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

ITF2 is a target of CXCR4 in MDA-MB-231 breast cancer cells and is associated with reduced survival in estrogen receptor-negative breast cancer

CXCR4, a chemokine receptor, plays an important role in breast cancer growth, invasion, and metastasis. The transcriptional targets of CXCR4 signaling are not known. Microarray analysis of CXCR4-enriched and CXCR4-low subpopulations of the MDA-MB-231 breast cancer cell line, which has a constitutively active CXCR4 signaling network, revealed differential expression of ∼ 200 genes in the CXCR4-enriched subpopulation. ITF2, upregulated in CXCR4-enriched cells, was investigated further. Expression array datasets of primary breast tumors revealed higher ITF2 expression in estrogen receptor negative tumors, which correlated with reduced progression free and overall survival and suggested its relevance in breast cancer progression. CXCL12, a CXCR4 ligand, increased ITF2 expression in MDA-MB-231 cells. ITF2 is a basic helixloop-helix transcription factor that controls the epithelial-to-mesenchymal transition and the function of the ID family (inhibitor-of-differentiation) of transcription factors, such as ID2. ID2 promotes differentiation of breast epithelial cells and its reduced expression in breast cancer is associated with an unfavorable prognosis. Both CXCR4 and ITF2 repressed ID2 expression. In xenograft studies, CXCR4-enriched cells formed large tumors and exhibited significantly elevated lung metastasis. Short interfering RNA against ITF2 reduced invasion of the CXCR4-enriched MDA-MB-231 subpopulation, whereas ITF2 overexpression restored the invasive capacity of MDA-MB-231 cells expressing CXCR4shRNA. Furthermore, overexpression of ITF2 in these cells enhanced tumor growth. We propose that ITF2 is one of the CXCR4 targets, which is involved in CXCR4-dependent tumor growth and invasion of breast cancer cells

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

Abstract 1175: Persistent upregulation of U6 small RNA in the serum of breast cancer patients

Abstract 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 disease. 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 RNA44 (SNORD44), were also analyzed for normalization. Sera from additional 14 healthy and 15 patients with overt metastasis were examined for U6, miR-16, and miR-195 levels. SNORD44 and miR-16 did not vary significantly in the sera of patients and healthy volunteers and thus served as normalization controls. Unexpectedly, we observed upregulation of U6 (2.4 fold, p=0.001) and 5S RNAs (1.7-fold, p=0.02) but not any of the other microRNAs analyzed in the sera of patients without active disease. U6 level was significantly elevated in the sera of patients with overt metastasis compared to healthy women. Elevated serum U6 levels in patients who are clinically cancer-free suggest persistently altered systemic RNAP-III activity in cancer patients and that cancer cells may not be the only primary source of serum U6. Additionally, U6 and 5S, to some extent, are of limited value for normalization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1175. doi:10.1158/1538-7445.AM2011-1175</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

Abstract 1344: HMGA2-targeted drug discovery for breast cancer brain metastasis

Abstract Breast cancer is a major health concern for women in the United States, with nearly 200,000 women diagnosed annually. An estimated 10-16% of women with breast cancer will develop symptomatic brain metastases and the incidence of brain metastasis has increased in recent years mainly due to the inability of current therapies to cross the blood-brain barrier (BBB). It is suggested that metastatic tumors contain a unique subpopulation of cancer cells with stem cell properties (CSCs), and these CSCs are enriched in metastatic tumors and express genes that enhance self-renewal and prevent differentiation. We used the MDA-MB-231 breast cancer cell line (MD-231) and its variant brain metastasis (231-BR) isolated from a xenograft model to characterize signaling molecules uniquely active in brain metastatic cells and to identify drugs that may target these molecules. 231-BR cells expressed lower levels of Let-7 microRNA, which is known to suppress levels of H-ras and high mobility group AT hook 2 (HMGA2) proteins, compared to parental MD-231 cells. H-ras is associated with self-renewal, whereas HMGA2 blocks differentiation of CSCs and effects the epithelial-to-mesenchymal transformation (EMT). HMGA2 binds to the minor grove of DNA and alters the expression of target genes, which include EMT-associated and NF-κB-driven genes. Reduced levels of Let-7 correlated with elevated HMGA2 in 231-BR cells, as evidenced by quantitative RT-PCR and western blot analysis. Although both MD-231 and 231-BR cells displayed CSC phenotype based on their CD44 and CD24 cell marker expression pattern (CD44+/CD24-), 231-BR cells contained higher numbers of a unique subset of CSCs compared to MD-231 cells based on CFSE-1 dye retention assay. Given these unique characteristics of 231-BR cells, we hypothesized that netropsin, a Streptomyces-derived antimicrobial agent with anti-tumoral properties, capable of crossing the BBB, and that interferes with the binding of proteins to DNA's minor groove, might be effective in targeting HMGA2 over-expressing cells. 231-BR cells, but not the MD-231 cells, were highly sensitive to netropsin, as evidenced by BrdU cell proliferation assay. Given that NF-κB is a downstream effector of HMGA2, we also tested LC-1, a parthenolide derived compound developed by this laboratory as an inhibitor of NF-κB, for its effects on 231-BR cells. As predicted, the 231-BR cells were more sensitive to LC-1 than the MD-231 cells. In contrast, salinomycin, a recently discovered drug that targets CD44+/CD24- CSCs, was effective in reducing proliferation of both cell types. Based on these results, we propose netropsin or an NF-κB inhibitor as potential new therapies for the growing problem of breast cancer brain metastases. Further testing is currently underway to establish the efficacy of netropsin and LC-1 in addressing the two main issues with brain metastases; cancer cell specificity, particularly CSCs, and ability to cross the BBB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1344. doi:10.1158/1538-7445.AM2011-1344</jats:p

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

Abstract Objective Management and diagnosis of multiple human cancers remains a challenge and search for a common biomarker is still debatable. In this manuscript we have evaluated the use of monoclonal antibody UNIVmAb, to detect the protein (H11) as a common biomarker for all cancers irrespective of the grade and origin. We have shown by both ELISA and Western Blot that the H11 protein, is a unique hyaluronan binding protein that has not been detected earlier. H11 protein was fractionated in an anion exchange column followed by cibacron blue gel exclusion chromatography. Hyaluronan binding H11 protein reacted with Monoclonal antibody UNIVmAb and b-HA inspite of b-Hyaluronan (biotinylated Hyaluronan) interaction and HA-Oligo (Hyaluronan oligosaccharides) competition from various grades of Human cancers sera. Results ELISA, Western blot and b-Hyaluronan interactions clearly showed an over-expression of UNIVmAb reacted H11 protein in all fifty cancer’s sera when compared with seventy normal sera. UNIVmAb reactive H11 protein can be used as a common biomarker. We believe, UNIVmAb detected H11 protein, is a unique hyaluronan binding protein, that can be used as a common biomarker for all cancers. </jats:sec