David W. Chan

Loss of MKP3 mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer cells

The RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) pathway plays a pivotal role in various cellular responses, including cellular growth, differentiation, survival and motility. Constitutive activation of the ERK pathway has been linked to the development and progression of human cancers. Here, we reported that mitogen-activated protein kinase phosphatase (MKP)-3, a negative regulator of ERK1/2, lost its expression particularly in the protein level, was significantly correlated with high ERK1/2 activity in primary human ovarian cancer cells using quantitative reverse transcription-polymerase chain reaction and western blot analyses. Intriguingly, the loss of MKP3 protein was associated with ubiquitination/proteosome degradation mediated by high intracellular reactive oxygen species (ROS) accumulation such as hydrogen peroxide in ovarian cancer cells. Functionally, short hairpin RNA knock down of endogenous MKP3 resulted in increased ERK1/2 activity, cell proliferation rate, anchorage-independent growth ability and resistance to cisplatin in ovarian cancer cells. Conversely, enforced expression of MKP3 in MKP3-deficient ovarian cancer cells significantly reduced ERK1/2 activity and inhibited cell proliferation, anchorage-independent growth ability and tumor development in nude mice. Furthermore, the enforced expression of MKP3 succeeded to sensitize ovarian cancer cells to cisplatin-induced apoptosis in vitro and in vivo. These results suggest a molecular mechanism by which the accumulation of ROS during ovarian cancer progression may cause the degradation of MKP3, which in turn leads to aberrant ERK1/2 activation and contributes to tumorigenicity and chemoresistance of human ovarian cancer cells.

Over-expression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis

The Forkhead Box M1 (FOXM1) transcription factor plays a crucial role in regulating expression of cell cycle genes which are essentially involved in cell proliferation, differentiation and transformation. Recent studies have reported that aberrant expression of FOXM1 in a variety of human cancers is associated with their aggressive behaviour. However, the functional significance of FOXM1 in human cervical cancer is not known. We have shown that FOXM1 was significantly over‐expressed in cervical squamous cell carcinoma (SCC) compared to normal cervical epithelium immunohistochemically (p < 0.001). In addition, intratumoural FOXM1 positivity was increased in cervical intraepithelial neoplasia (CIN) and carcinoma, compared with that in normal epithelium, indicating that FOXM1 is involved in tumour progression. Indeed, this is supported by clinicopathological analysis that the over‐expression of FOXM1 was significantly associated with tumour late stage (p = 0.012) and cell proliferation marker, Ki67 (p < 0.001). Functionally, enforced expression of FOXM1c in FOXM1‐deficient cervical cancer cells (C33A) remarkably enhanced cell proliferation and anchorage‐independent growth ability. Conversely, depletion of FOXM1 by RNA interference in FOXM1‐over‐expressing cervical cancer cells (SiHa) caused significant inhibition on cell proliferation and anchorage‐independent growth ability on soft agar. This inhibitory phenomenon was associated with the reduced expressions of cyclin B1, cyclinD1 and cdc25B but increased expression of p27Kip1 and p21Cip1. Our findings suggest a role for FOXM1 in the development and pathogenesis of human cervical SCC. Copyright

Activation of AMPK inhibits cervical cancer cell growth through AKT/FOXO3a/FOXM1 signaling cascade

