Different gene expression patterns in invasive lobular and ductal carcinomas of the breast.

Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two major histological types of breast cancer worldwide. Whereas IDC incidence has remained stable, ILC is the most rapidly increasing breast cancer phenotype in the United States and Western Europe. It is not clear whether IDC and ILC represent molecularly distinct entities and what genes might be involved in the development of these two phenotypes. We conducted comprehensive gene expression profiling studies to address these questions. Total RNA from 21 ILCs, 38 IDCs, two lymph node metastases, and three normal tissues were amplified and hybridized to approximately 42,000 clone cDNA microarrays. Data were analyzed using hierarchical clustering algorithms and statistical analyses that identify differentially expressed genes (significance analysis of microarrays) and minimal subsets of genes (prediction analysis for microarrays) that succinctly distinguish ILCs and IDCs. Eleven of 21 (52%) of the ILCs ("typical" ILCs) clustered together and displayed different gene expression profiles from IDCs, whereas the other ILCs ("ductal-like" ILCs) were distributed between different IDC subtypes. Many of the differentially expressed genes between ILCs and IDCs code for proteins involved in cell adhesion/motility, lipid/fatty acid transport and metabolism, immune/defense response, and electron transport. Many genes that distinguish typical and ductal-like ILCs are involved in regulation of cell growth and immune response. Our data strongly suggest that over half the ILCs differ from IDCs not only in histological and clinical features but also in global transcription programs. The remaining ILCs closely resemble IDCs in their transcription patterns. Further studies are needed to explore the differences between ILC molecular subtypes and to determine whether they require different therapeutic strategies.

A molecular 'signature' of primary breast cancer cultures; patterns resembling tumor tissue.

BACKGROUND: To identify the spectrum of malignant attributes maintained outside the host environment, we have compared global gene expression in primary breast tumors and matched short-term epithelial cultures. RESULTS: In contrast to immortal cell lines, a characteristic 'limited proliferation' phenotype was observed, which included over expressed genes associated with the TGFbeta signal transduction pathway, such as SPARC, LOXL1, RUNX1, and DAPK1. Underlying this profile was the conspicuous absence of hTERT expression and telomerase activity, a significant increase in TbetaRII, its cognate ligand, and the CDK inhibitor, p21CIP1/WAF1. Concurrently, tumor tissue and primary cultures displayed low transcript levels of proliferation-related genes, such as, TOP2A, ANKT, RAD51, UBE2C, CENPA, RRM2, and PLK. CONCLUSIONS: Our data demonstrate that commonly used immortal cell lines do not reflect some aspects of tumor biology as closely as primary tumor cell cultures. The gene expression profile of malignant tissue, which is uniquely retained by cells cultured on solid substrates, could facilitate the development and testing of novel molecular targets for breast cancer.

A streamlined platform for high-content functional proteomics of primary human specimens.

Achieving information content of satisfactory breadth and depth remains a formidable challenge for proteomics. This problem is particularly relevant to the study of primary human specimens, such as tumor biopsies, which are heterogeneous and of finite quantity. Here we present a functional proteomics strategy that unites the activity-based protein profiling and multidimensional protein identification technologies (ABPP-MudPIT) for the streamlined analysis of human samples. This convergent platform involves a rapid initial phase, in which enzyme activity signatures are generated for functional classification of samples, followed by in-depth analysis of representative members from each class. Using this two-tiered approach, we identified more than 50 enzyme activities in human breast tumors, nearly a third of which represent previously uncharacterized proteins. Comparison with cDNA microarrays revealed enzymes whose activity, but not mRNA expression, depicted tumor class, underscoring the power of ABPP-MudPIT for the discovery of new markers of human disease that may evade detection by other molecular profiling methods.

Phosphorylation-independent dimer-dimer interactions by the enhancer-binding activator NtrC of Escherichia coli: a third function for the C-terminal domain.

The response regulator NtrC transcriptionally activates genes of the nitrogen-regulated (Ntr) response. Phosphorylation of its N-terminal receiver domain stimulates an essential oligomerization of the central domain. Deletion of the central domain reduces, but does not eliminate, intermolecular interactions as assessed by cooperative binding to DNA. To analyze the structural determinants and function of this central domain-independent as well as phosphorylation-independent oligomerization, we randomly mutagenized DNA coding for an NtrC without its central domain and isolated strains containing NtrC with defective phosphorylation-independent cooperative binding. The alterations were primarily localized to helix B of the C-terminal domain. Site-specific mutagenesis that altered surface residues of helix B confirmed this localization. The purified NtrC variants, with or without the central domain, were specifically defective in phosphorylation-independent cooperative DNA binding and had little defect, if any, on other functions, such as non-cooperative DNA binding. We propose that this region forms an oligomerization interface. Full-length NtrC variants did not efficiently repress the glnA-ntrBC operon when NtrC was not phosphorylated, which suggests that phosphorylation-independent cooperative binding sets the basal level for glutamine synthetase and the regulators of the Ntr response. The NtrC variants in these cells generally, but not always, supported wild-type growth in nitrogen-limited media and wild-type activation of a variety of Ntr genes. We discuss the differences and similarities between the NtrC C-terminal domain and the homologous Fis, which is also capable of intermolecular interactions.