Katja Linher-Melville

Sestrin2 Modulates AMPK Subunit Expression and Its Response to Ionizing Radiation in Breast Cancer Cells

Background The sestrin family of stress-responsive genes (SESN1-3) are suggested to be involved in regulation of metabolism and aging through modulation of the AMPK-mTOR pathway. AMP-activated protein kinase (AMPK) is an effector of the tumour suppressor LKB1, which regulates energy homeostasis, cell polarity, and the cell cycle. SESN1/2 can interact directly with AMPK in response to stress to maintain genomic integrity and suppress tumorigenesis. Ionizing radiation (IR), a widely used cancer therapy, is known to increase sestrin expression, and acutely activate AMPK. However, the regulation of AMPK expression by sestrins in response to IR has not been studied in depth. Methods and Findings Through immunoprecipitation we observed that SESN2 directly interacted with the AMPKα1β1γ1 trimer and its upstream regulator LKB1 in MCF7 breast cancer cells. SESN2 overexpression was achieved using a Flag-tagged SESN2 expression vector or a stably-integrated tetracycline-inducible system, which also increased AMPKα1 and AMPKβ1 subunit phosphorylation, and co-localized with phosphorylated AMPKα-Thr127 in the cytoplasm. Furthermore, enhanced SESN2 expression increased protein levels of LKB1 and AMPKα1β1γ1, as well as mRNA levels of LKB1, AMPKα1, and AMPKβ1. Treatment of MCF7 cells with IR elevated AMPK expression and activity, but this effect was attenuated in the presence of SESN2 siRNA. In addition, elevated SESN2 inhibited IR-induced mTOR signalling and sensitized MCF7 cells to IR through an AMPK-dependent mechanism. Conclusions Our results suggest that in breast cancer cells SESN2 is associated with AMPK, it is involved in regulation of basal and IR-induced expression and activation of this enzyme, and it mediates sensitization of cancer cells to IR.

Expression of xCT and activity of system xc(-) are regulated by NRF2 in human breast cancer cells in response to oxidative stress.

Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc−, an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc−, is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc−. We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.

Ionizing radiation regulates the expression of AMP-activated protein kinase (AMPK) in epithelial cancer cells: Modulation of cellular signals regulating cell cycle and survival

PURPOSE To analyze the (i) expression of AMPK in a variety of epithelial cancer cells, (ii) regulation of AMPK subunit expression by ionizing radiation (IR) and (iii) impact of AMPK on signaling pathways regulating cell cycle and survival. METHODS AND MATERIALS Human lung, prostate, and breast normal and cancer cells were treated with 0 or 8 Gy IR and mRNA and protein levels of AMPK were evaluated by RT-PCR and immunoblotting 24 or 48 h later. Untreated and radiated wild type (WT) and AMPKα(-/-) mouse embryonic fibroblasts (MEFs) were analyzed by immunoblotting using total- and phosphorylation-specific antibodies. Histone H2Ax was examined by fluorescence microscopy. The cell cycle and survival of WT and AMPKα(-/-) MEFs was also evaluated following 8 Gy by IR. RESULTS AMPK subunits were found widely expressed in normal and cancer epithelial cells. IR increased subunit protein levels and stimulated gene transcription in cancer cells. AMPKα(-/-)-MEFs showed enhanced basal total levels of ATM and phosphorylation of its substrates histone H2Ax, but inhibited response of these markers and of checkpoint kinase Chk2 phosphorylation to IR. AMPKα(-/-)-MEFs showed increased basal levels of p53 and cyclin-dependent kinase inhibitors p21(cip1), but lack of response of both genes to IR. These cells had increased basal levels and activation of the Akt-mTOR-p70(S6K)/4-EBP1 signalling pathway. IR increased Akt, p70(S6K) and 4-EBP1 phosphorylation in WT-MEFs, but this was reduced in AMPKα(-/-)-MEFs. AMPKα(-/-)-MEFs failed to arrest at the G2-M checkpoint after IR and showed a trend for radio-resistance in proliferation assays. CONCLUSIONS AMPK is widely expressed in human normal and cancer epithelial cells and its gene transcription, protein levels, and enzymatic activity is stimulated by IR. Work with AMPKα knockout cells suggests that AMPK (i) may mediate a suppressive regulation on basal expression and activity of ATM and its downstream effector pathways Chk2/p53-p21(cip1) and Akt-mTOR, (ii) facilitates the normal response of these pathways to IR and, (iii) mediates the IR-induced G2-M checkpoint.

Liver kinase B1 expression (LKB1) is repressed by estrogen receptor alpha (ERα) in MCF-7 human breast cancer cells.

BACKGROUND Liver kinase 1 (LKB1) is emerging as a multifunctional protein, acting as a key metabolic enzyme, regulator of cell polarity, and transcription factor. Altered LKB1 expression has been linked with various cancers and may be a potential prognostic marker. While the functional role of LKB1 continues to undergo intensive investigation, the molecular mechanisms that regulate its expression remain to be defined more clearly. Recent reports have established a possible link between estrogen receptor alpha (ERα) signaling and LKB1 in MCF-7 human breast cancer cells. The current study aimed to investigate whether LKB1 is transcriptionally regulated by ERα in MCF-7 cells. METHODS siRNA transfections were used to transiently knock down LKB1 and ERα. LKB1 and ERα mRNA and protein levels were evaluated by real-time PCR and Western blotting, respectively. An approximately 3 kilobase pair human LKB1 promoter construct and various truncations were generated, transfected into MCF-7 cells, and luciferase reporter assays were performed. Cells were also treated with various doses of 17-β-estradiol (E2) to evaluate the effect on LKB1 and ERα mRNA levels. RESULTS LKB1 mRNA and protein levels were significantly lower in ERα-positive MCF-7 compared to ERα-negative MDA-MB-231 breast cancer cells, suggesting that ERα may act as a repressor. siRNA-mediated knock-down of ERα in MCF-7 cells significantly increased LKB1 promoter activity and expression at the mRNA and protein levels, and computational analysis revealed the presence of several putative estrogen response element (ERE) DNA binding sites in the LKB1 promoter region. In addition, treatment with E2 led to an increase in LKB1 expression, concomitant with decreased expression of ERα in MCF-7 cells. The E2-mediated increase was abrogated by pretreatment with actinomycin D, supporting that the observed changes in LKB1 levels were transcriptionally regulated. CONCLUSIONS ERα repressively modulates the expression of LKB1 at the transcriptional level. Targeting the expression of LKB1 by modulating ERα signaling may provide a potential approach to further evaluate its function in ERα-positive breast cancers.

Signal transducer and activator of transcription 3 and 5 regulate system Xc- and redox balance in human breast cancer cells

System Xc- is a cystine/glutamate antiporter that contributes to the maintenance of cellular redox balance. The human xCT (SLC7A11) gene encodes the functional subunit of system Xc-. Transcription factors regulating antioxidant defense mechanisms including system Xc- are of therapeutic interest, especially given that aggressive breast cancer cells exhibit increased system Xc- function. This investigation provides evidence that xCT expression is regulated by STAT3 and/or STAT5A, functionally affecting the antiporter in human breast cancer cells. Computationally analyzing two kilobase pairs of the xCT promoter/5′ flanking region identified a distal gamma-activated site (GAS) motif, with truncations significantly increasing luciferase reporter activity. Similar transcriptional increases were obtained after treating cells transiently transfected with the full-length xCT promoter construct with STAT3/5 pharmacological inhibitors. Knock-down of STAT3 or STAT5A with siRNAs produced similar results. However, GAS site mutation significantly reduced xCT transcriptional activity, suggesting that STATs may interact with other transcription factors at more proximal promoter sites. STAT3 and STAT5A were bound to the xCT promoter in MDA-MB-231 cells, and binding was disrupted by pre-treatment with STAT inhibitors. Pharmacologically suppressing STAT3/5 activation significantly increased xCT mRNA and protein levels, as well as cystine uptake, glutamate release, and total levels of intracellular glutathione. Our data suggest that STAT proteins negatively regulate basal xCT expression. Blocking STAT3/5-mediated signaling induces an adaptive, compensatory mechanism to protect breast cancer cells from stress, including reactive oxygen species, by up-regulating xCT expression and the function of system Xc-. We propose that targeting system Xc- together with STAT3/5 inhibitors may heighten therapeutic anti-cancer effects.

The complex roles of STAT3 and STAT5 in maintaining redox balance: Lessons from STAT-mediated xCT expression in cancer cells

STAT3 and STAT5 mediate diverse cellular processes, transcriptionally regulating gene expression and interacting with cytoplasmic proteins. Their canonical activity is stimulated by cytokines/growth factors through JAK-STAT signaling. As targets of oncogenes with intrinsic tyrosine kinase activity, STAT3 and STAT5 become constitutively active in hematologic neoplasms and solid tumors, promoting cell proliferation and survival and modulating redox homeostasis. This review summarizes reactive oxygen species (ROS)-regulated STAT activation and how STATs influence ROS production. ROS-induced effects on post-translational modifications are presented, and STAT3/5-mediated regulation of xCT, a redox-sensitive target up-regulated in numerous cancers, is discussed with regard to transcriptional cross-talk.

