Response Of Breast Cancer Cells Research Articles

Breast Tumour Kinase (Brk/PTK6) Contributes to Breast Tumour Xenograft Growth and Modulates Chemotherapeutic Responses In Vitro.

Breast tumour kinase (Brk/PTK6) is overexpressed in up to 86% of breast cancers and is associated with poorer patient outcomes. It is considered a potential therapeutic target in breast cancer, even though the full spectrum of its kinase activity is not known. This study investigated the role of the kinase domain in promoting tumour growth and its potential in sensitising triple negative breast cancer cells to standard of care chemotherapy. Triple negative human xenograft models revealed that both kinase-inactive and wild-type Brk promoted xenograft growth. Suppression of Brk activity in cells subsequently co-treated with the chemotherapy agents doxorubicin or paclitaxel resulted in an increased cell sensitivity to these agents. In triple negative breast cancer cell lines, the inhibition of Brk kinase activity augmented the effects of doxorubicin or paclitaxel. High expression of the alternatively spliced isoform, ALT-PTK6, resulted in improved patient outcomes. Our study is the first to show a role for kinase-inactive Brk in human breast tumour xenograft growth; therefore, it is unlikely that kinase inhibition of Brk, in isolation, would halt tumour growth in vivo. Breast cancer cell responses to chemotherapy in vitro were kinase-dependent, indicating that treatment with kinase inhibitors could be a fruitful avenue for combinatorial treatment. Of particular prognostic value is the ratio of ALT-PTK6:Brk expression in predicating patient outcomes.

Abstract PD3-09: Cd73 may influence zoledronate-induced b- and t-cell accumulation into triple-negative breast tumors

Abstract Background: CD73 is an ecto-5′-nucleotidase, which converts AMP to adenosine. In breast cancer, CD73 expression is associated with prognosis, treatment responses to anthracyclines and tumor immunity. Bisphosphonates (BP) inhibit osteoclast-mediated bone resorption. When given in the adjuvant setting, BPs increase survival among post-menopausal breast cancer patients. Since BPs also affect cells of the immune system, we hypothesized that CD73 expression may affect BP responses in triple negative breast cancer cells. Methods: Cell viability and IC50 BPs doses were determined by WST-8 kit. Apoptotic rate and cell cycle arrest were showed by FACS. The number of caspase3/7- positive cells was accessed by a red fluorescence emission via IncuCyteS3. The apoptosis-associated genes mRNA expression was determined by qPCR. To compare BP effect on TNBC tumoral responses in vivo, we used immune-competent mouse models of breast cancer. Tumors were stained by immunofluorescence and immunohistochemically.Results: Suppression of CD73 expression, but not of enzymatic activity significantly sensitized TNBC cells to nitrogen-containing BPs (n-BPs), especially to zoledronate in vitro. The stronger growth inhibitory effects of n-BPs in CD73-suppressed cells were associated with G1 cell cycle arrest and increased apoptosis, as compared with representative control cells in vitro. Zoledronate similarly inhibited both control and CD73-suppressed TNBC tumor growthin immune-competent mice. CD73 suppression in tumors led to the lower lung metastatic burden and metastases size. Zoledronate effect on increased apoptosis was similar in both groups, but significant in CD73-suppressed tumors. Impaired angiogenesis was shown by decreased CD34-positive cell number in CD73-suppressed tumors. Zoledronate significantly increased infiltration of CD45R/B220+ B cells and cytotoxic CD8+ T cells into CD73-suppressed tumors.Conclusions: Suppressed CD73 sensitizes TNBC cells to n-BP treatment, resulting inapoptosis activation and delayed proliferation in vitro. Zoledronate induces a more profound accumulation of CD45R/B220+ B cells and CD8+ T cells into TNBC tumors with the lack of CD73. Since tumor B-cell infiltration has been associated with favourable patient prognosis in breast cancer, the significance of this B-cell effect of zoledronate warrants further investigation. Citation Format: Nataliia Petruk, Arafat Siddiqui, Jorma Määttä, Jouko Sandholm, Katri Selander. Cd73 may influence zoledronate-induced b- and t-cell accumulation into triple-negative breast tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-09.

The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells That Leads to Paraptosis-like Cell Death.

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.

