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We have developed an integrated, multidisciplinary methodology, termed systems pathology, to generate highly accurate predictive tools for complex diseases, using prostate cancer for the prototype. To predict the recurrence of [...]

Author(s): María S. Soengas [1]; Paola Capodieci [2]; David Polsky [2]; Jaume Mora [2]; Manel Esteller [3]; Ximena Opitz-Araya [1]; Richard McCombie [1]; James G. Herman [3]; William L. Gerald [...]

Wnt/β-catenin signaling plays pivotal roles in cell proliferation and tissue homeostasis by maintaining somatic stem cell functions. The mammalian target of rapamycin (mTOR) signaling functions as an integrative rheostat that orchestrates various cellular and metabolic activities that shape tissue homeostasis. Whether these two fundamental signaling pathways couple to exert physiological functions still remains mysterious. Using a genome-wide CRISPRCas9 screening, we discover that mTOR complex 1 (mTORC1) signaling suppresses canonical Wnt/β-catenin signaling. Deficiency in tuberous sclerosis complex 1/2 (TSC1/2), core negative regulators of mTORC1 activity, represses Wnt/β-catenin target gene expression, which can be rescued by RAD001. Mechanistically, mTORC1 signaling regulates the cell surface level of Wnt receptor Frizzled (FZD) in a Dishevelled (DVL)-dependent manner by influencing the association of DVL and clathrin AP-2 adaptor. Sustained mTORC1 activation impairs Wnt/β-catenin signaling and causes loss of stemness in intestinal organoids ex vivo and primitive intestinal progenitors in vivo. Wnt/β-catenin-dependent liver metabolic zonation gene expression program is also down-regulated by mTORC1 activation. Our study provides a paradigm that mTORC1 signaling cell autonomously regulates Wnt/β-catenin pathway to influence stem cell maintenance. [ABSTRACT FROM AUTHOR]

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that two of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, two of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

c-Abl, a conserved nonreceptor tyrosine kinase, integrates genotoxic stress responses, acting as a transducer of both pro- and antiapoptotic effector pathways. Nuclear c-Abl seems to interact with the p53 homolog p73 to elicit apoptosis. Although several observations suggest that cytoplasmic localization of c-Abl is required for antiapoptotic function, the signals that mediate its antiapoptotic effect are largely unknown. Here we show that worms carrying an abl-1 deletion allele, abl-1(ok171), are specifically hypersensitive to radiation-induced apoptosis in the Caenorhabditis elegans germ line. Our findings delineate an apoptotic pathway antagonized by ABL-1, which requires sequentially the cell cycle checkpoint genes clk-2, hus-1 and mrt-2; the C. elegans p53 homolog, cep-1; and the genes encoding the components of the conserved apoptotic machinery, ced-3, ced-9 and egl-1. ABL-1 does not antagonize germline apoptosis induced by the DNA-alkylating agent ethylnitrosourea. Furthermore, worms treated with the c-Abl inhibitor STI-571 (Gleevec; used in human cancer therapy), two newly synthesized STI-571 variants or PD166326 had a phenotype similar to that generated by abl-1(ok171). These studies indicate that ABL-1 distinguishes proapoptotic signals triggered by two different DNA-damaging agents and suggest that C. elegans might provide tissue models for development of anticancer drugs. [ABSTRACT FROM AUTHOR]

The hormone αKlotho regulates lifespan in mice, as knockouts die early of what appears to be accelerated aging due to hyperphosphatemia and soft tissue calcification. In contrast, the overexpression of αKlotho increases lifespan. Given the severe mouse phenotype, we generated zebrafish mutants for αklotho as well as its binding partner fibroblast growth factor-23 (fgf23). Both mutations cause shortened lifespan in zebrafish, with abrupt onset of behavioral and degenerative physical changes at around 5 months of age. There is a calcification of vessels throughout the body, most dramatically in the outflow tract of the heart, the bulbus arteriosus (BA). This calcification is associated with an ectopic activation of osteoclast differentiation pathways. These findings suggest that the gradual loss of αKlotho found in normal aging might give rise to ectopic calcification. • Zebrafish αklotho mutants display reduced lifespans • The αklotho phenotype occurs later in zebrafish than in mice • Zebrafish αklotho mutants display adult-onset vascular calcification • Calcification coincides with an increase in osteoclast differentiation pathways αKlotho regulates mineral homeostasis and affects lifespans in mammals. Singh et al. show that a loss of αklotho in zebrafish results in reduced lifespans and vascular calcification in the outflow tract of the heart. Vascular calcification is associated with an upregulation of bone remodeling pathways and osteoclast differentiation. [ABSTRACT FROM AUTHOR]

Purpose: To characterize the expression profile of p73 in human normal tissues by immunohistochemistry (IHC) and to analyze the correlation between p73 expression and bladder cancer progression., Experimental Design: CJDp73 was characterized for p73alpha detection in Western blot and IHC through its application to isoform-transfected 293 cells. Normal tissues were analyzed by IHC with the CJDp73 antiserum. Transitional cell carcinoma (TCC)-derived cell lines were subjected to reverse transcription-PCR and Western blot. TCC tissue microarrays were analyzed for p73alpha expression by IHC, and the results were statistically analyzed., Results: p73 immunostaining was nuclear and restricted to epithelial cells of certain organs such as squamous epithelium of the epidermis and transitional epithelium of the bladder. The expression was also observed in certain specialized glandular epithelia such as acinar cells of breast and parotid gland. Four of seven TCC-derived cell lines had low to undetectable p73alpha protein levels. We found undetectable or low p73alpha expression in 104 of 154 (68%) TCC cases, this phenotype being more frequently observed in invasive tumors when compared with superficial lesions. This association was statistically significant (P < 0.0001). We also observed a significant association between p53, p63, and p73alpha alterations with bladder cancer progression (P < 0.0001)., Conclusions: p73alpha plays a tumor suppressor role in bladder cancer, and its inactivation occurs through an epigenetic mechanism, most probably involving protein degradation.