BackgroundAlthough advanced-stage cervical cancer can benefit from current treatments, approximately 30% patients may fail after definitive treatment eventually. Therefore, exploring alternative molecular therapeutic approaches is imperatively needed for this disease. We have recently shown that activation of AMP-activated protein kinase (AMPK), a metabolic sensor, hampers cervical cancer cell growth through blocking the Wnt/β-catenin signaling activity. Here, we report that activated AMPK (p-AMPK) also inhibits cervical cancer cell growth by counteracting FOXM1 function.MethodsEffect of the activation of AMPK on FOXM1 expression was examined by hypoxia and glucose deprivation, as well as pharmacological AMPK activators such as A23187, AICAR and metformin. RT Q-PCR and Western blot analysis were employed to investigate the activities of AMPK, FOXM1 and AKT/FOXO3a signaling.ResultsConsistent with our previous findings, the activation of AMPK by either AMPK activators such as AICAR, A23187, metformin, glucose deprivation or hypoxia significantly inhibited the cervical cancer cell growth. Importantly, we found that activated AMPK activity was concomitantly associated with the reduction of both the mRNA and protein levels of FOXM1. Mechanistically, we showed that activated AMPK was able to reduce AKT mediated phosphorylation of p-FOXO3a (Ser253). Interestingly, activated AMPK could not cause any significant changes in FOXM1 in cervical cancer cells in which endogenous FOXO3a levels were knocked down using siRNAs, suggesting that FOXO3a is involved in the suppression of FOXM1.ConclusionTaken together, our results suggest the activated AMPK impedes cervical cancer cell growth through reducing the expression of FOXM1.

Aberrant Activation of ERK/FOXM1 Signaling Cascade Triggers the Cell Migration/Invasion in Ovarian Cancer Cells

Forkhead box M1 (FOXM1) is a proliferation-associated transcription factor essential for cell cycle progression. Numerous studies have documented that FOXM1 has multiple functions in tumorigenesis and its elevated levels are frequently associated with cancer progression. Here, we characterized the role of ERK/FOXM1 signaling in mediating the metastatic potential of ovarian cancer cells. Immunohistochemical (IHC), immunoblotting and semi-quantitative RT-PCR analyses found that both phospho-ERK and FOXM1 were frequently upregulated in ovarian cancers. Intriguingly, the overexpressed phospho-ERK (p<0.001) and FOXM1 (p<0.001) were significantly correlated to high-grade ovarian tumors with aggressive behavior such as metastasized lymph node (5 out of 6). Moreover, the expressions of phospho-ERK and FOXM1 had significantly positive correlation (p<0.001). Functionally, ectopic expression of FOXM1B remarkably enhanced cell migration/invasion, while FOXM1C not only increased cell proliferation but also promoted cell migration/invasion. Conversely, inhibition of FOXM1 expression by either thiostrepton or U0126 could significantly impair FOXM1 mediated oncogenic capacities. However, the down-regulation of FOXM1 by either thiostrepton or U0126 required the presence of p53 in ovarian cancer cells. Collectively, our data suggest that over-expression of FOXM1 might stem from the constitutively active ERK which confers the metastatic capabilities to ovarian cancer cells. The impairment of metastatic potential of cancer cells by FOXM1 inhibitors underscores its therapeutic value in advanced ovarian tumors.

Knock-down of hepatitis B virus X protein reduces the tumorigenicity of hepatocellular carcinoma cells

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) in Southeast Asia and Hong Kong. Among the four proteins that are encoded by the HBV genome, HBV X (HBx) is the most potentially oncogenic factor. It is known that HBx plays an important role in hepatocarcinogenesis, but the exact functions and molecular mechanisms of HBx in HCC are not well understood. In this study, we constructed expression vectors for small hairpin RNAs (shRNA) against HBx and investigated their regulatory effects in PLC/PRF/5 HCC cells, which constitutively produce HBx. Our data show that this tool of RNA interference (RNAi) could successfully reduce the HBx mRNA and protein levels by 50–95%. RNAi targeting HBx in PLC/PRF/5 cells demonstrated significant reduction in cell proliferation, cell growth, anchorage‐independent growth in soft agar, and tumour development in nude mice. In addition, depletion of HBx expression increased cell sensitivity to TNFα‐mediated and serum‐free‐induced apoptosis, and reduced the expression levels of C‐myc and Bcl‐XL. These findings suggest that HBx plays an important role in tumorigenicity and anti‐apoptotic mechanisms in HCC. Copyright

AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

Recent evidence has suggested that AMPK activators may be applied as therapeutic drugs in suppressing cancer cell growth. However, the molecular mechanism of their suppressive function in cancer cells is still unclear. Here we show that AMPK activators impair cervical cancer cell growth through the reduction of DVL3, a positive regulator in Wnt/β-catenin signaling and an oncogenic player in cervical cancer tumorigenesis. By western blot and immunohistochemical analyses, we demonstrated that DVL3 was frequently upregulated and significantly associated with elevated β-catenin (P = 0.009) and CyclinD1 (P = 0.009) expressions in cervical cancer. Enforced expression of DVL3 elevated β-catenin and augmented cervical cancer cell growth, verifying that DVL3-mediated Wnt/β-catenin activation is involved in cervical cancer oncogenesis. On the other aspect, we noted that the cervical cancer cell growth was remarkably suppressed by AMPK activators and such cell growth inhibition was in concomitant with the reduction of DVL3 protein level in dose- and time-dependent manners. Besides, impaired mTOR signaling activity also reduced DVL3 expression. In contrast, co-treatment with Compound C (AMPK inhibitor) could significantly abrogate metformin induced DVL3 reduction. In addition, co-treatment with AM114 or MG132 (proteosomal inhibitors) could partially restore DVL3 expression under the treatment of metformin. Further in vivo ubiquitination assay revealed that metformin could reduce DVL3 by ubiquitin/proteasomal degradation. To our knowledge, this is the first report showing the probable molecular mechanisms of that the AMPK activators suppress cervical cancer cell growth by impairing DVL3 protein synthesis via AMPK/mTOR signaling and/or partially promoting the proteasomal degradation of DVL3.

Inhibition of Cervical Cancer Cell Growth through Activation of Upstream Kinases of AMP-Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a critical energy-balancing sensor in the regulation of cellular metabolism in response to external stimuli. Emerging evidence has suggested that AMPK is a potential therapeutic target for human cancers. AICAR, one of the pharmacological AMPK activators, has been widely used to suppress cancer cell growth through activation of LKB1, an upstream kinase of AMPK. However, frequent mutations and deletions of LKB1 found in some cancer cells limit the application of AICAR as an efficient therapeutic drug. Here we show that an alternative pharmacological AMPK activator, A23187, was able to inhibit cervical cancer cell growth through activation of Ca2+/calmodulin-dependent protein kinase kinase β, another upstream kinase of AMPK. Using cervical cancer cell models, we found that HeLa (LKB1-deficient cell) responded less to the anti-proliferative effect exerted by AICAR treatment (p < 0.001) compared with CaSki and C41 (LKB1-expressing cells). Conversely, the anti-proliferative effect was increased significantly in HeLa but not in CaSki and C41 cells under treatment by A23187 (p < 0.001). Moreover, co-treatment of AICAR and A23187 was able to further enhance the inhibitory effect on cell growth of Hela, CaSki and C41 cells. Notably, both AICAR and A23187 exerted the anti-proliferative effect on cervical cancer cells by suppressing AMPK/mTOR signalling activity. These data suggest that A23187 could be an alternative potential therapeutic drug used for anti-proliferation in LKB1-deficient cancer cells.

Genetic and epigenetic inactivation of T-cadherin in human hepatocellular carcinoma cells.

T‐cadherin is an atypical cadherin and growing evidence has indicated that T‐cadherin exerts tumor‐suppressive effects on cancers of epithelial cell type and also causes positive effects on tumor angiogenesis. Human hepatocellular carcinoma (HCC) is a hypervascular tumor and T‐cadherin has been shown to be overexpressed in intratumoral endothelial cells of HCCs. However, the expression status and functions of T‐cadherin in hepatocytes or HCC cells remain unclear. Here, we demonstrated that T‐cadherin was underexpressed in HCC cells (26.5%, 13/49 cases), but was frequently (77.6%, 38/49) overexpressed in intratumoral endothelial cells immunohistochemically. Semiquantitative RT‐PCR analysis also showed that the T‐cadherin gene was underexpressed in 7 of 11 HCC cell lines. Loss of heterozygosity analysis revealed that 32–38% of the 42 human HCC samples had allelic losses at this locus. Upon pharmacological treatment with demethylating agent 5‐aza‐2′‐deoxycytidine or histone deacetylase inhibitor trichostatin A, T‐cadherin promoter hypermethylation and/or histone deacetylation was frequently observed in HCC samples and cell lines. Functionally, enforced expression of T‐cadherin induced G2/M cell cycle arrest, reduced cell proliferation in low serum medium, suppressed anchorage‐independent growth in soft agar and increased sensitivity to TNFα‐mediated apoptosis in HCC cells. Intriguingly, we found that T‐cadherin significantly suppressed the activity of c‐Jun, a crucial oncoprotein constitutively activated in HCC cells. To conclude, T‐cadherin was differentially expressed in human HCCs. The underexpression of T‐cadherin in HCC cells suggests it may be another critical event in addition to T‐cadherin‐mediated angiogenesis during HCC development.

