Joshua C. Saldivar

Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.

Fibulin-3 Promotes Glioma Growth and Resistance through a Novel Paracrine Regulation of Notch Signaling

Malignant gliomas are highly invasive and chemoresistant brain tumors with extremely poor prognosis. Targeting of the soluble factors that trigger invasion and resistance, therefore, could have a significant impact against the infiltrative glioma cells that are a major source of recurrence. Fibulin-3 is a matrix protein that is absent in normal brain but upregulated in gliomas and promotes tumor invasion by unknown mechanisms. Here, we show that fibulin-3 is a novel soluble activator of Notch signaling that antagonizes DLL3, an autocrine inhibitor or Notch, and promotes tumor cell survival and invasion in a Notch-dependent manner. Using a strategy for inducible knockdown, we found that controlled downregulation of fibulin-3 reduced Notch signaling and led to increased apoptosis, reduced self-renewal of glioblastoma-initiating cells, and impaired growth and dispersion of intracranial tumors. In addition, fibulin-3 expression correlated with expression levels of Notch-dependent genes and was a marker of Notch activation in patient-derived glioma samples. These findings underscore a major role for the tumor extracellular matrix in regulating glioma invasion and resistance to apoptosis via activation of the key Notch pathway. More importantly, this work describes a noncanonical, soluble activator of Notch in a cancer model and shows how Notch signaling can be reduced by targeting tumor-specific accessible molecules in the tumor microenvironment.

Pathology and biology associated with the fragile FHIT gene and gene product

More than 12 years and >800 scientific publications after the discovery of the first gene at a chromosome fragile site, the FHIT gene at FRA3B, there are still questions to pursue concerning the selective advantage conferred to cells by loss of expression of FHIT, the most frequent target of allele deletion in precancerous lesions and cancers. These questions are considered in light of recent investigations of genetic and epigenetic alterations to the locus and in a retrospective consideration of biological roles of the Fhit protein discovered through functional studies. J. Cell. Biochem. 109: 858–865, 2010.

The essential kinase ATR: ensuring faithful duplication of a challenging genome

One way to preserve a rare book is to lock it away from all potential sources of damage. Of course, an inaccessible book is also of little use, and the paper and ink will continue to degrade with age in any case. Like a book, the information stored in our DNA needs to be read, but it is also subject to continuous assault and therefore needs to be protected. In this Review, we examine how the replication stress response that is controlled by the kinase ataxia telangiectasia and Rad3-related (ATR) senses and resolves threats to DNA integrity so that the DNA remains available to read in all of our cells. We discuss the multiple data that have revealed an elegant yet increasingly complex mechanism of ATR activation. This involves a core set of components that recruit ATR to stressed replication forks, stimulate kinase activity and amplify ATR signalling. We focus on the activities of ATR in the control of cell cycle checkpoints, origin firing and replication fork stability, and on how proper regulation of these processes is crucial to ensure faithful duplication of a challenging genome.

The FHIT gene product: tumor suppressor and genome “caretaker”

Abstract The FHIT gene at FRA3B is one of the earliest and most frequently altered genes in the majority of human cancers. It was recently discovered that the FHIT gene is not the most fragile locus in epithelial cells, the cell of origin for most Fhit-negative cancers, eroding support for past claims that deletions at this locus are simply passenger events that are carried along in expanding cancer clones, due to extreme vulnerability to DNA damage rather than to loss of FHIT function. Indeed, recent reports have reconfirmed FHIT as a tumor suppressor gene with roles in apoptosis and prevention of the epithelial–mesenchymal transition. Other recent works have identified a novel role for the FHIT gene product, Fhit, as a genome “caretaker.” Loss of this caretaker function leads to nucleotide imbalance, spontaneous replication stress, and DNA breaks. Because Fhit loss-induced DNA damage is “checkpoint blind,” cells accumulate further DNA damage during subsequent cell cycles, accruing global genome instability that could facilitate oncogenic mutation acquisition and expedite clonal expansion. Loss of Fhit activity therefore induces a mutator phenotype. Evidence for FHIT as a mutator gene is discussed in light of these recent investigations of Fhit loss and subsequent genome instability.

DNA replication stress underlies renal phenotypes in CEP290 -associated Joubert syndrome

Juvenile ciliopathy syndromes that are associated with renal cysts and premature renal failure are commonly the result of mutations in the gene encoding centrosomal protein CEP290. In addition to centrosomes and the transition zone at the base of the primary cilium, CEP290 also localizes to the nucleus; however, the nuclear function of CEP290 is unknown. Here, we demonstrate that reduction of cellular CEP290 in primary human and mouse kidney cells as well as in zebrafish embryos leads to enhanced DNA damage signaling and accumulation of DNA breaks ex vivo and in vivo. Compared with those from WT mice, primary kidney cells from Cep290-deficient mice exhibited supernumerary centrioles, decreased replication fork velocity, fork asymmetry, and increased levels of cyclin-dependent kinases (CDKs). Treatment of Cep290-deficient cells with CDK inhibitors rescued DNA damage and centriole number. Moreover, the loss of primary cilia that results from CEP290 dysfunction was rescued in 3D cell culture spheroids of primary murine kidney cells after exposure to CDK inhibitors. Together, our results provide a link between CEP290 and DNA replication stress and suggest CDK inhibition as a potential treatment strategy for a wide range of ciliopathy syndromes.

