SLUG/SNAI2 and Tumor Necrosis Factor Generate Breast Cells With CD44+/CD24- Phenotype

<p>Abstract</p> <p>Background</p> <p>Breast cancer cells with CD44+/CD24- cell surface marker expression profile are proposed as cancer stem cells (CSCs). Normal breast epithelial cells that are CD44+/CD24- express higher levels of stem/progenitor cell associated genes. We, amongst others, have shown that cancer cells that have undergone epithelial to mesenchymal transition (EMT) display the CD44+/CD24- phenotype. However, whether all genes that induce EMT confer the CD44+/CD24- phenotype is unknown. We hypothesized that only a subset of genes associated with EMT generates CD44+/CD24- cells.</p> <p>Methods</p> <p>MCF-10A breast epithelial cells, a subpopulation of which spontaneously acquire the CD44+/CD24- phenotype, were used to identify genes that are differentially expressed in CD44+/CD24- and CD44-/CD24+ cells. Ingenuity pathway analysis was performed to identify signaling networks that linked differentially expressed genes. Two EMT-associated genes elevated in CD44+/CD24- cells, SLUG and Gli-2, were overexpressed in the CD44-/CD24+ subpopulation of MCF-10A cells and MCF-7 cells, which are CD44-/CD24+. Flow cytometry and mammosphere assays were used to assess cell surface markers and stem cell-like properties, respectively.</p> <p>Results</p> <p>Two thousand thirty five genes were differentially expressed (p < 0.001, fold change ≥ 2) between the CD44+/CD24- and CD44-/CD24+ subpopulations of MCF-10A. Thirty-two EMT-associated genes including SLUG, Gli-2, ZEB-1, and ZEB-2 were expressed at higher levels in CD44+/CD24- cells. These EMT-associated genes participate in signaling networks comprising TGFβ, NF-κB, and human chorionic gonadotropin. Treatment with tumor necrosis factor (TNF), which induces NF-κB and represses E-cadherin, or overexpression of SLUG in CD44-/CD24+ MCF-10A cells, gave rise to a subpopulation of CD44+/CD24- cells. Overexpression of constitutively active p65 subunit of NF-κB in MCF-10A resulted in a dramatic shift to the CD44+/CD24+ phenotype. SLUG overexpression in MCF-7 cells generated CD44+/CD24+ cells with enhanced mammosphere forming ability. In contrast, Gli-2 failed to alter CD44 and CD24 expression.</p> <p>Conclusions</p> <p>EMT-mediated generation of CD44+/CD24- or CD44+/CD24+ cells depends on the genes that induce or are associated with EMT. Our studies reveal a role for TNF in altering the phenotype of breast CSC. Additionally, the CD44+/CD24+ phenotype, in the context of SLUG overexpression, can be associated with breast CSC "stemness" behavior based on mammosphere forming ability.</p

Validation and evaluation of common biomarker in human cancers sera protein detected by a monoclonal antibody UNIVMab

Abstract Objective: Management and diagnosis of multiple human cancers remain a challenge and search for a common biomarker are still debatable. We described a method and evaluated the use of monoclonal antibody UNIVMab, to detect the protein (H11) as a common biomarker for all cancer irrespective of grade and origin . H11 protein identified as a unique Hyaluronan binding protein not detected earlier. We applied this test both with ELISA, Western blot, fractionated in anion exchange, cibacron gel exclusion, b-Hyaluronan interaction and HA-Oligo competition from various grades of Human cancers sera and processed for the detection of hyaluronan binding protein H11, reacted with Monoclonal antibody UNIVMab and with b-HA. Results: Studies from ELISA, Western blot and b-Hyaluronan interactions showed a definitive positive over-expression of UNIVMab reacted H11 antigen in all fortyfive cancer sera compared to normal sera and can be used as a common biomarker. We believe, UNIVMab detected H11 antigen, a unique hyaluronan binding protein, is a common biomarker for all cancer sera. Keywords: ELISA, Western blot, Hyaluronic acid binding protein, H11 (sera antigen), UNIVMab. Common Biomarker, Cancers Sera</jats:p

Validation and evaluation of a common biomarker in human cancers sera protein detected by a monoclonal antibody UNIVMab

Abstract Abstract Objective: Management and diagnosis of multiple human cancers remain a challenge and search for a common biomarker are still debatable. We described a method and evaluated the use of monoclonal antibody UNIVMab, to detect the protein (H11) as a common biomarker for all cancer irrespective of grade and origin . H11 protein identified as a unique Hyaluronan binding protein not detected earlier. We applied this test both with ELISA, Western blot, fractionated in anion exchange, cibacron gel exclusion, b-Hyaluronan interaction and HA-Oligo competition from various grades of Human cancers sera and processed for the detection of hyaluronan binding protein H11, reacted with Monoclonal antibody UNIVMab and with b-HA. Results: Studies from ELISA, Western blot and b-Hyaluronan interactions showed a definitive positive over-expression of UNIVMab reacted H11 antigen in all fortyfive cancer sera compared to normal sera and can be used as a common biomarker. We believe, UNIVMab detected H11 antigen, a unique hyaluronan binding protein, is a common biomarker for all cancer sera. Keywords: ELISA, Western blot, Hyaluronic acid binding protein, H11 (sera antigen), UNIVMab. Common Biomarker, Cancers Sera</jats:p

