Quantification of cancer driver mutations in human breast and lung DNA using targeted, error-corrected CarcSeq.

There is a need for scientifically-sound, practical approaches to improve carcinogenicity testing. Advances in DNA sequencing technology and knowledge of events underlying cancer development have created an opportunity for progress in this area. The long-term goal of this work is to develop variation in cancer driver mutation (CDM) levels as a metric of clonal expansion of cells carrying CDMs because these important early events could inform carcinogenicity testing.

Akt Regulates a Rab11-Effector Switch Required for Ciliogenesis.

Serum starvation stimulates cilia growth in cultured cells, yet serum factors associated with ciliogenesis are unknown. Previously, we showed that starvation induces rapid Rab11-dependent vesicular trafficking of Rabin8, a Rab8 guanine-nucleotide exchange factor (GEF), to the mother centriole, leading to Rab8 activation and cilium growth. Here, we demonstrate that through the LPA receptor 1 (LPAR1), serum lysophosphatidic acid (LPA) inhibits Rab11a-Rabin8 interaction and ciliogenesis.

Analysis of Enzymatic Activity of Matrix Metalloproteinase (MMP) by Collagen Zymography in Melanoma.

Protein zymography is the most commonly used technique to study the enzymatic activity of matrix metalloproteinases (MMPs) and their inhibitors. MMPs are proteolytic enzymes that promote extracellular matrix degradation. MMPs are frequently mutated in malignant melanomas as well as other cancers and are linked to increasing incidence of tumor metastasis.

Oncogenic RAS-Induced Perinuclear Signaling Complexes Requiring KSR1 Regulate Signal Transmission to Downstream Targets.

The precise characteristics that distinguish normal and oncogenic RAS signaling remain obscure. Here, we show that oncogenic RAS and BRAF induce perinuclear relocalization of several RAS pathway proteins, including the kinases CK2 and p-ERK1/2 and the signaling scaffold KSR1. This spatial reorganization requires endocytosis, the kinase activities of MEK-ERK and CK2, and the presence of KSR1.

CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation.

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening.

Mutational and functional analysis of the tumor-suppressor PTPRD in human melanoma.

Protein tyrosine phosphatases (PTPs) tightly regulate tyrosine phosphorylation essential for cell growth, adhesion, migration, and survival. We performed a mutational analysis of the PTP gene family in cutaneous metastatic melanoma and identified 23 phosphatase genes harboring somatic mutations. Among these, receptor-type tyrosine-protein phosphatase delta (PTPRD) was one of the most highly mutated genes, harboring 17 somatic mutations in 79 samples, a prevalence of 21.

Loss of CLCA4 promotes epithelial-to-mesenchymal transition in breast cancer cells.

The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells downregulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In breast cancer, EMT facilitates invasion of surrounding tissues and correlates closely with cancer metastasis and relapse. We found previously that the candidate tumor suppressor CLCA2 is expressed in differentiated, growth-arrested mammary epithelial cells but is downregulated during tumor progression and EMT.

Delving into somatic variation in sporadic melanoma.

Melanoma, the most aggressive form of skin cancer, has increased in incidence more rapidly than any other cancer. The completion of the human genome project and advancements in genomics technologies has allowed us to investigate genetic alterations of melanoma at a scale and depth that is unprecedented. Here, we survey the history of the different approaches taken to understand the genomics of melanoma - from early candidate genes, to gene families, to genome-wide studies.

Exon capture analysis of G protein-coupled receptors identifies activating mutations in GRM3 in melanoma.

G protein-coupled receptors (GPCRs), the largest human gene family, are important regulators of signaling pathways. However, knowledge of their genetic alterations is limited. In this study, we used exon capture and massively parallel sequencing methods to analyze the mutational status of 734 GPCRs in melanoma.

Exome sequencing identifies GRIN2A as frequently mutated in melanoma.

The incidence of melanoma is increasing more than any other cancer, and knowledge of its genetic alterations is limited. To systematically analyze such alterations, we performed whole-exome sequencing of 14 matched normal and metastatic tumor DNAs. Using stringent criteria, we identified 68 genes that appeared to be somatically mutated at elevated frequency, many of which are not known to be genetically altered in tumors.

Micromanagement of the mitochondrial apoptotic pathway by p53.

It is now well established that p53 is the primary arbiter of stress-response and the principal barrier to neoplastic processes at the cellular level. Perhaps the most potent weapon in p53's tumor suppressive arsenal is apoptosis, enacted as a last resort when all other remedies are exhausted. Initially, the mechanism was thought to be simply activation or repression of Bcl-2 family members by p53.

Enrichment for breast cancer cells with stem/progenitor properties by differential adhesion.

Cancer stem cells are commonly isolated by cell sorting for surface antigens that typify stem cells. This technique is very expensive, requiring advanced, high-speed sorters and high-quality antibodies, and yields are often low. Some stem cells can be isolated based on ability to exclude dyes, conferred by expression of membrane transporters, but this property is not universal.

hCLCA2 Is a p53-Inducible Inhibitor of Breast Cancer Cell Proliferation.

hCLCA2 is frequently down-regulated in breast cancer and is a candidate tumor suppressor gene. We show here that the hCLCA2 gene is strongly induced by p53 in response to DNA damage. Adenoviral expression of p53 induces hCLCA2 in a variety of breast cell lines.

p53 represses c-Myc through induction of the tumor suppressor miR-145.

The tumor suppressor p53 negatively regulates a number of genes, including the proto-oncogene c-Myc, in addition to activating many other genes. One mechanism of the p53-mediated c-Myc repression may involve transcriptional regulation. However, it is not clear whether microRNAs (miRNAs) play a role in the p53-mediated posttranscriptional regulation of c-Myc.

p53 in breast cancer: mutation and countermeasures.

p53 is the primary arbiter of the mammalian cell's response to stress, the governor of life and death. It is the nexus upon which signals converge from an array of sensors that detect damage to DNA or to the mitotic spindle or the cytoskeleton, hypoxia, cell detachment, growth factor deprivation, oncogene expression and other forms of stress. Depending on the type, intensity and duration of the signals, p53 in turn transactivates batteries of genes specifying cell cycle arrest, DNA repair, apoptosis, or other anti-neoplastic functions.

The putative chloride channel hCLCA2 has a single C-terminal transmembrane segment.

Calcium-activated chloride channel (CLCA) proteins were first described as a family of plasma membrane Cl(-) channels that could be activated by calcium. Genetic and electrophysiological studies have supported this view. The human CLCA2 protein is expressed as a 943-amino-acid precursor whose N-terminal signal sequence is removed followed by internal cleavage near amino acid position 680.