Aikseng Ooi

Interests

Teaching

Biochemistry, Cell Signaling, Molecular genetics, Cancer biology

Research

NRF2 signaling, Iron metabolism, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC), Computational Biology.

Courses

Scholarly Contributions

Journals/Publications

• Giles, J., Martinez, K., Steiner, H., Klein, A., Ooi, A., Pryor, J., Sweitzer, N., Fuchs, D., & Karnes, J. (2024). Association of Metal Cations with the Anti-PF4/Heparin Antibody Response in Heparin-Induced Thrombocytopenia. Cardiovascular Toxicology, 24(9). doi:10.1007/s12012-024-09895-w
  More info

  Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against complexes of heparin and platelet factor 4 (PF4). The electrostatic interaction between heparin and PF4 is critical for the anti-PF4/heparin antibody response seen in HIT. The binding of metal cations to heparin induces conformational changes and charge neutralization of the heparin molecule, and cation-heparin binding can modulate the specificity and affinity for heparin-binding partners. However, the effects of metal cation binding to heparin in the context of anti-PF4/heparin antibody response have not been determined. Here, we utilized inductively coupled plasma mass spectrometry (ICP-MS) to quantify 16 metal cations in patient plasma and tested for correlation with anti-PF4/heparin IgG levels and platelet count after clinical suspicion of HIT in a cohort of heparin-treated patients. The average age of the cohort (n = 32) was 60.53 (SD = 14.31) years old, had a mean anti-PF4/heparin antibody optical density [OD405] of 0.93 (SD = 1.21) units, and was primarily female (n = 23). Patients with positive anti-PF4/heparin antibody test results (OD405 ≥ 0.5 units) were younger, had increased weight and BMI, and were more likely to have a positive serotonin release assay (SRA) result compared to antibody-negative patients. We observed statistical differences between antibody-positive and -negative groups for sodium and aluminum and significant correlations of anti-PF4/heparin antibody levels with sodium and silver. While differences in sodium concentrations were associated with antibody-positive status and correlated with antibody levels, no replication was performed. Additional studies are warranted to confirm our observed association, including in vitro binding studies and larger observational cohorts.
• Anandhan, A., Dodson, M., Shakya, A., Chen, J., Liu, P., Wei, Y., Tan, H., Wang, Q., Jiang, Z., Yang, K., Garcia, J. G., Chambers, S. K., Chapman, E., Ooi, A., Yang-Hartwich, Y., Stockwell, B. R., & Zhang, D. D. (2023). NRF2 controls iron homeostasis and ferroptosis through HERC2 and VAMP8. Science advances, 9(5), eade9585.
  More info

  Enhancing the intracellular labile iron pool (LIP) represents a powerful, yet untapped strategy for driving ferroptotic death of cancer cells. Here, we show that NRF2 maintains iron homeostasis by controlling HERC2 (E3 ubiquitin ligase for NCOA4 and FBXL5) and VAMP8 (mediates autophagosome-lysosome fusion). knockout cells have low expression, leading to a simultaneous increase in ferritin and NCOA4 and recruitment of apoferritin into the autophagosome. knockout cells also have low expression, which leads to ferritinophagy blockage. Therefore, deletion of results in apoferritin accumulation in the autophagosome, an elevated LIP, and enhanced sensitivity to ferroptosis. Concordantly, NRF2 levels correlate with HERC2 and VAMP8 in human ovarian cancer tissues, as well as ferroptosis resistance in a panel of ovarian cancer cell lines. Last, the feasibility of inhibiting NRF2 to increase the LIP and kill cancer cells via ferroptosis was demonstrated in preclinical models, signifying the impact of NRF2 inhibition in cancer treatment.
• Liu, P., Anandhan, A., Chen, J., Shakya, A., Dodson, M., Ooi, A., Chapman, E., White, E., Garcia, J. G., & Zhang, D. D. (2023). Decreased autophagosome biogenesis, reduced NRF2, and enhanced ferroptotic cell death are underlying molecular mechanisms of non-alcoholic fatty liver disease. Redox biology, 59, 102570.
  More info

  Caloric excess and sedentary lifestyles have led to an epidemic of obesity, metabolic syndrome, and non-alcoholic fatty liver disease (NAFLD). The objective of this study was to investigate the mechanisms underlying high fat diet (HFD)-induced NAFLD, and to explore NRF2 activation as a strategy to alleviate NAFLD.
• Shakya, A., Liu, P., Godek, J., McKee, N. W., Dodson, M., Anandhan, A., Ooi, A., Garcia, J. G., Costa, M., Chapman, E., & Zhang, D. D. (2023). The NRF2-p97-NRF2 negative feedback loop. Redox biology, 65, 102839.
  More info

  p97 is a ubiquitin-targeted ATP-dependent segregase that regulates proteostasis, in addition to a variety of other cellular functions. Previously, we demonstrated that p97 negatively regulates NRF2 by extracting ubiquitylated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex, facilitating proteasomal destruction. In the current study, we identified p97 as an NRF2-target gene that contains a functional ARE, indicating the presence of an NRF2-p97-NRF2 negative feedback loop that maintains redox homeostasis. Using CRISPR/Cas9 genome editing, we generated endogenous p97 ARE-mutated BEAS-2B cell lines. These p97 ARE-mutated cell lines exhibit altered expression of p97 and NRF2, as well as a compromised response to NRF2 inducers. Importantly, we also found a positive correlation between NRF2 activation and p97 expression in human cancer patients. Finally, using chronic arsenic-transformed cell lines, we demonstrated a synergistic effect of NRF2 and p97 inhibition in killing cancer cells with high NRF2 and p97 expression. Our study suggests dual upregulation of NRF2 and p97 occurs in certain types of cancers, suggesting that inhibition of both NRF2 and p97 could be a promising treatment strategy for stratified cancer patients.
• Anandhan, A., Chapman, E., Dai, W., Dodson, M., Galligan, J. J., Kitamura, N., Liu, P., Ooi, A., Schmidlin, C. J., Wei, Y., Wilson, N. C., & Zhang, D. D. (2022). CHML is an NRF2 target gene that regulates mTOR function.. Molecular oncology, 16(8), 1714-1727. doi:10.1002/1878-0261.13194
  More info

  The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is often highly expressed in non-small cell lung cancer (NSCLC). Through its target genes, NRF2 enhances cancer progression and chemo/radioresistance, leading to a poorer prognosis in patients with high NRF2 expression. In this study, we identified CHM-like Rab escort protein (CHML; encoding Rep2) as an NRF2 target gene with an antioxidant response element (ARE) in its promoter region (-1622 to -1612). Analysis of patient data curated by The Cancer Genome Atlas (TCGA) and Oncomine databases revealed that CHML mRNA expression was elevated in lung adenocarcinoma (LUAD) patient tumor tissues and correlated with decreased patient survival. Immunohistochemistry (IHC) analysis of normal versus lung cancer patient tissues revealed that Rep2 protein levels were higher in lung tumors compared with normal tissue, which also correlated with increased levels of NRF2. Importantly, siRNA-mediated knockdown of CHML/Rep2 in A549 NSCLC cells decreased their ability to proliferate. Mechanistically, Rep2 mediates mTOR function, as loss of Rep2 inhibited, whereas overexpression enhanced, mTOR translocation and activation at the lysosome. Our findings identify a novel NRF2-Rep2-dependent regulation of mTOR function.
• Bauman, J. E., Carew, J. S., Espitia, C. M., Jones, T. M., Nawrocki, S. T., & Ooi, A. (2022). Abstract 2640: Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism. Cancer Research, 82(12_Supplement), 2640-2640. doi:10.1158/1538-7445.am2022-2640
  More info

  Abstract Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. We, and others, have reported that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8 activating enzyme (NAE) inhibitor pevonedistat. Here, we report that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augments cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target, CUL4A, played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen. Citation Format: Trace M. Jones, Claudia M. Espitia, Aikseng Ooi, Julie E. Bauman, Jennifer S. Carew, Steffan T. Nawrocki. Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2640.
• Bauman, J. E., Carew, J. S., Espitia, C. M., Jones, T. M., Nawrocki, S. T., & Ooi, A. (2022). Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism.. Cell death & disease, 13(4), 350.
  More info

  Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. Here we report that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8-activating enzyme (NAE) inhibitor pevonedistat. Additional studies established that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augmented cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target CUL4A played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A or DDB2 enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.
• Dodson, M., Dai, W., Anandhan, A., Schmidlin, C. J., Liu, P., Wilson, N. C., Wei, Y., Kitamura, N., Galligan, J. J., Ooi, A., Chapman, E., & Zhang, D. D. (2022). Author response for "CHML is an NRF2 target gene that regulates mTOR function". Molecular Oncology. doi:10.1002/1878-0261.13194/v4/response1
• Dodson, M., Dai, W., Anandhan, A., Schmidlin, C., Liu, P., Wilson, N., Wei, Y., Kitamura, N., Galligan, J., Ooi, A., Chapman, E., & Zhang, D. (2022). CHML is an NRF2 target gene that regulates mTOR function. Molecular Oncology, 16(8). doi:10.1002/1878-0261.13194
  More info

  The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is often highly expressed in non-small cell lung cancer (NSCLC). Through its target genes, NRF2 enhances cancer progression and chemo/radioresistance, leading to a poorer prognosis in patients with high NRF2 expression. In this study, we identified CHM-like Rab escort protein (CHML; encoding Rep2) as an NRF2 target gene with an antioxidant response element (ARE) in its promoter region (–1622 to –1612). Analysis of patient data curated by The Cancer Genome Atlas (TCGA) and Oncomine databases revealed that CHML mRNA expression was elevated in lung adenocarcinoma (LUAD) patient tumor tissues and correlated with decreased patient survival. Immunohistochemistry (IHC) analysis of normal versus lung cancer patient tissues revealed that Rep2 protein levels were higher in lung tumors compared with normal tissue, which also correlated with increased levels of NRF2. Importantly, siRNA-mediated knockdown of CHML/Rep2 in A549 NSCLC cells decreased their ability to proliferate. Mechanistically, Rep2 mediates mTOR function, as loss of Rep2 inhibited, whereas overexpression enhanced, mTOR translocation and activation at the lysosome. Our findings identify a novel NRF2–Rep2-dependent regulation of mTOR function.
• Ergen, E., Kerimoglu, B., Lamb, C., McPherson, R. D., Ooi, A., & Stone, E. M. (2022). Cyst(e)inase–Rapamycin Combination Induces Ferroptosis in Both In Vitro and In Vivo Models of Hereditary Leiomyomatosis and Renal Cell Cancer. Molecular Cancer Therapeutics, 21(3), 419-426. doi:10.1158/1535-7163.mct-21-0661
  More info

