Regulation of ChREBP Transcription in Response to Glucose

14 (ChoRE) and their associated transcription factors MondoA and Max-like protein X (MLX). Thioredoxin interacting protein (TXNIP), the product of an immediate glucose response gene TXNIP, functions as a negative regulator for glucose uptake, and its expression is dysregulated in diabetes and cancer. We have observed that the ChoRE cis regulatory element is duplicated during vertebrate evolution, with one ChoRE in fish and two in mammals. In mammalian cells, both ChoREs are required for an optimal glucose response. With assistance by nuclear factor Y (NF-Y), MondoA/MLX complex is recruited to TXNIP promoter upon glucose stimulation, which in turn recruits general transcription factors and RNA polymerases to initiate gene transcription. In addition to glucose or its derived metabolites, MondoA/MLX activity and TXNIP expression is tightly correlated with status of mitochondrial oxidative phosphorylation (OXPHOS), and inhibition of OXPHOS by drugs such as metformin can dramatically repress TXNIP transcription by inducing glycolytic flux. Moreover, we have discovered that the expression of TXNIP is induced by an array of adenosine-containing molecules, and these molecules function as amplifiers of glucose signaling. Thus, MondoA/MLX complex serves as a hub integrating diverse upstream signals and a master regulator of glucose homeostasis. http://dx.doi.org/10.1016/j.gendis.2016.10.011

REGULATION OF SNAIL IN EPITHELIAL-MESENCHYMAL TRANSITION AND BREAST CANCER PROGRESSION AND METASTASIS Binhua P. Zhou Madeline F. James Gardner Endowed Chair Co-leader, Genomic, Epigenetic and Metabolism Program Markey Cancer Center and Department of Molecular and Cellular Biochemistry University of Kentucky School of Medicine, USA Triple-negative Breast Cancer (TNBC) is associated with an aggressive clinical history, development of recurrence, distant metastasis and shorter patient survival. Intriguingly, TNBC has activated the epithelial-mesenchymal transition (EMT) program, which is a cellular de-differentiation process that provides cells with the increased plasticity and stem cell-like properties required during embryonic development, tissue remodeling, wound healing and metastasis. Using unbiased protein purification coupled with mass spectrometry analysis, we identified that Snail and Twist, two key EMT-inducing transcriptional factors, act as transcriptional repressor and activator, respectively. Snail is a labile protein and is subjected to protein ubiquitination and degradation, and we have identified the protein kinase, phosphatase, ubiquitin E3 ligase and de-ubiquitinase in the regulation of Snail. Interestingly, the protein stability of Snail and its interaction with these effectors are controlled by signals from the tumor microenvironment, resulting in the EMT induction and invasive phenotypes that commonly observed at the tumor-stromal boundary. Our study provides new insights and opportunities for the development of effective therapeutic approaches against metastatic breast cancer. http://dx.doi.org/10.1016/j.gendis.2016.10.012

WHY ARE RED HAIRED INDIVIDUALS SO PRONE TO DEVELOPING MELANOMA? Rutao Cui Department of Pharmacology and Experimental Therapeutics, Boston University Cancer Center, Boston University School of Medicine, Boston, MA 02118, USA Melanoma is a highly aggressive cancer with an alarmingly increasing incidence. A major question in melanoma biology is why are red-haired individuals at a high risk of developing melanoma. Polymorphisms in the melanocortin-1-receptor (MC1R) gene, encoding a trimeric G proteincoupled receptor activated by a-melanocyte-stimulating hormone (aMSH), are frequently associated with red or blonde hair, fair skin, freckling and skin sensitivity to ultraviolet (UV) light, and several (RHCpolymorphisms) also associate with increased melanoma risk. However, some polymorphisms appear to affect melanoma risk independent of phenotype; using an in vivo model system we recently reported that some MC1R mutations synergize with UV to induce melanoma independently of their effects on melanogenesis. Understanding precisely how MC1R polymorphisms differentially affect melanoma biology is therefore a key issue. Importantly, we also found that UV irradiation triggered MC1Rinteraction with and degradation of PTEN, leading to increased PI3K-

