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XAF1 enhances temozolomide induced autophagic cell death through AMPK signaling pathway
abstracts
40P
Vascular endothelial growth factor in colorectal cancer pathology, survival and treatment
L. Baker, N. Robinson, D. Wilson, M. Tabaqchali, D. Leaper Surgery, North Tees and Hartlepool NHS Foundation Trust, Stockton On Tees, UK Background: Angiogenesis (the growth of new blood vessels) is important for tumour invasion and metastasis in order for a tumour to grow beyond a few millimetres in size. Vascular endothelial growth factor (VEGF) is thought to be the predominant angiogenic factor in colorectal and other cancers. As a result, numerous studies have looked at VEGF expression in colorectal cancer patients and several therapeutic agents have been trialled that target the VEGF pathway. Methods: VEGF levels were determined in 100 paired tumour and normal colorectal tissue samples (1998-2001) and 75 pre-operative plasma samples (2000-2002) by ELISA. Tissue (pg/mg protein) and plasma VEGF levels (pg/ml) were correlated with the tumour pathology. Survival analysis was performed for disease-free and overall 15year survival (Kaplan Meier, p < 0.05). The study had ethics approval. Results: VEGF levels were significantly up-regulated in colorectal tumour tissue compared to normal tissue. The median VEGF levels were 288 (range, 30-4,156) pg/mg protein for tumour, 38 (0-833) for normal colon and 90 (0-1,262) pg/ml for pre-operative plasma samples. Tumour levels of VEGF correlated with the tumour depth, differentiation and whether the tumour had undergone lymphatic invasion e.g. well differentiated, 230 (79-1,348), moderate 392 (30-4,156) and poorly differentiated 685 (882,612)pg/mg protein. Plasma VEGF levels significantly correlated with tumour differentiation and vascular invasion,e.g. 211 (14-889) vascular invasion and 80 (0-785)pg/ ml, no vascular invasion. The 15-year survival status for patients with tissue samples was 30% were alive and well, 1% was alive with recurrence and 69% had died. For the 75 plasma patients; 27% patients were alive and well with the remaining 67% having died. VEGF levels in plasma but not tissue samples significantly correlated with both disease-free and overall 15-year survival, with patients with low plasma VEGF levels having a better survival outcome (p < 0.05, Kaplan Meier). Conclusions: VEGF levels in both colorectal tumour tissue and pre-operative plasma samples correlated with the tumour pathology. However, only pre-operative plasma VEGF levels correlated with disease-free and overall 15-year survival. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
and high LCSCAFX expression serve as an independent prognostic marker in patients with NSCLC. Importantly, genetic depletion of LCSCAFX combined chemotherapy dramatically inhibited NSCLC progression in patient-derived xenograft (PDX) model. Conclusions: These findings demonstrated a previously unappreciated role of LCSCAFX in the regulation of LCSCs, assigning LCSCAFX as a promising therapeutic target for lung cancer treatments. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
43P
M. Lee, K-P. Ko, S-G. Chi College of Life Science, Korea University, Seoul, Republic of Korea Background: XIAP-associated factor 1 (XAF1) is a pro-apoptotic tumor suppressor whose expression is inactivated in many human malignancies. To explore the XAF1’s candidacy for a suppressor in the pathogenesis of human glioma, we investigated its expression and function in tumor cell lines and tissues. Methods: Expression study was performed using quantitative RT-PCR and immunoblot assays. Functional interplay between XAF1 and AMPK was determined by gene transfection, siRNA-mediated depletion. Results: XIAP-associated factor 1 (XAF1) is a pro-apoptotic tumor suppressor whose expression is inactivated in many human malignancies. In this study, we explored the XAF1’s candidacy for a suppressor in human glioma pathogenesis. XAF1 reduction is more common in high grade tumors versus low grade tumors and tightly associated with aberrant hypermethylation at 7 CpG sites in the 5’ proximal region of the promoter. XAF1 expression decreases proliferation and colony-forming ability of glioma cells while its depletion enhances cellular resistance to genotoxic drugs, such as temozolomide (TMZ), etoposide and cisplatin. The XAF1 promoter is activated in response to TMZ through JNK-IRF-1 signaling and its activation greatly increases cellular response to TMZ-induced cell death. Furthermore, XAF1 promotes autophagic cell death (ACD) by activating AMP-activated protein kinase (AMPK) in a XIAP-independent manner. Both AMPK-activating and ACD-inducing effects of XAF1 are linked to its activity to decrease intracellular ATP level, oxygen consumption, and mitochondrial membrane potential. XAF1 proteins translocate to the mitochondria and the zinc finger (ZF) 6 domain is essential for its mitochondrial distribution. Consistently, a mutant XAF1 lacking the ZF6 fails to decrease ATP level, activate AMPK, and trigger AMPKmediated autophagic cell death. Collectively, this study demonstrates that epigenetic inactivation of XAF1 contributes to the malignant progression of human glioma by rendering tumor cells a survival advantage via the attenuation of AMPK signaling. Conclusions: Epigenetic inactivation of XAF1 contributes to the malignant progression of human glioma by rendering tumor cells a survival advantage via the attenuation of AMPK signaling. Legal entity responsible for the study: The authors. Funding: National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2018R1D1A1B07041512). Disclosure: All authors have declared no conflicts of interest.