  Abstract Renal cell carcinomas associated with hereditary leiomyomatosis and renal cell cancer (HLRCC) are notoriously aggressive and represent the leading cause of death among patients with HLRCC. To date, a safe and effective standardized therapy for this tumor type is lacking. Here we show that the engineered synthetic therapeutic enzyme, Cyst(e)inase, when combined with rapamycin, can effectively induce ferroptosis in HLRCC cells in vivo. The drug combination promotes lipid peroxidation to a greater degree than cysteine deprivation or Cyst(e)inase treatment alone, while rapamycin treatment alone does not induce ferroptosis. Mechanistically, Cyst(e)inase induces ferroptosis by depleting the exogenous cysteine/cystine supply, while rapamycin reduces cellular ferritin level by promoting ferritins' destruction via ferritinophagy. Since both Cyst(e)inase and rapamycin are well tolerated clinically, the combination represents an opportunity to exploit ferroptosis induction as a cancer management strategy. Accordingly, using a xenograft mouse model, we showed that the combination treatment resulted in tumor growth suppression without any notable side effects. In contrast, both Cyst(e)inase only and rapamycin only treatment groups failed to induce a significant change when compared with the vehicle control group. Our results demonstrated the effectiveness of Cyst(e)inase–rapamycin combination in inducing ferroptotic cell death in vivo, supporting the potential translation of the combination therapy into clinical HLRCC management.
• Jones, T., Espitia, C., Ooi, A., Bauman, J., Carew, J., & Nawrocki, S. (2022). Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism. Cell Death and Disease, 13(4). doi:10.1038/s41419-022-04798-6
  More info

  Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. Here we report that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8-activating enzyme (NAE) inhibitor pevonedistat. Additional studies established that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augmented cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target CUL4A played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A or DDB2 enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.
• Kerimoglu, B., Lamb, C., McPherson, R., Ergen, E., Stone, E., & Ooi, A. (2022). Cyst(e)inase-Rapamycin Combination Induces Ferroptosis in Both In Vitro and In Vivo Models of Hereditary Leiomyomatosis and Renal Cell Cancer. Molecular Cancer Therapeutics, 21(3). doi:10.1158/1535-7163.MCT-21-0661
  More info

  Renal cell carcinomas associated with hereditary leiomyomatosis and renal cell cancer (HLRCC) are notoriously aggressive and represent the leading cause of death among patients with HLRCC. To date, a safe and effective standardized therapy for this tumor type is lacking. Here we show that the engineered synthetic therapeutic enzyme, Cyst(e)inase, when combined with rapamycin, can effectively induce ferroptosis in HLRCC cells in vivo. The drug combination promotes lipid peroxidation to a greater degree than cysteine deprivation or Cyst(e)inase treatment alone, while rapamycin treatment alone does not induce ferroptosis. Mechanistically, Cyst(e)inase induces ferroptosis by depleting the exogenous cysteine/ cystine supply, while rapamycin reduces cellular ferritin level by promoting ferritins' destruction via ferritinophagy. Since both Cyst (e)inase and rapamycin are well tolerated clinically, the combination represents an opportunity to exploit ferroptosis induction as a cancer management strategy. Accordingly, using a xenograft mouse model, we showed that the combination treatment resulted in tumor growth suppression without any notable side effects. In contrast, both Cyst(e)inase only and rapamycin only treatment groups failed to induce a significant change when compared with the vehicle control group. Our results demonstrated the effectiveness of Cyst(e) inase-rapamycin combination in inducing ferroptotic cell death in vivo, supporting the potential translation of the combination therapy into clinical HLRCC management.
• Sung, Y., Kerimoglu, B., Ooi, A., & Tomat, E. (2022). Aroylhydrazone Glycoconjugate Prochelators Exploit Glucose Transporter 1 (GLUT1) to Target Iron in Cancer Cells. ACS Medicinal Chemistry Letters, 13(9). doi:10.1021/acsmedchemlett.2c00250
  More info

  Glycoconjugation strategies in anticancer drug discovery exploit the high expression of glucose transporters in malignant cells to achieve preferential uptake and hence attractive pharmacological characteristics of increased therapeutic windows and decreased unwanted toxicity. Here we present the design of glycoconjugated prochelators of aroylhydrazone AH1, an antiproliferative scavenger that targets the increased iron demand of rapidly proliferating malignant cells. The constructs feature a monosaccharide (d-glucose, d-glucosamine, or glycolytic inhibitor 2-deoxy-d-glucose) connected at the C2 or C6 position via a short linker, which masks the chelator through a disulfide bond susceptible to intracellular reduction. Cellular assays showed that the glycoconjugates rely on the GLUT1 transporter for uptake, lead to intracellular iron deprivation, and present antiproliferative activity. Ectopic overexpression of GLUT1 in malignant and normal cells increased the uptake and toxicity of the glycoconjugated prochelators, demonstrating that these compounds are well suited for targeting cells overexpressing glucose transporters and therefore for selective iron sequestration in malignant cells.
• Sung, Y., Kerimoğlu, B., Ooi, A., & Tomat, E. (2022). Aroylhydrazone Glycoconjugate Prochelators Exploit Glucose Transporter 1 (GLUT1) to Target Iron in Cancer Cells. ACS Medicinal Chemistry Letters. doi:10.1021/acsmedchemlett.2c00250
  More info

  Glycoconjugation strategies in anticancer drug discovery exploit the high expression of glucose transporters in malignant cells to achieve preferential uptake and hence attractive pharmacological characteristics of increased therapeutic windows and decreased unwanted toxicity. Here we present the design of glycoconjugated prochelators of aroylhydrazone AH1, an antiproliferative scavenger that targets the increased iron demand of rapidly proliferating malignant cells. The constructs feature a monosaccharide (d-glucose, d-glucosamine, or glycolytic inhibitor 2-deoxy-d-glucose) connected at the C2 or C6 position via a short linker, which masks the chelator through a disulfide bond susceptible to intracellular reduction. Cellular assays showed that the glycoconjugates rely on the GLUT1 transporter for uptake, lead to intracellular iron deprivation, and present antiproliferative activity. Ectopic overexpression of GLUT1 in malignant and normal cells increased the uptake and toxicity of the glycoconjugated prochelators, demonstrating that these compounds are well suited for targeting cells overexpressing glucose transporters and therefore for selective iron sequestration in malignant cells.
• Dodson, M., Dai, W., Anandhan, A., Schmidlin, C. J., Liu, P., Wilson, N. C., Wei, Y., Kitamura, N., Galligan, J. J., Ooi, A., Chapman, E., & Zhang, D. D. (2021). Author response for "CHML is an NRF2 target gene that regulates mTOR function". Molecular Oncology. doi:10.1002/1878-0261.13194/v2/response1
• Goldfarb, A. N., Freeman, K. C., Sahu, R. K., Elagib, K. E., Holy, M., Arneja, A., Polanowska-Grabowska, R., Gru, A. A., White, Z., Khalil, S., Kerins, M. J., Ooi, A., Leitinger, N., Luckey, C. J., & Delehanty, L. L. (2021). Iron control of erythroid microtubule cytoskeleton as a potential target in treatment of iron-restricted anemia. Nature communications, 12(1), 1645.
  More info

  Anemias of chronic disease and inflammation (ACDI) result from restricted iron delivery to erythroid progenitors. The current studies reveal an organellar response in erythroid iron restriction consisting of disassembly of the microtubule cytoskeleton and associated Golgi disruption. Isocitrate supplementation, known to abrogate the erythroid iron restriction response, induces reassembly of microtubules and Golgi in iron deprived progenitors. Ferritin, based on proteomic profiles, regulation by iron and isocitrate, and putative interaction with microtubules, is assessed as a candidate mediator. Knockdown of ferritin heavy chain (FTH1) in iron replete progenitors induces microtubule collapse and erythropoietic blockade; conversely, enforced ferritin expression rescues erythroid differentiation under conditions of iron restriction. Fumarate, a known ferritin inducer, synergizes with isocitrate in reversing molecular and cellular defects of iron restriction and in oral remediation of murine anemia. These findings identify a cytoskeletal component of erythroid iron restriction and demonstrate potential for its therapeutic targeting in ACDI.
• Liu, P., Dodson, M., Li, H., Schmidlin, C. J., Shakya, A., Wei, Y., Garcia, J. G., Chapman, E., Kiela, P. R., Zhang, Q. Y., White, E., Ding, X., Ooi, A., & Zhang, D. D. (2021). Non-canonical NRF2 activation promotes a pro-diabetic shift in hepatic glucose metabolism. Molecular metabolism, 51, 101243.
  More info

  NRF2, a transcription factor that regulates cellular redox and metabolic homeostasis, plays a dual role in human disease. While it is well known that canonical intermittent NRF2 activation protects against diabetes-induced tissue damage, little is known regarding the effects of prolonged non-canonical NRF2 activation in diabetes. The goal of this study was to determine the role and mechanisms of prolonged NRF2 activation in arsenic diabetogenicity.
• Liu, P., Luo, G., Dodson, M., Schmidlin, C. J., Wei, Y., Kerimoglu, B., Ooi, A., Chapman, E., Garcia, J. G., & Zhang, D. D. (2021). The NRF2-LOC344887 signaling axis suppresses pulmonary fibrosis. Redox biology, 38, 101766.
  More info

  Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible disease characterized by an increase in differentiation of fibroblasts to myofibroblasts and excessive accumulation of extracellular matrix in lung tissue. Pharmacological activation of NRF2 has proved to be a valuable antifibrotic approach, however the detailed mechanisms of how NRF2 mediates antifibrotic function remain unclear. In this study, we found that the antifibrotic function of sulforaphane (SFN), an NRF2 activator, was largely dependent on LOC344887, a long noncoding RNA. Two functional AREs were identified in both the promoter and intron 1 of LOC344887, which defines LOC344887 as a novel anti-fibrotic NRF2 target gene. RNA-seq analysis revealed that LOC344887 controls genes and signaling pathways associated with fibrogenesis. Deletion or downregulation of LOC344887 enhanced expression of CDH2/N-cadherin, as well as a number of other fibrotic genes and blunted the antifibrotic effects of SFN. Furthermore, LOC344887-mediated downregulation of fibrotic genes may involve the PI3K-AKT signaling pathway, as pharmacologic inhibition of PI3K activity blocked the effects of LOC344887 knockdown. Our findings demonstrate that NRF2-mediated LOC344887 upregulation contributes to the antifibrotic potential of SFN by repressing the expression of CDH2 and other fibrotic genes, providing novel insight into how NRF2 controls the regulatory networks of IPF. This study provides a better understanding of the molecular mechanisms of NRF2 activators against pulmonary fibrosis and presents a novel therapeutic axis for prevention and intervention of fibrosis-related diseases.
• Ooi, A. (2020). Advances in hereditary leiomyomatosis and renal cell carcinoma (HLRCC) research.. Seminars in cancer biology, 61, 158-166. doi:10.1016/j.semcancer.2019.10.016
  More info

  Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an autosomal dominant hereditary cancer syndrome with incomplete penetrance. It is caused by a germline amorphic allele of the FH gene, which encodes the TCA cycle enzyme, fumarate hydratase (FH). HLRCC patients are genetically predisposed to develop skin leiomyomas, uterine fibroids, and the aggressive kidney cancer of type 2 papillary morphology. Loss-of-heterozygocity at the FH locus that cause a complete loss of FH enzymatic function is always detected in these tumor tissues. Molecular pathway elucidation, genomic studies, and systematic genetics screens reported over the last two decades have identified several FH-inactivation driven pathways alterations, as well as rationally conceived treatment strategies that specifically target FH-/- tumor cells. These treatment strategies include ferroptosis induction, oxidative stress promotion, and metabolic alteration. As the fundamental biology of HLRCC continues to be uncovered, these treatment strategies continue to be refined and may one day lead to a strategy to prevent disease onset among HLRCC patients. With a more complete picture of HLRCC biology, the safe translation of experimental treatment strategies into clinical practice is achievable in the foreseeable future.
• Chapman, E., Moghaddam, S. J., Ooi, A., Tao, S., Wang, S., Wong, P. K., & Zhang, D. D. (2019). Correction: Oncogenic KRAS Confers Chemoresistance by Upregulating NRF2.. Cancer research, 79(5), 1015. doi:10.1158/0008-5472.can-19-0070
  More info

  In the original version of [this article][1] ([1][2]), the HMOX1 Western blot band in Fig. 1B was inadvertently duplicated from the HMOX1 Western blot band in Fig. 3B. The error has been corrected in the latest online HTML and PDF versions of the article. The authors regret this error. 1. 1.[↵][
• Kerins, M. J., Liu, P., Tian, W., Mannheim, W., Zhang, D. D., & Ooi, A. (2019). Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor. Molecular and cellular biology, 39(13).
  More info

  The nuclear factor (erythroid 2)-like 2 (NRF2 or NFE2L2) transcription factor regulates the expression of many genes that are critical in maintaining cellular homeostasis. Its deregulation has been implicated in many diseases, including cancer and metabolic and neurodegenerative diseases. While several mechanisms by which NRF2 can be activated have gradually been identified over time, a more complete regulatory network of NRF2 is still lacking. Here we show through a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen that a total of 273 genes, when knocked out, will lead to sustained NRF2 activation. Pathway analysis revealed a significant overrepresentation of genes (18 of the 273 genes) involved in autophagy. Molecular validation of a subset of the enriched genes identified 8 high-confidence genes that negatively regulate NRF2 activity irrespective of cell type: , , , , , , , and the well-known negative regulator of NRF2, Of these, , , , and are known to be involved in autophagic processes. Our results present a comprehensive list of NRF2 negative regulators and reveal an intimate link between autophagy and NRF2 regulation.
• Liu, P., Kerins, M. J., Tian, W., Neupane, D., Zhang, D. D., & Ooi, A. (2019). Differential and overlapping targets of the transcriptional regulators NRF1, NRF2, and NRF3 in human cells. The Journal of biological chemistry, 294(48), 18131-18149.
  More info

  The nuclear factor (erythroid 2)-like (NRF) transcription factors are a subset of cap'n'collar transcriptional regulators. They consist of three members, NRF1, NRF2, and NRF3, that regulate the expression of genes containing antioxidant-response elements (AREs) in their promoter regions. Although all NRF members regulate ARE-containing genes, each is associated with distinct roles. A comprehensive study of differential and overlapping DNA-binding and transcriptional activities of the NRFs has not yet been conducted. Here, we performed chromatin immunoprecipitation (ChIP)-exo sequencing, an approach that combines ChIP with exonuclease treatment to pinpoint regulatory elements in DNA with high precision, in conjunction with RNA-sequencing to define the transcriptional targets of each NRF member. Our approach, done in three U2OS cell lines, identified 31 genes that were regulated by all three NRF members, 27 that were regulated similarly by all three, and four genes that were differentially regulated by at least one NRF member. We also found genes that were up- or down-regulated by only one NRF member, with 84, 84, and 22 genes that were regulated by NRF1, NRF2, and NRF3, respectively. Analysis of the ARE motifs identified in ChIP peaks revealed that NRF2 prefers binding to AREs flanked by GC-rich regions and that NRF1 prefers AT-rich flanking regions. Thus, sequence preference, likely in combination with upstream signaling events, determines NRF member activation under specific cellular contexts. Our analysis provides a comprehensive description of differential and overlapping gene regulation by the transcriptional regulators NRF1, NRF2, and NRF3.
• Ooi, A. (2019). Advances in hereditary leiomyomatosis and renal cell carcinoma (HLRCC) research. Seminars in cancer biology.
  More info

  Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an autosomal dominant hereditary cancer syndrome with incomplete penetrance. It is caused by a germline amorphic allele of the FH gene, which encodes the TCA cycle enzyme, fumarate hydratase (FH). HLRCC patients are genetically predisposed to develop skin leiomyomas, uterine fibroids, and the aggressive kidney cancer of type 2 papillary morphology. Loss-of-heterozygocity at the FH locus that cause a complete loss of FH enzymatic function is always detected in these tumor tissues. Molecular pathway elucidation, genomic studies, and systematic genetics screens reported over the last two decades have identified several FH-inactivation driven pathways alterations, as well as rationally conceived treatment strategies that specifically target FH tumor cells. These treatment strategies include ferroptosis induction, oxidative stress promotion, and metabolic alteration. As the fundamental biology of HLRCC continues to be uncovered, these treatment strategies continue to be refined and may one day lead to a strategy to prevent disease onset among HLRCC patients. With a more complete picture of HLRCC biology, the safe translation of experimental treatment strategies into clinical practice is achievable in the foreseeable future.
• Tao, S., Wang, S., Moghaddam, S., Ooi, A., Chapman, E., Wong, P., & Zhang, D. (2019). Correction: Oncogenic KRAS confers chemoresistance by upregulating Nrf2 (Cancer Research (2014) 74 (7430-7441) DOI: 10.1158/0008-5472.CAN-14-1439). Cancer Research, 79(5). doi:10.1158/0008-5472.CAN-19-0070
  More info

  In the original version of this article (1), the HMOX1 Western blot band in Fig. 1B was inadvertently duplicated from the HMOX1 Western blot band in Fig. 3B. The error has been corrected in the latest online HTML and PDF versions of the article. The authors regret this error.
• Kerins, M. J., & Ooi, A. (2018). A catalogue of somatic NRF2 gain-of-function mutations in cancer. Scientific reports, 8(1), 12846.
  More info

  Identification and characterization of somatic mutations in cancer have important prognostication and treatment implications. Genes encoding the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor and its negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), are frequently mutated in cancer. These mutations drive constitutive NRF2 activation and correlate with poor prognosis. Despite its apparent significance, a comprehensive catalogue of somatic NRF2 mutations across different tumor types is still lacking. Here, we catalogue NRF2 mutations in The Cancer Genome Atlas (TCGA) database. 226 unique NRF2-mutant tumors were identified from 10,364 cases. NRF2 mutations were found in 21 out of the 33 tumor types. A total of 11 hotspots were identified. Of these, mutation to the R34 position was most frequent. Notably, R34 and D29 mutations were overrepresented in bladder, lung, and uterine cancers. Analyses of corresponding RNA sequencing data using a de novo derived gene expression classifier showed that the R34 mutations drive constitutive NRF2 activation with a selection pressure biased against the formation of R34L. Of all R34 mutants, R34L conferred the least degree of protein stabilization, suggesting a pro-tumor NRF2 half-life threshold. Our findings offer a comprehensive catalogue of NRF2 mutations in cancer that can help prognostication and NRF2 research.
• Kerins, M. J., Milligan, J., Wohlschlegel, J. A., & Ooi, A. (2018). Fumarate hydratase inactivation in hereditary leiomyomatosis and renal cell cancer is synthetic lethal with ferroptosis induction. Cancer science, 109(9), 2757-2766.
  More info

  Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome characterized by inactivation of the Krebs cycle enzyme fumarate hydratase (FH). HLRCC patients are at high risk of developing kidney cancer of type 2 papillary morphology that is refractory to current radiotherapy, immunotherapy and chemotherapy. Hence, an effective therapy for this deadly form of cancer is urgently needed. Here, we show that FH inactivation (FH ) proves synthetic lethal with inducers of ferroptosis, an iron-dependent and nonapoptotic form of cell death. Specifically, we identified gene signatures for compound sensitivities based on drug responses for 9 different drug classes against the NCI-60 cell lines. These signatures predicted that ferroptosis inducers would be selectively toxic to FH cell line UOK262. Preferential cell death against UOK262-FH was confirmed with 4 different ferroptosis inducers. Mechanistically, the FH sensitivity to ferroptosis is attributed to dysfunctional GPX4, the primary cellular defender against ferroptosis. We identified that C93 of GPX4 is readily post-translationally modified by fumarates that accumulate in conditions of FH , and that C93 modification represses GPX4 activity. Induction of ferroptosis in FH-inactivated tumors represents an opportunity for synthetic lethality in cancer.
• Kerins, M. J., Vashisht, A., Liang, B., Duckworth, S. J., Praslicka, B. J., Wohlschlegel, J. A., & Ooi, A. (2017). Fumarate mediates a chronic proliferative signal in fumarate hydratase inactivated cancer cells by increasing transcription and translation of ferritin genes.. Mol. Cell. Biol..
• Kerins, M., & Ooi, A. (2018). The Roles of NRF2 in Modulating Cellular Iron Homeostasis. Antioxidants and Redox Signaling, 29(17). doi:10.1089/ars.2017.7176
  More info

