Disruption of the FA/BRCA pathway in bladder cancer

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.     

Technologically advanced cancer modeling in mice

Multiple approaches now exist for the generation of genetically engineered murine cancer models. These new models utilize latent, conditional and inducible alleles to better mimic the in vivo setting in which sporadic human cancers occur. The murine tumor models are beginning to reveal mysteries of tumorigenesis, such as the role of the tumor microenvironment and the dependence of tumors on continuous oncogenic stimulation.     

Disruption of the Man-6-P targeting pathway in mice impairs osteoclast secretory lysosome biogenesis

Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo-lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain two types of lysosomes. The major species is a secretory lysosome containing cathepsin K and tartrate-resistant acid phosphatase (TRAP), two hydrolases critical for bone resorption. These secretory lysosomes are shown to fuse with the plasma membrane, allowing the regulated release of acid hydrolases at the site of bone resorption. The other type of lysosome contains cathepsin D, but little cathepsin K or TRAP. Osteoclasts from Gnptab(-/-) (gene encoding GlcNAc-1-phosphotransferase α, β-subunits) mice, which lack a functional mannose 6-phosphate (Man-6-P) targeting pathway, show increased secretion of cathepsin K and TRAP and impaired secretory lysosome formation. However, cathepsin D targeting was intact, showing that osteoclasts have a Man-6-P-independent pathway for selected acid hydrolases.     

Alterations in components of the TGF-beta superfamily signaling pathways in human cancer

Signaling by transforming growth factor-beta (TGF-beta) superfamily ligands to the nucleus is mediated by type I and type II receptors and the intracellular signal transducers, the Smads. Alteration of some of the components of these pathways has been observed in human tumors. These alterations can be deletions or mutations, or downregulation of components that act positively in the pathway, or alternatively, amplification or overexpression of inhibitors of the pathways. The selection of these alterations during tumor progression and their correlation with clinical outcomes, such as survival, risk of recurrence after tumor resection or tendency for metastatic spread, suggest that many are involved in tumor progression. Here, we review the genetic alterations and epigenetic modifications that occur in different components of the TGF-beta superfamily signaling pathways in human tumors and we discuss their correlation with clinical outcome. The evidence suggests that not all alterations of the TGF-beta superfamily signaling pathway components in human cancer have an equivalent effect on tumor progression and we discuss what implications this has for our understanding of the role of TGF-beta signaling in human cancer.     

TGF-beta and cancer

The relationships between transforming growth factor-beta (TGF-beta) and cancer are varied and complex. The paradigm that is emerging from the experimental evidence accumulated over the past decade or so is that TGF-beta can play two different and opposite roles with respect to the process of malignant progression. During early stages of carcinogenesis, TGF-beta acts predominantly as a potent tumor suppressor and may mediate the actions of chemopreventive agents such as retinoids and nonsteroidal anti-estrogens. However, at some point during the development and progression of malignant neoplasms, bioactive TGF-betas make their appearance in the tumor microenvironment and the tumor cells escape from TGF-beta-dependent growth arrest. In many cases, this resistance to TGF-beta is the consequence of loss or mutational inactivation of the genes that encode signaling intermediates. These include the types I and II TGF-beta receptors, as well as receptor-associated and common-mediator Smads. The stage of tumor development or progression at which TGF-beta-resistant clones come to dominate the tumor cell population in different types of neoplasm remains to be defined. The phenotypic switch from TGF-beta-sensitivity to TGF-beta-resistance that occurs during carcinogenesis has several important implications for cancer prevention and treatment.     

TGF-beta and cancer

TGF-beta signaling regulates tumorigenesis and in human cancer its signaling pathways are often modified during tumor progression. Prior to initiation and early during progression TGF-beta acts upon the epithelium as a tumor suppressor, however at later stages it is often a tumor promoter. Over the years, many studies have focused on the epithelial cell autonomous role for TGF-beta, however, TGF-beta is not strictly limited to this compartment in vivo. Recent studies addressing TGF-beta mediated stromal-epithelial interactions have significantly improved our understanding related to the regulation of cancer. In addition, stromal fibroblast cell autonomous effects have been observed in response to TGF-beta stimulation. According to the current literature and experimental evidence, TGF-beta is a potent ligand that regulates carcinoma initiation, progression and metastasis through a broad and complex spectrum of interdependent interactions.     

Disruption of the hedgehog signaling pathway contributes to the hair follicle cycling deficiency in Vdr knockout mice

Mice null for the Vitamin D receptor (VdrKO) have a disrupted first hair follicle cycle and aborted subsequent hair follicle cycling. We examined the expression of different markers and mediators of hair follicle cycling in the hair follicle of the VdrKO mouse during days 13–22 when the hair follicle normally initiates and completes the first catagen. We compared the expression of those genes in mice with a nonsense mutation in hairless (Rhino), which have a similar alopecia phenotype, and to Cyp27b1 null mice which are deficient in the production of 1,25(OH)2D3, the Vdr ligand, but display normal hair follicle cycling. Our results demonstrate the down regulation of hair follicle markers and the alteration of expression of hedgehog (Hh), Wnt, Fgf, and Tgfβ pathways in VdrKO and Rhino mice, but not in Cyp27b1KO mice. Treatment of VdrKO mice with an agonist to the Hh pathway partially restored hair follicle cycling, suggesting a role of this pathway in the regulation of hair follicle cycling by VDR. These results suggest that Vdr regulates directly or indirectly the expression of genes required for hair follicle cycling, including Hh signaling, independent of 1,25(OH)2D3. J. Cell. Physiol. 225: 482–489, 2010. © 2010 Wiley‐Liss, Inc.     

Evidence for c-myc in the signaling pathway for TGF-beta in well-differentiated human colon carcinoma cells

Previously, we described a model culture system for comparing responsiveness of poorly differentiated and well-differentiated human colon carcinoma cells to exogenous growth factors. While polypeptide growth stimulators elicited an up-regulation of c-myc, as well as a mitogenic response in the well-differentiated cells, the poorly differentiated cells were insensitive to exogenous growth stimulators. We now show, by thymidine incorporation experiments and autoradiographic analysis, that transforming growth factor beta 1 (TGF-beta) abrogated the mitogenic responses to the growth factors epidermal growth factor + insulin + transferrin (IC50 = 0.8 ng/ml), as well as to nutrients (basal medium; IC50 = 0.2 ng/ml) in the well-differentiated cells. The poorly differentiated cells did not respond to TGF-beta. Moreover, TGF-beta (10 ng/ml) completely abrogated the growth factor-stimulated up-regulation of c-myc in the TGF-beta responsive, well-differentiated colon carcinoma cells. Addition of TGF-beta to the TGF-beta-responsive, well-differentiated cells, at a time after c-myc had been transiently up-regulated in response to growth stimulatory factors, resulted in a loss of responsiveness to TGF-beta. Addition of TGF-beta to these cells at increasing time periods after EIT stimulation also resulted in a loss of the TGF-beta-induced repression of c-myc. The results suggest an important role for c-myc in the mechanism of action of TGF-beta in well-differentiated human colon carcinoma cells.     

Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer

Transforming growth factor-beta (TGF-beta) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-beta inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-beta receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-beta receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-beta whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-beta induces epithelial-mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-beta acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-beta regulates angiogenesis. Finally, TGF-beta suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-beta pathway. In conclusion, TGF-beta signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.     

Disruption of the ugt1 locus in mice resembles human Crigler-Najjar type I disease

The 9 UDP-glucuronosyltranferases (UGTs) encoded by the UGT1 locus in humans are key enzymes in the metabolism of most drugs as well as endogenous substances such as bile acids, fatty acids, steroids, hormones, neurotransmitters, and bilirubin. Severe unconjugated hyperbilirubinemia in humans that suffer from Crigler-Najjar type I disease results from lesions in the UGT1A1 gene and is often fatal. To examine the physiological importance of the Ugt1 locus in mice, this locus was rendered non-functional by interrupting exon 4 to create Ugt1(-/-) mice. Because UGT1A1 in humans is responsible for 100% of the conjugated bilirubin, it followed that newborn Ugt1(-/-) mice developed serum levels of unconjugated bilirubin that were 40-60 times higher than Ugt1(+/-) or wild-type mice. The result of extreme unconjugated bilirubin in Ugt1(-/-) mice, comparable to the induced levels noted in patients with Crigler-Najjar type 1 disease, is fatal in neonatal Ugt1(-/-) mice within 2 weeks following birth. The extreme jaundice is present as a phenotype in skin color after 8 h. Neonatal Ugt1(-/-) mice exhibit no detectable UGT1A-specific RNA, which corresponds to a complete absence of UGT1A proteins in liver microsomes. Conserved glucuronidation activity attributed to the Ugt1 locus can be defined in Ugt1(-/-) mice, because UGT2-dependent glucuronidation activity is unaffected. Remarkably, the loss of UGT1A functionality in liver results in significant alterations in cellular metabolism as investigated through changes in gene expression. Thus, the loss of UGT1A function in Ugt1(-/-) mice leads to a metabolic syndrome that can serve as a model to further investigate the toxicities associated with unconjugated bilirubin and the impact of this disease in humans.     

Hypoxia induces the activation of human hepatic stellate cells LX-2 through TGF-beta signaling pathway

Hypoxia is a common environmental stress factor and is also associated with various physiological and pathological conditions such as fibrogenesis. The activation of hepatic stellate cells (HSCs) is the key event in the liver fibrogenesis. In this study, the behavior of human HSCs LX-2 in low oxygen tension (1% O2) was analyzed. Upon hypoxia, the expression of HIF-1alpha and VEGF gene was induced. The result of Western blotting showed that the expression of alpha-SMA was increased by hypoxic stimulation. Furthermore, the expression of MMP-2 and TIMP-1 genes was increased. Hypoxia also elevated the protein expression of the collagen type I in LX-2 cells. The analysis of TGF-beta/Smad signaling pathway showed that hypoxia potentiated the expression of TGF-beta1 and the phosphorylation status of Smad2. Gene expression profiles of LX-2 cells induced by hypoxia were obtained by using cDNA microarray technique.     

Disruption of microtubules induces an endogenous suicide pathway in human leukaemia HL-60 cells

Abstract. Terminally differentiated HL-60 cells undergoing programmed cell death (apoptosis) in culture were found to have a disrupted microtubular network. Treatment of undifferentiated HL-60 cells with microtubule-disrupting agents alone was found to induce apoptosis en masse in these cells. In contrast, disruption of microfilaments did not induce apoptosis; instead these cells underwent necrosis, the pathological mode of cell death. Apoptosis in response to microtubule disruption in HL-60 cells was characterized by cell shape changes, nuclear condensation followed by fragmentation and the separation of the cell into numerous intact fragments, termed apoptotic bodies. Apoptosis of these cells was further confirmed by DNA analysis, which demonstrated the activation of an endogenous endonuclease which cleaved the DNA of these cells into oligonucleosomal fragments. Microtubule disrupting agents were found to exert these effects over a wide range of doses. Apoptosis was also inducible in HL-60 cells, in a dose-dependant manner, by the calcium ionophore A23187. Since microtubules are known to be highly sensitive to intracellular calcium fluctuations, this suggests that calcium influx could act at the microtubule level in efTftctino annntnsis     

Disruption of microtubules induces an endogenous suicide pathway in human leukaemia HL-60 cells

Terminally differentiated HL-60 cells undergoing programmed cell death (apoptosis) in culture were found to have a disrupted microtubular network. Treatment of undifferentiated HL-60 cells with microtubule-disrupting agents alone was found to induce apoptosis en masse in these cells. In contrast, disruption of microfilaments did not induce apoptosis; instead these cells underwent necrosis, the pathological mode of cell death. Apoptosis in response to microtubule disruption in HL-60 cells was characterized by cell shape changes, nuclear condensation followed by fragmentation and the separation of the cell into numerous intact fragments, termed apoptotic bodies. Apoptosis of these cells was further confirmed by DNA analysis, which demonstrated the activation of an endogenous endonuclease which cleaved the DNA of these cells into oligonucleosomal fragments. Microtubule disrupting agents were found to exert these effects over a wide range of doses. Apoptosis was also inducible in HL-60 cells, in a dose-dependent manner, by the calcium ionophore A23187. Since microtubules are known to be highly sensitive to intracellular calcium fluctuations, this suggests that calcium influx could act at the microtubule level in effecting apoptosis.     

Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors

Ovarian tumor cells are often genomically unstable and hypersensitive to cisplatin. To understand the molecular basis for this phenotype, we examined the integrity of the Fanconi anemia-BRCA (FANC-BRCA) pathway in those cells. This pathway regulates cisplatin sensitivity and is governed by the coordinate activity of six genes associated with Fanconi anemia (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG) as well as BRCA1 and BRCA2 (FANCD1). Here we show that the FANC-BRCA pathway is disrupted in a subset of ovarian tumor lines. Mono-ubiquitination of FANCD2, a measure of the function of this pathway, and cisplatin resistance were restored by functional complementation with FANCF, a gene that is upstream in this pathway. FANCF inactivation in ovarian tumors resulted from methylation of its CpG island, and acquired cisplatin resistance correlated with demethylation of FANCF. We propose a model for ovarian tumor progression in which the initial methylation of FANCF is followed by FANCF demethylation and ultimately results in cisplatin resistance.