Sensitivity Of Breast Cancer Cells Research Articles

Baicalin, a Potent Inhibitor of NF-κB Signaling Pathway, Enhances Chemosensitivity of Breast Cancer Cells to Docetaxel and Inhibits Tumor Growth and Metastasis Both In Vitro and In Vivo.

ObjectiveThe aim of this study is to investigate the anti-cancer activity and sensibilization of baicalin (BA) against breast cancer (BC) cells.MethodsThe anti-proliferation of BA in BC cell lines was evaluated by MTT and colony formation assays. Apoptotic induction of BA was measured by flow cytometry. Wound-healing and transwell assays were exploited to assess migrated and invasive inhibition of BA. Western-blot and immunofluorescence were used to study mechanisms of anti-migration and sensibilization of BA. Anti-tumor and anti-metastasis effects of BA were evaluated in subcutaneous and pulmonary metastasis mouse model of BC cells.ResultsBA significantly suppressed proliferation and induced apoptosis of BC cells in a concentration- and time-dependent manner. Additionally, BA induced cell apoptosis via the mitochondria-mediated pathway, as evidenced by cellular induction of reactive oxygen species and upregulated expression of the Bax/Bcl-2 ratio. The overall expression and nuclear translocation of NF-κB signaling pathway in BC cells were dramatically inhibited by treatment with BA. BA significantly suppressed abilities of migration and invasion in BC cells. Notably, BA sensitized BC cells to docetaxel (DXL) by suppressing the expression of survivin/Bcl-2. BA also retarded tumor growth and triggered apoptosis of tumor cells in a tumor mouse model of 4T1 cells. Furthermore, pulmonary metastasis of BC cells was distinctly suppressed by BA in a tumor mouse model of 4T1 cells.ConclusionBA effectively triggered apoptosis, inhibited metastasis, and enhanced chemosensitivity of BC, implying that BA might serve as a promising agent for the treatment of BC.

Circle RNA circABCB10 Modulates PFN2 to Promote Breast Cancer Progression, as Well as Aggravate Radioresistance Through Facilitating Glycolytic Metabolism Via miR-223-3p.

Background: Breast cancer (BC) is a common tumor in women worldwide, and irradiation (IR) resistance is a major obstacle for BC therapy. Circle RNAs (circRNAs) were identified as implicated in the progression of cancer and IR resistance. However, the role of circABCB10 in BC progression and IR resistance is not well defined. Materials and Methods: The levels of circABCB10, miR-223-3p, and profilin-2 (PFN2) were detected by quantitative real-time polymerase chain reaction. The cell viability and survival rate were monitored by MTT assay. The glucose consumption, lactic acid production, LDH-A activity, and ATP production were evaluated to measure glycolysis. The protein levels of hypoxia inducible factor-1α (HIF-1α), hexokinase 2 (HK2), lactate dehydrogenase A chain (LDH-A), and PFN2 were estimated by Western blot assay. The colony formation rate was tested by colony formation assay. Dual-luciferase reporter assay was constructed to validate the interaction between miR-223-3p and circABCB10 or PFN2. The mice xenograft assay was performed to further verify the effects of circABCB10 on BC progression in vivo. Results: CircABCB10 and PFN2 were elevated, while miR-223-3p was reduced in BC tissues and cells. CircABCB10 sponged miR-223-3p, and PFN2 was a target of miR-223-3p in BC cells. CircABCB10 silencing inhibited cell proliferation, glycolysis, colony formation, and decreased IR resistance in BC cells by modulating miR-223-3p. Meanwhile, circABCB10 depletion restrained xenograft tumor growth in vivo. Also, miR-223-3p overexpression refrained cell proliferation, glycolysis, and colony formation while improving IR sensitivity in BC cells by regulating PFN2. Besides, circABCB10 knockdown declined PFN2 in BC cells via miR-223-3p. The glycolysis inhibitor 2-deoxy-D-glucose enhanced IR sensitivity in BC cells via circABCB10. Conclusion: CircABCB10 knockdown contributed to irradiation sensitivity by negatively regulating glycolysis via the miR-223-3p/PFN axis in breast cancer.