44P 41P
LCSCAF1 maintains cancer stem-like traits by stabilizing c-Myc protein and promotes metastasis and recurrence in lung cancer
T. Guo, L. Zhao, S. Zhao, C. Gu Department of Thoracic Surgery, 1st Affiliated Hospital of Dalian Medical University, Dalian, China Background: Aberrantly overexpressed lung cancer stem cell associated factor-X (LCSCAFX) has been implicated in non-small cell lung cancer (NSCLC) development. Methods: To explore the effect of LCSCAFXon the lung cancer stem-like traitsin vitro and in vivo. Results: We reported that enforced expression of LCSCAFX promoted NSCLC cells invasion and metastasis, drug resistance and recurrencein vitro and in vivo, whereas depletion of LCSCAFX suppressed metastasis and relapse in NSCLC. Moreover, we found that LCSCAFX expression was elevated in lung cancer stem cells (LCSCs). Indeed, knockdown of LCSCAFX impaired lung cancer stem-like traits. Conversely, ectopic overexpression of LCSCAFX enhanced the self-renewal ability of lung cancer cells in vitro. In addition, elevated LCSCAFX robustly enhanced tumor initiating frequencies, as well as growth rates of NSCLC cells derived tumor xenografts in immunodeficient mice. Furthermore, mechanistic investigations established that LCSCAFX stabilized c-Myc protein by suppressing its ubiquitylation and proteasomal degradation, which is required for lung cancer stem-like properties maintenance. Consistently, immunohistochemical analysis of clinical lung tumor tissues showed that LCSCAFX was elevated in NSCLC tissues
v12 | Basic Science
XAF1 enhances temozolomide induced autophagic cell death through AMPK signaling pathway
The effect of cortisol on methylation patterns in breast cancer cell lines
H. Intabli1, M.S. Flint1, A. Qattan2, M. Allen1, M. Yeoman1 Pharmacy and Biomolecular Sciences, University of Brighton-Moulsecoomb Campus, Brighton, UK, 2Molecular Oncology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
1
Background: Epigenetic changes are highly responsive to environmental changes, including stress. The release of stress hormones; such as glucocorticoids, in response to stress have been shown to induce epigenetic modifications in neuronal cells. However, the role of glucocorticoids on epigenetic changes underlying important processes such as cell cycle regulation, apoptosis, and proliferation in breast cancer are not yet established. Furthermore, it is not known if cortisol can induce irreversible epigenetic changes on these cellular processes and whether these changes relate to the duration of the stress response. In this study, cortisol-induced epigenetic changes and the potential involvement of DNA methylation was assessed. Methods: We analyzed the expression levels of maintenance DNA methyltrasferase (DNMT1) in MDA-MB-231, Hs-578T, MCF7, and T47D breast cancer cell lines by real-time PCR. We also used Qiagen Epitect Methyl ll Complete PCR array for Tumour Suppressor genes to analyse the level of methylation in 94 tumour suppressor genes. The methylation level on the Long Interspersed Nuclear Element (LINE-1) was used as surrogate marker for global DNA methylation. Results: Our results show that cortisol significantly decreased the expression of DNMT1 in the triple negative cells lines MDA-MB-231 (p < 0.005), and Hs-578T (p < 0.05). We also showed that cortisol induced aberrant methylation characterised by loss of methylation on promoter regions of key tumour suppressor genes in MDA-MB-
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz238.041/5576654 by guest on 26 October 2019
Results: RT-PCR analysis showed that LION is highly expressed in several cancer cells such as non-small cell lung cancer, cervical cancer and colorectal cancer compared with normal lung fibroblasts. Silencing LION by siRNA oligonucleotides inhibits the proliferation of H1299, HCT116 and HeLa cells. Q-RT-PCR analysis showed that silencing LION increases the p15 and p16 mRNA, suggesting that LION is involved in the transcriptional repression of INK4 locus. Cell cycle analysis showed that silencing LION causes G2/M phase arrest in cell cycle, suggesting that LION functions to promote G2/ M transition. Conclusions: LION is involved in the promotion of cancer cells proliferation such as H1299 and HCT116 cells via regulating p15, p16 and other genes related to G2/M phase control. Legal entity responsible for the study: The authors. Funding: JSPS KAKENHI. Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology
40P
Vascular endothelial growth factor in colorectal cancer pathology, survival and treatment
