Id-1 stimulates serum independent prostate cancer cell proliferation through inactivation of p16(INK4a)/pRB pathway

It has been suggested that the helix-loop-helix protein Id-1 plays an important role in tumourigenesis in certain types of human cancer. Previously, we reported that Id-1 was up-regulated during sex hormone-induced prostate carcinogenesis in a Noble rat model (Ouyang et al. (2001) Carcinogenesis, 22, 965-973). In the present study, we investigated the direct effect of Id-1 expression on human prostate cancer cell proliferation by transfecting an Id-1 expression vector into a prostate cancer cell line LNCaP. Ten stable transfectant clones were isolated and the ectopic Id-1 expression resulted in both increased DNA synthesis rate and the percentage of S phase cells. To study the possible mechanisms involved in the Id-1 induced prostate cancer cell growth, we examined the expression of several factors responsible for G(1) to S phase progression. We found that Id-1 expression induced phosphorylation of RB and down-regulation of p16(INK4a) but not p21(Waf1)or p27(Kip1). Our results indicate that the Id-1 induced inactivation of p16(INK4a)/pRB pathway may be responsible for the increased cell proliferation in prostate cancer cells. Given the fact that both Id-1 over-expression and inactivation of p16(INK4a)/pRB are common events in prostate cancer, our results provide a possible mechanism on the molecular basis of prostate carcinogenesis.     

Prostate cancer cells modulate osteoblast mineralisation and osteoclast differentiation through Id-1

Background:

Id-1 is overexpressed in and correlated with metastatic potential of prostate cancer. The role of Id-1 in this metastatic process was further analysed.

Methods:

Conditioned media from prostate cancer cells, expressing various levels of Id-1, were used to stimulate pre-osteoclast differentiation and osteoblast mineralisation. Downstream effectors of Id-1 were identified. Expressions of Id-1 and its downstream effectors in prostate cancers were studied using immunohistochemistry in a prostate cancer patient cohort (N=110).

Results:

We found that conditioned media from LNCaP prostate cancer cells overexpressing Id-1 had a higher ability to drive osteoclast differentiation and a lower ability to stimulate osteoblast mineralisation than control, whereas conditioned media from PC3 prostate cancer cells with Id-1 knockdown were less able to stimulate osteoclast differentiation. Id-1 was found to negatively regulate TNF-β and this correlation was confirmed in human prostate cancer specimens (P=0.03). Furthermore, addition of recombinant TNF-β to LNCaP Id-1 cell-derived media blocked the effect of Id-1 overexpression on osteoblast mineralisation.

Conclusion:

In prostate cancer cells, the ability of Id-1 to modulate bone cell differentiation favouring metastatic bone disease is partially mediated by TNF-β, and Id-1 could be a potential therapeutic target for prostate cancer to bone metastasis.     

Activation of MAPK signaling pathway is essential for Id-1 induced serum independent prostate cancer cell growth

The helix-loop-helix protein Id-1 has been suggested to play a positive role in cell proliferation and tumorigenesis of many types of human cancers. However, little is known about the molecular mechanism involved in the function of Id-1. In this study, using four stable Id-1 transfectant clones, we investigated the involvement of MAPK signaling pathway in the Id-1 induced serum independent prostate cancer cell growth. Our results demonstrated that both transient and stable ectopic Id-1 expression in prostate cancer LNCaP cells led to activation of the Raf/MEK1/2 signaling pathway. In addition, inhibition of MEK1/2 phosphorylation by one of its inhibitors, PD098059, resulted in the decreased cell cycle S phase fraction and cell growth rate, suggesting that activation of MAPK signaling pathway is essential for Id-1 induced prostate cancer cell proliferation. Furthermore, treatment with antisense oligonucleotide complementary to Id-1 mRNA in PC-3 and DU145 cells resulted in a decreased Id-1 expression which was accompanied by decreased Egr-1 protein. Our results suggest for the first time that the function of Id-1 is associated with MAPK signaling pathway activation and indicate a possible novel mechanism in which Id-1 regulates prostate cancer cell growth and tumorigenesis.     

