Y.C. Wong

Over expression of ID-1 in prostate cancer

PURPOSEnThe helix-loop-helix protein Id-1 serves to prevent basic helix-loop-helix transcription factors from binding to DNA, thus, inhibiting the transcription of differentiation associated genes. Over expression of Id-1 has been reported in certain tumors, such as breast, esophageal, pancreatic and medullary thyroid cancers. In Noble rats we have previously demonstrated that up-regulation of Id-1 is closely associated with the development of sex hormone induced prostate cancers. Therefore, we hypothesized that over expression of Id-1 would also occur in human prostate cancer and Id-1 protein may serve as a potential marker for prostate carcinogenesis. To test this hypothesis we analyzed Id-1 messenger RNA and protein expression by in situ hybridization and immunohistochemical study in human normal prostate, benign prostatic hyperplasia (BPH) and prostate cancer tissues.nnnMATERIALS AND METHODSnPathological specimens were obtained from 19 patients with BPH and 47 with prostate carcinoma, representing a complete range of Gleason grades. A total of 12 normal prostate tissue specimens were used for comparison. Immunohistochemical study was performed using the polyclonal antibody against human Id-1 protein and an RNA probe was generated from Id-1 complementary DNA for in situ hybridization.nnnRESULTSnNegative to weak expression of Id-1 in normal prostate or BPH tissue was observed on immunohistochemical study and in situ hybridization. In contrast, all prostate cancer biopsies showed significant positive Id-1 expression in tumor cells at the messenger RNA and protein levels. Furthermore, Id-1 expression was stronger in poorly differentiated than in well differentiated carcinomas, suggesting that the level of Id-1 expression may be associated with tumor malignancy.nnnCONCLUSIONSnOur results suggest that over expression of Id-1 may have important roles in the development of prostate cancer. The potential use of Id-1 protein as a marker for prostate cancer should be further explored.

Id-1 expression and cell survival

The Id (inhibitor of differentiation or DNA binding) helix-loop-helix (HLH) proteins are a group of dominant negative regulators of basic HLH transcriptional factors which promote cell differentiation. Recent evidence has revealed that Id proteins, especially Id-1, are also able to promote cell proliferation and cell cycle progression through inactivation of tumour suppressor and activation of growth promoting pathways in mammalian cells. In addition, upregulation of Id-1 has been found in many types of human cancer and its expression levels are also associated with advanced tumour stage. Furthermore, ectopic expression of Id-1 in human cancer cells is able to induce cell proliferation under sub-optimal conditions and protect the cells against apoptosis. These lines of evidence strongly indicate Id-1 as a positive regulator of cell growth and its expression may be a key factor required for tumour cell proliferation. This review will discuss recent evidence on the role of Id-1 in cell proliferation and survival, and its significance in malignant transformation. In addition, we will highlight the recent development in the understanding of the molecular mechanisms responsible for the action of Id-1 in promoting cell survival and tumourigenesis. Finally, the therapeutic implications through inactivation of Id-1 in the treatment of human cancer will also be addressed.

γ-Tocotrienol suppresses prostate cancer cell proliferation and invasion through multiple-signalling pathways

Tocotrienol-rich fraction (TRF) has demonstrated antiproliferative effect on prostate cancer (PCa) cells. To elucidate this anticancer property in PCa cells, this study aimed, first, to identify the most potent isomer for eliminating PCa cells; and second, to decipher the molecular pathway responsible for its activity. Results showed that the inhibitory effect of γ-tocotrienol was most potent, which resulted in induction of apoptosis as evidenced by activation of pro-caspases and the presence of sub-G1 cell population. Examination of the pro-survival genes revealed that the γ-tocotrienol-induced cell death was associated with suppression of NF-κB, EGF-R and Id family proteins (Id1 and Id3). Meanwhile, γ-tocotrienol treatment also resulted in the induction of JNK-signalling pathway and inhibition of JNK activity by a specific inhibitor (SP600125) was able to partially block the effect of γ-tocotrienol. Interestingly, γ-tocotrienol treatment led to suppression of mesenchymal markers and the restoration of E-cadherin and γ-catenin expression, which was associated with suppression of cell invasion capability. Furthermore, a synergistic effect was observed when cells were co-treated with γ-tocotrienol and Docetaxel. Our results suggested that the antiproliferative effect of γ-tocotrienol act through multiple-signalling pathways, and demonstrated for the first time the anti-invasion and chemosensitisation effect of γ-tocotrienol against PCa cells.

