A molecular approach to triple-negative breast cancer: targeting the Notch signaling pathway

ABSTRACT Introduction Triple-negative breast cancer is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. This phenotype renders triple-negative breast cancer cells refractory to conventional therapies, resulting in poor clinical outcomes and an urgent need for novel therapeutic approaches. Recent studies have implicated dysregulation of the Notch receptor signaling pathway in the development and progression of triple-negative breast cancer. Objective This study aimed to conduct a comprehensive literature review to identify potential therapeutic targets of the Notch pathway. Our analysis focused on the upstream and downstream components of this pathway to identify potential therapeutic targets. Results Modulating the Notch signaling pathway may represent a promising therapeutic strategy to treat triple-negative breast cancer. Several potential therapeutic targets within this pathway are in the early stages of development, including upstream (such as Notch ligands) and downstream (including specific molecules involved in triple-negative breast cancer growth). These targets represent potential avenues for therapeutic intervention in triple-negative breast cancer. Comments Additional research specifically addressing issues related to toxicity and improving drug delivery methods is critical for the successful translation of these potential therapeutic targets into effective treatments for patients with triple-negative breast cancer.

Down-regulation of METTL5 inhibits proliferation, migration and invasion of triple-negative breast cancer cells through Wnt/6-catenin signaling pathway / 中国药理学通报

Aim To investigate the role and potential mechanism of methyltransferase-like 5 (METTL5) in triple-negative breast cancer (TNBC) . Methods The expression of METTL5 in TNBC tumor tissues and cell lines was detected by immunohistochemistry and Western blot. After shRNA targeting METTL5 (shRNAMETTL5) was transfected into TNBC cells, cell proliferation, migration and invasion were detected by CCK-8, colony formation, wound healing and Transwell assays, respectively. Western blot was used to detect the expression of Wnt/p-catenin signaling-related key proteins. A xenograft tumor model was constructed to verify the effect of METTL5 knockdown on the growth of TNBC cells and Wnt/p-catenin signaling activity in vivo. Results The expression of METTL5 was up-regulated in TNBC tumor tissues and cell lines (P < 0. 01) . Knockdown of METTL5 significantly inhibited the proliferation, migration and invasion of TNBC cells and reduced the expression of Wnt/p-catenin signaling molecules (3-catenin, cyclin Dl, matrix metalloproteinase (MMP) -2 and MMP-7 (all P < 0. 01) . Knockdown of METTL5 reduced tumor growth and Wnt/pcatenin signaling activity in vivo. Conclusions Knockdown of METTL5 can inhibit the proliferation, migration and invasion of TNBC cells, which may be related to the inhibition of Wnt/p-catenin signaling pathway.

Programmed cell death protein 1 is a marker for neoadjuvant chemotherapy response in triple-negative breast cancer

SUMMARY OBJECTIVE: Tumor-infiltrating lymphocytes are detectable in up to 75% of triple-negative breast cancer. The composition of these infiltrates may influence prognosis and is not known regarding regulatory or effector lymphocytes. The objectives of this study were to describe and quantify the composition of the tumor-infiltrating lymphocytes before and after chemotherapy (neoadjuvant chemotherapy) and to evaluate their association with complete pathological response and overall survival. METHODS: This was a retrospective observational study. Clinical and pathological data from 38 triple-negative breast cancer patients treated with neoadjuvant chemotherapy at the University Hospital (HUCFF/UFRJ), between November 2004 and November 2018, were analyzed. The Stromal tumor-infiltrating lymphocytes (Stromal tumor-infiltrating lymphocytes) have been identified on hematoxylin and eosin-stained sections according to the guidelines of the "International tumor-infiltrating lymphocytes Working Group." Immunohistochemistry studies were performed to identify T-cell subsets (i.e., CD3, CD4, CD8, and FOXP3) and T-cell exhaustion (i.e., programmed cell death protein 1). RESULTS: Statistically significant changes in stromal tumor-infiltrating lymphocyte categories were observed before and post-neoadjuvant chemotherapy, with 32% of intermediate cases becoming high. The correlation between pre-neoadjuvant chemotherapy stromal tumor-infiltrating lymphocytes and pathological response, pre-neoadjuvant chemotherapy and post-neoadjuvant chemotherapy, and stromal tumor-infiltrating lymphocytes and overall survival was not statistically significant. However, we noticed an increase of cells that favor the antitumor activity (i.e., CD3, CD8, and CD8/FOXP3 ratio) and decreased levels of cells inhibiting tumor activities (i.e., FOXP3 and programmed cell death protein 1) post-neoadjuvant chemotherapy. Importantly, programmed cell death protein 1 expression pre-neoadjuvant chemotherapy showed an association with pathological response. CONCLUSION: In this study, we observed that chemotherapy significantly increases stromal tumor-infiltrating lymphocytes, CD8 T cells, as well as CD8/FoxP3 ratio. Most importantly, programmed cell death protein 1 expression before neoadjuvant chemotherapy positively correlates with pathological response suggesting the use of programmed cell death protein 1 as a prognostic marker before neoadjuvant chemotherapy.

