Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines

11109 Background: Gene silencing is universally observed in cancer and involves promoter DNA methylation. We found that a cohesin-related stem cell regulator, APRIN (Pds5B) was silenced in breast cancer clinical samples. Surprisingly, in 40% of these samples DNA methylation was not involved. Furthermore, in some breast cancer cell lines the APRIN protein was silenced without transcript downregulation or promoter methylation. This “translational disequilibrium” has been frequently reported with other proteins, but without mechanistic explanations. Recent results with RNA interference indicate that gene repression through microRNAs (typically mismatched) is mostly translational without transcript degradation. We propose, therefore, that the puzzling translational disequilibrium phenomenon is a new form of epigenetic silencing by miRNA mechanisms. We aim (i) to verify miRNA epigenetics of APRIN silencing in breast cancer cell lines; (ii) to study clinical breast cancer samples for methylation vs. miRNAs mechanisms in APRIN translational disequilibrium; and (iii) to investigate if miRNA silencing of APRIN affects a fetal embryonic stem cell pool in breast cancer (microchimerism). Methods: (i) We used miRNA mimics and miRNA inhibitors in breast cancer cell lines to verify specific miRNA involvement in APRIN silencing. (ii) We used immunohistochemistry with bisulfite converted DNA for methylation and microdissected RNA for microRNA interference studies from 56 clinical breast cancer samples. (iii) We used Y-chromosome markers on microdissected DNA for fetal microchimerism studies. Results: (i) We found that in breast cancer cell lines with APRIN translational disequilibrium a set of microRNAs correlate with APRIN silencing. (ii) We found miRNA related mechanisms in about 35 percent of breast cancer samples where APRIN was silenced and (iii) APRIN may specifically affect stem cells of fetal origin in the mother's mammary gland and contribute to cancer. Conclusions: The novel miRNA-based mechanism maybe a new epigenetic factor of gene silencing in cancer. We experimentally confirmed a set of APRIN specific miRNAs and established preliminary correlations with fetal microchimerism in breast cancer. No significant financial relationships to disclose.

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Human Arylamine N-Acetyltransferase 1 (NAT1) Knockout in MDA-MB-231 Breast Cancer Cell Lines Leads to Transcription of NAT2

Frontiers in Pharmacology ◽

10.3389/fphar.2021.803254 ◽

2022 ◽

Vol 12 ◽

Author(s):

Samantha M. Carlisle ◽

Patrick J. Trainor ◽

Mark A. Doll ◽

David W. Hein

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Differential Expression ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Global Gene Expression ◽

Cancer Cell Lines ◽

Breast Cancer Cell Lines ◽

Metabolizing Enzyme

Many cancers, including breast cancer, have shown differential expression of human arylamine N-acetyltransferase 1 (NAT1). The exact effect this differential expression has on disease risk and progression remains unclear. While NAT1 is classically defined as a xenobiotic metabolizing enzyme, other functions and roles in endogenous metabolism have recently been described providing additional impetus for investigating the effects of varying levels of NAT1 on global gene expression. Our objective is to further evaluate the role of NAT1 in breast cancer by determining the effect of NAT1 overexpression, knockdown, and knockout on global gene expression in MDA-MB-231 cell lines. RNA-seq was utilized to interrogate differential gene expression (genes correlated with NAT1 activity) across three biological replicates of previously constructed and characterized MDA-MB-231 breast cancer cell lines expressing parental (Scrambled), increased (Up), decreased (Down, CRISPR 2–12), or knockout (CRISPR 2–19, CRISPR 5–50) levels of NAT1. 3,889 genes were significantly associated with the NAT1 N-acetylation activity of the cell lines (adjusted p ≤ 0.05); of those 3,889 genes, 1,756 were positively associated with NAT1 N-acetylation activity and 2,133 were negatively associated with NAT1 N-acetylation activity. An enrichment of genes involved in cell adhesion was observed. Additionally, human arylamine N-acetyltransferase 2 (NAT2) transcripts were observed in the complete NAT1 knockout cell lines (CRISPR 2–19 and CRISPR 5–50). This study provides further evidence that NAT1 functions as more than just a drug metabolizing enzyme given the observation that differences in NAT1 activity have significant impacts on global gene expression. Additionally, our data suggests the knockout of NAT1 results in transcription of its isozyme NAT2.

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P1-04-03: The Effect of Survivin Downregulation on Radiosensitization of Breast Cancer Cell Lines Grown under Adherent and Stem Cell Promoting Culture Conditions.

10.1158/0008-5472.sabcs11-p1-04-03 ◽

2011 ◽

Author(s):

BG Debeb ◽

R Larson ◽

W Xu ◽

L Lacerda ◽

JM Reuben ◽

...

Keyword(s):

Breast Cancer ◽

Stem Cell ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Cancer Cell Lines ◽

Culture Conditions ◽

Breast Cancer Cell Lines

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CRIPTO antagonist ALK4L75A-Fc inhibits breast cancer cell plasticity and adaptation to stress

Breast Cancer Research ◽

10.1186/s13058-020-01361-z ◽

2020 ◽

Vol 22 (1) ◽

Author(s):

Ozlen Balcioglu ◽

Richard E. Heinz ◽

David W. Freeman ◽

Brooke L. Gates ◽

Berhane M. Hagos ◽

...

Keyword(s):

Breast Cancer ◽

Stem Cell ◽

Cancer Cells ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Breast Cancer Cells ◽

Nutrient Deprivation ◽

Stem Cell Markers ◽

Cell Markers ◽

Cancer Stem Cell Markers

Abstract Background CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells. Methods We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4L75A-Fc, which represents a candidate therapeutic approach. Results ALK4L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden. Conclusions Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

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