Annie L.M. Cheung

Id‐1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway

Id‐1 (inhibitor of differentiation or DNA binding) is a helix‐loop‐helix protein that is overexpressed in many types of cancer including esophageal squamous cell carcinoma (ESCC). We previously reported that ectopic Id‐1 expression activates the phosphatidylinositol‐3‐kinase (PI3K)/protein kinase B (AKT) signaling pathway in human esophageal cancer cells. In this study, we confirmed a positive correlation between Id‐1 and phospho‐AKT (Ser473) expressions in ESCC cell lines, as well as in ESCC on a tissue microarray. To investigate the significance of Id‐1 in esophageal cancer progression, ESCC cells with stable ectopic Id‐1 expression were inoculated subcutaneously into the flank of nude mice and were found to form larger tumors that showed elevated Ki‐67 proliferation index and increased angiogenesis, as well as reduced apoptosis, compared with control cells expressing the empty vector.The Id‐1‐overexpressing cells also exhibited enhanced metastatic potential in the experimental metastasis assay. Treatment with the PI3K inhibitor LY294002 attenuated the tumor promotion effects of Id‐1, indicating that the effects were mediated by the PI3K/AKT signaling pathway. In addition, our in vitro experiments showed that ectopic Id‐1 expression altered the expression levels of markers associated with epithelial–mesenchymal transition and enhanced the migration ability of esophageal cancer cells. The Id‐1‐overexpressing ESCC cells also exhibited increased invasive potential, which was in part due to PI3K/AKT‐dependent modulation of matrix metalloproteinase‐9 expression. In conclusion, our results provide the first evidence that Id‐1 promotes tumorigenicity and metastasis of human esophageal cancer in vivo and that the PI3K inhibitor LY294002 can attenuate these effects.

Identification of a Novel Inhibitor of Differentiation-1 (ID-1) Binding Partner, Caveolin-1, and Its Role in Epithelial-Mesenchymal Transition and Resistance to Apoptosis in Prostate Cancer Cells

Recently, ID-1 (inhibitor of differentiation/DNA binding) is suggested as an oncogene and is reported to promote cell proliferation, invasion, and survival in several types of human cancer cells through multiple signaling pathways. However, how Id-1 interacts with these pathways and the immediate downstream effectors of the Id-1 protein are not known. In this study, using a yeast two-hybrid screening technique, we identified a novel Id-1-interacting protein, caveolin-1 (Cav-1), a cell membrane protein, and a positive regulator of cell survival and metastasis in prostate cancer. Using an immunoprecipitation method, we found that the helix-loop-helix domain of the Id-1 protein was essential for the physical interaction between Id-1 and Cav-1. In addition, we also demonstrated that the physical interaction between Id-1 and Cav-1 played a key role in the epithelial-mesenchymal transition and increased cell migration rate as well as resistance to taxol-induced apoptosis in prostate cancer cells. Furthermore, our results revealed that this effect was regulated by Id-1-induced Akt activation through promoting the binding activity between Cav-1 and protein phosphatase 2A. Our study demonstrates a novel Id-1 binding partner and suggests a molecular mechanism that mediates the function of Id-1 in promoting prostate cancer progression through activation of the Akt pathway leading to cancer cell invasion and resistance to anticancer drug-induced apoptosis.

Targeting NF-κB signaling pathway suppresses tumor growth, angiogenesis, and metastasis of human esophageal cancer

Esophageal cancer is the eighth most common malignancy, and one of the leading causes of cancer-related deaths worldwide. The overall 5-year survival rate of patients with esophageal cancer remains low at 10% to 40% due to late diagnosis, metastasis, and resistance of the tumor to radiotherapy and chemotherapy. NF-κB is involved in the regulation of cell growth, survival, and motility, but little is known about the role of this signaling pathway in the tumorigenesis of human esophageal squamous cell carcinoma (ESCC), the most common form of esophageal cancer. This study aims to explore the functions of NF-κB in human ESCC progression and to determine whether targeting the NF-κB signaling pathway might be of therapeutic value against ESCC. Our results from human ESCC cell lines and ESCC tissue indicated that NF-κB is constitutively active in ESCC. Exposure of ESCC cells to two NF-κB inhibitors, Bay11-7082 and sulfasalazine, not only reduced cancer cell proliferation, but also induced apoptosis and enhanced sensitivity to chemotherapeutic drugs, 5-fluorouracil, and cisplatin. In addition, Bay11-7082 and sulfasalazine suppressed the migration and invasive potential of ESCC cells. More importantly, the results from tumor xenograft and experimental metastasis models showed that Bay11-7082 had significant antitumor effects on ESCC xenografts in nude mice by promoting apoptosis, and inhibiting proliferation and angiogenesis, as well as reduced the metastasis of ESCC cells to the lungs without significant toxic effects. In summary, our data suggest that NF-κB inhibitors may be potentially useful as therapeutic agents for patients with esophageal cancer. [Mol Cancer Ther 2009;8(9):2635–44]

A New Method for Improving Metaphase Chromosome Spreading

The success of complex molecular cytogenetic studies depends on having properly spread chromosomes. However, inconsistency of optimum chromosome spreading remains a major problem in cytogenetic studies.