  Significance: Iron and oxygen are intimately linked: iron is an essential nutrient utilized as a cofactor in enzymes for oxygen transport, oxidative phosphorylation, and metabolite oxidation. However, excess labile iron facilitates the formation of oxygen-derived free radicals capable of damaging biomolecules. Therefore, biological utilization of iron is a tightly regulated process. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor, which can respond to oxidative and electrophilic stress, regulates several genes involved in iron metabolism. Recent Advances: The bulk of NRF2 transcription factor research has focused on its roles in detoxification and cancer prevention. Recent works have identified that several genes involved in heme synthesis, hemoglobin catabolism, iron storage, and iron export are under the control of NRF2. Constitutive NRF2 activation and subsequent deregulation of iron metabolism have been implicated in cancer development: NRF2-mediated upregulation of the iron storage protein ferritin or heme oxygenase 1 can lead to enhanced proliferation and therapy resistance. Of note, NRF2 activation and alterations to iron signaling in cancers may hinder efforts to induce the iron-dependent cell death process known as ferroptosis. Critical Issues: Despite growing recognition of NRF2 as a modulator of iron signaling, exactly how iron metabolism is altered due to NRF2 activation in normal physiology and in pathologic conditions remains imprecise; moreover, the roles of NRF2-mediated iron signaling changes in disease progression are only beginning to be uncovered. Future Directions: Further studies are necessary to connect NRF2 activation with physiological and pathological changes to iron signaling and oxidative stress. Antioxid. Redox Signal. 00, 000-000.
• Liu, P., Rojo de la Vega, M., Sammani, S., Mascarenhas, J. B., Kerins, M., Dodson, M., Sun, X., Wang, T., Ooi, A., Garcia, J. G., & Zhang, D. D. (2018). RPA1 binding to NRF2 switches ARE-dependent transcriptional activation to ARE-NRE-dependent repression. Proceedings of the National Academy of Sciences of the United States of America, 115(44), E10352-E10361.
  More info

  NRF2 regulates cellular redox homeostasis, metabolic balance, and proteostasis by forming a dimer with small musculoaponeurotic fibrosarcoma proteins (sMAFs) and binding to antioxidant response elements (AREs) to activate target gene transcription. In contrast, NRF2-ARE-dependent transcriptional repression is unreported. Here, we describe NRF2-mediated gene repression via a specific seven-nucleotide sequence flanking the ARE, which we term the NRF2-replication protein A1 (RPA1) element (NRE). Mechanistically, RPA1 competes with sMAF for NRF2 binding, followed by interaction of NRF2-RPA1 with the ARE-NRE and eduction of promoter activity. Genome-wide in silico and RNA-seq analyses revealed this NRF2-RPA1-ARE-NRE complex mediates negative regulation of many genes with diverse functions, indicating that this mechanism is a fundamental cellular process. Notably, repression of , which encodes the nonmuscle myosin light chain kinase, by the NRF2-RPA1-ARE-NRE complex disrupts vascular integrity in preclinical inflammatory lung injury models, illustrating the translational significance of NRF2-mediated transcriptional repression. Our findings reveal a gene-suppressive function of NRF2 and a subset of negatively regulated NRF2 target genes, underscoring the broad impact of NRF2 in physiological and pathological settings.
• Ooi, A., Schmidlin, C. J., Tian, W., Vega, M. R., & Zhang, D. D. (2018). Kelch-like ECH-associated protein 1 (KEAP1) differentially regulates nuclear factor erythroid-2-related factors 1 and 2 (NRF1 and NRF2).. The Journal of biological chemistry, 293(6), 2029-2040. doi:10.1074/jbc.ra117.000428
  More info

  Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology-like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1+/+ than in KEAP1-/- isogenic cell lines, whereas NRF2 is dramatically stabilized in KEAP1-/- cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.
• Tao, S., Rojo de la Vega, M., Chapman, E., Ooi, A., & Zhang, D. D. (2018). The effects of NRF2 modulation on the initiation and progression of chemically and genetically induced lung cancer. Molecular carcinogenesis, 57(2), 182-192.
  More info

  Targeting the transcription factor NRF2 has been recognized as a feasible strategy for cancer prevention and treatment, but many of the mechanistic details underlying its role in cancer development and progression are lacking. Therefore, careful mechanistic studies of the NRF2 pathway in cancer initiation and progression are needed to identify which therapeutic avenue-activation or inhibition-is appropriate in a given context. Moreover, while numerous reports confirm the protective effect of NRF2 activation against chemical carcinogenesis little is known of its role in cancer arising from spontaneous mutations. Here, we tested the effects of NRF2 modulation (activation by sulforaphane or inhibition by brusatol) in lung carcinogenesis using a chemical (vinyl carbamate) model in A/J mice and a genetic (conditional KrasG12D oncogene expression, to simulate spontaneous oncogene mutation) model in C57BL/6J mice. Mice were treated with NRF2 modulators before carcinogen exposure or KrasG12D expression to test the role of NRF2 in cancer initiation, or treated after tumor development to test the role of NRF2 in cancer progression. Lung tissues were analyzed to determine tumor burden, as well as status of NRF2 and KRAS pathways. Additionally, proliferation, apoptosis, and oxidative DNA damage were assessed. Overall, NRF2 activation prevents initiation of chemically induced cancer, but promotes progression of pre-existing tumors regardless of chemical or genetic etiology. Once tumors are initiated, NRF2 inhibition is effective against the progression of chemically and spontaneously induced tumors. These results have important implications for NRF2-targeted cancer prevention and intervention strategies.
• Tian, W., De La Vega, M., Schmidlin, C., Ooi, A., & Zhang, D. (2018). Kelch-like ECH-Associated protein 1 (KEAP1) differentially regulates nuclear factor erythroid-2-related factors 1 and 2 (NRF1 and NRF2). Journal of Biological Chemistry, 293(6). doi:10.1074/jbc.RA117.000428
  More info

  Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-Associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology- like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1+/+ than in KEAP1-/-isogenic cell lines, whereas NRF2 is dramatically stabilized in KEAP1-/-cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.
• Chapman, E., Clair, J. J., Harder, B., Ooi, A., Tan, A. C., Tian, W., & Zhang, D. D. (2017). Brusatol overcomes chemoresistance through inhibition of protein translation.. Molecular carcinogenesis, 56(5), 1493-1500. doi:10.1002/mc.22609
  More info

  The NRF2 pathway activates a cell survival response when cells are exposed to xenobiotics or are under oxidative stress. Therapeutic activation of NRF2 can also be used prior to insult as a means of disease prevention. However, prolonged expression of NRF2 has been shown to protect cancer cells by inducing the metabolism and efflux of chemotherapeutics, leading to both intrinsic and acquired chemoresistance to cancer drugs. This effect has been termed the "dark side" of NRF2. In an effort to combat this chemoresistance, our group discovered the first NRF2 inhibitor, the natural product brusatol, however the mechanism of inhibition was previously unknown. In this report, we show that brusatol's mode of action is not through direct inhibition of the NRF2 pathway, but through the inhibition of both cap-dependent and cap-independent protein translation, which has an impact on many short-lived proteins, including NRF2. Therefore, there is still a need to develop a new generation of specific NRF2 inhibitors with limited toxicity and off-target effects that could be used as adjuvant therapies to sensitize cancers with high expression of NRF2.
• Chapman, E., Ooi, A., Rojo de la Vega, M., Tao, S., & Zhang, D. D. (2017). The effects of NRF2 modulation on the initiation and progression of chemically and genetically induced lung cancer. Molecular Carcinogenesis, 57(2), 182-192. doi:10.1002/mc.22745
  More info

  Targeting the transcription factor NRF2 has been recognized as a feasible strategy for cancer prevention and treatment, but many of the mechanistic details underlying its role in cancer development and progression are lacking. Therefore, careful mechanistic studies of the NRF2 pathway in cancer initiation and progression are needed to identify which therapeutic avenue—activation or inhibition—is appropriate in a given context. Moreover, while numerous reports confirm the protective effect of NRF2 activation against chemical carcinogenesis little is known of its role in cancer arising from spontaneous mutations. Here, we tested the effects of NRF2 modulation (activation by sulforaphane or inhibition by brusatol) in lung carcinogenesis using a chemical (vinyl carbamate) model in A/J mice and a genetic (conditional Kras G12D oncogene expression, to simulate spontaneous oncogene mutation) model in C57BL/6J mice. Mice were treated with NRF2 modulators before carcinogen exposure or Kras G12D expression to test the role of NRF2 in cancer initiation, or treated after tumor development to test the role of NRF2 in cancer progression. Lung tissues were analyzed to determine tumor burden, as well as status of NRF2 and KRAS pathways. Additionally, proliferation, apoptosis, and oxidative DNA damage were assessed. Overall, NRF2 activation prevents initiation of chemically induced cancer, but promotes progression of pre‐existing tumors regardless of chemical or genetic etiology. Once tumors are initiated, NRF2 inhibition is effective against the progression of chemically and spontaneously induced tumors. These results have important implications for NRF2‐targeted cancer prevention and intervention strategies.
• Harder, B., Tian, W., La Clair, J., Tan, A., Ooi, A., Chapman, E., & Zhang, D. (2017). Brusatol overcomes chemoresistance through inhibition of protein translation. Molecular Carcinogenesis, 56(5). doi:10.1002/mc.22609
  More info

  The NRF2 pathway activates a cell survival response when cells are exposed to xenobiotics or are under oxidative stress. Therapeutic activation of NRF2 can also be used prior to insult as a means of disease prevention. However, prolonged expression of NRF2 has been shown to protect cancer cells by inducing the metabolism and efflux of chemotherapeutics, leading to both intrinsic and acquired chemoresistance to cancer drugs. This effect has been termed the “dark side” of NRF2. In an effort to combat this chemoresistance, our group discovered the first NRF2 inhibitor, the natural product brusatol, however the mechanism of inhibition was previously unknown. In this report, we show that brusatol's mode of action is not through direct inhibition of the NRF2 pathway, but through the inhibition of both cap-dependent and cap-independent protein translation, which has an impact on many short-lived proteins, including NRF2. Therefore, there is still a need to develop a new generation of specific NRF2 inhibitors with limited toxicity and off-target effects that could be used as adjuvant therapies to sensitize cancers with high expression of NRF2.
• Kerins, M. J., & Ooi, A. (2017). The Roles of NRF2 in Modulating Cellular Iron Homeostasis. Antioxidants & redox signaling.
  More info

