Cancer is one of the leading causes of human death worldwide. Throughout history, humankind has won the battle against deadly diseases, including small pox and polio, by defeating them through prevention and treatment. By focusing on the molecular mechanisms explaining how normal cells can undergo neoplastic transformation induced by carcinogens and tumor promoters, we have discovered that key protein kinases, transcriptional factors, and other signaling molecules are critical factors in cancer development and are significant targets for cancer prevention and treatment.
Skin cancer is the most common human malignancy in the world and is the number one cancer in terms of incidence in the USA. The major etiological factor of human skin cancer is the chronic exposure to UV light from the sun. Numerous oncogenic and/or protective signaling pathways are activated in UV-induced carcinogenesis. We found that the UV-induced signal transduction pathways are mediated primarily through signaling cascades involving the mitogen-activated protein (MAP) kinases, epidermal growth factor receptor (EGFR), CB1/2, transient receptor potential channel vanilloid subfamily 1 (TRPV1), TOPK, ribosomal S6 kinase 2 (RSK2), and mitogen and stress activated protein kinase 1/2 (MSK1/2), which results in the modification of transcription factors, including activator protein-1 (AP-1), nuclear factor-kappaB (NF-κB), signal transducer and activator of transcription (STATs), p53 and nuclear factor of activated T cells (NFAT). We also found that histone phosphorylation is critical to mediate UV or other tumor promoter induced apoptosis and cancer formation.
RSK2 plays an important role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor (EGF) mediated through the N-terminal kinase domain of RSK2 in JB6 Cl41 mouse skin epidermal cells in vitro. We examined the effect of knocking down RSK2 expression on EGF-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma (MM) cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of MM cells. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth.
The serine-threonine mitogen-activated protein kinase kinase family member T-LAK celloriginated protein kinase (TOPK/PBK) is heavily involved in tumor development and is highly overexpressed in many cancers such as colorectal cancer and skin cancer. Despite the identification of TOPK as a promising novel therapeutic target, no inhibitor of TOPK has yet been reported. We screened 36 drug candidates and identified the novel TOPK inhibitor HITOPK- 032. HI-TOPK-032 strongly suppressed TOPK kinase activity but had little effect on extracellular signal-regulated kinase 1 (ERK1), c-jun-NH2-kinase 1, or p38 kinase activities. HI-TOPK-032 also inhibited anchorage-dependent and -independent colon cancer cell growth by reducing ERK-RSK phosphorylation as well as increasing colon cancer cell apoptosis. Administration of HI-TOPK-032 suppressed tumor growth in a colon cancer xenograft model. Our findings show that HI-TOPK-032 is a specific inhibitor of TOPK both in vitro and in vivo that may be further developed as a potential therapeutic against colorectal cancer.
TRAF2 has an important function in mediating the TNF-R signaling pathway toward activation of NF-κB and JNKs. We have revealed a novel function of TRAF2 in the epidermal growth factor (EGF) signaling pathway. Knockdown of TRAF2 blocked EGF-induced AP-1 activity and anchorage- independent cell transformation. Notably, we showed that EGF induces RSK2 ubiquitination, and knocking down TRAF2 suppresses ubiquitination of RSK2 induced by EGF. We also found that TRAF2 affects RSK2 activity through RSK2 ubiquitination. RSK2 plays a critical role in AP-1 activity mediated through CREB and c-Fos, which regulates anchorage-independent cell transformation.
Activator protein-1 (AP-1) regulates a wide range of cellular processes including proliferation, differentiation, and apoptosis. As a transcription factor, AP-1 is commonly found as a heterodimer comprised of c-Jun and c-Fos proteins. However, other heterodimers may also be formed. But their function is not known. A gain-of-function study performed by overexpressing junD and c-fos and a loss-offunction study using morpholino junD demonstrate a critical role for AP-1JunD/c-Fos in hematopoiesis during Xenopus embryogenesis. Our findings identify AP-1JunD/c-Fos as a novel hematopoietic transcription factor and the requirement of AP-1JunD/c-Fos in BMP-4-induced hematopoiesis during Xenopus hematopoiesis.
