STAT3 as a promising chemoresistance biomarker associated with the CD44 +/high /CD24 -/low /ALDH + BCSCs-like subset of the triple-negative breast cancer (TNBC) cell line

Abstract Sprouty (Spry) proteins have been implicated in cancer progression, but their role in triple-negative breast cancer (TNBC), a subtype of lethal and aggressive breast cancer, is unknown. Here, we reported that Spry1 is significantly expressed in TNBC specimen and MDA-MB-231 cells. To understand Spry1 regulation of signaling events controlling breast cancer phenotype, we used lentiviral delivery of human Spry1 shRNAs to suppress Spry1 expression in MDA-MB-231, an established TNBC cell line. Spry1 knockdown MDA-MB-231 cells displayed an epithelial phenotype with increased membrane E-cadherin expression. Knockdown of Spry1 impaired MDA-MB-231 cell migration, Matrigel invasion, and anchorage-dependent and -independent growth. Tumor xenografts originating from Spry1 knockdown MDA-MB-231 cells grew slower, had increased E-cadherin expression, and yielded fewer lung metastases compared to control. Furthermore, suppressing Spry1 in MDA-MB-231 cells impaired the induction of Snail and Slug expression by EGF, and this effect was associated with increased EGFR degradation and decreased EGFR/Grb2/Shp2/Gab1 signaling complex formation. The same phenotype was also observed in the TNBC cell line MDA-MB-157. Together, our results show that unlike in some tumors, where Spry may mediate tumor suppression, Spry1 plays a selective role in at least a subset of TNBC to promote the malignant phenotype via enhancing EGF-mediated mesenchymal phenotype.

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Abstract P5-06-09: Cell Cycle Effects of Iniparib, a PARP Inhibitor, in Combination with Gemcitabine and Carboplatin in the MDA-MB-468(-) Triple-Negative Breast Cancer (TNBC) Cell Line

10.1158/0008-5472.sabcs10-p5-06-09 ◽

2010 ◽

Cited By ~ 2

Author(s):

V Ossovskaya ◽

C-u Lim ◽

G Schools ◽

S Kalurupalle ◽

IB Roninson ◽

...

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Cell Line ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Parp Inhibitor ◽

Tnbc Cell ◽

Tnbc Cell Line

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TMEPAI genome editing in triple negative breast cancer cells

Medical Journal of Indonesia ◽

10.13181/mji.v26i1.1871 ◽

2017 ◽

Vol 26 (1) ◽

pp. 14-8 ◽

Cited By ~ 3

Author(s):

Bantari W.K. Wardhani ◽

Meidi U. Puteri ◽

Yukihide Watanabe ◽

Melva Louisa ◽

Rianto Setiabudy ◽

...

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Genome Editing ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

High Efficiency ◽

Knock Out ◽

Tnbc Cell ◽

A Genome ◽

Tnbc Cell Line

Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line.Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.

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Knock-out transmembrane prostate androgen-induced protein gene suppressed triple-negative breast cancer cell proliferation

Medical Journal of Indonesia ◽

10.13181/mji.v26i3.1823 ◽

2017 ◽

Vol 26 (3) ◽

pp. 178-82 ◽

Cited By ~ 1

Author(s):

Bantari W.K. Wardhani ◽

Meidi U. Puteri ◽

Yukihide Watanabe ◽

Melva Louisa ◽

Rianto Setiabudy ◽

...

Keyword(s):

Breast Cancer ◽

Cell Proliferation ◽

Cell Line ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Ki 67 ◽

Knock Out ◽

Tnbc Cell ◽

Tnbc Cell Line ◽

Mrna Expression Levels

Background: Triple negative breast cancer (TNBC) tends to grow more rapidly and has poorer prognosis compared to others. High expression of transmembrane prostate androgen-induced protein (TMEPAI) correlates with poor prognosis in TNBC patients. However, the mechanistic role of TMEPAI in tumorigenic remains unknown. This study aimed to knock-out TMEPAI in TNBC cell line to determine its function further in cells proliferation.Methods: CRISPR-Cas9 has been used previously to knock-out TMEPAI in Hs857T TNBC cell line. Hs587T TNBC parental cell line (wild-type/WT) and TMEPAI knock out Hs 586T cell lines were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and amphotericin B. Both cell lines were seeded in 24-well plates and counted every two days, then proliferation rates were plotted. Afterwards, total RNA were isolated from the cells and Ki-67, and TGF-β mRNA expression levels as proliferation markers were determined.Results: Cell proliferation rates as displayed in growth curve plots showed that WT-TMEPAI cell line grew more rapidly than KO-TMEPAI. In accordance, mRNA expression levels of Ki-67 and TGF-β were significantly decreased KO-TMEPAI as compare to TMEPAI-WT.Conclusion: Knock-out of TMEPAI attenuates cell proliferation in TNBC.

