Duo Zheng

Direct Inhibition of Insulin-Like Growth Factor-I Receptor Kinase Activity by (−)−Epigallocatechin-3-Gallate Regulates Cell Transformation

Insulin-like growth factor-I receptor (IGF-IR) has been implicated in cancer pathophysiology. Furthermore, impairment of IGF-IR signaling in various cancer cell lines caused inhibition of the transformed phenotype as determined by the inhibition of colony formation in soft agar and the inhibition of tumor formation in athymic nude mice. Thus, the IGF-IR might be an attractive target for cancer prevention. We showed that the tea polyphenol, (−)−epigallocatechin-3-gallate (EGCG), is a small-molecule inhibitor of IGF-IR activity (IC50 of 14 μmol/L). EGCG abrogated anchorage-independent growth induced by IGF-IR overexpression and also prevented human breast and cervical cancer cell phenotype expression through inhibition of IGF-IR downstream signaling. Our findings are the first to show that the IGF-IR is a novel binding protein of EGCG and thus may help explain the chemopreventive effect of EGCG on cancer development. (Cancer Epidemiol Biomarkers Prev 2007;16(3):598–605)

Ribosomal S6 Kinase 2 Is a Key Regulator in Tumor Promoter–Induced Cell Transformation

The ribosomal S6 kinase 2 (RSK2), a member of the p90(RSK) (RSK) family of proteins, is a widely expressed serine/threonine kinase that is activated by extracellular signal-regulated kinase 1/2 and phosphoinositide-dependent kinase 1 in response to many growth factors and peptide hormones. Its activation signaling enhances cell survival. However, the roles of RSK2 in cell transformation have not yet been elucidated. Here, we found that RSK2 is a critical serine/threonine kinase for the regulation of cell transformation. When cells were stimulated with tumor promoters, such as epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), phosphorylation of RSK was increased within 5 min. Cell proliferation was suppressed in RSK2(-/-) mouse embryonic fibroblasts (MEFs) compared with RSK2(+/+) MEFs. Moreover, RSK2(-/-) MEFs accumulated at the G(1) phase of the cell cycle under normal cell culture conditions as well as after stimulation with EGF or TPA. In the anchorage-independent cell transformation assay (soft agar), stable expression of RSK2 in JB6 cells significantly enhanced colony formation in either the presence or absence of tumor promoters. Furthermore, knockdown of RSK2 with small interfering RNA-RSK2 suppressed constitutively active Ras (Ras(G12V))-induced foci formation in NIH3T3 cells. In addition, kaempferol, an inhibitor of RSK2, suppressed EGF-induced colony formation of JB6 Cl41 cells in soft agar, which was associated with inhibition of histone H3 phosphorylation (Ser(10)). These results showed that RSK2 is a key regulator for cell transformation induced by tumor promoters such as EGF and TPA.

The cannabinoid receptors are required for ultraviolet-induced inflammation and skin cancer development

Solar UV irradiation is an important carcinogen that leads to the development of skin cancer, which is the most common human cancer. However, the receptors that mediate UV-induced skin carcinogenesis have not yet been unequivocally identified. Here we showed that UV irradiation directly activates cannabinoid receptors 1 and 2 (CB1/2). Notably, our data indicated that the absence of the CB1/2 receptors in mice results in a dramatic resistance to UVB-induced inflammation and a marked decrease in UVB-induced skin carcinogenesis. A marked attenuation of UVB-induced activation of mitogen-activated protein kinases and nuclear factor- kappaB was associated with CB1/2 deficiency. These data provide direct evidence indicating that the CB1/2 receptors play a key role in UV-induced inflammation and skin cancer development.

Transient Receptor Potential Type Vanilloid 1 Suppresses Skin Carcinogenesis

Blockade of the transient receptor potential channel vanilloid subfamily 1 (TRPV1) is suggested as a therapeutic approach to pain relief. However, TRPV1 is a widely expressed protein whose function might be critical in various nonneuronal physiologic conditions. The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is overexpressed in many human epithelial cancers and is a potential target for anticancer drugs. Here, we show that TRPV1 interacts with EGFR, leading to EGFR degradation. Notably, the absence of TRPV1 in mice results in a striking increase in skin carcinogenesis. The TRPV1 is the first membrane receptor shown to have a tumor-suppressing effect associated with the down-regulation of another membrane receptor. The data suggest that, although a great deal of interest has focused on TRPV1 as a target for pain relief, the chronic blockade of this pain receptor might increase the risk for cancer development.