Id-1 expression promotes cell survival through activation of NF-kappaB signalling pathway in prostate cancer cells

The growth-promoting effect of Id-1 (inhibitor of differentiation/DNA binding) has been demonstrated in a number of human cancers. However, the mechanisms responsible for its action are not clear. In this study, we report that in prostate cancer cells, Id-1 promotes cell survival through activation of nuclear factor-kappaB (NF-kappaB) signalling pathway. After stable expression of Id-1 protein in LNCaP cells, we found that the Id-1 transfectants showed increased resistance to apoptosis induced by TNFalpha through inactivation of Bax and caspase 3. In addition, in the LNCaP cells expressing ectopic Id-1 protein, we also observed increased NF-kappaB transactivation activity and nuclear translocation of the p65 and p50 proteins, which was accompanied by upregulation of their downstream effectors Bcl-xL and ICAM-1. These results indicate that the Id-1-induced antiapoptotic effect may be via NF-kappaB signalling transduction pathway in these cells. In addition, inactivation of Id-1 by its antisense oligonucleotide and retroviral construct in DU145 cells resulted in the decrease of nuclear level of p65 and p50 proteins, which was associated with increased sensitivity to TNFalpha-induced apoptosis. Our results strongly suggest that Id-1 may be one of the upstream regulators of NF-kappaB and activation of NF-kappaB signalling pathway may be essential for Id-1 induced cell proliferation through protection against apoptosis. Our findings also suggest a potential therapeutic strategy in which inactivation of Id-1 may lead to sensitization of prostate cancer cells to chemotherapeutic drug-induced apoptosis.     

Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor

This study discovered that Id-1 expression in androgen-dependent prostate cancer decreased immediately after androgen deprivation but increased after longer androgen deprivation both in vivo and in vitro. Id-1 expression in androgen-independent LNCaP cells was about 6 fold as that in their parental cells. As was the case with LNCaP cells, when androgen receptor (AR) was introduced into AR-negative PC-3 cells, dihydrotestosterone inhibited while flutamide increased Id-1 expression. Thus, Id-1 expression in androgen-dependent prostate cancer was negatively regulated by androgen in a receptor-dependent way. The re-increased Id-1 might partially contribute to the emergence of androgen-independent prostate cancer after longer androgen deprivation therapy.     

Inactivation of Id-1 in prostate cancer cells: A potential therapeutic target in inducing chemosensitization to taxol through activation of JNK pathway

Resistance to anticancer drugs is the major problem in the treatment of many advanced cancers, including androgen-independent prostate cancer. Recently, increased expression of Id-1, a basic helix-loop-helix protein, is reported in several types of advanced cancer. It is suggested that high expression of Id-1 may provide an advantage for cancer cell survival and inactivation of Id-1 may be able to increase cancer cells' susceptibility to apoptosis. To test this hypothesis, in this study, by using RNA interfering technology, we inactivated the Id-1 gene in 2 androgen-independent prostate cancer cell lines, DU145 and PC3, and investigated whether downregulation of Id-1 could lead to increased sensitivity to a commonly used anticancer drug, taxol. By using colony forming assay and MTT assay, we found that inactivation of Id-1 resulted in both decreased colony forming ability and cell viability in prostate cancer cells, after taxol treatment. In addition, the si-Id-1-induced sensitization to taxol was associated with activation of apoptosis pathway, which is demonstrated by increased apoptotic index, DNA laddering, sub-G1 phase of the cell cycle, as well as cleaved-PARP and Caspase 3. Furthermore, c-Jun N-terminal kinase (JNK), one of the common pathways responsible for taxol-induced apoptosis, was also activated in the si-Id-1 transfected cells. Inhibition of JNK activity by a specific inhibitor, SP600125, blocked the si-Id-1-induced sensitivity to taxol. These results indicate that increased Id-1 expression in prostate cancer cells may play a protective role against apoptosis, and downregulation of Id-1 may be a potential target to increase sensitivity of taxol-induced apoptosis in prostate cancer cells.     

