SKBR3 Breast Cancer Cells Research Articles (Page 2)

The advent of pH-sensitive liposomes (pHLips) has opened new opportunities for the improved and targeted delivery of antitumor drugs as well as gene therapeutics. Comprising fusogenic dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS), these nanosystems harness the acidification in the tumor microenvironment and endosomes to deliver drugs effectively. pH-responsive liposomes that are internalized through endocytosis encounter mildly acidic pH in the endosomes and thereafter fuse or destabilize the endosomal membrane, leading to subsequent cargo release into the cytoplasm. The extracellular tumor matrix also presents a slightly acidic environment that can lead to the enhanced drug release and improved targeting capabilities of the nano-delivery system. Recent studies have shown that folic acid (FA) and iRGD-coated nanocarriers, including pH-sensitive liposomes, can preferentially accumulate and deliver drugs to breast tumors that overexpress folate receptors and αvβ3 and αvβ5 integrins. This study focuses on the development and characterization of 5-Fluorouracil (5-FU)-loaded FA and iRGD surface-modified pHLips (FA-iRGD-5-FU-pHLips). The novelty of this research lies in the dual targeting mechanism utilizing FA and iRGD peptides, combined with the pH-sensitive properties of the liposomes, to enhance selective targeting and uptake by cancer cells and effective drug release in the acidic tumor environment. The prepared liposomes were small, with an average diameter of 152 ± 3.27 nm, uniform, and unilamellar, demonstrating efficient 5-FU encapsulation (93.1 ± 2.58%). Despite surface functionalization, the liposomes maintained their pH sensitivity and a neutral zeta potential, which also conferred stability and reduced aggregation. Effective pH responsiveness was demonstrated by the observation of enhanced drug release at pH 5.5 compared to physiological pH 7.4. (84.47% versus 46.41% release at pH 5.5 versus pH 7.4, respectively, in 72 h). The formulations exhibited stability for six months and were stable when subjected to simulated biological settings. Blood compatibility and cytotoxicity studies on MDA-MB-231 and SK-BR3 breast cancer cell lines revealed an enhanced cytotoxicity of the liposomal formulation that was modified with FA and iRGD compared to free 5-FU and minimal hemolysis. Collectively, these findings support the potential of FA and iRGD surface-camouflaged, pH-sensitive liposomes as a promising drug delivery strategy for breast cancer treatment.

Estrogen receptor (ER)-negative breast cancers are known to be aggressive and unresponsive to antiestrogen therapy, and triple-negative breast cancers are associated with poor prognosis and metastasis. Thus, new targeted therapies are needed. Forkhead box M1 (FOXM1) is abundantly expressed in human cancers and implicated in protecting tumor cells from oxidative stress by reducing the levels of intracellular reactive oxygen species (ROS). Aspirin, a prototypical anticancer agent with deleterious side effects that has been modified to release nitric oxide and hydrogen sulfide is called nitric oxide-hydrogen sulfide-releasing aspirin (NOSH-aspirin, NOSH-ASA), generating a "safer" class of new anti-inflammatory agents. We evaluated NOSH-ASA against ER-negative breast cancer using cell lines and a xenograft mouse model. NOSH-ASA strongly inhibited growth of MDA-MB-231 and SKBR3 breast cancer cells with low IC50s of 90 ± 5 and 82 ± 5 nM, respectively, with marginal effects on a normal breast epithelial cell line. NOSH-ASA inhibited cell proliferation, caused G0/G1 phase arrest, increased apoptosis, and was associated with increases in ROS. In MDA-MB-231 cell xenografts, NOSH-ASA reduced tumor size markedly, which was associated with reduced proliferation (decreased proliferating cell nuclear antigen expression), induction of apoptosis (increased terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells), and increased ROS, whereas nuclear factor κ-light-chain-enhancer of activated B cells and FoxM1 that were high in untreated xenografts were significantly reduced. mRNA data for FoxM1, p21, and cyclin D1 corroborated with the respective protein expressions and arrest of cells. Taken together, these molecular events contribute to NOSH-ASA-mediated growth inhibition and apoptotic death of ER-negative breast cells invitro and invivo. Additionally, as a ROS inducer and FOXM1 inhibitor, NOSH-ASA has potential as a targeted therapy. SIGNIFICANCE STATEMENT: We examined the cellular effects and xenograft tumor inhibitory potential of NOSH-aspirin, a nitric oxide- and hydrogen sulfide-donating hybrid, against estrogen receptor-negative breast cancer, which currently lacks effective therapeutic options. Inducing reactive oxygen species and downregulating forkhead box M1 are plausible mechanisms contributing to decreased cell proliferation and increased apoptosis. NOSH-aspirin reduced tumor size by 90% without inducing any observable gross toxicity, underscoring its promising translational potential.