Abstract B19: Role of cell-cell communication in endocrine therapy-resistant breast cancer

Abstract Endocrine therapy is the mainstay for the treatment of estrogen-receptor positive (ER+) breast cancer. However, frequent emergence of endocrine resistance is a major clinical challenge. Cell-cell cooperation in the tumor microenvironment plays a vital role in acquiring resistance, but its role is poorly understood. We studied the interactions among endocrine-resistant (R) and -sensitive (S) breast cancer cells in the presence and absence of ER-targeted endocrine therapy. We measured changes in admixed cell population remodeling by dual fluorescent labeling of LCC1 (S) with eGFP (green fluorescent protein) and their derived resistant variant (selected against the antiestrogen fulvestrant) LCC9 (R) with mCherry (red fluorescent protein), respectively. Thus, unlike most prior studies, these are genetically related cells, rather than ER+ and ER- cell lines obtained from different patients. Our results revealed that “S”cells (LCC1) acquired the phenotype of “R” cells when cultured in a mixed population. A ratio of 5:1 of S:R cells in mixed population was sufficient to confer resistance to fulvestrant to the S cells within a six-day period. Furthermore, we show that acquirement of this resistant phenotype for the S cells was partially blocked by inhibition of either gap junctions or exosome secretion, suggesting a role of cell-cell contact as well as extracellular vesicles. Global proteomic analysis of pure and admixed S and R cell populations showed that R and S can alter key molecular features of each other when present in mixed cell populations. We used a Bayesian approach to fit single culture cell populations to infer density-dependent growth parameters (growth rate, carrying capacity) and a generalized Lotka-Volterra model to understand how S and R coculture populations may be depressing or supporting growth of the other. Our results identify a net mutualistic interaction between the susceptible and resistant cancer strains, demonstrating that there are ecological dynamics to cancer resistance. In some tumors, ecological dynamics may be more mechanistically relevant than simple Darwinian-based clonal selection in driving tumor heterogeneity and determining response to treatment. Our study strongly indicates that different tumor cell types that may exist in tumors are able to influence each other, which leads to alteration of phenotype. Note: This abstract was not presented at the conference. Citation Format: Surojeet Sengupta, Rong Hu, Zachary Susswein, Adeola Fagunloye, Shweta Bansal, Robert Clarke. Role of cell-cell communication in endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B19.

Abstract B29: Modeling the oxygen-deprived breast cancer tumor microenvironment within a three-dimensional bioengineered platform that exhibits hypoxia-driven immune evasion

Abstract Introduction: Oxygen deprivation within tumors has been associated with enhanced cancer cell survival, altered extracellular matrix (ECM) production, and increased immune evasion in breast cancer (BCa). These outcomes collectively contribute to chemotherapeutic challenges. Current models do not adequately mimic physiologic oxygen levels relevant to breast tissue and tumor-immune interactions. Taking this into consideration, we developed a plasma-derived three-dimensional (3D) model that supports growth of BCa cells, generates physio- and pathophysiologic oxygen levels, and supports prevailing hypoxia-driven tumor outcomes such as immune evasion. Methods: BCa cells (MCF7 and MDA-MB-231) were embedded into 3D scaffolds formed through the cross-linking of patient-derived plasma. The oxygen content within these 3D matrices was engineered and further characterized to recapitulate physiologic blood and breast tissue (3D physiologic, gradient 14-10 kPa) or pathophysiologic (3D tumorous, gradient 9.6-2.4 kPa) levels by the manipulation of height, culture time, oxygen incubation environment, and cellular concentration. BCa cells grown within 3D physiologic and tumorous scaffolds were analyzed by flow cytometry, confocal imaging, or immunohistochemistry for the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha), proliferation capabilities, and ECM production. Peripheral blood mononuclear cells were incorporated on the top of 3D physiologic and tumorous scaffolds, and the differences in the tumor-infiltrating capabilities of T cells into 3D physiologic and tumorous scaffolds were assessed by flow cytometry. Lastly, hypoxic inhibition was validated as a target to sensitize BCa cells in order to overcome immune surveillance. Results: BCa cells grown in oxygen-deprived 3D tumorous environments showed a significant increase in the expression of HIF-1 alpha and a reduced cell proliferation compared to the BCa cells in 3D physiologic scaffolds. Increased secretion of collagen I, collagen III, laminin, and fibronectin was demonstrated by BCa cells in 3D tumorous compared to 3D physiologic scaffolds. T-cell infiltration (especially CD8 T cells) was significantly impaired within the oxygen-deprived breast cancer tumor microenvironment (TME), and hypoxia inhibition was further confirmed to resensitize BCa cells to cytotoxic CD8 T cells. Conclusions: Modeling of the oxygen-deprived breast cancer TME in our engineered 3D physiologic and tumorous scaffolds supported known intratumoral hypoxia characteristics such as reduced BCa cell proliferation, increased ECM expression, and hindered immune infiltration. Furthermore, hypoxic inhibition was validated as a target to sensitize BCa cells to immune surveillance. These models could serve as a promising platform for the evaluation of immunologic events as well as a drug-screening platform tool to overcome hypoxia-driven immune evasion. Citation Format: Somshuvra Bhattacharya, Kristin Calar, Claire Evans, Pilar de la Puente. Modeling the oxygen-deprived breast cancer tumor microenvironment within a three-dimensional bioengineered platform that exhibits hypoxia-driven immune evasion [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B29.

