Case with Triple-Negative Breast Cancer Shows Overexpression of Both Cfos and Tgf-β1 in Node-Positive Tissue

Ivanović, Vesna; Dedović-Tanić, Nasta; Milovanović, Zorka; Lukic, Silvana; Nikolić, Srdjan; Baltic, Vladimir; Stojiljković, Bratislav; Demajo, Miroslav; Mandušić, Vesna; Dimitrijević, Bogomir

doi:10.2217/pme-2016-0032

Cited by 3 publications

(6 citation statements)

References 21 publications

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“…Considerable research attention has been focussed on a role of deregulation of Transforming Growth Factor β1 (TGFβ1) as tumor promoter step favoring BC invasion and metastasis [ 7 ]. Moreover, accumulating evidence shows that FOS transcription factor binding motifs are critical for the regulation of TGFβ1 expression [ 8 ]. Thus, cFOS elevation may have utility as a complementary candidate biomarker of BC invasiveness, co-expressed with TGFβ1.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, cFOS elevation may have utility as a complementary candidate biomarker of BC invasiveness, co-expressed with TGFβ1. Consequently, we have previously proposed that cFOS and TGFβ1 proteins may be considered as a pair of biomarkers of an early assessment of invasive BC [ 7 , 8 ], providing adequate invasive BC specimens are available. In the past, primary tumor tissue had been fractionated into nuclear [ 9 ] or cytosolic [ 10 , 11 ] extract to assess specific biomarkers of interest.…”

Section: Introductionmentioning

confidence: 99%

“…Using this experimental design, we have previously observed overexpression of TGFβ1 protein in cytosolic extracts of mALN [ 7 ]. Likewise, we encountered a case of an advanced Triple Negative Breast Cancer (TNBC) patient with overexpressed both cytosolic TGFβ1 and nuclear cFOS proteins as a pair of mALN biomarkers for an early assessment of TNBC poor prognosis [ 8 ]. However, in above mentioned studies [ 7 , 8 ], apart from the outlined methods used, specific experimental protocols were not described.…”

Section: Introductionmentioning

confidence: 99%

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Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

et al. 2022

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Background Metastatic Axillary Lymph Node (mALN) status is currently the most important prognostic factor in the management of primary breast cancer (BC). Thus, development of specimens which enable identification of new mALN markers, involved in the progression of the disease, are of considerable interest. The specific aim of this work was to describe the method of establishment of Metastatic Axillary Nodal Cell Suspension and its fractionation, termed Fractionated Nodal Cell Suspension (FNCS), into nuclear and cytosolic extracts to enable determination of protein expression levels of nuclear cFOS and cytosolic Transforming Growth Factor β1 (TGFβ1) in BC patients. Results To standardize the procedure, HeLa cells were successfully fractionated into nuclear/cytosolic extracts with confirmed presence of nuclear cFOS and cytosolic TGFβ1 proteins. Subsequently, the ALN Cell Suspension specimens were obtained and further fractionated from a pilot sample of six ALN tissue pairs, mALN versus autologous normal ALN (nALN), dissected from invasive BC patients. The mALN/nALN results revealed overexpression of both nuclear cFOS and cytosolic TGFβ1 protein levels. However, only the TGFβ1 data exhibited statistically significant overexpression, which was proportional to the respective values of mALN diameter of tumor deposits. Conclusions Detailed protocol for establishment and fractionation of mALN cell suspension specimens, termed FNCS, into nuclear and cytosolic extracts is here described for the first time. This approach might be a convenient ex vivo model for simultaneous analysis of protein, RNA and DNA biomarkers from nuclear/cytosolic extracts of the same mALN tissue sample. It might have potential to enable, in the age of genomics and personalized medicine, an identification of novel mALN biomarkers and thus improve the screening, diagnosis and prognosis of invasive BC.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

et al. 2022

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“…Additionally, we reported that pS134-GR species are elevated in TNBC relative to luminal breast cancer subtypes [16]. Since TGFβ1 expression is also elevated in TNBC and associated with poor outcome, we predicted tight linkage between TGFβ1 signaling and phosphorylation of GR Ser134 [42,43]. MDA-MB-231 cells were treated with 10 ng/mL TGFβ1 for 30 min, 1 h, and 2 h; phosphorylation of GR Ser134 was induced by TGFβ1 at 1 h and 2 h. Additionally, p38 MAPK activity, as measured by active-site phosphorylation, was also elevated at these timepoints (Fig.…”

