Chien-Chen Lai

IKKβ Suppression of TSC1 Links Inflammation and Tumor Angiogenesis via the mTOR Pathway

TNFalpha has recently emerged as a regulator linking inflammation to cancer pathogenesis, but the detailed cellular and molecular mechanisms underlying this link remain to be elucidated. The tuberous sclerosis 1 (TSC1)/TSC2 tumor suppressor complex serves as a repressor of the mTOR pathway, and disruption of TSC1/TSC2 complex function may contribute to tumorigenesis. Here we show that IKKbeta, a major downstream kinase in the TNFalpha signaling pathway, physically interacts with and phosphorylates TSC1 at Ser487 and Ser511, resulting in suppression of TSC1. The IKKbeta-mediated TSC1 suppression activates the mTOR pathway, enhances angiogenesis, and results in tumor development. We further find that expression of activated IKKbeta is associated with TSC1 Ser511 phosphorylation and VEGF production in multiple tumor types and correlates with poor clinical outcome of breast cancer patients. Our findings identify a pathway that is critical for inflammation-mediated tumor angiogenesis and may provide a target for clinical intervention in human cancer.

ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation

The RAS–ERK pathway is known to play a pivotal role in differentiation, proliferation and tumour progression. Here, we show that Erk downregulates Forkhead box O 3a (FOXO3a) by directly interacting with and phosphorylating FOXO3a at Ser 294, Ser 344 and Ser 425, which consequently promotes cell proliferation and tumorigenesis. The ERK-phosphorylated FOXO3a degrades via an MDM2-mediated ubiquitin-proteasome pathway. However, the non-phosphorylated FOXO3a mutant is resistant to the interaction and degradation by murine double minute 2 (MDM2), thereby resulting in a strong inhibition of cell proliferation and tumorigenicity. Taken together, our study elucidates a novel pathway in cell growth and tumorigenesis through negative regulation of FOXO3a by RAS–ERK and MDM2.

CDK1-dependent phosphorylation of EZH2 suppresses methylation of H3K27 and promotes osteogenic differentiation of human mesenchymal stem cells.

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2) and catalyses the trimethylation of histone H3 on Lys 27 (H3K27), which represses gene transcription. EZH2 enhances cancer-cell invasiveness and regulates stem cell differentiation. Here, we demonstrate that EZH2 can be phosphorylated at Thr 487 through activation of cyclin-dependent kinase 1 (CDK1). The phosphorylation of EZH2 at Thr 487 disrupted EZH2 binding with the other PRC2 components SUZ12 and EED, and thereby inhibited EZH2 methyltransferase activity, resulting in inhibition of cancer-cell invasion. In human mesenchymal stem cells, activation of CDK1 promoted mesenchymal stem cell differentiation into osteoblasts through phosphorylation of EZH2 at Thr 487. These findings define a signalling link between CDK1 and EZH2 that may have an important role in diverse biological processes, including cancer-cell invasion and osteogenic differentiation of mesenchymal stem cells.

The Crosstalk of mTOR/S6K1 and Hedgehog Pathways

Esophageal adenocarcinoma (EAC) is the most prevalent esophageal cancer type in the United States. The TNF-α/mTOR pathway is known to mediate the development of EAC. Additionally, aberrant activation of Gli1, downstream effector of the Hedgehog (HH) pathway, has been observed in EAC. In this study, we found that an activated mTOR/S6K1 pathway promotes Gli1 transcriptional activity and oncogenic function through S6K1-mediated Gli1 phosphorylation at Ser84, which releases Gli1 from its endogenous inhibitor, SuFu. Moreover, elimination of S6K1 activation by an mTOR pathway inhibitor enhances the killing effects of the HH pathway inhibitor. Together, our results established a crosstalk between the mTOR/S6K1 and HH pathways, which provides a mechanism for SMO-independent Gli1 activation and also a rationale for combination therapy for EAC.

KEAP1 E3 Ligase-Mediated Downregulation of NF-κB Signaling by Targeting IKKβ

IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.

Down-regulation of Myeloid Cell Leukemia-1 through Inhibiting Erk/Pin 1 Pathway by Sorafenib Facilitates Chemosensitization in Breast Cancer

Myeloid cell leukemia-1 (Mcl-1), a Bcl-2-like antiapoptotic protein, plays a role in cell immortalization and chemoresistance in a number of human malignancies. A peptidyl-prolyl cis/trans isomerase, Pin1 is involved in many cellular events, such as cell cycle progression, cell proliferation, and differentiation through isomerizing prophosphorylated substrates. It has been reported that down-regulation of Pin1 induces apoptosis, and that Erk phosphorylates and up-regulates Mcl-1; however, the underlying mechanisms for the two phenomena are not clear yet. Here, we showed that Pin 1 stabilizes Mcl-1, which is required for Mcl-1 posphorylation by Erk. First, we found expression of Mcl-1 and Pin1 were positively correlated and associated with poor survival in human breast cancer. We then showed that Erk could phosphorylate Mcl-1 at two consensus residues, Thr 92 and 163, which is required for the association of Mcl-1 and Pin1, resulting in stabilization of Mcl-1. Moreover, Pin1 is also required for the up-regulation of Mcl-1 by Erk activation. Based on this newly identified mechanism of Mcl-1 stabilization, two strategies were used to overcome Mcl-1-mediated chemoresistance: inhibiting Erk by Sorafenib, an approved clinical anticancer drug, or knocking down Pin1 by using a SiRNA technique. In conclusion, the current report not only unravels a novel mechanism to link Erk/Pin1 pathway and Mcl-1-mediated chemoresistance but also provides a plausible combination therapy, Taxol (Paclitaxel) plus Sorafenib, which was shown to be effective in killing breast cancer cells.