Abstract B066: SCUBE3 inhibition improves doxorubicin response in breast cancer

Abstract The development of novel targeted therapies is urgently required for improving the outcome of breast cancer patients. Chemotherapy is the common treatment option for malignant breast cancer. However, resistance and toxicity remain the major obstacles hindering the effectiveness of chemotherapeutic agents in cancer patients. Therefore, identifying genes/factors that sensitize breast cancer cells to chemotherapeutic agents could improve treatment outcome in patients. Using an unbiased high-throughput screen, we identified Signal peptide CUB domain EGF-like 3 (SCUBE3) genes as a novel therapeutic adjuvant that can improve the efficacy of doxorubicin, a chemotherapeutic agent commonly used in treating breast cancer patients. Our findings demonstrated that SCUBE3 promotes breast cancer cells' progression as knockdown of SCUBE3 inhibited the ability of breast cancer cells to form colony, migrate, and invade, while overexpression of SCUBE3 promoted tumor growth in preclinical mouse models. Our results revealed that SCUBE3 mediates its protumor effects by regulating genes involved in growth and survival in the MAPK pathway, DNA damage surveillance pathway including RAD51 and FOXM1, and apoptotic pathway including Mcl-1. Using interaction studies, we demonstrated that EGFR is a true receptor of SCUBE3 as EGFR and SCUBE3 interact and this interaction mediated progrowth signaling of SCUBE3. These findings underline the importance of SCUBE3 as a potent therapeutic target for treating breast cancer patients. Citation Format: Benjamin C. Onyeagucha, Kashish Dhillon, Subapriya Rajamanickam, Subbarayalu Panneerdoss, Vijay K. Eedunuuri, Tabrez A. Mohammad, Santosh Timilsina, Yidong Chen, Manjeet K. Rao. SCUBE3 inhibition improves doxorubicin response in breast cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B066.

MiR-27a ameliorates chemoresistance of breast cancer cells by disruption of reactive oxygen species homeostasis and impairment of autophagy

In patients with breast cancer, primary chemotherapy often fails due to survival of chemoresistant breast cancer stem cells (BCSCs) which results in recurrence and metastasis of the tumor. However, the factors determining the chemoresistance of BCSCs have remained to be investigated. Here, we profiled a series of differentially expressed microRNAs (miRNAs) between parental adherent breast cancer cells and BCSC-mimicking mammosphere-derived cancer cells, and identified hsa-miR-27a as a negative regulator for survival and chemoresistance of BCSCs. In the mammosphere, we found that the expression of hsa-miR-27a was downregulated, and ectopic overexpression of hsa-miR-27a reduced both number and size of mammospheres. In addition, overexpression of hsa-miR-27a sensitized breast cancer cells to anticancer drugs by downregulation of genes essential for detoxification of reactive oxygen species (ROS) and impairment of autophagy. Therefore, enhancing the hsa-miR-27a signaling pathway can be a potential therapeutic modality for breast cancer.

