Inactivation Of Id-1 Research Articles

Abstract 5433: Functional involvement of ribosomal protein L9 to colorectal cancer stemness

Abstract We have previously demonstrated that targeting of ribosomal protein L9 (RPL9) suppresses the growth of colorectal cancer (CRC) via the inactivation of Id-1/NF-κB signaling axis which is known to augment tumor stemness. Thus we aimed in this study to investigate whether the function of RPL9 was associated with CRC stemness properties. It was initially evidenced that inhibition of RPL9 expression reduces the migration and invasion abilities of HT29 parental cell population. We have then sorted out CD133+ cancer stem cells (CSCs) from HT-29 parental cell culture and treated RPL9-specific siRNAs to the isolated CSCs to observe the effect of RPL9 targeting on stemness. As results, knockdown of RPL9 significantly suppressed the proliferation potential and sphere forming capacity of CD133+ HT29 CSCs accompanying with the reduction in CD133 and Id-1 levels. Reflecting these molecular alterations, targeting RPL9 also inhibited the abilities of migration and invasion in CD133+ HT29 CSCs population. Taken together, these findings suggest that RPL9 could be a therapeutic target for both primary CRC treatment and the prevention of metastasis and/or recurrence. Citation Format: Eun-Hye Jeon, Keun Uk Park, Hun-Mo Ryoo, Ilseon Hwang, Yun-Han Lee. Functional involvement of ribosomal protein L9 to colorectal cancer stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5433.

Knockdown of RPL9 expression inhibits colorectal carcinoma growth via the inactivation of Id-1/NF-κB signaling axis.

Ribosomal protein L9 (RPL9), a component of the 60S subunit for protein synthesis, is upregulated in human colorectal cancer. In the present study, we investigated whether RPL9 gained extraribosomal function during tumorigenesis and whether targeting of RPL9 with small interfering (si) RNA could alter the course of colorectal cancer progression. Our results showed that siRNA knockdown of RPL9 suppresses colorectal cancer (CRC) cell growth and long-term colony formation through an increase in sub-G1 cell population and a strong induction of apoptotic cell death. To obtain insights into the molecular changes in response to RPL9 knockdown, global changes in gene expression were examined using RNA sequencing. It revealed that RPL9-specific knockdown led to dysregulation of 918 genes in HCT116 and 3178 genes in HT29 cells. Among these, 296 genes showed same directional regulation (128 upregulated and 168 downregulated genes) and were considered as a common RPL9 knockdown signature. Particularly, we found through a network analysis that Id-1, which is functionally associated with activation of NF-κB and cell survival, was commonly downregulated. Subsequent western blot analysis affirmed that RPL9 silencing induced the decrease in the levels of Id-1 and phosphorylated IκBα in both HCT116 and HT29 cells. Also, the same condition decreased the levels of PARP-1 and pro-caspase-3, accelerating apoptosis. Furthermore, inhibition of RPL9 expression significantly suppressed the growth of human CRC xenografts in nude mice. These findings indicate that the function of RPL9 is correlated with Id-1/NF-κB signaling axis and suggest that targeting RPL9 could be an attractive option for molecular therapy of colorectal cancer.

Id1 promotes lung cancer cell proliferation and tumor growth through Akt-related pathway

Overexpression of Id family proteins inhibits cell differentiation and enhances cell proliferation and invasiveness. Although Id1 is the Id family member mostly linked to tumorigenesis, its role in lung cancer is unclear. An elevated Id1 expression was observed in lung cancer cell lines as well as lung cancer tissues. Id1 overexpression increased cell proliferation while Id1 knockdown decreased cell proliferation, mostly through Akt-related pathway. Nude mice study further confirmed an increased tumor growth in Id1-overexpressing cells and a decreased tumor growth in Id1-knockdowned cells. In conclusion, inactivation of Id1 may provide a novel strategy for treatment of lung cancer patients.

Downregulation of Id1 by small interfering RNA in prostate cancer PC3 cells in vivo and in vitro

Overexpression of helix-loop-helix protein-Id1 in prostate cancer correlates with tumor differentiation, and may play a key role in the development of prostate cancer. Hence, we inactivated the Id1 gene in prostate cancer cells in vitro and in vivo to determine whether the Id1 gene has therapeutic potential in the treatment of prostate cancer. A modified small interfering RNA (siRNA) was used to inactivate the Id1 gene in androgen-independent prostate cancer cell line PC3 and its xenografts in nude mice. Downregulation of Id1 by siRNA was confirmed by real-time PCR. The changes of cell viability, apoptosis and senescence rate in PC3 were individually detected. The mRNA and protein expression of Id1, PCNA and MMP2 in xenografted tumors was further individually assayed by real-time PCR and immunohistochemistry. The mRNA and protein expression of Id1 were obviously inhibited by the siRNA strategy in PC3 cells and their xenografts (P<0.01). The cell viability of PC3 was suppressed obviously (P<0.01); meanwhile, the apoptosis and senescence rates of PC3 were significantly increased by siRNA (P<0.01). Moreover, tumor growth was inhibited by the gene silencing of Id1. Two genes involved in proliferation (PCNA) and tumor invasion (MMP2) were found significantly decreased by siRNA in PC3 xenografts (P<0.01). Our results show that inactivation of Id1 can suppress cell proliferation, induce apoptosis and senescence in PC3. Silencing of the Id1 gene has an in-vivo preventive effect against the development of prostate cancer in a mouse model.