Characterization of multifunctional β-cyclodextrin-coated Bi2O3 nanoparticles conjugated with curcumin for CT imaging-guided synergetic chemo-radiotherapy in breast cancer

Discovery of acetophenone/piperazin-2-one hybrids as selective anti-TNBC cancer agents by causing DNA damage

Our study focused on assessing the effects of three newly identified BRCA1 exon 11 variants (c.1019T>C, c.2363T>G, and c.3192T>C) on breast cancer susceptibility. Using computational predictions and experimental splicing assays, we evaluated their potential as pathogenic mutations. Our in silico analyses suggested that the c.2363T>G and c.3192T>C variants could impact both splicing and protein function, resulting in the V340A and V788G mutations, respectively. We further examined their splicing effects using minigene assays in MCF7 and SKBR3 breast cancer cell lines. Interestingly, we found that the c.2363T>G variant significantly altered splicing patterns in MCF7 cells but not in SKBR3 cells. This finding suggests a potential influence of cellular context on the variant's effects. While attempts to correlate in silico predictions with RNA binding factors were inconclusive, this observation underscores the complexity of splicing regulation. Splicing is governed by various factors, including cellular contexts and protein interactions, making it challenging to predict outcomes accurately. Further research is needed to fully understand the functional consequences of the c.2363T>G variant in breast cancer pathogenesis. Integrating computational predictions with experimental data will provide valuable insights into the role of alternative splicing regulation in different breast cancer types and stages.

Abstract Introduction Due to the unique conditions of the tumor microenvironment, such as hypoxia, poor angiogenesis, and mitochondrial dysfunction, cancer cells face challenges in obtaining nutrients and energy supply. However, monounsaturated fatty acids have been identified as preferred substrates for energy generation and maintenance of cellular structures in cancer cells. These fatty acids can be obtained through de novo fatty acid synthesis. Stearoyl-CoA desaturase 1 (SCD1) is the primary enzyme involved in the biosynthesis of monounsaturated fatty acids from saturated fatty acids and is known to be upregulated in various malignancies, including breast cancer. This study aimed to assess the expression and activity index of SCD1 in well-characterized MCF-7, SK-BR3, and MDA-MB231 breast cancer cell lines, representing luminal A/B, HER2-enriched, and triple-negative subtypes, respectively. Methods The breast cancer cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum at 37°C, 5% CO2, and 95% humidity for 72 hours. After harvesting the cells, SCD1 expression was analyzed using real-time quantitative PCR, and the activity index was determined by measuring the ratio of monounsaturated fatty acids to saturated fatty acids using capillary gas-liquid chromatography. Results MCF-7 cell line showed a significantly higher SCD1 expression compared to MDA-MB231 (250-fold, p< 0.0001) and SK-BR3 (83-fold, p< 0.0001) cell lines. The activity index of SCD1 was significantly higher in both MCF-7 and MDA-MB231 cell lines with fold changes of 2.6 and 2.5, respectively, compared to SK-BR3 cell line (p=0.004 for both). Conclusion Cell model assays demonstrated that the luminal A/B and triple-negative subtypes showed a higher capacity to convert saturated fatty acids to monounsaturated fatty acids through SCD1 enzyme activity, indicating a different lipid metabolism profile in the HER2-enriched subtype. These new findings reveal differences in SCD1 expression and activity among breast cancer subclasses. They highlight vulnerabilities in metabolism and identify potential therapeutic targets with clinical implications for managing breast cancer. Citation Format: Hamid-Reza Feizi, Amir Mehdizadeh, Zohreh Sanaat, Khadijeh Abbasi, Frank Gieseler, Masoud Darabi. Comparative analysis of stearoyl-CoA desaturase 1 expression and activity index in model cell lines of breast cancer subclasses [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-08.

