RalA but Not RalB Enhances Polarized Delivery of Membrane Proteins to the Basolateral Surface of Epithelial Cells

RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins

Heterogeneity for Stem Cell-Related Markers According to Tumor Subtype and Histologic Stage in Breast Cancer

Purpose: To evaluate the expression of stem cell-related markers at the cellular level in human breast tumors of different subtypes and histologic stage. Experimental Design: We performed immunohistochemical analyses of 12 proteins [CD44, CD24, ALDH1, vimentin, osteonectin, EPCR, caveolin 1, connexin 43, cytokeratin 18 (CK18), MUC1, claudin 7, and GATA3] selected based on their differential expression in breast cancer cells with more differentiated and stem cell-like characteristics in 47 cases of invasive ductal carcinoma (IDC) only, 135 cases of IDC with ductal carcinoma in situ (DCIS), 35 cases of DCIS with microinvasion, and 58 cases of pure DCIS. We also analyzed 73 IDCs with adjacent DCIS to determine the differences in the expression of markers by histology within individual tumors. CD44+/CD24- and CD24-/CD24+ cells were detected using double immunohistochemistry. Results: CD44 and EPCR expression was different among the four histologic groups and was lower in invasive compared with in situ tumors, especially in luminal A subtype. The expression of vimentin, osteonectin, connexin 43, ALDH1, CK18, GATA3, and MUC1 differed by tumor subtype in some histologic groups. ALDH1-positive cells were more frequent in basal-like and HER2+ than in luminal tumors. CD44+/CD24- cells were detected in 69% of all tumors with 100% of the basal-like and 52% of HER2+ tumors having some of these cells. Conclusions: Our findings suggest that in breast cancer, the frequency of tumor cells positive for stem cell-like and more differentiated cell markers varies according to tumor subtype and histologic stage. Clin Cancer Res; 16(3); 876-87. (C) 2010 AACR.Shackleton M, 2009, CELL, V138, P822, DOI 10.1016/j.cell.2009.08.017Creighton CJ, 2009, P NATL ACAD SCI USA, V106, P13820, DOI 10.1073/pnas.0905718106Lim E, 2009, NAT MED, V15, P907, DOI 10.1038/nm.2000Rosen JM, 2009, SCIENCE, V324, P1670, DOI 10.1126/science.1171837Hennessy BT, 2009, CANCER RES, V69, P4116, DOI 10.1158/0008-5472.CAN-08-3441Charafe-Jauffret E, 2009, CANCER RES, V69, P1302, DOI 10.1158/0008-5472.CAN-08-2741Visvader JE, 2008, NAT REV CANCER, V8, P755, DOI 10.1038/nrc2499Korkaya H, 2008, ONCOGENE, V27, P6120, DOI 10.1038/onc.2008.207Bloushtain-Qimron N, 2008, P NATL ACAD SCI USA, V105, P14076, DOI 10.1073/pnas.0805206105Xiao Y, 2008, AM J PATHOL, V173, P561, DOI 10.2353/ajpath.2008.071214Raouf A, 2008, CELL STEM CELL, V3, P109, DOI 10.1016/j.stem.2008.05.018Mylona E, 2008, HUM PATHOL, V39, P1096, DOI 10.1016/j.humpath.2007.12.003Mani SA, 2008, CELL, V133, P704, DOI 10.1016/j.cell.2008.03.027Hyun CL, 2008, J CLIN PATHOL, V61, P317, DOI 10.1136/jcp.2007.050336Sarrio D, 2008, CANCER RES, V68, P989, DOI 10.1158/0008-5472.CAN-07-2017Allred DC, 2008, CLIN CANCER RES, V14, P370, DOI 10.1158/1078-0432.CCR-07-1127Fillmore CM, 2008, BREAST CANCER RES, V10, DOI 10.1186/bcr1982Honeth G, 