Necroptosis is an alternative programmed cell death pathway that is unleashed in the absence of apoptosis and mediated by signaling complexes containing receptor-interating protein kinase 1 (RIPK1) and RIPK3. This form of cell death has recently been implicated in host defense system to eliminate pathogen-infected cells. However, only a few viral species such as herpes simplex virus (HSV) and cytomegalovirus (CMV) have evolved mechanisms inhibiting necroptosis to overcome host antiviral defense, which is important for successful pathogenesis. Here, we show that the γ-herpesvirus Epstein–Barr virus (EBV) blocks necroptosis in EBV-infected human nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells. Our findings indicate that EBV-encoded latent membrane protein 1 (LMP1), which lacks an RIP homotypic interaction motif (RHIM) domain, has mechanisms distinct from RHIM signaling competition to inhibit this necroptotic pathway. Intriguingly, LMP1 interacts directly with both RIPK1 and RIPK3 through its C-terminal activation region. More importantly, LMP1 can modulate the post-translational modification of the two receptor-interacting proteins. We then show that LMP1-mediated promotion of K63-polyubiquitinated RIPK1, suppression of RIPK1 protein expression and inhibition of K63-polyubiquitinated RIPK3 induced a switch in cell fate from necroptotic death to survival. These findings provide direct evidence for the suppression of necroptosis by EBV and define a mechanism of LMP1 to interrupt the initiation process of necroptosis before necrosome formation.

Aberrant DNA methylation mediated by deregulation of DNA methyltransferases (DNMT) is a key hallmark of acute myeloid leukemia (AML), yet efforts to target DNMT deregulation for drug development have lagged. We previously demonstrated that upregulation of fatty acid-binding protein 4 (FABP4) promotes AML aggressiveness through enhanced DNMT1-dependent DNA methylation. Here, we demonstrate that FABP4 upregulation in AML cells occurs through vascular endothelial growth factor (VEGF) signaling, thus elucidating a crucial FABP4-DNMT1 regulatory feedback loop in AML biology. We show that FABP4 dysfunction by its selective inhibitor BMS309403 leads to downregulation of DNMT1, decrease of global DNA methylation and re-expression of p15 INK4B tumor suppressor gene by promoter DNA hypomethylation in vitro , ex vivo and in vivo . Functionally, BMS309403 suppresses cell colony formation, induces cell differentiation, and, importantly, impairs leukemic disease progression in mouse models of leukemia. Our findings highlight AML-promoting properties of the FABP4-DNMT1 vicious loop, and identify an attractive class of therapeutic agents with a high potential for clinical use in AML patients. The results will also assist in establishing the FABP4-DNMT1 loop as a target for therapeutic discovery to enhance the index of current epigenetic therapies.

Obesity is becoming more prevalent worldwide and is a major risk factor for cancer development. Acute myeloid leukemia (AML), the most common acute leukemia in adults, remains a frequently fatal disease. Here we investigated the molecular mechanisms by which obesity favors AML growth and uncovered the fatty acid-binding protein 4 (FABP4) and DNA methyltransferase 1 (DNMT1) regulatory axis that mediates aggressive AML in obesity. We showed that leukemia burden was much higher in high-fat diet-induced obese mice, which had higher levels of FABP4 and interleukin (IL)-6 in the sera. Upregulation of environmental and cellular FABP4 accelerated AML cell growth in both a cell-autonomous and cell-non-autonomous manner. Genetic disruption of FABP4 in AML cells or in mice blocked cell proliferation in vitro and induced leukemia regression in vivo. Mechanistic investigations showed that FABP4 upregulation increased IL-6 expression and signal transducer and activator of transcription factor 3 phosphorylation leading to DNMT1 overexpression and further silencing of the p15INK4B tumor-suppressor gene in AML cells. Conversely, FABP4 ablation reduced DNMT1-dependent DNA methylation and restored p15INK4B expression, thus conferring substantial protection against AML growth. Our findings reveal the FABP4/DNMT1 axis in the control of AML cell fate in obesity and suggest that interference with the FABP4/DNMT1 axis might be a new strategy to treat leukemia.