MST1 promotes apoptosis through phosphorylation of histone H2AX

MST1 (mammalian STE20-like kinase 1) is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. Overexpression of MST1 induces apoptotic morphological changes such as chromatin condensation, but the mechanism is not clear. Here we show that MST1 induces apoptotic chromatin condensation through its phosphorylation of histone H2AX at Ser-139. During etoposide-induced apoptosis in Jurkat cells, the cleavage of MST1 directly corresponded with strong H2AX phosphorylation. In vitro kinase assay results showed that MST1 strongly phosphory-lates histone H2AX. Western blot and kinase assay results with a mutant S139A H2AX confirmed that MST1 phos-phorylates H2AX at Ser-139. Direct binding of MST1 and H2AX can be detected when co-expressed in HEK293 cells and was also confirmed by an endogenous immunoprecipitation study. When overexpressed in HeLa cells, both the MST1 full-length protein and the MST1 kinase domain (MST1-NT), but not the kinase-negative mutant (MST1-NT-KN), could induce obvious endogenous histone H2AX phosphorylation. The caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (Z-DEVD-fmk) attenuates phosphorylation of H2AX by MST1 but cannot inhibit MST1-NT-induced histone H2AX phosphorylation, indicating that cleaved MST1 is responsible for H2AX phosphory-lation during apoptosis. Histone H2AX phosphorylation and DNA fragmentation were suppressed in MST1 knockdown Jurkat cells after etoposide treatment. Taken together, our data indicated that H2AX is a substrate of MST1, which functions to induce apoptotic chromatin condensation and DNA fragmentation.

Extracellular Signal-Regulated Kinase 8-Mediated c-Jun Phosphorylation Increases Tumorigenesis of Human Colon Cancer

Extracellular signal-regulated kinase 8 (ERK8), a recently discovered member of the mitogen-activated protein kinase protein family, has been less studied than other family members, leaving its physiologic functions mostly unknown. The biological consequences of overexpression of ERK8 in JB6 Cl41 epidermal skin cells or knockdown of ERK8 in HCT15 colorectal cancer cells was studied. Kinase assays and transient transfection experiments were performed to study the signaling pathway between ERK8 and c-Jun. We found that ERK8 is relatively highly expressed in HCT15 human colorectal cancer cells and plays an important role in the promotion and progression of colorectal cancer. ERK8 promoted neoplastic transformation, and knockdown of ERK8 in HCT15 colorectal cancer cells reduced the tumorigenic properties of these cell lines. Furthermore, a direct interaction between ERK8 and c-Jun was shown. With epidermal growth factor treatment, overexpression of ERK8 in JB6 Cl41 cells caused an increased phosphorylation of c-Jun at Ser(63) and Ser(73), resulting in increased activator protein-1 transactivation. In contrast, knockdown of ERK8 in HCT15 colorectal cancer cells blocked c-Jun phosphorylation. The interaction between ERK8 and c-Jun seems to increase the tumorigenic properties of HCT15 colorectal cancer cells. Thus, ERK8-regulated signaling might serve as a potential therapeutic target in colorectal cancer.

Cyclin-Dependent Kinase-3–Mediated c-Jun Phosphorylation at Ser63 and Ser73 Enhances Cell Transformation

c-Jun is a component of the activator protein-1 (AP-1) complex, which plays a crucial role in the regulation of gene expression, cell proliferation, and cell transformation, as well as cancer development. Herein, we found that cyclin-dependent kinase (Cdk)-3, but not Cdk2 or c-Jun NH(2)-terminal kinase, is a novel kinase of c-Jun induced by stimulation with growth factors such as epidermal growth factor (EGF). Cdk3 was shown to phosphorylate c-Jun at Ser63 and Ser73 in vitro and ex vivo. EGF-induced Cdk3 activation caused c-Jun phosphorylation at Ser63 and Ser73, resulting in increased AP-1 transactivation. Ectopic expression of Cdk3 resulted in anchorage-independent cell transformation of JB6 Cl41 cells induced by EGF and foci formation stimulated by constitutively active Ras (Ras(G12V)), which was mediated by AP-1 in NIH3T3 cells. These results showed that the Cdk3/c-Jun signaling axis plays an important role in EGF-stimulated cell proliferation and cell transformation.

