Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2-KO counterparts. The Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation, in comparison with WT and Sos2-KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Cellular proliferation and migration were impaired in Sos1-KO and Sos1/2-DKO MEFs in comparison with WT and Sos2-KO MEFs, whereas cell adhesion was only impaired upon depletion of both Sos isoforms. RasGTP formation was practically absent in Sos1/2-DKO MEFs as compared with the other genotypes and extracellular signal-regulated kinase phosphorylation showed only significant reduction after combined Sos1/2 depletion. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1-KO and the Sos1/2-DKO cells as compared with Sos2-KO and WT MEFs. Interestingly, treatment of the MEF cultures with antioxidants corrected the altered phenotypes of Sos1-KO and Sos1/2-DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2-KO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability.

Colchicine site ligands with indole B rings are potent tubulin polymerization inhibitors. Structural modifications at the indole 3-position of 1-methyl-5-indolyl-based isocombretastatins (1,1-diarylethenes) and phenstatins endowed them with anchors for further derivatization and resulted in highly potent compounds. The substituted derivatives displayed potent cytotoxicity against several human cancer cell lines due to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy, and tubulin polymerization inhibitory activity studies and promoted cell killing mediated by caspase-3 activation. Binding at the colchicine site was confirmed by means of fluorescence measurements of MTC displacement. Molecular modeling suggests that the tropolone-binding region of the colchicine site of tubulin can adapt to hosting small polar substituents. Isocombretastatins accepted substitutions better than phenstatins, and the highest potencies were achieved for the cyano and hydroxyiminomethyl substituents, with TPI values in the submicromolar range and cytotoxicities in the subnanomolar range. A 3,4,5-trimethoxyphenyl ring usually afforded more potent derivatives than a 2,3,4-trimethoxyphenyl ring.

Using Sos1-KO, Sos2-KO and Sos1/2-DKO mice, we assessed the functional role of Sos1 and Sos2 in skin homeostasis under physiological and/or pathological conditions. Sos1 depletion resulted in significant alterations of skin homeostasis including reduced keratinocyte proliferation, altered hair follicle and blood vessel integrity in dermis, and reduced adipose tissue in hypodermis. These defects worsened significantly when both Sos1 and Sos2 were absent. Simultaneous Sos1/2 disruption led to severe impairment of the ability to repair skin wounds as well as to almost complete ablation of the neutrophil-mediated inflammatory response in the injury site. Furthermore, Sos1 disruption delayed the onset of tumor initiation, decreased tumor growth and prevented malignant progression of papillomas in a DMBA/TPA-induced skin carcinogenesis model. Finally, Sos1 depletion in preexisting chemically-induced papillomas resulted also in decreased tumor growth, probably linked to significantly reduced underlying keratinocyte proliferation.Our data unveil novel, distinctive mechanistic roles of Sos 1 and Sos2 in physiological control of skin homeostasis and wound repair as well as in pathological development of chemically induced skin tumors. These observations underscore the essential role of Sos proteins in cellular proliferation and migration and support the consideration of these RasGEFs as potential biomarkers/therapy targets in Ras-driven epidermal tumors.