Malignant Breast Cancer Cells Research Articles

A tumor targeted antifouling upconversion nanoplatform for fluorescence imaging and precise photodynamic therapy triggered by NIR laser

Photodynamic therapy (PDT) has been considered as a promising treatment in the biomedical field because of low toxicity to normal tissues and minor trauma area. However, the PDT effect of materials is greatly affected by many factors, such as nonspecific adsorption and poor light penetration, etc. In this work, an intelligent nano platform has been constructed based on upconversion nanoparticles (UCNPs) loaded with a large amount of photosensitizers Ce6, which could specifically light up tumor tissues and stimulate the production of reactive oxygen species (ROS) under 980 nm near-infrared (NIR) irradiation, exhibiting a conspicuous imaging and therapeutic effect of PDT treatment for deep tumors. An excellent anti-fouling performance in complex biological substrate was obtained upon the judicious introduction of anti-fouling peptide, which also contributed to the improved PDT efficiency. In addition, the specificity of nanoplatform to malignant breast cancer cells was realized by modification of polypeptide targeting for HER2. This anti-fouling nanoplatform provided an original paradigm for the development of fluorescence imaging and PDT for deep tumor tissue with high targeting and therapeutic efficacy, promising to be used in the early therapy of malignant breast cancer specifically.

ERα prevents tumorigenesis of both liver and breast cancer cells through CCN5

Estrogen receptor alpha (ERα)-mediated estrogen signaling has also shown to prevent hepatic tumorigenesis in mice. Consistent with this, hormone replacement therapy with estrogen supplementation dramatically reduced the risk of hepatocellular carcinoma. Silencing of ERα is also a key event for the transformation of ERα-positive breast cancer cells into malignant triple-negative breast cancer cells. However, the mechanisms underlying ERα-mediated prevention of both hepatic and mammary tumorigenesis in humans are still unclear. Here, we present a functional genomics study of ERα targeting by comparing human liver cancer cells with human breast cancer cells using “loss or gain of function” genetic assays of ERα in vitro and in vivo. We discover that cellular communication network factor 5 (CCN5) is a direct downstream target of ERα; ERα suppresses growth and prevents tumorigenesis and malignant transformation of both liver and breast cancer cells through CCN5 in humans. The ERα-CCN5 regulatory axis functions as suppressors for both hepatic and mammary tumors, which is a common mechanism of preventing tumorigenesis for both liver cancer and breast cancer in humans.

Multi-Omics Analysis Revealed Increased De Novo Synthesis of Serine and Lower Activity of the Methionine Cycle in Breast Cancer Cell Lines.

A pipeline for metabolomics, based on UPLC-ESI-MS, was tested on two malignant breast cancer cell lines of the sub-types ER(+), PR(+), and HER2(3+) (MCF-7 and BCC), and one non-malignant epithelial cancer cell line (MCF-10A). This allowed us to quantify 33 internal metabolites, 10 of which showed a concentration profile associated with malignancy. Whole-transcriptome RNA-seq was also carried out for the three mentioned cell lines. An integrated analysis of metabolomics and transcriptomics was carried out using a genome-scale metabolic model. Metabolomics revealed the depletion of several metabolites that have homocysteine as a precursor, which was consistent with the lower activity of the methionine cycle caused by lower expression of the AHCY gene in cancer cell lines. Increased intracellular serine pools in cancer cell lines appeared to result from the over-expression of PHGDH and PSPH, which are involved in intracellular serine biosynthesis. An increased concentration of pyroglutamic acid in malignant cells was linked to the overexpression of the gene CHAC1.

Abstract 5022: Atypical protein kinase C inhibitors abrogate malignant breast cancer

