F1012-2 Induced ROS-Mediated DNA Damage Response through Activation of MAPK Pathway in Triple-Negative Breast Cancer

We have previously reported that F1012-2, a sesquiterpene lactone isolated from the Chinese herbal medicine Eupatorium lindleyanum DC., exhibits strong effects against Triple Negative Breast Cancer (TNBC). In this study, we found F1012-2 effectively inhibited cell migration and invasion detected by wound healing and transwell assays. In order to elucidate the potential mechanisms of F1012-2, we further studied its effect on DNA damage in TNBC cell lines. Using single cell gel electrophoresis (comet assay), immunofluorescence, and western blotting assays, we found that F1012-2 treatment induced significant DNA strand breaks and γ-H2AX activation. Moreover, exposure to F1012-2 led to overproduction of reactive oxygen species (ROS). NAC treatment completely eliminated ROS, which may be due to the interaction between NAC and F1012-2. A further study of the molecular mechanisms demonstrated that the MAPK signaling pathway participated in the anti-TNBC effect of F1012-2. Pretreatment with specific inhibitors targeting JNK (SP600125) and ERK (PD98059) could rescue the decrease in cell viability and inhibit expressions of JNK and ERK phosphorylation, but SB203580 had no effects. Finally, in the acute toxicity experiment, there were no obvious symptoms of poisoning in the F1012-2 treatment group. An in vivo study demonstrated that F1012-2 significantly suppressed the tumor growth and induced DNA damage. In conclusion, the activity of F1012-2-induced DNA damage in TNBC was found in vivo and in vitro, which might trigger the MAPK pathway through ROS accumulation. These results indicate that F1012-2 may be an effective anti-TNBC therapeutic agent.

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Salvia Chinensia Benth Inhibits Triple Negative Breast Cancer Progression Through Inducing DNA Damage Pathway

10.21203/rs.3.rs-662543/v1 ◽

2021 ◽

Author(s):

Kainan Wang ◽

Ye Hu ◽

Lingzhi Xu ◽

Shanshan Zhao ◽

Chen Song ◽

...

Keyword(s):

Breast Cancer ◽

Dna Damage ◽

Triple Negative Breast Cancer ◽

Antitumor Effect ◽

Triple Negative ◽

Bioinformatic Analysis ◽

Migration And Invasion ◽

Recombination Pathway

Abstract Background: Salvia chinensia Benth (SCH) is an herb that has extensively demonstrated antitumor attributes in multiple tumors. However, there are only marginal reports about the impacts of SCH on TNBC-triple negative breast cancer tumorigenesis. Herein, our current research reported the in vitro effects as well as the in vivo antitumor effect, and the fundamental mechanisms of SCH on TNBC. Methods: In vitro, we measured the antitumor effect of SCH on TNBC through clonogenic assay, cell viability, flow cytometry, apoptosis assay, migration and invasion assay. Meanwhile, breast cancer xenografts with the aid of MDA-MB-231 cells were established to evaluate the therapeutic effect of SCH on TNBC in vivo. To investigate the molecular mechanisms of SCH, bioinformatic analysis, specifically network pharmacology-based analysis, was performed. Then, the comet assay and western blot analyses were applied to confirm the influence of SCH on the DNA damage and repair pathways.Results: Our results demonstrated that the application of SCH could lead to the reticence of cell proliferation, migration, invasion and the induction of cellular apoptosis caused in TNBC cells. Based on the bioinformatic analysis, we discovered two key compounds of SCH, quercetin and beta-sitosterol, and detected significant synergistic effects of the combined treatment of them on TNBC, especially on the DNA damage of TNBC cells as judged by comet assays. Moreover, substantial inductions of DNA damage were examined after SCH treatment in TNBC cells, as well as quercetin, and beta-sitosterol treatment. Apart from inducing DNA damage, SCH specifically decreased inter-strand crosslink repair pathway and the double-strand breach repair perforated by the homologous recombination pathway. Similar results were also obtained with quercetin and beta-sitosterol treatment. Conclusion: Taken together, the current investigation elucidates the novel discernment into the therapeutic function of SCH as a DNA damage agent for treatment of TNBC.

