- Email: [email protected]
Development of MDA-MB-231-3D-Spheroid as a reliable model for studying Nav1.5 and nNav1.5-mediated breast cancer metastasis
abstracts Conclusions: The schedule of 2-week administration period followed by 1-week rest seems to be safe and effective for primary treatment of metastatic and recurrent breast cancer. Legal entity responsible for the study: Kobe Breast Cancer Oncology Group. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
Development of MDA-MB-231-3D-Spheroid as a reliable model for studying Nav1.5 and nNav1.5-mediated breast cancer metastasis
A.H. Murtadha, N.F. Mokhtar Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Kubang Kerian, Malaysia Background: Nav1.5 and nNav1.5 are potent metastatic markers for breast cancer with aggressive phenotype; detected in tumour tissues positive for lymph node metastasis. In vitro, most of the studies involving Nav1.5 and nNav1.5 were evaluated in monolayer cultured cells. Subsequently, since 3D-spheroid culture is now known for its ability to mimic multiple in vivo tumour microenvironment, there is a need to re-evaluate the potential role of Nav1.5 and nNav1.5 in a reliable tumour-accurate model. This study aimed to develop MDA-MB-231-3D-spheroid culture and assess the mRNA gene expression of Nav1.5 and nNav1.5 including several other potent markers for aggressive breast cancer, MMP1, MMP13 and fibronectin. Methods: MDA-MB-231-3D-spheroids were cultured using scaffold-free liquid overlay method (via agarose layered 96-well flat bottomed plate) for 15 days. The total RNA was conventionally extracted and converted to cDNA. The mRNA expression for Nav1.5 and nNav1.5, MMP1, MMP13 and fibronectin were measured using real-time PCR and the expression were analysed via the 2-DDCt method in which the monolayer culture was used as a normalizing control. Results: MDA-MB-231-3D-spheroid was successfully developed. mRNA expression of Nav1.5, nNav1.5, MMP1 and fibronectin were significantly higher (p < 0.05) in MDAMB-231-3D-spheroid compared to the monolayer culture; 4.8-fold, 2.1-fold, 62.1-fold and 5.3-fold, respectively. Conclusions: Evaluating the role of tumour markers in a reliable tumour-accurate model is very important. MDA-MB-231-3D-spheroid was able to increase the expression of Nav1.5 and nNav1.5 and can be utilized to re-evaluate the role of Nav1.5 and nNav1.5 in cancer metastasis. Legal entity responsible for the study: Institute for Research in Molecular Medicine, Universiti Sains Malaysia. Funding: Universiti Sains Malaysia, FRGS (203/CIPPM/6171212). Disclosure: All authors have declared no conflicts of interest.
49P
Biochemical study on modifying role of variants of leptin gene and its receptor on serum leptin levels in breast cancer
A.M. Alhanafy1, S. Tayel2, S. Ahmed3, A. Altorgoman3, I. Elsayed3 1 Clinical oncology and nuclear medicine, Faculty of Medicine - Menoufia University, Sheben Elkom, Egypt, 2Medical Biochemistry and Molecular Biology, Faculty of Medicine - Menoufia University, Sheben Elkom, Egypt, 3Chemistry Department, Faculty of Science - Menoufia University, Sheben Elkom, Egypt Background: Leptin is produced by adipose tissue of breast and is overexpressed in Breast cancer. Association of leptin gene (LEP) and leptin receptor gene (LEPR) polymorphisms with Breast cancer are inconsistent. Only a very few studies have examined this biochemical and genetic variables so we planned to investigate this association and its impact on leptin level and disease characteristics. Methods: Study included 70 females (40 women with pathological prove of invasive Breast cancer patients and 30 controls). Staging was done according to the American Joint Committee on Cancer 8th edition. LEP and LEPR polymorphisms were estimated by real time PCR. Serum leptin was measured by ELISA. Results: Both LEPR rs 1137101 and LEP rs 7799039 increase risk of breast cancer where GG genotype and G allele frequencies of LEPR rs 1137101 were significantly higher in patients than control [52.5% vs 16.7% and 68.8% vs 36.7%] (p ¼ 0.002) and (p < 0.001) respectively. GG genotype increase risk of BC with [OR 9.1: 95% CI 2.3035.94] while G allele predispose to disease with [OR 3.8: 95% CI 1.87 – 7.70]. Furthermore, LEP rs 7799039 A allele was markedly elevated in patients (61.2%) than healthy ones (43.3%) (p ¼ 0.037) with [OR 2.06: 95% CI 1.05 – 4.08]. Leptin levels were markedly elevated than healthy ones (p < 0.001). Conclusions: Results clarified interesting relation of circulating leptin level and LEP and LEPR polymorphisms with predicting Breast cancer that may be implicated in pathogenesis of disease. Legal entity responsible for the study: Menoufia University. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