Non-homogeneous dispersion of graphene in polyacrylonitrile substrates induces a migrastatic response and epithelial-like differentiation in MCF7 breast cancer cells

BackgroundRecent advances from studies of graphene and graphene-based derivatives have highlighted the great potential of these nanomaterials as migrastatic agents with the ability to modulate tumor microenvironments. Nevertheless, the administration of graphene nanomaterials in suspensions in vivo is controversial. As an alternative approach, herein, we report the immobilization of high concentrations of graphene nanoplatelets in polyacrylonitrile film substrates (named PAN/G10) and evaluate their potential use as migrastatic agents on cancer cells.ResultsBreast cancer MCF7 cells cultured on PAN/G10 substrates presented features resembling mesenchymal-to-epithelial transition, e.g., (i) inhibition of migratory activity; (ii) activation of the expression of E-cadherin, cytokeratin 18, ZO-1 and EpCAM, four key molecular markers of epithelial differentiation; (iii) formation of adherens junctions with clustering and adhesion of cancer cells in aggregates or islets, and (iv) reorganization of the actin cytoskeleton resulting in a polygonal cell shape. Remarkably, assessment with Raman spectroscopy revealed that the above-mentioned events were produced when MCF7 cells were preferentially located on top of graphene-rich regions of the PAN/G10 substrates.ConclusionsThe present data demonstrate the capacity of these composite substrates to induce an epithelial-like differentiation in MCF7 breast cancer cells, resulting in a migrastatic effect without any chemical agent-mediated signaling. Future works will aim to thoroughly evaluate the mechanisms of how PAN/G10 substrates trigger these responses in cancer cells and their potential use as antimetastatics for the treatment of solid cancers.Graphical

Abstract P014: Driving immune-dependent metabolic vulnerabilities in the breast tumor microenvironment

Abstract The tumor microenvironment (TME) is a complex arms race composed of host stroma and rapidly adapting cancer cells. This symbiosis is further complicated when dissecting the influence of inflammation, due to its multifaceted function within the TME as a driver of both pro- and anti-tumor responses. Comprising a major component of the TME, targeting or exploiting tumor-associated inflammation has long been sought for therapeutic purposes. However, the optimal amount and composition of inflammation for such purposes remains elusive. We have recently identified an interferon (IFN)-dependent transcriptional response in breast cancer cells that renders a unique sensitivity to oxidative stress produced by the biguanide-class complex I inhibitor, phenformin. Using syngeneic murine models representing luminal B (PyMT) and basal (4T1) breast cancer, we identified a modest sensitivity to phenformin administration in vivo. However, phenformin effectiveness is dramatically enhanced when used in combination with the toll-like receptor 3 (TLR3) agonist, polyinosinic:polycytidylic acid (poly (I:C)). Critically, the effectiveness of this combination treatment was lost when performed in immune-deficient mice (SCID-beige), indicating that an immune component was essential. These findings highlight a novel role for inflammation and the immune system to promote sensitivity to oxidative stress in the TME. By identifying cell types responsible for cultivating a microenvironment conducive to biguanide sensitivity in breast cancer, we can identify a novel immune-biomarker of complex I sensitivity. Therefore, using high-parameter flow cytometry, we are analyzing systemic and tumor-infiltrating leukocyte diversity and enumeration in both our genetically and phenotypically distinct breast cancer models. Combination-therapy-induced immune populations will be further characterized in their molecular and cellular response to TLR agonism and complex I inhibition. Functional responses such as inflammatory-mediator production, immunomodulatory activity, and direct and indirect tumor cytotoxicity will be assessed, ex vivo. Furthermore, monoclonal antibody-mediated depletion of candidate cell types will be used to validate their requirement and contribution to the observed synergy to the combination therapy. Examples of tumoricidal synergy between biguanides and the inflammatory TLR agonists have not yet been described, establishing these findings as novel additions to the field of tumor biology. Furthermore, identifying how sensitivity to oxidative stress can become situationally immune-dependent can greatly advance our understanding of how inflammation and metabolism intersect within the TME. Citation Format: John Heath, Stephanie Totten, Young Kyuen Im, Valerie Sabourin, Kathryn Hunt, Josie Ursini-Siegel. Driving immune-dependent metabolic vulnerabilities in the breast tumor microenvironment [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P014.