CLINICOPATHOLOGICAL STUDY IN TRIPLE NEGATIVE BREAST CANCER

Background and Objectives: - Carcinoma Breast is most common malignancy in females in USA and second among cases deaths in females (after lung cancer). There is considerable geographic , ethnic and racial variability in Breast cancer in evidence with about 5 fold variation throughout the world. Triple negative breast cancer is a heterogonous disease diagnosed by Immune Histo Chemistry (IHC).Triple Negative Breast cancer is characterized by tumor that do not express ER or PR and HER2neu . Proto typical Triple Negative Brest cancer is aggressive in nature and associate with poor prognosis. The Objectives of this study is to analyse the clinical and Pathological features of Triple Negative Breast Cancer and compare the result with similar studies in literature. Fifty Methods:- cases Triple negative Breast Cancer were included in this study. Clinical and pathological features and treatment were noted. Incidence Result:- of Triple Negative Breast Cancer was 35%. The median age of presentation was 45yrs. There were 4% males Triple negative Breast cancer cases out of female patients, most of patients were Pre (or) Perimenopausal(65%). 4% patients had family history of malignantly. Most common stage of presentation was stage III (46%). In Stage IV, Lung and bone metastasis was common. Ten Patients received Neoadjuvant chemo therapy (NACJ) and disease progressed in 4% while on Neoadjuvant chemotherapy, Even though 45 patients had surgery only 34 were eligible to received Adjuvant Radiotherapy. Total of 18% Patients had either progressive disease while on treatment (8%) (or) recurrence 10%. Eighteen percent patients died due to the disease. 33% patients on follow up. There were more Invasive Duct Cell carcinoma (IDCC) cases with medullary differentiations (or) Purse medullary Carcinomas (12%). No deaths Occur in the medullary variants TNBC. Majority of the tumor were high grade margins were negative in most of the cases. Incidence of Tri Inclusion:- ple negative breast cases was higher than western literature but comparable to Indian Studies. The age of Presentation was about 10 years younger than western data. Triple Negative Breast cancer was more common in young, pre (or) perimenopausal women. Small number of patients had family history, majority were state II (or) III. There was high number of progressive disease, recurrence and death while on the study (or) within less than 1 yr of treatment. Triple Negative Breast cancer is very aggressive disease with relatively better prognosis in the medullary variant Triple Negative Breast Cancer.

Carcinoma de mama triple negativo. Estudio anatomo-estadístico / Triple- negative breast cancer. An anatomical and statistical study

Introducción: El carcinoma de mama triple negativo se asocia a un comportamiento biológico más agresivo y de desfavorable pronóstico. Objetivo: Determinar la incidencia del subtipo molecular triple negativo en carcinomas mamarios y su relación con otras variables clínico-patológicas de valor pronóstico. Método: Se realizó un estudio descriptivo y retrospectivo, en el Hospital Universitario Docente «Celestino Hernández Robau» de Villa Clara, en el período comprendido de enero de 2017 a junio de 2019, con el fin de determinar la incidencia de los tumores triples negativos y su relación con las variables edad, talla tumoral, tipo y grado histológicos e índice de proliferación. Resultados: Se determinó la incidencia del subtipo molecular triple negativo en carcinomas mamarios y su relación con las formas histológicas moderada y poco diferenciadas. Conclusiones: El subtipo molecular triple negativo en carcinomas mamarios está asociado con frecuencia a: la edad posmenopáusica, el tipo histológico ductal, el grado histológico alto, altos índices de Ki-67 y talla tumoral mayor de 2 cm.