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CircRNA circ-ERBB2 elevates Warburg effect and facilitates triple-negative breast cancer growth by the miR-136-5p/PDK4 axis

Molecular and Cellular Biology ◽

10.1128/mcb.00609-20 ◽

2021 ◽

Author(s):

Yihong Huang ◽

Shuo Zheng ◽

Ying Lin ◽

Liming Ke

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Warburg Effect ◽

Triple Negative ◽

Pyruvate Dehydrogenase Kinase ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Loss Of Function

Triple negative breast cancer (TNBC) is an aggressive histological subtype of breast cancer. It has been reported that that circRNA circ-ERBB2 (circBase ID: hsa_circ_0007766) is mainly distributed in the cytoplasm of TNBC cells and promotes the proliferation and invasion of TNBC cells. This study aimed to explore the molecular mechanism of circ-ERBB2 regulating the progression of TNBC. Expression of circ-ERBB2 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to investigate the function of circ-ERBB2 in TNBC cells in vitro and in vivo . The regulatory mechanism of circ-ERBB2 was surveyed by bioinformatics analysis, dual-luciferase reporter and RNA immunoprecipitation (RIP) or RNA pull-down assays. We observed that Circ-ERBB2 was overexpressed in TNBC, and TNBC patients with high circ-ERBB2 expression had a poor prognosis. Functionally, circ-ERBB2 knockdown constrained TNBC growth in vivo and reduced Warburg effect, accelerated apoptosis, repressed proliferation, migration, and invasion of TNBC cell in vitro . Mechanically, circ-ERBB2 sponged miR-136-5p to elevate pyruvate dehydrogenase kinase 4 (PDK4) expression. In conclusion, circ-ERBB2 facilitated Warburg effect and malignancy of TNBC cells by the miR-136-5p/PDK4 pathway, at least in part. This study supported circ-ERBB2 as a prognostic indicator for TNBC.

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Mitotic kinesin-like protein 1, regulated by FOXM1, promotes triple negative breast cancer progression via activating Wnt/β-catenin pathway

10.21203/rs.3.rs-1228723/v1 ◽

2022 ◽

Author(s):

Zhi Li ◽

Hai-Yan Yang ◽

Xiao-Lan Zhang ◽

Xu Zhang ◽

Yu-Zhou Huang ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Progression ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Underlying Mechanism ◽

Migration And Invasion ◽

Cancer Proliferation ◽

Tumor Growth And Metastasis

Abstract Background: Triple negative breast cancer (TNBC) is highly malignant and has a worse prognosis, compared with other subtypes of breast cancer due to the absence of therapeutic targets. MKLP1 plays a crucial role in tumorigenesis and cancer progression. However, the role of MKLP1 in triple negative breast cancer and the underlying mechanism remain unknown. The study aimed to elucidate the biological function regulatory mechanism of MKLP1 in triple negative breast cancerMethods: Quantitative real-time PCR and Western blotting were used to determine the MKLP1 expression in breast cancer tissues and cell lines. Then, functional experiments in vitro and in vivo were performed to investigate the effects of MKLP1 on tumor growth and metastasis in triple negative breast cancer. Chromatin immunoprecipitation assay was conducted to illustrate the potential regulatory mechanisms of MKLP1 in triple negative breast cancer.Results: We found that MKLP1 was significantly up-regulated and associated with poor prognosis in triple negative breast cancer. MKLP1 could promote triple negative breast cancer proliferation, migration and invasion in vitro and in vivo. MKLP1 could activate Wnt/β-catenin pathway and promote EMT progression. In addition, FOXM1, upregulated by WDR5 via H3K4me3 modification, directly bound to the promoter of MKLP1 gene to promote its transcription and accelerated TNBC progression via Wnt/β-catenin pathway. Both of small inhibitor of FOXM1 and WDR5 could inhibit TNBC progression. Conclusions: Our findings elucidate WDR5/FOXM1/MKLP1/Wnt/β-catenin axis is associated with TNBC progression and may provide a novel and promising therapeutic target for TNBC treatment.

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LncRNA PCIR Is an Oncogenic Driver via Strengthen the Binding of TAB3 and PABPC4 in Triple Negative Breast Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.630300 ◽

2021 ◽

Vol 11 ◽

Author(s):

Wenhui Guo ◽

Jingyi Li ◽

Haobo Huang ◽

Fangmeng Fu ◽

Yuxiang Lin ◽

...