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.

Down-regulation of Id-1 expression is associated with TGFβ1-induced growth arrest in prostate epithelial cells

Transforming growth factor beta1 (TGF beta 1) plays important roles in the regulation of cell growth and differentiation in both normal and malignant prostate epithelial cells. Although certain pathways have been suggested, the mechanisms responsible for the action of TGF beta 1 are not well understood. In the present study, using a human papilloma virus 16 E6/E7 immortalized prostate epithelial cell line, HPr-1, we report that TGF beta 1 was able to suppress the expression of Id-1, a helix-loop-helix (HLH) protein, which plays important roles in the inhibition of cell differentiation and growth arrest. In addition, a decrease at both Id-1 mRNA and protein expression levels was associated with TGF beta 1-induced growth arrest and differentiation, indicating that Id-1 may be involved in TGF beta 1 signaling pathway. The fact that up-regulation of p21(WAF1), one of the downstream effectors of Id-1, was observed after exposure to TGF beta 1 further indicates the involvement of Id-1 in the TGF beta 1-induced growth arrest in HPr-1 cells. However, increased expression of p27(KIP1) was also observed in the TGF beta 1-treated cells, suggesting that in addition to down-regulation of Id-1, other factors may be involved in the TGF beta 1-induced cell growth arrest and differentiation in prostate epithelial cells. Our results provide evidence for the first time that TGF beta 1 may be one of the upstream regulators of Id-1.

Mitotic checkpoint defects in human cancers and their implications to chemotherapy

The mitotic checkpoint, also known as spindle assembly checkpoint, is to ensure accurate chromosome segregation by inducing mitotic arrest when errors occur in the spindle structure or in the alignment of the chromosomes on the spindle. Loss of mitotic checkpoint control is a common event in human cancer cells, which is thought to be responsible for chromosome instability frequently observed in cancer cells. Several reports have shown that cells with a defective mitotic checkpoint are more resistant to several types of anticancer drugs from microtubule disruptors to DNA damaging agents. In addition, inactivation of key mitotic checkpoint proteins such as BUB (budding uninhibited by benzimidazole) and MAD (mitotic arrest deficient ) is influential in drug resistance in mitotic checkpoint defective cancer cells. The mitotic checkpoint has also been linked to DNA damage response and a defective mitotic checkpoint confers cancer cells resistance to certain DNA damaging anticancer drugs. This review presents recent evidence on mitotic checkpoint defects in human cancers and their association with resistance to anticancer drugs. In addition, the clinical importance and potential therapeutic implications of targeting the mitotic checkpoint to reverse drug resistance in cancer cells are also discussed.

Evidence of increased Id-1 expression and its role in cell proliferation in nasopharyngeal carcinoma cells

Inhibitor of differentiation or DNA binding (Id‐1), a helix‐loop‐helix transcription factor, has recently been shown to inactivate the retinoblastoma (RB)/p16INK4a pathway through down‐regulation of p16INK4a and increasing phosphorylation of RB in certain cell types. Nasopharyngeal carcinoma (NPC) is a common cancer in Hong Kong, and inactivation of the tumor suppressor RB at transcription level is a rare event in NPC. The objective of this study was to investigate the role of Id‐1 in NPC cell proliferation and its expression in NPC samples. An NPC cell line, CNE1, was transfected with a retroviral vector containing a full‐length Id‐1 cDNA, and six stable transfectant clones were isolated with differential Id‐1 expression levels. The effect of ectopic Id‐1 expression on serum‐independent cell growth, cell‐cycle distribution, and expression of proteins associated with RB pathway was studied. The Id‐1 expression in five NPC samples was also investigated using immunohistochemistry. Ectopic Id‐1 expression in CNE1 cells resulted in an increase in serum‐independent cell growth, percentage of cells in S phase, and phosphorylation of RB and cyclin‐dependent kinase 2 proteins. In addition, immunohistochemical studies on NPC samples showed that expression of Id‐1 was present in NPC cells but absent in normal tissues. This study demonstrates that Id‐1 plays an important role in cell proliferation in NPC cells, and our results provide evidence for the first time of the significance of Id‐1 expression in NPC cells and suggest a possible role of Id‐1 expression in the inactivation of RB and development of NPC.