Abstract Breast cancer is the most common cancer type with expected cases of approximately 284,200 according to the National Cancer Institute. Atypical Protein Kinase C (aPKC) plays a significant role in many cancer types. A positive correlation between elevated PKC levels and both the invasive and chemotactic potential of human breast cancer cell lines has been suggested in in-vitro studies. In addition, the atypical protein kinase C isoforms (aPKC), PKC- and PKC- are involved in the increased proliferation of several cancer types, including breast cancer and ovarian cancer. Hence, this project focused on utilizing aPKC inhibitors, ICA-1S (5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide and - Stat (8-hydroxy-1,3,6- naphthalenetrisulfonic acid), on two malignant breast cancer cell lines (T47D and BT549) to observe the effect on cell proliferation. To observe the effects of the aPKC inhibitors on the malignant breast cancer cells, cell proliferation assays were conducted, where cells were treated with ICA-1S, and - Stat for 72 hours (about 3 days). Furthermore, to observe if ICA-1S induces apoptosis in the malignant breast cancer cells, both the cell lines were treated with ICA-1S for 120 hours (about 5 days), and western blotting techniques were utilized. The results indicated that in T47D cell line, treatment with ICA-1S (50uM) and - Stat (10uM) caused 52% and 75% reduction in cell proliferation, respectively. Moreover, with BT549 cell line, treatment with - Stat (1uM) decreased the cell proliferation by 40%. Additionally, it was observed by western blotting that ICA-1S induces apoptosis in BT549 cell line. This was shown by an increase in the cleavage of caspase 3 and PARP, along with a decrease in survivin, and phospho-BAD. However, ICA-1S did not induce apoptosis in T47D cell line. Further investigation will be conducted on determining the effect of aPKC inhibitors on the proliferation, invasion, and migration of the malignant breast cancer cell lines. In addition, siRNA treatment will be applied to knockdown PKC- and PKC- and subsequent effect will be observed. In conclusion, PKC- and PKC- plays a significant role on breast cancer, and further investigation will be conducted to analyze these roles, which will lead to the determination of a signaling pathway via which atypical protein Kinase C functions in breast cancer. Citation Format: Nuzhat Nowshin Oishee, Khandker Mohammad Khalid, Dr. Mildred Acevedo-Duncan. Atypical protein kinase C inhibitors abrogate malignant breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5022.

Abstract 5989: Targeting RRM2 & cell cycle for breast cancer treatment

Abstract Endocrine treatment is an effective first-line therapy for targeting ER positive, HER2 negative breast cancer. But success is limited by development of acquired resistance due to long-term therapy. Cyclin D1 and cyclin dependent kinase 4/6 (CDK4/6) complex causes phosphorylation and subsequent inactivation of retinoblastoma (Rb) tumor suppressor protein which promotes progression of the cell cycle from G1 to S phase. This observation led the development of the first CDK4/6 inhibitor palbociclib (Ibrance; Pfizer) which induces cell cycle arrest at G1 phase in cancer cells. Owing to intrinsic and acquired drug resistance development, success is limited despite promising clinical outcomes. This situation necessitates the development of potential combination strategies to overcome drug resistance. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. Didox inhibits ribonucleotide reductase subunit 2 (RRM2) which ultimately blocks DNA synthesis. Combination of didox with palbociclib is a potential strategy to target ER positive and ER negative/triple negative breast cancer. Previously, we have shown that didox (DDX) can significantly halt malignant breast cancer cell division in combination with the anthracycline drug doxorubicin by targeting RRM2, mutant p53 and NFkB regulatory proteins. Recently, we confirmed that didox in combination with palbociclib significantly lowers ER positive and ER negative breast cancer and their palbociclib resistant counterparts’ growth compared to no treatment or palbociclib treatment. We also confirmed that ER positive MCF7 and ER negative MDA-MB-468 parental breast cancer cells IC50 of palbociclib drug is lower than their palbociclib resistant counterparts. Here, we are reporting that didox decreases cell cycle proteins alone or in combination with palbociclib in ER positive MCF7 and ER negative MDA-MB-468 parental and palbociclib resistant breast cancer cells. This finding opens a novel approach for targeting both ER positive as well as ER negative breast cancer treatment. We are also reporting that didox treatment modulates cyclin D1 (CCND1) and RRM2 expression in MCF7 and MDA-MB-468 breast cancer along with their palbociclib resistant counterparts. Additionally, we observed that didox alone or in combination with palbociclib alters the cell cycle of MCF7 and MDA-MB-468 parental and palbociclib resistant breast cancer cells. Our data presents a novel and promising approach for the treatment of ER positive and ER negative breast cancer that involves inhibition of RRM2 and cell cycle that merits further clinical investigation in animal and human models. Citation Format: Nahid Sultana, Howard L. Elford, Jesika S. Faridi. Targeting RRM2 & cell cycle for breast cancer treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5989.

