Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo

OBJECTIVE To discover a small molecule targeting ULK1-modulated cell death of triple negative breast cancer and exploreits potential mechanisms.METHODS ULK1 expression was analyzed by The Cancer Genome Atlas(TCGA)analysis and tissue microarray(TMA)analysis.ULK1agonist was designed by using in silico screening,as well as modified by chemical synthesis and screened by kinase and anti-proliferative activities.The amino acid residues that key to the activation site of LYN-1604 were determined by site-directed mutagenesis,as well as in vitro kinase assay and ADP-Glo kinase assay.The mechanisms of LYN-1604 induced cell death were investigated by fluorescence microscope,western blotting,flow cytometry analysis,immunocytochemistry,as well as si RNA and GFP-m RFP-LC3 plasmid transfections.Potential ULK1 interactors were discovered by performing comparative microarray analysis and the therapeutic effect of LYN-1604 was assessed by xenograft breast cancer mouse model.RESULTS We found that ULK1 was remarkably downregulated in breast cancer tissue samples,especial y in triple negative breast cancer(TNBC).32 candidate smal molecules were synthesized,and we discovered a small molecule named LYN-1604 as the best candidate ULK1agonist.Additionally,we identified that three amino acid residues(LYS50,LEU53 and TYR89)were key to the activation site of LYN-1604 and ULK1.Subsequently,we demonstrated that LYN-1604 could induce autophagy-associated cell death via ULK complex(ULK1-m ATG13-FIP200-ATG101)in MDA-MB-231 cells.We also found that LYN-1604 induced cell death involved in ATF3,RAD21 and caspase 3,accompanied with autophagy and apoptosis.Moreover,we demonstrated that LYN-1604 had a good therapeutic potential on TNBC by targeting ULK1-modulated cell death in vivo.CONCLUSION We discovered a small molecule(LYN-1604)has therapeutic potential by targeting ULK1-modulated cell death associated with autophagy and apoptosis of TNBC in vitro and in vivo,which could be utilized as a new anti-TNBC drug candidate.

Inhibition of growth and metastasis of triple-negative breast cancer targeted by Traditional Chinese Medicine Tubeimu in orthotopic mice models

Objective： Triple-negative breast cancer（TNBC） is highly invasive and metastatic, which is in urgent need of transformative therapeutics. Tubeimu（TBM）, the rhizome of Bolbostemma paniculatum（Maxim.） Franquet, is one of the Chinese medicinal herbs used for breast diseases since the ancient times. The present study evaluated the efficacy, especially the anti-metastatic effects of the dichloromethane extract of Tubeimu（ETBM） on TNBC orthotopic mouse models and cell lines.Methods： We applied real-time imaging on florescent orthotopic TNBC mice model and tested cell migration and invasion abilities with MDA-MB-231 cell line. Digital gene expression sequencing was performed and Kyoto Encyclopedia of Genes and Genomes（KEGG） analysis applied to explore the pathways influenced by ETBM.Moreover, quantitative real-time polymerase chain reactions（q RT-PCR） and Western blot were delivered to confirm the gene expression changes.Results： ETBM exhibited noticeable control on tumor metastasis and growth of TNBC tumors with no obvious toxicity. In compliance with this, it also showed inhibition of cell migration and invasion in vitro. Its impact on the changed biological behavior in TNBC may be a result of decreased expression of integrin β1（ITGβ1）, integrin β8（ITGβ8） and Rho GTPase activating protein 5（ARHGAP5）, which disabled the focal adhesion pathway and caused change in cell morphology.Conclusions： This study reveals that ETBM has anti-metastatic effects on MDA-MB-231-GFP tumor and may lead to a new therapeutic agent for the integrative treatment of highly invasive TNBC.

The effect and side effects of Gemcitabine plus Vinorelbine in patients with triple-negative metastatic breast cancer

Objective： The aim of this study was to evaluate the anti-tumor activity and safety of Gemcitabine （GEM） combined with Vinorelbine （NVB） in patients with advanced TNABC after chemotherapy. Methods： Thirty-seven patients with immunohistochemical proved TNABC were enrolled. The patients received 21-day cycles of NVB 25mg/m^2 i.v. with GEM 1000 mg/m^2 i.v. on days 1 and 8. Results： A total of 136 cycles were given to 37 patients（median 4 cycles, ranged 2-6 cycles）. The treatment response was evaluable in all patients. Of the 37 patients, 1 received complete remission （CR）, 8 received partial remission （PR）, 20 had stable disease （SD）, 9 had progressive disease （PD）. Overall objective response （CR＋ PR） were 24.3 %. The median time to progress （TTP） was 6 months （95% CI, 4-6 months）. The median overall survival was 24 months （95% CI, 11-37 months）. The median 1-year survival rate was （66.24±8.43）%. The median 3-year survival rate was （28.77±11.96）%. The major adverse events were grade Ⅰ-Ⅱ myelosuppression, peripheral neurologic toxicities, nausea and vomiting. Some patients had rash and hepatic dysfunction. A total of 40% of patients experienced flu-like symptoms. Alopecia and diarrhea were rare. Conclusion： The combination of GEM and NVB is an effective and well tolerated regimen for the patients with TNABC.

ALDH 1A1 and caveolin-1 expression in triple negative breast cancer

Objective Triple negative breast cancer(TNBC) contains a high proportion of breast cancer stem cells(BCSCs) and exhibits resistance to chemotherapy treatments. Therefore, the identification of BCSCs that are novel molecular targets may improve patient survival. Aldehyde dehydrogenase-1(ALDH 1 A1) has been considered a cancer stem cell marker in different tumors. Caveolin-1(Cav-1), a membrane transporter protein, regulates cancer chemo-resistance and stem cell signaling. Thus, the aim of this study was to evaluate the expression of ALDH 1 A1 and Cav-1 in patients with TNBC by immunohistochemistry(IHC) and to correlate their expression with clinical and pathological parameters.Methods Paraffin blocks of 30 breast cancer patients who underwent modified radical mastectomy between January 2013 and December 2016 in Zagazig University Hospitals(Egypt) were evaluated. Antibodies to ALDH 1 A1 and Cav-1 were used. Results ALDH 1 A1 and Cav-1 significantly correlated with tumor size. A significant association between ALDH 1 A1/Cav-1 IHC staining and relapse was found. Cav-1 and ALDH 1 A1-positive expression correlated with a short 3-year disease-free survival rate and a 3-year overall survival rate(P < 0.001). Conclusion ALDH 1 A1 and Cav-1 expression in TNBC was significantly positively correlated with poor clinicopathological parameters and shortened survival. Expression of both markers was significantly positively correlated with each other(P < 0.001). ALDH 1 A1 and Cav-1 could be potential therapeutic targets in breast cancer.

P53 gene could be a new effective therapeutic target in triple-negative breast cancer: a Meta-analysis

Objective： The aim of this study was to explore the relationship between p53 gene and triple-negative breast cancer （TNBC）, and determine that whether p53 gene could be a new effective therapeutic target. Methods： We identified studies with quantitative data on the relation of p53 gene and TNBC through searching 12 databases online （Oct. 1999-Oct. 2012） and reviewing the references, which were written in English or Chinese. Summary estimates of odds ratio （OR） was calculated using the fixed-effects model or the random-effects model as appropriate. Results： We identified 12 eligible stud- ies with 1532 cases of TNBC patients and 6329 controls of non-TNBC patients. The test for homogeneity resulted in X^2 = 200.16 （P 〈 0.05）, it showed significant heterogeneity so that a random effect model was applied. Our results showed that the expression of p53 gene could be much stronger in TNBC group than that in non-TNBC group [OR = 2.10, 95% confidence interval （CI） = 1.21-3.65]. In ethnicity-subgroup analysis, we found that in Caucasian group, the expression of p53 gene were stronger in TNBC group （OR = 2.60, 95% CI = 1.21-5.57）, but there was no statistical significance in Asian group （OR = 1.69, 95% CI = 0.83-3.45）. Conclusion： P53 gene could be an effective predictor and a good therapeutic target for TNBC patients in the future, especially in Caucasian. Further researches focusing on p53 gene would gain a breakthrough in the treatment of TNBC.

COX-2与MVD在三阴性乳腺癌中的表达及相关性研究

目的:研究COX-2在三阴性乳腺癌中的表达及意义,探讨COX-2与CD31标记的新生微血管密度(MVD)的相关性。方法:采用免疫组化法检测60例三阴性乳腺癌及518例非三阴性乳腺癌组织石蜡标本中的COX-2和MVD的表达情况,并对三阴性乳腺癌与非三阴性乳腺癌的临床病理特征进行比较。结果:①与非三阴性乳腺癌组相比,三阴性乳腺癌组患者年龄偏轻、绝经前患者比例明显升高;三阴性乳腺癌组织分化程度低,组织学分级为Ⅲ级的比例明显升高;②COX-2在三阴性乳腺癌及非三阴性乳腺癌组织中的阳性率分别为73.3%、58.0%,两者存在差异(P<0.05);③三阴性乳腺癌的MVD为183.73±25.64,非三阴性乳腺癌组织中MVD为168.84±24.07,两者间具有显著差异(P<0.01);④COX-2与MVD在三阴性乳腺癌中的表达具有显著相关性(P<0.01)。结论:三阴性乳腺癌组织分化差,恶性程度高,COX-2表达和MVD在三阴性乳腺癌中显著增高,两者可能共同参与三阴性乳腺癌的发生、发展,检测COX-2和MVD可作为反映三阴性乳腺癌临床及病理学特点的参考指标,同时可考虑将COX-2作为三阴性乳腺癌治疗的新靶点。

三阴性乳腺癌与基底细胞样乳腺癌临床病理分析

目的研究CK5/6、CK14在三阴性乳腺癌(triple negativebreast carcinoma,TNBC)中的表达情况,并探讨基底细胞样乳腺癌(basal-like breast carcinoma,BLBC)与TNBC的关系。方法利用免疫组化方法从浸润性乳腺癌中筛选TNBC病例,然后利用CK5/6和CK14从TNBC中筛选出BLBC的病例,分析两者的临床与病理资料及免疫组化表达情况并复习有关文献。结果TNBC的发病率占浸润性乳腺癌的16.1%。TNBC中的CK5/6和CK14表达有正相关性(γ=0.463)。应用CK5/6、CK14从TNBC中筛选出的BLBC的百分率为54%。结论BLBC与TNBC有大部分交叉。CK5/6和CK14可以用来从TNBC中筛选出大部分的BLBC的病例。BLBC相对其他类型乳腺癌,预后不良,有必要将其从TN-BC中鉴别出来。

PTEN、p53和BAG-1在三阴乳腺癌中的表达及意义

为了研究PTEN、p53和BAG-1在三阴乳腺癌中的表达情况及临床病理意义,并讨论三者之间的关系。采用免疫组织化学SP法检测89例三阴乳腺癌中PTEN、p53、BAG-1的表达,其阳性表达率分别为44.9%、47.2%、73.0%,三者的表达均与患者的病理组织学分级、淋巴结转移情况有关(P<0.05)。PTEN的表达与p53、BAG-1表达呈负相关;BAG-1与p53的表达呈正相关,提示三者在三阴乳腺癌中的表达存在一定关联性,临床上联合检测三者可能为TNBC预后评估、患者的个体化治疗提供一定的参考。

TAC与TP方案治疗三阴性乳腺癌的临床观察

目的比较新辅助化疗TAC与TP方案治疗三阴性乳腺癌(TNBC)的疗效和安全性。方法 102例三阴性乳腺癌患者均经病理组织学确诊,分为TAC组(52例)和TP组(50例)。TAC组:多西他赛75mg/m2或紫杉醇(紫杉醇脂质体)135mg/m2iv,d1;吡柔比星40mg/m2或表柔比星75mg/m2iv,d2;环磷酰胺600mg/m2iv,d1。TP组:多西他赛75mg/m2或紫杉醇(紫杉醇脂质体)135mg/m2iv,d1;顺铂30mg/m2iv,d2～d4。21天为1周期,化疗2～4个周期后行手术治疗。评价两组近期疗效和毒副反应。结果 TAC组获pCR 5例(9.6%),PR 35例(67.3%),SD 9例(17.3%),RR为76.9%;TP组获pCR 4例(8.0%),PR 32例(64.0%),SD 5例(10.0%),RR为72.0%,两组pCR率和RR差异均无统计学意义(P>0.05)。102例患者中12例经2个周期化疗后肿瘤进展,其中TAC组3例,TP组9例。TAC组有2例发生房性前期收缩,TP组3例发生2级肾功能损伤。TAC组3～4级血液学毒性和脱发的发生率明显高于TP组,但TP组的3～4级胃肠道反应发生率高于TAC组。结论 TAC方案与TP方案在三阴性乳腺癌新辅助化疗中均具有一定疗效,不良反应尚可耐受。