FAK competes for Src to promote migration against invasion in melanoma cells

Melanoma is one of the most deadly cancers because of its high propensity to metastasis, a process that requires migration and invasion of tumor cells driven by the regulated formation of adhesives structures like focal adhesions (FAs) and invasive structures like invadopodia. FAK, the major kinase of FAs, has been implicated in many cellular processes, including migration and invasion. In this study, we investigated the role of FAK in the regulation of invasion. We report that suppression of FAK in B16F10 melanoma cells led to increased invadopodia formation and invasion through Matrigel, but impaired migration. These effects are rescued by FAK WT but not by FAK^Y397F reexpression. Invadopodia formation requires local Src activation downstream of FAK and in a FAK phosphorylation-dependant manner. FAK deletion correlates with increased phosphorylation of Tks-5 (tyrosine kinase substrate with five SH3 domain) and reactive oxygen species production. In conclusion, our data show that FAK is able to mediate opposite effects on cell migration and invasion. Accordingly, beneficial effects of FAK inhibition are context dependent and may depend on the cell response to environmental cues and/or on the primary or secondary changes that melanoma experienced through the invasion cycle.Patients with spreading melanoma diseases have a very poor prognosis with a 5-year survival rate <5%. The metastatic spread of melanoma is a complex process involving several genetic alterations. In melanoma,¹ as in many highly invasive cancer cell types like head and neck squamous cell carcinoma² or breast carcinoma,³ specialized matrix-degrading organelles termed invadopodia have been identified. Invadopodia consist of dynamic actin-based protrusions of 0, 1 to 2 μm in diameter emanating from the ventral edge of tumor cells.⁴ Besides their actin scaffold, these structures are enriched in proteolytic enzymes such as matrix metalloproteinases (MMPs), which mediate extracellular matrix (ECM) degradation. Indeed, MMP are upregulated in invasive melanoma and there is extensive evidence that they have a role in promoting the dissemination of melanoma.^{5, 6, 7} Several proteins like integrins, Src and paxillin, found at sites of cell adhesion to the matrix, are also present in invadopodia.^{8, 9} On the other hand, other proteins like the Src substrate proteins cortactin¹⁰ and the tyrosine kinase substrate with five SH3 domain (Tks-5)¹¹ are specifically localized at invadopodia and not found at focal adhesion (FA). In addition, reactive oxygen species (ROS)¹² have been localized at invadopodia and are supposed to have a prominent role in inducing invadopodia function.^{13, 14} Although significant efforts have been made to characterize components of invadopodia, the precise mechanisms of their regulation, especially in a melanoma context, remain poorly understood.Tumor invasion is a multistep process that requires cell adhesion to the environing substratum, migration and invasion. In many cell types, migration requires fine control of FA turn-over. FAs are formed by the cluster of up to 200 proteins¹⁵ ensuring cell anchorage to the ECM. The cyclic process of FA formation and disruption is crucial for cell migration. Because both anchorage and migration involve cellular interactions with ECM components, FAs are endowed with transmembrane ECM receptor proteins such as integrins that interact with ECM molecules and intra-cellular proteins composed of scaffold proteins, as well as signal-transducing molecules. Among those, focal adhesion kinase (FAK) is a crucial signaling protein that integrates signals from integrins to the actin filaments during cell migration.¹⁶ Structurally, FAK is a 125-kDa protein that contains an N-terminal 4.1-ezrin–radixin–moesin domain, a central kinase domain and a C-terminal domain that contains the focal adhesion targeting site.¹⁷ The phosphorylation of FAK at Y397 creates a binding site for Src, which can phosphorylate other tyrosines on the FAK sequence, thus creating new binding sites for SH2 domain-containing proteins.FAK is involved in many aspects of the metastatic process and thus, overexpression, hyperphosphorylation and/or elevated activity of FAK have been reported in a variety of human cancers, including sarcomas and carcinomas of the breast, colon, thyroid, prostate, oral cavity, liver, stomach and ovary.¹⁸ In human melanoma cell lines, early studies reported high FAK expression and requirement of FAK for cell substrate adhesion.¹⁹ Later, it was reported that FAK promotes the aggressive melanoma phenotype.²⁰ Indeed, immunohistochemical analyses revealed high levels of FAK phosphorylation at Tyr397 and Tyr576, a marker of FAK kinase activity, in late-stage cutaneous and uveal melanoma, which correlated with their increased invasion and migration properties.²¹ Furthermore, melanoma differentiation-associated gene-9 (mda-9)/syntenin was also reported to mediate adhesion-dependant activation of protein kinase Cα (PKCα) and FAK in melanoma cells. Thus, inhibiting either mda-9/syntenin or PKCα suppressed fibronectin-induced formation of integrin-β1/FAK/c-Src signaling complexes and reduced migration and invasion toward fibronectin.²² Therefore, FAK appears to be a major player of melanoma invasion, but how this kinase controls the formation and proteolytic activity of invadopodia in melanoma cells was never investigated.In this study, we uncovered a surprising negative regulation of invadopodia activity in B16F10 cells by FAK. The depletion of FAK was associated with increased ROS production and Tks-5 phosphorylation. Using mutation of FAK at Tyr397, a binding site for Src, we found that these sites are implicated in FAK-mediated inhibition of invadopodia activity. In addition, we report that this mutation induced decreased migration speed but increased invasive properties. Taken together, our data suggest a competition between FA and invadopodia substrates for Src phosphorylation that might depend on environmental cues, thus leading to the engagement of either migration or degradation pathways.     