Abstract 1104: Gene expression profile analysis of hyperthermia-induced radiosensitivity in breast cancer

Abstract Hyperthermia treatment of tumors (43-47°C) is used to sensitize breast cancer tumors to radiotherapy. Thermoradiotherapy significantly increases complete response rate and provides a treatment modality for more aggressive forms of the disease for which treatment options are generally more limited. However, the cellular mechanism underlying hyperthermia-induced radiosensitivity remains unclear. To better understand this phenomenon, we analyzed microarray data from four cell lines following treatment at 45°C for 30 minutes (Gene Expression Omnibus GSE48398). Three of the cell lines, MDA231 (37ºC, n=6; 45ºC, n=3), MDA486 (37ºC, n=6; 45ºC, n=3), and MCF7 (37ºC, n=6; 45ºC, n=6), were malignant breast cancer cell lines. The fourth cell line, MCF10A (37ºC, n=3; 45ºC, n=3), was from non-malignant mammary epithelial tissue. We identified 164 DEGs (adjP < 0.05, Fold change > 2) shared between the four cell lines and 182 DEGS shared by the three cancerous cell lines. Genes involved in radiosensitivity regulation as identified by dbCRSR, a database of radioresistance genes, were significantly dysregulated (Chi-squared test, p = 0.04). Gene ontology analysis using Cytoscape (v3.8.2) found 53 significantly enriched GO terms from an analysis off the DEGs (Benjamini-Hochberg adjP < 0.05). In particular, DEGs were enriched in processes involving G2 cell cycle arrest, apoptosis, and DNA damage checkpoints (adjP<0.005). We identified nine significantly enriched KEGG pathways, which included the ErbB (adjP=0.02), p53 (adjP=0.03), and PD-1 (adjP=0.02) cancer signaling pathways. We constructed a STRING protein-protein interaction network and then identified the 20 DEGs which had the greatest number of interactions. Identification of key genes in regulating hyperthermia-induced radiosensitivity were identified out of these 20 genes using two criteria: 1) overlap with two cancer signaling pathways and 2) involvement in radiosensitivity regulation. PTEN, a tumor suppressor overexpressed by up to 3.2-fold in vitro following hyperthermia, was identified as a key gene that may regulate hyperthermia-induced radiosensitivity in breast cancer. Distinct patterns of gene expression existed between breast cancer cells treated with hyperthermia and non-treated cells. Our data suggest that hyperthermia may exert its effects through arresting cells in the G2 phase, a particularly radiosensitive section of the cell cycle. Understanding the genes and pathways responsible for the heat-induced sensitization of breast cancer will allow us to exploit genetic alterations in tumors and develop non-invasive thermoradiotherapy regimens that more specifically target the tumor. Our study provides the basis for further investigation of genes regulating heat-induced radiosensitization and its molecular mechanism in breast cancer. Citation Format: Kevin Jili Tu, Hong Zhang, Dario Rodrigues, Jason K. Molitoris, Amit Sawant, Hem D. Shukla. Gene expression profile analysis of hyperthermia-induced radiosensitivity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1104.

Aberrant accumulation of NIK promotes tumor growth by dysregulating translation and post-translational modifications in breast cancer

BackgroundIn vivo investigations with cancer cells have powerful tools to discover cancer progression mechanisms and preclinical candidate drugs. Among these in vivo experimental models, the establishment of highly malignancy cell lines with xenograft has been frequently used. However, few previous researches targeted malignancy-related genes whose protein levels translationally changed. Therefore, this study aimed to identify malignancy-related genes which contributed to cancer progression and changed at the protein level in the in vivo selected cancer cell lines.MethodsWe established the high malignancy breast cancer cell line (LM05) by orthotopic xenograft as an in vivo selection method. To explore the altered genes by translational or post-translational regulation, we analyzed the protein production by western blotting in the highly malignant breast cancer cell line. Functional analyses of the altered genes were performed by in vitro and in vivo experiments. To reveal the molecular mechanisms of the regulation with protein level, we evaluated post-translational modification by immunoprecipitation. In addition, we evaluated translational production by click reaction-based purification of nascent protein.ResultsAs a result, NF-κB inducing kinase (NIK) increased at the protein level and promoted the nuclear localization of NF-κB2 (p52) and RelB in the highly malignant breast cancer cell line. The functional analyses indicated the NIK upregulation contributed to tumor malignancy via cancer-associated fibroblasts (CAFs) attraction and partially anti-apoptotic activities. Additionally, the immunoprecipitation experiment revealed that the ubiquitination of NIK decreased in LM05 cells. The decline in NIK ubiquitination was attributed to the translational downregulation of cIAP1.ConclusionsOur study identified a dysregulated mechanism of NIK production by the suppression of NIK post-modification and cIAP1 translation. The aberrant NIK accumulation promoted tumor growth in the highly malignant breast cancer cell line.