Abstract Background Trastuzumab has shown great effectiveness in HER2 positive breast cancer treatment, but about 50% patients would undergo resistance during or after treatment. Although previous research has suggested several potential reasons for trastuzumab resistance, the metabolic reprogramming during resistance formation remains largely unclear. Here we identified aberrant ferroptosis associated cysteine metabolism in trastuzumab resistant HER2 positive breast cancer, which might become a novel target for overcoming resistance. Methods Trastuzumab sensitive HER2 positive breast cancer cell SKBR3 and resistant JITM1 were obtained for transcriptomics, proteomics, metabolomics and epigenomics analysis. Gene silencing was mediated by siRNAs. CUT&Tag was applied to compare H3K4me3 and H3K27me3 binding regions. DNA methylation levels and different methylated regions were evaluated by WGBS-seq. CRISPRi with dCas9-DNMT3A was applied to regulate specific DNA methylation in CpG islands. Lipid ROS was measured by flow cytometry with BODIPY-C11. Results Joint analyses of transcriptomics and proteomics according to ferroptosis pathways revealed downregulated glutathione metabolism, glutamate transmembrane and homocysteine metabolism processes, as well as upregulated fatty acid metabolism and iron metabolism pathways in JIMT1. Metabolomics verified that JIMT1 increased cysteine metabolism and decreased glutathione metabolism. SLC7A11 expression and GSH/GSSG ratio were increased in JIMT1, while no difference was observed in free cysteine. JIMT1 featured significant higher UGC codon usage bias and increased cysteinyl-tRNA synthetase. The abundance of H3K4me3 other than H3K27me3 in SLC7A11 promoter region was found increased in JIMT1, and the 5-mC level of CpG islands in SLC7A11 promoter region was shown decreased. Using dCas9-DNMT3A, the methylation of SLC7A11 promoter was enhanced and SLC7A11 expression was reduced in JIMT1. Inhibition of SLC7A11 by siRNAs, CRISPRi or Erastin all indicated a higher ferroptosis sensitivity in JIMT1. Conclusion Trastuzumab resistant HER2 positive breast cancer features aberrant cysteine metabolism resulting from altered H3K4me3 modification and DNA methylation in SLC7A11 promoter region. This might provide novel targets for further anti-HER2 treatment. Citation Format: Yongmei Yin, Yijia Hua, Ningjun Duan, Chunxiao Sun, Fan Yang. Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-05.

Breast cancer is a significant global health concern, with the overexpression of human epidermal growth factor receptor 2 (HER2/ERBB2) being a driver oncogene in 20%-30% of cases. Indeed, HER2/ERBB2 plays a crucial role in regulating cell growth, differentiation, and survival via a complex signaling network. Overexpression of HER2/ERBB2 is associated with more aggressive behavior and increased risk of brain metastases, which remains a significant clinical challenge for treatment. Recent research has highlighted the role of breast cancer secretomes in promoting tumor progression, including excessive proliferation, immune invasion, and resistance to anti-cancer therapy, and their potential as cancer biomarkers. In this study, we investigated the impact of ERBB2+ breast cancer SKBR-3 cell line compared with MCF10-A mammary non-tumorigenic cell conditioned medium on the electrophysiological activity and morphology of neural networks derived from neurons differentiated from human induced pluripotent stem cells. Our findings provide evidence of active modulation of neuronal-glial networks by SKBR-3 and MCF10-A conditioned medium. These results provide insights into the complex interactions between breast cancer cells and the surrounding microenvironment. Further research is necessary to identify the specific factors within breast cancer conditioned medium that mediate these effects and to develop targeted therapies that disrupt this interaction.