Molecular cytogenetic characterization of a constitutional complex intrachromosomal 4q rearrangement in a patient with multiple congenital anomalies

Constitutional Complex Chromosomal Rearrangements (CCRs) are very rare. While the vast majority of CCRs involve more than one chromosome, only seven cases describe CCRs with four or more breakpoints within a single chromosome. Here, we present a patient with multiple congenital anomalies and mental retardation. Array Comparative Genomic Hybridisation (array CGH), FISH and Multicolour Banding FISH revealed a de novo complex rearrangement with two deletions, a duplication and an inversion of 4q. This CCR involving at least seven breakpoints is one of the most complex rearrangements of a single chromosome reported thus far. Potential mechanisms generating such complex rearrangements are discussed.     

Taxa distribution and RAPD markers indicate different origin and regional differentiation of hybrids in the invasive Fallopia complex in central-western Europe

Interspecific hybridization can be a driving force for evolutionary processes during plant invasions, by increasing genetic variation and creating novel gene combinations, thereby promoting genetic differentiation among populations of invasive species in the introduced range. We examined regional genetic structure in the invasive Fallopia complex, consisting of F. japonica var. japonica , F.   sachalinensis and their hybrid F.  ×  bohemica , in seven regions in Germany and Switzerland using RAPD analysis and flow cytometry. All individuals identified as F. japonica var . japonica had the same RAPD phenotype, while F. sachalinensis (11 RAPD phenotypes for 11 sampled individuals) and F.  ×  bohemica (24 RAPD phenotypes for 32 sampled individuals) showed high genotypic diversity. Bayesian cluster analysis revealed three distinct genetic clusters. The majority of F . ×  bohemica individuals were assigned to a unique genetic cluster that differed from those of the parental species, while the other F . ×  bohemica individuals had different degrees of admixture to the three genetic clusters. At the regional scale, the occurrence of male-fertile F. sachalinensis coincided with the distribution of F . ×  bohemica plants showing a high percentage of assignment to both parental species, suggesting that they originated from hybridization between the parental species. In contrast, in regions where male-fertile F. sachalinensis were absent, F . ×  bohemica belonged to the non-admixed genetic group, indicating multiple introductions of hybrids or sexual reproduction among hybrids. We also found regional differentiation in the gene pool of F.  ×  bohemica , with individuals within the same region more similar to each other than to individuals from different regions.     

Assessment of real-time PCR for quantification of Legionella spp. in spa water

Aim: To identify media and environmental conditions suitable for rapid mycelial growth and sporulation of Diplocarpon mali. Methods and Results: Liquid shake cultures were used to evaluate effects of media and environmental conditions on mycelial growth and conidial production of D. mali. Carrot sucrose broth (CSB), potato and carrot dextrose broth (PCDB) and potato and carrot sucrose broth (PCSB) were most favourable for rapid mycelial growth. PCDB, PCSB, PCB (potato and carrot broth) and carrot dextrose broth (CDB) were favourable for conidial production. All carbon sources tested and peptone favoured for mycelial growth. Carbon and nitrogen sources tested did not significantly stimulate conidial production. The optimum temperature for mycelial growth and conidial production was 25°C. No mycelial growth occurred at 5 or 30°C, but D. mali survived at these temperatures. Active mycelial growth occurred at pH 5–7, and pH 5–8 was favourable for sporulation. Conclusions: PCDB and PCSB incubated at 25°C for 14 day are recommended for mycelial growth and conidial production of D. mali. Significance and Impact of the Study: The information generated in this study will facilitate mycological and pathological research on D. mali and Marssonina leaf blotch of apple caused by D. mali.     

Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes

Xenopus oocyte maturation is analogous to G2/M transition and characterized by germinal vesicle breakdown (GVBD), spindle formation, activation of MPF and Mos-Xp42(Mpk1) pathways. It is accompanied prior to GVBD by a transient increase in intracellular pH. We determined that a well known acidifying compound, NH(4)Cl, delayed progesterone-induced GVBD in a dose-dependent manner. GVBD(50) was delayed up to 2.3-fold by 10 mM NH(4)Cl. Cyclin B2 phosphorylation, Cdk1 Tyr15 dephosphorylation as well as p39(Mos) accumulation, Xp42(Mpk1) and p90(Rsk) phosphorylation induced by progesterone were also delayed by incubation of oocyte in NH(4)Cl. The delay induced by NH(4)Cl was prevented by injection of MOPS buffer pH 7.7. In contrast to acidifying medium, alkalyzing treatment such as Tris buffer pH 9 injections, accelerated GVBD, MPF and Xp42(Mpk1) activation, indicating that pHi changes control early steps of G2/M dynamics. When injected in an immature recipient oocyte, egg cytoplasm triggers GVBD through MPF auto-amplification, independently of protein synthesis. In these conditions, GVBD and Xp42(Mpk1) activation were delayed by high concentration of NH(4)Cl, which never prevented or delayed MPF activation. Strickingly, NH(4)Cl strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or active MAPK-induced GVBD. Taken together, our results suggest that dynamic of early events driving Xp42(Mpk1) and MPF activation induced by progesterone may be negatively or positively regulated by pH(i) changes. However Xp42(Mpk1) pathway was inhibited by acidification alone. Finally, MPF auto-amplification loop was not sensitive to pH(i) changes.     

PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress

Endoplasmic reticulum stress is emerging as an important modulator of different pathologies and as a mechanism contributing to cancer cell death in response to therapeutic agents. In several instances, oxidative stress and the onset of endoplasmic reticulum (ER) stress occur together; yet, the molecular events linking reactive oxygen species (ROS) to ER stress-mediated apoptosis are currently unknown. Here, we show that PERK (RNA-dependent protein kinase (PKR)-like ER kinase), a key ER stress sensor of the unfolded protein response, is uniquely enriched at the mitochondria-associated ER membranes (MAMs). PERK^−/− cells display disturbed ER morphology and Ca²⁺ signaling as well as significantly weaker ER-mitochondria contact sites. Re-expression of a kinase-dead PERK mutant but not the cytoplasmic deletion mutant of PERK in PERK^−/− cells re-establishes ER-mitochondria juxtapositions and mitochondrial sensitization to ROS-mediated stress. In contrast to the canonical ER stressor thapsigargin, during ROS-mediated ER stress, PERK contributes to apoptosis twofold by sustaining the levels of pro-apoptotic C/EBP homologous protein (CHOP) and by facilitating the propagation of ROS signals between the ER and mitochondria through its tethering function. Hence, this study reveals an unprecedented role of PERK as a MAMs component required to maintain the ER-mitochondria juxtapositions and propel ROS-mediated mitochondrial apoptosis. Furthermore, it suggests that loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress.     

Heparan sulfate mimetics modulate calpain activity during rat Soleus muscle regeneration.