  Iron and oxygen are intimately linked: iron is an essential nutrient utilized as a cofactor in enzymes for oxygen transport, oxidative phosphorylation, and metabolite oxidation. However, excess labile iron facilitates the formation of oxygen-derived free radicals capable of damaging biomolecules. Therefore, biological utilization of iron is a tightly regulated process. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor, which can respond to oxidative and electrophilic stress, regulates several genes involved in iron metabolism. Recent Advances: The bulk of NRF2 transcription factor research has focused on its roles in detoxification and cancer prevention. Recent works have identified that several genes involved in heme synthesis, hemoglobin catabolism, iron storage, and iron export are under the control of NRF2. Constitutive NRF2 activation and subsequent deregulation of iron metabolism have been implicated in cancer development: NRF2-mediated upregulation of the iron storage protein ferritin or heme oxygenase 1 can lead to enhanced proliferation and therapy resistance. Of note, NRF2 activation and alterations to iron signaling in cancers may hinder efforts to induce the iron-dependent cell death process known as ferroptosis.
• Kerins, M. J., Vashisht, A. A., Liang, B. X., Duckworth, S. J., Praslicka, B. J., Wohlschlegel, J. A., & Ooi, A. (2017). Fumarate Mediates a Chronic Proliferative Signal in Fumarate Hydratase-Inactivated Cancer Cells by Increasing Transcription and Translation of Ferritin Genes. Molecular and cellular biology, 37(11).
  More info

  Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase () cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how inactivation can endow cells with a growth advantage.
• Praslicka, B., Harmson, J. S., Kim, J., Rangaraj, V. R., Ooi, A., & Gissendanner, C. R. (2017). BINDING SITE ANALYSIS OF THE CAENORHABDITIS ELEGANS NR4A NUCLEAR RECEPTOR NHR-6 DURING DEVELOPMENT. Nuclear receptor research, 4.
  More info

  Members of the NR4A subfamily of nuclear receptors make up a highly conserved, functionally diverse group of transcription factors implicated in a multitude of cellular processes such as proliferation, differentiation, apoptosis, metabolism and DNA repair. The gene nhr-6, which encodes the sole C. elegans NR4A nuclear receptor homolog, has a critical role in organogenesis and regulates the development of the spermatheca organ system. Our previous work revealed that nhr-6 is required for spermatheca cell divisions in late L3 and early L4 and spermatheca cell differentiation during the mid L4 stage. Here, we utilized chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify NHR-6 binding sites during both the late L3/early L4 and mid L4 developmental stages. Our results revealed 30,745 enriched binding sites for NHR-6, ~70% of which were within 3 kb upstream of a gene transcription start site. Binding sites for a cohort of candidate target genes with probable functions in spermatheca organogenesis were validated through qPCR. Reproductive and spermatheca phenotypes were also evaluated for these genes following a loss-of-function RNAi screen which revealed several genes with critical functions during spermatheca organogenesis. Our results uncovered a complex nuclear receptor regulatory network whereby NHR-6 regulates multiple cellular processes during spermatheca organogenesis.
• Praslicka, B., Harmson, J. S., Kim, J., Rangaraj, V. R., Ooi, A., & Gissendanner, C. R. (2017). Binding Site Analysis of the Caenorhabditis elegans NR4A Nuclear Receptor NHR-6 During Development. Nuclear receptor research. doi:10.11131/2017/101288
  More info

  Members of the NR4A subfamily of nuclear receptors make up a highly conserved, functionally diverse group of transcription factors implicated in a multitude of cellular processes such as proliferation, differentiation, apoptosis, metabolism and DNA repair. The gene nhr-6, which encodes the sole C. elegans NR4A nuclear receptor homolog, has a critical role in organogenesis and regulates the development of the spermatheca organ system. Our previous work revealed that nhr-6 is required for spermatheca cell divisions in late L3 and early L4 and spermatheca cell differentiation during the mid L4 stage. Here, we utilized chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify NHR-6 binding sites during both the late L3/early L4 and mid L4 developmental stages. Our results revealed 30,745 enriched binding sites for NHR-6, ~70% of which were within 3 kb upstream of a gene transcription start site. Binding sites for a cohort of candidate target genes with probable functions in spermatheca organogenesis were validated through qPCR. Reproductive and spermatheca phenotypes were also evaluated for these genes following a loss-of-function RNAi screen which revealed several genes with critical functions during spermatheca organogenesis. Our results uncovered a complex nuclear receptor regulatory network whereby NHR-6 regulates multiple cellular processes during spermatheca organogenesis.
• Tian, W., Rojo de la Vega, M., Schmidlin, C. J., Ooi, A., & Zhang, D. D. (2017). Kelch-like ECH-associated protein 1 (KEAP1) differentially regulates nuclear factor erythroid-2-related factors 1 and 2 (NRF1 and NRF2). The Journal of biological chemistry.
  More info

  Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2, and mediates ubiquitylation and degradation of NRF2 through its Neh2 domain. Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1+/+ than in KEAP1-/- isogenic cell lines, while NRF2 is dramatically stabilized in KEAP1-/- cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.
• Harder, B., Tian, W., La Clair, J. J., Tan, A. C., Ooi, A., Chapman, E., & Zhang, D. D. (2016). Brusatol overcomes chemoresistance through inhibition of protein translation. Molecular carcinogenesis.
  More info

  The NRF2 pathway activates a cell survival response when cells are exposed to xenobiotics or are under oxidative stress. Therapeutic activation of NRF2 can also be used prior to insult as a means of disease prevention. However, prolonged expression of NRF2 has been shown to protect cancer cells by inducing the metabolism and efflux of chemotherapeutics, leading to both intrinsic and acquired chemoresistance to cancer drugs. This effect has been termed the "dark side" of NRF2. In an effort to combat this chemoresistance, our group discovered the first NRF2 inhibitor, the natural product brusatol, however the mechanism of inhibition was previously unknown. In this report, we show that brusatol's mode of action is not through direct inhibition of the NRF2 pathway, but through the inhibition of both cap-dependent and cap-independent protein translation, which has an impact on many short-lived proteins, including NRF2. Therefore, there is still a need to develop a new generation of specific NRF2 inhibitors with limited toxicity and off-target effects that could be used as adjuvant therapies to sensitize cancers with high expression of NRF2.
• Kerins, M. J., Ooi, A., & Praslicka, B. J. (2016). The complex role of NRF2 in cancer: A genomic view. Current Opinion in Toxicology, 1, 37-45. doi:10.1016/j.cotox.2016.09.003
  More info

  Abstract Heterotrophs are regularly exposed to harmful xenobiotics from their diet and from environmental exposure. As such, systems to counteract the negative consequences brought on by this exposure have evolved since the dawn of heterotrophic nutrition. In mammalian cells, the CNC transcription factor, NRF2, plays a crucial role in counteracting the harmful effects of xenobiotics exposure. In response to increased electrophilic and oxidative stresses, NRF2 upregulates a wide array of target genes involved in various cellular processes to restore cellular homeostasis. Consequently, NRF2 activation is traditionally regarded as cancer preventative and cytoprotective. However, recent cancer genome sequencing efforts have uncovered a significant overrepresentation of somatic mutations that drive a sustained NRF2 activation phenotype in cancer. These mutations share similar features to somatic mutations in tumor suppressors and oncogenes, indicating that NRF2 activation is positively selected for during tumor growth. Additionally, genome wide determination of NRF2 target genes identified new target genes involved in proliferation and differentiation, metabolism, apoptosis, and DNA damage repair. NRF2 activation in cancer cells has also been correlated with chemo- and radio- therapy resistance and an overall poorer patient prognosis. Taken together, these characteristics of NRF2 function have established a need to better understand the full scope of the NRF2 regulatory network. In this review we discuss recent discoveries in the NRF2 field that were enabled by advances in genomic and computational biology methods and outline possible future endeavors.
• Knatko, E. V., Praslicka, B., Higgins, M., Evans, A., Purdie, K. J., Harwood, C. A., Proby, C. M., Ooi, A., & Dinkova-Kostova, A. T. (2016). Whole-Exome Sequencing Validates a Preclinical Mouse Model for the Prevention and Treatment of Cutaneous Squamous Cell Carcinoma. Cancer prevention research (Philadelphia, Pa.), 10(1), 67-75.
  More info

  Cutaneous squamous cell carcinomas (cSCC) are among the most common and highly mutated human malignancies. Solar UV radiation is the major factor in the etiology of cSCC. Whole-exome sequencing of 18 microdissected tumor samples (cases) derived from SKH-1 hairless mice that had been chronically exposed to solar-simulated UV (SSUV) radiation showed a median point mutation (SNP) rate of 155 per Mb. The majority (78.6%) of the SNPs are C.G>T.A transitions, a characteristic UVR-induced mutational signature. Direct comparison with human cSCC cases showed high overlap in terms of both frequency and type of SNP mutations. Mutations in Trp53 were detected in 15 of 18 (83%) cases, with 20 of 21 SNP mutations located in the protein DNA-binding domain. Strikingly, multiple nonsynonymous SNP mutations in genes encoding Notch family members (Notch1-4) were present in 10 of 18 (55%) cases. The histopathologic spectrum of the mouse cSCC that develops in this model resembles very closely the spectrum of human cSCC. We conclude that the mouse SSUV cSCCs accurately represent the histopathologic and mutational spectra of the most prevalent tumor suppressors of human cSCC, validating the use of this preclinical model for the prevention and treatment of human cSCC. Cancer Prev Res; 10(1); 67-75. ©2016 AACR.
• Cutcutache, I., Suzuki, Y., Tan, I. B., Ramgopal, S., Zhang, S., Ramnarayanan, K., Gan, A., Lee, H. H., Tay, S. T., Ooi, A., Ong, C. K., Bolthouse, J. T., Lane, B. R., Anema, J. G., Kahnoski, R. J., Tan, P., Teh, B. T., & Rozen, S. G. (2015). Exome-wide Sequencing Shows Low Mutation Rates and Identifies Novel Mutated Genes in Seminomas. European urology, 68(1), 77-83.
  More info

  Testicular germ cell tumors are the most common cancer diagnosed in young men, and seminomas are the most common type of these cancers. There have been no exome-wide examinations of genes mutated in seminomas or of overall rates of nonsilent somatic mutations in these tumors.
• Perrier-Trudova, V., Huimin, B. W., Kongpetch, S., Huang, D., Ong, P., LE Formal, A., Poon, S. L., Siew, E. Y., Myint, S. S., Gad, S., Gardie, B., Couvé, S., Foong, Y. M., Choudhury, Y., Poh, J., Ong, C. K., Toh, C. K., Ooi, A., Richard, S., , Tan, M., et al. (2015). Fumarate Hydratase-deficient Cell Line NCCFH1 as a New In Vitro Model of Hereditary Papillary Renal Cell Carcinoma Type 2. Anticancer research, 35(12), 6639-53.
  More info

  Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare autosomal dominant disorder characterized by fumarate hydratase (FH) gene mutation. It is associated with the development of very aggressive kidney tumors, characterized by early onset and high metastatic potential, and has no effective therapy. The aim of the study was to establish a new preclinical platform for investigating morphogenetic and metabolic features, and alternative therapy of metastatic hereditary papillary renal cell carcinoma type 2 (PRCC2).
• Sandhu, I. S., Maksim, N. J., Amouzougan, E. A., Gallion, B. W., Raviele, A. L., & Ooi, A. (2015). Sustained NRF2 activation in hereditary leiomyomatosis and renal cell cancer (HLRCC) and in hereditary tyrosinemia type 1 (HT1). Biochemical Society transactions, 43(4), 650-6.
  More info

  The nuclear erythroid 2-like 2 transcription factor (NRF2), is a major regulator of cellular redox balance. Although NRF2 activation is generally regarded as beneficial to human health, recent studies have identified that sustained NRF2 activation is over-represented in many cancers. This raises the question regarding the role of NRF2 activation in the development and progression of those cancers. This review focuses on the mechanisms and the effects of NRF2 activation in two hereditary cancer predisposition syndromes: hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary tyrosinemia type 1 (HT1). Because the cancer initiating mutations in these hereditary syndromes are well defined, they offer a unique opportunity to explore the roles of NRF2 activation in the early stages of carcinogenesis. Over the years, a variety of approaches have been utilized to study the biology of HLRCC and HT1. In HLRCC, in vitro studies have demonstrated the importance of NRF2 activation in sustaining cancer cell proliferation. In the mouse model of HT1 however, NRF2 activation seems to protect cells from malignant transformation. In both HT1 and HLRCC, NRF2 activation promotes the clearance of electrophilic metabolites, enabling cells to survive cancer-initiating mutations. Biological insights gained from the hereditary syndromes' studies may shed light on to the roles of NRF2 activation in sporadic tumours.
• Chapman, E., Elisa, B. H., Guinea, R. D., Jiang, T., Ooi, A., Tao, S., Tian, W., Wu, T., & Zhang, D. D. (2014). P8 - Nrf2 REGULATION AND ITS DUAL ROLE IN CANCER. Free Radical Biology and Medicine, 76, S6. doi:10.1016/j.freeradbiomed.2014.10.018
• Tao, S., Wang, S., Moghaddam, S. J., Ooi, A., Chapman, E., Wong, P. K., & Zhang, D. D. (2014). Oncogenic KRAS confers chemoresistance by upregulating NRF2. Cancer research, 74(24), 7430-41.
  More info

  Oncogenic KRAS mutations found in 20% to 30% of all non-small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically, KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in a regulatory region in exon 1 of NRF2. In a mouse model of mutant KrasG12D-induced lung cancer, we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Cotreatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS mutations with NRF2 pathway inhibitors.
• Wong, M. H., Tan, C. S., Lee, S. C., Yong, Y., Ooi, A. S., Ngeow, J., & Tan, M. H. (2014). Potential genetic anticipation in hereditary leiomyomatosis-renal cell cancer (HLRCC). Familial cancer, 13(2), 281-9.
  More info

  Hereditary leiomyomatosis-renal cell cancer (HLRCC) is an autosomal dominant disorder characterised by cutaneous leiomyomas, symptomatic uterine leiomyomas and aggressive type II papillary renal cell carcinoma. It is caused by heterozygous mutations in the fumarate hydratase (FH) gene on chromosome 1q43. We present evidence of genetic anticipation in HLRCC syndrome. A comprehensive literature review was performed to determine the potential for genetic anticipation in HLRCC syndrome. The normal random effects model was used to evaluate for genetic anticipation to ensure reduction in bias. A total of 11 FH kindreds with available multi-generational data were identified for analysis. The mean difference in age at diagnosis of RCC between the first and second generation was -18.6 years (95 % CI -26.6 to -10.6, p 
• Ooi, A., Dykema, K., Ansari, A., Petillo, D., Snider, J., Kahnoski, R., Anema, J., Craig, D., Carpten, J., Teh, B., & Furge, K. A. (2013). CUL3 and NRF2 mutations confer an NRF2 activation phenotype in a sporadic form of papillary renal cell carcinoma. Cancer research, 73(7), 2044-51.
  More info

  Sustained activation of the stress-regulated transcription factor NRF2 (NFE2L2) is a prominent feature of many types of cancer, implying that mutations driving NRF2 may be important to tumor progression. In hereditary type 2 papillary renal cell carcinoma (PRCC2, also known as hereditary leiomyomatosis and renal cell cancer), NRF2 activation is a direct consequence of the accumulation of intracellular fumarate, a result of fumarate hydratase (FH) inactivation, but it is not clear how NRF2 may be activated in sporadic forms of PRCC2. Here we show that somatic mutations in NRF2, CUL3, and SIRT1 are responsible for driving the NRF2 activation phenotype in sporadic PRCC2. Transcriptome sequencing revealed the expression pattern of mutant alleles of NRF2, CUL3, and SIRT1 and also confirmed NRF2 activation in clinical specimens. Our results show a convergence in somatic mutations in sporadic PRCC2 with FH mutation in hereditary PRCC2.
• Chong, L. Y., Cheok, P. Y., Tan, W., Thike, A. A., Allen, G., Ang, M. K., Ooi, A. S., Tan, P., Teh, B. T., & Tan, P. H. (2012). Keratin 15, transcobalamin I and homeobox gene Hox-B13 expression in breast phyllodes tumors: novel markers in biological classification. Breast cancer research and treatment, 132(1), 143-51.
  More info

  Breast phyllodes tumors are rare neoplasms which present challenges for histological classification. Microscopic features are not always predictive of clinical behavior, and scarce data exist on the prognostic role of biological markers. Our study evaluated a series of 145 phyllodes tumors diagnosed at the Department of Pathology, Singapore General Hospital between 2006 and 2009, incorporating 91 (62.8%) benign, 40 (27.6%) borderline, and 14 (9.7%) malignant phyllodes tumors. Antibodies to keratin 15 (KRT15), transcobalamin I (TCN1), and homeobox gene Hox-B13 (HOXB13) were applied to sections cut from tissue microarray blocks. KRT15 and TCN1 positivity was defined when there was reactivity of 1% or more stromal cells, while HOXB13 positivity was defined using a H-score of 100 and above. Positive immunohistochemical expression for KRT15, TCN1, and HOXB13 was seen in 21 (14.5%), 96 (66.2%), and 66 (45.5%) of tumors, respectively. Stromal expression of KRT15, TCN1, and HOXB13 was significantly correlated with tumor grade (P < 0.001, P < 0.001, P = 0.012), stromal hypercellularity (P = 0.005, P < 0.001, P = 0.023), mitotic activity (P < 0.001), and microscopic borders (P = 0.006, P < 0.001, P = 0.011). Co-expression of TCN1 and HOXB13 was seen in 21 of 91 (23.1%) benign, 18 of 40 (45.0%) borderline, and 11 of 14 (78.6%) malignant tumors, suggesting that the dual-marker panels of TCN1 and HOXB13 might be helpful in classifying borderline and malignant tumors. Although expression of TCN1 alone was present in all malignant and 34 of 40 (85.0%) borderline tumors, a combined panel with HOXB13 excluded some benign cases and was a better discriminant for a significant proportion of borderline and malignant tumors.
• Furge, K. A., Ooi, A., Richard, S., Teh, B. T., Whitten, D., Yang, X. J., & Zhou, M. (2012). Abstract 1134: Fumarate activates NRF2 in tissues with fumarate hydratase mutation. Cancer Research, 72, 1134-1134. doi:10.1158/1538-7445.am2012-1134
  More info

  Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Fumarate hydratase (FH) mutation is associated with hereditary type 2 papillary renal cell carcinoma. Recently, we as well as others found that nuclear factor (erythroid-derived 2)-like 2 (NRF2) activation is a dominant feature of FH mutation. We demonstrate that in hereditary type 2 papillary renal cell carcinoma, fumarate covalently modified KEAP1 and leads to its ubiquitination. Consequently, NRF2 become activated. This predicts that similar mode of NRF2 activation may also be present in other tissues with FH mutation; overexpression of NRF2 target genes in various FH mutated tissues support the prediction that NRF2 is activated in these tissues. Variation in NRF2 targets expression may account for phenotypic differences among tumors associated with FH mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1134. doi:1538-7445.AM2012-1134
• Lee, S. Y., Chao-Nan, Q., Seng, O. A., Peiyi, C., Bernice, W. H., Swe, M. S., Chii, W. J., Jacqueline, H. S., & Chee, S. K. (2012). Changes in specialized blood vessels in lymph nodes and their role in cancer metastasis. Journal of translational medicine, 10, 206.
  More info

  High endothelial venules (HEV) have been recognized to play a role in metastasis by its changes seen in the cancer microenvironment of lymph nodes (LN) and solid cancers. Squamous cell carcinoma (SCC) of the tongue is a prevalent tumor of the head and neck region with high propensity for LN metastasis. The extent of LN metastasis is the most reliable adverse prognostic factor. Primary tumors can induce vasculature reorganization within sentinel LN before the arrival of tumor cells and HEV represents these remodelled vessels. This study aims to evaluate the cancer induced vascular changes in regional lymph nodes (LN) of patients by studying the morphological and functional alterations of HEV and its correlation with clinical outcome and pathological features.
• Lee, S. Y., Qian, C. N., Ooi, A. S., Chen, P., Tan, V. K., Chia, C. S., Hwang, J. S., Teh, B. T., & Soo, K. C. (2012). 2011 Young Surgeon's Award Winner: high endothelial venules: a novel prognostic marker in cancer metastasis and the missing link?. Annals of the Academy of Medicine, Singapore, 41(1), 21-8.
  More info