Long Noncoding RNA LINC00261 Induces Chemosensitization to Tamoxifen in Human Breast Cancer

Breast cancer (BC) is one of the most prevalent and mortal malignancies in women worldwide, and tamoxifen is the mainstay treatment of breast cancer and the development of resistance represents a major obstacle for a cure. Long non-coding RNAs (LncRNAs) LINC00261 have been identified to serve a key role in the development of several tumors. However, the role of LINC00261 in breast cancer and chemotherapy resistance remains largely unknown. To investigate the role of LINC00261 in BC cells, LINC00261 was upregulated in MCF-7-TAM cells by transfecting with LINC00261 plasmid (pcDNA-LINCC00261). Subsequently, cell viability and drug sensitivity were measured using the CCK-8 assay. Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) was performed to detect the level of LINC00261 in BC cells. Cell migration, invasion, and apoptosis were detected by Transwell, Scratch Test and Flow cytometry, respectively. Additionally, the associated protein expression was detected using Western blot. The results demonstrated that LINC00261 was significantly down-regulated in BC cells, especially in MCF-7-TAM cells. Overexpression of LINC00261 inhibited cell proliferation, migration, and invasion in MCF-7-TAM cells. Further, an abundant of LINC00261 sensitized breast cancer cells to tamoxifen and reduced tamoxifen-induced apoptosis in MCF-7-TAM cells. Finally, LINC00261 significantly regulated the protein expression of drug-resistant genes and the protein expression related to tumor metastasis and cell apoptosis. Therefore, this study revealed that LINC00261 induces chemosensitization to tamoxifen in human breast cancer, it may be a useful biomarker and potential therapeutic target.

Linc‐ROR promotes the progression of breast cancer and decreases the sensitivity to rapamycin through miR‐194‐3p targeting MECP2

linc‐ROR is reported to be a potential biomarker of breast cancer, but the detailed mechanism of linc‐ROR‐mediated breast cancer regulation has not been fully studied. We aimed to explore how linc‐ROR affects proliferation, metastasis, and drug sensitivity in breast cancer. Cell lines in which linc‐ROR was overexpressed or knocked down were constructed, and the cell proliferation, colony formation, cell migration, and invasion abilities of these lines were explored. A CCK‐8 assay was performed to determine the sensitivity of the breast cancer cells to rapamycin. Next‐generation sequencing was conducted to explore the detailed regulatory mechanism of linc‐ROR; differentially expressed RNAs in the linc‐ROR‐overexpressing cell line compared with the negative control were screened out, and their target genes were chosen to perform Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, protein–protein interaction network analysis, and competing endogenous RNA (ceRNA) network analysis. The ceRNA mechanism of linc‐ROR for miR‐194‐3p, which targets MECP2, was determined through dual‐luciferase reporter assay, RT–qPCR, western blot, and rescue experiments. Finally, we found that linc‐ROR was upregulated in breast tumor tissues. linc‐ROR promoted the cell proliferation, colony formation, cell migration, and invasion of breast cancer and decreased the sensitivity of breast cancer cells to rapamycin. The overexpression of linc‐ROR triggered changes in the whole transcriptome of breast cancer cells, and a total of 85 lncRNAs, 414 microRNAs, 490 mRNAs, and 92 circRNAs were differentially expressed in the linc‐ROR‐overexpressing cell line compared with the negative control. Through a series of bioinformatic analyses, the ‘linc‐ROR/miR‐194‐3p/MECP2’ ceRNA regulatory axis was confirmed to be involved in the linc‐ROR‐mediated progression and drug sensitivity of breast cancer. In conclusion, linc‐ROR serves as an onco‐lncRNA in breast cancer and promotes the survival of breast cancer cells during rapamycin treatment by functioning as a ceRNA sponge for miR‐194‐3p, which targets MECP2.

Administration with hyperoside sensitizes breast cancer cells to paclitaxel by blocking the TLR4 signaling

