Yakun Chen

Nuclear receptors in the multidrug resistance through the regulation of drug-metabolizing enzymes and drug transporters.

Chemotherapy is one of the three most common treatment modalities for cancer. However, its efficacy is limited by multidrug resistant cancer cells. Drug metabolizing enzymes (DMEs) and efflux transporters promote the metabolism, elimination, and detoxification of chemotherapeutic agents. Consequently, elevated levels of DMEs and efflux transporters reduce the therapeutic effectiveness of chemotherapeutics and, often, lead to treatment failure. Nuclear receptors, especially pregnane X receptor (PXR, NR1I2) and constitutive androstane activated receptor (CAR, NR1I3), are increasingly recognized for their role in xenobiotic metabolism and clearance as well as their role in the development of multidrug resistance (MDR) during chemotherapy. Promiscuous xenobiotic receptors, including PXR and CAR, govern the inducible expressions of a broad spectrum of target genes that encode phase I DMEs, phase II DMEs, and efflux transporters. Recent studies conducted by a number of groups, including ours, have revealed that PXR and CAR play pivotal roles in the development of MDR in various human carcinomas, including prostate, colon, ovarian, and esophageal squamous cell carcinomas. Accordingly, PXR/CAR expression levels and/or activation statuses may predict prognosis and identify the risk of drug resistance in patients subjected to chemotherapy. Further, PXR/CAR antagonists, when used in combination with existing chemotherapeutics that activate PXR/CAR, are feasible and promising options that could be utilized to overcome or, at least, attenuate MDR in cancer cells.

G-protein-coupled receptor for short-chain fatty acids suppresses colon cancer

GPR43 is a G‐protein‐coupled receptor for short‐chain fatty acids (SCFAs). Expression of GPR43 is detected in hematopoietic tissues and the large intestine. SCFAs are derived from bacterial fermentation and metabolism of undigested dietary fibers and have been recognized for their cancer prevention activities in the colon. The role of SCFAs, particularly butyrate, in colon cancer therapy has been extensively studied, and its tumor suppressive functions are believed to be due to their intracellular actions, notably inhibition of histone deacetylase. In our study, we show that SCFAs also exert their antitumor effects via receptor GPR43 and that GPR43 is frequently lost in colon cancer cells. Immunohistostaining revealed that GPR43 immunoreactivity was high in normal colon tissues (N = 31) but was markedly reduced or completely lost in most colorectal adenocarcinoma tissues (N = 70) and their corresponding lymph node metastatic adenocarcinomas (N = 38). RT‐PCR analysis detected the presence of full length GPR43 mRNA in only one (HT‐29) of nine established human colon cancer cell lines. Restoration of GPR43 expression in HCT8 human colonic adenocarcinoma cells induced G0/G1 cell cycle arrest and activated caspases, leading to increased apoptotic cell death after propionate/butyrate treatment. Restored GPR43 expression, coupled with propionate treatment, induced an upregulation of p21 and a decrease in the levels of cyclin D3 and cyclin‐dependent kinases (CDKs) 1 and 2, while the CDK4 and CDK6 levels remained unchanged. Our results suggest that GPR43 functions as a tumor suppressor by mediating SCFA‐induced cell proliferation inhibition and apoptotic cell death in colon cancer.

Human Pregnane X Receptor and Resistance to Chemotherapy in Prostate Cancer

Resistance to chemotherapy is a significant barrier to the effective management of prostate cancer. Human pregnane X receptor (hPXR), an orphan nuclear receptor known for its activation by many important clinical drugs, interacts with many cellular signaling pathways during carcinogenesis and is a major transcription factor regulating the expression of drug metabolism enzymes, including transporters. It is unknown whether hPXR is a determinant of drug resistance in prostate cancer. In this study, we first detected the expression of hPXR in both normal and cancerous prostate tissues. Pretreatment with SR12813, a potent and selective agonist of hPXR, led to nuclear translocation of PXR in PC-3 cells and increased expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance 1 (MDR1). SR12813 pretreatment increased resistance of PC-3 cells to Taxol and vinblastine, as assessed by viability and clonogenic survival. To further study the role of hPXR in prostate cancer drug resistance, hPXR expression was knocked down using PXR-targeting short hairpin RNAs. The activities of hPXR toward the promoter of CYP3A4 in hPXR-ablated clones decreased when compared with that of wild-type PC-3 cells. Their sensitivities to Taxol and vinblastine were enhanced by hPXR ablation. Our data here suggest that hPXR may play an important role in prostate cancer resistance to chemotherapeutics.

The role of short-chain fatty acids in orchestrating two types of programmed cell death in colon cancer.