Analysis of the molecular alterations in cancer cells following nanotechnology-assisted targeted radiotherapy using Raman spectroscopy

Abstract Homeobox genes are regulatory genes that encode nuclear proteins acting as transcription factors during normal development and differentiation. One such gene, HOXB7, plays a role in various developmental processes, including hematopoietic differentiation, lymphoid development, and mammary gland development. However, the role of HOX genes in breast cancer development remains largely unexplored. Our previous studies revealed that HOXB7 expression was significantly elevated in primary cancer and distant metastasis. HOXB7 overexpression promoted cell proliferation in SKBR3 breast cancer cells, leading to robustly vascularized xenografts in immunodeficient mice. Furthermore, HOXB7 enhanced tumor growth through TGF beta signaling and recruitment of macrophages, indicating the role of HOXB7 in the crosstalk of stromal components. We found evidence that HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes. By screening secreted factors in adipocytes induced by TCM of MCF-7-HOXB7 cells, it was also found that CCL5 is highly expressed in HOXB7-overexpressing MCF-7 cells. The CCR5 inhibitor, maraviroc, sensitized MCF-7-HOXB7 cells compared to control cells. These findings suggest that blocking the interaction between CCL5 and CCR5 signaling significantly inhibits ER+ breast cancer with HOXB7 overexpression. We aim to further investigate if HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes and the role of CCL5 in breast cancer cell growth and migration. Citation Format: Ahone Gina Akume, Kideok Jin. The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocyte [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 294.

Abstract Her-2 is a well-known driver of carcinogenesis that is overexpressed in breast, ovarian and other tumors. Immunotherapy with bispecific antibodies is a promising cancer therapy approach. Her-2 (Herceptin) antibody was used to test bispecific antibody against ovarian cancer cells. First, Her-2-CAR-T cells with Her-2 (4D5) ScFv and 4-1BB costimulatory domain were generated and tested using in vitro killing and IFN-gamma assays, and in vivo efficacy SKOV-3 xenograft NSG mouse model. Her-2-CAR-T cells killed SKBR-3 breast and SKOV-3 ovarian cancer cells and stopped SKOV-3 xenograft tumor growth. This Her-2-ScFv was used to generate bispecific Her-2 ScFv-CD3-ScFv human Fc antibody using in vitro transcribed mRNA. The 5’capped Her-2-CD3-hFc mRNA with 5’UTR, 3’UTR and poly A tail was generated by in vitro transcription using DNA template. The mRNA was embedded into lipid nanoparticles (LNP) using microfluidic PreciGenome Flex S NanoGenerator. Western blotting detected >160 kDa bispecific antibody secreted from A1847 and 293 cells transfected with mRNA-LNPs. The bispecific antibody was binding Her-2-positive A1847 ovarian cancer cell lines and T cells by FACS analysis. The Her-2-CD3-hFc bispecific antibody with T cells effectively killed Her-2-positive target cells in a dose-dependent manner in RTCA assay and produced high level of IFN-gamma. Moreover, intratumoral delivery of Her-2-CD3-hFc mRNA-LNP with intravenous injection of T cells completely blocked A1847 xenograft tumor growth. In conclusion. intratumoral delivery of Her-2-CD3 mRNA-LNP blocks ovarian xenograft tumor growth and provides a solid basis for future pre-clinical and clinical studies. Citation Format: Vita M. Golubovskaya, John Sienkiewicz, Jinying Sun, Shiming Zhang, Yanwei Huang, Liang Hu, Hua Zhou, Hizkia Harto, Shirley Xu, Robert Berahovich, Lijun Wu. Intratumoral delivery of HER-2-CD3hFc mRNA-LNP inhibits ovarian tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2368.

