Cancer cell vascular invasion is a crucial step in the malignant progression toward metastasis. Here we used a genome-wide RNA interference screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these Krtaps have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/β4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a Krtap can serve as a switch toward malignant progression.

Metastasis of cancer cells involves multiple steps, including their dissociation from the primary tumor and invasion through the endothelial cell barrier to enter the circulation and finding their way to distant organ sites where they extravasate and establish metastatic lesions. Deficient contact inhibition is a hallmark of invasive cancer cells, yet surprisingly the vascular invasiveness of commonly studied cancer cell lines is regulated by the density at which cells are propagated in culture. Cells grown at high density were less effective at invading an endothelial monolayer than cells grown at low density. This phenotypic difference was also observed in a zebrafish model of vascular invasion of cancer cells after injection into the yolk sac and extravasation of cancer cells into tissues from the vasculature. The vascular invasive phenotypes were reversible. A kinome-wide RNA interference screen was used to identify drivers of vascular invasion by panning small hairpin RNA (shRNA) library-transduced noninvasive cancer cell populations on endothelial monolayers. The selection of invasive subpopulations showed enrichment of shRNAs targeting the large tumor suppressor 1 (LATS1) kinase that inhibits the activity of the transcriptional coactivator yes-associated protein (YAP) in the Hippo pathway. Depletion of LATS1 from noninvasive cancer cells restored the invasive phenotype. Complementary to this, inhibition or depletion of YAP inhibited invasion in vitro and in vivo. The vascular invasive phenotype was associated with a YAP-dependent upregulation of the cytokines IL6, IL8 and C-X-C motif ligand 1, 2 and 3. Antibody blockade of cytokine receptors inhibited invasion and confirmed that they are rate-limiting drivers that promote cancer cell vascular invasiveness and could provide therapeutic targets.

Objective—In a previous study we identified metallothionein (MT) as a candidate gene potentially influencing collaterogenesis. In this investigation, we determined the effect of MT on collaterogenesis and examined the mechanisms contributing to the effects we found.Methods and Results—Collateral blood flow recovery was assessed using laser Doppler perfusion imaging, and angiogenesis was measured using a Matrigel plug assay. Smooth muscle cells were isolated from MT knockout (KO) mice for functional assays. Gene expression of matrix metalloproteinase-9, platelet-derived growth factor, vascular endothelial growth factor, and Fat cadherin in smooth muscle cells was measured by real-time polymerase chain reaction, and protein levels of vascular endothelial growth factor and matrix metalloproteinase-9 were determined using enzyme-linked immunosorbent assay and Western blot. CD11b⁺macrophages were tested for invasiveness using a real-time impedance assay. Both flow recovery and angiogenesis were impaired in MT KO mice. Proliferation, migration, and invasion were decreased in MT KO smooth muscle cells, and matrix metalloproteinase-9, platelet-derived growth factor, and vascular endothelial growth factor expression were also decreased, whereas FAT-1 cadherin expression was elevated. MT KO CD11b⁺cells were more invasive than wild-type cells.Conclusion—MT plays an important role in collateral flow recovery and angiogenesis, an activity that appears to be mediated, in part, by the effects of MT on the functionality of 3 cell types essential for these processes: endothelial cells, smooth muscle cells, and macrophages.

SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized. We found that at 4 degrees C SecA homodimer binds one ADP molecule with high affinity. This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true. Azi- and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA. Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA. The level of Azi- and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity. These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export.