Extracellular Matrix Enhances Heregulin-Dependent BRCA1 Phosphorylation and Suppresses BRCA1 Expression through Its C Terminus

ABSTRACT Germ line mutations in the breast cancer susceptibility gene BRCA1 account for the increased risk of early onset of familial breast cancer, whereas overexpression of the ErbB family of receptor tyrosine kinases has been linked to the development of nonfamilial or sporadic breast cancer. To analyze whether there is a link between these two regulatory molecules, we studied the effects of ErbB-2 activation by heregulin (HRG) on BRCA1 function. It was previously demonstrated that HRG induced the phosphorylation of BRCA1, which was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Since altered interaction between cells and the surrounding extracellular matrix (ECM) is a common feature in a variety of tumors and since ECM modulates intracellular signaling, we hypothesized that ECM may affect the expression and HRG-dependent phosphorylation of BRCA1. Following stimulation by HRG, a strong increase in [3H]thymidine incorporation was observed in human T47D breast cancer cells seeded on plastic (PL). When T47D cells were seeded on laminin (LAM) or Matrigel, HRG induced a significantly higher proliferation than it did in cells seeded on PL. T47D cells seeded on poly-l-lysine had an abrogated mitogenic response, indicating the involvement of integrins in this process. HRG treatment induced a transient phosphorylation of BRCA1 that was enhanced in T47D cells grown on LAM. LAM-enhanced BRCA1 phosphorylation was mediated through α6 integrin upon HRG stimulation. Accordingly, T47D cells grown on LAM had the greatest increase in ErbB-2 activation, PI3K activity, and phosphorylation of Akt. A similar pattern of BRCA1 mRNA expression was observed when T47D cells were seeded on PL, LAM, or COL4. There was a significant decrease in the steady state of the BRCA1 mRNA level on both the LAM and COL4 matrices compared to that for cells seeded on PL. In addition, HRG stimulation caused a significant decrease in BRCA1 mRNA expression that was dependent on protein synthesis. Pretreatment with both the calpain inhibitor ALLN (N-acetyl-Leu-Leu-norleucinal) and the proteosome inhibitor lactacystin inhibited the HRG-induced down-regulation of BRCA1 mRNA expression. Likewise, there was a strong decrease in the protein level of BRCA1 in T47D cells 4 h after treatment with HRG compared to its level in control nontreated T47D cells. Pretreatment with the proteosome inhibitors ALLN, lactacystin, and PSI [N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal] inhibited also the HRG-induced down-regulation of BRCA1 protein in breast cancer cells. Interestingly, BRCA1 mRNA expression in HCC-1937 breast cancer cells, which express C-terminally truncated BRCA1, was not affected by either LAM or CL4. No phosphorylation of BRCA1 from HCC-1937 cells was observed in response to HRG. While Cdk4 phosphorylated wild-type BRCA1 in response to HRG in T47D cells, Cdk4 failed to phosphorylate the truncated form of BRCA1 in HCC-1937 cells. Furthermore, overexpression of wild-type BRCA1 in HCC-1937 cells resulted in the phosphorylation of BRCA1 and decreased BRCA1 expression upon HRG stimulation while overexpression of truncated BRCA1 in T47D cells resulted in a lack of BRCA1 phosphorylation and restoration of BRCA1 expression. These findings suggest that ECM enhances HRG-dependent BRCA1 phosphorylation and that ECM and HRG down-regulate BRCA1 expression in breast cancer cells. Furthermore, ECM suppresses BRCA1 expression through the C terminus of BRCA1.

Download Full-text

The progestin ORG2058 but not retinoic acid stimulates BRCA1 mRNA expression in MCF-7 human breast cancer cells

International Journal of Oncology ◽

10.3892/ijo.10.2.253 ◽

1997 ◽

Author(s):

M Caligo ◽

A Bellachene ◽

G Cipollini ◽

G Bevilacqua ◽

V Castronovo

Keyword(s):

Breast Cancer ◽

Retinoic Acid ◽

Mrna Expression ◽

Cancer Cells ◽

Human Breast Cancer ◽

Breast Cancer Cells ◽

Human Breast Cancer Cells ◽

Human Breast ◽

Brca1 Mrna ◽

Mcf 7

Download Full-text

1, 25-Dihydroxyvitamin D3 Altered Metabolism of MCF10A-ras Cells Decreases Viability in Extracellular Matrix Detached Conditions (FS13-05-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz030.fs13-05-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Madeline Sheeley ◽

Dorothy Teegarden ◽

Katie Wong

Keyword(s):

Breast Cancer ◽

Vitamin D ◽

Extracellular Matrix ◽

Cell Viability ◽

Mrna Expression ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Tca Cycle ◽

Dihydroxyvitamin D3 ◽

Glutamine Deprivation

Abstract Objectives Metastasis is the main cause of most breast cancer related deaths. Throughout the process of metastasis, cancer cells strive to survive in conditions where they are detached from the extracellular matrix (ECM). Vitamin D may play a role in preventing breast cancer metastasis by modulating metabolism in breast cancer cells detached from ECM. Here we hypothesize that the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3(1,25D), regulates the metabolism of breast cancer cells, and decreases their viability in ECM detached conditions. Methods The Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) were utilized for these studies. Poly-HEMA coated plates were used to model ECM detachment paired with MTT assays to measure cell viability. Viability is measured in cells treated with vehicle (ethanol) or 10 nM 1,25D and maintained in varying nutrient concentrations. mRNA expression of metabolic genes is quantified using qPCR and, triacylglycerol (TAG) is quantified using a Wako Diagnostics colorimetric assay. Results Results show that 1,25D treatment decreases viability in detached conditions by 23%. Glutamine deprivation also decreases viability in detached conditions by 40%. In addition, in combination 1,25D treatment and glutamine deprivation results in a 71% decrease in viability. These results imply that while glutamine is required for ECM survival, 1,25D's modulation of metabolism is targeted elsewhere. Further, when compared to vehicle, 1,25D treated cells have a 45% increased TAG accumulation prior to plating in detached conditions. A 50% decrease was observed in mRNA expression of the enzyme pyruvate carboxylase (PC), and replenishing the product of PC and tricarboxylic acid (TCA) cycle intermediate oxaloacetate rescues detached cell viability in glutamine deprived conditions by 84%. Conclusions These results demonstrate the important role energy metabolism plays in cell survival during ECM detachment. Furthermore, our results suggest that 1,25D regulation of metabolism may sensitize breast cancer cells to ECM detachment death. We conclude that cells detached from ECM require flux into the TCA cycle in order to survive, and 1,25D's downregulation of PC contributes to the observed decrease in viability following 1,25D treatment. Funding Sources National Institute of Health and USDA.

Download Full-text

Role of Sialyl-O-Acetyltransferase CASD1 on GD2 Ganglioside O-Acetylation in Breast Cancer Cells

Cells ◽

10.3390/cells10061468 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1468

Author(s):

Sumeyye Cavdarli ◽

Larissa Schröter ◽

Malena Albers ◽

Anna-Maria Baumann ◽

Dorothée Vicogne ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Cancer Cell Line ◽

Wild Type ◽

Cellular Models ◽

Promising Therapeutic Target ◽

Plasmid Transfection ◽

Ganglioside Species

The O-acetylated form of GD2, almost exclusively expressed in cancerous tissues, is considered to be a promising therapeutic target for neuroectoderm-derived tumors, especially for breast cancer. Our recent data have shown that 9-O-acetylated GD2 (9-OAcGD2) is the major O-acetylated ganglioside species in breast cancer cells. In 2015, Baumann et al. proposed that Cas 1 domain containing 1 (CASD1), which is the only known human sialyl-O-acetyltransferase, plays a role in GD3 O-acetylation. However, the mechanisms of ganglioside O-acetylation remain poorly understood. The aim of this study was to determine the involvement of CASD1 in GD2 O-acetylation in breast cancer. The role of CASD1 in OAcGD2 synthesis was first demonstrated using wild type CHO and CHOΔCasd1 cells as cellular models. Overexpression using plasmid transfection and siRNA strategies was used to modulate CASD1 expression in SUM159PT breast cancer cell line. Our results showed that OAcGD2 expression was reduced in SUM159PT that was transiently depleted for CASD1 expression. Additionally, OAcGD2 expression was increased in SUM159PT cells transiently overexpressing CASD1. The modulation of CASD1 expression using transient transfection strategies provided interesting insights into the role of CASD1 in OAcGD2 and OAcGD3 biosynthesis, and it highlights the importance of further studies on O-acetylation mechanisms.

Download Full-text

Lobetyolin inhibits the proliferation of breast cancer cells via ASCT2 down-regulation-induced apoptosis

Human & Experimental Toxicology ◽

10.1177/09603271211021476 ◽

2021 ◽

pp. 096032712110214

Author(s):

Yansong Chen ◽

Ye Tian ◽

Gongsheng Jin ◽

Zhen Cui ◽

Wei Guo ◽

...

Keyword(s):

Breast Cancer ◽

Mrna Expression ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Glutamine Metabolism ◽

Down Regulation ◽

Anti Cancer ◽

Induced Apoptosis ◽

Glutamine Uptake

This study aimed to investigate the anti-cancer effect of lobetyolin on breast cancer cells. Lobetyolin was incubated with MDA-MB-231 and MDA-MB-468 breast cancer cells for 24 h. Glucose uptake and the mRNA expression of GLUT4 ( SLC2A4), HK2 and PKM2 were detected to assess the effect of lobetyolin on glucose metabolism. Glutamine uptake and the mRNA expression of ASCT2 ( SLC1A5), GLS1, GDH and GLUL were measured to assess the effect of lobetyolin on glutamine metabolism. Annexin V/PI double staining and Hoechst 33342 staining were used to investigate the effect of lobetyolin on cell apoptosis. Immunoblot was employed to estimate the effect of lobetyolin on the expression of proliferation-related markers and apoptosis-related markers. SLC1A5 knockdown with specific siRNA was performed to study the role of ASCT2 played in the anti-cancer effect of lobetyolin on MDA-MB-231 and MDA-MB-468 breast cancer cells. C-MYC knockdown with specific siRNA was performed to study the role of c-Myc played in lobetyolin-induced ASCT2 down-regulation. Myr-AKT overexpression was performed to investigate the role of AKT/GSK3β signaling played in lobetyolin-induced down-regulation of c-Myc and ASCT2. The results showed that lobetyolin inhibited the proliferation of both MDA-MB-231 and MDA-MB-468 breast cancer cells. Lobetyolin disrupted glutamine uptake via down-regulating ASCT2. SLC1A5 knockdown attenuated the anti-cancer effect of lobetyolin. C-MYC knockdown attenuated lobetyolin-caused down-regulation of ASCT2 and Myr-AKT overexpression reversed lobetyolin-caused down-regulation of both c-Myc and ASCT2. In conclusion, the present work suggested that lobetyolin exerted anti-cancer effect via ASCT2 down-regulation-induced apoptosis in breast cancer cells.

Download Full-text

Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22158165 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8165

Author(s):

Amanda Chantziou ◽

Kostas Theodorakis ◽

Hara Polioudaki ◽

Eelco de Bree ◽

Marilena Kampa ◽

...

Keyword(s):

Breast Cancer ◽

Plasma Membrane ◽

Subcellular Localization ◽

Cancer Cells ◽

Membrane Trafficking ◽

Breast Cancer Cells ◽

Endocytic Pathway ◽

Cell Periphery ◽

Wild Type ◽

Mcf 7

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

Download Full-text

The Role of Multicellular Aggregation in the Survival of ErbB2-positive Breast Cancer Cells during Extracellular Matrix Detachment

Journal of Biological Chemistry ◽

10.1074/jbc.m114.612754 ◽

2015 ◽

Vol 290 (14) ◽

pp. 8722-8733 ◽

Cited By ~ 23

Author(s):

Raju R. Rayavarapu ◽

Brendan Heiden ◽

Nicholas Pagani ◽

Melissa M. Shaw ◽

Sydney Shuff ◽

...

Keyword(s):

Breast Cancer ◽

Extracellular Matrix ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Positive Breast Cancer

Download Full-text

TK1 in Cancer Progression: elucidating its influence on cellular invasion and survival

10.21203/rs.2.17532/v1 ◽

2019 ◽

Author(s):

Eliza E. Bitter ◽

Michelle H. Townsend ◽

Kary Y.F. Tsai ◽

Carolyn I. Allen ◽

Rachel I. Erickson ◽

...

Keyword(s):

Breast Cancer ◽

Cell Survival ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Progression ◽

Breast Cancer Cells ◽

S Phase ◽

Wild Type ◽

Cellular Invasion ◽

Cancer Pathogenesis

Abstract 1. Background: The salvage pathway enzyme thymidine kinase 1 (TK1) is elevated in the serum of several different cancer types and higher expression is associated with more aggressive tumor grade. As a result, it has potential as a biomarker for diagnosis and prognosis. Recent studies indicate that TK1 may be involved in cancer pathogenesis; however, its direct involvement has not been identified. We propose to evaluate the effects of TK1 on cancer progression in vitro through measuring cellular invasion and survival of breast cancer cells.2.Methods: Breast cancer cells MDA-MB-231, HCC 1806, and MCF7 were cultured according to standard techniques. We employed the use of TK1 target siRNA and a CRISPR-Cas9 TK1 knockout plasmid to compare transfected cell lines to wild type cell lines. Protein factors in survival and invasive pathways were also tested for correlations to TK1 in BRCA RNA-seq patient data (n=1095) using the TIMER program. Cellular invasion was quantified in cell index (factor of impedance) over a 24-hour period. Cell survival was measured by apoptosis under metabolic and DNA stress using flow cytometry. All results were statistically assessed using an ANOVA or t-test in GraphPad PRISM®.3.Results: Cellular invasion assays assessing wild type and TK1 knockdown/knockout (TK1-/-) cell types showed TK1-/- cell lines had increased invasion potential (p= 0.0001). Bioinformatically, we saw a strong overall negative correlation between apoptotic factors and TK1 (p ≤ 0.05). When testing TK1 effects on cell survival we saw a protective affect under DNA stress (p ≤ 0.05), but not under metabolic stress (p= 0.0001).4.Conclusion From cell cycle analysis, we observed a shift towards S phase in TK1-/- cells. This shift to S phase would promote growth and account for the increased cellular invasion and decrease in metabolic induced stress in TK1-/- cells. We propose that cancer cells still may elicit a cancer progressive phenotype based on effects of TK1, but that a system which isolates TK1 is not effective to understand the effects. Instead, identifying protein networks inclusive of TK1 will help to elucidate its effects on cancer progression.

Download Full-text