Matthew R. Young

Activator protein 1 (AP-1)– and nuclear factor κB (NF-κB)–dependent transcriptional events in carcinogenesis

Abstract Generation of reactive oxygen species (ROS) during metabolic conversion of molecular oxygen imposes a constant threat to aerobic organisms. Other than the cytotoxic effects, many ROS and oxidants are also potent tumor promoters linking oxidative stress to carcinogenesis. Clonal variants of mouse epidermal JB6 cells originally identified for their differential susceptibility to tumor promoters also show differential reduction-oxidation (redox) responses providing a unique model to study oxidative events in tumor promotion. AP-1 and NF-κB, inducible by tumor promoters or oxidative stimuli, show differential protein levels or activation in response to tumor promoters in JB6 cells. We further demonstrated that AP-1 and NF-κB are both required for maintaining the transformed phenotypes where inhibition of either activity suppresses transformation response in JB6 cells as well as human keratinocytes and transgenic mouse. NF-κB proteins or extracellular signal–regulated kinase (ERK) but not AP-1 proteins are shown to be sufficient for conversion from transformation-resistant to transformation-susceptible phenotype. Insofar as oxidative events regulate AP-1 and NF-κB transactivation, these oxidative events can be important molecular targets for cancer prevention.

Promising molecular targets for cancer prevention: AP-1, NF-κB and Pdcd4

Abstract There are still many unanswered questions regarding the processes by which extracellular signals are transduced from plasma-membrane receptors to the transcription machinery in the nucleus and the translation machinery in the cytoplasm. Some of these gene expression events become misregulated as a result of environmental or endogenous exposure to agents that cause multistage carcinogenesis. We are now beginning to identify and validate the crucial molecular events that drive the rate-limiting steps of carcinogenesis and to target these events for cancer prevention. Transcription factors AP-1 and nuclear factor κB can be specifically targeted to prevent cancer induction in mouse models. A protein known as programmed-cell-death-4 is a new potential molecular target that has a surprising mode of action.

Expression of dominant negative Erk2 inhibits AP-1 transactivation and neoplastic transformation

The mitogen activated protein (MAP) kinases or extracellular signal-regulated kinases (Erks) are activated in response to Ras expression or exposure to tumor promoters or to growth factors, and have been implicated in AP-1 transactivation in some models. We have shown that tumor promoter induced activation of the transcription factor AP-1 is required for induced neoplastic transformation in the Balb/C JB6 cell model. Jun and Fos family protein levels have been found not to be limiting for AP-1 response. The present study asks whether activation of Erks1 and 2 is required for AP-1 transactivation and transformation of JB6 cells and whether Erks might be targeted for cancer prevention. Expression of either of two different dominant negative kinase inactive Erk2 mutants in transformation sensitive (P+) JB6 cells substantially inhibited the tumor promoter induced activation of Erks1 and 2 and of AP-1 measured by a collagenase-luciferase reporter. Multiple mutant Erk2 expressing clonal lines were also rendered non-responsive to induced neoplastic transformation. These observations, together with our recent finding attributing AP-1 non-responsiveness to Erk deficiency in a clonal line of transformation resistant (P−) cells, argue for a requirement for Erks1 and/or 2 activation in AP-1 transactivation in the mouse JB6 neoplastic progression model, and suggest the utility of Erks as a prevention target.

Transactivation of Fra-1 and consequent activation of AP-1 occur extracellular signal-regulated kinase dependently

ABSTRACT Mitogen-activated protein (MAP) kinase, extracellular-signal-regulated kinases (ERKs) play an important role in activating AP-1-dependent transcription. Studies using the JB6 mouse epidermal model and a transgenic mouse model have established a requirement for AP-1-dependent transcription in tumor promotion. Tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor induce activator protein 1 (AP-1) activity and neoplastic transformation in JB6 transformation-sensitive (P+) cells, but not in transformation-resistant (P−) variants. The resistance in one of the P− variants can be attributed to the low levels of the MAP kinases, ERKs 1 and 2, and consequent nonresponsiveness to AP-1 activation. The resistant variant is not deficient in c-fos transcription. The purpose of these studies was to define the targets of activated ERK that lead to AP-1 transactivation. The results establish that the transactivation domain of Fra-1 can be activated, that activation of Fra-1 is ERK dependent, and that a putative ERK phosphorylation site must be intact for activation to occur. Fra-1 was activated by TPA in ERK-sufficient P+ cells but not in ERK-deficient P− cells. A similar activation pattern was seen for c-Fos but not for Fra-2. Gel shift analysis identified Fra-1 as distinguishing mitogen-activated (P+) from nonactivated (P−) AP-1 complexes. A second AP-1-nonresponsive P− variant that underexpresses Fra-1 gained AP-1 response upon introduction of a Fra-1 expression construct. These observations suggest that ERK-dependent activation of Fra-1 is required for AP-1 transactivation in JB6 cells.