L. Baker, N. Robinson, D. Wilson, M. Tabaqchali, D. Leaper Surgery, North Tees and Hartlepool NHS Foundation Trust, Stockton On Tees, UK Background: Angiogenesis (the growth of new blood vessels) is important for tumour invasion and metastasis in order for a tumour to grow beyond a few millimetres in size. Vascular endothelial growth factor (VEGF) is thought to be the predominant angiogenic factor in colorectal and other cancers. As a result, numerous studies have looked at VEGF expression in colorectal cancer patients and several therapeutic agents have been trialled that target the VEGF pathway. Methods: VEGF levels were determined in 100 paired tumour and normal colorectal tissue samples (1998-2001) and 75 pre-operative plasma samples (2000-2002) by ELISA. Tissue (pg/mg protein) and plasma VEGF levels (pg/ml) were correlated with the tumour pathology. Survival analysis was performed for disease-free and overall 15year survival (Kaplan Meier, p < 0.05). The study had ethics approval. Results: VEGF levels were significantly up-regulated in colorectal tumour tissue compared to normal tissue. The median VEGF levels were 288 (range, 30-4,156) pg/mg protein for tumour, 38 (0-833) for normal colon and 90 (0-1,262) pg/ml for pre-operative plasma samples. Tumour levels of VEGF correlated with the tumour depth, differentiation and whether the tumour had undergone lymphatic invasion e.g. well differentiated, 230 (79-1,348), moderate 392 (30-4,156) and poorly differentiated 685 (882,612)pg/mg protein. Plasma VEGF levels significantly correlated with tumour differentiation and vascular invasion,e.g. 211 (14-889) vascular invasion and 80 (0-785)pg/ ml, no vascular invasion. The 15-year survival status for patients with tissue samples was 30% were alive and well, 1% was alive with recurrence and 69% had died. For the 75 plasma patients; 27% patients were alive and well with the remaining 67% having died. VEGF levels in plasma but not tissue samples significantly correlated with both disease-free and overall 15-year survival, with patients with low plasma VEGF levels having a better survival outcome (p < 0.05, Kaplan Meier). Conclusions: VEGF levels in both colorectal tumour tissue and pre-operative plasma samples correlated with the tumour pathology. However, only pre-operative plasma VEGF levels correlated with disease-free and overall 15-year survival. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
and high LCSCAFX expression serve as an independent prognostic marker in patients with NSCLC. Importantly, genetic depletion of LCSCAFX combined chemotherapy dramatically inhibited NSCLC progression in patient-derived xenograft (PDX) model. Conclusions: These findings demonstrated a previously unappreciated role of LCSCAFX in the regulation of LCSCs, assigning LCSCAFX as a promising therapeutic target for lung cancer treatments. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
43P
M. Lee, K-P. Ko, S-G. Chi College of Life Science, Korea University, Seoul, Republic of Korea Background: XIAP-associated factor 1 (XAF1) is a pro-apoptotic tumor suppressor whose expression is inactivated in many human malignancies. To explore the XAF1’s candidacy for a suppressor in the pathogenesis of human glioma, we investigated its expression and function in tumor cell lines and tissues. Methods: Expression study was performed using quantitative RT-PCR and immunoblot assays. Functional interplay between XAF1 and AMPK was determined by gene transfection, siRNA-mediated depletion. Results: XIAP-associated factor 1 (XAF1) is a pro-apoptotic tumor suppressor whose expression is inactivated in many human malignancies. In this study, we explored the XAF1’s candidacy for a suppressor in human glioma pathogenesis. XAF1 reduction is more common in high grade tumors versus low grade tumors and tightly associated with aberrant hypermethylation at 7 CpG sites in the 5’ proximal region of the promoter. XAF1 expression decreases proliferation and colony-forming ability of glioma cells while its depletion enhances cellular resistance to genotoxic drugs, such as temozolomide (TMZ), etoposide and cisplatin. The XAF1 promoter is activated in response to TMZ through JNK-IRF-1 signaling and its activation greatly increases cellular response to TMZ-induced cell death. Furthermore, XAF1 promotes autophagic cell death (ACD) by activating AMP-activated protein kinase (AMPK) in a XIAP-independent manner. Both AMPK-activating and ACD-inducing effects of XAF1 are linked to its activity to decrease intracellular ATP level, oxygen consumption, and mitochondrial membrane potential. XAF1 proteins translocate to the mitochondria and the zinc finger (ZF) 6 domain is essential for its mitochondrial distribution. Consistently, a mutant XAF1 lacking the ZF6 fails to decrease ATP level, activate AMPK, and trigger AMPKmediated autophagic cell death. Collectively, this study demonstrates that epigenetic inactivation of XAF1 contributes to the malignant progression of human glioma by rendering tumor cells a survival advantage via the attenuation of AMPK signaling. Conclusions: Epigenetic inactivation of XAF1 contributes to the malignant progression of human glioma by rendering tumor cells a survival advantage via the attenuation of AMPK signaling. Legal entity responsible for the study: The authors. Funding: National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2018R1D1A1B07041512). Disclosure: All authors have declared no conflicts of interest.