Inorganic arsenic is a well-documented human carcinogen associated with cancers of the skin, lung, liver, and bladder. The underlying mechanisms explaining the tumorigenic role of arsenic are not well understood. Exposure to a low dose of arsenic trioxide (As2O3) caused transformation of BALB/c 3T3 cells. In addition, in a xenograft mouse model, tumor growth of the arsenicinduced transformed cells was dramatically increased. In arsenic-induced transformed cells, polycomb group (PcG) proteins, including BMI1 and SUZ12, were activated resulting in enhanced histone H3K27 tri-methylation levels. Histone H3K27 tri-methylation returned to normal in BMI1- or SUZ12-knockdown BALB/c 3T3 cells compared with BMI1- or SUZ12-wildtype cells after arsenic exposure. As a consequence, the expression of p16INK4a and p19ARF was recovered in arsenic-treated BMI1- or SUZ12-knockdown cells. Thus, arsenic-induced cell transformation was blocked by inhibition of PcG function. Taken together, these results strongly suggest that the polycomb proteins, BMI1 and SUZ12 are required for cell transformation induced by organic arsenic exposure.
Nutritional or dietary factors have attracted a great deal of interest because of their perceived effective chemopreventive cancer activity. They are perceived as being generally safe and may have the efficacy for preventing or reversing premalignant lesions and reducing second tumor incidency. We have studied the effects of these compounds such as (-)-epigallocatechin-3-gallate (EGCG), theaflavins, caffeine, myricetin, gingerol, luteolin, and resveratrol.
We have developed in silico computer screening and kinase profiling methods to identify the novel molecular targets of chemopreventing agents and tools to develop novel inhibitors/drugs for these specific cancer targets. For example, in silico computer screening and kinase profiling results suggest that the EGF receptor (EGFR), phosphinositide 3-kinase (PI3K), and Src are potential targets for taxifolin. Pull-down assay results showed that EGFR, PI3K, and Src directly interacted with taxifolin in vitro, whereas taxifolin bound to EGFR and PI3K, but not to Src in cells. ATP competition and in vitro kinase assay data revealed that taxifolin interacted with EGFR and PI3K at the ATPbinding pocket and inhibited their kinase activities. Western blot analysis showed that taxifolin suppressed UVB-induced phosphorylation of EGFR and Akt, and subsequently suppressed their signaling pathways in JB6 P+ mouse skin epidermal cells. The effect of taxifolin on UVB-induced signaling pathways and PGE2 generation was reduced in EGFR knockout murine embryonic fibroblasts (MEF) compared with EGFR wild-type MEFs. Taxifolin also inhibited EGF-induced cell transformation. Importantly, topical treatment of taxifolin to the dorsal skin significantly suppressed tumor incidence, volume, and multiplicity in a solar UV (SUV)-induced skin carcinogenesis mouse model. Further analysis showed that the taxifolin-treated group had a substantial reduction in SUVinduced phosphorylation of EGFR and Akt in mouse skin. These results suggest that taxifolin exerts chemopreventive activity against UV-induced skin carcinogenesis by targeting EGFR and PI3K.
We showed that quercetin-3-methyl ether and noratyriol are novel ERKs inhibitors. Quercetin-3-methyl ether inhibited proliferation of mouse skin epidermal JB6 P+ cells by inducing cell cycle G2-M phase accumulation. It also suppressed 12-Otetradecanoylphorbol- 13-acetate-induced neoplastic cell transformation. Western blot and kinase assay data revealed that quercetin-3-methyl ether inhibited ERKs kinase activity and attenuated phosphorylation of ERKs. Pull-down assays revealed that quercetin-3-methyl ether directly binds with ERKs. Furthermore, a loss-of-function ERK2 mutation inhibited the effectiveness of the quercetin-3-methyl ether.
We also found that norathyriol inhibits ERK1/2 kinase activities and attenuates UVB-induced phosphorylation of the mitogen-activated protein kinase (MAPK) cascades. Direct binding of norathyriol with ERK2 was confirmed by a co-crystal structure. Norathyriol inhibited cell growth in mouse skin epidermal JB6 P+ cells by inducing G23-M phase arrest. Mouse skin tumorigenesis data clearly showed that treatment with norathyriol significantly suppressed solar UV-induced skin carcinogenesis in vivo.