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Migration And Invasion ◽

Tnf Α ◽

Associated Proteins ◽

Rna Immunoprecipitation ◽

Underlying Mechanisms

Long non-coding RNAs (LncRNA) as the key regulators in all stages of tumorigenesis and metastasis. However, the underlying mechanisms are largely unknown. Here, we report a lncRNA RP11-214F16.8, which renamed Lnc-PCIR, is upregulated and higher RNA level of Lnc-PCIR was positively correlated to the poor survival of patients with triple negative breast cancer (TNBC) tissues. Lnc-PCIR overexpression significantly promoted cell proliferation, migration, and invasion in vitro and in vivo. RNA pulldown, RNA immunoprecipitation (RIP) and RNA transcriptome sequencing technology (RNA-seq) was performed to identify the associated proteins and related signaling pathways. Mechanistically, higher Lnc-PCIR level of blocks PABPC4 proteasome-dependent ubiquitination degradation; stable and highly expressed PABPC4 can further increase the stability of TAB3 mRNA, meanwhile, overexpression of Lnc-PCIR can disrupt the binding status of TAB3 and TAB2 which lead to activate the TNF-α/NF-κB pathway in TNBC cells. Our findings suggest that Lnc-PCIR promotes tumor growth and metastasis via up-regulating the mRNA/protein level of TAB3 and PABPC4, activating TNF-α/NF-κB signaling pathway in TNBC.

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Long noncoding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple-negative breast cancer

Cell Death and Disease ◽

10.1038/s41419-021-03917-z ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Xiaohui Zhang ◽

Fangyuan Li ◽

Yidong Zhou ◽

Feng Mao ◽

Yan Lin ◽

...

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Noncoding Rna ◽

Triple Negative ◽

Quantitative Polymerase Chain Reaction ◽

Cell Counting ◽

Luciferase Reporter ◽

Migration And Invasion

AbstractLong noncoding ribonucleic acids (LncRNAs) have been found to be involved in the proliferation, apoptosis, invasion, migration, and other pathological processes of triple-negative breast cancer (TNBC). Expression of the lncRNA actin filament-associated protein 1 antisense RNA1 (AFAP1-AS1) has been found to be significantly higher in TNBC than in other subtypes or in normal tissue samples, but the specific mechanism by which AFAP1-AS1 affects the occurrence and development of TNBC is yet to be revealed. In this study, we used Cell Counting Kit-8 (CCK-8), colony formation, wound healing migration, Transwell invasion, and nude mouse xenograft assays to confirm the role of AFAP1-AS1 in the proliferation, migration of TNBC cells in vitro and in vivo. In addition, we performed bioinformatics analyses, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), western blot (WB), and dual-luciferase reporter assays (dual-LRA) to confirm interaction among AFAP1-AS1, micro-RNA 2110 (miR-2110), and Sp1 transcription factor (Sp1). We found that silencing AFAP1-AS1 and Sp1 or upregulating miR-2110 suppressed the proliferation, migration, and invasion of MDA–MB-231 and MDA–MB-468 cells in vitro as well as tumor growth in vivo. Mechanistically, the dual-LRA highlighted that miR-2110 was an inhibitory target of AFAP1-AS1, and that AFAP1-AS1 functioned as a miR-2110 sponge to increase Sp1 expression. AFAP1-AS1 silencing led to a reduction in Sp1 mRNA and protein levels, which could be reversed by joint transfection with miR-2110 inhibitor. Our findings demonstrated that AFAP1-AS1 could modulate the progression of breast cancer cells and affect tumorigenesis in mice by acting as a miR-2110 sponge, resulting in regulation of Sp1 expression. Therefore, AFAP1-AS1 could play a pivotal role in the treatment of TNBC.

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MLK4 regulates DNA damage response and promotes triple-negative breast cancer chemoresistance

Cell Death and Disease ◽

10.1038/s41419-021-04405-0 ◽

2021 ◽

Vol 12 (12) ◽

Author(s):

Dawid Mehlich ◽

Michał Łomiak ◽

Aleksandra Sobiborowicz ◽

Alicja Mazan ◽

Dagmara Dymerska ◽

...

Keyword(s):

Breast Cancer ◽

Dna Damage ◽

Dna Damage Response ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Chemotherapeutic Agents ◽

Response Factors ◽

Damage Response

AbstractChemoresistance constitutes a major challenge in the treatment of triple-negative breast cancer (TNBC). Mixed-Lineage Kinase 4 (MLK4) is frequently amplified or overexpressed in TNBC where it facilitates the aggressive growth and migratory potential of breast cancer cells. However, the functional role of MLK4 in resistance to chemotherapy has not been investigated so far. Here, we demonstrate that MLK4 promotes TNBC chemoresistance by regulating the pro-survival response to DNA-damaging therapies. We observed that MLK4 knock-down or inhibition sensitized TNBC cell lines to chemotherapeutic agents in vitro. Similarly, MLK4-deficient cells displayed enhanced sensitivity towards doxorubicin treatment in vivo. MLK4 silencing induced persistent DNA damage accumulation and apoptosis in TNBC cells upon treatment with chemotherapeutics. Using phosphoproteomic profiling and reporter assays, we demonstrated that loss of MLK4 reduced phosphorylation of key DNA damage response factors, including ATM and CHK2, and compromised DNA repair via non-homologous end-joining pathway. Moreover, our mRNA-seq analysis revealed that MLK4 is required for DNA damage-induced expression of several NF-кB-associated cytokines, which facilitate TNBC cells survival. Lastly, we found that high MLK4 expression is associated with worse overall survival of TNBC patients receiving anthracycline-based neoadjuvant chemotherapy. Collectively, these results identify a novel function of MLK4 in the regulation of DNA damage response signaling and indicate that inhibition of this kinase could be an effective strategy to overcome TNBC chemoresistance.