Chronic Inhibition of STAT3/STAT5 in Treatment-Resistant Human Breast Cancer Cell Subtypes: Convergence on the ROS/SUMO Pathway and Its Effects on xCT Expression and System xc- Activity

Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This “ying-yang” effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient’s breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers.

Functional effects of TrkA inhibition on system xC−-mediated glutamate release and cancer-induced bone pain:

Breast cancer cells release the signalling molecule glutamate via the system xC− antiporter, which is upregulated to exchange extracellular cystine for intracellular glutamate to protect against oxidative stress. Here, we demonstrate that this antiporter is functionally influenced by the actions of the neurotrophin nerve growth factor on its cognate receptor tyrosine kinase, TrkA, and that inhibiting this complex may reduce cancer-induced bone pain via its downstream actions on xCT, the functional subunit of system xC−. We have characterized the effects of the selective TrkA inhibitor AG879 on system xC− activity in murine 4T1 and human MDA-MB-231 mammary carcinoma cells, as well as its effects on nociception in our validated immunocompetent mouse model of cancer-induced bone pain, in which BALB/c mice are intrafemorally inoculated with 4T1 murine carcinoma cells. AG879 decreased functional system xC− activity, as measured by cystine uptake and glutamate release, and inhibited nociceptive and physiologically relevant responses in tumour-bearing animals. Cumulatively, these data suggest that the activation of TrkA by nerve growth factor may have functional implications on system xC−-mediated cancer pain. System xC−-mediated TrkA activation therefore presents a promising target for therapeutic intervention in cancer pain treatment.

TUMOUR-DERIVED GLUTAMATE: LINKING ABERRANT CANCER CELL METABOLISM TO PERIPHERAL SENSORY PAIN PATHWAYS.

Background Chronic pain is a major symptom that develops in cancer patients, most commonly emerging during advanced stages of the disease. The nature of cancer-induced pain is complex, and the efficacy of current therapeutic interventions is restricted by the dose-limiting side-effects that accompany common centrally targeted analgesics. Methods This review focuses on how up-regulated glutamate production and export by the tumour converge at peripheral afferent nerve terminals to transmit nociceptive signals through the transient receptor cation channel, TRPV1, thereby initiating central sensitization in response to peripheral disease-mediated stimuli. Results Cancer cells undergo numerous metabolic changes that include increased glutamine catabolism and over-expression of enzymes involved in glutaminolysis, including glutaminase. This mitochondrial enzyme mediates glutaminolysis, producing large pools of intracellular glutamate. Up-regulation of the plasma membrane cystine/glutamate antiporter, system xc-, promotes aberrant glutamate release from cancer cells. Increased levels of extracellular glutamate have been associated with the progression of cancer-induced pain and we discuss how this can be mediated by activation of TRPV1. Conclusion With a growing population of patients receiving inadequate treatment for intractable pain, new targets need to be considered to better address this largely unmet clinical need for improving their quality of life. A better understanding of the mechanisms that underlie the unique qualities of cancer pain will help to identify novel targets that are able to limit the initiation of pain from a peripheral source–the tumour.

The Disrupted Steady-State: Tipping the Balance in Favour of Cancer

Genetic changes, such as the activation of oncogenes or the repression of tumour suppressors, contribute to the development of cancer, imparting malignant cells with the potential for self-promoting growth and survival in the presence of anti-growth or pro-apoptotic signals. However, while these changes may initiate the process of cancer development, they are not necessarily sufficient for disease progression, given the body’s intrinsic ability to regain homeostasis. Cancer initiation, promotion, and eventual progression depend on disruptions in normal homeostasis, as well as subsidiary processes imparted by cells of the tumour microenvironment. Recurring players that have been linked with disrupted homeostasis include inflammation and oxidative stress, which have both been strongly associated with the development of cancer. This chapter discusses the intricate relationship between the body and cancer, and how disruptions in normal physiological processes impact the maintenance of homeostasis and tissue repair, providing a framework for understanding the connection between dysregulated homeostatis and a complex disease such as cancer.