Breast cancer is a malignancy and one of the most frequent causes of cancer death among women worldwide. Paclitaxel is a common chemotherapeutic drug and has recently been shown to facilitate tumor cell escape during cytotoxic chemotherapy by inducing inflammatory mediators and pro-survival protein expression. Hyperoside is a flavonoid glycoside compound and exerts anti-inflammation, and anti-tumor growth properties. However, its function in breast cancer chemosensitivity remains poorly elucidated. In this study, hyperoside exhibited little cytotoxicity to normal human breast mammary epithelial cell lines, and also protected against paclitaxel-induced cytotoxicity in MCF-10A. Importantly, treatment with hyperoside engendered not only inhibition of cell viability, but also potentiated cancer cell sensitivity to paclitaxel in TLR4-positive breast cancer MDA-MB-231 cells by suppressing cell viability, and increasing cell apoptosis and caspase-3 activity. Nevertheless, although hyperoside exposure restrained cell viability, its treatment presented little effects to paclitaxel sensitivity in TLR4-null HCC1806 cells. Intriguingly, paclitaxel stimulation activated the TLR4-NF-κB signaling, which was reversed after hyperoside administration. Concomitantly, hyperoside also attenuated paclitaxel-mediated anti-apoptotic Bcl-2 expression, but enhanced the effects of paclitaxel on pro-apoptotic Bax expression, and pro-inflammatory cytokine IL-6 and IL-6 levels in MDA-MB-231 cells. Importantly, restoring the TLR4 pathway overturned hyperoside-evoked chemosensitivity to paclitaxel in MDA-MB-231 cells. Thus, hyperoside may elevate breast cancer cell sensitivity to paclitaxel by blocking TLR4 activation-mediated pro-inflammatory and pro-survival approaches, thereby endorsing its usefulness as a promising therapeutic combination to overcome chemosensitivity in breast cancer.

MiR‑187‑3p increases gemcitabine sensitivity in breast cancer cells by targeting FGF9 expression

Breast cancer is the most common type of malignancy in women, which remains a significant health concern worldwide. Gemcitabine is a frequently applied anticancer pharmacological agent. However, the efficacy of gemcitabine is limited by chemoresistance. In the present study, a combination of reverse transcription quantitative-PCR, cell viability, flow cytometry, luciferase reporter assay and western blot analysis were performed to elucidate the potential effects of miR-187-3p on gemcitabine sensitivity in the breast cancer cell line, MDA-MB-231. The results revealed that miR-187-3p was significantly decreased in the breast cancer tumor tissues. Moreover, the overexpression of miR-187-3p significantly inhibited cell viability and promoted apoptosis in MDA-MB-231 cells. In addition, miR-187-3p overexpression enhanced the anti-proliferative and pro-apoptotic effects of gemcitabine, indicating that miR-187-3p regulated gemcitabine sensitivity in breast cancer cells. Mechanistically, miR-187-3p negatively regulated the expression of fibroblast growth factor 9 (FGF9) by binding to its 3'-untranslated region. Overexpression of FGF9 reversed the aforementioned effects of miR-187-3p overexpression on cell viability and apoptosis in the presence of gemcitabine. In conclusion, the present study indicated that miR-187-3p increased gemcitabine sensitivity in breast cancer cells by targeting FGF9 expression.

LncRNA TROJAN promotes proliferation and resistance to CDK4/6 inhibitor via CDK2 transcriptional activation in ER+ breast cancer

BackgroundEstrogen receptor-positive (ER+) breast cancers represent approximately two-thirds of all breast cancers and have a sustained risk of late disease recurrence. Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown significant efficacy in ER+ breast cancer. However, their effects are still limited by drug resistance. In this study, we aim to explore the role of long noncoding RNA TROJAN in ER+ breast cancer.MethodsThe expression level of TROJAN in breast cancer tissue and cell lines was determined by quantitative real-time PCR. In vitro and in vivo assays as well as patient derived organoid were preformed to explore the phenotype of TROJAN in ER+ breast cancer. The TROJAN-NKRF-CDK2 axis were screened and validated by RNA pull-down, mass spectrometry, RNA immunoprecipitation, microarray, dual-luciferase reporter and chromatin immunoprecipitation assays.ResultsHerein, we showed that TROJAN was highly expressed in ER+ breast cancer. TROJAN promoted cell proliferation and resistance to a CDK4/6 inhibitor and was associated with poor survival in ER+ breast cancer. TROJAN can bind to NKRF and inhibit its interaction with RELA, upregulating the expression of CDK2. The inhibition of TROJAN abolished the activity of CDK2, reversing the resistance to CDK4/6 inhibitor. A TROJAN antisense oligonucleotide sensitized breast cancer cells and organoid to the CDK4/6 inhibitor palbociclib both in vitro and in vivo.ConclusionsTROJAN promotes ER+ breast cancer proliferation and is a potential target for reversing CDK4/6 inhibitor resistance.

Upregulation of microRNA‑1 inhibits proliferation and metastasis of breast cancer.