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Efficacy and mechanism of the combination of PARP and CDK4/6 inhibitors in the treatment of triple-negative breast cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01930-w ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Xiuzhi Zhu ◽

Li Chen ◽

Binhao Huang ◽

Xiaoguang Li ◽

Liu Yang ◽

...

Keyword(s):

Breast Cancer ◽

Dna Damage ◽

Cell Lines ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Single Agent ◽

Wnt Signalling ◽

Tnbc Cell

Abstract Background PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients with BRCA mutations, and their efficacy is even more limited in triple-negative breast cancer (TNBC) due to clinical primary and acquired resistance. Here, we found that the efficacy of the PARPi olaparib in TNBC can be improved by combination with the CDK4/6 inhibitor (CDK4/6i) palbociclib. Methods We screened primary olaparib-sensitive and olaparib-resistant cell lines from existing BRCAmut/TNBC cell lines and generated cells with acquired olaparib resistance by gradually increasing the concentration. The effects of the PARPi olaparib and the CDK4/6i palbociclib on BRCAmut/TNBC cell lines were examined in both sensitive and resistant cells in vitro and in vivo. Pathway and gene alterations were assessed mechanistically and pharmacologically. Results We demonstrated for the first time that the combination of olaparib and palbociclib has synergistic effects against BRCAmut/TNBC both in vitro and in vivo. In olaparib-sensitive MDA-MB-436 cells, the single agent olaparib significantly inhibited cell viability and affected cell growth due to severe DNA damage. In olaparib-resistant HCC1937 and SUM149 cells, single-agent olaparib was ineffective due to potential homologous recombination (HR) repair, and the combination of olaparib and palbociclib greatly inhibited HR during the G2 phase, increased DNA damage and inhibited tumour growth. Inadequate DNA damage caused by olaparib activated the Wnt signalling pathway and upregulated MYC. Further experiments indicated that the overexpression of β-catenin, especially its hyperphosphorylation at the Ser675 site, activated the Wnt signalling pathway and mediated olaparib resistance, which could be strongly inhibited by combined treatment with palbociclib. Conclusions Our data provide a rationale for clinical evaluation of the therapeutic synergy of the PARPi olaparib and CDK4/6i palbociclib in BRCAmut/TNBCs with high Wnt signalling activation and high MYC expression that do not respond to PARPi monotherapy.

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XJP Inhibits Apoptosis, Invasion, EMT, and Wnt/Β-Catenin Pathway in Triple-Negative Breast Cancer

10.21203/rs.3.rs-898479/v1 ◽

2021 ◽

Author(s):

Dandan Feng ◽

Hongzhi Chen ◽

Guangxi Shi ◽

Mengdi Zhang ◽

Hongyi Liang ◽

...

Keyword(s):

Breast Cancer ◽

Flow Cytometry ◽

Triple Negative Breast Cancer ◽

Molecular Mechanisms ◽

Triple Negative ◽

Epithelial Mesenchymal Transition ◽

Cell Counting ◽

Migration And Invasion ◽

Mesenchymal Transition

Abstract Background: Triple-negative breast cancer (TNBC) progresses at a rapid pace. Chemotherapy is a major clinical application. However, resistance and metastases are key barriers to chemotherapy. Xiaojin pills (XJP) have been used clinically for treating TNBC for decades. However, the potential molecular mechanisms of the effect of XJP on breast cancer is still not understood.Methods: The cell viability was analyzed using Cell Counting Kit-8 (CCK-8). Flow cytometry was used to detect apoptosis, and the migration and invasion abilities of TNBC were assessed using Transwell assay. For molecular mechanisms, the protein expression levels were determined by Western blot analysis. The expression of β-catenin in the Wnt/β-serial protein (β-catenin) pathway was detected with immunofluorescence (IF).Results: XJP inhibited the viability and proliferation of the TNBC cell line in vitro. Flow cytometry analysis showed that apoptosis increased in both MDA-MB-231 and MDA-MB-468 cells induced by XJP. The expression of the proteins associated with invasion, for example, matrix metalloproteinase (MMP) and MMP9, was reduced. Among epithelial–mesenchymal transition markers, E-cadherin was upregulated and N-cadherin was downregulated. The apoptosis-related proteins caspase-8, caspase-3, caspase-9, and Parp were all upregulated. Additionally, XJP effectively suppressed the expression of β-catenin, which belonged to the Wnt/β-catenin pathway.Conclusions: These results suggested that XJP suppressed the progression of TNBC cells by suppressing apoptosis, invasion, EMT, and Wnt/β-catenin pathway.