Abstract P4-08-13: The Use of Reverse Phase Protein Arrays (RPPA), Principal Component Analysis (PCA), and CScape Pathway Mapping to Identify New Tumor Vulnerability and Actionable Targets in Human Malignant Triple-Negative Breast cancer cell lines

Abstract Breast cancer is the major leading cause of cancer-related fatalities nationally and globally. There will be 287,850 new breast cancer cases and 43,250 breast cancer related deaths in the United States in 2022. The relapsed, treatment-resistant, undruggable, and incurable breast cancers are often associated with EGFR/HER2/K-RAS/SIAH pathway activation. The EGFR/HER2/K-RAS/SIAH pathway is a major tumor-driver whose hyperactivation is associated malignant tumor growth, multidrug-resistant phenotypes, early tumor relapse, and systematic metastasis. Seven-In-Absentia (SINA) homologues (SIAH) are extraordinarily evolutionarily-conserved E3 ubiquitin ligases that play a critical gatekeeper role downstream of the EGFR/HER2/K-RAS pathway. SIAH is a major tumor vulnerability that is ideally positioned to become an attractive target for innovative targeted therapy development against metastatic breast cancer (MBC). Prior studies have shown that tumor growth was abolished in malignant tumor cell lines such as MDA-MB-231, MDA-MB-468, MiaPaCa, A459, and HeLa following SIAH inhibition in xenograft models; however, the underpinning molecular mechanisms that give rise to this striking anti-EGFR/K-RAS and anticancer phenotype remain unclear. Specific objectives: to delineate the molecular mechanism(s) of why anti-SIAH2PD targeted therapy is so effective in impeding and eradicating the stage IV and aggressive tumors, we conducted reverse phase protein array (RPPA)-based kinomic analysis to delineate how major cancer signaling pathways and EGFR/HER2/K-RAS/SIAH-dependent signaling networks are rewired and remodeled in response to anti-SIAH2 targeted therapy. Brief statement of methods: About 300 proteins/phosphoproteins were quantititatively measured by the RPPA platform to identify new tumor vulnerabilities and actionable targets, compensatory signaling network activation/inhibition in response to anti-SIAH targeted therapies in five highly malignant cancer cell lines. Doxycycline (DOX)-inducible Tet-ON MDA-MB-231, MDA-MB-468, MiaPaCa, HeLa and A459 cell lines were amplified from single cell and DOX-induced SIAH2PD expression was confirmed. Each of the cell lines was then subjected to one of four experimental conditions: Tet-ON control cells without DOX induction (group A), Tet-ON control cells with DOX induction (group B), Tet-ON-SIAH2PD cancer cells without DOX-induction (no SIAH2PD inhibitor) (group C), Tet-ON-SIAH2PD cancer cells with DOX-induction (SIAH2PD inhibitor) (group D). Reverse Phase Protein Array (RPPA) in conjunction with Principal Component Analysis (PCA) was conducted to quantify fold-changes of proteins/phosphoproteins in response to SIAH inhibition. The ratios of D/C/B/A, D/C, D/B, C/A, and B/A were calculated using GAPDH normalized data. Summary of results: Supported by statistical analyses, we identified 6 unique phospho-proteins that were either up- or down-regulated in response to SIAHLoss-of-function. Many have known roles in controlling and regulating cell growth, cell death, NFκB signaling, stress response, DNA damage, and cell attachment pathways, supporting a tumor eradication phenotype in the absence of SIAH function in these cancer cell lines. Conclusion: Cancer landscape (CScape) functional protein pathway mapping has categorized the synergistic feedforward, feedback, and compensatory signaling pathway activation/inactivation in response to SIAH blockade in these EGFR/HER2/K-RAS-driven malignant human cell lines. Further validation analysis will be conducted to gain better insight into the global cancer pathway alterations to reveal the molecular mechanism(s) of why SIAH inhibition works so effectively to shut down malignant tumor growth in the preclinical models. Citation Format: Andrew P. Howell, Julia Wulfkuhle, Rosa I. Gallagher, Emanuel F. Petricoin, Amy H. Tang. The Use of Reverse Phase Protein Arrays (RPPA), Principal Component Analysis (PCA), and CScape Pathway Mapping to Identify New Tumor Vulnerability and Actionable Targets in Human Malignant Triple-Negative Breast cancer cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-13.