Abstract Background: Estrogen receptor (ER) positive breast cancer is the most common subtype of breast cancer. Elucidation of the regulatory network in estrogen receptor pathway is critical to develop and deliver potent targeted therapy for ER-positive breast cancer. The Mir-99a-let-7c cluster host gene MIR99AHG is a long non-coding RNA involving in a variety of tumor formation and disease progression processes. However, the biological functions of MIR99AHG in ER-positive breast cancer have not been reported. Methods: The Western-blot, qRT-PCR, Immunofluorescence and fluorescence in-situ hybridization were used to identify potential target genes and regulatory pathway of estrogen receptor α (ERa), which is a key proliferative driver of ER-positive breast cancer cells. The cell proliferation assay, clone formation assay, xenograft tumor model and rescue assay were used to investigate the effect of differential expression of MIR99AHG on the proliferative capacity of ER-positive breast cancer cells MCF7 and T47D. In addition, correlation analysis was performed to verify the relation of the expression of MIR99AHG, LATS1, LATS2, CYR61, CTGF and ESR1 from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts. And survival analysis was used to predict the prognostic impact of MIR99AHG in breast cancer patients. Results: MIR99AHG expression was significantly higher in ER-positive breast cancer cells MCF7 and T47D than in ER-negative breast cancer cells SKBR3, MDA-MB-231 and BT549. The high expression of MIR99AHG predicted poor prognosis in ER-positive breast cancer patients. In vitro and in vivo assays demonstrated that MIR99AHG positively regulated the proliferation of MCF7 and T47D cells. Importantly, the Hippo signaling was identified to be down-stream mediators of MIR99AHG in modulating ERα transcription. Furthermore, XMU-MP-1, which targets the Hippo signaling upstream kinase MST1/2, was able to reverse the effect of MIR99AHG on MCF7 cell proliferation. Conclusions: MIR99AHG up-regulates ERα expression and promotes proliferation in ER-positive breast cancer cells through activating Hippo signaling pathway. Targeting MIR99AHG/Hippo pathway is a potential strategy for the treatment of ER-positive breast cancer. Citation Format: Wende Wang, Danping Lin, Ziyang Lin, Yangyang Yan, Yusen Qin, Yuanke Liang, Haoyu Lin, De Zeng. MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2994.

Silver nanoparticles (AgNPs) are known as one of the highly utilized NPs owing to their unique characteristics in the field of cancer research. The goal of this research was to explore the oxidative stress, apoptosis, and angiogenesis in SKBR3 breast cancer cells after exposure to AgNPs. The survival rate of SKBR3 cancer cells and MCF-10A normal breast cells was assessed under the effects of different concentrations (0, 32, 64, 128, and 250 μg/ml) by MTT method. The oxidative condition was assessed by measuring reactive oxygen species (ROS) production, total oxidant status (TOS), total antioxidant capacity (TAC), malondialdehyde (MDA), and antioxidant enzyme activity (CAT, GPx, and CAT) using colorimetric-based kits. Flow cytometry and Hoechst 33258 staining were performed to investigate the induction of apoptosis. Furthermore, the expression of Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), and caspase 3 and 7 activity was measured. The cell migration and vascular endothelial growth factor-A (VEGF-A) gene expression, protein kinase B (AKT), phosphatidylinositol 3-kinase (PI3K) were also studied. The MTT results indicated that AgNPs inhibit the SKBR3 cells' viability in a concentration-dependent way. Besides, AgNPs markedly induced oxidative stress via increasing TOS content, MDA production, reduction of TAC, and regulation of antioxidant enzyme level. Additionally, AgNPs promoted apoptosis as revealed by an enhancement in Bax/Bcl-2 expression ratio. Findings also indicated that AgNPs suppress the expression of genes (VEGF-A, AKT, and PI3K) involved in angiogenesis. Altogether, our data revealed that AgNPs initiate oxidative stress and apoptosis in SKBR3 breast cancer cells, dose dependently.