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Vitamin C Inhibits Triple-Negative Breast Cancer Metastasis by Affecting the Expression of YAP1 and Synaptopodin 2

Nutrients ◽

10.3390/nu11122997 ◽

2019 ◽

Vol 11 (12) ◽

pp. 2997 ◽

Cited By ~ 6

Author(s):

Liping Gan ◽

Vladimir Camarena ◽

Sushmita Mustafi ◽

Gaofeng Wang

Keyword(s):

Breast Cancer ◽

Vitamin C ◽

Triple Negative Breast Cancer ◽

Cancer Metastasis ◽

Triple Negative ◽

Breast Cancer Metastasis ◽

Migration And Invasion ◽

Synaptopodin 2

Vitamin C supplementation has been shown to decrease triple-negative breast cancer (TNBC) metastasis. However, the molecular mechanism whereby vitamin C inhibits metastasis remains elusive. It has been postulated that vitamin C reduces the levels of HIF-1α, the master regulator of metastasis, by promoting its hydroxylation and degradation. Here, we show that vitamin C at 100 µM, a concentration achievable in the plasma in vivo by oral administration, blocks TNBC cell migration and invasion in vitro. The protein level of HIF-1α remains largely unchanged in cultured TNBC cells and xenografts, partially due to its upregulated transcription by vitamin C, suggesting that HIF-1α unlikely mediates the action of vitamin C on metastasis. Vitamin C treatment upregulates the expression of synaptopodin 2 and downregulates the expression of the transcription coactivator YAP1, both genes in the Hippo pathway. The changes in SYNPO2 and YAP1 expression were subsequently validated at mRNA and protein levels in cultured TNBC cells and xenografts. Further experiments showed that vitamin C treatment inhibits F-actin assembly and lamellipodia formation, which correlates with the changes in SYNPO2 and YAP1 expression. Overall, these results suggest that vitamin C inhibits TNBC metastasis by affecting the expression of SYNPO2 and YAP1. Vitamin C may thus have a potential role in the prevention and treatment of TNBC metastasis.

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Long non-coding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple negative breast cancer

10.21203/rs.3.rs-103653/v1 ◽

2020 ◽

Author(s):

Xiaohui Zhang ◽

Fangyuan Li ◽

Yidong Zhou ◽

Feng Mao ◽

Yan Lin ◽

...

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Quantitative Polymerase Chain Reaction ◽

Cell Counting ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Bioinformatics Analyses

Abstract Background: LncRNAs have been proved to be involved in the proliferation, apoptosis, invasion, migration and other pathological processes of triple negative breast cancer (TNBC). And the expression level of LncRNA AFAP1-AS1 in TNBC was found to be significantly higher than that in other subtypes and normal tissue samples, but the specific mechanism of LncRNA AFAP1-AS1 affecting the occurrence and development of TNBC needs to be revealed.Methods: Cell Counting Kit-8 assays, colony formation assays, wound-healing migration, transwell invasion assays and nude mouse xenograft assays were used to confirm the role of LncRNA AFAP1-AS1 in the proliferation, migration of TNBC cells in vitro and in vivo. Bioinformatics analyses, quantitative polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase assays were performed to confirm the interaction between between LncRNA AFAP1-AS1, miR-2110 and Sp1.Results: In the present study, the silencing of AFAP1-AS1 and Sp1 or the upregulation of miR-2110 would result in the suppression of proliferation, migration and invasion of MDA-MB-231 and MDA-MB-468 cells in vitro as well as tumor growth in vivo. Mechanistically, the dual-luciferase reporter assay highlighted that AFAP1-AS1 functioned as a miR-2110 sponge to increase Sp1 expression. AFAP1-AS1 silencing led to a reduction in Sp1 mRNA and protein levels, which could be reverse by the joint transfection of miR-2110 inhibitor.Conclusions: Our findings demonstrated that AFAP1-AS1 acts as a miR-2110 sponge in TNBC cells, resulting in the regulation of Sp1 expression. And the AFAP1-AS1/miR-2110/Sp1 axis modulated the proliferation, migration and invasion of breast cancer cells and affected the tumorigenesis in mice.

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