Overexpression of FOXG1 contributes to TGF-β resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer

Background:Loss of growth inhibitory response to transforming growth factor-β (TGF-β) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-β/Smads signalling cascade contribute to the TGF-β resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-β/Smads signalling in ovarian cancer.Methods:FOXG1 and p21WAF1/CIP1 expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT–PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21WAF1/CIP1 transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells.Results:Quantitative RT–PCR and western blot analyses showed that FOXG1 was upregulated and inversely associated with the expression levels of p21WAF1/CIP1 in ovarian cancer. The overexpression of FOXG1 was significantly correlated with high-grade ovarian cancer (P=0.025). Immunohistochemical analysis on ovarian cancer tissue array was further evidenced that FOXG1 was highly expressed and significantly correlated with high-grade ovarian cancer (P=0.048). Functionally, enforced expression of FOXG1 selectively blocked the TGF-β-induced p21WAF1/CIP1 expressions and increased cell proliferation in ovarian cancer cells. Conversely, FOXG1 knockdown resulted in a 20–26% decrease in cell proliferation together with 16–33% increase in p21WAF1/CIP1 expression. Notably, FOXG1 was able to inhibit the p21WAF1/CIP1 promoter activity in a p53-independent manner by transient reporter assays.ConclusionOur results suggest that FOXG1 acts as an oncoprotein inhibiting TGF-β-mediated anti-proliferative responses in ovarian cancer cells through suppressing p21WAF1/CIP1 transcription.

Over-expressions of AMPK subunits in ovarian carcinomas with significant clinical implications

BackgroundAMP-activated protein kinase (AMPK) has recently been considered as a potential target for cancer therapy. However, the expression status of various subunits of the heterotrimeric AMPK in human cancers is rarely reported. We decided to determine their expressions in ovarian carcinomas and their relationships with the disease.MethodsExpressions and locations of the AMPK-α1, -α2, -β1, -β2, -γ1 and -γ2 were detected by quantitative PCR (Q-PCR) and immunohistochemical staining (IHC). Their expression levels in ovarian tumors were compared with normal controls and also correlated with clinicopathological parameters.ResultsExcept AMPK-α1, expressions of the other five AMPK subunits are significantly higher in ovarian carcinomas as determined by Q-PCR. Although IHC detection of AMPK-γ1 and -γ2 were not successful, over-expressions of AMPK-α2, -β1, and -β2 were further confirmed by IHC. Over-expressions of various AMPK subunits occurred independently and were mainly detected in the cytoplasm. Interestingly, AMPK-α2 and -β1 were also detected in the nucleus and cell membrane, respectively. Clinical correlation analyses indicate that expressions of different AMPK subunits are associated with different subtypes of carcinoma. High expression of AMPK-α2 is significantly associated with endometrioid carcinomas. On the other hand, high expressions of AMPK-β and -γ subunits are associated with mucinous and serous carcinomas, respectively. Furthermore, high expressions of AMPK-β1 and -γ2 are also associated with early and late stages of disease, respectively. Finally, patients with high expression of AMPK-α2 had better prognosis.ConclusionsAberrant expressions of AMPK subunits may play important roles in ovarian carcinogenesis. Each AMPK subunit may have its own function other than just a component of the AMPK molecule. Correlations with clinical parameters suggest that expressions of AMPK subunits have different clinical implications in ovarian cancer development.