Viral load of HPV in esophageal squamous cell carcinoma

We previously reported the presence of HPV DNA in esophageal squamous cell carcinoma (ESCC) cases from Hong Kong and Sichuan. The role of HPV in the carcinogenesis of ESCC remains unclear, partly due to the large variations in infection rates reported by different studies. While some of these variations may truly reflect different HPV infection rates in ESCC among different geographic regions, differences in sensitivity and specificity of the detection methods used also contribute. In the present study, we used quantitative real‐time PCR to determine the copy numbers of HPV‐16 and HPV‐18 in ESCC from 5 different regions of China with different incidence rates of ESCC. Conforming to our previous reports, HPV infection was detected in 2–22.2% of samples. Infection with HPV‐16 was again shown to be more common than that with HPV‐18 among Chinese ESCC patients. The copy number of HPV‐16 in these ESCC cases ranged from ≤1 to 157 copies/genome equivalent, with 65% of samples harboring fewer than 10 copies/genome equivalent. The median copy number of HPV‐18 was 4.9/genome equivalent. Assays were validated using cervical carcinoma cell lines with known copy numbers of HPV‐16 or HPV‐18. The relatively low HPV copy number and infection rate in ESCC suggest that HPV is unlikely to play as essential a role in the carcinogenesis of ESCC as in cervical cancer. However, with the consistent detection of oncogenic HPVs in ESCC from some regions of China, the possibility of HPV infection being one of the multiple risk factors of ESCC in some geographic areas cannot be excluded.

Distinct profiles of critically short telomeres are a key determinant of different chromosome aberrations in immortalized human cells: whole-genome evidence from multiple cell lines.

Chromosomal aberrations are common in cancers. However, the search for chromosomal aberrations leading to development of specific solid tumors has been severely hindered because the majority of solid tumors have complex chromosomal aberrations that differ within the same tumor types. A similar phenomenon exists in immortalized cell lines. The underlying mechanisms driving these diverse aberrations are largely unknown. Telomeres play crucial roles in protecting the integrity of eucaryotic chromosomes and maintaining genomic stability of human cells. Telomere lengths on individual chromosomes in normal human somatic cells are heterogeneous and undergo progressive shortening with aging process. In this study, for the first time, a molecular cytogenetic method using sequential telomere quantitative fluorescence in situ hybridization and spectral karyotyping on the same human metaphases was applied successfully to examine the dynamic profiles of individual telomere shortening and their relationship to chromosome aberrations in multiple human cell lines undergoing immortalization. Human ovarian surface epithelial cells and esophageal epithelial cells were immortalized by the expression of HPV16 E6 and E7, which drive cells to proliferate by inactivating p53 and Rb genes. In these cell lines, we consistently detected large-scale differences in telomere signal intensities not only among nonhomologous chromosome arms but also between some homologous chromosome arms. The cell lines derived from different donors had different profiles of critically short telomeres (lacking telomere signals). Strikingly, the different profiles of chromosomal structural aberrations in multiple immortalized cell lines were highly significantly associated with the distinct distributions of critically short telomeres in whole-genome. Since cellular immortalization is one of the hallmarks of cancer, our findings suggest that distinct profiles of critically short telomeres in different human individuals might play an essential role in determining the complex and individual-specific chromosomal structural aberrations in human solid tumors.

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)‐α‐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.

Amplification and overexpression of aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells

Immortalization is an early and essential step of human carcinogenesis. Amplification of chromosome 20q has been shown to be a common event in immortalized cells and cancers. We have previously reported that gain and amplification of chromosome 20q is a non‐random and common event in immortalized human ovarian surface epithelial (HOSE) cells. The chromosome 20q harbors genes including TGIF2 (20q11.2‐q12), AIB1 (20q12), PTPN1 (20q13.1), ZNF217 (20q13.2), and AURKA (20q13.2‐q13.3), which were previously reported to be amplified and overexpressed in ovarian cancers. Some of these genes may be involved in immortalization of HOSE cells and represent crucial premalignant changes in ovarian surface epithelium. Investigation of the involvement of these genes was examined in four pairs of pre‐crisis (preimmortalized) and post‐crisis (immortalized) HOSE cells. Overexpression of AURKA (Aurora kinase A), also known as BTAK and STK15, by both real time‐quantitative polymerase chain reaction (RT‐QPCR) and Western blotting was detected in all the four immortalized HOSE cells examined while overexpression of AIB1 and ZNF217 was observed in two of four immortalized HOSE cells examined. Overexpression of TGIF2 and PTPN1 was not significant in our immortalized HOSE cell systems. The degree of overexpression of AURKA was shown to be closely associated with the amplification of chromosome 20q in immortalized HOSE cells. Fluorescence in situ hybridization (FISH) with labeled P1 artificial clone (PAC) confirmed the amplification of the chromosomal region (20q13.2‐13.3) where AURKA resides. DNA amplification of AURKA was also confirmed using semi‐quantitative PCR. Our study showed that amplification and overexpression of AURKA is a common and significant event during immortalization of HOSE cells and may represent an important premalignant change in ovarian carcinogenesis.

Id1-Induced IGF-II and Its Autocrine/Endocrine Promotion of Esophageal Cancer Progression and Chemoresistance—Implications for IGF-II and IGF-IR–Targeted Therapy

Purpose: To investigate the autocrine/endocrine role of Id1-induced insulin-like growth factor-II (IGF-II) in esophageal cancer, and evaluate the potential of IGF-II- and IGF-type I receptor (IGF-IR)-targeted therapies. Experimental Design: Antibody array-based screening was used to identify differentially secreted growth factors from Id1-overexpressing esophageal cancer cells. In vitro and in vivo assays were performed to confirm the induction of IGF-II by Id1, and to study the autocrine and endocrine effects of IGF-II in promoting esophageal cancer progression. Human esophageal cancer tissue microarray was analyzed for overexpression of IGF-II and its correlation with that of Id1 and phosphorylated AKT (p-AKT). The efficacy of intratumorally injected IGF-II antibody and intraperitoneally injected cixutumumab (fully human monoclonal IGF-IR antibody) was evaluated using in vivo tumor xenograft and experimental metastasis models. Results: Id1 overexpression induced IGF-II secretion, which promoted cancer cell proliferation, survival, and invasion by activating AKT in an autocrine manner. Overexpression of IGF-II was found in 21 of 35 (60%) esophageal cancer tissues and was associated with upregulation of Id1 and p-AKT. IGF-II secreted by Id1-overexpressing esophageal cancer xenograft could instigate the growth of distant esophageal tumors, as well as promote metastasis of circulating cancer cells. Targeting IGF-II and IGF-IR had significant suppressive effects on tumor growth and metastasis in mice. Cixutumumab treatment enhanced the chemosensitivity of tumor xenografts to fluorouracil and cisplatin. Conclusions: The Id1–IGF-II–IGF-IR–AKT signaling cascade plays an important role in esophageal cancer progression. Blockade of IGF-II/IGF-IR signaling has therapeutic potential in the management of esophageal cancer. Clin Cancer Res; 20(10); 2651–62. ©2014 AACR.

Transforming Growth Factor β1 Promotes Chromosomal Instability in Human Papillomavirus 16 E6E7–Infected Cervical Epithelial Cells

Uterine cervical cancer, the second most frequently occurring cancer in women worldwide, is tightly associated with the expression of high-risk human papillomavirus [mainly human papillomavirus (HPV)-16 and HPV18] oncogenes E6 and E7 and characteristically exhibits chromosomal instability. However, the mechanisms underlying chromosomal instability in cervical cancer are still not fully understood. In this study, we observed that two of three human cervical epithelial cell lines expressing HPV16 E6E7 became immortalized without extensive chromosomal instability and crisis. The introduction of transforming growth factor (TGF)-beta1, a multiple functional cytokine/growth factor, in the culture medium induced crisis, which was associated with massive chromosomal end-to-end fusions and other structural aberrations. The distributions of structural aberrations on individual chromosomes were significantly correlated with the profiles of telomere signal-free ends. The immortalized cells that emerged from the TGF-beta1-induced crisis showed multiple clonal structural aberrations that were not observed in cells without TGF-beta1 treatment. Overexpression of the catalytic subunit of telomerase (hTERT) abolished the effects of TGF-beta1 on chromosomal instability. Interestingly, another HPV16 E6E7-expressing cervical cell line that experienced crisis and telomere dysfunction under ordinary culture condition had a higher level of autocrine TGF-beta1 production than the other two crisis-free immortalized cell lines. Blocking the TGF-beta1 pathway by an inhibitor of TGF-beta1 receptor type I prevented the crisis and telomere-mediated chromosomal instability. In addition, more dramatic telomere shortening was observed in cervical intraepithelial neoplasias having higher expression of TGF-beta1 in vivo. These results together suggest an important role of TGF-beta1 in the early process of cervical carcinogenesis.