Skeletal muscle regenerates after injury. Tissue remodelling, which takes place during muscle regeneration, is a complex process involving proteolytic enzymes. It is inferred that micro and milli calpains are involved in the protein turnover and structural adaptation associated with muscle myolysis and reconstruction. Using a whole-crush injured skeletal muscle, we previously have shown that in vivo muscle treatment with synthetic heparan sulfate mimetics, called RGTAs (for ReGeneraTing Agents), greatly accelerates and improves muscle regeneration after crushing. This effect was particularly striking in the case of the slow muscle Soleus that otherwise would be atrophied. Therefore, we used this regeneration model to study milli and micro calpain expressions in the regenerating Soleus muscle and to address the question of a possible effect of RGTAs treatment on calpain levels. Micro and milli calpain contents increased by about five times to culminate at days 7 and 14 after crushing respectively, thus during the phases of fibre reconstruction and reinnervation. After 64 days of regeneration, muscles still displayed higher levels of both calpains than an intact uninjured muscle. Milli calpain detected by immunocytochemistry was shown in the cytoplasm whereas micro calpain was in both nuclei and cytoplasm in small myofibres but appeared almost exclusively in nuclei of more mature fibres. Interestingly, the treatment of muscles with RGTA highly reduced the increase of both milli and micro calpain contents in Soleus regenerating muscles. These results suggest that the improvement of muscle regeneration induced by RGTA may be partly mediated by minimising the consequences of calpain activity.     

Hypoxia tolerance and air-breathing ability correlate with habitat preference in coral-dwelling fishes

Hypoxia tolerance and air-breathing occur in a range of freshwater, estuarine and intertidal fishes. Here it is shown for the first time that coral reef fishes from the genera Gobiodon, Paragobiodon and Caracanthus, which all have an obligate association with living coral, also exhibit hypoxia tolerance and a well-developed air-breathing capacity. All nine species maintained adequate respiration in water at oxygen concentrations down to 15–25% air saturation. This hypoxia tolerance is probably needed when the oxygen levels in the coral habitat drops sharply at night. Air-breathing abilities of the species correlated with habitat association, being greatest (equaling oxygen uptake in water) in species that occupy corals extending into shallow water, where they may become air exposed during extreme low tides. Air-breathing was less well-developed or absent in species inhabiting corals from deeper waters. Loss of scales and a network of subcutaneous capillaries appear to be key adaptations allowing cutaneous respiration in air. While hypoxia tolerance may be an ancestral trait in these fishes, air-breathing is likely to be a more recent adaptation exemplifying convergent evolution in the unrelated genera Gobiodon and Caracanthus in response to coral-dwelling lifestyles.     

Orally active microtubule-targeting agent,MPT0B271, for the treatment of human non-small cell lung cancer,alone and in combination with erlotinib

Microtubule-binding agents, such as taxanes and vinca alkaloids, are used in the treatment of cancer. The limitations of these treatments, such as resistance to therapy and the need for intravenous administration, have encouraged the development of new agents. MPT0B271 (N-[1-(4-Methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-1-oxy-isonicotinamide), an orally active microtubule-targeting agent, is a completely synthetic compound that possesses potent anticancer effects in vitro and in vivo. Tubulin polymerization assay and immunofluorescence experiment showed that MPT0B271 caused depolymerization of tubulin at both molecular and cellular levels. MPT0B271 reduced cell growth and viability at nanomolar concentrations in numerous cancer cell lines, including a multidrug-resistant cancer cell line NCI/ADR-RES. Further studies indicated that MPT0B271 is not a substrate of P-glycoprotein (P-gp), as determined by flow cytometric analysis of rhodamine-123 (Rh-123) dye efflux and the calcein acetoxymethyl ester (calcein AM) assay. MPT0B271 also caused G2/M cell-cycle arrest, accompanied by the up-regulation of cyclin B1, p-Thr161 Cdc2/p34, serine/threonine kinases polo-like kinase 1, aurora kinase A and B and the downregulation of Cdc25C and p-Tyr15 Cdc2/p34 protein levels. The appearance of MPM2 and the nuclear translocation of cyclin B1 denoted M phase arrest in MPT0B271-treated cells. Moreover, MPT0B271 induced cell apoptosis in a concentration-dependent manner; it also reduced the expression of Bcl-2, Bcl-xL, and Mcl-1 and increased the cleavage of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP). Finally, this study demonstrated that MPT0B271 in combination with erlotinib significantly inhibits the growth of the human non-small cell lung cancer A549 cells as compared with erlotinib treatment alone, both in vitro and in vivo. These findings identify MPT0B271 as a promising new tubulin-binding compound for the treatment of various cancers.