Section: Tgfβ1 Induces Tnbc Cell Migration Via Phosphorylation Of Gr Ser134mentioning

confidence: 83%

Glucocorticoid receptors are required effectors of TGFβ1-induced p38 MAPK signaling to advanced cancer phenotypes in triple-negative breast cancer

et al. 2020

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Background Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor sensors. We previously reported that phospho-Ser134-GR (pS134-GR) species are elevated in triple-negative breast cancer (TNBC) and cooperate with hypoxia-inducible factors, providing a novel avenue for activation of GR in response to local or cellular stress. Methods We probed GR regulation by factors (cytokines, growth factors) that are rich within the tumor microenvironment (TME). TNBC cells harboring endogenous wild-type (wt) or S134A-GR species were created by CRISPR/Cas knock-in and subjected to transwell migration, invasion, soft-agar colony formation, and tumorsphere assays. RNA-seq was employed to identify pS134-GR target genes that are regulated both basally (intrinsic) or by TGFβ1 in the absence of exogenously added GR ligands. Regulation of selected basal and TGFβ1-induced pS134-GR target genes was validated by qRT-PCR and chromatin immunoprecipitation assays. Bioinformatics tools were used to probe public data sets for expression of pS134-GR 24-gene signatures. Results In the absence of GR ligands, GR is transcriptionally activated via p38-dependent phosphorylation of Ser134 as a mechanism of homeostatic stress-sensing and regulated upon exposure of TNBC cells to TME-derived agents. The ligand-independent pS134-GR transcriptome encompasses TGFβ1 and MAPK signaling gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TNBC cell anchorage-independent growth in soft-agar, migration, invasion, and tumorsphere formation, an in vitro readout of cancer stemness properties. Both pS134-GR and expression of the MAPK-scaffolding molecule 14-3-3ζ were essential for a functionally intact p38 MAPK signaling pathway downstream of MAP3K5/ASK1, indicative of a feedforward signaling loop wherein self-perpetuated GR phosphorylation enables cancer cell autonomy. A 24-gene pS134-GR-dependent signature induced by TGFβ1 predicts shortened overall survival in breast cancer patients. Conclusions Phospho-S134-GR is a critical downstream effector of p38 MAPK signaling and TNBC migration/invasion, survival, and stemness properties. Our studies define a ligand-independent role for GR as a homeostatic “sensor” of intrinsic stimuli as well as extrinsic factors rich within the TME (TGFβ1) that enable potent activation of the p38 MAPK stress-sensing pathway and nominate pS134-GR as a therapeutic target in aggressive TNBC.

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“…Consequently, we have, previously proposed that cFOS and TGFβ1 proteins may be considered as a pair of biomarkers of an early assessment of invasive BC (7,8), providing adequate invasive BC specimens are available. In the past, primary tumor tissue had been fractionated into nuclear (9) or cytosolic (10,11) extract to assess speci c biomarkers of interest. Until recently, however, the protocol for fractionation of mALN nuclear and cytosolic extracts has not been available, presumably due to the speci c tough, brous nature of mALN tissue.…”

Section: Considerable Research Attention Has Been Focused On a Role Of Deregulation Of Transforming Growthmentioning

confidence: 99%

Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

Ivanović

Dedović-Tanić

Milovanović

et al. 2021

Preprint

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Background. Metastatic Axillary Lymph Node (mALN) status is currently the most important prognostic factor in the management of primary breast cancer (BC). Thus, development of specimens which enable identification of new mALN markers, involved in the progression of the disease, are of considerable interest. The specific aim of this work was to describe the method of establishment of Metastatic Axillary Nodal Cell Suspension and its fractionation, termed Fractionated Nodal Cell Suspension (FNCS), into nuclear and cytosolic extracts to enable determination of protein expression levels of nuclear cFOS and cytosolic Transforming Growth Factor β1 (TGFβ1) in BC patients. Results. To standardize the procedure, HeLa cells were successfully fractionated into nuclear/cytosolic extracts with confirmed presence of nuclear cFOS and cytosolic TGFβ1 proteins. Subsequently, the ALN Cell Suspension specimens were obtained and further fractionated from a pilot sample of six ALN tissue pairs, mALN versus autologous normal ALN (nALN), dissected from invasive BC patients. The mALN results revealed statistically significant overexpression in nuclear cFOS and cytosolic TGFβ1 protein levels, which was proportional to the respective values of mALN diameter of tumor deposits. Conclusions. Detailed protocol for establishment and fractionation of mALN cell suspension specimens, termed FNCS, into nuclear and cytosolic extracts is here described for the first time. This approach might be a convenient ex vivo model for simultaneous analysis of protein, RNA and DNA biomarkers from nuclear/cytosolic extracts of the same mALN tissue sample. It might have potential to enable, in the age of genomics and personalized medicine, an identification of novel mALN biomarkers and thus improve the screening, diagnosis and prognosis of invasive BC.

show abstract

Case with Triple-Negative Breast Cancer Shows Overexpression of Both Cfos and Tgf-β1 in Node-Positive Tissue

Cited by 3 publications

References 21 publications

Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

Glucocorticoid receptors are required effectors of TGFβ1-induced p38 MAPK signaling to advanced cancer phenotypes in triple-negative breast cancer

Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

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