Response of MCF-7 Breast Cancer Cells Overexpressed with P-Glycoprotein to Apoptotic Induction after Photodynamic Therapy.

Multidrug resistance (MDR) has posed a significant threat to cancer treatment and has led to the emergence of a new therapeutic regime of photodynamic therapy (PDT) to curb the menace. The PDT modality employs a photosensitiser (PS), excited at a specific wavelength of light to kill cancer cells. In the present study, we used a zinc phthalocyanine tetrasulfonic acid PS to mediate the photodynamic killing of MCF-7 cells overexpressed with P-glycoprotein (P-gp) and investigate the response to cell death induction. After photodynamic treatment, MCF-7 cells undergo cell death, and indicators like Annexin V/PI staining, DNA fragmentation, and measurement of apoptotic protein expression were investigated. Results showed increased externalisation of phosphatidylserine protein, measured as a percentage in flow cytometry indicative of apoptotic induction. This expression was significant (p < 0.006) for the untreated control cells, and there was no detection of DNA fragments after a laser fluence of 20 J/cm2. In addition, a statistically significant difference (p < 0.05) was seen in caspase 8 activity and Bax protein expression. These findings were indicative of apoptotic induction and thus seem to represent the extrinsic apoptotic pathway. This study shows the role of PDT in the treatment of a resistant phenotype breast cancer.

A set of accessible enhancers enables the initial response of breast cancer cells to physiological progestin concentrations

Here, we report that in T47D breast cancer cells 50 pM progestin is sufficient to activate cell cycle entry and the progesterone gene expression program. At this concentration, equivalent to the progesterone blood levels found around the menopause, progesterone receptor (PR) binds only to 2800 genomic sites, which are accessible to ATAC cleavage prior to hormone exposure. These highly accessible sites (HAs) are surrounded by well-organized nucleosomes and exhibit breast enhancer features, including estrogen receptor alpha (ERα), higher FOXA1 and BRD4 (bromodomain containing 4) occupancy. Although HAs are enriched in RAD21 and CTCF, PR binding is the driving force for the most robust interactions with hormone-regulated genes. HAs show higher frequency of 3D contacts among themselves than with other PR binding sites, indicating colocalization in similar compartments. Gene regulation via HAs is independent of classical coregulators and ATP-activated remodelers, relying mainly on MAP kinase activation that enables PR nuclear engagement. HAs are also preferentially occupied by PR and ERα in breast cancer xenografts derived from MCF-7 cells as well as from patients, indicating their potential usefulness as targets for therapeutic intervention.

Micro RNA-34a sensitizes MCF-7 breast cancer cells to carboplatin through the apoptosis induction

AimBreast cancer (BC) has been classified among the main causes of death owing to females' cancer. Carboplatin is a platinum-based chemotherapeutic drug that is an important treatment option for BC. But high and frequent doses of carboplatin usually reducing the reaction of cancer cells to medication. There is an immediate need to establish methods for increasing the carboplatin susceptibility to BC cells. For instance, micro RNAs (miRNAs) such as MiR34a demonstrate significant potential. Considering that, this research was planned to explore the better clinical effect and underlying mechanism of miR-34a as a possible tumor inhibitor and drug resistance regulator in compared with carboplatin chemotherapy drug in the cell lines of BC in humans. MethodsMCF-7 cell line was transfected with miR-34a to perform functional analyses. Subsequently, the MTT assay was applied to assess cell viability. Cell viability and cell death-associated gene expression amounts including Bax, Bcl-2, caspase-3, MDR1, P53, and mir34-a, were examined through quantitative real-time PCR. ResultsFindings showed that miR-34a upregulation significantly decreased MCF7 cell viability in comparison with the control group. Furthermore, the single treatment of cells with miR-34a mimics and carboplatin could significantly increase Bax, Caspase-3, P53, and decrease in Bcl-2 mRNA expression levels compared to the non-treated group. Moreover, by reducing the expression levels of the MDR1 gene, BC cells' reaction to carboplatin has been increased via miR-34a. ConclusionThis study showed, that miR-34a restoration might improve the responsiveness of breast cancer cells to carboplatin chemotherapy through the downregulation of MDR1.

