mTORC1 signaling suppresses Wnt/ß-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level.

Wnt/ß-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 CRISPR-Cas9 screening, we discover that mTOR complex 1 (mTORC1) signaling suppresses canonical Wnt/ß-catenin signaling. Deficiency in tuberous sclerosis complex 1/2 (TSC1/2), core negative regulators of mTORC1 activity, represses Wnt/ß-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/ß-catenin signaling and causes loss of stemness in intestinal organoids ex vivo and primitive intestinal progenitors in vivo. Wnt/ß-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/ß-catenin pathway to influence stem cell maintenance.

NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Caenorhabditis elegans ABL-1 antagonizes p53-mediated germline apoptosis after ionizing irradiation.

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.

Improved prediction of prostate cancer recurrence through systems pathology.

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 prostate cancer following radical prostatectomy, defined by rising serum prostate-specific antigen (PSA), we used machine learning to develop a model based on clinicopathologic variables, histologic tumor characteristics, and cell type-specific quantification of biomarkers. The initial study was based on a cohort of 323 patients and identified that high levels of the androgen receptor, as detected by immunohistochemistry, were associated with a reduced time to PSA recurrence. The model predicted recurrence with high accuracy, as indicated by a concordance index in the validation set of 0.82, sensitivity of 96%, and specificity of 72%. We extended this approach, employing quantitative multiplex immunofluorescence, on an expanded cohort of 682 patients. The model again predicted PSA recurrence with high accuracy, concordance index being 0.77, sensitivity of 77% and specificity of 72%. The androgen receptor was selected, along with 5 clinicopathologic features (seminal vesicle invasion, biopsy Gleason score, extracapsular extension, preoperative PSA, and dominant prostatectomy Gleason grade) as well as 2 histologic features (texture of epithelial nuclei and cytoplasm in tumor only regions). This robust platform has broad applications in patient diagnosis, treatment management, and prognostication.

Androgen receptor expression is associated with prostate cancer-specific survival in castrate patients with metastatic disease.

OBJECTIVE: To investigate whether baseline (before treatment) clinical variables and tumour specimen characteristics (including the androgen receptor, AR) from patients with castrate-resistant metastatic prostate cancer can be used to predict the time to prostate cancer-specific mortality and overall survival, as AR levels in prostate cancer have been associated with disease progression, including prostate-specific antigen (PSA) recurrence and systemic metastasis. PATIENTS AND METHODS: Haematoxylin and eosin (H&E) slides/blocks and outcome data from a 104 castrate patients with metastatic disease (43 prostatectomy and 61 prostate needle biopsy samples), were independently reviewed; H&E morphometry and quantitative immunofluorescence were used to assess the samples. Sections were analysed with a multiplex quantitative immunofluorescence (IF) assay for cytokeratin-18 (epithelial cells), 4',6-diamidino-2-phenylindole (nuclei), p63/high molecular weight keratin (basal cells), AR and alpha-methyl CoA-racemase. Images were acquired with spectral imaging software and processed for quantification with IF algorithms. RESULTS: The median follow-up was 12 years from diagnosis; 49 men (47%) baseline PSA levels of > or = 20 ng/mL, 55 (53%) had a Gleason sum of 8, 63 (60%) died from the disease and 40% were alive (censored). In all, 66 patients had evaluable IF features, and the association with outcome was evaluated by univariate Cox modelling and support-vector regression. PSA was the only clinical variable associated with outcome (concordance index, CoI, 0.41; P < 0.05, log-rank test). The amount of AR present within tumour nuclei (regardless of tissue provenance and primary treatment) significantly correlated with a greater risk of a shorter time to prostate cancer-specific mortality (CoI 0.36; P < 0.05 log-rank test). There were no H&E features that correlated with mortality. CONCLUSION: By univariate analysis, increased nuclear AR expression in either the diagnostic biopsy and/or radical prostatectomy specimen, from patients with advanced disease, was associated with a reduced time to prostate cancer-specific mortality.