Short-chain fatty acids are the major by-products of bacterial fermentation of undigested fibers in the colon. SCFAs, mostly propionate and butyrate, induce differentiation, growth arrest, and apoptosis in colon cancer cells. The anticancer effect of SCFAs is also supported by epidemiological studies suggesting an inverse relationship between the level of dietary fibers and the incidence of human colon cancer. Dietary components influence the risk of human colon cancer including colon cancer through diverse mechanisms, which include the activation or inhibition of autophagy (type II programmed cell death (PCD)). Herein we demonstrate that propionate and butyrate induce autophagy in human colon cancer cells to dampen apoptosis, whereas inhibition of autophagy potentiates SCFA-induced apoptosis. The propionate-induced autophagy originates from mitochondria defects associated with cellular ATP depletion and ROS generation, both of which contribute to AMPK activation and consequential mTOR inhibition. Remarkably, when autophagy is suppressed through either pharmacological or genetic approaches, the colon cancer cells become sensitized toward propionate-induced apoptotic cell death (type I PCD). Our study is the first report characterizing this novel role of SCFAs in orchestrating two types of programmed cell death. The observed pro-survival effects of autophagy in retarding mitochondria-mediated apoptosis suggest that application of an autophagy inhibitor might improve the therapeutic efficacy of SCFAs in inducing colon cancer suppression.

Impairment of mitochondrial respiration in mouse fibroblasts by oncogenic H-RAS(Q61L).

A common metabolic change in cancer is the acquisition of glycolytic phenotypes. Increased expression of glycolytic enzymes is considered as one contributing factor. The role of mitochondrial defects in acquisition of glycolytic phenotypes has been postulated but remains controversial. Here we show that functional defects in mitochondrial respiration could be induced by oncogenic H-RasQ61L transformation, even though the mitochondrial contents or mass was not reduced in the transformed cells. First, mitochondrial respiration, as measured by mitochondrial oxygen consumption, was suppressed in NIH-3T3 cells transformed with H-RasQ61L. Second, oligomycin or rotenone did not reduce the cellular ATP levels in the H-RasQ61L transformed cells, suggesting a diminished role of mitochondrial respiration in the cellular energy metabolism. Third, inhibition of glycolysis with iodoacetic acid reduced ATP levels at a much faster rate in H-RasQ61L transformed cells than in the vector control cells. The reduction of cellular ATP levels was reversed by exogenously added pyruvate in the vector control cells but not in H-RasQ61L transformed cells. Finally when compared to the H-RasQ61L transformed cells, the vector control cells had increased resistance toward glucose deprivation. The increased resistance was dependent on mitochondrial oxidative phosphorylation since rotenone or oligomycin abolished the increased survival of the vector control cells under glucose deprivation. The results also suggest an inability of the H-RasQ61L transformed cells to reactivate mitochondrial respiration under glucose deprivation. Taken together, the data suggest that mitochondrial respiration can be impaired during transformation of NIH-3T3 cells by oncogeneic H-RasQ61L.

Camptothecin attenuates cytochrome P450 3A4 induction by blocking the activation of human pregnane X receptor.

Differential regulation of drug-metabolizing enzymes (DMEs) is a common cause of adverse drug effects in cancer therapy. Due to the extremely important role of cytochrome P450 3A4 (CYP3A4) in drug metabolism and the dominant regulation of human pregnane X receptor (hPXR) on CYP3A4, finding inhibitors for hPXR could provide a unique tool to control drug efficacies in cancer therapy. Camptothecin (CPT) was demonstrated as a novel and potent inhibitor (IC50 = 0.58 μM) of an hPXR-mediated transcriptional regulation on CYP3A4 in this study. In contrast, one of its analogs, irinotecan (CPT-11), was found to be an hPXR agonist in the same tests. CPT disrupted the interaction of hPXR with steroid receptor coactivator-1 but had effects on neither the competition of ligand binding nor the formation of the hPXR and retinoid X receptor α heterodimer, nor the interaction between the regulatory complex and DNA-responsive elements. CPT treatment resulted in delayed metabolism of nifedipine in human hepatocytes treated with rifampicin, suggesting a potential prevention of drug-drug interactions between CYP3A4 inducers and CYP3A4-metabolized drugs. Because CPT is the leading compound of topoisomerase I inhibitors, which comprise a quickly developing class of anticancer agents, the findings indicate the potential of a new class of compounds to modify hPXR activity as agonists/inhibitors and are important in the development of CPT analogs.

Downregulation of vascular endothelial growth factor and induction of tumor dormancy by 15-lipoxygenase-2 in prostate cancer

The enzyme 15‐lipoxygenase‐2 (15‐LOX‐2) utilizes arachidonic acid, a polyunsaturated fatty acid, to synthesize 15(S)‐hydroxyeicosatetraenoic acid. Abundantly expressed in normal prostate epithelium but frequently suppressed in the cancerous tissues, 15‐LOX‐2 has been suggested as a functional suppressor of prostate cancer, but the mechanism(s) involved remains unknown. To study the functional role of 15‐LOX‐2 in prostate cancer, we expressed 15‐LOX‐2 as a fusion protein with GFP in DU145 and PC‐3 cells and found that 15‐LOX‐2 increased cell cycle arrest at G0/G1 phase. When injected into athymic nu/nu mice, prostate cancer cells with 15‐LOX‐2 expression could still form palpable tumors without significant changes in tumorigenicity. But, the tumors with 15‐LOX‐2 expression grew significantly slower than those derived from vector controls and were kept dormant for a long period of time. Histological evaluation revealed an increase in cell death in tumors derived from prostate cancer cells with 15‐LOX‐2 expression, while in vitro cell culture conditions, no such increase in apoptosis was observed. Further studies found that the expression of vascular endothelial growth factor A (VEGF‐A) was significantly reduced in prostate cancer cells with 15‐LOX‐2 expression restored. Our studies suggest that 15‐LOX‐2 suppresses VEGF gene expression and sustains tumor dormancy in prostate cancer. Loss of 15‐LOX‐2 functionalities, therefore, represents a key step for prostate cancer cells to exit from dormancy and embark on malignant progression in vivo.

Promotion of tumor development in prostate cancer by progerin.

Progerin is a truncated form of lamin A. It is identified in patients with Hutchinson-Gilford progeria syndrome (HGPS), a disease characterized by accelerated aging. The contribution of progerin toward aging has been shown to be related to increased DNA damages. Since aging is one major risk factor for carcinogenesis, and genomic instability is a hallmark of malignant cancers, we investigated the expression of progerin in human cancer cells, and whether its expression contributes to carcinogenesis. Using RT-PCR and Western blotting, we detected the expression of progerin in prostate PC-3, DU145 and LNCaP cells at mRNA and protein levels. Ectopic progerin expression did not cause cellular senescence in PC-3 or MCF7 cells. PC-3 cells progerin transfectants were sensitized to DNA damage agent camptothecin (CPT); and persistent DNA damage responses were observed, which might be caused by progerin induced defective DNA damage repair. In addition, progerin transfectants were more tumorigenic in vivo than vector control cells. Our study for the first time describes the expression of progerin in a number of human cancer cell lines and its contributory role in tumorigenesis.

Abstract C8: Short‐chain fatty acids induce autophagy through inhibition of mTOR pathway

Short‐chain fatty acid (SCFA), mainly propionate and butyrate are major by‐products of the bacterial fermentation of undigested dietary fiber in the human large intestine. Evidence to date implicates propionate/butyrate as inhibitors of colon cancer cell proliferation and inducers of apoptotic cell death. In this study, we demonstrated that propionate/butyrate was able to induce another type of programmed cell death named autophagy in human colon cancer HCT116 cells. Propionate/butyrate‐treated cells exhibited extensive characteristics of autophagy, namely the conversion of LC3‐I into LC3‐II, reduced expression of p62/ SQSTM1; redistribution of GFPLC3 fusion protein from a diffuse pattern towards autophagosomes, which became visible as cytoplasmic puncta; and increased acidic vesicular organelle development as manifested by dramatically increased quantity of strongly stained punctate areas reflected by LysoTracker staining and monodansylcadaverine (MDC) incorporation. Propionate/butyrate‐induced autophagy was based on inhibition of its negative regulator‐mTOR pathway as evidenced by dephosphorylation of p70S6K and 4E‐BP1 and was marked by increased phosphorylation of AMP kinase, two events known to be linked to autophagy. Activation of AMPK was associated with a fluctuation of intracellular ATP, during which the level of ATP transiently increased following propionate/butyrate exposure, reaching maximum at 5 hours, and then declined in slow but gradual manner. The decline of ATP may be due to compromised mitochondria membrane potential and enhanced cellular reactive oxygen species generation. We also showed that this autophagic increase was associated with a series of changes in metabolic pathways, in which energy‐demanding program (fatty acids composition and glycogen synthesis inhibition) was downregulated whereas energy‐producing program (mitochondria biogenesis) was initiated to cope with the energy crisis. Finally, we showed that increased autophagy and mTOR inhibition by propionate/butyrate are not dependent on p53 activation. Altogether, this study demonstrates autophagy as a novel programmed cell death pathway for propionate/butyrate induced cell death. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C8.

Abstract 4837: Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Short-chain fatty acids (SCFAs) are the major by-products of the bacterial fermentation of undigested dietary fiber in the human large intestine. The role of SCFAs, mostly propionate and butyrate, in colon cancer apoptotic cell death and cell proliferation inhibition has been well studied. Herein we demonstrate that, in addition to apoptotic cell death, propionate and butyrate induce another type of cell death termed “autophagy” in human colon cancer cells. Propionate-treated cells exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion and reduced expression of p62/SQSTM1; increased acidic vesicular organelle development as manifested by dramatic increase in punctate GFPLC3, LysoTracker and MDC staining. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity, two events known to be linked to autophagy. The elevated AMPKa phosphorylation is believed to have been caused by cellular ATP depletion due to mitochondrial dysfunction involving induction of mitochondrial permeability transition, as evidenced by the loss of mitochondrial membrane potential and overproduction of reactive oxygen species. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented at an early stage by 3-methyladenine, or when autophagic degradation was inhibited at a late stage by Chloroquine, the colon cancer cells became sensitized toward propionate induced apoptosis through activation of caspase 7 and its downstream effector caspase-3. Potentiation of cell death was also observed when autophagy was abolished using shRNA against the autophagic gene ATG5. These observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death. As SCFAs widely exist in the human large intestine, application of an autophagy inhibitor, such as Chloroquine, is expected to enhance the therapeutic efficacy of SCFAs in inducing tumor cell apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4837.