We have studied the novel use of FDA-approved drugs for cancer treatment. For example, ceftriaxone, an FDA-approved third-generation cephalosporin antibiotic, has antimicrobial activity against both gram-positive and gram-negative organisms. Generally, ceftriaxone is used for a variety of infections such as pneumonia, meningitis and gonorrhea. Its primary molecular targets are the penicillin-binding proteins. However, other activities of ceftriaxone remain unknown. We reported for the first time that ceftriaxone has antitumor activity. Kinase profiling results predicted that Aurora B might be a potential ‘off’ target of ceftriaxone. Pull-down assay data confirmed that ceftriaxone could bind with Aurora B cells. Furthermore, ceftriaxone suppressed anchorage-independent cell growth by targeting lung cancer cells. Importantly, in vivo xenograft animal model results showed that ceftriaxone effectively suppressed lung tumor growth by inhibiting Aurora B. These data suggest the anticancer efficacy of ceftriaxone for the treatment of lung cancers through its inhibition of Aurora B.
N-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) is a synthetic retinoid that has been tested in clinical trials as a cancer therapeutic and chemopreventive agent. Until now, no direct cancer-related molecular target has been reported to be involved in the antitumor activities of 4HPR. We found that 4HPR inhibited mammalian target of rapamycin (mTOR) kinase activity by directly binding with mTOR, which suppressed the activities of both the mTORC1 and the mTORC2 complexes. The predicted binding mode of 4HPR with mTOR was based on a homology computer model, which showed that 4HPR could bind in the ATP-binding pocket of the mTOR protein through hydrogen bonds and hydrophobic interactions. In vitro studies also showed that 4HPR attenuated mTOR downstream signaling in a panel of non-smallcell lung cancer cells, resulting in growth inhibition. The expression of mTOR downstream signaling molecules in tumor tissues was also decreased after 4HPR treatment. Our results identified mTOR as a direct antitumor target of 4HPR, providing a valuable rationale for guiding the clinical uses of 4HPR.
Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. We used a ligand docking method to explore the novel scaffold of potential Aurora B inhibitors. One thousand compounds from our in-house compound library were screened against the Aurora B structure and one compound, designated as HOI-07, was selected for further study. HOI-07 potently inhibited Aurora B kinase activity, without obvious inhibition of another 49 kinases, including Aurora A. This compound suppressed Aurora B kinase activity in lung cancer cells, resulting in apoptosis induction, G(2)-M arrest, polyploidy cells, and attenuation of cancer cell anchorage-independent growth. Moreover, knocking down the expression of Aurora B effectively reduced the sensitivity of cancer cells to HOI-07. HOI-07 treatment effectively suppressed the growth of A549 xenografts, without affecting the body weight of mice. The expression of phospho-histone H3, phospho-Aurora B, and Ki-67 was also suppressed in the HOI-07 treated group. Taken together, we identified HOI-07 as a specific Aurora B inhibitor, which deserves further investigation.
In summary, we address fundamental questions concerning the response of animal and/or human cells to carcinogens and tumor promoters such as UV light, arsenic, TPA and growth factors. We have established a series of necessary models and systems, such as the over-agar assay for cell transformation, gene knockout mice, transcription factor/luciferase promoter stably transfected cells and transgenic mice, as well as gene knockdown (siRNA) or dominant negative mutant stably transfected cell lines. These models have been extensively utilized to examine the tumor promoter-induced signal transduction pathways and their role in cell neoplastic transformation. We have systematically studied the signal transduction networks induced by solar UV, UVA, UVB and UVC. Such studies have provided the basis for the carcinogenic process caused by environmental carcinogens and molecular mechanisms for cancer prevention.
Many nutritional/dietary compounds appear to act on multiple tumor promoterinduced carcinogenesis processes and thus can be used for chemopreventing agents. Large-scale animal and molecular biology studies are needed to address the bioavailability, toxicity, molecular target, signal transduction pathways, and side effects of dietary factors. Clinical trials based on clear mechanistic studies are also needed to assess the effectiveness of these dietary factors in the human population. A continuing emphasis on obtaining rigorous research data and critical analysis of those data regarding these and other food factors is vital to determine the molecular basis and long-term effectiveness and safety of these compounds as chemopreventive agents. Articles on our work using the supercomputer and molecular modeling and simulation were published in Nature Reviews Cancer and other top tier journals in the last few years.
Other Professional Activities
Grant Reviewer, National Institutes of Health
Editor, Associate Editor or Editing Board Member
Cancer Prevention Research
International Journal of Biological Sciences