Long non-coding RNAs (lncRNAs) have been found to play important roles in occurrence, development, and metastasis of various tumors. We aimed to screen long non-coding RNAs (lncRNAs) that promote invasion and metastasis of breast cancer cells under hypoxia, and investigate the relationship between lncRNA expression and clinicopathological features and prognosis in invasive breast cancer. LncRNA microarray was used to screen the differentially expressed lncRNAs in MCF7, MDA-MB-231, and SKBR3 breast cancer cell lines cultured under normoxia and hypoxia, respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to verify the microarray results. CCK8 and Transwell experiments were performed to identify the lncRNA that promote proliferation, migration, and invasion of breast cancer cells. Expression of the lncRNA and HIF-1α in invasive breast cancer was detected by RNAscope and immunohistochemistry, respectively. Correlation between the lncRNA expression and baseline characteristics was analyzed. Prognostic value of the lncRNA was evaluated using univariate and multivariate Cox regression. Expression of lncRNA TCONS_I2_00001955 in all the three breast cancer cells was increased under hypoxia. Overexpression of TCONS_I2_00001955 significantly enhanced proliferation, migration, and invasion of SKBR3 cells. Positive expression of TCONS_I2_00001955 was associated with recurrence, metastasis, and high expression of HIF-1α (P < 0.05), and it was an independent risk factor for poor disease-free survival of breast cancer. Hypoxia-induced lncRNA TCONS_I2_00001955 was associated with aggressive feature and poor prognosis of breast cancer.

Abstract New therapies targeting metastatic spread are greatly needed to improve breast cancer patient survival. Recently, obscurin, an ~800kDa protein localized to the cell membrane, has risen to the spotlight as an overlooked metastasis suppressor commonly lost in breast cancer. Specifically, breast epithelial cells lacking obscurin undergo epithelial-mesenchymal transition (EMT) and demonstrate increased migration and invasion. Biochemical studies in our lab have linked breast epithelial obscurin loss to increased activation of the pro-metastatic PI3K/Akt signaling pathway. Mechanistically, the obscurin pleckstrin homology (PH)-domain directly binds the SH3-domain of the p85 regulatory subunit of PI3K. Thus far, due to obscurin’s massive size, gain of function/rescue experiments of obscurin have not been feasible. To combat this challenge, we have generated a mini-obscurin protein construct composed of an obscurin core functional unit: a myristilated obscurin PH-domain (Myr-PH). We hypothesized that expression of Myr-PH in obscurin-low breast cancer cells will reduce PI3K/Akt pathway activity and suppress metastasis. Remarkably, we report that ectopic expression of Myr-PH in the obscurin-low MDA-MB-231 (triple negative) and SKBr3 (HER2 positive) breast cancer cell lines diminishes PI3K/Akt pathway activity. Furthermore, Myr-PH sequesters p85 to the cell membrane, anchoring actin at the cell periphery. Functionally, we demonstrate that expression of Myr-PH in both cell line receptor subtypes: 1) reduces cellular migration collectively through decreased scratch assay wound closure and decreased transwell assay 8um pore migration or at the single cell level, through reduced 3um diameter microchannel migration, 2) diminishes cell seeding on multiple extracellular matrix (ECM) substrates known to populate metastatic microenvironments, and 3) decreases cell dissemination and tumorsphere invasion into a 3D collagen-1 matrix. Underlying these functional changes, we show that Myr-PH inhibits actin-dependent filopodia and invadopodia formation and decreases the expression of downstream PI3K/Akt target matrix metalloproteinases, all of which are potent drivers of metastatic dissemination. To this end, we are currently delivering the obscurin-PH domain in animals via lipid nanoparticles, aiming to examine its anti-metastatic efficacy in vivo. If successful, this work would pioneer the first gene therapy-based, targeted use of a single metastasis suppressor protein domain to block PI3K signaling and inhibit breast cancer metastasis. Citation Format: Matthew K Eason, Se-Jong Lee, Poornima Dubey, Anthony Kim, Konstantinos Konstantopoulos, Aikaterini Kontrogianni-Konstantopoulos. Ectopic Expression of the Obscurin PH-domain in Aggressive Breast Cancer Cells Modulates PI3K/Akt Activity and Inhibits Cellular Migration and Invasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B023.