ix18 | Breast cancer, metastatic
50P
Prognostic factors of recurrence or distant metastasis in elderly breast cancer patients
S. Lee Breast Center, Pusan National University Yangsan Hospital, Yangsan, Republic of Korea Background: As we enter an aging society, the number of elderly breast cancer patients is increasing. We assessed the prognostic factors of recurrence or metastasis for breast cancer patients over the age of 65 by analyzing the characteristics of tumor and longterm clinical outcomes. Methods: Data were retrospectively analyzed for 286 breast cancer patients aged 65 years and older who underwent treatment in the Pusan national university hospital and Pusan national university Yangsan hospital from 2008 to 2014. Dividing two groups as the patients with event such as recurrence or metastasis and those who without it, the Cox regression model was used to examine the risk factors of recurrence or metastasis. Results: Among 286 patients with invasive breast cancer, forty three patients (15.0%) have been found to have recurrence or distant metastasis during median follow-up period of 61 months. The poor prognosis was found in the advanced stage of breast cancer, patients without endocrine therapy and low Allred scores of estrogen receptor.
Table: 50P Variable
Hazard ratio
P-value
95% CI
Stage Endocrine therapy Estrogen receptor
1.78 0.04 1.34
<0.0001 0.0002 0.0076
(1.39, 2.28) (0.01, 0.23) (1.08, 1.66)
Conclusions: The advanced stage of breast cancer was poor prognosis in the elderly breast cancer patients. Moreover, endocrine therapy should be performed on elderly breast cancer patients with high Allred score of estrogen receptor because it is good for prognosis. Legal entity responsible for the study: The author. Funding: Has not received any funding. Disclosure: The author has declared no conflicts of interest.
51P
Enhancing the anti-breast tumour activity of STING through a novel sting transcriptional regulator
H. Xiong, W. Zheng, X-F. Yu Cancer Institution of Zhejiang University, The Second Affiliated Hospital - Zhejiang University School of Medicine, Hangzhou, China Background: STING (STimulator of INterferon Genes) is a pattern recognition receptor detecting cytoplasmic nucleic acids and transmits signals that activate host innate immune responses. It has been found to be involved in anti-microbial immunity, autoimmune disorder and tumorigenesis. Thus, the acknowledgement of the cellular regulation of STING is important. However, transcriptional regulation of STING and its role in tumor development has not been reported. Methods: STNF expressing vector was constructed and used to identify the STNF regulatory function of STING. Small interfering RNA was used to silence STNF expression. For the first time, full length of STING promoter was constructed with luciferase reporter which enabled quick and semi-quantitative of STING promoter activity. The bc-GenExMiner v4.2 database was used to evaluate the expression and prognostic merit of STNF and STING in breast cancer (BC). Western blot, RT-qPCR and CCK-8 assay were used to detect STING expression and BC cell growth. Results: We have identified a novel negative regulator of STING (STNF). The effect of STNF appeared to be at the transcriptional level of STING since STNF could suppress the promoter activity of STING. STNF was up-regulated in breast cancer (BC) and associated with poor prognosis of BC patients. Silencing STNF or pharmacologically inhibiting STNF promoted STING expression and suppressed BC growth. STING down-regulation was observed in different types of BC and restoration of STING expression resulted in broad BC inhibition. Conclusions: Our study demonstrated an unprecedented strategy used by breast tumor to escape from STING mediated tumor suppression. The identification of a novel STING regulator will provide insights for novel anti-tumor strategy against BC. Legal entity responsible for the study: Xiao-Fang Yu. Funding: National Youth Science Fund Project: 81701988(C0025181). Disclosure: All authors have declared no conflicts of interest.