Cell viability and electrical response of breast cancer cell treated in aqueous graphene oxide solution deposition on interdigitated electrode

Breast cancer is one of the most reported cancers that can lead to death. Despite the advances in diagnosis and treatment procedures, the possibility of cancer recurrences is still high in many cases. With that in consideration, researchers from all over the world are showing interest in the unique features of Graphene oxide (GO), such as its excellent and versatile physicochemical properties, to explore further its potential and benefits towards breast cancer cell treatment. In this study, the cell viability and electrical response of GO, in terms of resistivity and impedance towards the breast cancer cells (MCF7) and normal breast cells (MCF10a), were investigated by varying the pH and concentration of GO. Firstly, the numbers of MCF7 and MCF10a were measured after being treated with GO for 24 and 48 h. Next, the electrical responses of these cells were evaluated by using interdigitated gold electrodes (IDEs) that are connected to an LCR meter. Based on the results obtained, as the pH of GO increased from pH 5 to pH 7, the number of viable MCF7 cells decreased while the number of viable MCF10a slightly increased after the incubation period of 48 h. Similarly, the MCF7 also experienced higher cytotoxicity effects when treated with GO concentrations of more than 25 µg/mL. The findings from the electrical characterization of the cells observed that the number of viable cells has corresponded to the impedance of the cells. The electrical impedance of MCF7 decreased as the number of highly insulating viable cell membranes decreased. But in contrast, the electrical impedance of MCF10a increased as the number of highly insulating viable cell membranes increased. Hence, it can be deduced that the GO with higher pH and concentration influence the MCF7 cancer cell line and MCF10a normal breast cell.

HPIP protooncogene differentially regulates metabolic adaptation and cell fate in breast cancer cells under glucose stress via AMPK and RNF2 dependent pathways

While cancer cells rewire metabolic pathways to sustain growth and survival under metabolic stress in solid tumors, the molecular mechanisms underlying these processes remain largely unknown. In this study, cancer cells switched from survival to death during the early to late phases of metabolic stress by employing a novel signaling switch from AMP activated protein kinase (AMPK)-Forkhead box O3 (FOXO3a)-hematopoietic PBX1-interacting protein (HPIP) to the ring finger protein 2 (RNF2)-HPIP-ubiquitin (Ub) pathway. Acute metabolic stress induced proto-oncogene HPIP expression in an AMPK-FOXO3a-dependent manner in breast cancer (BC) cells. HPIP depletion reduced cell survival and tumor formation in mouse xenografts, which was accompanied by diminished intracellular ATP levels and increased apoptosis in BC cells in response to metabolic (glucose) stress. Glutamine flux (13C-labeled) analysis further suggested that HPIP rewired glutamine metabolism by controlling the expression of the solute carrier family 1 member 5 (SLC1A5) and glutaminase (GLS) genes by acting as a coactivator of MYC to ensure cell survival upon glucose deprivation. However, in response to chronic glucose stress, HPIP was ubiquitinated by the E3-Ub ligase, RNF2, and was concomitantly degraded by the proteasome-mediated pathway, ensuring apoptosis. In support of these data, clinical analyses further indicated that elevated levels of HPIP correlated with AMPK activation in BC. Taken together, these data suggest that HPIP is a signal coordinator during metabolic stress and thus serves as a potential therapeutic target in BC.

Kinome-wide RNAi screening for mediators of ABT-199 resistance in breast cancer cells identifies Wee1 as a novel therapeutic target

Antiapoptotic and proapoptotic BCL-2 protein family members regulate mitochondrial apoptotic pathway. Small molecule inhibitors of antiapoptotic BCL-2 proteins including BCL-2-specific inhibitor ABT-199 (Venetoclax) are in clinical development. However, the efficiency of ABT-199 as a single agent in solid tumors is limited. We performed a high-throughput RNAi kinome screen targeting 691 kinases to identify potentially targetable kinases to enhance ABT-199 response in breast cancer cells. Our studies identified Wee1 as the primary target kinase to overcome resistance to ABT-199. Depletion of Wee1 by siRNA-mediated knockdown or inhibition of Wee1 by the small molecule Wee1 inhibitor AZD1775 sensitized SKBR3, MDA-MB-468, T47D and CAMA-1 breast cancer cells to ABT-199 along with decreased MCL1. BH3-only proteins PUMA and BIM functionally contribute to apoptosis signaling following co-targeting BCL-2 and Wee1. Suppression of Wee1 function increased mitochondrial cell death priming. Furthermore, we found that Wee1 inhibition altered MCL1 phosphorylation and protein stability, which led to HUWE1-mediated MCL1 degradation. Our findings suggest that Wee1 inhibition can overcome resistance to ABT-199 and provide a rationale for further translational investigation of BCL-2 inhibitor/Wee1 inhibitor combination in breast cancer.

Abstract 2053: Molecular characterization of BRCA1c.5339T&amp;gt;C missense mutation in DNA damage response of triple-negative breast cancer cells

Abstract Purpose Recent studies re-classified the c.5339T-&amp;gt;C; p.Leu1780Pro (L1780P) BRCA1 missense mutation as “likely pathogenic” in ACMG classification, which shows high prevalence in Korean population. This study aims to reveal the molecular mechanisms and therapeutic relevance of BRCA1 L1780P mutation in DNA damaging response of triple-negative breast cancer (TNBC) cells. Method We established MDA-MB 231 and HCC1937 TNBC cell lines expressing BRCA1 wild-type and BRCA1 L1780P by stable transfection of plasmid DNAs. We performed cell proliferation assays of the TNBC cell lines with treatment of olaparib and carboplatin. We compared the protein and mRNA expression level of RAD51 in BRCA1 wild-type and L1780 transfected TNBC cell lines by western blot and RT-PCR, and BRCA1-mediated DNA damaging responses were evaluated by immunofluorescence staining of RAD51 upon olaparib and carboplatin treatment. The growth inhibitory potential of RAD51 inhibitor, RI-1 was tested in BRCA1 L1780P transfected TNBC cell lines. Results BRCA1 L1780P transfected TNBC cells showed higher migration and invasion capacity, as well as increased sensitivity to olaparib and carboplatin compared to BRCA1 wild-type transfected cells. The induction of RAD51 expression on carboplatin treatment was significantly decreased in BRCA1 L1780P TNBC cells compared with BRCA1 wild-type cells. BRCA1 L1780P transfected TNBC cells showed also showed reduced nuclear RAD51 foci formation on carboplatin and olaparib treatment. The molecular interaction between p-BRCA1 and ATM was decreased by BRCA1 L1780P introduction in TNBC cells, suggesting BRCA1 L1780P mutation disrupts binding of p-BRCA1-ATM. In a cell proliferation assay, BRCA1 L1780P transfected TNBC cells were highly sensitive to RAD51 inhibitor (IC50 = 4.15μM). Conclusion This study shows BRCA1 L1780P mutation causes defects in DNA damage responses of TNBC cells by disrupting molecular interaction between p-BRCA1 and ATM, resulting in RAD51 downregulation. The combinatory inhibition of RAD51 and PARP is a candidate strategy for TNBCs with BRCA1 L1780P mutation. Keywords: Triple-negative Breast Cancer; BRCA1 L1780 Mutation; Homologous Recombination Deficiency; RAD51 Citation Format: Jeong Dong Lee, Min Hwan Kim, Yeon A Choi, Won-Ji Ryu, Hyun Ju Han, Ae Ran Choi, Tae Yeong Kim, Joo Hyuk Sohn. Molecular characterization of BRCA1c.5339T&amp;gt;C missense mutation in DNA damage response of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2053.

Effects of Epstein-Barr Virus Infection on the Response of Human Breast Cancer Cells to Nicotine.

The purpose of our study was to test whether EBV infection affects the response of human breast cancer cells to nicotine. In addition, the underlying signaling mechanisms were evaluated. EBV-infected MDA-MB-231 and LMP1-transfected MDA-MB-231 breast cancer cells were established. Reverse transcription-polymerase chain reaction and western blotting were performed to evaluate nicotine receptor expression. To verify the functional role and underlying signaling mechanism of nicotine receptor expression induced by EBV infection, morphologic analysis, and proliferation and inhibition assays were performed. Both EBV infection and LMP1 transfection increased cell proliferation and induced the up-regulation of α9-nAChR expression. Additionally, nicotine treatment induced tumorigenic activity in both EBV-infected and LMP1-transfected MDA-MB-231 breast cancer cells. Western blot and inhibitor assays showed that the nicotine-induced signaling was mediated through MAPK/ERK and AKT signaling pathways in EBV-infected and LMP1-transfected breast cancer cells, respectively. These results suggest that EBV infection and EBV-related LMP1 may act as potential molecular targets for breast cancer risk associated with nicotine, and provide a novel insight into the mechanism of nicotine stimulation in EBV-positive breast cancer cells.