Id-1 regulates Bcl-2 and Bax expression through p53 and NF-κB in MCF-7 breast cancer cells

Although increasing evidence supports the protective role of inhibitor of differentiation and DNA binding-1 (Id-1) against anticancer drug-induced apoptosis, the underlying molecular mechanisms seem to vary depending on the tumor system. Here, we examined the direct role of Id-1 in MCF-7 breast cancer cells by ectopically overexpressing Id-1 under serum-free condition, where the endogenous Id-1 expression was suppressed. Id-1 expression resulted in increased number of viable cells, reduced Bax expression, enhanced Bcl-2 expression, but no change in Bcl-xL expression. The expression of nuclear factor-kappaB (NF-kappaB) was augmented, while those of p53 and IkappaB were reduced. Such changes in p53 and NF-kappaB pathways were also functional, as assessed by real-time polymerase chain reactions and reporter assays of their known downstream targets, p21 and Il-6, as well as Bax and Bcl-2 genes. Finally, Id-1 played a protective role against taxol-induced apoptosis in breast cancer cells as assessed by MTT assay and apoptotic cell count upon taxol treatment (0-200 nM). Reduced Bax expression and enhanced Bcl-2 expression by Id-1 were also noted in the presence of taxol. Taken together, we present a molecular mechanism where Id-1 regulates p53 and NF-kappaB pathways, which in turn regulates Bax and Bcl-2 genes, thus providing a survival advantage under exogenous stress such as serum-free or taxol treatment in MCF-7 breast cancer cells. In this regard, inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of breast cancer progression and anti-cancer drug resistance.

Id-1 activation of PI3K/Akt/NF B signaling pathway and its significance in promoting survival of esophageal cancer cells

Inhibitor of differentiation or DNA binding (Id-1) is a helix-loop-helix protein that is over-expressed in many types of cancer including esophageal cancer. This study aims to investigate its effects on the phosphatidylinositol-3-kinase (PI3K)/Akt/ nuclear factor kappa B (NFkappaB) signaling pathway and the significance in protecting esophageal cancer cells against apoptosis. We found elevated expression of phosphorylated forms of Akt, glycogen synthase kinase 3beta and inhibitor of kappa B, as well as increased nuclear translocation of NFkappaB subunit p65 and NFkappaB DNA-binding activity, in esophageal cancer cells with stable ectopic Id-1 expression. Transient transfection of Id-1 into HEK293 cells confirmed activation of PI3K/Akt/NFkappaB signaling and the effects were counteracted by the PI3K inhibitor LY294002. Treatment with tumor necrosis factor-alpha (TNF-alpha) elicited a significantly weaker apoptotic response, following a marked and sustained activation of Akt and NFkappaB in the Id-1-over-expressing cells, compared with the vector control. The effects of Id-1 on the PI3K/Akt/NFkappaB signaling pathway and apoptosis were reversed in esophageal cancer cells transfected with siRNA against Id-1. In addition, inhibition of PI3K or NFkappaB signaling using the PI3K inhibitor LY294002 or the NFkappaB inhibitor Bay11-7082 increased the sensitivity of Id-1-over-expressing esophageal cancer cells to TNF-alpha-induced apoptosis. Our results provide the first evidence that Id-1 induces the activation of PI3K/Akt/NFkappaB signaling pathway, and protects esophageal cancer cells from TNF-alpha-induced apoptosis in vitro. Inactivation of Id-1 may provide us with a novel strategy to improve the treatment and survival of patients with esophageal cancer.

RETRACTED ARTICLE: 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.

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.

Id-1 expression induces androgen-independent prostate cancer cell growth through activation of epidermal growth factor receptor (EGF-R)

The failure of prostate cancer treatment is largely due to the development of androgen independence, since the androgen depletion therapy remains the front-line option for this cancer. Previously, we reported that over-expression of the helix-loop-helix protein Id-1 was associated with progression of prostate cancer and ectopic expression of Id-1 induced serum-independent proliferation in prostate cancer cells. In the present study, we investigated if exogenous Id-1 expression in the androgen sensitive LNCaP cells had any effect on androgen-dependent cell growth and studied the molecular mechanisms involved. Using stable Id-1 transfectants, we found that expression of Id-1 was able to reduce androgen-stimulated growth and S phase fraction of the cell cycle in LNCaP cells, indicating that Id-1 may be involved in the development of androgen independence in these cells. The Id-1-induced androgen-independent prostate cancer cell growth was correlated with up-regulation of EGF-R (epidermal growth factor-receptor) and PSA (prostate specific antigen) expression, as confirmed by western blotting analysis and luciferase assays. In contrast, down-regulation of Id-1 in androgen-independent DU145 cells by its antisense oligonucleotides resulted in suppression of EGF-R expression at both transcriptional and protein levels. In addition, the results from immunohistochemistry study showed that Id-1 expression was significantly elevated in hormone refractory prostate cancer tissues when compared with the hormone-dependent tumours. Our results suggest that up-regulation of Id-1 in prostate cancer cells may be one of the mechanisms responsible for developing androgen independence and this process may be regulated through induction of EGF-R expression. Inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of androgen-independent prostate cancer cell growth.

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.