P21 Activated Kinase-1 (Pak1) Promotes Prostate Tumor Growth and Microinvasion via Inhibition of Transforming Growth Factor β Expression and Enhanced Matrix Metalloproteinase 9 Secretion

P21-activated kinases (Paks) are major effectors downstream of the small Rho family of GTPases. Among the six isoforms, Pak1 is the most ubiquitous and the best characterized member. Previous studies have shown that inhibition of Pak6, which is predominantly present in the prostate compared with other tissues, inhibits prostate tumor growth in vivo. Even though Pak1 has been identified in normal prostatic epithelial cells and cancer cells, its specific role in the development of prostate cancer remains unclear. We report here that highly invasive prostate cancer cells express significantly higher levels of Pak1 protein compared with non-invasive prostate cancer cells. Furthermore, prostate tumor tissues and prostate cancer metastasized to lungs showed a higher expression of Pak1 compared with normal tissues. Interestingly, Pak6 protein expression levels did not change with the invasive/metastatic potential of the cancer cells or tumors. Although inhibition of Pak1, and not Pak6, resulted in impaired PC3 cell migration, the effects of Pak1 knockdown on transendothelial migration (microinvasion), tumor growth, and tumor angiogenesis was higher compared with Pak6 knockdown. Finally, gene array data revealed reduced expression of matrix metalloproteinase 9 with the ablation of either Pak1 or Pak6 gene expression in PC3 cells, whereas protein levels of TGFβ was elevated significantly with specific modulation of Pak1 activity or ablation of the Pak1 gene. Our observations suggest that although some level of functional redundancy exists between Pak1 and Pak6 in prostate cancer cells, targeting Pak1 is a potential option for the management of prostate tumor growth, microinvasion, and metastasis.     

Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex

Death receptors (DRs) induce apoptosis but also stimulate proinflammatory "non-apoptotic" signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIP(S), cFLIP(L), or mutants of cFLIP(L) (cFLIP(D376N) and cFLIP(p43)). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIP(L) induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIP(S) or cFLIP(p43) blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin.     

Sustained JNK activation in response to tumor necrosis factor is mediated by caspases in a cell type-specific manner

In most cell types, tumor necrosis factor (TNF) induces a transient activation of the JNK pathway. However, in NFkappaB-inhibited cells, TNF stimulates also a second sustained phase of JNK activation, which has been implicated in cell death induction. In the present study, we have analyzed the relationship of cell death induction, caspase activity, JNK, and NFkappaB stimulation in the context of TNF signaling in four different cellular systems. In all cases, NFkappaB inhibition enhanced TNF-induced cell death and primed most, but not all, cells for sustained JNK activation. The caspase inhibitor Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-fmk) and overexpression of the antiapoptotic proteins FLIP-L and Bcl2 differentially blocked transient and sustained JNK activation in NFkappaB-inhibited KB and HaCaT cells, indicating that the two phases of TNF-induced JNK activation occur at least in these cellular models by different pathways. Although the broad range caspase inhibitor Z-VAD-fmk and the antioxidant butylated hydroxyanisole interfered with TNF-induced cell death to a varying extent in a cell type-specific manner, inhibition of JNK signaling had no or only a very moderate effect. Notably, the JNK inhibitory effect of neither Z-VAD-fmk nor butylated hydroxyanisole was strictly correlated with the capability of these compounds to rescue cells from TNF-induced cell death. Thus, sustained JNK activation by TNF has no obligate role in TNF-induced cell death and is mediated by caspases and reactive oxygen species in a cell type-specific manner.     

Molecular mechanisms to sense soil overlay and optimize emergence in plants

The famous Mexican proverb says “They tried to bury us, they did not know we were seeds”. Seeds buried under the soil find their way through the soil particles and emerge out. The mechanical pressure of the soil cover induces the production of the gaseous hormone ethylene in plants. Ethylene promotes formation of an apical hook to protect the plant tip when seedlings try to penetrate through the soil. The dark environment under the soil cover also promotes apical hook formation. Once the seedlings reach the soil surface the apical hook opens to expose the meristem and initiate the development of aerial parts. Recent studies suggest the integration of the ethylene and light signaling pathway to regulate soil emergence. Here we summarize our current understanding of how light and ethylene signals coordinate to optimize seedling establishment. This might contribute towards crop improvement programs as successful emergence is critical for survival of seeds plants in natural environments and agricultural fields.