Introduction: triple-negative breast cancer has a more aggressive biological behaviour and is associated with an unfavourable prognosis. Objective: to determine the incidence of triple- negative breast cancer molecular subtypes and its relationship with other clinical and pathological variables of prognostic value. Methods: a descriptive and retrospective study was carried out at "Celestino Hernández Robau" University Teaching Hospital from Villa Clara between January 2017 and June 2019 in order to determine the incidence of triple- negative tumors and its relationship with the variables: age, tumor size, histological type and grade as well as proliferative index. Results: the incidence of triple- negative breast cancer molecular subtype and its relationship with moderate and poorly differentiated histological forms were determined. Conclusions: triple- negative breast cancer molecular subtype is frequently associated with postmenopausal age, ductal histological type, high histological grade, high Ki-67 indices and tumor size greater than 2 cm.

BRCA1 promoter methylation & its immunohistochemical correlation in sporadic breast cancer

Background & objectives: Studies have shown that apart from hereditary breast carcinomas, breast cancer susceptibility gene 1 (BRCA1) mutations conferring to its loss are seen in sporadic breast carcinomas (SBC) as well. The aim of the present study was to assess BRCA1 methylation in females presenting at King George’s Medical University, Lucknow, with SBC by both immunohistochemistry (IHC) and methylation PCR with respect to hormonal profile and various morphological prognostic parameters. The primary objective was to look for the association between BRCA1 protein expression and DNA promoter methylation. Methods: 81 mastectomy specimens from SBC of invasive breast carcinoma (no special type) were included in this study. After a detailed morphological assessment, formalin fixed paraffin embedded tissue from a representative tumour area was selected for BRCA1 IHC by heat-mediated antigen retrieval under high pH and DNA extraction and further bisulphate treatment. BRCA1 was studied for methylation by methylated and unmethylated PCR-specific primers. Results: BRCA1 promoter methylation was present in 42/81 (51.9%) participants, with significant BRCA1 protein loss (72.7%; P=0.002). A significant association between BRCA1 loss and hormonal profile was found (P=0.001); maximum in triple negative breast carcinoma (TNBC) (72%; 18/25). Most of the TNBC also harboured methylation (68%). Although not significant grade II and III tumours, lymph vascular invasion, ductal carcinoma in situ, and nodal metastasis (?3) were seen in a higher percentage in methylated tumours. Mortality in SBC was significantly associated with BRCA1 loss (30.3%; P=0.024). Interpretation & conclusions: Study results highlight the concept of “BRCAness” in SBC as well. Hence, we can confer that identification of BRCA1 loss in SBC can make it a perfect candidate for poly ADP- ribose polymerase inhibitors or cisplatin-based therapy like hereditary ones.

Inhibitory effect of chikusetsusaponin IVa on proliferation of triple negative breast cancer cells / 药学学报

Chikusetsusaponin IVa (CsIVa) is a natural active monomer of triterpene saponins in the Chinese herbal medicine of Panax japonicus, which has anti-inflammatory, anti-tumor and other effects. However, its function and mechanism in triple negative breast cancer (TNBC) remain unclear. This study investigated the inhibitory effect and mechanisms of CsIVa on the proliferation of triple negative breast cancer cell line MDA-MB-231. In this study, we found that CsIVa could significantly inhibit the proliferation of MDA-MB-231 cells and eliminate its potential toxic effect on normal breast cells (MCF-10A). The transcriptome sequencing results showed that the inhibition of proliferation of MDA-MB-231 cells by CsIVa was closely related to cell cycle and the pathway regulating cell cycle. Further studies confirmed that CsIVa blocked the cell cycle in G2/M phase by down-regulating the expression of cyclin dependent kinase 1 (CDK1), cyclin B1 and up-regulating the expression of cyclin dependent kinase inhibitor 1A (p21). Moreover, CsIVa can block cell cycle through inhibiting PI3K/AKT signal pathway. In conclusion, CsIVa regulates the expression of cell cycle related proteins (p21, CDK1, cyclin B1) via inhibiting the activity of PI3K/AKT signaling pathway, blocks TNBC cell cycle, and thus exerts its anti-tumor activity.