Volume 30 | Supplement 9 | November 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_9/mdz418.011/5638769 by Macquarie University user on 08 December 2019
48P
Annals of Oncology
Development of MDA-MB-231-3D-Spheroid as a reliable model for studying Nav1.5 and nNav1.5-mediated breast cancer metastasis
A.H. Murtadha, N.F. Mokhtar Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Kubang Kerian, Malaysia Background: Nav1.5 and nNav1.5 are potent metastatic markers for breast cancer with aggressive phenotype; detected in tumour tissues positive for lymph node metastasis. In vitro, most of the studies involving Nav1.5 and nNav1.5 were evaluated in monolayer cultured cells. Subsequently, since 3D-spheroid culture is now known for its ability to mimic multiple in vivo tumour microenvironment, there is a need to re-evaluate the potential role of Nav1.5 and nNav1.5 in a reliable tumour-accurate model. This study aimed to develop MDA-MB-231-3D-spheroid culture and assess the mRNA gene expression of Nav1.5 and nNav1.5 including several other potent markers for aggressive breast cancer, MMP1, MMP13 and fibronectin. Methods: MDA-MB-231-3D-spheroids were cultured using scaffold-free liquid overlay method (via agarose layered 96-well flat bottomed plate) for 15 days. The total RNA was conventionally extracted and converted to cDNA. The mRNA expression for Nav1.5 and nNav1.5, MMP1, MMP13 and fibronectin were measured using real-time PCR and the expression were analysed via the 2-DDCt method in which the monolayer culture was used as a normalizing control. Results: MDA-MB-231-3D-spheroid was successfully developed. mRNA expression of Nav1.5, nNav1.5, MMP1 and fibronectin were significantly higher (p < 0.05) in MDAMB-231-3D-spheroid compared to the monolayer culture; 4.8-fold, 2.1-fold, 62.1-fold and 5.3-fold, respectively. Conclusions: Evaluating the role of tumour markers in a reliable tumour-accurate model is very important. MDA-MB-231-3D-spheroid was able to increase the expression of Nav1.5 and nNav1.5 and can be utilized to re-evaluate the role of Nav1.5 and nNav1.5 in cancer metastasis. Legal entity responsible for the study: Institute for Research in Molecular Medicine, Universiti Sains Malaysia. Funding: Universiti Sains Malaysia, FRGS (203/CIPPM/6171212). Disclosure: All authors have declared no conflicts of interest.
49P
Biochemical study on modifying role of variants of leptin gene and its receptor on serum leptin levels in breast cancer
A.M. Alhanafy1, S. Tayel2, S. Ahmed3, A. Altorgoman3, I. Elsayed3 1 Clinical oncology and nuclear medicine, Faculty of Medicine - Menoufia University, Sheben Elkom, Egypt, 2Medical Biochemistry and Molecular Biology, Faculty of Medicine - Menoufia University, Sheben Elkom, Egypt, 3Chemistry Department, Faculty of Science - Menoufia University, Sheben Elkom, Egypt Background: Leptin is produced by adipose tissue of breast and is overexpressed in Breast cancer. Association of leptin gene (LEP) and leptin receptor gene (LEPR) polymorphisms with Breast cancer are inconsistent. Only a very few studies have examined this biochemical and genetic variables so we planned to investigate this association and its impact on leptin level and disease characteristics. Methods: Study included 70 females (40 women with pathological prove of invasive Breast cancer patients and 30 controls). Staging was done according to the American Joint Committee on Cancer 8th edition. LEP and LEPR polymorphisms were estimated by real time PCR. Serum leptin was measured by ELISA. Results: Both LEPR rs 1137101 and LEP rs 7799039 increase risk of breast cancer where GG genotype and G allele frequencies of LEPR rs 1137101 were significantly higher in patients than control [52.5% vs 16.7% and 68.8% vs 36.7%] (p ¼ 0.002) and (p < 0.001) respectively. GG genotype increase risk of BC with [OR 9.1: 95% CI 2.3035.94] while G allele predispose to disease with [OR 3.8: 95% CI 1.87 – 7.70]. Furthermore, LEP rs 7799039 A allele was markedly elevated in patients (61.2%) than healthy ones (43.3%) (p ¼ 0.037) with [OR 2.06: 95% CI 1.05 – 4.08]. Leptin levels were markedly elevated than healthy ones (p < 0.001). Conclusions: Results clarified interesting relation of circulating leptin level and LEP and LEPR polymorphisms with predicting Breast cancer that may be implicated in pathogenesis of disease. Legal entity responsible for the study: Menoufia University. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
ix18 | Breast cancer, metastatic
50P
Prognostic factors of recurrence or distant metastasis in elderly breast cancer patients
S. Lee Breast Center, Pusan National University Yangsan Hospital, Yangsan, Republic of Korea Background: As we enter an aging society, the number of elderly breast cancer patients is increasing. We assessed the prognostic factors of recurrence or metastasis for breast cancer patients over the age of 65 by analyzing the characteristics of tumor and longterm clinical outcomes. Methods: Data were retrospectively analyzed for 286 breast cancer patients aged 65 years and older who underwent treatment in the Pusan national university hospital and Pusan national university Yangsan hospital from 2008 to 2014. Dividing two groups as the patients with event such as recurrence or metastasis and those who without it, the Cox regression model was used to examine the risk factors of recurrence or metastasis. Results: Among 286 patients with invasive breast cancer, forty three patients (15.0%) have been found to have recurrence or distant metastasis during median follow-up period of 61 months. The poor prognosis was found in the advanced stage of breast cancer, patients without endocrine therapy and low Allred scores of estrogen receptor.
Table: 50P Variable
Hazard ratio
P-value
95% CI
Stage Endocrine therapy Estrogen receptor
1.78 0.04 1.34
<0.0001 0.0002 0.0076
(1.39, 2.28) (0.01, 0.23) (1.08, 1.66)
Conclusions: The advanced stage of breast cancer was poor prognosis in the elderly breast cancer patients. Moreover, endocrine therapy should be performed on elderly breast cancer patients with high Allred score of estrogen receptor because it is good for prognosis. Legal entity responsible for the study: The author. Funding: Has not received any funding. Disclosure: The author has declared no conflicts of interest.
51P
Enhancing the anti-breast tumour activity of STING through a novel sting transcriptional regulator
H. Xiong, W. Zheng, X-F. Yu Cancer Institution of Zhejiang University, The Second Affiliated Hospital - Zhejiang University School of Medicine, Hangzhou, China Background: STING (STimulator of INterferon Genes) is a pattern recognition receptor detecting cytoplasmic nucleic acids and transmits signals that activate host innate immune responses. It has been found to be involved in anti-microbial immunity, autoimmune disorder and tumorigenesis. Thus, the acknowledgement of the cellular regulation of STING is important. However, transcriptional regulation of STING and its role in tumor development has not been reported. Methods: STNF expressing vector was constructed and used to identify the STNF regulatory function of STING. Small interfering RNA was used to silence STNF expression. For the first time, full length of STING promoter was constructed with luciferase reporter which enabled quick and semi-quantitative of STING promoter activity. The bc-GenExMiner v4.2 database was used to evaluate the expression and prognostic merit of STNF and STING in breast cancer (BC). Western blot, RT-qPCR and CCK-8 assay were used to detect STING expression and BC cell growth. Results: We have identified a novel negative regulator of STING (STNF). The effect of STNF appeared to be at the transcriptional level of STING since STNF could suppress the promoter activity of STING. STNF was up-regulated in breast cancer (BC) and associated with poor prognosis of BC patients. Silencing STNF or pharmacologically inhibiting STNF promoted STING expression and suppressed BC growth. STING down-regulation was observed in different types of BC and restoration of STING expression resulted in broad BC inhibition. Conclusions: Our study demonstrated an unprecedented strategy used by breast tumor to escape from STING mediated tumor suppression. The identification of a novel STING regulator will provide insights for novel anti-tumor strategy against BC. Legal entity responsible for the study: Xiao-Fang Yu. Funding: National Youth Science Fund Project: 81701988(C0025181). Disclosure: All authors have declared no conflicts of interest.
Volume 30 | Supplement 9 | November 2019
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_9/mdz418.011/5638769 by Macquarie University user on 08 December 2019
48P
Annals of Oncology