2008, BREAST CANCER RES, V10, DOI 10.1186/bcr2108Wright MH, 2008, BREAST CANCER RES, V10, DOI 10.1186/bcr1855Charafe-Jauffret E, 2008, PATHOBIOLOGY, V75, P75, DOI 10.1159/000123845Ginestier C, 2007, CELL STEM CELL, V1, P555Polyak K, 2007, J CLIN INVEST, V117, P3155, DOI 10.1172/JCI33295Campbell LL, 2007, CELL CYCLE, V6, P2332Shipitsin M, 2007, CANCER CELL, V11, P259, DOI 10.1016/j.ccr.2007.01.013Beaulieu LM, 2007, EXP CELL RES, V313, P677, DOI 10.1016/j.yexcr.2006.11.019Asselin-Labat ML, 2007, NAT CELL BIOL, V9, P201, DOI 10.1038/ncb1530Chaffer CL, 2007, CELLS TISSUES ORGANS, V185, P7, DOI 10.1159/000101298Kouros-Mehr H, 2006, CELL, V127, P1041, DOI 10.1016/j.cell.2006.09.048Carey LA, 2006, JAMA-J AM MED ASSOC, V295, P2492Sorlie T, 2006, BMC GENOMICS, V7, DOI 10.1186/1471-2164-7-127Clarke MF, 2006, CELL, V124, P1111, DOI 10.1016/j.cell.2006.03.011Polyak K, 2006, NAT MED, V12, P296, DOI 10.1038/nm1379Sheridan C, 2006, BREAST CANCER RES, V8, DOI 10.1186/bcr1610Baumann P, 2005, CANCER RES, V65, P10783, DOI 10.1158/0008-5472.CAN-05-0619Abraham BK, 2005, CLIN CANCER RES, V11, P1154Blanpain C, 2004, CELL, V118, P635Nielsen TO, 2004, CLIN CANCER RES, V10, P5367Thiery JP, 2003, CURR OPIN CELL BIOL, V15, P740, DOI 10.1016/j.ceb.2003.10.006Kristiansen G, 2003, CLIN CANCER RES, V9, P4906Al-Hajj M, 2003, P NATL ACAD SCI USA, V100, P3983, DOI 10.1073/pnas.0530291100Park SY, 2002, HUM PATHOL, V33, P1078, DOI 10.1053/hupa.2002.129422Ramalho-Santos M, 2002, SCIENCE, V298, P597, DOI 10.1126/science.1072530Ivanova NB, 2002, SCIENCE, V298, P601, DOI 10.1126/science.1073823Sorlie T, 2001, P NATL ACAD SCI USA, V98, P10869Perou CM, 2000, NATURE, V406, P747Graff JR, 2000, J BIOL CHEM, V275, P2727HEPPNER GH, 1983, CANCER METAST REV, V2, P5NOWELL PC, 1976, SCIENCE, V194, P234

Gene expression profiling of human breast tissue samples using SAGE-Seq

We present a powerful application of ultra high-throughput sequencing, SAGE-Seq, for the accurate quantification of normal and neoplastic mammary epithelial cell transcriptomes. We develop data analysis pipelines that allow the mapping of sense and antisense strands of mitochondrial and RefSeq genes, the normalization between libraries, and the identification of differentially expressed genes. We find that the diversity of cancer transcriptomes is significantly higher than that of normal cells. Our analysis indicates that transcript discovery plateaus at 10 million reads/sample, and suggests a minimum desired sequencing depth around five million reads. Comparison of SAGE-Seq and traditional SAGE on normal and cancerous breast tissues reveals higher sensitivity of SAGE-Seq to detect less-abundant genes, including those encoding for known breast cancer-related transcription factors and G protein–coupled receptors (GPCRs). SAGE-Seq is able to identify genes and pathways abnormally activated in breast cancer that traditional SAGE failed to call. SAGE-Seq is a powerful method for the identification of biomarkers and therapeutic targets in human disease

The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells

SummaryThe epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties