Redundant Canonical and Noncanonical Caenorhabditis elegans p21-Activated Kinase Signaling Governs Distal Tip Cell Migrations

p21-activated kinases (Paks) are prominent mediators of Rac/Cdc42-dependent and -independent signaling and regulate signal transduction and cytoskeletal-based cell movements. We used the reproducible migrations of the Caenorhabditis elegans gonadal distal tip cells to show that two of the three nematode Pak proteins, MAX-2 and PAK-1, function redundantly in regulation of cell migration but are regulated by very different mechanisms. First, we suggest that MAX-2 requires CED-10/Rac function and thus functions canonically. Second, PIX-1 and GIT-1 function in the same role as PAK-1, and PAK-1 interaction with PIX-1 is required for PAK-1 activity; thus, PAK-1 functions noncanonically. The human Pak-Pix-Git complex is central to noncanonical Pak signaling and requires only modest Rac/CDC-42 input. Unlike the human complex, our results suggest that the C. elegans Pak-Pix-Git complex requires PAK-1 kinase domain activity. This study delineates signaling network relationships in this cell migration model, thus providing potential further mechanistic insights and an assessment of total Pak contribution to cell migration events

Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032

To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells

Lapatinib in Combination With Radiation Diminishes Tumor Regrowth in HER2+ and Basal-Like/EGFR+ Breast Tumor Xenografts

To determine whether lapatinib, a dual epidermal growth factor receptor (EGFR)/HER2 kinase inhibitor, can radiosensitize EGFR+ or HER2+ breast cancer xenografts

A prognostic signature of defective p53-dependent G1 checkpoint function in melanoma cell lines: A signature of defective p53 function in melanoma

Melanoma cell lines and normal human melanocytes were assayed for p53-dependent G1 checkpoint response to ionizing radiation-induced DNA damage. Sixty six percent of melanoma cell lines displayed a defective G1 checkpoint. Checkpoint function was correlated with sensitivity to ionizing radiation with checkpoint-defective lines being radio-resistant. Microarray analysis identified 316 probes whose expression was correlated with G1 checkpoint function in melanoma lines (P≤0.007) including p53 transactivation targets CDKN1A, DDB2 and RRM2B. The 316 probe list predicted G1 checkpoint function of the melanoma lines with 86% accuracy using a binary analysis and 91% accuracy using a continuous analysis. When applied to microarray data from primary melanomas, the 316 probe list was prognostic of four year distant metastases-free survival. Thus, p53 function, radio-sensitivity and metastatic spread may be estimated in melanomas from a signature of gene expression

Targeted next generation sequencing identifies clinically actionable mutations in patients with melanoma

Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here we describe a NextGen sequencing approach to fully analyze 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high-throughput sequencing, and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard-of-care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma

Developmental fidelity is imposed by genetically separable RalGEF activities that mediate opposing signals

International audienceThe six C. elegans vulval precursor cells (VPCs) are induced to form the 3°-3°-2°-1°-2°-3° pattern of cell fates with high fidelity. In response to EGF signal, the LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK canonical MAP kinase cascade is necessary to induce 1° fate and synthesis of DSL ligands for the lateral Notch signal. In turn, LIN-12/Notch receptor is necessary to induce neighboring cells to become 2°. We previously showed that, in response to graded EGF signal, the modulatory LET-60/Ras-RGL-1/RalGEF-RAL-1/Ral signal promotes 2° fate in support of LIN-12. In this study, we identify two key differences between RGL-1 and RAL-1. First, deletion of RGL-1 confers no overt developmental defects, while previous studies showed RAL-1 to be essential for viability and fertility. From this observation, we hypothesize that the essential functions of RAL-1 are independent of upstream activation. Second, RGL-1 plays opposing and genetically separable roles in VPC fate patterning. RGL-1 promotes 2° fate via canonical GEF-dependent activation of RAL-1. Conversely, RGL-1 promotes 1° fate via a non-canonical GEF-independent activity. Our genetic epistasis experiments are consistent with RGL-1 functioning in the modulatory 1°-promoting AGE-1/PI3-Kinase-PDK-1-AKT-1 cascade. Additionally, animals lacking RGL-1 experience 15-fold higher rates of VPC patterning errors compared to the wild type. Yet VPC patterning in RGL-1 deletion mutants is not more sensitive to environmental perturbations. We propose that RGL-1 functions to orchestrate opposing 1°- and 2°-promoting modulatory cascades to decrease developmental stochasticity. We speculate that such switches are broadly conserved but mostly masked by paralog redundancy or essential functions