Role of MEK/ERK pathway in the MAD2-mediated cisplatin sensitivity in testicular germ cell tumour cells

Testicular germ cell tumour (TGCT) is the most common malignancy in young males. Although most TGCTs are sensitive to cisplatin-based chemotherapy, significant numbers of TGCT patients still relapse and die each year because of the development of resistance to cisplatin. Previously, we first reported that a key regulator of the mitotic checkpoint, mitotic arrest deficient-2 (MAD2), was a mediator of cisplatin sensitivity in human cancer cells. In this study, we investigated whether MAD2 played a role in cellular sensitivity to cisplatin in TGCT cells and the underlying molecular mechanisms responsible. Using 10 TGCT cell lines, we found that increased MAD2 expression was correlated with cellular sensitivity to cisplatin, which was associated with activation of the MEK pathway. Treatment of cells expressing high levels of MAD2 with an MEK inhibitor, U0126, led to cellular protection against cisplatin-induced apoptosis. Inactivation of MAD2 by transfecting a dominant-negative construct in TGCT cells with high levels of MAD2 resulted in the suppression of MEK pathway and resistance to cisplatin-induced cell death. These results support previous suggestion on the involvement of mitotic checkpoint in DNA damage response in human cancer cells and demonstrate a possible molecular mechanism responsible for the MAD2-mediated sensitivity to cisplatin in TGCT cells. Our results also suggest that downregulation of MAD2 may be an indicator for identification of TGCT cancer cells that are potentially resistant to cisplatin-based therapy.

Id-1 promotes chromosomal instability through modification of APC/C activity during mitosis in response to microtubule disruption.

Id-1 (Inhibitor of DNA binding/differential-1) plays a positive role in tumorigenesis through regulation of multiple signaling pathways. Recently, it is suggested that upregulation of Id-1 in cancer cells promotes chromosomal instability. However, the underlying molecular mechanism is not known. In this study, we report a novel function of Id-1 in regulation of mitosis through physical interaction with Cdc20 (cell division cycle protein 20) and Cdh1 (Cdc20 homolog 1). During early mitosis, Id-1 interacts with Cdc20 and RASSF1A (Ras association domain family 1A), leading to enhanced APCCdc20 activity, which in turn promotes cyclin B1/securin degradation and premature mitosis. During late mitosis, Id-1 binds to Cdh1 and disrupts the interaction between Cdh1 and APC, resulting in suppression of APCCdh1 activity. On the other hand, overexpression of Cdh1 leads to Id-1 protein degradation, suggesting that Id-1 may also act as a substrate of APCCdh1. The negative effect of Id-1 on APCCdh1 results in suppression of APCCdh1-induced Aurora A and Cdc20 degradation, leading to failure in cytokinesis. As a result, overexpression of Id-1 in human prostate epithelial cells leads to polyploidy in response to microtubule disruption, and this effect is abolished when Id-1 expression is suppressed using antisense technology. These results demonstrate a novel function of Id-1 in promoting chromosomal instability through modification of APC/C activity during mitosis and provide a novel molecular mechanism accounted for the function of Id-1 as an oncogene.

Id-1 stimulates cell proliferation through activation of EGFR in ovarian cancer cells

Increased EGFR (epidermal growth factor receptor) expression has been reported in many types of human cancer and its levels are positively associated with advanced cancers. Recently, upregulation of Id-1 (inhibitor of differentiation or DNA binding) protein was found in over 70% of ovarian cancer samples and correlated with poor survival of ovarian cancer patients. However, the molecular mechanisms responsible for the role of Id-1 in ovarian cancer are not clear. The aim of this study was to investigate the effect of Id-1 on ovarian cancer proliferation and its association with the EGFR pathway. To achieve this, we transfected an Id-1 expression vector into three ovarian cancer cell lines and examined cell proliferation rate by flow cytometry and bromodeoxyuridine staining. We found that ectopic Id-1 expression led to increased cell proliferation demonstrated by increased BrdU incorporation rate and S-phase fraction. The Id-1-induced cell growth was associated with upregulation of EGFR at both transcriptional and protein levels. In contrast, inactivation of Id-1 through transfection of an Id-1 antisense vector resulted in downregulation of EGFR. Our results indicate that increased Id-1 in ovarian cancer cells may promote cancer cell proliferation through upregulation of EGFR. Our findings also implicate that Id-1 may be a potential target for the development of novel strategies in the treatment of ovarian cancer.