Cellular and Molecular Biology
Ann Bode, PhD
Cancer is one of the leading causes of human death worldwide. By focusing on molecular mechanisms, we continue to discover the key molecular events in cancer development as well as agents for cancer prevention and therapy.
1. Discovery of key molecular events in cancer development.
a. The discovery of circulating prostaglandin biosynthesis in colorectal cancer. We found that the prostaglandin thromboxane A2 (TXA2) level is unexpectedly correlated with colorectal cancer progression. The TXA2 pathway is constitutively activated during colorectal tumorigenesis and required for anchorage-independent growth of colon cancer cells. Our work lays the foundation for introducing a TXA2-targeting strategy for the prevention, early detection and therapy of colon cancer. We further discovered that human colorectal cancer progression is accompanied by an elevation in epidermal growth factor receptor (EGFR) levels. These high levels of EGFR can be attenuated by aspirin intake. The widespread over-expression of EGFR occurs as a consequence of COX-2 activation in familial adenomatous polyposis (FAP) patients. This study revealed a functional association between COX-2 and EGFR expression during colon carcinogenesis and provided new strategies for colon cancer prevention and therapy. b. Discovery of novel mechanisms of RNF2 in cell death. RNF2, also known as Ring1B/Ring2, is a component of the polycomb repression complex 1. RNF2 is highly expressed in many tumors, suggesting it might have an oncogenic function, but the mechanism of action is unknown. We showed that knocking down RNF2 expression significantly inhibits both cell proliferation and colony formation in soft agar and induces apoptosis in cancer cells. Knockdown of RNF2 in HCT116 p53+/+ cells resulted in significantly more apoptosis than was observed in RNF2 knockdown HCT116 p53./. cells, indicating that RNF2 knockdown-induced apoptosis is at least partially, dependent on p53. Various p53-targeted genes were increased in RNF2 knockdown cells. Further studies revealed that in RNF2 knockdown cells, the p53 protein level was increased, the half-life of p53 was prolonged, and p53 ubiquitination was decreased. In contrast, cells over-expressing RNF2 showed a decreased p53 protein level, a shorter p53 half-life, and increased p53 ubiquitination. Importantly, we found that RNF2 directly binds with both p53 and MDM2 as well as promotes MDM2-mediated p53 ubiquitination. RNF2 over-expression also could increase the half-life of MDM2 and inhibit its ubiquitination. The regulation of p53 and MDM2 stability by RNF2 also was observed during the etoposide-induced DNA damage response. These results provide a possible mechanism explaining the oncogenic function of RNF2, and, because RNF2 is important for cancer cell survival and proliferation, it might be an ideal target for cancer therapy or prevention.
2. Discovery of novel targets and agents for skin cancer prevention and therapy.
Solar UV (SUV) irradiation is a major factor in skin carcinogenesis, the most common form of cancer in the United States. The mitogen-activated protein kinase (MAPK) cascades are activated by SUV irradiation. We found that p38 signaling is critical for skin carcinogenesis. The 90 kDa ribosomal S6 kinase (RSK) and mitogen and stress-activated protein kinase (MSK) proteins constitute a family of protein kinases that mediate signal transduction downstream of the MAPK cascades. Phosphorylation of RSK and MSK1 was upregulated in human squamous cell carcinoma (SCC) and SUV-treated mouse skin. Kaempferol . a natural flavonol found in tea, broccoli, grapes, apples, and other plant sources . is known to have anticancer activity, but its mechanisms and direct target(s) in cancer chemoprevention are unclear. Kinase array results revealed that kaempferol inhibited RSK2 and MSK1. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that kaempferol interacts with RSK2 and MSK1 at the ATP-binding pocket and inhibits their respective kinase activities. Mechanistic investigations showed that kaempferol suppresses RSK2 and MSK1 kinase activities to attenuate SUV-induced phosphorylation of cAMP-responsive element binding protein (CREB) and histone H3 in mouse skin cells. Kaempferol was a potent inhibitor of SUV-induced mouse skin carcinogenesis. Further analysis showed that skin from the kaempferol-treated mice exhibited a substantial reduction in SUVinduced phosphorylation of CREB, c-Fos, and histone H3. Overall, our results identify kaempferol as a safe and novel chemopreventive agent against SUVinduced skin carcinogenesis that acts by targeting RSK2 and MSK1.
Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. The anti-cancer activity of chrysin observed in vivo studies, however, has been disappointing. A chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P+ cells compared with chrysin. It attenuated cell-cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser795 and Ser807/811. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, that acts by binding inside the Cdk2 allosteric pocket.
Caffeic acid (3,4-dihydroxycinnamic acid) is a well-known phenolic phytochemical in coffee that reportedly has anti-cancer activities. The underlying molecular mechanisms and targeted proteins involved in the suppression of carcinogenesis by caffeic acid, however, are not fully understood. We reported that caffeic acid significantly inhibits colony formation of human skin cancer cells and EGF-induced neoplastic transformation of HaCaT cells dose-dependently. Caffeic acid topically applied to dorsal mouse skin significantly suppressed tumor incidence and volume in a solar UV-induced skin carcinogenesis mouse model. A substantial reduction of phosphorylation in mitogen-activated protein kinase signaling was observed in mice treated with caffeic acid either before or after solar UV exposure. Caffeic acid directly interacted with ERK1/2 and inhibited ERK1/2 activities in vitro. Importantly, we resolved the co-crystal structure of ERK2 complexed with caffeic acid. Caffeic acid interacted directly with ERK2 at amino acid residues Q105, D106 and M108. Moreover, A431 cells expressing knockdown of ERK2 lost sensitivity to caffeic acid in a skin cancer xenograft mouse model. Taken together, our results suggest that caffeic acid exerts chemopreventive activity against solar UV-induced skin carcinogenesis by targeting ERK1 and 2.
Many proteins are overexpressed only in cancer. The epidermal growth factor (green) is highly expressed in skin tumors and is a major chemotherapy target in breast cancer. The Pim-1 kinase regulates cell survival, proliferation, and differentiation, and it is overexpressed frequently in many malignancies, including leukemia and skin cancer. We used kinase profiling analysis to demonstrate that 2.-hydroxycinnamicaldehyde (2.-HCA), a compound found in cinnamon, specifically inhibits Pim-1. Co-crystallography studies determined the hydrogen bonding pattern between 2.-HCA and Pim-1. Notably, 2.-HCA binding altered the apo kinase structure in a manner that shielded the ligand from solvent, thereby acting as a gatekeeper loop. Biologically, 2.-HCA inhibited the growth of human erythroleukemia or squamous epidermoid carcinoma cells by inducing apoptosis. The compound also was effective as a chemopreventive agent against EGF-mediated neoplastic transformation. Lastly, 2.-HCA potently suppressed the growth of mouse xenografts representing human leukemia or skin cancer. Overall, our results offered preclinical proof of concept for 2.-HCA as a potent anti-cancer principle arising from direct targeting of the Pim-1 kinase.
3. Discovery of novel agents for lung cancer prevention and therapy. Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Despite progress in developing chemotherapeutics for the treatment of NSCLC, primary and secondary resistance limits therapeutic success. NSCLC cells exhibit multiple mutations in the epidermal growth factor receptor (EGFR), which causes aberrant activation of diverse cell signaling pathways. Suppression of the inappropriate amplification of EGFR downstream signaling cascades, therefore, is considered to be a rational therapeutic and preventive strategy for the management of NSCLC. Our initial molecular target.oriented virtual screening revealed that the ginger components . including -shogaol, -paradol, and -gingerol, and butein . a USP8 inhibitor, and 3,6,2.,4.,5.-pentahydroxy-flavone seem to be potential candidates for the prevention and treatment of NSCLC. Among the compounds, -shogaol showed the greatest inhibitory effects against NSCLC cell proliferation and anchorage-independent growth. -Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore, -shogaol inhibited Akt kinase activity, a downstream mediator of EGFR signaling, by binding with an allosteric site of Akt. Other inhibitors, such as butein, a USP8 inhibitor and 3,6,2.,4.,5.-pentahydroxy-flavone, all showed potent inhibitory effects against lung cancer cells in vitro and in vivo. These inhibitors can overcome EGFR inhibitor resistance in lung cancer.
We also investigated the anti-cancer effect of isoliquiritigenin (ILQ), a chalcone derivative. We first studied the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)- polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild-type and double-mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild-type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild-type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu762 and Met793) with wild-type EGFR and three hydrogen bonds (Lys745, Met793, and Asp855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild-type or mutant EGFR.