Recent studies have shown that microRNAs (miRs) play a key role in the regulation of cancer development. In the present study, reverse transcription-quantitative PCR was used to detect the expression of miR-1 in breast cancer and adjacent tissues, and survival analysis was performed to compare the low-expression groups with the Kaplan-Meier method. Overexpression of miR-1 was used to observe the effects on the proliferation, migration and invasion of breast cancer cells in vitro and in vivo. Moreover, Bcl-2 expression was measured by western blotting and luciferase assays after the overexpression of miR-1. The present study reported that miR-1 is expressed at low levels in breast cancer and that cell proliferation, migration and invasion are inhibited in miR-1-overexpressing cells. Enhanced miR-1 expression can also increase cell apoptosis. The present study also demonstrated that Bcl-2 is a potential target of miR-1. In vivo studies indicate that overexpression of miR-1 decreases tumor volume and weight in nude mice. The data from the present study demonstrated for the first time that overexpression of miR-1 increases the sensitivity of breast cancer cells to paclitaxel and cisplatin. The present study provided new evidence for the important role of miR-1 in the tumorigenesis and drug sensitivity of breast cancer.

Correction: Senescence Sensitivity of Breast Cancer Cells Is Defined by Positive Feedback Loop between CIP2A and E2F1

Correction: Senescence Sensitivity of Breast Cancer Cells Is Defined by Positive Feedback Loop between CIP2A and E2F1

<p>MiR-153-5p Enhances the Sensitivity of Triple-Negative Breast Cancer Cells to Paclitaxel by Inducing G2M Phase Arrest</p>

BackgroundPaclitaxel (PTX) resistance is a main obstacle for the treatment of triple-negative breast cancers (TNBC). Evidences have shown that miR-153-5p could induce the apoptosis of breast cancer cells. Thus, this study aimed to investigate the effect of miR-153-5p on PTX-resistance TNBC cells.MethodsCell Counting Kit-8, flow cytometry and wound healing assays were used to detect the viability, apoptosis and migration of MDA-MB-231/PTX cells, respectively. The luciferase reporter assay was used to explore the potential binding targets of miR-153-5p. The expressions of CDK1, cyclin B1 and p-Akt in MDA-MB-231/PTX cells were detected with Western blot. In vivo animal study was performed finally.ResultsIn this study, the inhibitory effects of PTX on the proliferation and migration of MDA-MB-231/PTX cells were significantly enhanced following transfection with miR-153-5p. In addition, overexpression of miR-153-5p markedly enhanced the pro-apoptotic effect of PTX on MDA-MB-231/PTX cells. Luciferase reporter assay validated that cyclin-dependent kinase 1 (CDK1) was a potential binding target of miR-153-5p. Moreover, overexpression of miR-153-5p prominently increased PTX-induced cell cycle arrest at G2/M phase in MDA-MB-231/PTX cells via downregulation of CDK1, cyclin B1 and p-Akt. In vivo experiments confirmed that overexpression of miR-153-5p notably enhanced PTX sensitivity in MDA-MB-231/PTX xenograft model.ConclusionWe found that overexpression of miR-153-5p could reverse PTX resistance in PTX-resistant TNBC cells via inducing G2/M phase arrest, indicating that miR‑153-5p may be a promising agent for patients with PTX-resistant TNBC.

LncRNA SNORD3A specifically sensitizes breast cancer cells to 5-FU by sponging miR-185-5p to enhance UMPS expression

Breast cancer is the most common cancer type in women. Long non-coding RNAs (lncRNAs) have been reported as potential new diagnostic markers, prognostic factors, and therapeutic targets in cancer. However, the specific roles and mechanisms of lncRNAs in breast cancer remain to be elucidated. Here we demonstrated the downregulation of lncRNA SNORD3A in breast cancer cells and tissues and verified its non-protein-coding property. SNORD3A overexpression had no effect on cell proliferation but specifically sensitized breast cancer cells to 5-fluorouracil (5-FU) in vitro and in vivo. Mechanistically, SNORD3A exerts its effect via enhancing uridine monophosphate synthetase (UMPS) protein expression. SNORD3A acts as a competing endogenous RNA for miR-185-5p, leading to UMPS protein upregulation. miR-185-5p overexpression disrupted the effect of SNORD3A on chemosensitization to 5-FU in vitro and in vivo. Moreover, Meis1 overexpression transcriptionally promotes SNORD3A expression, and Meis1 is downregulated in breast cancer cells and tissues. In breast cancer tissues, SNORD3A level positively correlates with Meis1 and UMPS protein levels, whereas miR-185-5p level negatively correlates with UMPS protein level. High SNORD3A transcript and Meis1 and UMPS protein levels predicts a better outcome, but high miR-185-5p level predicts a worse outcome in breast cancer patients receiving 5-FU-based chemotherapy. Our findings indicate that Meis1-regulated SNORD3A specifically sensitizes breast cancer cells to 5-FU via enhancing UMPS expression. The SNORD3A–UMPS axis may serve as a potential biomarker and therapeutic target to improve the efficacy of 5-FU-based chemotherapy for breast cancer patients.

Checkpoint Kinase 1 (CHK1) Inhibition Enhances the Sensitivity of Triple-Negative Breast Cancer Cells to Proton Irradiation via Rad51 Downregulation.

Due to a superior dose conformity to the target, proton beam therapy (PBT) continues to rise in popularity. Recently, considerable efforts have been directed toward discovering treatment options for use in combination with PBT. This study aimed to investigate the targeting of checkpoint kinase 1 (CHK1), a critical player regulating the G2/M checkpoint, as a promising strategy to potentiate PBT in human triple-negative breast cancer (TNBC) cells. Protons induced cell-cycle arrest at the G2/M checkpoint more readily in response to increased CHK1 activation than X-rays. A clonogenic survival assay revealed that CHK1 inhibition using PF-477736 or small interfering RNA (siRNA) enhanced the sensitivity toward protons to a greater extent than toward X-rays. Western blotting demonstrated that PF-477736 treatment in the background of proton irradiation increased the pro-apoptotic signaling, which was further supported by flow cytometry using annexin V. Immunofluorescence revealed that proton-induced DNA double-strand breaks (DSBs) were further enhanced by PF-477736, which was linked to the downregulation of Rad51, essential for the homologous recombination repair of DSBs. Direct inactivation of Rad51 resulted in enhanced proton sensitization. Collectively, these data suggest that targeting CHK1 may be a promising approach for improving PBT efficacy in the treatment of TNBC.

Integrative bioinformatics analysis reveals miR-494 and its target genes as predictive biomarkers of trastuzumab-resistant breast cancer

BackgroundThe focus of trastuzumab resistance biomarkers in recent decades has been on epigenetic and non-coding RNA-based mechanisms. In this study, the potential of miR-494 and its target genes as predictive biomarkers for breast cancer (BC) resistance to trastuzumab was identified. The microarray data were obtained from the GEO database, including GSE101841, GSE75669, and GSE66305. Data processing was conducted using GEO2R to obtain differentially expressed genes (DEGs).ResultsThe data analysis using GEO2R revealed that DEGs from GSE101841 and GSE75669 consisted of 3 and 135 upregulated miRNAs, respectively. On the other hand, the same analysis revealed 8 and 226 downregulated miRNAs for DEGs from GSE101841 and GSE75669, respectively. A Venn diagram showed that one miR was detectable in serum and tissue samples, namely miR-494. The miR-494 target was predicted using the miRecords database and resulted in 69 target genes. A Venn diagram between miR-494 target genes from miRecords and the mRNA array from GSE66305 revealed three potential targets of CNR1, RBM39, and ZNF207. A Kaplan–Meier survival plot showed that BC patients with a high miR-494 level and a low ZNF207 mRNA level had significantly worse overall survival. Validation of target genes in BC samples and trastuzumab-resistant and -sensitive BC cells with GEPIA and ONCOMINE highlighted the potential of CNR1, RBM39, and ZNF207 as predictive biomarkers of trastuzumab resistance in BC cells.ConclusionTaken together, these results suggest that miR-494 plays a role in the mechanism of BC resistance to trastuzumab by involving its target genes CNR1, RBM39, and ZNF207.