Fhit Deficiency-Induced Global Genome Instability Promotes Mutation and Clonal Expansion

Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome ‘caretaker’ whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit -/- MEFs escape senescence to become immortal more rapidly than Fhit +/+ MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a ‘mutator’ phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.

Characterization of the role of Fhit in suppression of DNA damage

The fragile histidine triad protein, Fhit, has a number of reported tumor suppressive functions which include signaling of apoptosis in cancer cells in vitro and in vivo, modulation of the DNA damage response, down-regulation of target oncogene expression, suppression of tumor growth in vivo, and suppression of cancer cell invasion and metastasis. Most of these functions of Fhit have been observed on exogenous re-expression of Fhit in Fhit-negative cancer cells. However, little is known about the tumorigenic changes that occur in normal or precancerous cells following loss of Fhit expression. Recently, we have shown that shortly after loss of Fhit expression, cells exhibit signs of DNA replication stress-induced DNA damage and develop genomic instability. Here, we extend these findings through investigation of different factors that affect Fhit function to prevent DNA damage. We found that Fhit activity is dependent upon a functional HIT domain and the tyrosine-114 residue, previously shown to be required for tumor suppression by Fhit. Furthermore, Fhit function was shown to be independent of exogenous and endogenous sources of oxidative stress. Finally, Fhit function was shown to be dependent upon Chk1 kinase activity, but independent of Atr or Atm kinases. Evidence suggests that Fhit and Chk1 kinase cooperate to prevent replication stress-induced DNA damage. These findings provide important and unexpected insights into the mechanism whereby loss of Fhit expression contributes to cell transformation.

Response of subtype-specific human breast cancer-derived cells to poly(ADP-ribose) polymerase and checkpoint kinase 1 inhibition

When DNA damage is detected, checkpoint signal networks are activated to stop the cell cycle, and DNA repair processes begin. Inhibitory compounds targeting components of DNA damage response pathways have been identified and are being used in clinical trials, in combination with chemotherapeutic agents, to enhance cancer therapy. Inhibitors of checkpoint kinases, Chk1 and Chk2, have been shown to sensitize tumor cells to DNA damaging agents, and treatment of BRCA1/2‐deficient tumor cells, as well as triple negative breast cancers, with poly(ADP‐ribose) polymerase (PARP) inhibitors has shown promise. But systematic studies to determine which tumor subtypes are likely to respond to these specific inhibitors have not been reported. The current study was designed to test sensitivity of specific breast cancer subtype‐derived cells to two classes of these new inhibitory drugs, PARP and Chk1 inhibitors. Luminal, HER2 overexpressing, and triple negative breast cancer‐derived cells were tested for sensitivity to killing by PARP inhibitors, ABT‐888 and BSI‐201, and Chk1 inhibitor, PF‐00477736, alone or in combination with gemcitabine or carboplatin. Each of the triple negative breast cancer cell lines showed strong sensitivity to the Chk1 inhibitor, but only the BRCA1‐deficient breast cancer cell lines showed sensitivity to the PARP inhibitors, suggesting that in vitro testing of cancer cell lines of specific subtypes, with panels of the different PARP and Chk1 inhibitors, will contribute to stratification of patients for clinical trials using these classes of inhibitors. (Cancer Sci 2011; 102: 1882–1888)

Exome-wide single-base substitutions in tissues and derived cell lines of the constitutive Fhit knockout mouse.

Loss of expression of Fhit, a tumor suppressor and genome caretaker, occurs in preneoplastic lesions during development of many human cancers. Furthermore, Fhit‐deficient mouse models are exquisitely susceptible to carcinogen induction of cancers of the lung and forestomach. Due to absence of Fhit genome caretaker function, cultured cells and tissues of the constitutive Fhit knockout strain develop chromosome aneuploidy and allele copy number gains and losses and we hypothesized that Fhit‐deficient cells would also develop point mutations. On analysis of whole exome sequences of Fhit‐deficient tissues and cultured cells, we found 300 to >1000 single‐base substitutions associated with Fhit loss in the 2% of the genome included in exomes, relative to the C57Bl6 reference genome. The mutation signature is characterized by increased C>T and T>C mutations, similar to the “age at diagnosis” signature identified in human cancers. The Fhit‐deficiency mutation signature also resembles a C>T and T>C mutation signature reported for human papillary kidney cancers and a similar signature recently reported for esophageal and bladder cancers, cancers that are frequently Fhit deficient. The increase in T>C mutations in −/− exomes may be due to dNTP imbalance, particularly in thymidine triphosphate, resulting from decreased expression of thymidine kinase 1 in Fhit‐deficient cells. Fhit‐deficient kidney cells that survived in vitro dimethylbenz(a)anthracene treatment additionally showed increased T>A mutations, a signature generated by treatment with this carcinogen, suggesting that these T>A transversions may be evidence of carcinogen‐induced preneoplastic changes.