  The extent of lymph nodes (LNs) metastasis is a major determinant for the staging and the most reliable adverse prognostic factor. Primary tumours can induce lymphatics and vasculature reorganisations within sentinel LN before the arrival of cancer cells and these key blood vessels are identified as high endothelial venules (HEV). The alterations of HEV in the presence of cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift of HEV from immune response mediator to blood flow carrier. We aim to evaluate tumour-induced vascularisation in regional LN of cancer patients by studying the morphological and functional alterations of HEV and its correlation to clinico-pathological features.
• Ong, C. K., Subimerb, C., Pairojkul, C., Wongkham, S., Cutcutache, I., Yu, W., McPherson, J. R., Allen, G. E., Ng, C. C., Wong, B. H., Myint, S. S., Rajasegaran, V., Heng, H. L., Gan, A., Zang, Z. J., Wu, Y., Wu, J., Lee, M. H., Huang, D., , Ong, P., et al. (2012). Exome sequencing of liver fluke-associated cholangiocarcinoma. Nature genetics, 44(6), 690-3.
  More info

  Opisthorchis viverrini-related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini-related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8-3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini-related CCA.
• Ooi, A., & Furge, K. A. (2012). Fumarate hydratase inactivation in renal tumors: HIF1α, NRF2, and "cryptic targets" of transcription factors. Chinese journal of cancer, 31(9), 413-20.
  More info

  Biallelic inactivation of fumarate hydratase(FH) causes type 2 papillary renal cell carcinoma (PRCC2), uterine fibroids, and cutaneous leimyomas, a condition known as hereditary leiomyomatosis and renal cell cancer(HLRCC). The most direct effect of FH inactivation is intracellular fumarate accumulation. A majority of studies on FH inactivation over the past decade have focused on the theory that intracellular fumarate stabilizes hypoxia-inducible factor 1α(HIF1A) through competitive inhibition of HIF prolyl hydroxylases. Recently, a competing theory that intracellular fumarate activates nuclear factor (erythroid-derived 2)-like 2(NRF2) through post-translational modification of its negative regulator. Kelch-like ECH-associated protein 1(KEAP1) has emerged from a computational modeling study and mouse model studies. This review dissects the origin of these two governing theories and highlights the presence of chromatin-structure-regulated targets of transcription factors, which we refer to as "cryptic targets" of transcription factors. One such cryptic target is heme oxygenase I(HMOX1), the expression of which is known to be modulated by the gene product of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4, also known as BRG1).
• Wondergem, B., Zhang, Z., Huang, D., Ong, C. K., Koeman, J., Hof, D. V., Petillo, D., Ooi, A., Anema, J., Lane, B., Kahnoski, R. J., Furge, K. A., & Teh, B. T. (2012). Expression of the PTTG1 oncogene is associated with aggressive clear cell renal cell carcinoma. Cancer research, 72(17), 4361-71.
  More info

  The pituitary tumor transforming gene (PTTG1) is a recently discovered oncogene implicated in malignant progression of both endocrine and nonendocrine malignancies. Clear cell renal cell carcinoma (ccRCC) is cytogenetically characterized by chromosome 3p deletions that harbor the ccRCC-related von Hippel-Lindau, PBRM1, BAP1, and SETD2 tumor suppressor genes, along with chromosome 5q amplifications where the significance has been unclear. PTTG1 localizes to the chromosome 5q region where amplifications occur in ccRCC. In this study, we report a functional role for PTTG1 in ccRCC tumorigenesis. PTTG1 was amplified in ccRCC, overexpressed in tumor tissue, and associated with high-grade tumor cells and poor patient prognosis. In preclinical models, PTTG1 ablation reduced tumorigenesis and invasion. An analysis of gene expression affected by PTTG1 indicated an association with invasive and metastatic disease. PTTG1-dependent expression of the RhoGEF proto-oncogene ECT2 was observed in a number of ccRCC cell lines. Moreover, ECT2 expression correlated with PTTG1 expression and poor clinical features. Together, our findings reveal features of PTTG1 that are consistent with its identification of an oncogene amplified on chromsome 5q in ccRCC, where it may offer a novel therapeutic target of pathologic significance in this disease.
• Ang, M. K., Ooi, A. S., Thike, A. A., Tan, P., Zhang, Z., Dykema, K., Furge, K., Teh, B. T., & Tan, P. H. (2011). Molecular classification of breast phyllodes tumors: validation of the histologic grading scheme and insights into malignant progression. Breast cancer research and treatment, 129(2), 319-29.
  More info

  Phyllodes tumors of the breast are rare fibroepithelial neoplasms with a potential for recurrence. Current histological classification is not always predictive of clinical behavior. The aim of this study was to identify genetic changes associated with the development of borderline and malignant phyllodes tumors in an Asian population, and to assess if genetic data supported the categorization of these tumors into the existing three grades of benign, borderline, and malignant. Expression profiling of 21 phyllodes tumors (6 benign, 10 borderline, 5 malignant) was performed using Affymetrix U133Plus 2.0 GeneChips(®). Gene expression among benign, borderline, and malignant tumors was compared and a 29 gene list was able to classify them according to their histologic grade. Among these 29 genes are those responsible for matrix formation, cell adhesion, epidermis formation, and cell proliferation. Comparative genomic microarray analysis showed that the most common chromosomal alteration associated with borderline and malignant tumors was 1q gain, and an increasing number of chromosomal changes was noted with increasing histological grade. Upregulation of HOXB13 was seen in malignant relative to borderline phyllodes tumors and further investigated by immunohistochemistry in a corresponding set of formalin-fixed, paraffin-embedded tumors. HOXB13 protein overexpression was found to be correlated with stromal hypercellularity and atypia (P = 0.03, P = 0.039, respectively) and may be implicated in the development of malignant phyllodes tumors.
• Furge, K. A., Hm, B. W., Ooi, A., Perrier-trudova, V., Petillo, D., Richard, S., Roossien, D., Tan, M., Teh, B. T., Wong, J., Yang, X. J., Zhang, Z., & Zhou, M. (2011). Abstract 3820: Deregulation of KEAP1-NRF axis in phenotypically type 2 papillary renal cell carcinoma. Cancer Research, 71, 3820-3820. doi:10.1158/1538-7445.am2011-3820
  More info

  Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Type 2 papillary renal cell carcinoma (PRCC2) is an aggressive subtype of kidney cancer that has no known effective treatment modality. The hereditary form of the tumor is known to associate with the loss of fumarate hydratase (FH) gene function. However, FH gene mutation has yet to be found in sporadic PRCC2. Herein, we report the overexpression of antioxidant response element (ARE) controlled genes as a common feature that is shared between the hereditary and sporadic form of PRCC2. We demonstrate that fumarate stabilizes NRF1 and NRF2, which drive the upregulation of ARE controlled genes. We propose that fumarate stabilizes NRF1 and NRF2 through direct suppression of KEAP1. This finding better explains the phenotypical differences that are apparent between PRCC2 and CCRCC and may lead to the development of an effective therapeutic against PRCC2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3820. doi:10.1158/1538-7445.AM2011-3820
• Li, X., Ong, C. K., Cao, Y., Xiang, Y., Shao, J., Ooi, A., Peng, L., Lu, W., Zhang, Z., Petillo, D., Qin, L., Bao, Y., Zheng, F., Chia, C. S., Iyer, N. G., Kang, T., Zeng, Y., Soo, K. C., Trent, J. M., , Teh, B. T., et al. (2011). Serglycin is a theranostic target in nasopharyngeal carcinoma that promotes metastasis. Cancer research, 71(8), 3162-72.
  More info

  Nasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparative genomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed the serglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibition of serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducing metastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motile behavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorly metastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting a critical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression of vimentin but not other epithelial-mesenchymal transition proteins. In clinical specimens, serglycin expression was elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated the prognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patients followed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorable independent indicator of distant metastasis-free and disease-free survival. Our findings establish that glycosylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as an independent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients.
• Ooi, A., Wong, J., Petillo, D., Roossien, D., Perrier-Trudova, V., Whitten, D., Min, B. W., Tan, M., Zhang, Z., Yang, X. J., Zhou, M., Gardie, B., Molinié, V., Richard, S., Tan, P. H., Teh, B. T., & Furge, K. A. (2011). An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma. Cancer cell, 20(4), 511-23.
  More info

  Fumarate hydratase (FH) mutation causes hereditary type 2 papillary renal cell carcinoma (PRCC2). The main effect of FH mutation is fumarate accumulation. The current paradigm posits that the main consequence of fumarate accumulation is HIF-α stabilization. Paradoxically, FH mutation differs from other HIF-α stabilizing mutations, such as VHL and SDH mutations, in its associated tumor types. We identified that fumarate can directly up-regulate antioxidant response element (ARE)-controlled genes. We demonstrated that aldo-keto reductase family 1 member B10 (AKR1B10) is an ARE-controlled gene and is up-regulated upon FH knockdown as well as in FH null cell lines. AKR1B10 overexpression is also a prominent feature in both hereditary and sporadic PRCC2. This phenotype better explains the similarities between hereditary and sporadic PRCC2.
• Tan, S., Ooi, A., Ang, M., Koh, M., Wong, J., Dykema, K., Ngeow, J., Loong, S., Gatter, K., Tan, L., Lim, L., Furge, K., Tao, M., Lim, S., Loong, F., Cheah, P., & Teh, B. (2011). Nuclear expression of MATK is a novel marker of type II enteropathy-associated T-cell lymphoma. Leukemia, 25(3). doi:10.1038/leu.2010.295
• Ang, P. C., Ooi, A. S., Poon, E., Sim, H. G., Tan, P. H., Tan, M. H., Tan, H. L., Tay, M. H., Teh, B. T., Toh, C. K., Wong, C. F., & Yang, J. (2010). A unique pair of monozygotic twins with concordant clear cell renal cell carcinoma: a case report.. Annals of the Academy of Medicine, Singapore, 39(1), 61-3.
  More info

  Genetic predisposition to clear cell renal cell carcinoma (ccRCC) has been linked to disorders such as von Hippel-Lindau (VHL) syndrome. While twin research is a classic approach for elucidating genetic and environmental contributions to disease, no monozygotic twin-pair concordant for ccRCC in the absence of VHL syndrome has been previously reported in the literature or in major twin registries..We describe a unique monozygotic twin-pair concordant for ccRCC, with discordant but early ages of onset of 25 and 38 respectively. Cytogenetic studies and direct sequencing for VHL gene mutations in the second twin proved unremarkable..This is the fi rst reported case of monozygotic twins concordant for ccRCC in the absence of VHL gene mutation. The early yet discordant, age of onset of disease in both twins suggests both genetic and environmental contributions to ccRCC.
• Huang, D. C., Ong, C. K., Ooi, A. S., Qian, M. C., Teh, B. T., Wong, J. J., & Wong, B. H. (2010). Abstract 3314: Side population in renal cell carcinoma cell line is enriched with drug resistant, but not tumor initiating cells. Cancer Research, 70, 3314-3314. doi:10.1158/1538-7445.am10-3314
  More info

  Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Several lines of evidences suggested the presence of tumor initiating cells in renal cell carcinoma (RCC) and side population (SP) may be enriched with these “cancer stem cells (CSC)”. Herein we demonstrated the presence of Side population in 10 RCC cell lines ranging from 0.19 - 36.1%. Lineage tracing experiments showed that the SP can be enriched through sequential sorting and can differentiate into non-SP (NSP) which constitutes the bulk of the cells. The NSP can also formed back the original population of the SP in CRL1611 cells. Interestingly, the presence of NSP seem to prevent the SP from differentiating into NSP. The SP cells have about 15% higher proliferation rate and 50% greater colony forming efficiency which are reported characteristics of CSC. SP expresses higher level of cMyc, but lower level of CD105 as compared to the NSP. On the contrary, there is no difference in tumorigenic potential between SP and NSP using both correlation and in vivo studies. However, we verified that SP are 10% more resistance to mTOR inhibitor, RAD001 which coincide with higher expression of drug resistance membrane transporters ABCB1 and ABCG2 in SP cells. Treatment with RAD001 increases the amount of SP concomitantly up regulate ABCB1 and ABCC1 significantly. Taken together, Hoechst dye exclusion did not define tumor initiating cells, but is a valuable technique for identifying drug resistance cells in RCC. 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 3314.
• Hwang, S. G., Lee, S. Y., Ooi, A. S., Qian, C., Soo, K. C., & Teh, B. T. (2010). Abstract 2238: A novel prognostic marker in patients with squamous cell carcinoma of the tongue: high endothelial venules. Cancer Research, 70, 2238-2238. doi:10.1158/1538-7445.am10-2238
  More info

  AIMS: To evaluate tumor-induced vascularization in regional lymph nodes (LN) of patients with tongue carcinoma by studying the morphological and functional alterations of high endothelial venules (HEV) and its correlation to clinico-pathological features. BACKGROUND: Squamous cell carcinoma (SCC) of the tongue is one of the most prevalent tumors of the head and neck region. The extent of LN metastasis is a major determinant for the staging and the most reliable adverse prognostic factor. Primary tumors can induce lymphatics and vasculature reorganizations within sentinel LN before the arrival of cancer cells and these key blood vessels are identified as HEV. The morphological altera[[Unsupported Character - Codename s]]tion of HEV in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift of HEV from immune response mediator to blood-flow carrier. METHODS: This study is based on 65 consecutive patients with tongue SCC who underwent primary surgical treatment at Singapore General Hospital from January 2001 to December 2005. The patients were categorized into 2 groups (Cases and Controls) based on the presence of malignancy in their cervical lymph nodes. A review of the patients’ pathological and clinical data was performed from a prospective database. Immunohistochemical staining of the tissue blocks for HEV and high-power-field image analysis were performed and analyzed with correlation to the patients’ clinico-pathological features. RESULTS: The total number of HEVs is significantly associated to disease-free interval when controlling for the group (p=0.022) as well as combining both groups as one cohort (p=0.023). There also a similar association comparing the HEV parameters to overall survival. CONCLUSION: Our results suggest that the HEV possibly plays a key role in the pathogenesis of lymphatic and subsequent distant metastases. Confirmation of this hypothesis would offer a novel therapeutic approach to preventing metastasis by blocking the remodeling processes of HEV in LN. 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 2238.
• Tan, F. L., Ooi, A., Huang, D., Wong, J. C., Qian, C., Chao, C., Ooi, L., Tan, Y., Chung, A., Cheow, P., Zhang, Z., Petillo, D., Yang, X. J., & Teh, B. T. (2010). p38delta/MAPK13 as a diagnostic marker for cholangiocarcinoma and its involvement in cell motility and invasion. International journal of cancer. Journal international du cancer, 126(10), 2353-61.
  More info

  Cholangiocarcinoma (CC) and hepatocellularcarcinoma (HCC) are two main forms of liver malignancies, which exhibit differences in drug response and prognosis. Immunohistotochemical staining for cytokeratin markers has been used to some success in the differential diagnosis of CC from HCC. However, there remains a need for additional markers for increased sensitivity and specificity of diagnosis. In this study, we have identified a p38 MAP kinase, p38delta (also known as MAPK13 or SAPK4) as a protein that is upregulated in CC relative to HCC and to normal biliary tract tissues. We performed microarray gene expression profiling on 17 cases of CC, 12 cases of adjacent normal liver tissue, and three case of normal bile duct tissue. p38delta was upregulated in 16 out of 17 cases of CC relative to normal tissue. We subsequently performed immunohistochemical staining of p38delta in 54 cases of CC and 54 cases of HCC. p38delta staining distinguished CC from HCC with a sensitivity of 92.6% and a specificity of 90.7%. To explore the possible functional significance of p38delta expression in CC, we examined the effects of overexpression and knockdown of p38delta expression in human CC cell lines. Our results indicate that p38delta is important for motility and invasion of CC cells, suggesting that p38delta may play an important role in CC metastasis. In summary, p38delta may serve as a novel diagnostic marker for CC and may also serve as a new target for molecular based therapy of this disease.
• Tan, M., Yang, J., Tan, H., Wong, C., Tan, P., Sim, H., Ang, P., Toh, C., Tay, M., Poon, E., Ooi, A., & Teh, B. (2010). A unique pair of monozygotic twins with concordant clear cell renal cell carcinoma: A case report. Annals of the Academy of Medicine Singapore, 39(1). doi:10.47102/annals-acadmedsg.v39n1p61
  More info

  Introduction: Genetic predisposition to clear cell renal cell carcinoma (ccRCC) has been linked to disorders such as von Hippel-Lindau (VHL) syndrome. While twin research is a classic approach for elucidating genetic and environmental contributions to disease, no monozygotic twin-pair concordant for ccRCC in the absence of VHL syndrome has been previously reported in the literature or in major twin registries. Clinical Picture: We describe a unique monozygotic twin-pair concordant for ccRCC, with discordant but early ages of onset of 25 and 38 respectively. Cytogenetic studies and direct sequencing for VHL gene mutations in the second twin proved unremarkable. Conclusions: This is the first reported case of monozygotic twins concordant for ccRCC in the absence of VHL gene mutation. The early yet discordant, age of onset of disease in both twins suggests both genetic and environmental contributions to ccRCC.
• Tan, F., Ooi, A., Huang, D., Wong, J. C., Qian, C. N., Chao, C., Ooi, L., Tan, Y. M., Chung, A. Y., Cheow, P. C., Zhang, Z., Petillo, D., Yang, X. J., & Teh, B. T. (2009). p38delta/MAPK13 as a diagnostic marker for cholangiocarcinoma and its involvement in cell motility and invasion. International Journal of Cancer. doi:10.1002/ijc.24944
  More info

  Abstract Cholangiocarcinoma (CC) and hepatocellularcarcinoma (HCC) are two main forms of liver malignancies, which exhibit differences in drug response and prognosis. Immunohistotochemical staining for cytokeratin markers has been used to some success in the differential diagnosis of CC from HCC. However, there remains a need for additional markers for increased sensitivity and specificity of diagnosis. In this study, we have identified a p38 MAP kinase, p38δ (also known as MAPK13 or SAPK4) as a protein that is upregulated in CC relative to HCC and to normal biliary tract tissues. We performed microarray gene expression profiling on 17 cases of CC, 12 cases of adjacent normal liver tissue, and three case of normal bile duct tissue. p38δ was upregulated in 16 out of 17 cases of CC relative to normal tissue. We subsequently performed immunohistochemical staining of p38δ in 54 cases of CC and 54 cases of HCC. p38δ staining distinguished CC from HCC with a sensitivity of 92.6% and a specificity of 90.7%. To explore the possible functional significance of p38δ expression in CC, we examined the effects of overexpression and knockdown of p38δ expression in human CC cell lines. Our results indicate that p38δ is important for motility and invasion of CC cells, suggesting that p38δ may play an important role in CC metastasis. In summary, p38δ may serve as a novel diagnostic marker for CC and may also serve as a new target for molecular based therapy of this disease.
• Teoh, P., Ooi, A., AbuBakar, S., & Othman, R. Y. (2009). Virus-specific read-through codon preference affects infectivity of chimeric cucumber green mottle mosaic viruses displaying a dengue virus epitope. Journal of biomedicine & biotechnology, 2009, 781712.
  More info

  A Cucumber green mottle mosaic virus (CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct.
• Ooi, A., Tan, S., Mohamed, R., Rahman, N. A., & Othman, R. Y. (2006). The full-length clone of cucumber green mottle mosaic virus and its application as an expression system for Hepatitis B surface antigen. Journal of biotechnology, 121(4), 471-81.
  More info

  A cucumber green mosaic mottle virus (CGMMV) full-length clone was developed for the expression of Hepatitis B surface antigen (HBsAg). The expression of the surface displayed HBsAg by the chimeric virus was confirmed through a double antibody sandwich ELISA. Assessment of the coat protein composition of the chimeric virus particles by SDS-PAGE analysis showed that 50% of the coat proteins were fused to the HBsAg. Biological activity of the expressed HBsAg was assessed through the stimulation of in vitro antibody production by cultured peripheral blood mononuclear cells (PBMC). PBMC that were cultured in the presence of the chimeric virus showed up to an approximately three-fold increase in the level of anti HBsAg immunoglobulin thus suggesting the possible use of this new chimeric virus as an effective Hepatitis B vaccine.

Reviews

• Praslicka, B. J., Kerins, M. J., & Ooi, A. (2016. The complex role of NRF2 in cancer: A genomic view(pp 37-45).

Case Studies

• Tan, M. H., Yang, J., Tan, H. L., Wong, C. F., Tan, P. H., Sim, H. G., Ang, P., Toh, C. K., Tay, M. H., Poon, E., Ooi, A. S., & Teh, B. T. (2010. A unique pair of monozygotic twins with concordant clear cell renal cell carcinoma: a case report(pp 61-3).
  More info

  Genetic predisposition to clear cell renal cell carcinoma (ccRCC) has been linked to disorders such as von Hippel-Lindau (VHL) syndrome. While twin research is a classic approach for elucidating genetic and environmental contributions to disease, no monozygotic twin-pair concordant for ccRCC in the absence of VHL syndrome has been previously reported in the literature or in major twin registries.