BackgroundTriple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems.MethodsTo understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis.ResultsWe isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A).ConclusionsTaken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.

Structural features of the integral membrane protein flavocytochrome b (Cyt b) were discovered using an antibody "imprint" of the Cyt b surface. Amino acid sequences were selected from a random nonapeptide phage-display library by their affinity for the monoclonal antibody 44.1 binding site, which recognizes the native conformation of the p22 phox subunit of Cyt b. Transferred nuclear Overhauser effect spectroscopy and rotating frame Overhauser effect spectroscopy NMR were used to study the antibody-bound conformation of a synthetic peptide derived from phage-displayed sequences. The NMR data supported the phagedisplay analysis suggesting the existence of a complex epitope and allowed the modeling of the close spatial proximity of the epitope components 29 TAGRF 33 and 183 PQVNPI 188 from discontinuous regions of p22 phox . Although these regions are separated by two putative membrane-spanning domains and are 150 residues apart in the sequence, they appear to combine to form a complex epitope on the cytosolic surface of the transmembrane protein. NMR constraints, measured from the antibody-bound conformation of a composite peptide mimetic of the Cyt b epitope, and one constraint inferred from the phage-display results, were used to demonstrate the close proximity of these two regions. This information provides a low resolution view of the tertiary structure of the native discontinuous epitope on the Cyt b surface. Given additional antibodies, such imprint analysis has the potential for producing structural constraints to help support molecular modeling of this and other low abundance or noncrystallizable proteins.Human phagocyte flavocytochrome b (Cyt b) 1 is an electron transferase that directs metabolic electrons across the plasma membrane to reduce molecular oxygen and form superoxide (O 2 . ). The production of superoxide by phagocytes, which involves modulation of Cyt b structure, is essential for antimicrobial host defense (1), plays a central role in inflammatory tissue injury (2), and may play a more general role in cellular regulation (3). Individuals with a defective Cyt b have insufficient superoxide production, suffer from chronic granulomatous disease (4), and sustain repeated life-threatening infections (5). Despite its cardinal role in many inflammatory processes and possibly in growth regulation, few experimental methods have been able to provide information on the structure of Cyt b. Therefore, we have developed an alternative method to describe topological features of the Cyt b surface. Because antibodies and their cognate antigens form complementary surfaces of interaction (6), we have sought structural information about Cyt b from an antibody binding site, specific for the Cyt b surface. We recently reported mapping of monoclonal antibody epitopes and functional sites of Cyt b, using random sequence phage-display peptide libraries (7;8). mAb 44.1 recognizes Cyt b in situ in permeabilized human neutrophils and thus contains information about the three-dimensional structure of the protein sur...

Cdc42Hs is a member of the Ras superfamily of GTPases and initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously used a 46 amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs [Guo et al. (1998) Biochemistry 37, 14030-14037]. Here we describe the three-dimensional solution structure of the Cdc42Hs. GMPPCP-PBD46 complex. Heteronuclear NMR methods were used to assign resonances in the complex, and approximately 2400 distance and dihedral restraints were used to calculate a set of 20 structures using a combination of distance geometry, simulated annealing, and chemical shift and Ramachandran refinement. The overall structure of Cdc42Hs in the complex differs from the uncomplexed structure in two major aspects: (1) the first alpha helix is reoriented to accommodate the binding of the peptide and (2) the regions corresponding to switch I and switch II are less disordered. As suggested by our previous work (Guo et al., 1998) and similar to the complex between Cdc42Hs and fACK [Mott et al. (1999) Nature 399, 384-388], PBD46 forms an intermolecular beta-sheet with beta2 of Cdc42Hs and contacts both switch I and switch II. The extensive binding surface between PBD46 and Cdc42Hs can account for both the high affinity of the complex and the inhibition by PBD46 of GTP hydrolysis.