Abstract P1-13-01: Targeting RRM2 for the treatment of palbociclib resistant breast cancer

Abstract Hormone receptor (HR) positive breast cancer (BC) is a prevalent disease accounting for almost 2 million new cases globally. Almost 70-80% of breast cancer patients are women with a positive score for the estrogen receptor (ER). HR positive BCs of all stages are selectively treated with endocrine therapy targeting ER activity. Intrinsic and acquired resistance are common phenomena, impacting patient outcomes negatively. Palbociclib is an FDA approved checkpoint inhibitor for the treatment of HR+/HER2- breast cancer in combination with aromatase inhibitors or selective estrogen receptor degraders. However, patients responding well to the therapy in the beginning, lose sensitivity to palbociclib with time. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. In our previous study, we have shown that RRM2 is upregulated in ER positive tamoxifen resistant BC. Previously, we have also reported that didox (DDX) which is a unique RRM2 enzyme inhibitor can significantly halt malignant breast cancer cell division in combination with an anthracycline drug doxorubicin by targeting RRM2, mutant p53 and NFkB regulatory proteins. In this study, we target palbociclib resistant BC with DDX to circumvent palbociclib resistance. For this purpose, we have developed palbociclib resistant ER positive MCF7 and ER negative MDA-MB-468 breast cancer cell lines. Here, we report morphological changes in parental as compared to palbociclib resistant cells after treatment with palbociclib alone, DDX alone or with palbociclib as compared to non-treated cells. Cell proliferation studies confirm the synergistic combinatorial effect of palbociclib and DDX compared to palbociclib or DDX alone. We also report that DDX alone or in combination with palbociclib downregulates cell cycle and NFkB proteins in palbociclib resistant ER positive MCF7 and ER negative MBA-MB-468 breast cancer cell lines. Citation Format: Nahid Sultana, Howard L. Elford, Jesika S. Faridi. Targeting RRM2 for the treatment of palbociclib resistant breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-01.

Ubiquitin-Related Modifier 1 (URM-1) Modulates Cx43 in Breast Cancer Cell Lines.

Gap-junction-forming connexins are exquisitely regulated by post-translational modifications (PTMs). In particular, the PTM of connexin 43 (Cx43), a tumor suppressor protein, regulates its turnover and activity. Here, we investigated the interaction of Cx43 with the ubiquitin-related modifier 1 (URM-1) protein and its impact on tumor progression in two breast cancer cell lines, highly metastatic triple-negative MDA-MB-231 and luminal breast cancer MCF-7 cell lines. To evaluate the subsequent modulation of Cx43 levels, URM-1 was downregulated in these cells. The transcriptional levels of epithelial-to-mesenchymal transition (EMT) markers and the metastatic phenotype were assessed. We demonstrated that Cx43 co-localizes and interacts with URM-1, and URMylated Cx43 was accentuated upon cellular stress. The significant upregulation of small ubiquitin-like modifier-1 (SUMO-1) was also observed. In cells with downregulated URM-1, Cx43 expression significantly decreased, and SUMOylation by SUMO-1 was affected. The concomitant expression of EMT markers increased, leading to increased proliferation, migration, and invasion potential. Inversely, the upregulation of URM-1 increased Cx43 expression and reversed EMT-induced processes, underpinning a role for this PTM in the observed phenotypes. This study proposes that the URMylation of Cx43 in breast cancer cells regulates its tumor suppression properties and contributes to breast cancer cell malignancy.

Cancer-educated mammary adipose tissue-derived stromal/stem cells in obesity and breast cancer: spatial regulation and function

BackgroundBreast cancer is the most frequently diagnosed cancer and a common cause of cancer-related death in women. It is well recognized that obesity is associated with an enhanced risk of more aggressive breast cancer as well as reduced patient survival. Breast adipose tissue-derived mesenchymal stromal/stem cells (bASCs) are crucial components of the tumor microenvironment. A key step initially involved in this process might be the de-differentiation of bASCs into tumor supporting phenotypes.MethodsIn the present work, we isolated bASCs from adipose tissues adjacent to the tumor (aT bASCs) from lean- (ln-aT bASCs, BMI ≤ 25) and breast cancer patients with obesity (ob-aT bASCs, BMI ≥ 35), and analyzed their phenotypes with functional assays and RNA sequencing, compared to their counterparts isolated from adipose tissues distant from the tumor (dT bASCs).ResultsWe show that ln-aT bASCs are susceptible to be transformed into an inflammatory cancer-associated phenotype, whereas ob-aT bASCs are prone to be cancer-educated into a myofibroblastic phenotype. Both ln-aT- and ob-aT bASCs compromise their physiological differentiation capacity, and upregulate metastasis-promoting factors. While ln-aT bASCs stimulate proliferation, motility and chemoresistance by inducing epithelial-mesenchymal transition of low malignant breast cancer cells, ob-aT bASCs trigger more efficiently a cancer stem cell phenotype in highly malignant breast cancer cells.ConclusionBreast cancer-associated bASCs are able to foster malignancy of breast cancer cells by multiple mechanisms, especially, induction of epithelial-mesenchymal transition and activation of stemness-associated genes in breast cancer cells. Blocking the de-differentiation of bASCs in the tumor microenvironment could be a novel strategy to develop an effective intervention for breast cancer patients.SignificanceThis study provides mechanistic insights into how obesity affects the phenotype of bASCs in the TME. Moreover, it highlights the molecular changes inside breast cancer cells upon cell-cell interaction with cancer-educated bASCs.Graphical abstract

Acoustofluidic lysis of cancer cells and Raman spectrum profiling.