Immunohistochemical detection of tyrosine phosphatase SHP‐1 predicts outcome after radical prostatectomy for localized prostate cancer

The protein tyrosine kinase (PTK) receptors and cytosolic signaling proteins as well as the protein tyrosine phosphatases (PTPs) have important roles in regulation of growth of the benign and malignant prostate gland. Here, we studied expression of the protein tyrosine phosphatase SHP‐1 in prostate cancer cell lines and in human prostatic tissues. SHP‐1 is expressed at a high level in LNCaP prostate cancer cells compared with PC3 cells. Silencing of SHP‐1 expression with siRNA in LNCaP cells led to an increased rate of proliferation, whereas overexpression of SHP‐1 by means of transient and stable transfection in PC3 cells led to a decrease in proliferation. Corresponding changes were observed in cyclin D1 expression. We further demonstrate that LNCaP and PC3 cells respond differently to IL‐6 stimulation. SHP‐1 overexpression in PC3 cells reversed IL‐6 stimulation of proliferation, whereas in SHP‐1‐silenced LNCaP cells, IL‐6 inhibition of proliferation was not affected. In addition, IL‐6 treatment led to higher levels of phosphorylated STAT3 in SHP‐1‐silenced LNCaP cells than in control cells. Next, SHP‐1 expression in human prostate cancer was analyzed by immunohistochemical staining of tissue microarrays comprising tumor specimens from 100 prostate cancer patients. We found an inverse correlation between the tumor level of SHP‐1 expression and time to biochemical recurrence and clinical progression among prostate cancer patients. In conclusion, our results suggest that a decreased level of SHP‐1 expression in prostate cancer cells is associated with a high proliferation rate and an increased risk of recurrence or clinical progression after radical prostatectomy for localized prostate cancer.     

PC-1基因表达对前列腺癌细胞迁移能力的影响

背景与目的:PC-1基因在雄激素非依赖和高转移能力的前列腺癌C4-2细胞中高表达,在雄激素依赖及不转移的前列腺癌LNCaP细胞中低表达。本实验旨在研究PC-1对前列腺癌细胞迁移能力的影响。方法:构建PC-1稳定高表达的LNCaP细胞株和反义核酸调低内源性PC-1表达的C4-2细胞株。利用体外迁移系统检测PC-1表达对LNCaP和C4-2细胞迁移运动能力的影响。结果:体外迁移实验表明稳定转染提高PC-1的表达水平并未使LNCaP迁移细胞数增多(P>0.05),而反义核酸降低PC-1表达则使C4-2迁移细胞数降低(P<0.05)。PC-1蛋白水平升高不能提高LNCaP细胞迁移能力,但降低内源性PC-1表达则使C4-2细胞迁移能力明显降低。结论:PC-1可能在前列腺癌细胞侵袭过程中起一定作用。     

Expression and functional role of CCR9 in prostate cancer cell migration and invasion.

PURPOSE: Metastasis is responsible for most cancer-related deaths; hence, therapies designed to minimize metastasis are greatly needed. The precise cellular and molecular mechanisms used by cancer cells for metastasis are not fully understood; however, the metastatic spread of neoplastic cells is probably related to the ability of these cells to migrate, invade, home, and survive locally. The migration of tumor cells shares many similarities with leukocyte trafficking, which is regulated by chemokine receptor-ligand interactions. The current study evaluates the molecular mechanisms of CCL25 and CCR9 in prostate cancer cell migration and invasion. EXPERIMENTAL DESIGN: In the current study, real-time quantitative polymerase chain reaction, flow cytometry analysis, and in vitro migration as well as invasion chamber analysis (with and without antibody-mediated inhibition) were used to ascertain the biological and functional significance of CCR9 expression by normal prostatic epithelial cells (PrEC) or prostate cancer cell lines (LNCaP-10995 and PC3). RESULTS: We report that functional CCR9 is highly expressed by LNCaP cells and modestly, yet significantly, expressed by PC3 cells when compared with PrEC cells. Neutralization of CCL25-CCR9 interactions impaired the migration and invasion potential of the LNCaP and PC3 cell lines. CCL25 differentially modulated the expression of collagenase-1 or matrix metalloproteinase (MMP)-1, collagenase-3 (MMP-13), stromalysin-2 (MMP-10), stromalysin-3 (MMP-11), and gelatinase-A (MMP-2), but not MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, or MMP-14 in prostate cancer cells. CONCLUSIONS: These studies suggest that the expression and activation of CCR9 affect cancer cell migration, invasion, and MMP expression, which together may affect prostate cancer metastasis.     