The nuclear factor of activated T cells (NFAT) family proteins are transcription factors that regulate the expression of pro-inflammatory cytokines and other genes during the immune response. Although the NFAT proteins have been extensively investigated in the immune system, their role in cancer progression remains controversial. Here, we report that NFAT3 is highly expressed in various skin cancer cell lines and tumor tissues. Knockdown of endogenous NFAT3 expression by short hairpin RNA (shRNA) significantly inhibited tumor cell proliferation, colony formation and anchorage-independent cell growth. Furthermore, results of the mammalian two-hybrid assay showed that cyclin-dependent kinase 3 (CDK3) directly interacted with NFAT3 and phosphorylated NFAT3 at serine 259 (Ser259), which enhanced the transactivation and transcriptional activity of NFAT3. The phosphorylation site of NFAT3 was critical for epidermal growth factor (EGF)-stimulated cell transformation of the HaCaT immortalized skin cell line and mutation of NFAT3 at Ser259 led to a reduction of colony formation in soft agar. We also found that overexpressing wildtype NFAT3, but not mutant NFAT3-S259A, promoted A431 xenograft tumor growth. Importantly, we showed that CDK3, NFAT3 and phosphorylated NFAT3-Ser259 were highly expressed in skin cancer compared with normal skin tissues. These results provided evidence supporting the oncogenic potential of NFAT3 and suggested that CDK3-mediated phosphorylation of NFAT3 has an important role in skin tumorigenesis.

Solar ultraviolet (UV) light is a major etiological factor in skin carcinogenesis, with solar UV-stimulated signal transduction inducing pathological changes and skin damage. The primary sensor of solar UV-induced cellular signaling has not been identified. We use an experimental system of solar simulated light (SSL) to mimic solar UV and we demonstrate that Fyn is a primary redox sensor involved in SSL-induced signal transduction. Reactive oxygen species (ROS) generated by SSL exposure directly oxidize Cys488 of Fyn, resulting in increased Fyn kinase activity. Fyn oxidation was increased in mouse skin after SSL exposure and Fyn-knockout mice formed larger and more tumors compared with Fyn wild-type mice when exposed to SSL for an extended period of time. Murine embryonic fibroblasts (MEFs) lacking Fyn and cells in which Fyn expression was knocked down were resistant to SSL-induced apoptosis. Furthermore, cells expressing mutant Fyn (C448A) were resistant to SSL-induced apoptosis. These findings suggest that Fyn acts as a regulatory nexus between solar UV, ROS and signal transduction during skin carcinogenesis.

Reactive oxygen species (ROS) are chemically reactive molecules that perform essential functions in living organisms. Accumulating evidence suggests that many types of cancer cells exhibit elevated levels of ROS. Conversely, generation of ROS has become an effective method to kill cancer cells. (E)-3-hydroxy-3-(4-(4-nitrophenyl)-2-oxobut-3-en-1-yl) indolin-2-one, which is an NO2 group-containing compound designated herein as HOI-02, generated ROS and, in a dose-dependent manner, decreased esophageal cancer cell viability and inhibited anchorage-independent growth, followed by apoptosis and G2-M arrest. Moreover, results of an in vivo study using a patient-derived xenograft mouse model showed that HOI-02 treatment suppressed the growth of esophageal tumors, without affecting the body weight of mice. The expression of Ki-67 was significantly decreased with HOI-02 treatment. In addition, the phosphorylation of c-Jun, and expression of p21, cleaved caspase 3, and DCFH-DA were increased in the HOI-02-treated group compared with the untreated control group. In contrast, treatment of cells with (E)-3-(4-(4-aminophenyl)-2-oxobut-3-en-1-yl)-3-hydroxyindolin-2-one, which is an NH2 group-containing compound designated herein as HOI-11, had no effect. Overall, we identified HOI-02 as an effective NO2 group-containing compound that was an effective therapeutic or preventive agent against esophageal cancer cell growth.