Cyclin-dependent kinase 3-mediated activating transcription factor 1 phosphorylation enhances cell transformation

Cyclin-dependent kinase (cdk)-3, a member of the cdk family of kinases, plays a critical role in cell cycle regulation and is involved in G(0)-G(1) and G(1)-S cell cycle transitions. However, the role of cdk3 in cell proliferation, as well as cell transformation, is not yet clearly understood. Here, we report that the protein expression level of cdk3 is higher in human cancer cell lines and human glioblastoma tissue compared with normal brain tissue. Furthermore, we found that cdk3 phosphorylates activating transcription factor 1 (ATF1) at serine 63 and enhances the transactivation and transcriptional activities of ATF1. Results also indicated that siRNA directed against cdk3 (si-cdk3) suppresses ATF1 activity, resulting in inhibition of proliferation and growth of human glioblastoma T98G cells in soft agar. Importantly, we showed that cdk3 enhances epidermal growth factor-induced transformation of JB6 Cl41 cells and si-cdk3 suppresses Ras(G12V)/cdk3/ATF1-induced foci formation in NIH3T3 cells. These results clearly showed that the cdk3-ATF1 signaling axis is critical for cell proliferation and transformation.

Phosphorylation of H2AX at Ser139 and a New Phosphorylation Site Ser16 by RSK2 Decreases H2AX Ubiquitination and Inhibits Cell Transformation

Histone H2AX is a histone H2A variant that is ubiquitously expressed throughout the genome. It plays a key role in the cellular response to DNA damage and has been designated as the histone guardian of the genome. Histone H2AX deficiency decreases genomic stability and increases tumor susceptibility of normal cells and tissues. However, the role of histone H2AX phosphorylation in malignant transformation and cancer development is not totally clear. Herein, we found that ribosomal S6 kinase 2 (RSK2) directly phosphorylates histone H2AX at Ser139 and also at a newly discovered site, Ser16. Epidermal growth factor (EGF)-induced phosphorylation of histone H2AX at both sites was decreased in RSK2 knockout cells. Phosphorylated RSK2 and histone H2AX colocalized in the nucleus following EGF treatment, and the phosphorylation of histone H2AX by RSK2 enhanced the stability of histone H2AX and prevented cell transformation induced by EGF. RSK2 and DNA-PK, but not ATM or ATR, are required for EGF-induced phosphorylation of H2AX at Ser139; however, only RSK2 is required for phosphorylation of H2AX at Ser16. Phosphorylation of histone H3 was suppressed in cells expressing wild-type H2AX compared with H2AX knockout (H2AX-/-) cells. EGF-associated AP-1 transactivation activity was dramatically lower in H2AX-/- cells overexpressing wild-type H2AX than H2AX-/- cells expressing mutant H2AX-AA. Thus, the RSK2/H2AX signaling pathway negatively regulates the RSK2/histone H3 pathway and therefore maintains normal cell proliferation.

Phosphorylation of Histone H2B Serine 32 Is Linked to Cell Transformation

Various types of post-translational modifications of the histone tails have been revealed, but a few modifications have been found within the histone core sequences. Histone core post-translational modifications have the potential to modulate nucleosome structure and DNA accessibility. Here, we studied the histone H2B core domain and found that phosphorylation of H2B serine 32 occurs in normal cycling and mitogen-stimulated cells. Notably, this phosphorylation is elevated in skin cancer cell lines and tissues compared with normal counterparts. The JB6 Cl41 mouse skin epidermal cell line is a well established model for tumor promoter-induced cell transformation and was used to study the function of H2B during EGF-induced carcinogenesis. Remarkably, cells overexpressing a nonphosphorylatable H2BS32A mutant exhibited suppressed growth and EGF-induced cell transformation, possibly because of decreased activation of activator protein-1, compared with control cells overexpressing wild type H2B. We identified ribosomal S6 kinase 2 (RSK2) as the kinase responsible for H2BS32 phosphorylation. Serum-starved JB6 cells contain very little endogenous H2BS32 phosphorylation, and EGF treatment induced this phosphorylation. The phosphorylation was attenuated in RSK2 knock-out MEFs and RSK2 knockdown JB6 cells. Taken together, our results demonstrate a novel role for H2B phosphorylation in cell transformation and show that H2BS32 phosphorylation is critical for controlling activator protein-1 activity, which is a major driver in cell transformation.