AP-1 a Target for Cancer Prevention

The transcription factor activator protein 1 (AP-1) plays a pivotal role in tumorigenesis. AP-1 activity is increased in multiple human tumor types. Inhibitors of AP-1 have been shown to block tumor promotion, transformation, progression and invasion. Chronic inflammation and tumor development are linked. AP-1 may act, in part, by perpetuating the inflammatory signal. AP-1 is a recognized molecular target of many antioxidant and anti-inflammatory chemopreventive compounds. This review focuses on the AP-1 family proteins as targets for chemoprevention.

Bioactive Food Components, Inflammatory Targets, and Cancer Prevention

Various dietary components may modify chronic inflammatory processes at the stage of cytokine production, amplification of nuclear factor-κB–mediated inflammatory gene expression, and the release of anti-inflammatory cytokine, transforming growth factor-β. This review provides a synopsis of the strengths and weaknesses of the evidence that specific bioactive food components influence inflammation-related targets linked to cancer. A target repeatedly surfacing as a site of action for several dietary components is transforming growth factor β. Whereas the use of dietary intervention strategies offers intriguing possibilities for maintaining normal cell function by modifying a process that is essential for cancer development and progression, more information is needed to characterize the minimum quantity of the bioactive food components required to bring about a change in inflammation-mediated cancer, the ideal time for intervention, and the importance of genetics in determining the response. Unquestionably, the societal benefits of using foods and their components to prevent chronic inflammation and associated complications, including cancer, are enormous.

Fluorescence endoscopic detection of murine colitis-associated colon cancer by topically applied enzymatically rapid-activatable probe

Objectives Screening colonoscopy to monitor for early colitis-associated colon cancer (CAC) is difficult due to the aberrant mucosal patterns associated with long-standing colitis. The aim of this study was to develop a rapid fluorescent detection method for use during colonoscopy for improving the detection of CAC utilising a topically applied enzymatically activatable probe (gGlu-HMRG) which fluoresces in the presence of γ-glutamyltranspeptidase (GGT), an enzyme associated with cancer. Methods Expression of GGT in colon cell lines was examined with fluorescence microscopy and flow cytometry. A mouse model (azoxymethane/dextran sulphate sodium) of CAC was used and mice were examined with white light and fluorescence colonoscopy before and after topical gGlu-HMRG administration. Results Expression of GGT, although variable, was higher in human colon cancer cells than normal human colon cells. Using fluorescence colonoscopy in mice, gGlu-HMRG fluorescent lesions were detected 5 min after topical administration and fluorescence persisted for at least 30 min. Fluorescence guided biopsy revealed all fluorescent lesions that contained cancer or dysplasia (n=16), whereas three out of 12 non-fluorescent lesions contained low grade dysplasia and others did not contain neoplastic histology. Microscopic inflammatory infiltration also had variable fluorescence but in general was much lower (∼10-fold) in signal than cancer. Repeat fluorescence endoscopy allowed individual tumours to be monitored. Conclusion These results suggest that gGlu-HMRG can improve endoscopic detection of CAC with a higher target to background ratio than conventional white light colonoscopy. This could be of benefit to patients with long-standing colitis who must undergo repeated screening colonoscopies.

