, IL-6-induced G1 arrest appears to be due to the accumulation of p27(Kip1). In addition, IL-6-treated LNCaP cells induced neuron-like morphological changes. Since neuroendocrine differentiation is observed in most prostate carcinomas, these findings raise the possibility that IL-6 may be involved in neuroendocrine differentiation in vivo.     

Combining p53 stabilizers with metformin induces synergistic apoptosis through regulation of energy metabolism in castration-resistant prostate cancer

Since altered energy metabolism is a hallmark of cancer, many drugs targeting metabolic pathways are in active clinical trials. The tumor suppressor p53 is often inactivated in cancer, either through downregulation of protein or loss-of-function mutations. As such, stabilization of p53 is considered as one promising approach to treat those cancers carrying wild type (WT) p53. Herein, SIRT1 inhibitor Tenovin-1 and polo-like kinase 1 (Plk1) inhibitor BI2536 were used to stabilize p53. We found that both Tennovin-1 and BI2536 increased the anti-neoplastic activity of metformin, an inhibitor of oxidative phosphorylation, in a p53 dependent manner. Since p53 has also been shown to regulate metabolic pathways, we further analyzed glycolysis and oxidative phosphorylation upon drug treatments. We showed that both Tennovin-1 and BI2536 rescued metformin-induced glycolysis and that both Tennovin-1 and BI2536 potentiated metformin-associated inhibition of oxidative phosphorylation. Of significance, castration-resistant prostate cancer (CRPC) C4-2 cells show a much more robust response to the combination treatment than the parental androgen-dependent prostate cancer LNCaP cells, indicating that targeting energy metabolism with metformin plus p53 stabilizers might be a valid approach to treat CRPC carrying WT p53.