Abstracts signallingedriven senescence in melanocytes, but senescence bypass in BRaf mutant melanoma. Importantly, WT MC1R but not red-hair associated MC1R mutants could interact with PTEN. Furthermore, we used newly generated MC1R conditional RHC-polymorphism mouse models to dissect the tumor suppressive functions of MC1R in melanoma initiation in vivo and specifically its role in controlling PI3K signaling via PTEN degradation. Our studies identify intracellular molecular targets of MC1R in suppressing melanoma initiation that are directed towards identifying novel strategies for melanoma prevention and therapeutic intervention. http://dx.doi.org/10.1016/j.gendis.2016.10.013

CYSTEINE PROMOTION OF BREAST CANCER TUMORIGENESIS IS DEPENDENT OF THE SOLUTE CARRIER SLC3A1 Yuan Cao Shanghai Jiaotong University School of Medicine, Shanghai 200240, China Cysteine as well as glutamine and alanine are critical substrates for synthesis of glutathione (GSH). Glutamine and alanine, but not cysteine, are most upregulated intermediate metabolites in cancer cells, suggesting cysteine uptake is increased. However, it’s not clear how the uptake of cysteine is regulated. Here, we report that the cysteine carrier SLC3A1 is upregulated in breast cancer cells, and its expression levels are correlated with clinical stages and patients’ survival. In breast cancer cells, overexpression of SLC3A1 accelerates the uptake of cysteine, which in turn increases the concentration of reductive GSH and the GSH/GSSH ratio and concomitantly decreases the cellular level of ROS (reactive oxygen species). Consequently, overexpression of SLC3A1 promotes the tumorigenesis of breast cancer cells, whereas knocking-down or inhibition of SLC3A1 decreases tumorigenesis of breast cancer cells in mice. Moreover, SLC3A1 inhibitor Sulfasazine suppressed tumor growth and abolished dietary NAC-promoted tumor growth. Mechanistically, our data manifest that ROS catalyzes thiol into sulfenic acid at the HC(X)4EXV motif of serine/ threonine phosphatase PP2Ac which is similar as the ROS-induced sulfenylation at the HC(X)5RS/T motif of protein tyrosine phosphatases (PTPs); however, unlike PTPs, sulfenylation increases the stability and activity of PP2Ac rather than deactivating PTPs. SLC3A1 activates the Akt pathway through decreasing the sulfenylation and stability of protein phosphatase PP2Ac. Collectively, our data demonstrate that SLC3A1-mediated increase of cysteine uptake promotes the tumorigenesis of breast cancer cells, and SLC3A1 determines breast cancer cell response to antioxidant N-acetylcysteine, suggesting SLC3A1 is a potential therapeutic target for breast cancer. http://dx.doi.org/10.1016/j.gendis.2016.10.014

REGULATION OF CHREBP TRANSCRIPTION IN RESPONSE TO GLUCOSE Jian Meng, Ming Feng, Xuemei Tong Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China Transcription factor carbohydrate responsive element binding protein (ChREBP) is abundant in liver and adipose tissues. ChREBP promotes glycolysis and lipogenesis in metabolic tissues and cancer cells. ChREBP-a and ChREBPb are two isoforms of ChREBP transcribed from different promoters. Both ChREBP-a and ChREBP-b are transcriptionally induced by glucose. However, the mechanism by which glucose promotes ChREBP transcription remains unclear. Here we report that hepatocyte nuclear factor 4 alpha (HNF-4a) mediates transcription of ChREBP-a and ChREBP-b induced by glucose. Ectopic HNF-4a expression promoted ChREBP transcription while knockdown of HNF-4a reduced ChREBP mRNA levels in liver cancer cells and mouse primary hepatocytes. We found that the expression of HNF-4a and ChREBP was positively correlated by analyzing levels of ChREBP and HNF-4a in the liver of mice under fasting and feeding conditions. HNF-4a directly bound to an E-box-containing region in intron 12 of the ChREBP gene, in addition to directly binding to DR1 sites of the ChREBP-b promoter. Moreover, HNF-4a interacted with ChREBP-a and synergistically promoted ChREBP-b transcription. Interestingly, HNF-4a knockdown decreased glucose-dependent ChREBP induction in liver cancer cells. Glucose increased expression and nuclear abundance of HNF-4a and its binding to cis-elements of ChREBP gene, which contributed to glucose-induced ChREBP transcription in liver cancer cells.

Abstracts Taken together, our results revealed the novel mechanism by which HNF-4a increased ChREBP transcription in response to glucose in liver cancer cells. http://dx.doi.org/10.1016/j.gendis.2016.10.015

CANCER-ASSOCIATED FIBROBLASTS PROMOTES TUMOR RELAPSE AFTER RADIOTHERAPY Guifang Gan Shanghai Jiaotong University School of Medicine, Shanghai 200240, China Tumor relapse after radiotherapy is a big challenge to oncologists. Here, we discovered that cancer-associated fibroblasts (CAFs), a major component of cancer stromal cells, promote cancer cell recovery and tumor relapse postradiation. We provided evidences that CAFs-produced cytokines/chemokines and intermediate metabolites are capable of inducing autophagy in cancer cells post-radiation and promoting cancer cells recovery from radiationinduced damage in vitro and in mice, which was consistent with our clinical observation that the newly developed image-guided stereotactic body radiation therapy (SBRT) was less effective than the conventional external beam radiotherapy (EBRT) in term of recurrence and survival of cancer patients. Mechanistically, CAF-derived cytokines maintained the redox homeostasis post-radiation and increased ROS level compared to control irradiated cancer cells, ROS enhanced the activity of PP2Ac through a reversible sulfenylation on the cysteine residue in the motif of HC(X)4EXV, similar as the motif of HC(X)5RS/T in PTPs (protein tyrosine phosphatases), distinct from the sulfenylation of HC(X)5RS/T motif, which inactivates the activity of PTPs. Moreover, the inhibition of autophagy abolished the CAF-promoted tumor relapse post-radiotherapy. Taken together, our findings demonstrate that CAFs promote cancer cell recovery and tumor regrowth post-radiation, suggesting CAFs are critical for tumor recurrence after radiation and autophagy pathway is a therapeutic target for radiotherapy sensitization. http://dx.doi.org/10.1016/j.gendis.2016.10.016

THE ROLE OF AUTOPHAGIC DEGRADATION IN CANCER TREATMENT Xiao-Feng Zhu State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou 510060, China Autophagy is an evolutionarily conserved protein degradation pathway in eukaryotes that plays key role in a number of pathological conditions including cancer. Understanding the mechanism of autophagy will offer hope for the development of new therapeutic approaches for cancer treatment. To elucidate the role of autophagy in tumorigenesis or tumor progression, we explored the expression of autophagy-associated proteins in tumor tissues. Our results showed that low expression of Beclin 1 was significantly associated with poor prognosis in ovarian tumors, colon cancers with stage IIIB, lymphoma. We also found that the patients with lower level of ULK1 had a significant shorter cancer-related survival time and distant metastasis-free survival time. To understand whether anticancer drugs induces autophagy in cancer cells, topotecan and ceramide were employed. The results showed that activation of JNK pathway can phosphorylate c-Jun and promote c-Jun binding to the promoters of Beclin 1, LC3 and Sestrin 2 to upregulate Beclin 1, LC3 and Sestrin 2 expression, which plays a key role in anticancer agentsinduced autophagy in cancer cells. We also found that Beclinacetylated by p300 at lysine residues 430 and 437. The phosphorylation of Beclin 1 at S409 by CK1 is required for the subsequent p300 binding and Beclin. Beclin 1 acetylation inhibits autophagosome maturation and endocytic trafficking by promoting the recruitment of Rubicon. In tumor xenografts, the expression of 2KR mutant Beclin 1 leads to enhanced autophagosome maturation and tumor growth suppression. In order to explore the role of autophagy in cancer treatment, our investigation showed that Rhabdastrellic acid-A, an isomalabaricane triterpenoid isolated from the sponge Rhabdastrella globostellata, induced autophagy of cancer cells. SYUIQ-5, previously identified by us as a novel G-quadruplex stabilizer and potent telomerase inhibitor, inhibited proliferation of cancer cells, triggered a rapid and potent telomere