4. Discovery of Src as a novel potential off-target of RXR agonists, 9-cis-UAB30 and Targretin, in human breast cancer cells 9-cis-UAB30 (UAB30) and Targretin are well-known retinoid X receptor (RXR) agonists. They were highly effective in decreasing the incidence of methylnitrosourea (MNU)-induced mammary cancers. It.s unclear, however, whether the anti-mammary cancer effects of UAB30 or Targretin originate from the activation of RXR. We hypothesized that UAB30 and Targretin not only affect RXR, but likely influence one or more off-target proteins. Virtual screening results suggest that Src is a potential target for UAB30 and Targretin that regulates extracellular matrix (ECM) molecules and cell motility and invasiveness. In vitro kinase assay data revealed that UAB30 or Targretin interacted with Src and attenuated its kinase activity. We found that UAB30 or Targretin substantially inhibited invasiveness and migration of MCF-7 and SKBR- 3 human breast cancer cells. We examined the effects of UAB30 and Targretin on the expression of matrix metalloproteinases (MMP)-9, which are known to play an essential role in tumor invasion. We showed that activity and expression of MMP-9 were decreased by UAB30 or Targretin. Western blot data showed that UAB30 or Targretin decreased AKT and its substrate molecule p70s6k, which are downstream of Src in MCF-7 and SK-BR-3 cells. Moreover, knocking down the expression of Src effectively reduced the sensitivity of SK-BR-3 cells to the inhibitory effects of UAB30 and Targretin on invasiveness. Taken together, our results demonstrate that UAB30 and Targretin each inhibit invasion and migration by targeting Src in human breast cancer cells.
5. Discovery of novel targets and agents for inhibition of colon cancer. Recent clinical trials raised concerns regarding the cardiovascular toxicity of selective cyclooxygenase-2 (COX-2) inhibitors, and cyclooxygenase-1 (COX- 1) now is being reconsidered as a chemoprevention target. Our aims were to determine whether selective COX-1 inhibition could delay or prevent cancer development as well as clarify the underlying mechanisms. We showed that COX-1 was required for maintenance of malignant characteristics of colon cancer cells or tumor promoter-induced transformation of preneoplastic cells. We also successfully applied a ligand-docking computational method to identify a novel selective COX-1 inhibitor, 6-C-(E-phenylethenyl)-naringenin (designated herein as 6CEPN). 6CEPN could bind to COX-1 and specifically inhibited its activity both in vitro and ex vivo. In colorectal cancer cells, it potently suppressed anchorage-independent growth by inhibiting COX-1 activity. 6CEPN also effectively suppressed tumor growth in a 28-day colon cancer xenograft model without any obvious systemic toxicity. Taken together, COX-1 plays a critical role in human colorectal carcinogenesis, and this specific COX-1 inhibitor merits further investigation as a potential preventive agent against colorectal cancer.
Further, we found that naproxen, a COX1 and COX2 inhibitor, induces cell-cycle arrest and apoptosis by downregulation of Bcl-2 and upregulation of Bax.
Importantly, we found the direct cellular target of curcumin. Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Given that curcumin induced cell-cycle arrest, we investigated the anti-proliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Due to the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantial relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells.
The c-Jun N-terminal kinases (JNKs) play an important role in many physiologic processes induced by numerous stress signals. Each JNK protein appears to have a distinct function in cancer, diabetes, and Parkinson.s disease. We found that licochalcone A . a major phenolic constituent isolated from licorice root . suppressed JNK1 activity but had little effect on JNK2 in vitro activity. Although licochalcone A binds with JIP1 competitively with either JNK1 or JNK2, a computer simulation model showed that after licochalcone A binding, the ATPbinding cleft of JNK1 was distorted more substantially than that of JNK2. This could reduce the affinity of JNK1 more than JNK2 for ATP binding. Furthermore, licochalcone A inhibited JNK1-mediated, but not JNK2-mediated, c-Jun phosphorylation in both ex vivo and in vitro systems. We also observed that in colon and pancreatic cancer cell lines, JNK1 is highly expressed compared with normal cell lines. In cancer cell lines, treatment with licochalcone A or knocking down JNK1 expression suppressed colon and pancreatic cancer cell proliferation and colony formation. The inhibition resulted in G1 phase arrest and apoptosis. Moreover, an in vivo xenograft mouse study showed that licochalcone A treatment effectively suppressed the growth of HCT116 xenografts, without affecting the body weight of mice. These results show that licochalcone A is a selective JNK1 inhibitor. We, therefore, suggest that because of the critical role of JNK1 in colon cancer and pancreatic carcinogenesis, licochalcone A might have preventive or therapeutic potential against these devastating diseases.