SUMMARY Carcinomas typically invade as a cohesive multicellular unit, a process termed collective invasion. It remains unclear how different subpopulations of cancer cells contribute to this process. We developed three-dimensional (3D) organoid assays to identify the most invasive cancer cells in primary breast tumors. Collective invasion was led by specialized cancer cells that were defined by their expression of basal epithelial genes, such as cytokeratin-14 (K14) and p63. Furthermore, K14+ cells led collective invasion in the major human breast cancer subtypes. Importantly, luminal cancer cells were observed to convert phenotypically to invasive leaders following induction of basal epithelial genes. Although only a minority of cells within luminal tumors expressed basal epithelial genes, knockdown of either K14 or p63 was sufficient to block collective invasion. Our data reveal that heterotypic interactions between epithelial subpopulations are critical to collective invasion. We suggest that targeting the basal invasive program could limit metastatic progression.

LATS (large tumor suppressor) or warts is a Ser/Thr kinase that belongs to the Ndr/LATS subfamily of AGC (protein kinase A/PKG/PKC) kinases. It is a tumor suppressor gene originally isolated from Drosophila and recently isolated from mice and humans. Drosophila or mice mutant for LATS develop tumors in various tissues. Recent studies in Drosophila demonstrate that LATS is a central player of an emerging tumor suppressor pathway called the Hippo-LATS/Warts pathway that suppresses tumor growth by regulating cell proliferation, cell growth, and cell death. Although tremendous progress has been made toward understanding the roles of LATS in tumorigenesis, the kinase substrates of LATS or downstream target proteins mediating LATS function remain largely unknown. In this study, we have provided convincing evidence that the LATS1 tumor suppressor can bind to and phosphorylate transcription regulator and oncogene YAP in vitro and in vivo. We have also identified HX(R/H/K)XX(S/T) as the consensus phosphorylation sequence for LATS/Ndr kinase substrates. Significantly, we have discovered that LATS1 inactivates YAP oncogenic function by suppressing its transcription regulation of cellular genes via sequestration of YAP in the cytoplasm after phosphorylation of YAP. Finally, by using microarray analysis, we have also identified many oncogenes or tumor suppressor genes up-regulated or down-regulated by YAP. These research findings will have profound impacts on our understanding of the molecular mechanism of the LATS tumor suppressor and the emerging Hippo-LATS/Warts pathway.

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14 + cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14+ epithelial tumor cell clusters disseminate collectively to colonize distant organs.uring metastasis, cancer cells escape the primary tumor, travel through the circulation, and colonize distant organs. Conventional models of cancer progression propose that each metastasis arises from the clonal outgrowth of a single tumor cell and this conceptual framework is a foundation for models, such as epithelial-mesenchymal transition (EMT) and migratory cancer stem cells (1).Challenging the generality of the single-cell/single-metastasis model are long-standing clinical observations that tumor cell clusters (also termed "tumor clumps") are also observed across the stages of metastasis. Tumor cell clusters are detected in the bloodstream of cancer patients (2), clusters can efficiently seed metastases (3), and though rare, circulating tumor cell (CTC) clusters have prognostic significance (4, 5). Furthermore, metastases are composed of multiple genetically distinct tumor cell clones, in mouse models of breast, pancreas, and small cell carcinoma (5-7), and in human metastatic prostate cancer patients (8). Taken together, these observations provide accumulating evidence that tumor cell clusters contribute to metastasis. However, they leave unresolved two important questions: how do tumor cell clusters emerge from the primary tumor, and which molecular features identify cell clusters that metastasize?An important clinical observation is that cancer cells invade the surrounding stroma as cohesive clusters in the majority of ...

Despite decades of study, there are still many unanswered questions about metastasis, the process by which a localized cancer becomes a systemic disease. One of these questions is the nature of the tumor cells that give rise to metastases. Although conventional models suggest that metastases are seeded by single cells from the primary tumor, there is growing evidence that seeding requires the collective action of tumor cells traveling together in clusters. Here, we review this evidence, which comes from analysis of both experimental models and patient samples. We present a model of metastatic dissemination that highlights the activities of clusters of tumor cells that retain and require their epithelial properties.