αKlotho Regulates Age-Associated Vascular Calcification and Lifespan in Zebrafish.

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.

The RSPO-LGR4/5-ZNRF3/RNF43 module controls liver zonation and size.

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/ß-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/ß-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/ß-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/ß-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/ß-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.

Expression of p63 in diffuse large B-cell lymphoma.

The p63 gene, a homolog of the tumor suppressor gene TP53, maps to chromosome 3q27-28, a region frequently displaying genomic amplification in squamous cell carcinomas. p63 is expressed in a variety of epithelial tissues and has been reported to be critical for the normal development of stratified epithelia, including skin epidermis. In a previous study, the authors reported the expression of p63 in occasional cells in the germinal center of lymph nodes and also observed p63 expression in B-cell lymphomas, among other tumor types surveyed in that analysis. The present study was conducted to further analyze the potential clinical significance of identifying p63 expression, assessing a larger cohort of well-characterized patients with diffuse large B-cell lymphoma (DLBCL) (n = 172 cases) and a panel of established lymphoma cell lines. p63 expression at the microanatomic detail was examined by immunohistochemistry using a monoclonal antibody (clone 4A4), while distinction of p63 isoforms was analyzed by Western blotting and reverse transcription-polymerase chain reaction using isoform-specific primers. The authors found that a subset of DLBCL (32% of cases) expressed p63 in the nuclei of neoplastic lymphocytes. Examination of the different p63 isoforms revealed that the DeltaNp63 species was expressed by only one cell line, while the other p63 isoforms were found in most cell lines analyzed. The authors also observed that p63 expression correlated with high proliferative index, as assessed by Ki-67 immunostaining. Even though in univariate analysis p63 expression did not correlate with overall survival, the association of p63 with increased proliferative index suggests its involvement in DLBCL tumor progression.

p73 Expression in human normal and tumor tissues: loss of p73alpha expression is associated with tumor progression in bladder cancer.

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.

Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung.

The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells. To identify mechanisms regulating basal cell fate, we developed a screenable 3D culture system of airway epithelial morphogenesis. We performed a high-throughput screen using a collection of secreted proteins and identified inflammatory cytokines that specifically biased basal cell differentiation toward a goblet cell fate, culminating in enhanced mucus production. We also demonstrate a specific requirement for Notch2 in cytokine-induced goblet cell metaplasia in vitro and in vivo. We conclude that inhibition of Notch2 prevents goblet cell metaplasia induced by a broad range of stimuli and propose Notch2 neutralization as a therapeutic strategy for preventing goblet cell metaplasia in airway diseases.

Expression of SSX genes in the neoplastic cells of Hodgkin's lymphoma.

The cancer/testis antigen (CTA) group of tumor-associated proteins have been reported to be expressed in various cancers and in adult testis but they are essentially not found in any other normal adult nonneoplastic tissues. Prompted by the frequent detection of SSX1 in a previous comprehensive expression profile of the Hodgkin's lymphoma (HL) cell line L428, we analyzed SSX expression by nonnested reverse-transcription polymerase chain reaction (RT-PCR) in 4 HL cell lines (L428, L540, HD-MY-Z, and KM-H2) and 32 tumor samples of HL. The cellular localization of SSX expression in the tumor samples was further analyzed by in situ hybridization (ISH). All 4 HL cell lines were positive by RT-PCR using SSX consensus primers. Using primers specific to individual SSX genes, all 4 cell lines expressed multiple SSX family members. Five tumor samples (15.6%) were positive by RT-PCR using SSX consensus primers and direct sequencing of the RT-PCR products showed that 4 of 5 expressed more than 1 SSX family member. ISH confirmed that SSX expression originated in HL cells in all 5 RT-PCR-positive tumor samples. Furthermore, ISH demonstrated SSX-positive HL cells in 6 of 11 cases (55%) that were negative by RT-PCR. Our results suggest that members of the SSX family of CTA are expressed in most HL. This subset of HL may be a candidate for immunotherapy approaches directed at SSX proteins.