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circGFRA1 affects the sensitivity of triple negative breast cancer cells to paclitaxel via the miR-361-5p/TLR4 pathway

The Journal of Biochemistry ◽

10.1093/jb/mvaa148 ◽

2021 ◽

Author(s):

Shu-Rong Zheng ◽

Qi-di Huang ◽

Zhi-Hai Zheng ◽

Zhong-Tao Zhang ◽

Gui-Long Guo

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Luciferase Reporter ◽

Circular Rnas ◽

Luciferase Reporter Gene ◽

Tnbc Cell ◽

Gene Assay ◽

Tnbc Cell Line

Abstract In recent years, the role of circular RNAs (circRNAs) in tumors has attracted widespread attention. Some circRNAs have been reported to play a role in triple negative breast cancer (TNBC), however, circRNAs have rarely been reported in terms of TNBC resistance. This study aimed to clarify that circGFRA1 affects the sensitivity of TNBC cells to paclitaxel (PTX) by the miR-361-5p/TLR4 pathway. Compared with the non-PTX-resistant TNBC cell line MDA-MB-231, the expression of circGFRA1 in the PTX-resistant TNBC cell line MDA-MB-231.PR was significantly increased. The small hairpin RNA-mediated circGFRA1 knockdown inhibited the resistance of TNBC cells to PTX. RNA pull-down assay and luciferase reporter gene assay confirmed the binding between circGFRA1 and miR-361-5p and between miR-361-5p and TLR4. It has been proven that circGFRA1 knockdown can inhibit the resistance of TNBC cells to PTX by promoting the expression of miR-361-5p, and subsequently reduce the expression of TLR4.

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Substance P Antagonism as a Novel Therapeutic Option to Enhance Efficacy of Cisplatin in Triple Negative Breast Cancer and Protect PC12 Cells against Cisplatin-Induced Oxidative Stress and Apoptosis

Cancers ◽

10.3390/cancers13153871 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3871

Author(s):

Emma Rodriguez ◽

Guangsheng Pei ◽

Sang T. Kim ◽

Alexis German ◽

Prema Robinson

Keyword(s):

Breast Cancer ◽

Flow Cytometry ◽

Cell Line ◽

Pc12 Cells ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Therapeutic Option ◽

Neuronal Cell ◽

Receptor Antagonism ◽

Tnbc Cell

Although cisplatin is very effective as a treatment strategy in triple-negative breast cancer (TNBC), it has unwarranted outcomes owing to recurrence, chemoresistance and neurotoxicity. There is critically important to find new, effective and safe therapeutics for TNBC. We determined if SP-receptor antagonism in combination with cisplatin may serve as a novel, more efficacious and safer therapeutic option than existing therapies for TNBC. We used a neuronal cell line (PC12) and two TNBC cell lines (Sum 185 and Sum 159) for these studies. We determined that the levels of cells expressing the high-affinity SP-receptor (neurokinin 1 receptor (NK1R)), as determined by flow-cytometry was significantly elevated in response to cisplatin in all three cells. We determined that treatment with aprepitant, an SP-receptor antagonist decreased cisplatin-induced, loss of viability (studied by MTT assay), production of reactive oxygen species (by DCFDA assay) and apoptosis (by flow-cytometry) in PC12 cells while it was increased in the two TNBC cells. Furthermore, we demonstrated that important genes associated with metastases, inflammation, chemoresistance and cell cycle progression are attenuated by SP-receptor antagonism in the TNBC cell line, Sum 185. These studies implicate that SP-receptor antagonism in combination with cisplatin may possibly serve as a novel, more efficacious and safer therapeutic option than existing therapies for TNBC.

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Effective treatment of triple-negative breast cancer with targeted cytotoxic somatostatin analogue AN-162 (AEZS-124)

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.619 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 619-619

Author(s):

S. Seitz ◽

A. V. Schally ◽

S. Gluck ◽

F. Rick ◽

L. Szalontay ◽

...