KEAP1 E3 ligase-mediated downregulation of NF-kappaB signaling by targeting IKKbeta.

IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.

Analysis of urinary nucleosides as potential tumor markers in human breast cancer by high performance liquid chromatography/electrospray ionization tandem mass spectrometry

BACKGROUND Breast cancer is the most common female cancer and the fourth leading cause of cancer death among women in Taiwan. We measured urinary nucleoside levels in female breast cancer patients (n=36) to evaluate the diagnostic value of nucleosides as potential tumor markers. METHODS Purification of urinary nucleosides was performed using a 96-well solid phase extraction (SPE, cation-exchange column) procedure to decrease the variation between the single column preparations and to shorten the pretreatment time. Cation-exchange allows for the comprehensive purification of modified nucleosides, such as 2-deoxynucleosides, that are not purifiable by phenylboronic acid-based SPE. High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) in selected reaction monitoring (SRM) mode was used to quantify multiple nucleosides. Tubercidin was used as an internal standard. The qualitative parameters, retention time, and the parent and daughter ions used revealed that the method was more specific and sensitive than traditional UV detection. RESULTS Urinary levels of 3 nucleosides, cytidine, 3-methylcytidine, and inosine were significantly higher in breast cancer patients than in normal controls (p<0.01). The discriminative powers of cytidine, 3-methylcytidine, and inosine were 58%, 58%, and 62%, respectively. CONCLUSIONS LC/MS/MS is a highly specific and sensitive method for rapidly screening a large number of urinary nucleosides that may be potential cancer markers. The 3-methylcytidine may be a candidate marker for breast cancer.

Acid/Base‐ and Anion‐Controllable Organogels Formed From a Urea‐Based Molecular Switch

Low-molecular-weight organogels have applications in several fields, including molecular sensing, nanostructure assembly, and drug delivery. Ideally, these materials would switch reversibly between their solution and gel states through the addition or removal of heat, electrons, or ions. Although these modes of operation are similar to those employed for switches based on interlocked molecules, organogels formed from pseudorotaxaneor rotaxane-type gelators are rare. Indeed, we are aware of only a few previously reported examples, all of which feature long alkyl chains or cholesterol units incorporated into the molecular structures to assist the gelation process. Predicting the molecular structures of potential gelators and their preferred solvents remains difficult, and developing new rotaxane-based gelators that do not feature commonly used types of gelation units (e.g., long alkyl chains, steroids) in their structures is particularly challenging. Herein we report the serendipitous discovery of a urea-based [2]rotaxane that behaves as both a molecular switch and an organogelator; both functions are mediated by acid/base and anion control. The reaction of the macrocycle 1, the amino-terminated salt [2-H][PF6], [7] and the isocyanate 3 in CH3NO2 gave the dumbbell-shaped salt [4-H][PF6] and the [2]rotaxane [5-H][PF6] in 49 and 46% yield, respectively (Scheme 1). The binding constant for the assembly formed from the macrocycle 1 and dibenzylammonium hexafluorophosphate ((DBA)PF6) in CD3NO2 is (300 30)m , and 1 interacts only negligibly with diphenylurea derivatives in this solvent. 8] Therefore we suspected that the interlocked macrocycle in the [2]rotaxane [5-H][PF6] would prefer to encircle the DBA station, rather than the diphenylurea station, when dissolved in CD3NO2. Indeed, the 2D NOESY spectrum of the [2]rotaxane [5-H][PF6] in CD3NO2 shows cross-signals between the ethylene glycol protons of the macrocyclic unit and the aromatic protons of the 3,5-di-tert-butylphenyl stopper adjacent to the DBA center, however, no crosssignals are seen between the macrocyle and the stopper unit adjacent to the urea station. As expected, addition of potassium tert-butoxide (1 equivalent) to a solution of the [2]rotaxane [5-H][PF6] (CD3NO2, 13.6 mm) resulted in significant shifts in the locations of many of the signals in the H NMR spectrum (Figure 1). The significant downfield shift of the signal for the macrocyle NH protons, and the appearance of signals for the formerly severely broadened urea protons suggested the formation of hydrogen bonds to the carbonyl group of the urea station (Figure 1b). The addition of perchloric acid (70% in H2O, 1 equivalent) to this solution afforded a spectrum similar to that of the original [2]rotaxane. These observations suggest that the [2]rotaxane [5-H][X] is an acid/base-controllable molecular switch; the interlocked macrocyclic unit can be Scheme 1. Synthesis and switching of the [2]rotaxane [5-H][PF6].

A Molecular Cage-Based [2]Rotaxane That Behaves as a Molecular Muscle

We report a molecular cage-based [2]rotaxane that functions as an artificial molecular muscle through the control of the addition and removal of fluoride anions. The percentage change in molecular length of the [2]rotaxane is about 36% between the stretched and contracted states, which is larger than the percentage change (approximately 27%) in human muscle.