Cyclin-dependent kinase 2 (CDK2) is a known regulator in the cell cycle control of the G1/S and S/G2 transitions. However, the role of CDK2 in tumorigenesis is controversial. Evidence from knockout mice as well as colon cancer cell lines indicated that CDK2 is dispensable for cell proliferation. In this study, we found that ectopic CDK2 enhances Ras (G12V)-induced foci formation and knocking down CDK2 expression markedly decreases epidermal growth factor (EGF)-induced cell transformation mediated through the downregulation of c-fos expression. Interestingly, CDK2 directly phosphorylates ELK4 at Thr194 and Ser387 and regulates the ELK4 transcriptional activity, which serves as a mechanism to regulate c-fos expression. In addition, ELK4 is overexpressed in melanoma and knocking down the ELK4 or CDK2 expression significantly attenuated the malignant phenotype of melanoma cells. Taken together, our study reveals a novel function of CDK2 in EGF-induced cell transformation and the associated signal transduction pathways. This indicates that CDK2 is a useful molecular target for the chemoprevention and therapy against skin cancer.

Resveratrol (trans-3,5,4′-truhydroxystilbene) possesses a strong anticancer activity exhibited as the induction of apoptosis through p53 activation. However, the molecular mechanism and direct target(s) of resveratrol-induced p53 activation remain elusive. Here, the Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) was identified as a potential target of resveratrol, and in vitro binding assay results using resveratrol-conjugated Sepharose 4B beads confirmed their direct binding. Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis. We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53. Disruption of the interaction of p53 with USP10 by G3BP1 interference leads to the suppression of p53 deubiquitination. Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53, and consequently increased p53 expression. These findings disclose a novel mechanism of resveratrol-induced p53 activation and resveratrol-induced apoptosis by direct targeting of G3BP1.

Our goal in this work was to illustrate the Epstein-Barr virus (EBV)-modulated global biochemical profile and provide a novel metabolism-related target to improve the therapeutic regimen of nasopharyngeal carcinoma (NPC). We used a metabolomics approach to investigate EBV-modulated metabolic changes, and found that the exogenous overexpression of the EBV-encoded latent membrane protein 1 (LMP1) significantly increased glycolysis. The deregulation of several glycolytic genes, including hexokinase 2 (HK2), was determined to be responsible for the reprogramming of LMP1-mediated glucose metabolism in NPC cells. The upregulation of HK2 elevated aerobic glycolysis and facilitated proliferation by blocking apoptosis. More importantly, HK2 was positively correlated with LMP1 in NPC biopsies, and high HK2 levels were significantly associated with poor overall survival of NPC patients following radiation therapy. Knockdown of HK2 effectively enhanced the sensitivity of LMP1-overexpressing NPC cells to irradiation. Finally, c-Myc was demonstrated to be required for LMP1-induced upregulation of HK2. The LMP1-mediated attenuation of the PI3-K/Akt-GSK3beta-FBW7 signaling axis resulted in the stabilization of c-Myc. These findings indicate a close relationship between EBV and glycolysis in NPC. Notably, LMP1 is the key regulator of the reprogramming of EBV-mediated glycolysis in NPC cells. Given the importance of EBV-mediated deregulation of glycolysis, anti-glycolytic therapy might represent a worthwhile avenue of exploration in the treatment of EBV-related cancers.