Keyword(s):

Breast Cancer ◽

Fluorescence Microscopy ◽

Tumor Growth ◽

Cell Line ◽

Triple Negative ◽

Somatostatin Analogue ◽

Tnbc Cell ◽

Tnbc Cell Line ◽

Fluorescence Assays

619 Background: Triple negative breast cancers (TNBC) represent a distinct subtype of breast cancer being negative to ER, PR and HER2 and are associated with poor prognosis. Limited systemic treatment options exist for TNBC. TNBC cells express somatostatin receptors (SSTR). Therefore, to investigate preclinical characteristics of TNBC we used a novel targeted cytotoxic somatostatin analogue AN-162 containing doxorubicin (DOX) which binds to the subtypes 2, 3 and 5 of SSTR. Methods: The expression of SSTR in HCC 1806 human TNBC cell line was detected by RT-PCR. Cytotoxic effect of AN-162 in vitro was visualized by ethidium bromide staining fluorescence microscopy. Internalization of AN-162 into HCC 1806 cells was tested by 125Iodide-labeled AN-162 uptake assays and the presence of DOX in the nucleus was measured by fluorescence assays after separating the nucleus from the cytoplasm. For in vivo experiments, HCC 1806 TNBC cells were xenografted subcutaneously into nude mice which were then randomized into four groups receiving AN-162, DOX, an unconjugated mixture of DOX and somatostatin analogue RC-160 at the same equimolar dose of 2.5 μmol/kg (1.45 mg/kg Dox equivalent) i.v. (q7d 4x) and vehicle solution control. Results: HCC 1806 TNBC cell line was positive for the expression of all five SSTR receptor subtypes. Ethidum bromide staining of cells treated with 2.5 μM of AN-162 for 30 min demonstrated cell death after 24h by fluorescence microscopy. Uptake assays with AN-162 showed specific internalization of AN-162 into the cells mediated through the sstrs. After treatment of the cells with 2.5 μM AN-162 for 10 or 30 min, DOX could be detected in the nucleus by fluorescence assays. In vivo, AN-162 significantly (p<0.05) inhibited tumor growth of HCC 1806 xenografts compared to Control, DOX and the unconjugated mixture of DOX+RC-160 from day 14 and the inhibition remained significant until the end of the study on day 35. Conclusions: Our results indicate that treatment with targeted cytotoxic somatostatin analogue AN-162 produces a greater inhibition of tumor growth than DOX alone in somatostatin receptor positive TNBC. Our findings support the concept of targeted chemotherapy based on cytotoxic peptide analogues for the treatment of breast cancer and other cancers. No significant financial relationships to disclose.

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The oncogenic kinase NEK2 regulates an RBFOX2-dependent pro-mesenchymal splicing program in triple-negative breast cancer cells

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02210-3 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Chiara Naro ◽

Monica De Musso ◽

Francesca Delle Monache ◽

Valentina Panzeri ◽

Pierre de la Grange ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Triple Negative Breast Cancer ◽

Epithelial To Mesenchymal Transition ◽

Triple Negative ◽

Splice Variants ◽

The Cancer Genome Atlas ◽

Mesenchymal Transition ◽

Tnbc Cell ◽

Tnbc Cell Line

Abstract Background Triple-negative breast cancer (TNBC) is the most heterogeneous and malignant subtype of breast cancer (BC). TNBC is defined by the absence of expression of estrogen, progesterone and HER2 receptors and lacks efficacious targeted therapies. NEK2 is an oncogenic kinase that is significantly upregulated in TNBC, thereby representing a promising therapeutic target. NEK2 localizes in the nucleus and promotes oncogenic splice variants in different cancer cells. Notably, alternative splicing (AS) dysregulation has recently emerged as a featuring trait of TNBC that contributes to its aggressive phenotype. Methods To investigate whether NEK2 modulates TNBC transcriptome we performed RNA-sequencing analyses in a representative TNBC cell line (MDA-MB-231) and results were validated in multiple TNBC cell lines. Bioinformatics and functional analyses were carried out to elucidate the mechanism of splicing regulation by NEK2. Data from The Cancer Genome Atlas were mined to evaluate the potential of NEK2-sensitive exons as markers to identify the TNBC subtype and to assess their prognostic value. Results Transcriptome analysis revealed a widespread impact of NEK2 on the transcriptome of TNBC cells, with 1830 AS events that are susceptible to its expression. NEK2 regulates the inclusion of cassette exons in splice variants that discriminate TNBC from other BC and that correlate with poor prognosis, suggesting that this kinase contributes to the TNBC-specific splicing program. NEK2 elicits its effects by modulating the expression of the splicing factor RBFOX2, a well-known regulator of epithelial to mesenchymal transition (EMT). Accordingly, NEK2 splicing-regulated genes are enriched in functional terms related to cell adhesion and contractile cytoskeleton and NEK2 depletion in mesenchymal TNBC cells induces phenotypic and molecular traits typical of epithelial cells. Remarkably, depletion of select NEK2-sensitive splice-variants that are prognostic in TNBC patients is sufficient to interfere with TNBC cell morphology and motility, suggesting that NEK2 orchestrates a pro-mesenchymal splicing program that modulates migratory and invasive properties of TNBC cells. Conclusions Our study uncovers an extensive splicing program modulated by NEK2 involving splice variants that confer an invasive phenotype to TNBCs and that might represent, together with NEK2 itself, valuable therapeutic targets for this disease.

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