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Abstract 1301: Real-time single-cell drug response assay in metastatic breast cancer cell lines using quantitative phase imaging

Abstract Introduction: The ability for oncologists to predict a cancer's response to therapy is limited to a few biomarkers used for histologic diagnosis and targeted therapy. Often, in advanced and metastatic disease these biomarkers provide no alternative options for next step systemic treatments. There is a need in oncology for functional assays that can determine a tumor's response to a drug, regardless of its tissue of origin, previous treatments, or mutation status. Quantitative phase imaging (QPI) can measure changes in single cell mass in response to drug treatment in vitro and ex vivo. This platform offers advantages over other functional/metabolic assays in that it monitors changes in real-time and on a single cell basis, revealing heterogeneity in drug response. Methods: Here, we describe the validation of QPI for the measurement of breast cancer cell response to therapy versus CellTiterGlo (CTG), an endpoint ATP assay. We ran a series of 3-day drug response assays using QPI alongside CTG. We used a 96 well plate with a 6-point dose response between 1.6 nM and 20 μM for multiple cell lines spanning a range of receptor statuses (MCF7, MDA-MB-231, BT-474) with two controls in triplicate. We analyzed single-cell data to measure the heterogeneity of response and assessed how cell-to-cell heterogeneity is affected by dose. Our response data were fitted to a four-parameter Hill equation to compute the IC50 and depth of response. Results: We found that QPI can determine IC50s for effective treatments as validated by concordance to CTG. As measured by QPI, doxorubicin has a substantial depth of response, indicating cytotoxic effects. Doxorubicin data also show a tighter range of growth rates at high concentration than control, which implies low heterogeneity of response. As expected, ER positive MCF7 cells responded to hydroxy-tamoxifen. This response shows a similar reduction in heterogeneity to doxorubicin but with a reduced depth of response indicating a cytostatic effect. MDA-MB-231 response to palbociclib exhibits a wide range of growth rates, indicating an increase in heterogeneity as measured by QPI. Fluorouracil response shows no significant difference in heterogeneity from control. Conclusion: In summary, QPI is a useful tool for functional assays that can capture IC50, depth of response, and single-cell heterogeneity of response. In particular, this additional information about single-cell behavior and heterogeneity cannot be measured using a typical endpoint assay. Future work is needed to prove the clinical utility of functional assays from QPI. Citation Format: Tarek E. Moustafa, Edward R. Polanco, Andrew Butterfield, Sandra D. Scherer, Bryan E. Welm, Philip S. Bernard, Thomas A. Zangle. Real-time single-cell drug response assay in metastatic breast cancer cell lines using quantitative phase imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1301.

Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised.

Molecular chaperones, such as Hsp70, prevent proteotoxicity and maintain homeostasis. This is perhaps most evident in cancer cells, which overexpress Hsp70 and thrive even when harboring high levels of misfolded proteins. To define the response to proteotoxic challenges, we examined adaptive responses in breast cancer cells in the presence of an Hsp70 inhibitor. We discovered that the cells bin into distinct classes based on inhibitor sensitivity. Strikingly, the most resistant cells have higher autophagy levels, and autophagy was maximally activated only in resistant cells upon Hsp70 inhibition. In turn, resistance to compromised Hsp70 function required the integrated stress response transducer, GCN2, which is commonly associated with amino acid starvation. In contrast, sensitive cells succumbed to Hsp70 inhibition by activating PERK. These data reveal an unexpected route through which breast cancer cells adapt to proteotoxic insults and position GCN2 and autophagy as complementary mechanisms to ensure survival when proteostasis is compromised.

Distinct Modulatory Effects of Fever-Range Hyperthermia on the Response of Breast Cancer Cells and Macrophages to Mistletoe (Viscum album L.) Extract.