A hypomorphic lsd1 allele results in heart development defects in mice.

Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. Lsd1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that Lsd1-interacting proteins regulate the activity and specificity of Lsd1, the significance and roles of such interactions in developmental processes remain largely unknown. Here we describe a hypomorphic Lsd1 allele that contains two point mutations in the tower domain, resulting in a protein with reduced interaction with known binding partners and decreased enzymatic activity. Mice homozygous for this allele die perinatally due to heart defects, with the majority of animals suffering from ventricular septal defects. Molecular analyses revealed hyperphosphorylation of E-cadherin in the hearts of mutant animals. These results identify a previously unknown role for Lsd1 in heart development, perhaps partly through the control of E-cadherin phosphorylation.

The spatial order of transcription in mammalian cells.

We have previously developed technology for multiplexing probes for the detection of transcription of many genes simultaneously within single cells. This has allowed us to determine the spatial localization of multiple genes with respect to each other in the nucleus, and ultimately the expression profile of the cell with respect to surrounding cells in a tissue. Six parameters of transcriptional organization in individual cells from culture and tissue were used to characterize significant differences in intracellular and intercellular expression patterns while preserving cellular morphology and histological context. We found that, unlike yeast, mammalian expression is excluded from the periphery and in addition, a subtle but complex organization underlies the transcriptional activity of these cells, both intra- and intercellularly. The approach has sufficient spatial resolution to be applied to the detection of chromosomal translocations or the identification of cancer cells.

Gene expression profiling in single cells within tissue.

We developed a robust multiplex fluorescent in situ hybridization (FISH) technique in archival formalin-fixed, paraffin-embedded (FFPE) human tissue sections while preserving the microanatomical context. This identifies single-cell gene expression patterns by probing multiple, unique nascent RNA transcripts and yields predictive quantitative gene expression signatures.

Tumor suppressor role of KiSS-1 in bladder cancer: loss of KiSS-1 expression is associated with bladder cancer progression and clinical outcome.

The expression profiles of nine bladder cancer cell lines were compared against a pool containing equal total RNA quantities of each of them. Lower expression of KiSS-1 was revealed in cells derived from the most advanced bladder tumors. When comparing 15 primary bladder tumors versus a pool of four bladder cancer cell lines, lower transcript levels of KiSS-1 were observed in the invasive bladder carcinomas as compared to superficial tumors. KiSS-1 expression ratios provided prognostic information. The expression pattern of KiSS-1 transcripts was analyzed using in situ hybridization in nine bladder cancer cells, paired normal urothelium and bladder tumor samples (n = 25), and tissue microarrays of bladder tumors (n = 173). We observed complete loss of KiSS-1 in all invasive tumors under study as compared to their respective normal urothelium. The expression of KiSS-1 was found to be significantly associated with histopathological stage. Patients with lower KiSS-1 expression showed a direct correlation with overall survival in a subset of bladder tumors whose follow-up was available (n = 69). We did not observe any significant differential KiSS-1 expression along cell cycle by sorting analysis. A potential tumor suppressor role in bladder cancer was revealed for KiSS-1. Moreover, it showed predictive value by identifying patients with poor outcome.

Loss of the tumor suppressor PML in human cancers of multiple histologic origins.

BACKGROUND: The PML gene is fused to the RARalpha gene in the vast majority of acute promyelocytic leukemias (APL) and has been implicated in the control of key tumor-suppressive pathways. However, its role in the pathogenesis of human cancers other than APL is still unclear. We therefore assessed the status and expression of the PML gene in solid tumors of multiple histologic origins. METHODS: We created tumor tissue microarrays (TTMs) with samples from patients with colon adenocarcinoma (n = 109), lung carcinoma (n = 19), prostate adenocarcinoma (n = 36), breast carcinoma (n = 38), central nervous system (CNS) tumors (n = 51), germ cell tumors (n = 60), thyroid carcinoma (n = 32), adrenal cortical carcinoma (n = 12), and non-Hodgkin's lymphoma (n = 251) and from normal tissue corresponding to each histotype and analyzed PML protein and mRNA expression by immunohistochemistry and in situ hybridization, respectively. Tumor cell lines (n = 64) of various histologic origins were analyzed for PML protein and mRNA expression by immunofluorescence and northern blotting, respectively. DNA from microdissected tumor samples and cell lines was analyzed for PML mutations and loss of heterozygosity (LOH). For some tumor types, the association between PML expression and tumor stage and grade was analyzed. Statistical tests were two-sided. RESULTS: All normal tissues expressed PML protein. PML protein expression was reduced or abolished in prostate adenocarcinomas (63% [95% confidence interval [CI] = 48% to 78%] and 28% [95% CI = 13% to 43%], respectively), colon adenocarcinomas (31% [95% CI = 22% to 40%] and 17% [95% CI = 10% to 24%]), breast carcinomas (21% [95% CI = 8% to 34%] and 31% [95% CI = 16% to 46%]), lung carcinomas (36% [95% CI = 15% to 57%] and 21% [95% = 3% to 39%]), lymphomas (14% [95% CI = 10% to 18%] and 69% [95% CI = 63% to 75%]), CNS tumors (24% [95% CI = 13% to 35%] and 49% [95% CI = 36% to 62%]), and germ cell tumors (36% [95% CI = 24% to 48%] and 48% [95% CI = 36% to 60%]) but not in thyroid or adrenal carcinomas. Loss of PML protein expression was associated with tumor progression in prostate cancer (the progression from prostatic intraepithelial neoplasia to invasive carcinoma was associated with complete PML loss; P<.001), breast cancer (complete PML loss was associated with lymph node metastasis; P =.01), and CNS tumors (complete PML loss was associated with high-grade tumors; P =.003). PML mRNA was expressed in all tumor and cell line samples. The PML gene was rarely mutated and was not subject to LOH. CONCLUSIONS: PML protein expression is frequently lost in human cancers of various histologic origins, and its loss associates with tumor grade and progression in some tumor histotypes.

Mutations of the PML tumor suppressor gene in acute promyelocytic leukemia.

The promyelocytic leukemia (PML) tumor suppressor of acute promyelocytic leukemia (APL) is essential for a number of proapoptotic and growth-suppressive pathways as well as for the activity of differentiating agents such as retinoic acid (RA). In human APL, the dose of PML is reduced to heterozygosity given that one allele is involved in the chromosomal translocation while the status of the remaining PML allele is unknown. We have therefore used single-strand conformational polymorphism (SSCP) and sequencing analysis to screen DNA from APL patients for mutations at the PML locus. We identified DNA sequence variations resulting in a truncated PML protein in APL cases that displayed RA resistance and a very poor prognosis. Mutation analysis also led to the identification of aberrant PML sequence variations in other hematopoietic malignancies. Complete functional loss of PML is therefore selected by the APL phenotype and associates with poor prognosis and RA unresponsiveness.

Characterization and expression of mammalian cyclin b3, a prepachytene meiotic cyclin.

We report the identification and expression pattern of a full-length human cDNA and a partial mouse cDNA encoding cyclin B3. Cyclin B3 (CCNB3) is conserved from Caenorhabditis elegans to Homo sapiens and has an undefined meiotic function in female, but not male Drosophila melanogaster. We show that H. sapiens cyclin B3 interacts with cdk2, is localized to the nucleus, and is degraded during anaphase entry after the degradation of cyclin B1. Degradation is dependent on sequences conserved in a destruction box motif. Overexpression of nondegradable cyclin B3 blocks the mitotic cell cycle in late anaphase, and at higher doses it can interfere with progression through G(1) and entry into S phase. H. sapiens cyclin B3 mRNA and protein are detected readily in developing germ cells in the human testis and not in any other tissue. The mouse cDNA has allowed us to further localize cyclin B3 mRNA to leptotene and zygotene spermatocytes. The expression pattern of mammalian cyclin B3 suggests that it may be important for events occurring in early meiotic prophase I.