Sulfiredoxin–Peroxiredoxin IV axis promotes human lung cancer progression through modulation of specific phosphokinase signaling

Oxidative stress is known to cause tumorigenesis through induction of DNA and lipid damage. It also promotes cancer progression through a largely unknown mechanism. Sulfiredoxin (Srx) is a novel oxidative stress-induced antioxidant protein whose function in tumorigenesis and cancer progression has not been well studied. We report that Srx is highly expressed in human lung cancer. Knockdown of Srx reduces anchorage-independent colony formation, cell migration, and invasion of human lung cancer cells. Srx preferentially interacts with Peroxiredoxin (Prx) IV relative to other Prxs due to its intrinsic higher binding affinity. Knockdown of Prx IV recapitulates the phenotypic changes of depleting Srx. Disruption or enhancement of the Srx–Prx IV axis leads respectively to reduction or acceleration of tumor growth and metastasis formation in vivo. Through identification and validation of the downstream mediators we unraveled the Srx-mediated signaling network that traverses AP-1–activating and other phosphokinase signaling cascades. Our work reveals that the Srx–Prx IV axis is critical for lung cancer maintenance and metastasis, suggesting that targeting the Srx–Prx IV axis may provide unique effective strategies for cancer prevention and treatment.

Epigenetic Regulation of SOX9 by the NF‐κB Signaling Pathway in Pancreatic Cancer Stem Cells

Pancreatic cancer is the fourth leading cause of cancer‐related mortality in the world. Pancreatic cancer can be localized, locally advanced, or metastatic. The median 1‐ and 5‐year survival rates are 25% and 6%, respectively. Epigenetic modifications such as DNA methylation play a significant role during both normal human development and cancer progression. To investigate epigenetic regulation of genes in the tumor‐initiating population of pancreatic cancer cells, which are also termed cancer stem cells (CSCs), we conducted epigenetic arrays in PANC1 and HPAC pancreatic cancer cell lines and compared the global DNA methylation status of CpG promoters in invasive cells, demonstrated to be CSCs, to their noninvasive counterparts, or non‐CSCs. Our results suggested that the NF‐κB pathway is one of the most activated pathways in pancreatic CSCs. In agreement with this, we determined that upon treatment with NF‐κB pathway inhibitors, the stem cell‐like properties of cells are significantly disrupted. Moreover, SOX9, demethylated in CSCs, is shown to play a crucial role in the invasion process. Additionally, we found a potential NF‐κB binding site located in the SOX9 promoter and determined that the NF‐κB subunit p65 positively regulates SOX9 expression by binding to its promoter directly. This interaction can be efficiently blocked by NF‐κB inhibitors. Thus, our work establishes a link between the classic NF‐κB signaling transduction pathway and the invasiveness of pancreatic CSCs, which may result in the identification of novel signals and molecules that function at an epigenetic level, and could potentially be targeted for pharmaceutical investigations and clinical trials. STEM Cells 2013;31:1454–1466

Chemopreventive activity of plant flavonoid isorhamnetin in colorectal cancer is mediated by oncogenic Src and β-catenin

Analysis of the Polyp Prevention Trial showed an association between an isorhamnetin-rich diet and a reduced risk of advanced adenoma recurrence; however, the mechanism behind the chemoprotective effects of isorhamnetin remains unclear. Here, we show that isorhamnetin prevents colorectal tumorigenesis of FVB/N mice treated with the chemical carcinogen azoxymethane and subsequently exposed to colonic irritant dextran sodium sulfate (DSS). Dietary isorhamnetin decreased mortality, tumor number, and tumor burden by 62%, 35%, and 59%, respectively. MRI, histopathology, and immunohistochemical analysis revealed that dietary isorhamnetin resolved the DSS-induced inflammatory response faster than the control diet. Isorhamnetin inhibited AOM/DSS-induced oncogenic c-Src activation and β-catenin nuclear translocation, while promoting the expression of C-terminal Src kinase (CSK), a negative regulator of Src family of tyrosine kinases. Similarly, in HT-29 colon cancer cells, isorhamnetin inhibited oncogenic Src activity and β-catenin nuclear translocation by inducing expression of csk, as verified by RNA interference knockdown of csk. Our observations suggest the chemoprotective effects of isorhamnetin in colon cancer are linked to its anti-inflammatory activities and its inhibition of oncogenic Src activity and consequential loss of nuclear β-catenin, activities that are dependent on CSK expression.