The epidermal growth factor receptor 2 (HER2) is overexpressed in 30% of breast cancers, and this overexpression is strongly correlated with a poor prognosis. Herceptin is a common treatment for HER2-positive breast cancer; however, cancer cells tend to adapt gradually to the drug, rendering it ineffective. The study revealed an association between the methylation status of the Homeobox C8 (HOXC8) gene and tumor development. Therefore, it is of paramount importance to delve into the interaction between HOXC8 and HER2-positive breast cancer, along with its molecular mechanisms. This exploration holds significant implications for a deeper understanding of the pathophysiological processes underlying HER2-positive breast cancer. Tumor tissue and pathological data from patients with HER2-positive breast cancer were systematically collected. Additionally, the human HER2-positive breast cancer cell line, SKBR3, was cultured in vitro to assess both the expression level of HOXC8 and the degree of DNA methylation. The study aimed to explore the relationship between the relative expression of HOXC8 and the clinical characteristics of breast cancer patients. The expression level of HOXC8 and the promoter methylation of HOXC8 were verified by methylation treatment of SKBR3 breast cancer cells. The regulation of HOXC8 was meticulously carried out, leading to the division of the cells into distinct groups. The study further analyzed the expression levels and biological capabilities within each group. Finally, the in vitro and in vivo sensitivity of the cells to Herceptin, a common treatment for HER2-positive breast cancer, was measured to assess the efficacy of the drug. In HER2-positive breast cancer cases characterized by poor methylation, there was an up-regulation of HOXC8. Its expression was found to be correlated with key clinical factors such as tumor size, lymph node status, clinical tumor, node, metastasis (cTNM) staging, and Herceptin resistance (p < 0.05). Upon methylation of breast cancer cells, there was a significant decrease in HOXC8 expression (p < 0.05). The study revealed that overexpression of HOXC8 resulted in increased proliferation, cloning, and metastasis of HER2-positive breast cancer cells, along with a reduced apoptosis rate (p < 0.05). Conversely, interference with HOXC8 expression reversed this scenario (p < 0.05). A Herceptin-resistant substrain, POOL2, was established using SKBR3 cells. Animal studies demonstrated that overexpressing HOXC8 accelerated tumor development and enhanced POOL2 cells' resistance to Herceptin (p < 0.05). However, following interference with HOXC8, POOL2 cells exhibited increased responsiveness to Herceptin, leading to a gradual reduction in tumor size (p < 0.05). In HER2-positive breast cancer, the expression of HOXC8 is elevated in a manner dependent on DNA methylation, and this elevated expression is closely linked to the pathology of the patient. Interfering with HOXC8 expression demonstrates the potential to partially inhibit the development and spread of breast cancer, as well as to alleviate resistance to Herceptin.

BackgroundThe influences of Oxycodone (OXY) combined with Paclitaxel (PTX) on breast cancer cells are unclear. The present study aimed to examine the effects of OXY combined with PTX on the proliferation, apoptosis, and migration of human breast cancer SKBR3 cells and the underlying mechanism. MethodsThe proliferation, apoptosis and invasion of SKBR3 cells were assessed by CCK-8, colony formation assay, flowcytometric, Transwell assay and scratch assays, respectively. In addition, Western blotting was used to detect the expression of related proteins in these cells. The autophagic bodies were observed under a transmission electron microscope. ResultsOXY (0.25, 0.5 and 1 mM) significantly inhibited the viability, colony-forming, migration, and invasion of SKBR3 cells as compared to the control group. Furthermore, OXY (0.25, 0.5 and 1 mM) markedly induced the apoptosis of SKBR3 cells and the levels of apoptosis-related proteins. In addition, OXY (0.25, 0.5 and 1 mM) and PTX inhibited the proliferation of SKBR3 cells synergistically as compared to PTX group in vitro. Moreover, OXY (0.25, 0.5 and 1 mM) significantly elevated the PTX-induced apoptosis in SKBR3 cells via downregulating the expression of N-cadherin, Becline-1 LC3-Ⅱ, p-Akt and p-mTOR and upregulating E-cadherin expression. Compared with the control group, OXY (1 mM) treatment induced autophagy in SKBR3 cells. ConclusionsThe present study indicates that OXY can enhance the antitumor effect of PTX on breast cancer in vitro. Hence, the combination of OXY with PTX may serve as a potential strategy for the treatment of breast cancer.