The lysis of cancer cells inside a sessile droplet was performed using traveling surface acoustic waves (SAWs) without any chemical reagents. Raman spectrum profiling was then carried out to explore detailed cell-derived data. The Rayleigh waves formed by an interdigital transducer were made to propagate along the surface of an LiNbO3 substrate. Polystyrene microparticles (PSMPs) were used to establish mechanical cell lysis effectively, and gold nanoparticles (AuNPs) were added to enhance the Raman signals from the lysed cells by SAWs. The lysis efficiency was evaluated according to the size and concentration of the PSMPs in experiments where the frequency was varied. Lysis occurred mainly by mechanical collision using PSMPs in a high-frequency domain, and the lysis efficiency was improved by increasing the application time and the energy density of the SAWs. Raman signals from the lysed cells were greatly enhanced by nanogaps formed by the AuNPs, which were evenly distributed irrespective of the SAWs through the frequency-independent behavior of the AuNPs. Finally, detailed Raman spectra of MDA-MB-231, malignant breast cancer cells, were acquired, and various organic matter-derived peaks were observed. The 95% confidence region for cells subjected to lysis was more widely distributed than that of cells not subjected to lysis. The proposed SAW platform is expected to facilitate the detection of small quantities and to be applied in biomedical applications.

LINC01140 Hinders the Development of Breast Cancer Through Targeting miR-200b-3p to Downregulate DMD

Long non-coding RNAs (lncRNAs) are frequently reported to be involved in breast cancer (BC) oncogenicity. The goal of this study was to probe lncRNA LINC01140’s role and action mechanism in BC. Relative LINC01140, miR-200b-3p, and dystrophin (DMD) levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR). DMD protein levels in BC cells were quantified using Western blotting, and the targeting relationships were validated by luciferase reporter assays and RNA immunoprecipitation experiments. The proliferative potential of the cells was evaluated using CCK-8 and colony formation tests, while the migratory and invasive abilities of the cells were assessed using scratch and transwell assays. Apoptosis was assessed by flow cytometry. Nude mouse models have been established to allow the examination of tumor growth in vivo. Pronounced downregulation of LINC01140 and DMD, as well as upregulation of miR-200b-3p, was observed in BC. LINC01140 binds directly to miR-200b-3p to downregulate DMD expression. Ectopic LINC01140 expression not only limited tumor growth in vivo but also diminished the proliferation, migration, and invasion abilities of BC cells in vitro, however, it induced apoptosis in BC cells. Elevated miR-200b-3p expression stimulated the tumorigenic potential of BC cells and attenuated the suppressive effect of LINC01140 or DMD overexpression on BC cell malignancy, whereas DMD overexpression restricted the tumorigenic potential of BC cells. Overall, LINC01140 prevents BC development via the miR-200b-3p–DMD axis. These findings support the latent potential and usefulness of the LINC01140-miR-200b-3p–DMD network as a target for BC therapy.

Regulation of micropatterned curvature-dependent FA heterogeneity on cytoskeleton tension and nuclear DNA synthesis of malignant breast cancer cells.

Breast cancer is considered as a worldwide disease due to its high incidence and malignant metastasis. Although numerous techniques have been developed well to conduct breast cancer therapy, the influence of micropattern-induced interfacial heterogeneity on the molecular mechanism and nuclear signalling transduction of carcinogenesis is rarely announced. In this study, PDMS stencil-assisted micropatterns were fabricated on tissue culture plates to manage cell clustering colony by adjusting initial cell seeding density and the size of microholes. The curvature of each microholes was controlled to construct the interfacial heterogeneity of MDA-MB231 cancer cells at the periphery of micropatterned colony. The distinguished focal adhesion (FA) and cytoskeleton distribution at the central and peripheral regions of the cell colony were regulated by heterogeneous properties. The interfacial heterogeneity of FA and cytoskeleton would induce the biased tension force to encourage more ezrin expression at the periphery and further promote DNA synthesis, therefore disclosing a stem-like phenotype in heterogeneous cells. This study will provide a value source of information for the development of micropattern-induced heterogeneity and the interpretation of metastatic mechanism in malignant breast cancer cells.