44P 41P
LCSCAF1 maintains cancer stem-like traits by stabilizing c-Myc protein and promotes metastasis and recurrence in lung cancer
T. Guo, L. Zhao, S. Zhao, C. Gu Department of Thoracic Surgery, 1st Affiliated Hospital of Dalian Medical University, Dalian, China Background: Aberrantly overexpressed lung cancer stem cell associated factor-X (LCSCAFX) has been implicated in non-small cell lung cancer (NSCLC) development. Methods: To explore the effect of LCSCAFXon the lung cancer stem-like traitsin vitro and in vivo. Results: We reported that enforced expression of LCSCAFX promoted NSCLC cells invasion and metastasis, drug resistance and recurrencein vitro and in vivo, whereas depletion of LCSCAFX suppressed metastasis and relapse in NSCLC. Moreover, we found that LCSCAFX expression was elevated in lung cancer stem cells (LCSCs). Indeed, knockdown of LCSCAFX impaired lung cancer stem-like traits. Conversely, ectopic overexpression of LCSCAFX enhanced the self-renewal ability of lung cancer cells in vitro. In addition, elevated LCSCAFX robustly enhanced tumor initiating frequencies, as well as growth rates of NSCLC cells derived tumor xenografts in immunodeficient mice. Furthermore, mechanistic investigations established that LCSCAFX stabilized c-Myc protein by suppressing its ubiquitylation and proteasomal degradation, which is required for lung cancer stem-like properties maintenance. Consistently, immunohistochemical analysis of clinical lung tumor tissues showed that LCSCAFX was elevated in NSCLC tissues
v12 | Basic Science
XAF1 enhances temozolomide induced autophagic cell death through AMPK signaling pathway
The effect of cortisol on methylation patterns in breast cancer cell lines
H. Intabli1, M.S. Flint1, A. Qattan2, M. Allen1, M. Yeoman1 Pharmacy and Biomolecular Sciences, University of Brighton-Moulsecoomb Campus, Brighton, UK, 2Molecular Oncology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
1
Background: Epigenetic changes are highly responsive to environmental changes, including stress. The release of stress hormones; such as glucocorticoids, in response to stress have been shown to induce epigenetic modifications in neuronal cells. However, the role of glucocorticoids on epigenetic changes underlying important processes such as cell cycle regulation, apoptosis, and proliferation in breast cancer are not yet established. Furthermore, it is not known if cortisol can induce irreversible epigenetic changes on these cellular processes and whether these changes relate to the duration of the stress response. In this study, cortisol-induced epigenetic changes and the potential involvement of DNA methylation was assessed. Methods: We analyzed the expression levels of maintenance DNA methyltrasferase (DNMT1) in MDA-MB-231, Hs-578T, MCF7, and T47D breast cancer cell lines by real-time PCR. We also used Qiagen Epitect Methyl ll Complete PCR array for Tumour Suppressor genes to analyse the level of methylation in 94 tumour suppressor genes. The methylation level on the Long Interspersed Nuclear Element (LINE-1) was used as surrogate marker for global DNA methylation. Results: Our results show that cortisol significantly decreased the expression of DNMT1 in the triple negative cells lines MDA-MB-231 (p < 0.005), and Hs-578T (p < 0.05). We also showed that cortisol induced aberrant methylation characterised by loss of methylation on promoter regions of key tumour suppressor genes in MDA-MB-
Volume 30 | Supplement 5 | October 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz238.041/5576654 by guest on 26 October 2019
Results: RT-PCR analysis showed that LION is highly expressed in several cancer cells such as non-small cell lung cancer, cervical cancer and colorectal cancer compared with normal lung fibroblasts. Silencing LION by siRNA oligonucleotides inhibits the proliferation of H1299, HCT116 and HeLa cells. Q-RT-PCR analysis showed that silencing LION increases the p15 and p16 mRNA, suggesting that LION is involved in the transcriptional repression of INK4 locus. Cell cycle analysis showed that silencing LION causes G2/M phase arrest in cell cycle, suggesting that LION functions to promote G2/ M transition. Conclusions: LION is involved in the promotion of cancer cells proliferation such as H1299 and HCT116 cells via regulating p15, p16 and other genes related to G2/M phase control. Legal entity responsible for the study: The authors. Funding: JSPS KAKENHI. Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology