TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell–like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest–derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.

Breast cancer is accompanied by increased oxidative stress and induction of polymorphic cytochrome P-450 mixed oxidase enzymes (CYP). Both processes affect the abundance of volatile organic compounds (VOCs) in the breath because oxidative stress causes lipid peroxidation of polyunsaturated fatty acids in membranes, producing alkanes and methylalkanes which are catabolized by CYP. We performed a pilot study of breath VOCs, a potential new marker of disease in women with breast cancer. This was a combined case-control and cross-sectional study of women with abnormal mammograms scheduled for a breast biopsy. Breath samples were analyzed by gas chromatography and mass spectroscopy in order to determine the breath methylated alkane contour (BMAC), a three-dimensional display of the alveolar gradients (abundance in breath minus abundance in room air) of C4-C20 alkanes and monomethylated alkanes. BMACs in women with and without breast cancer were compared using forward stepwise discriminant analysis. Two hundred one breath samples were obtained from women with abnormal mammograms and biopsies read by two pathologists. There were 51 cases of breast cancer in 198 concordant biopsies. The breath test distinguished between women with breast cancer and healthy volunteers with a sensitivity of 94.1% (48/51) and a specificity of 73.8% (31/42) (cross-validated sensitivity 88.2% (45/51), specificity 73.8% (31/42)). Compared to women with abnormal mammograms and no cancer on biopsy, the breath test identified breast cancer with a sensitivity of 62.7% (32/51) and a specificity of 84.0% (42/50) (cross-validated sensitivity of 60.8% (31/51), specificity of 82.0% (41/50)). The negative predictive value (NPV) of a screening breath test for breast cancer was superior to a screening mammogram (99.93% versus 99.89%); the positive predictive value (PPV) of a screening mammogram was superior to a screening breath test (4.63% versus 1.29%). A breath test for markers of oxidative stress accurately identified women with breast cancer, with an NPV superior to a screening mammogram. This breath test could potentially be employed as a primary screen for breast cancer. Confirmatory studies in larger groups are required.

We evaluated a breath test for volatile organic compounds (VOCs) as a predictor of breast cancer. Breath VOCs were assayed in 51 asymptomatic women with abnormal mammograms and biopsy-proven breast cancer, and 42 age-matched healthy women. A fuzzy logic model predicted breast cancer with accuracy superior to previously reported findings. Following random assignment to a training set (64) or a prediction set (29), a model was constructed in the training set employing five breath VOCs that predicted breast cancer in the prediction set with 93.8% sensitivity and 84.6% specificity. The same model predicted no breast cancer in 16/50 (32.0%) women with abnormal mammograms and no cancer on biopsy. A two-minute breath test could potentially provide a safe, accurate and painless screening test for breast cancer, but prospective validation studies are required.

KRASis the most frequently mutatedrasfamily member in lung carcinomas 1,2 , whereasHRASmutations are common in tumours from stratified epithelia such as bladder or skin (www.sanger.ac.uk/genetics/CGP/cosmic/). Using a mouse model (HrasKI) 3 in which theHrascoding sequence was inserted into theKraslocus, we demonstrate that specificity forKrasmutations in lung andHras mutations in skin tumours is determined by local regulatory elements in the targetrasgenes. We further show that, whileKras-4Ais dispensable for mouse development 4,5 , it is necessary both for lung carcinogenesisin vivoand for the previously reported 6,7 inhibitory effect of wild-type (WT)Krason the transforming properties of the mutant allele. Kras-4A expression is detected in a sub-population of normal lung epithelial cells, but at very low levels in lung tumours, suggesting a role in tumour initiation rather than in tumour maintenance. The two Kras isoforms undergo different post-translational modifications 8 , therefore these findings can have important implications for the design of therapeutic strategies for inhibiting oncogenic Kras activity in the prevention and treatment of cancer.Kras deficiency in mice leads to embryonic lethality 4,5 , thus it has not been possible to determine whether the selection for Kras mutations in lung tumours reflects a specific oncogenic function that is required for lung carcinogenesis and cannot be compensated for by mutant Hras or Nras. The HrasKI mouse provides a viable model that completely lacks Kras expression 3 (Fig. 1), but carries a "knock-in" Hras cDNA expressed under the control of Kras regulatory elements. We reasoned that HrasKI mice should be resistant to lung tumour development if Kras protein is essential for lung carcinogenesis. To control for the possibility of structural alterations affecting experimental outcomes, we used KrasKI mice in which the coding sequence corresponding to the Kras-4B isoform was knocked back into the Kras locus in the exact manner used to generate the HrasKI mouse 3 . HrasKI mice expressed elevated levels of Hras protein, consistent with the increase in Hras gene dosage from two copies to four in these animals ( Fig. 1). KrasKI mice express similar levels of Kras protein as WT mice, as expected since Kras-4B is the major splice isoform 9,10 . Levels of Akt and Erk activation in these animals were similar to WT mice (Fig. 1). There was also no difference in levels of p38α, recently shown to affect ras mediated lung carcinogenesis 11 . The elevated level of Hras We further pursued the specificity of ras gene mutations in lung and skin tumours by inducing both tumour types in the same HrasKI homozygous mice. A single dose of urethane was injected intraperitoneally to induce lung tumours, followed by a single topical treatment on the skin with urethane and subsequent biweekly application of tetradecanoyl-phorbol acetate (TPA) to induce papilloma development. All lung tumours (n = 28) analyzed have the CAA>CTA mutation in the HrasKI allele. Of the papillomas (n = ...

Reactive oxygen species (ROS) play a central role in aqueous-phase processing and health effects of atmospheric aerosols. Although hydroxyl radical (•OH) and hydrogen peroxide (H2O2) are regarded as major oxidants associated with secondary organic aerosols (SOA), the kinetics and reaction mechanisms of superoxide (O2 •–) formation are rarely quantified and poorly understood. Here, we demonstrate a dominant formation of O2 •– with molar yields of 0.01–0.03% from aqueous reactions of biogenic SOA generated by •OH photooxidation of isoprene, β-pinene, α-terpineol, and d-limonene. The temporal evolution of •OH and O2 •– formation is elucidated by kinetic modeling with a cascade of aqueous reactions including the decomposition of organic hydroperoxides, •OH oxidation of primary or secondary alcohols, and unimolecular decomposition of α-hydroxyperoxyl radicals. Relative yields of various types of ROS reflect a relative abundance of organic hydroperoxides and alcohols contained in SOA. These findings and mechanistic understanding have important implications on the atmospheric fate of SOA and particle-phase reactions of highly oxygenated organic molecules as well as oxidative stress upon respiratory deposition.

Epithelial-mesenchymal transition (EMT) is thought to be an important, possibly essential, component of the process of tumor dissemination and metastasis. About 20%-30% of Hras mutant mouse skin carcinomas induced by chemical initiation/promotion protocols have undergone EMT. Reduced exposure to TPA-induced chronic inflammation causes a dramatic reduction in classical papillomas and squamous cell carcinomas (SCCs), but the mice still develop highly invasive carcinomas with EMT properties, reduced levels of Hras and Egfr signaling, and frequent Ink4/Arf deletions. Deletion of Hras from the mouse germline also leads to a strong reduction in squamous tumor development, but tumors now acquire activating Kras mutations and exhibit more aggressive metastatic properties. We propose that invasive carcinomas can arise by different genetic and biological routes dependent on exposure to chronic inflammation and possibly from different target cell populations within the skin. Our data have implications for the use of inhibitors of inflammation or of Ras/Egfr pathway signaling for prevention or treatment of invasive cancers.

BACKGROUND: Molecular testing of lung adenocarcinomas (ADCs) is crucial for therapy stratification of patients.Because of the often limited diagnostic material, the authors aimed to explore the suitability of cytology smears for nextgeneration sequencing (NGS) and compared the results with concurrent histological specimens or cell blocks. METHODS:A total of 16 formalin-fixed paraffin-embedded (FFPE) ADCs with known genetic alterations were used as the first cohort for targeted DNA and RNA sequencing. In the second cohort of 8 cases, 8 cytological smears were compared with matching histological specimens or cell blocks for the study. For NGS library amplification, commercially available panels for DNA and RNA sequencing were applied. The Ion Torrent Personal Genome Machine and the Ion Reporter workflow (version 5.0) were used for sequencing. RESULTS: All DNA libraries derived from FFPE and non-formalin-fixed cytological smear samples produced acceptable quality metrics, thereby enabling successful targeted DNA sequencing (100% performance). Targeted RNA sequencing failed in 1 FFPE case and 1 cytology probe by not reaching enough mapped fusion reads (92% performance rate). All previously detected mutations and gene rearrangements could be confirmed (sensitivity of 100%), whereas specificity of the DNA-based NGS assay reached 96%. CONCLUSIONS: The results of the current study demonstrated the suitability of non-formalin cytology specimens for the simultaneous NGS testing of lung ADCs using amplicon resequencing panels. These assays allowed for the input of cytological smears equal to concurrent histology or cell blocks and proved to be accurate in the detection of therapeutically actionable somatic mutations and gene rearrangements.