15 DNA damage response and obviously induced autophagy. These phenomena may primarily depend on the delocalization of TRF2 from telomere. We found that wild-type p53 can activate AMPK, inhibit mTORC1 and promote colon cancer cells survival by enabling cytoprotective autophagy in response to topotecan treatment. In contrast, the inhibition of autophagy alleviated the anti-tumour effect of topotecan treatment in p53 mutant or knockout colon cancer cells both in vitro and in vivo. We also found that topotecan, a topoisomerase I inhibitor, and cisplatin induced DNA damage and activated ATM, which phosphorylates PTEN at serine 113 and further regulates PTEN nuclear translocation in A549 and HeLa cells. After nuclear translocation, PTEN induces autophagy, in association with the activation of the p-JUNSESN2/AMPK pathway, in response to topotecan. Our researches show that decreased autophagy-related protein expression is associated with tumor progression and poor prognosis in ovarian cancer, breast cancer, colon cancer. We also found that some anticancer agents-mediated autophagyassociated cell death or survival dependent of cancer cell context. Therefore, understanding the mechanisms of autophagy and the role of autophagy in cancers will facilitate novel therapeutics for cancer. http://dx.doi.org/10.1016/j.gendis.2016.10.017

SIRT1 IN TUMOR METABOLISM AND TUMORIGENESIS Xiaoling Li Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA SIRT1, the most conserved NAD+-dependent protein deacetylase, is an important cellular metabolic and stress sensor. However, the role of this critical factor in cancer development remains unclear and inconclusive. SIRT1 has been shown to directly maintain genome stability and repress inflammation, thereby decreasing tumor growth. On the other hand, SIRT1 also has been reported to inhibit activities of tumor suppressors, promoting growth and survival of cancer cells. As a result, whether SIRT1 is an oncogene or tumor suppressor remains controversial. We recently explored the possibility that SIRT1 regulates cancer development in a quantitative dose-dependent manner. We hypothesized that different SIRT1-regulated cellular pathways have distinct sensitivities to changes of SIRT1 dosages. These distinct sensitivities may differentiate the outcomes in cancer cell proliferation and growth, contributing to observed dual functions of SIRT1 in tumor development. To test this hypothesis, we generated immortalized mouse embryonic fibroblasts (MEFs) and human colorectal cancer cell lines carrying two copies (WT), one copy (Het), or no copy (KO) of SIRT1 gene. Consistent with our hypothesis, SIRT1 Het cells displayed elevated proliferation in culture, increased colony formation on soft agar, and enhanced tumor formation in nude mice in a xenograft model, whereas SIRT1 KO cells exhibited reduced proliferation, colony formation, and cancer formation. Further mechanistic studies revealed that deletion of one copy of SIRT1 gene is sufficient to activate NF-kB and induce c-Myc expression, promoting cell proliferation, autophagy, and stress resistance in a glutamine-dependent manner. Deletion of both copies of SIRT1 gene, on the other hand, triggers cellular apoptotic pathways, leading to increased cell death, diminished autophagy, and reduced cancer formation. Consistently, intestine-specific SIRT1 heterozygous mice have enhanced intestinal tumor formation, whereas intestinespecific SIRT1 homozygous knockout mice have reduced development of colon cancer. Finally, expression levels of SIRT1 are reduced in human colorectal tumors, and reduced tumor SIRT1 expression correlates with poor prognosis in colorectal cancer patients. In summary, our findings indicate that the dose-dependent regulation of tumor metabolism and possibly apoptosis by SIRT1 mechanistically contributes to the observed dual roles of SIRT1 in tumorigenesis. Our study highlights the importance of maintenance of a suitable SIRT1 dosage for metabolic and tissue homeostasis, which will have important implications in SIRT1 small molecule activators/inhibitors based therapeutic strategies for cancers. http://dx.doi.org/10.1016/j.gendis.2016.10.018