WWOX possesses N‐terminal cell surface‐exposed epitopes WWOX7‐21 and WWOX7‐11 for signaling cancer growth suppression and prevention in vivo

Membrane hyaluronidase Hyal‐2 supports cancer cell growth. Inhibition of Hyal‐2 by specific antibody against Hyal‐2 or pY216‐Hyal‐2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal‐2. Alternatively, WWOX undergoes self‐polymerization and localizes in the cell membrane. Proapoptotic pY33‐WWOX binds Hyal‐2, and TGF‐β induces internalization of the pY33‐WWOX/Hyal‐2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14‐WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface‐exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3‐dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7‐21 peptide, or truncation to 5‐amino‐acid WWOX7‐11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time‐lapse microscopy revealed that WWOX7‐21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33‐WWOX, downregulation of prosurvival pERK, prompt increases in Ca+2 influx, and disruption of the IκBα/WWOX/ERK prosurvival signaling. In contrast, pS14‐WWOX7‐21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib‐mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14‐WWOX significantly enhanced the ceritinib‐induced apoptosis. Together, the N‐terminal epitopes WWOX7‐21 and WWOX7‐11 are potent in blocking cancer growth in vivo. WWOX7‐21 and WWOX7‐11 peptides and pS14‐WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.Support or Funding InformationMinistry of Science and Technology, Taiwan, National Health Research Institute, Taiwan, and Department of Defense, USAWWOX peptides regulate breast cancer cell sensitivity to ceritinib.Figure 1

Combination of cisplatin, Hedyotis corymbosa L. and Tinospora crispa extracts as a new therapy for breast cancer cells 4T1 through in vitro induction and cell cycle modulation

Cisplatin is a chemotherapeutic agent with varying side effects. Berberine compounds in Tinospora crispa and ursolic acids in Hedyotis corymbosa L are known to have high cytotoxic activities. This study was aimed to examine the effects of the combination of cisplatin, the ethanolic extract of Hedyotis corymbosa L.(EEHC), and the ethanolic extract of Tinospora crispa roots (EETC) on enhanced breast cancer cell sensitivity by apoptotic induction and cell cycle modulation. Cytotoxic effects were tested using the MTT assay in breast cancer cells, 4T1. The combination test of cisplatin and the extracts yielded combination index (CI) and cell viability, and its effects on the induction of apoptosis and cell cycle modulation were observed by the flow cytometry method. The results of the cytotoxic test showed CI of less than one for 1 mg/mL EEHC, 6 mg/mL EETC, and 1.68 mg/mL cisplatin. The EEHC-EETC-cisplatin combination accumulated cells in the S phase (29.98%) and induced apoptosis in 4T1 cells. It proves that EEHC and EETC can be developed as co-agents with cisplatin to improve the effectiveness of breast cancer chemotherapeutic treatment.

Dexamethasone simulates the anticancer effect of nano-formulated paclitaxel in breast cancer cells.

Although the chemosensitizing effect of Dexamethasone (DEX) and its ability to increase the sensitivity of breast cancer cells to chemotherapy were previously reported, this study aimed to explore how far cotreatment of breast cancer cells with paclitaxel (PTX) and DEX mimics the anticancer effect of nanoformulated PTX. To establish this goal, PTX was nanoformulated with poly (lactic-co-glycolic acid) (PLGA) and the nanoparticles (PTX-NPs) were physically authenticated. Breast cancer cells (MCF-7) were treated with PTX or PTX-NPs in presence or absence of low concentration (10nM) of DEX. Cells viability (assessed by MTT assay), apoptosis (assessed by flow cytometry) and the expression of PTX resistance gene (TRX1) and PTX metabolizing genes (CYP2C8 and CYP3A4) were investigated. The results showed that nanoformulated PTX was validated by nano-size assessment, increased the anionic surface charge and prober conjugation with the biodegradable carrier (PLGA), as indicated by the FTIR spectroscopy. Initially, the IC50 value of PTX was 19.3μg/ml and cotreatment with DEX minimized it to 5.22μg/ml, whereas PTX-NPs alone inhibited cell proliferation with IC50 6.67μg/ml. Also, in presence of DEX, PTX-NPs further decreased the IC50 to 5μg/ml. In parallel, DEX has increased the responsiveness of cells to PTX without potentiating its apoptotic effect. Moreover, the glucocorticoid (with PTX or PTX-NPs) downregulated TXR1 gene by 26% (P<0.01) and 28.4% (P<0.05) respectively. Similarly, the mRNA level of CYP3A4 significantly decreased in presence of DEX. The main PTX metabolizing gene CYP2C8, in contrast, was upregulated, especially in cells cotreated with PTX/DEX (P<0.001). Conclusively, the study reports that cotreatment of breast cancer cells with submolar concentration of DEX acts as similar as the nanoformulated PTX, possibly through its modulatory effects on the expression of the main PTX metabolizing gene (CYP2C8) and downregulating Taxol resistance gene.