Adipose Cells Induce Escape from an Engineered Human Breast Microtumor Independently of their Obesity Status.

Obesity is associated with increased breast cancer incidence, recurrence, and mortality. Adipocytes and adipose-derived stem cells (ASCs), two resident cell types in adipose tissue, accelerate the early stages of breast cancer progression. It remains unclear whether obesity plays a role in the subsequent escape of malignant breast cancer cells into the local circulation. We engineered models of human breast tumors with adipose stroma that exhibited different obesity-specific alterations. We used these models to assess the invasion and escape of breast cancer cells into an empty, blind-ended cavity (as a mimic of a lymphatic vessel) for up to sixteen days. Lean and obese donor-derived adipose stroma hastened escape to similar extents. Moreover, a hypertrophic adipose stroma did not affect the rate of adipose-induced escape. When admixed directly into the model tumors, lean and obese donor-derived ASCs hastened escape similarly. This study demonstrates that the presence of adipose cells, independently of the obesity status of the adipose tissue donor, hastens the escape of human breast cancer cells in multiple models of obesity-associated breast cancer. The online version contains supplementary material available at 10.1007/s12195-022-00750-y.

Comparative docking analysis of tyrosine kinase inhibitors with HER2 and HER4 receptors.

Tyrosine kinase receptors promote the growth and differentiation of normal breast and malignant human breast cancer cells, known as ERBB receptors. Various ERBB receptors are EGFR/ErbB1 and ErbB2/neu, which get over expressed in different solid tumors that activate upon binding of ligand to the extra cellular domain of these receptors. Of note, the epidermal growth factor receptor (EGFR) is a prime contributor to cancer through the involvement of four receptor tyrosine kinases (RTKs), namely, HER1, HER2, HER3, and HER4. Among them, HER2 and HER4 are majorly associated with breast cancer. Non-peptide quinazoline compounds homologous of the adenosine triphosphate (ATP) are competitively inhibited to RTKs to prevent cancer growth and metastasis. Various small drug molecule that targets the RTKs having the same scaffold, includes Lapatinib, Tivozanib, Erlotinib, Gefitinib, Crizotinib, and Ceritinib. The present study aims to investigate the comparative potential of structurally similar TKIs against HER2 and HER4 receptor receptors-silico molecular docking using FlexX software (LeadIT 2.3.2). Each docked complex's interaction profile was performed using BIOVIA Discovery Studio Visualizer 4.0. Molecular docking analysis was performed in order to get deeper insights into the interaction and binding pattern of the ligands with HER2 and HER4 receptors. The docking results revealed the Lapatinib compound acquired the relatively highest binding score of -32.36 kcal/mol and -35.76 kcal/mol with HER2 and HER4 proteins, respectively, concerning other compounds. Lapatinib is identified as a potential inhibitor for both the RTKs. Our study thus suggests the probable direction that could be further explored in inhibiting EGFR protein harboring breast cancer.

Regulatory mechanisms and function of hypoxia-induced long noncoding RNA NDRG1-OT1 in breast cancer cells

Hypoxia is a classic feature of the tumor microenvironment that has profound effects on cancer progression and is tightly associated with poor prognosis. Long noncoding RNAs (lncRNAs), a component of the noncoding genome, have been increasingly investigated due to their diverse roles in tumorigenesis. Previously, a hypoxia-induced lncRNA, NDRG1-OT1, was identified in MCF-7 breast cancer cells using next-generation sequencing. However, the regulatory mechanisms of NDRG1-OT1 remain elusive. Therefore, the purpose of this study was to investigate the regulatory mechanisms and functional roles of NDRG1-OT1 in breast cancer cells. Expression profiling of NDRG1-OT1 revealed that it was upregulated under hypoxia in different breast cancer cells. Overexpression and knockdown of HIF-1α up- and downregulated NDRG1-OT1, respectively. Luciferase reporter assays and chromatin immunoprecipitation assays validated that HIF-1α transcriptionally activated NDRG1-OT1 by binding to its promoter (−1773 to −1769 and −647 to −643 bp). Next, to investigate whether NDRG1-OT1 could function as a miRNA sponge, results of in silico analysis, expression profiling of predicted miRNAs, and RNA immunoprecipitation assays indicated that NDRG1-OT1 could act as a miRNA sponge of miR-875-3p. In vitro and in vivo functional assays showed that NDRG1-OT1 could promote tumor growth and migration. Lastly, a small peptide (66 a.a.) translated from NDRG1-OT1 was identified. In summary, our findings revealed novel regulatory mechanisms of NDRG1-OT1 by HIF-1α and upon miR-875-3p. Also, NDRG1-OT1 promoted the malignancy of breast cancer cells and encoded a small peptide.

ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model

The tumor microenvironment represents one of the main obstacles in breast cancer treatment owing to the presence of heterogeneous stromal cells, such as adipose-derived stem cells (ADSCs), that may interact with breast cancer cells and promote cancer development. Resistin is an adipocytokine associated with adverse breast cancer progression; however, its underlying mechanisms in the context of the breast tumor microenvironment remain largely unidentified. Here, we utilized a transwell co-culture model containing patient-derived ADSCs and breast cancer cell lines to investigate their potential interaction, and observed that breast cancer cells co-cultured with resistin-treated ADSCs (R-ADSCs) showed enhanced cancer cell growth and metastatic ability. Screening by proteome arrays revealed that C-X-C motif chemokine ligand 5 (CXCL5) was released in the conditioned medium of the co-culture system, and phosphorylated ERK was increased in breast cancer cells after co-culture with R-ADSCs. Breast cancer cells treated with the recombinant proteins of CXCL5 showed similarly enhanced cell migration and invasion ability as occurred in the co-culture model, whereas application of neutralizing antibodies against CXCL5 reversed these phenomena. The orthotopic xenograft in mice by breast cancer cells after co-culture with R-ADSCs had a larger tumor growth and more CXCL5 expression than control. In addition, clinical analysis revealed a positive correlation between the expression of resistin and CXCL5 in both tumor tissues and serum specimens of breast cancer patients. The current study suggests that resistin-stimulated ADSCs may interact with breast cancer cells in the tumor microenvironment via CXCL5 secretion, leading to breast cancer cell malignancy.

Cytotoxic Activity of Carrageenan on Malignant MCF-7 Breast Cancer and the non-Malignant SVCT Breast Epithelial Cell Lines

Objective: Recent studies have shown that sulfated polysaccharides obtained from marine algae have many biological and physiological activities. Kappa-carrageenan, a sulfated polysaccharide obtained from red marine algae, is known to have anti-proliferative effects in various cancer cell lines. In this study, the cytotoxic effect of kappa-carrageenan on two different cell lines, namely the malignant MCF-7 breast cancer cell line and the non-malignant SVCT breast epithelial cell lines, was investigated in an in vitro model. Materials & Methods: Three different concentrations of carrageenan were prepared for each cell in its own medium, with an initial dose of 1000 µg/ml (Dilution I: 1000 µg/ml; Dilution II: 250 µg/ml; Dilution III: 62.5 µg/ml). Cells were incubated with carrageenan at the doses set for each experimental group. Cells incubated in a carrageenan-free medium comprised the control group. Results: To measure cell viability, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) analysis was performed. Cell morphology was investigated by the acridine orange (AO)/propidium iodide (PI) fluorescent staining method. The present results indicated that carrageenan caused cytotoxic effects on both malignant and non-malignant cell lines. Conclusion: Considering that the different phenotypic features of the subtypes of the MCF-7 cell line may affect cell viability and cell proliferation, cell selection should be performed very carefully in cytotoxicity studies. We suggest that using the MCF-7 cell line for cytotoxicity experiments needs to contemplate this important phenomenon for further experimental setups.

Breast cancer cell-derived extracellular vesicles promote CD8+ T cell exhaustion via TGF-β type II receptor signaling

Cancer immunotherapies have shown clinical success in various types of tumors but the patient response rate is low, particularly in breast cancer. Here we report that malignant breast cancer cells can transfer active TGF-β type II receptor (TβRII) via tumor-derived extracellular vesicles (TEV) and thereby stimulate TGF-β signaling in recipient cells. Up-take of extracellular vesicle-TβRII (EV-TβRII) in low-grade tumor cells initiates epithelial-to-mesenchymal transition (EMT), thus reinforcing cancer stemness and increasing metastasis in intracardial xenograft and orthotopic transplantation models. EV-TβRII delivered as cargo to CD8+ T cells induces the activation of SMAD3 which we demonstrated to associate and cooperate with TCF1 transcription factor to impose CD8+ T cell exhaustion, resulting in failure of immunotherapy. The levels of TβRII+ circulating extracellular vesicles (crEV) appears to correlate with tumor burden, metastasis and patient survival, thereby serve as a non-invasive screening tool to detect malignant breast tumor stages. Thus, our findings not only identify a possible mechanism by which breast cancer cells can promote T cell exhaustion and dampen host anti-tumor immunity, but may also identify a target for immune therapy against the most devastating breast tumors.