Senescence evasion in melanoma progression: uncoupling of DNA‐damage signaling from p53 activation and p21 expression

The best‐established function of the melanoma‐suppressor p16 is mediation of cell senescence, a permanent arrest following cell proliferation or certain stresses. The importance of p16 in melanoma suggests indolence of the other major senescence pathway through p53. Little or no p53 is expressed in senescent normal human melanocytes, but p16‐deficient melanocytes can undergo p53‐mediated senescence. As p16 expression occurs in nevi but falls with progression toward melanoma, we here investigated whether p53‐dependent senescence occurs at some stage and, if not, what defects were detectable in this pathway, using immunohistochemistry. Phosphorylated checkpoint kinase 2 (CHEK2) can mediate DNA‐damage signaling, and under some conditions senescence, by phosphorylating and activating p53. Remarkably, we detected no prevalent p53‐mediated senescence in any of six classes of lesions. Two separate defects in p53 signaling appeared common: in nevi, lack of p53 phosphorylation by activated CHEK2, and in melanomas, defective p21 upregulation by p53 even when phosphorylated.     

TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.     

Intrasexual competition underlies sexual selection on male breeding coloration in the orangethroat darter,Etheostoma spectabile

Elaborate, sexually dimorphic traits are widely thought to evolve under sexual selection through female preference, male–male competition, or both. The orangethroat darter (Etheostoma spectabile) is a sexually dichromatic fish in which females exhibit no preferences for male size or coloration. We tested whether these traits affect individual reproductive success in E. spectabile when multiple males are allowed to freely compete for a female. The quality and quantity of male coloration were associated with greater success in maintaining access to the female and in spawning as the primary male (first male to participate). On the other hand, sneaking behavior showed little correlation with coloration. Male breeding coloration in E. spectabile may therefore demonstrate how intrasexual competition can be a predominant factor underlying the evolution of male ornaments.     

Glypican-5 is a tumor suppressor in non-small cell lung cancer cells

Glypican-5 (GPC5) belongs to the glypican family of proteoglycans that have been implicated in a variety of physiological processes, ranging from cell proliferation to morphogenesis. However, the role of GPC5 in human cancer remains poorly understood. We report that knockdown of GPC5 in bronchial epithelial cells promoted, and forced expression of GPC5 in non-small lung cancer (NSCLC) cells suppressed, the anchorage-independent cell growth. In vivo, expression of GPC5 inhibited xenograft tumor growth of NSCLC cells. Furthermore, we found that GPC5 was expressed predominantly as a membrane protein, and its expression led to diminished phosphorylation of several oncogenic receptor tyrosine kinases, including the ERBB family members ERBB2 and ERBB3, which play critical roles in lung tumorigenesis. Collectively, our results suggest that GPC5 may act as a tumor suppressor, and reagents that activate GPC5 may be useful for treating NSCLC.     

Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

Plasma membrane Ca²⁺-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca²⁺]_i). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca²⁺]_i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Yeast GPCR signaling reflects the fraction of occupied receptors,not the number

According to receptor theory, the effect of a ligand depends on the amount of agonist–receptor complex. Therefore, changes in receptor abundance should have quantitative effects. However, the response to pheromone in Saccharomyces cerevisiae is robust (unaltered) to increases or reductions in the abundance of the G‐protein‐coupled receptor (GPCR), Ste2, responding instead to the fraction of occupied receptor. We found experimentally that this robustness originates during G‐protein activation. We developed a complete mathematical model of this step, which suggested the ability to compute fractional occupancy depends on the physical interaction between the inhibitory regulator of G‐protein signaling (RGS), Sst2, and the receptor. Accordingly, replacing Sst2 by the heterologous hsRGS4, incapable of interacting with the receptor, abolished robustness. Conversely, forcing hsRGS4:Ste2 interaction restored robustness. Taken together with other results of our work, we conclude that this GPCR pathway computes fractional occupancy because ligand‐bound GPCR–RGS complexes stimulate signaling while unoccupied complexes actively inhibit it. In eukaryotes, many RGSs bind to specific GPCRs, suggesting these complexes with opposing activities also detect fraction occupancy by a ratiometric measurement. Such complexes operate as push‐pull devices, which we have recently described.