Study on the effects of 17-hydroxy-jolkinolide B on the proliferation and apoptosis of human triple-negative breast cancer cells / 中国药房

OBJECTIVE To study the effects of the active component 17-hydroxy-jolkinolide B （HJB） of Euphorbia fischeriana on the proliferation and apoptosis of human triple-negative breast cancers （TNBC） MDA-MB-231 and MDA-MB-468 cells. METHODS MTT assay was adopted to detect the inhibitory rate of MDA-MB-231 and MDA-MB-468 cells proliferation after treated with 0 （blank control），5，10，20，40，80 μmol/L HJB for 24， 48 and 72 h. Laser confocal microscope and flow cytometry were adopted to detect the apoptosis， mitochondrial membrane potential（MMP） and reactive oxygen species （ROS） of above 2 kinds of cells after treated with 0 （blank control）， 10，20，40 μmol/L HJB for 24 h. Western blot assay was used to detect the expressions of B cell lymphoma-2（ Bcl-2）， Bcl-2-associated X protein （Bax）， cytochrome-C （Cyt-C）， caspase-3， cleaved caspase- 3， caspase-9 and cleaved caspase-9. RESULTS Compared with blank control group， 5，10，20，40，80 μmol/L HJB could significantly increase the inhibitory rate of MDA-MB-231 and MDA-MB-468 cells proliferation （P＜0.05）， in dose- and time- dependent trend. After 24 h treatment of HJB （10，20，40 μmol/L）， the apoptosis of above 2 kinds of cells increased， and the total apoptotic rate increased significantly （P＜0.05）； the mitochondrial membrane potential decreased significantly （P＜0.05）； the level of ROS increased significantly （P＜0.05）； the protein expressions of Bcl-2， caspase-3 and caspase-9 were decreased significantly （P＜ 0.05）， while the protein expressions of Cyt-C， Bax， cleaved caspase-3 and cleaved caspase-9 were increased significantly （P＜0.05）. CONCLUSIONS HJB can inhibit the proliferation of MDA-MB-231 and MDA-MB-468 cells， and induce their apoptosis.

Silencing RAB27a inhibits proliferation, invasion and adhesion of triple-negative breast cancer cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells.@*METHODS@#Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated.@*RESULTS@#Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing.@*CONCLUSION@#RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.

Ginsenoside Rg_3 based liposomes target delivery of dihydroartemisinin and paclitaxel for treatment of triple-negative breast cancer / 中国中药杂志

Ginsenoside Rg_3, an active component of traditional Chinese medicine(TCM), was used as the substitute for cholesterol as the membrane material to prepare the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin and paclitaxel. The effect of the prepared drug-loading liposomes on triple-negative breast cancer in vitro was evaluated. Liposomes were prepared with the thin film hydration method, and the preparation process was optimized by single factor experiments. The physicochemical properties(e.g., particle size, Zeta potential, and stability) of the liposomes were characterized. The release behaviors of drugs in different media(pH 5.0 and pH 7.4) were evaluated. The antitumor activities of the liposomes were determined by CCK-8 on MDA-MB-231 and 4T1 cells. The cell scratch test was carried out to evaluate the effect of the liposomes on the migration of MDA-MB-231 and 4T1 cells. Further, the targeting ability of liposomes and the mechanism of lysosome escape were investigated. Finally, H9c2 cells were used to evaluate the potential cardiotoxicity of the preparation. The liposomes prepared were spheroid, with uniform particle size distribution, the ave-rage particle size of(107.81±0.01) nm, and the Zeta potential of(2.78±0.66) mV. The encapsulation efficiency of dihydroartemisinin and paclitaxel was 57.76%±1.38% and 99.66%±0.07%, respectively, and the total drug loading was 4.46%±0.71%. The accumulated release of dihydroartemisinin and paclitaxel from the liposomes at pH 5.0 was better than that at pH 7.4, and the liposomes could be stored at low temperature for seven days with good stability. Twenty-four hours after administration, the inhibition rates of the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin(70 μmol·L~(-1)) and paclitaxel on MDA-MB-231 and 4T1 cells were higher than those of the positive control(adriamycin) and free drugs(P<0.01). Compared with free drugs, liposomes inhibited the migration of MDA-MB-231 and 4T1 cells(P<0.05). Liposomes demonstrated active targeting and lysosome escape. In particular, liposomes showed lower toxicity to H9c2 cells than free drugs(P<0.05), which indicated that the preparation had the potential to reduce cardiotoxicity. The findings prove that ginsenoside Rg_3 characterized by the combination of drug and excipient is an ideal substitute for lipids in liposomes and promoted the development of innovative TCM drugs for treating cancer.

Research Progress in Androgen Receptor and Triple Negative Breast Cancer / 中国医学科学院学报

The research on androgen receptor (AR) in breast cancer is advancing.Although the prognostic value of AR in triple negative breast cancer (TNBC) is controversial,a variety of studies have demonstrated that the lack of AR expression exacerbates disease progression.Moreover,the TNBC subtype of AR(-) is more aggressive than that of AR(+) due to the lack of prognostic biomarkers and therapeutic targets.With the discovery and deepening research of novel therapeutic targets such as phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin and S-phase kinase-associated protein 2 signaling pathways,as well as the emerging of immunotherapies,the treatment options for TNBC are increasing.Regarding the role of AR in TNBC,the studies about the tumor biology of AR(-)TNBC and novel biomarkers for improved management of the disease remain insufficient.In this review,we summarize the research progress of AR in TNBC,put forward avenues for future research on TNBC,and propose potential biomarkers and therapeutic strategies that warrant investigation.

Effect of Chaihu Guizhitang on Triple-negative Breast Cancer Cells： Based on HIF-1α/VEGFA Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Chaihu Guizhitang on triple-negative breast cancer （TNBC） cells based on hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor A （VEGFA） signaling pathway. MethodTNBC xenograft model was established and the cells were randomized into model group， capecitabine group （0.2 mg·kg-1）， Chaihu Guizhitang low-dose group， medium-dose group， and high-dose group （10.62， 21.23， 42.46 g·kg-1）， with 10 mice in each group. After 21 days of medication， the content of tumor necrosis factor-α （TNF-α） in serum was detected by enzyme-linked immunosorbent assay （ELISA）. The expression of HIF-1α mRNA was detected by real-time fluorogenic quantitative polymerase chain reaction （real-time PCR）. Immunohistochemistry （IHC） was employed to detect the expression of HIF-1α， TNF-α， and VEGFA in tumor tissues， and CD34 staining to examine the angiogenesis in tumor tissues. Microvessel density （MVD） was calculated， and the protein expression of HIF-1α， VEGFA， and epidermal growth factor receptor （EGFR） in tumor tissues was measured by Western blot. ResultCompared with the model group， the rest four groups showed low levels of TNF-α （P<0.01）， HIF-1α mRNA （P<0.01）， expression of HIF-1α， TNF-α， VEGFA， and CD34 in cells， and MVD （P<0.05， P<0.01）， and low protein levels of HIF-1α， VEGFA， and EGFR （P<0.01）. Compared with capecitabine group， medium-dose and high-dose Chaihu Guizhitang decreased the level of TNF-α （P<0.01）， HIF-1α mRNA （P<0.01）， expression of HIF-1α， TNF-α， and VEGFA in cells （P<0.01）， CD34 expression， MVD， and protein levels of HIF-1α， VEGFA， and EGFR （P<0.01）. ConclusionChaihu Guizhitang may inhibit the angiogenesis in TNBC cells by regulating the expression of HIF-1α/VEGFA signaling pathway， thus exerting anti-tumor effect.

Research Progress on Immunization and Targeted Therapy for Triple-negative Breast Cancer / 肿瘤防治研究

Triple-negative breast cancer (TNBC) accounts for approximately 15%–20% of all breast cancers. Patients with TNBC have a rapidly progressive clinical course, an earlier age of onset, faster distant recurrence, and more common visceral metastases as compared with other subtypes. However, treatment of TNBC is often limited to chemotherapy and has a poor prognosis. Therefore, developing the best treatment strategy for patients is essential to reduce the burden of disease caused by TNBC. Various potential available drug targets have been discovered, as well as precision treatment and classified treatment are changing the clinical practice of TNBC, thereby indicating a new therapeutic area for TNBC in addition to traditional chemotherapy. This article reviews the systemic treatment options for TNBC in recent years, including immunotherapy and targeted therapy.

Comparison of Clinicopathological Characteristics Between Primary and Contralateral Cancers in BRCA1/2 Carriers with Metachronous Bilateral Breast Cancers / 肿瘤防治研究

Objective To compare the clinicopathological characteristics between primary and contralateral cancers in patients with metachronous bilateral breast cancer (MBBC) who carried a BRCA1/2 germline pathogenic variant. Methods A total of 496 BRCA1/2 carriers with primary unilateral breast cancer were included (196 with BRCA1 and 300 with BRCA2). Clinicopathological information of patients was collected, and the median follow-up for the entire cohort was 10.4 years (0.4-20.8 years). Results Among all patients, 31 (15.8%) of the 196 BRCA1 carriers and 49 (16.3%) of the 300 BRCA2 carriers had MBBC, respectively. Among the 31 BRCA1 carriers who developed MBBC, the proportion of triple-negative breast cancer (TNBC) in primary cancer and contralateral cancer was 61.3% and 67.7%, respectively. If the primary cancer of BRCA1-mutated MBBC was TNBC, the probability of the contralateral breast cancer with TNBC was 89.5% (17/19), which was significantly higher than that if the primary cancer was non-TNBC (33.3%, 4/12) (P=0.004). Among the 49 BRCA2 carriers who developed MBBC, the predominant molecular phenotype of the primary and contralateral cancers was HR+ & HER2- (77.6% and 67.3%, respectively; P=0.53). Conclusion Approximately 60% of BRCA1 carriers exhibit TNBC. If a BRCA1 carrier with a TNBC primary breast cancer had an MBBC, the probability of the contralateral breast cancer being TNBC phenotype is almost 89.5%.

Research Progress on Genetic Susceptibility to Triple-Negative Breast Cancer / 肿瘤防治研究

Triple-negative breast cancer (TNBC) is a type of breast cancer that is difficult to treat, has a poor prognosis, and is prone to recurrence and metastasis in the early postoperative period. The age of patients is tending younger, and the racial difference is large. It is also related to family history, and genetic susceptibility is obvious. So, elucidating the genetic risk factors of TNBC and obtaining precise therapeutic targets are urgent tasks. Obtaining reliable characteristic genes and their polymorphisms between TNBC of different subtypes is difficult. This review summarizes the susceptibility genes and the polymorphisms of TNBC susceptibility genes of different molecular subtypes, in order to develop effective TNBC prevention strategies and find effective therapeutic targets. This review provides a theoretical basis for promoting the study of TNBC from the perspective of genetics.

Hydnocarpin inhibits malignant progression of triple negative breast cancer via CNOT4-mediated ubiquitination and degradation of YAP / 中国中药杂志

This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.

Stapled anoplin peptide combined with photothermal therapy enhances oncolytic immunotherapy of triple-negative breast cancer / 中国中药杂志

This study constructed a nano-drug delivery system, A3@GMH, by co-delivering the stapled anoplin peptide(Ano-3, A3) with the light-harvesting material graphene oxide(GO), and evaluated its oncolytic immunotherapy effect on triple-negative breast cancer(TNBC). A3@GMH was prepared using an emulsion template method and its physicochemical properties were characterized. The in vivo and in vitro photothermal conversion abilities of A3@GMH were investigated using an infrared thermal imager. The oncoly-tic activity of A3@GMH against TNBC 4T1 cells was evaluated through cell counting kit-8(CCK-8), lactate dehydrogenase(LDH) release, live/dead cell staining, and super-resolution microscopy. The targeting properties of A3@GMH on 4T1 cells were assessed using a high-content imaging system and flow cytometry. In vitro and in vivo studies were conducted to investigate the antitumor mechanism of A3@GMH in combination with photothermal therapy(PTT) through inducing immunogenic cell death(ICD) in 4T1 cells. The results showed that the prepared A3@GMH exhibited distinct mesoporous and coated structures with an average particle size of(308.9±7.5) nm and a surface potential of(-6.79±0.58) mV. The encapsulation efficiency and drug loading of A3 were 23.9%±0.6% and 20.5%±0.5%, respectively. A3@GMH demonstrated excellent photothermal conversion ability and biological safety. A3@GMH actively mediated oncolytic features such as 4T1 cell lysis and LDH release, as well as ICD effects, and showed enhanced in vitro antitumor activity when combined with PTT. In vivo, A3@GMH efficiently induced ICD effects with two rounds of PTT, activated the host's antitumor immune response, and effectively suppressed tumor growth in 4T1 tumor-bearing mice, achieving an 88.9% tumor inhibition rate with no apparent toxic side effects. This study suggests that the combination of stapled anoplin peptide and PTT significantly enhances the oncolytic immunotherapy for TNBC and provides a basis for the innovative application of anti-tumor peptides derived from TCM in TNBC treatment.

Discovery of a first-in-class ANXA3 degrader for the treatment of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a nasty disease with extremely high malignancy and poor prognosis. Annexin A3 (ANXA3) is a potential prognosis biomarker, displaying an excellent correlation of ANXA3 overexpression with patients' poor prognosis. Silencing the expression of ANXA3 effectively inhibits the proliferation and metastasis of TNBC, suggesting that ANXA3 can be a promising therapeutic target to treat TNBC. Herein, we report a first-in-class ANXA3-targeted small molecule (R)-SL18, which demonstrated excellent anti-proliferative and anti-invasive activities to TNBC cells. (R)-SL18 directly bound to ANXA3 and increased its ubiquitination, thereby inducing ANXA3 degradation with moderate family selectivity. Importantly, (R)-SL18 showed a safe and effective therapeutic potency in a high ANXA3-expressing TNBC patient-derived xenograft model. Furthermore, (R)-SL18 could reduce the β-catenin level, and accordingly inhibit the Wnt/β-catenin signaling pathway in TNBC cells. Collectively, our data suggested that targeting degradation of ANXA3 by (R)-SL18 possesses the potential to treat TNBC.

Expression of long non-coding RNA FRAPT in triple-negative breast cancer and its biological significance / 中国生物制品学杂志

@#Objective To investigate the expression of long non-coding RNA FRAPT(lncRNA FRAPT) in triple-negative breast cancer(TNBC) cells and analyze its effects on proliferation,cell cycle and drug resistance of TNBC cells.Methods Using scRNASeqDB and TCGA on-line bioinformatics database,the expression of lncRNA FRAPT in TNBC and its correlation with prognosis were detected;TNBC cell line MDA-MB-231 was transfected with si-lncRNA FRAPT and si-Ctrl(negative control) by using Lipofectamine~(TM) 2000 respectively,of which the proliferation and cell cycle changes after transfection were detected by SRB test and flow cytometry,and the drug resistance to chemotherapy drug paclitaxel(PTX)was detected.Results The expression of lncRNA FRAPT was up-regulated in TNBC tissues and cells,and its high expression was positively correlated with the poor prognosis of TNBC patients(Hazard Ratio=1.66,P=0.047).Silencing lncRNA FRAPT significantly inhibited the proliferation of TNBC cells,with the cell cycle arrested in G1 phase,while increased the sensitivity of TNBC cells to PTX.Conclusion LncRNA FRAPT was highly expressed in TNBC and related to tumor prognosis-Silencing lncRNA FRAPT inhibited proliferation and cell cycle of TNBC,and increased the sensitivity to chemotherapy drug PTX.

Effect of transcription factor NFIB on cell proliferation and invasion in breast cancer / 中国医师杂志

Objective:To investigate the effect of transcription factor nuclear factor IB (NFIB) on cell proliferation and invasion in breast cancer.Methods:The lentivirus pLKO.1-shNFIB plasmid was constructed, packaged and infected with human estrogen receptor positive (ER + ) breast cancer cell line MCF-7 and triple-negative breast cancer (TNBC) cell line MDA-MB-231, respectively, NFIB was stably knocked down and verified by Western blot method; Cell count test (CCK-8) and clone formation test were used to investigate the effect of knockdown NFIB on the growth and proliferation of breast cancer cells; The transwell experiment and Western blot method were performed to detect the expression of epithelial mesenchymal transition protein markers. The effect of knockdown NFIB on the invasive ability of triple-negative breast cancer cells was explored; Kaplan-Meier survival was used to analyze web data (http: //kmplot.com/analysis/) to explore the effect of NFIB on the prognosis of ER + breast cancer and triple-negative breast cancer patients. Results:In MCF-7 and MDA-MB-231 breast cancer cells, knocking down NFIB inhibited cell growth and proliferation; In triple-negative breast cancer MDA-MB-231 cells, knocking down NFIB promoted the expression of interstitial marker fibronectin and promoted cell invasion; The lower the expression of NFIB, the worse the prognosis of triple negative breast cancer patients, while the expression of NFIB had no effect on the prognosis of ER + breast cancer patients. Conclusions:Knocking down NFIB inhibits the proliferation of MCF-7 cells, and the expression level of NFIB is not related to the prognosis of ER + breast cancer patients; Knocking down NFIB inhibits the proliferation of MDA-MB-231 cells but promotes their invasion; The low expression of NFIB is associated with the poor prognosis of triple-negative breast cancer patients.