Many natural compounds derived from plants or microbes show promising potential for anticancer treatment, but few have been found to target energy-relevant regulators. In this study, we report that neoalbaconol (NA), a novel small-molecular compound isolated from the fungus, Albatrellus confluens, could target 3-phosphoinositide-dependent protein kinase 1 (PDK1) and inhibit its downstream phosphoinositide-3 kinase (PI3-K)/Akt-hexokinase 2 (HK2) pathway, which eventually resulted in energy depletion. By targeting PDK1, NA reduced the consumption of glucose and ATP generation, activated autophagy and caused apoptotic and necroptotic death of cancer cells through independent pathway. Necroptosis was remarkably induced, which was confirmed by several necroptosis-specific markers: the activation of autophagy, presence of necrotic morphology, increase of receptor-interacting protein 1 (RIP1)/RIP3 colocalization and interaction and rescued by necroptosis inhibitor necrostatin-1. The possibility that Akt overexpression reversed the NA-induced energy crisis confirmed the importance of the PDK1-Akt-energy pathway in NA-mediated cell death. Moreover, NA shows the capability to inhibit PI3-K/Akt signaling and suppress tumor growth in the nasopharyngeal carcinoma (NPC) nude mouse model. These results supported the feasibility of NA in anticancer treatments.

RNF2, also known as Ring1B/Ring2, is a component of the polycomb repression complex 1. RNF2 is highly expressed in many tumors, suggesting that it might have an oncogenic function, but the mechanism is unknown. Here, we show that knockdown of RNF2 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 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 overexpressing 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 and promotes MDM2-mediated p53 ubiquitination. RNF2 overexpression could also increase the half-life of MDM2 and inhibit its ubiquitination. The regulation on p53 and MDM2 stability by RNF2 was also 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.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that contributes to the generation of chronic inflammation in response to chemical carcinogens and environmental stresses, including ultraviolet B (UVB) irradiation. Although post-translational histone modifications are believed to have an important role in modulating transcriptional regulation of UVB-induced COX-2, the underlying biochemical mechanisms are completely unknown. Here, we show that UVB activates the p38 MAPK/MSK1 kinase cascade to phosphorylate histone H3 at Ser10 and Ser28, contributing to UVB-induced COX-2 expression. UVB has no effect on the global tri-methylation level of histone H3 (H3K4me3, H3K9me3, and H3K27me3). We observed that selected mammalian 14-3-3 proteins bind to UVB-induced phosphorylated histone H3 (Ser10 and Ser28). In particular, 14-3-3ɛ is critical for recruiting MSK1 and Cdk9 to the chromatin and subsequently phosphorylating the C-terminal domain of RNA polymerase II in the cox-2 promoter. We propose that histone H3 phosphorylation at Ser10 and Ser28 serve as critical switches to promote cox-2 gene expression by facilitating the recruitment of MSK1 and Cdk9 to the cox-2 promoter, thereby promoting RNA polymerase II phosphorylation.

The c-Jun transcription factor is a highly unstable oncoprotein. Several ubiquitin ligases mediate c-Jun degradation. However, c-Jun can be stabilized once it is phosphorylated at the N-terminus by c-Jun N-terminal kinases (JNKs) or other protein kinases. This phosphorylation decreases c-Jun ubiquitination and degradation. The underlying mechanism for this phenomenon is still unknown. Here, we show that receptor for activated C-kinase 1 (Rack1) can bind with c-Jun and ubiquitin ligase Fbw7 to form a complex. When c-Jun is phosphorylated at the N-terminus, c-Jun is released from the complex and cannot be ubiquitinated by Fbw7, which leads to increased stabilization and accumulation of c-Jun. These results reveal that Rack1 has a very important role in tumorigenesis by maintaining the stability of c-Jun that has been phosphorylated at its N-terminus by JNKs or other kinases.