Heat utility as a critical component of fever is often ignored, although the symptom is observed in many medical conditions. Mistletoe extract (ME) is an adjunctive medication prescribed to cancer patients. The increase in body temperature is frequently observed in patients following ME administration. Nevertheless, the impact of this fever on the effectiveness of therapy is unknown. Therefore, we aimed to investigate the effect of fever-range temperatures on ME-treated breast cancer cells and macrophages. The cells were simultaneously stimulated with ME and subjected to fever-range hyperthermia (FRH; 39 °C or 41 °C). After co-treatment, the cell viability, generation of reactive oxygen species (ROS), cell cycle distribution, and production of pro-inflammatory factors (interleukin (IL)-1β, IL-6, and cyclooxygenase (COX)-2) were evaluated. The results showed that the exposure of ME-treated breast cancer cells to FRH at 39 °C resulted in a slight decrease in their viability, whereas FRH of 41 °C enhanced this effect. Only FRH of 41 °C induced minor changes in ROS level in ME-treated breast cancer cell lines. In ME-treated macrophages, FRH stimulated cell proliferation. The cell cycle distribution analysis showed a difference between cells cultured at 39 °C and 41 °C in all examined cell lines. Moreover, hyperthermia at 41 °C completely inhibited the ME-induced increase in IL-1β and IL-6 expression in MCF-7 breast cancer cells, whereas this effect was not observed in 4T1 breast cancer cells. In contrast, in ME-treated macrophages, FRH of 41 °C strongly up-regulated expression of the pro-inflammatory factors. We conclude that fever is an important component of ME therapy that differentially affects cancer and immune cells.

Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning.

SUMMARYWhile the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.

Characterising the Response of Human Breast Cancer Cells to Polyamine Modulation.

Breast cancer is a complex heterogeneous disease with multiple underlying causes. The polyamines putrescine, spermidine, and spermine are polycationic molecules essential for cell proliferation. Their biosynthesis is upregulated in breast cancer and they contribute to disease progression. While elevated polyamines are linked to breast cancer cell proliferation, there is little evidence to suggest breast cancer cells of different hormone receptor status are equally dependent on polyamines. In this study, we characterized the responses of two breast cancer cells, ER+ (oestrogen receptor positive) MCF-7 and ER- MDA-MB-231 cell lines, to polyamine modulation and determined the requirement of each polyamine for cancer cell growth. The cells were exposed to DFMO (a polyamine pathway inhibitor) at various concentrations under different conditions, after which several growth parameters were determined. Exposure of both cell lines to DFMO induced differential growth responses, MCF-7 cells showed greater sensitivity to polyamine pathway inhibition at various DFMO concentrations than the MDA-MB-231 cells. Analysis of intracellular DFMO after withdrawal from growth medium showed residual DFMO in the cells with concomitant decreases in polyamine content, ODC protein level, and cell growth. Addition of exogenous polyamines reversed the cell growth inhibition, and this growth recovery appears to be partly dependent on the spermidine content of the cell. Similarly, DFMO exposure inhibits the global translation state of the cells, with spermidine addition reversing the inhibition of translation in the breast cancer cells. Taken together, these data suggest that breast cancer cells are differentially sensitive to the antitumour effects of polyamine depletion, thus, targeting polyamine metabolism might be therapeutically beneficial in breast cancer management based on their subtype.

Cytotoxic and molecular assessment against breast (MCF-7) cancer cells with cobalt oxide nanoballs

Among various types of cancer, the breast cancer is a very common and widely affected to the human beings especially female candidate. To keep this view the present work was designed to access the cytotoxic and apoptotic responses of breast (MCF-7) cancer cells with cobalt oxide nanoparticles (Co3O4NPs). The Co3O4NPs was prepared via solution process and well characterized. The materials crystallinity, size and phase were determined via XRD, whereas the morphology of Co3O4NPs was analyzed via SEM and TEM respectively. The Co3O4NPs were spherical in shape with an average diameter of (~33±1). The cytotoxicity rate of breast (MCF-7) cells was much influenced and dose dependent (1, 2, 5, 10, 25, 50 and 100 μL/mL) with Co3O4NPs confirmed via the MTT and NRU assays. The rate of ROS was increased gradually from 105, 124,132 and 148 with different concentrations of Co3O4NPs (10, 25, 50 and 100 μL/mL) with control. The mRNA level of apoptotic markers with Co3O4NPs was up-regulated and it shows the apoptosis in cells. The study describes the cytotoxicity with Co3O4NPs in breast (MCF-7) cancer cell were apoptotic gene expression with possible discussion.