CBL458 Sigma-AldrichAnti-CD49f Antibody, clone 4F10

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Recent molecular profiling has identified six major subtypes of breast cancer: basal-like, ErbB2-overexpressing, normal breast epithelial-like, luminal A and B, and claudin-low subtypes. To help understand the relationship among mammary stem/progenitor cells and breast cancer subtypes, we have recently derived distinct hTERT-immortalized human mammary stem/progenitor cell lines: a K5(+)/K19(-) type, and a K5(+)/K19(+) type. Under specific culture conditions, bipotent K5(+)/K19(-) stem/progenitor cells differentiated into stable clonal populations that were K5(-)/K19(-) and exhibit self-renewal and unipotent myoepithelial differentiation potential in contrast to the parental K5(+)/K19(-) cells which are bipotent. These K5(-)/K19(-) cells function as myoepithelial progenitor cells and constitutively express markers of an epithelial to mesenchymal transition (EMT) and show high invasive and migratory abilities. In addition, these cells express a microarray signature of claudin-low breast cancers. The EMT characteristics of an un-transformed unipotent mammary myoepithelial progenitor cells together with claudin-low signature suggests that the claudin-low breast cancer subtype may arise from myoepithelial lineage committed progenitors. Availability of immortal MPCs should allow a more definitive analysis of their potential to give rise to claudin-low breast cancer subtype and facilitate biological and molecular/biochemical studies of this disease.

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell surface accumulation of urokinase and plasminogen and enhanced expression of alphaV integrins. Genetic rescue experiments and inhibitor studies revealed that the enhanced plasminogen activation was due to a direct effect of M6P/IGF2R on uPAR, whereas increased cell adhesion to vitronectin was dependent on alphaV integrin expression and not uPAR. Increased cell invasion of M6P/IGF2R knockdown cells was rescued by cosilencing both uPAR and alphaV integrin. Furthermore, we found that M6P/IGF2R expression accelerates the cleavage of uPAR. M6P/IGF2R silencing resulted in an increased ratio of full-length uPAR to the truncated D2D3 fragment, incapable of binding most uPAR ligands. We conclude that M6P/IGF2R controls cell invasion by regulating alphaV integrin expression and by accelerating uPAR cleavage, leading to the loss of the urokinase/vitronectin/integrin-binding site on uPAR.

The laminin family of proteins is critical for managing a variety of cellular activities including migration, adhesion, and differentiation. In bone, the roles of laminins in controlling osteogenic differentiation of human mesenchymal stem cells (hMSC) are unknown. We report here that laminin-5 is found in bone and expressed by hMSC. hMSC isolated from bone synthesize laminin-5 and adhere to exogenous laminin-5 through alpha3beta1 integrin. Adhesion to laminin-5 activates extracellular signal-related kinase (ERK) within 30 min and leads to phosphorylation of the osteogenic transcription factor Runx2/CBFA-1 within 8 d. Cells plated on laminin-5 for 16 d express increased levels of osteogenic marker genes, and those plated for 21 d deposit a mineralized matrix, indicative of osteogenic differentiation. Addition of the ERK inhibitor PD98059 mitigates these effects. We conclude that contact with laminin-5 is sufficient to activate ERK and to stimulate osteogenic differentiation in hMSC.

The integrins are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. To identify the pattern of expression of the beta 1, beta 3, and beta 4 integrins and their accessory adhesion molecules in relation to the malignant phenotype of invasive breast cancer, we performed an immunohistochemical study for the alpha 2 beta 1 (VLA-2), alpha 6 beta 1 (VLA-6), alpha v and alpha v beta 3 (vitronectin receptor), alpha 6 beta 4, carcinoembryonic antigen, and carcinoembryonic antigen-related molecules in a series of 37 invasive breast carcinomas. All integrin chains examined showed similar patterns in nonneoplastic breast tissue, with strong membrane staining of the myoepithelial cells and weak to moderate staining on the basolateral surfaces of the luminal cells. We found that downregulation of the alpha 2 chain of VLA-2 occurs more frequently in poorly differentiated grade III invasive ductal carcinomas (IDCs) (P = .048). Loss of alpha 6 beta 4 seems also to occur more frequently in grade III IDC (seven of 11 cases, 63.6%) than in grade I/II IDC (two of eight cases, 25%), although this did not reach statistical significance. Carcinoembryonic antigen and carcinoembryonic antigen-related antigens, which are known to function as accessory adhesion molecules, were found mainly in the cytoplasm of neoplastic cells and there was reduced membrane polarization in poorly organized tumors. In contrast the alpha v beta 3, vitronectin receptor heterodimer recognized by the 23C6 monoclonal antibody was weak or absent in normal breast epithelium, and was weakly expressed in two of 19 (10%) IDCs and in nine of 18 (50%) invasive lobular carcinomas (P = .008). However, the alpha v chain detected with the antibody 13C2 was weakly to moderately expressed on nonneoplastic epithelium and at a similar intensity in 13 of 19 IDCs and 15 of 17 invasive lobular carcinomas, suggesting that in IDC the alpha v chain may be associated with a different beta chain (possibly beta 1 or beta 5). No correlation between integrin expression and estrogen/progesterone receptor status was found. These data provide further evidence that in invasive breast carcinomas there is a widespread deregulated expression of integrins and their accessory adhesion molecules with loss of polarization. Changes in the expression and function of cell adhesion molecules, which control growth and differentiation, may have clinical relevance in the behavior of breast cancer.