Abstract Background: Lapatinib, an oral, small-molecule, reversible inhibitor of both EGFR and HER2, is highly active in HER2 positive breast cancer as a single agent and in combination with other therapeutics. However, resistance against lapatinib is an unresolved problem in clinical oncology. Recently, interest in the use of natural compounds to prevent or treat cancers has gained increasing interest because of presumed low toxicity. Quercetin-3-methyl ether, a naturally occurring compound present in various plants, has potent anticancer activity. Material and Methods: Quercetin-3-methyl ether was obtained from Analyticon Discovery (Potsdam, Germany). Lapatinib-resistant SK-Br-3 (SK-Br-3-Lap R) cells were isolated in the laboratory of Cell Biology and Biotherapy at the Istituto Nazionale dei Tumori, Naples, Italy. Parental SK-Br-3 and SK-Br-3-Lap R cells were cultured in monolayers at 37 °C in a 5% CO2 incubator in 10% FBS/McCoy supplemented with penicillin/streptomycin (100 units/ml; Invitrogen). SK-Br-3 Lap R cells were routinely maintained in 1 μM lapatinib. Anchorage-dependent and -independent growth and cell cycle were assessed in the presence and absence of quercetin-3-methyl ether. Results: Here, we found that quercetin-3-methyl ether caused in a significant growth inhibition of lapatinib-sensitive and -resistant breast cancer cells. Western blot data showed that quercetin-3-methyl ether had no effect on Akt or MAPKs signaling in resistant cells. However, quercetin-3-methyl ether caused a pronounced G2/M block mainly through the Chk1-Cdc25c-cyclin B1/Cdk1 pathway in lapatinib-sensitive and -resistant cells. In contrast, lapatinib produced an accumulation of cells in the G1 phase mediated through cyclin D1, but only in lapatinib-sensitive cells. Moreover, quercetin-3-methyl ether induced significant apoptosis, accompanied with an increase the levels of in cleaved caspase 3, caspase 7 and poly(ADP-ribose) polymerase (PARP) in both cell lines. Conclusion: Overall, these results suggested that quercetin-3-methyl ether might be a novel and promising therapeutic agent in lapatinib-sensitive or -resistant breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-11.

Abstract Phase II breast cancer prevention trials examine agent activity by sampling and imaging the breast through core needle biopsy, fine needle aspiration, or imaging (e.g., mammography). The biomarker endpoints in normal or at risk breast tissue employed with biopsies or fine needle aspirates (e.g., Ki67 or apoptosis) have often not been formally validated relative to a tumor endpoint. In this study, we have attempted to examine these endpoints in a preclinical model for which we have final tumor data. In the methylnitrosourea (MNU)-induced rat mammary cancer model, estrogen receptor positive cancers develop that are histologically similar to a large majority of human tumors. Here we present a protocol for correlating surrogate endpoint biomarkers and agent efficacy in the same animal. Biopsy samples are taken of the normal mammary gland after MNU treatment, and a week later the animals are started on the preventive agent. After 2 weeks, a second biopsy is taken and the animals are followed for tumor development. To test the protocol, we examined surrogate endpoints of tamoxifen efficacy at human equivalent dosages (5mg/d and 20mg/d). Both doses of tamoxifen prevented the development of tumors in the MNU-treated rats. The pre/post biopsy analysis revealed a statistically significant reduction in Ki-67 consistent with the clinical trial data. Additional biomarkers of apoptosis and the chromosome condensation pathway will be presented. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-11-07.

The EGFR inhibitors are effective in treatment of lung and pancreatic cancers (erlotinib, gefitinib) and Neu overexpressing breast cancer (lapatinib) in humans; as well as preventing multiple cancers in animal models. However, the development of toxicities (Iressa, skin rashes; Lapatinib, diarrhea) limit their potential use in prevention; and perhaps even in an adjuvant setting. We examined whether alternative dosing regimens which might reduce toxicity would still achieve preventive and therapeutic efficacy. Female Sprague-Dawley rats were administered a single IV dose of methylnitrosourea (MNU) at 50 days of age. In a prevention study, MNU treated rats administered Gefitinib daily (10 mg/kg BW/day, 7x/week) or Gefitinib (70 mg/kg BW, 1x/week) beginning 5 days after MNU resulted in 94 and 75% reductions, respectively, in cancer multiplicity. Simultaneous measurements of tumor load (number of tumors x tumor weight) showed that both regimens resulted in greater than a 90% decrease. In the therapeutic study (initiating treatment when animals developed a small palpable cancer), both regimens were again highly effective. A prevention study was also performed with Lapatinib (75 mg/kg BW/day, 7x/week or 525 mg/kg BW, 1x/week). While the daily dose reduced cancer multiplicity 90%, the weekly dose caused a 70% reduction. Finally, we examined the effects of daily or weekly dosing with Iressa (100 mg/kg BW/day, 5x/week or 500 or 250 mg/kg BW, 1x/week) in an ER− mouse model (using MMTV-Neu; p53+/− mice). This study showed that while daily dosing with Iressa decreased tumor multiplicity roughly 80%, weekly dosing at either dose caused roughly a 50% decrease. These data show that even a significant alteration in the dosing of Gefitinib (EGFR 1) or Lapatinib (EGFR 2/1) still resulted in a large reduction in mammary cancers. The important clinical question will be whether this altered dosing will diminish the toxicities associated with these agents. We cannot determine this in animal models since, at the effective doses employed, the typical toxicities observed in humans are not observed. Supported by NCI contract number HHSN261200433001C. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-10-01.

Abstract Background: Activation of tyrosine kinase receptors, including EGFR, HER2, HER3 and HER4, plays a key role in the prognosis of mammary cancer. EGFR is a validated therapeutic target; but unfortunately, only a small percentage of patients with EGFR-overexpressing tumors respond to therapy, and resistance develops even in responsive patients. Iressa is a small molecule tyrosine kinase inhibitor that suppresses the activation of EGFR. Completely understanding the molecular mechanisms and protein targets involved in the effects of Iressa can help determine why efficacy varies. This requires the simultaneous identification of specific molecular markers in the complex network of signaling pathways that are secondarily modulated by Iressa in mammary cancer. Methods and Materials: In this study, female Sprague-Dawley rats (50 days old) were given methylnitrosourea (MNU) by IV injection through the jugular vein (75 mg/kg BW). The rats were palpated for mammary cancers 2 times per week. When a palpable mammary cancer of approximately 200-250 mm2 was present, the rat was administered Iressa (6 mg/kg BW/day) by gavage for 2 days. The tumor was then biopsied. The rats were treated with Iressa at the same dose for an additional 40 days and tumors were harvested. At sacrifice, the mammary cancer was rapidly removed and frozen for protein array analysis. The tumors were sorted into two groups based on sensitivity to treatment with Iressa and analyzed by a phospho-protein Proteome Profiler™ Array (R&D, Minneapolis, MN). Three tumors that regressed 27, 39, and 59% were compared to 4 tumors that continued to grow, increasing in size by 56, 106, 210, and 236%. Results and Discussion: Most notably, tumors that continued to grow in spite of Iressa treatment consistently showed a marked increased phosphorylation of CREB (Ser133) and Src (Tyr419) compared to tumors that regressed. CREB phosphorylation at Ser133 enhances its transactivation and transcriptional activities, resulting in increased expression of many downstream target genes involved in cell proliferation and cancer development. In addition, tumors that continued to grow also showed increased phosphorylation of p27 (Thr157) compared to regressed tumors. Phosphorylation of p27 at Thr157 is known to impair its nuclear localization, suggesting an acceleration of the G1/S cell cycle transition, resulting in enhanced cell proliferation. In contrast, tumors that regressed showed marked increased phosphorylation of Hck (Tyr411) compared to tumors that continued to grow. The hck gene is located at 20q11-q12, which is a region affect by interstitial deletions in some acute myeloid leukemias and myeloproliferative disorders and damage to hck might contribute to the pathogenesis of these conditions, suggesting that autophosphorylation of Hck (Tyr411) might be involved in tumor suppression. Conclusions: These results indicated that Iressa resistance in mammary cancer induced by MNU is closely related with activation of the signaling axis of the cytosolic tyrosine kinase, CREB, and that increased p27 phosphorylation could contribute to continuous proliferation. Supported by NCI Contract Number HHSN-261200433009C - NO1-CN-55006-72. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-07-03.