Validation and evaluation of a common biomarker in human cancers sera protein detected by a monoclonal antibody UNIVMab

Abstract Abstract Objective: Management and diagnosis of multiple human cancers remain a challenge and search for a common biomarker are still debatable. We described a method and evaluated the use of monoclonal antibody UNIVMab, to detect the protein (H11) as a common biomarker for all cancer irrespective of grade and origin . H11 protein identified as a unique Hyaluronan binding protein not detected earlier. We applied this test both with ELISA, Western blot, fractionated in anion exchange, cibacron gel exclusion, b-Hyaluronan interaction and HA-Oligo competition from various grades of Human cancers sera and processed for the detection of hyaluronan binding protein H11, reacted with Monoclonal antibody UNIVMab and with b-HA. Results: Studies from ELISA, Western blot and b-Hyaluronan interactions showed a definitive positive over-expression of UNIVMab reacted H11 antigen in all fortyfive cancer sera compared to normal sera and can be used as a common biomarker. We believe, UNIVMab detected H11 antigen, a unique hyaluronan binding protein, is a common biomarker for all cancer sera. Keywords: ELISA, Western blot, Hyaluronic acid binding protein, H11 (sera antigen), UNIVMab. Common Biomarker, Cancers Sera</jats:p

Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients

Introduction Serum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease. Methods Sera from 40 age-matched healthy women and 39 breast cancer patients without clinical disease at the time of serum collection were analyzed for microRNAs let-7f, miR-16, miR-21 and miR-155 using quantitative real-time PCR. U6 and 5S, which are transcribed by RNA polymerase III (RNAP-III) and the small nucleolar RNU44 (SNORD44), were also analyzed for normalization. Significant results from the initial study were verified using a second set of sera from 15 healthy patients, 15 breast cancer patients without clinical disease and 15 with metastatic disease, and a third set of 12 healthy and 18 patients with metastatic disease. U6 was further verified in the extended second cohort of 75 healthy and 68 breast cancer patients without clinical disease. Results U6:SNORD44 ratio was consistently higher in breast cancer patients with or without active disease (fold change range 1.5-6.6, p value range 0.0003 to 0.05). This increase in U6:SNORD44 ratio was observed in the sera of both estrogen receptor-positive (ER+) and ER-negative breast cancer patients. MiR-16 and 5S, which are often used as normalization controls for microRNAs, showed remarkable experimental variability and thus are not ideal for normalization. Conclusions Elevated serum U6 levels in breast cancer patients irrespective of disease activity at the time of serum collection suggest a new paradigm in cancer; persistent systemic changes during cancer progression, which result in elevated activity of RNAP-III and/or the stability/release pathways of U6 in non-cancer tissues. Additionally, these results highlight the need for developing standards for normalization between samples in microRNA-related studies for healthy versus cancer and for inter-laboratory reproducibility. Our studies rule out the utility of miR-16, U6 and 5S RNAs for this purpose

Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients

Introduction Serum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease. Methods Sera from 40 age-matched healthy women and 39 breast cancer patients without clinical disease at the time of serum collection were analyzed for microRNAs let-7f, miR-16, miR-21 and miR-155 using quantitative real-time PCR. U6 and 5S, which are transcribed by RNA polymerase III (RNAP-III) and the small nucleolar RNU44 (SNORD44), were also analyzed for normalization. Significant results from the initial study were verified using a second set of sera from 15 healthy patients, 15 breast cancer patients without clinical disease and 15 with metastatic disease, and a third set of 12 healthy and 18 patients with metastatic disease. U6 was further verified in the extended second cohort of 75 healthy and 68 breast cancer patients without clinical disease. Results U6:SNORD44 ratio was consistently higher in breast cancer patients with or without active disease (fold change range 1.5-6.6, p value range 0.0003 to 0.05). This increase in U6:SNORD44 ratio was observed in the sera of both estrogen receptor-positive (ER+) and ER-negative breast cancer patients. MiR-16 and 5S, which are often used as normalization controls for microRNAs, showed remarkable experimental variability and thus are not ideal for normalization. Conclusions Elevated serum U6 levels in breast cancer patients irrespective of disease activity at the time of serum collection suggest a new paradigm in cancer; persistent systemic changes during cancer progression, which result in elevated activity of RNAP-III and/or the stability/release pathways of U6 in non-cancer tissues. Additionally, these results highlight the need for developing standards for normalization between samples in microRNA-related studies for healthy versus cancer and for inter-laboratory reproducibility. Our studies rule out the utility of miR-16, U6 and 5S RNAs for this purpose