Breast cancer progression involves genetic changes and changes in the extracellular matrix (ECM). To test the importance of the ECM in tumor cell dissemination, we cultured epithelium from primary human breast carcinomas in different ECM gels. We used basement membrane gels to model the normal microenvironment and collagen I to model the stromal ECM. In basement membrane gels, malignant epithelium either was indolent or grew collectively, without protrusions. In collagen I, epithelium from the same tumor invaded with protrusions and disseminated cells. Importantly, collagen I induced a similar initial response of protrusions and dissemination in both normal and malignant mammary epithelium. However, dissemination of normal cells into collagen I was transient and ceased as laminin 111 localized to the basal surface, whereas dissemination of carcinoma cells was sustained throughout culture, and laminin 111 was not detected. Despite the large impact of ECM on migration strategy, transcriptome analysis of our 3D cultures revealed few ECM-dependent changes in RNA expression. However, we observed many differences between normal and malignant epithelium, including reduced expression of cell-adhesion genes in tumors. Therefore, we tested whether deletion of an adhesion gene could induce sustained dissemination of nontransformed cells into collagen I. We found that deletion of P-cadherin was sufficient for sustained dissemination, but exclusively into collagen I. Our data reveal that metastatic tumors preferentially disseminate in specific ECM microenvironments. Furthermore, these data suggest that breaks in the basement membrane could induce invasion and dissemination via the resulting direct contact between cancer cells and collagen I.

Subgenic-resolution oligonucleotide microarrays were used to study global RNA degradation in wild-type Escherichia coli MG1655. RNA chemical half-lives were measured for 1036 open reading frames (ORFs) and for 329 known and predicted operons. The half-life of total mRNA was 6.8 min under the conditions tested. We also observed significant relationships between gene functional assignments and transcript stability. Unexpectedly, transcription of a single operon (tdcABCDEFG) was relatively rifampicin-insensitive and showed significant increases 2.5 min after rifampicin addition. This supports a novel mechanism of transcription for the tdc operon, whose promoter lacks any recognizable binding sites. Probe by probe analysis of all known and predicted operons showed that the 5Ј ends of operons degrade, on average, more quickly than the rest of the transcript, with stability increasing in a 3Ј direction, supporting and further generalizing the current model of a net 5Ј to 3Ј directionality of degradation. Hierarchical clustering analysis of operon degradation patterns revealed that this pattern predominates but is not exclusive. We found a weak but highly significant correlation between the degradation of adjacent operon regions, suggesting that stability is determined by a combination of local and operon-wide stability determinants. The 16 ORF dcw gene cluster, which has a complex promoter structure and a partially characterized degradation pattern, was studied at high resolution, allowing a detailed and integrated description of its abundance and degradation. We discuss the application of subgenic resolution DNA microarray analysis to study global mechanisms of RNA transcription and processing.Gene regulation is a dynamic process which can be controlled by a number of mechanisms as genetic information flows from nucleic acids to proteins. The study of gene regulation in the steady state, while informative, overlooks the underlying dynamics of the processes. Steady-state transcript levels are a result of both RNA synthesis and degradation, and as such, measurements of degradation rates can be used to determine their rates of synthesis (if their steady-state levels are known) as well as reveal regulation which occurs via changes in RNA stability.For the genetic regulatory network of Escherichia coli to be understood and eventually modeled, all means of regulation in use by the cell must be given due attention. RNA degradation in eubacteria was once viewed as a nonspecific, unregulated process. Today it is known to involve multiple degradation pathways, a multisubunit protein complex (the degradosome), and to be an important regulatory mechanism for the expression of some genes (for reviews, see GrunbergManago 1999;Rauhut and Klug 1999;Regnier and Arraiano 2000). A small number of large-scale RNA degradation analyses have recently been reported in budding yeast (Wang et al. 2002), humans (Lam et al. 2001), and E. coli (Bernstein et al. 2002).RNA expression analysis with DNA microarrays has allowed transcription to be st...

Ultrasound and sestamibi-SPECT are similar in ability to preoperatively localize abnormal parathyroid glands in pHPT. Accuracy may be improved with 4D-CT; however, further investigation is required. Choice of preoperative imaging strategy depends on numerous patient, institutional, and economic factors of which the surgeon must be aware.

We have developed a high-resolution "genome array" for the study of gene expression and regulation in Escherichia coli. This array contains on average one 25-mer oligonucleotide probe per 30 base pairs over the entire genome, with one every 6 bases for the intergenic regions and every 60 bases for the 4,290 open reading frames (ORFs). Twofold concentration differences can be detected at levels as low as 0.2 messenger RNA (mRNA) copies per cell, and differences can be seen over a dynamic range of three orders of magnitude. In rich medium we detected transcripts for 97% and 87% of the ORFs in stationary and log phases, respectively. We found that 1, 529 transcripts were differentially expressed under these conditions. As expected, genes involved in translation were expressed at higher levels in log phase, whereas many genes known to be involved in the starvation response were expressed at higher levels in stationary phase. Many previously unrecognized growth phase-regulated genes were identified, such as a putative receptor (b0836) and a 30S ribosomal protein subunit (S22), both of which are highly upregulated in stationary phase. Transcription of between 3,000 and 4,000 predicted ORFs was observed from the antisense strand, indicating that most of the genome is transcribed at a detectable level. Examples are also presented for high-resolution array analysis of transcript start and stop sites and RNA secondary structure.