Id-1 promotes proliferation of p53-deficient esophageal cancer cells

The helix-loop-helix protein inhibitor of differentiation and DNA binding (Id-1) is known to promote cellular proliferation in several types of human cancer. Although it has been reported that Id-1 is over-expressed in esophageal squamous cell carcinoma (ESCC), its function and signaling pathways in esophageal cancer are unknown. In our study, we investigated the direct effects of Id-1 on esophageal cancer cell growth by transfecting an Id-1 expression vector into an ESCC cell line (HKESC-3), which showed serum-dependent Id-1 expression. Ectopic Id-1 expression resulted in increased serum-independent cell growth and G1-S phase transition, as well as up-regulation of mouse double minute 2 (MDM2) and down-regulation of p21Waf1/Cip1 protein expressions in the transfectant clones in a p53-independent manner. However, overexpression of Id-1 had no effect on the pRB, CDK4 and p16INK4A expressions. Stable transfection of Id-1 antisense expression vector to inhibit the expression of endogenous Id-1 in another ESCC cell line (HKESC-1) reversed the effects on MDM2 and p21Waf1/Cip1. In addition, Id-1 expression protected ESCC cells from Tumor Necrosis Factor (TNF)-alpha-induced apoptosis by up-regulating and activating Bcl-2. In conclusion, our study provides evidence for the first time that Id-1 plays a role in both proliferation and survival of esophageal cancer cells. Our findings also suggest that unlike prostate, hepatocellular and nasopharyngeal carcinomas in which Id-1 induces cell proliferation through inactivation of p16INK4A/RB pathway, the increased cell proliferation observed in ESCC cells may be mediated through a different mechanism.     

Deregulation of the Rho GTPase, Rac1, suppresses cyclin-dependent kinase inhibitor p21(CIP1) levels in androgen-independent human prostate cancer cells

Abnormally suppressed levels of cyclin-dependent kinase inhibitors (CKIs) are associated with aggressive androgen-independent prostate cancer and contribute to uncontrolled proliferation. The androgen-independent human prostate cancer cell lines, LNCaP-104R1, ALVA31 and PC-3, express low levels of the CKI, p21(CIP1), compared to the less-malignant, androgen-dependent LNCaP cells. We investigated the mechanism underlying this suppression by examining the role of Rho GTPases, signaling proteins that play important roles in cell cycle progression, at least in part through regulation of CKIs. Inhibition of Rac1 induced p21 expression in androgen-independent lines but had no effect on the higher p21 levels characteristic of LNCaP cells. This induction of p21 was functionally significant as evidenced by inhibition of cyclin-dependent kinase 2 activity and decreased cell proliferation. Conversely, overexpression of constitutively active Rac1 suppressed the higher p21 levels seen in LNCaP cells. Thus, Rac1 activity is both necessary and sufficient for suppression of p21 in prostate cancer cells. Furthermore, Rac1 activity was significantly higher in all three androgen-independent cell lines compared to LNCaP cells. Thus in three models of aggressive human prostate cancer, hyperactivity of Rac1 corresponds to suppressed levels of p21. These results are unique in describing a role for Rac1 in p21 regulation and may implicate the Rac1 signaling pathway as a potential therapeutic target for controlling prostate cancer cell growth following progression to androgen independence.