Though the physiological effects of adenosine and adenine nucleotides on purinergic receptors in cancer cells have been well studied, the influence of extracellular guanosine and guanine nucleotides on breast cancer cells remains unclear. Here, we show that extracellular guanosine and guanine nucleotides decrease the viability and proliferation of human breast cancer SKBR-3 cells. Treatment with guanosine or guanine nucleotides increased mitochondrial production of reactive oxygen species (ROS), and modified the cell cycle. Guanosine-induced cell death was suppressed by treatment with adenosine or the equilibrium nucleoside transporter (ENT) 1/2 inhibitor dipyridamole, but was not affected by adenosine receptor agonists or antagonists. These results suggest that guanosine inhibits adenosine uptake through ENT1/2, but does not antagonize adenosine receptors. In contrast, guanosine triphosphate (GTP)-induced cell death was suppressed not only by adenosine and dipyridamole, but also by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), suggesting that GTP-induced cell death is mediated in part by an antagonistic effect on adenosine A1 receptor. Thus, both guanosine and GTP induce apoptosis of breast cancer cells, but via at least partially different mechanisms.

Background: The main reason for treatment failure and the primary cause of breast cancer deaths is metastasis. Cancer features, such as epithelial to mesenchymal transition (EMT), invasiveness, stemness, and ability to metastasize, are significantly influenced by oxidative stress. Objectives: The primary objective of this work was to evaluate the effects of human Wharton’s jelly mesenchymal stem cell secretomes (hWJMSCs-Se) on oxidant contents and development of the breast cancer SK-BR3 cell line and alterations in EMT markers genes after treatment. Methods: SK-BR3 cells received 48 hours of treatment with 10, 25, or 50 μg/mL hWJMSCs-Se. The MTT test and colony formation were used to evaluate the SK-BR3 cells’ viability and proliferation capability. By using annexin V/propidium iodide (PI) staining, apoptosis was determined. The messenger ribonucleic acid (mRNA) expression levels in genes associated with antioxidants were additionally assessed. Antioxidant enzyme activity was checked after SK-BR3 treatment with hWJMSCs-Se. Results: In the hWJMSCs-Se-treated SK-BR3 cells, colony counts, and viability percentages decreased significantly with time and concentration. The treated cells displayed considerably greater apoptotic indices when compared to the control. Catalase (CAT), superoxide dismutase (SOD) activities, and glutathione (GSH) content were significantly greater in the hWJMSCs-Se-exposed SK-BR3 cells. The Vimentin gene and N-cadherin gene were significantly elevated in the treated cells, and E-cadherin and β-catenin decreased conversely. Conclusions: The present study suggests that the use of hWJMSCs in the treatment of human epidermal growth factor receptor 2 (HER2)-positive malignancies provides an innovative solution for cancer therapy. As the oxidant level and EMT pathway decreased, breast cancer cell growth was significantly restricted, and mortality increased.

The main aim of this study is to report basic knowledge on how a protein corona (PC) could affect or modify the way in which multifunctionalized nanoparticles interact with cells. With this purpose, we have firstly optimized the development of a target-specific nanocarrier by coupling a specific fluorescent antibody on the surface of functionalized lipid liquid nanocapsules (LLNCs). Thus, an anti-HER2-FITC antibody (αHER2) has been used, HER2 being a surface receptor that is overexpressed in several tumor cells. Subsequently, the in vitro formation of a PC has been developed using fetal bovine serum supplemented with human fibrinogen. Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), Laser Doppler Electrophoresis (LDE), and Gel Chromatography techniques have been used to assure a complete physico-chemical characterization of the nano-complexes with (LLNCs-αHER2-PC) and without (LLNCs-αHER2) the surrounding PC. In addition, cellular assays were performed to study the cellular uptake and the specific cellular-nanocarrier interactions using the SKBR3 (high expression of HER2) breast cancer cell line and human dermal fibroblasts (HDFa) (healthy cell line without expression of HER2 receptors as control), showing that the SKBR3 cell line had a higher transport rate (50-fold) than HDFa at 60 min with LLNCs-αHER2. Moreover, the SKBR3 cell line incubated with LLNCs-αHER2-PC suffered a significant reduction (40%) in the uptake. These results suggest that the formation of a PC onto LLNCs does not prevent specific cell targeting, although it does have an important influence on cell uptake.