Fhit Delocalizes Annexin A4 from Plasma Membrane to Cytosol and Sensitizes Lung Cancer Cells to Paclitaxel

Fhit protein is lost or reduced in a large fraction of human tumors, and its restoration triggers apoptosis and suppresses tumor formation or progression in preclinical models. Here, we describe the identification of candidate Fhit-interacting proteins with cytosolic and plasma membrane localization. Among these, Annexin 4 (ANXA4) was validated by co-immunoprecipitation and confocal microscopy as a partner of this novel Fhit protein complex. Here we report that overexpression of Fhit prevents Annexin A4 translocation from cytosol to plasma membrane in A549 lung cancer cells treated with paclitaxel. Moreover, paclitaxel administration in combination with AdFHIT acts synergistically to increase the apoptotic rate of tumor cells both in vitro and in vivo experiments.     

A Fhit-ing Role in the DNA Damage Checkpoint Response

The FHIT gene encompasses the most active common fragile site of the human genome and is thus exquisitely sensitive to intragenic alterations by DNA damaging agents, alterations that can lead to FHIT allele loss very early in the preneoplastic phase of cancer development, before or coincident with activation of the DNA damage checkpoint. Fhit protein expression is lost or reduced in many preneoplastic lesions and in >50% of cancers, Fhit knockout mice are highly susceptible to carcinogen induction of tumors and Fhit replacement in these mice by gene therapy induces apoptosis and significantly reduces tumor burden. But learning how Fhit induces apoptosis and suppresses tumors has been a challenge because interacting proteins, effectors of Fhit signals, have not been discovered.Nevertheless, the study of Fhit deficient mouse and human tissue-derived and cancer-derived cells in vitro has led to several important conclusions: repair protein-deficient cancers are more likely to be Fhit-deficient; Fhit-deficient cells show enhanced resistance to UVC, mitomycin C, camptothecin and ionizing radiation-induced cell killing, possibly due to strong activation of the ATR pathway following DNA damage; Fhit-deficient cells show higher efficiency of homologous recombination repair, a double-strand break repair pathway in mammalian cells; Fhit protein indirectly affects S-phase checkpoint and DNA repair. Finally, results of a recent study have suggested that the DNA damage-susceptible FRA3B/FHIT chromosome fragile region, paradoxically, encodes a protein, Fhit, that is necessary for protecting cells from accumulation of DNA damage, through modulation of checkpoint proteins Hus1 and phosphoChk1. Thus, inactivation of Fhit contributes to accumulation of abnormal checkpoint phenotypes in cancer development. It will be very important to determine mechanisms employed by Fhit in modulating checkpoint pathways, and to define consequences of Fhit loss in specific preneoplastic and neoplastic tissues, to provide rationales for effective replacement or reactivation of endogenous Fhit pathways in novel therapeutic or preventive approaches.     

Protein expression profiling identifies cyclophilin A as a molecular target in Fhit-mediated tumor suppression

Loss of fragile histidine triad (Fhit) expression is often associated with human malignancies, and Fhit functions as a tumor suppressor in controlling cell growth and apoptosis, although specific signal pathways are still undefined. We have used a proteomic approach to define proteins in the Fhit-mediated tumor suppression pathway. Because substitution of Tyr(114) (Y114) with phenylalanine (Y114F) diminishes Fhit functions, we did protein expression profiling to identify proteins differentially expressed in Fhit-negative H1299 lung cancer cells infected with wild-type (Ad-FHIT-wt) and Y114 mutant FHIT-expressing (Ad-FHIT-Y114F) adenoviruses. Among 12 distinct proteins that exhibited 4-fold differences in expression on comparison of the two infected cell lysates, cyclophilin A, the intracellular reporter of the immunosuppressive drug cyclosporine A, showed a remarkably decreased protein level in cells infected with Ad-FHIT-wt versus Ad-FHIT-Y114F. Conversely, loss of Fhit expression resulted in increased cyclophilin A expression in mouse tissues and cell lines. Restoration of Fhit expression led to down-regulated cyclophilin A protein expression and subsequently prevented cyclophilin A-induced up-regulation of cyclin D1, Cdk4, and resultant cell cycle progression (G(1)-S transition), which was independent of Ca(2+)/calmodulin-dependent kinase inhibitor, KN-93. Interestingly, Fhit down-modulation of phosphatase activity of calcineurin, which controls cyclin D1/Cdk4 activation, was reversed by cyclophilin A treatment in a concentration-dependent manner, a reversal that was inhibited by additional cyclosporine A treatment. Thus, cyclophilin A is a downstream target in Fhit-mediated cessation of cell cycle progression at late G(1) phase. Elucidation of the protein effectors of Fhit signaling may lead to identification of targets for lung cancer therapy.     

Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers

BACKGROUND: The nucleotide-binding protein Fhit, among the earliest and most frequently inactivated proteins in lung cancer, suppresses tumor formation by inducing apoptosis. In invertebrates, Fhit is encoded as a fusion protein with Nit, a member of the nitrilase superfamily. In mice, the Nit1 and Fhit genes have nearly identical expression profiles. According to the Rosetta Stone hypothesis, if the separate Nit and Fhit genes could be shown to occur in the same subset of genomes (that is, to share a phylogenetic profile), then the existence of a fusion protein in invertebrates and the coordinated expression of separate mRNAs in mouse suggest that Nit and Fhit function in the same pathway and that the structure of invertebrate NitFhit may reflect the nature of Nit-Fhit interactions. RESULTS: To satisfy the phylogenetic profile criterion for functional significance of protein fusion events, we cloned additional Nit homologs from organisms with Fhit homologs. We used fluorescent nucleotide analogs of ApppA to follow the purification and to characterize the nucleotide specificity of NitFhit from Caenorhabditis elegans, crystallized the 200 kDa tetrameric complex, and solved the structure of NitFhit from a single mercury derivative phased by two-wavelength anomalous diffraction. CONCLUSIONS: Nit monomers possess a new alpha-beta-beta-alpha sandwich fold with a presumptive Cys-Glu-Lys catalytic triad. Nit assembles into a tetrameric, 52-stranded beta box that binds Fhit dimers at opposite poles and displays Nit active sites around the middle of the complex. The most carboxy-terminal beta strand of each Nit monomer exits the core of the Nit tetramer and interacts with Fhit. Residence in the NitFhit complex does not alter the nucleotide specificity of Fhit dimers, which are oriented with ApppA-binding surfaces away from Nit.     

Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

Heat shock protein 27 (Hsp27) is emerging as a promising therapeutic target for treatment of various cancers. Although the role of Hsp27 in protection from stress-induced intrinsic cell death has been relatively well studied, its role in Fas (death domain containing member of the tumor necrosis factor receptor superfamily)-induced apoptosis and cell proliferation remains underappreciated. Here, we show that Hsp27 silencing induces dual coordinated effects, resulting in inhibition of cell proliferation and sensitization of cells to Fas-induced apoptosis through regulation of PEA-15 (15-kDa phospho-enriched protein in astrocytes). We demonstrate that Hsp27 silencing suppresses proliferation by causing PEA-15 to bind and sequester extracellular signal-regulated kinase (ERK), resulting in reduced translocation of ERK to the nucleus. Concurrently, Hsp27 silencing promotes Fas-induced apoptosis by inducing PEA-15 to release Fas-associating protein with a novel death domain (FADD), thus allowing FADD to participate in death receptor signaling. Conversely, Hsp27 overexpression promotes cell proliferation and suppresses Fas-induced apoptosis. Furthermore, we show that Hsp27 regulation of PEA-15 activity occurs in an Akt-dependent manner. Significantly, Hsp27 silencing in a panel of phosphatase and tensin homolog on chromosome 10 (PTEN) wild-type or null cell lines, and in LNCaP cells that inducibly express PTEN, resulted in selective growth inhibition of PTEN-deficient cancer cells. These data identify a dual coordinated role of Hsp27 in cell proliferation and Fas-induced apoptosis via Akt and PEA-15, and indicate that improved clinical responses to Hsp27-targeted therapy may be achieved by stratifying patient populations based on tumor PTEN expression.     

Frequent altered expression of fragile histidine triad protein in human colorectal adenomas

Fragile histidine triad (FHIT) gene is involved in deletions on the short arm of chromosome 3 in various human cancers. We found that 47% of colorectal adenomas, which is a higher frequency than that of K-ras, showed altered expression of the Fhit protein by Western blot analysis. The amount of Fhit protein was inversely correlated with the degree of dysplasia. Importantly, 27% of low-grade dysplastic adenomas showed altered expression of Fhit protein. Additionally, expression of human Fhit protein in human colon carcinoma cell line SW480 exhibited a marked inhibition of growth and rendered SW480 cells highly susceptible to undergo apoptosis compared with control cells. These findings suggest that altered expression of the FHIT gene is a quite early aberration in the development of colorectal tumors and that Fhit protein may act as a tumor suppressor.     

Diadenosines as FHIT-ness instructors

FHIT is a tumor suppressor gene that is frequently inactivated in human cancer. Although the Fhit protein is known to hydrolyze diadenosine triphosphate (Ap(3)A), this hydrolase activity is not required for Fhit-mediated oncosuppression. Indeed, the molecular mechanisms and the regulatory elements of Fhit oncosuppression are largely unknown. Here, we review physiological and pathological aspects of Fhit in the context of the Ap(n)A family of signaling molecules, as well as the involvement of Fhit in apoptosis and the cell cycle in cancer models. We also discuss recent findings of novel Fhit interactions that may lead to new hypotheses about biochemical mechanisms underlying the oncosuppressor activity of this gene.     

Evaluation of influence of Ap4A analogues on Fhit-positive HEK293T cells; cytotoxicity and ability to induce apoptosis

Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 μM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy.     

Involvement of the Fhit gene in the ionizing radiation-activated ATR/CHK1 pathway

Fragile Histidine Triad (Fhit) gene deletion, methylation, and reduced Fhit protein expression occur in about 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified, although it has been shown that Fhit overexpression leads to apoptosis in many cancer cell lines. We report in this study that Fhit-/- cells derived from gene knockout mice show much stronger S and G2 checkpoint responses than their wild type counterparts. The strong checkpoint responses are regulated by the ATR/CHK1 pathway, which contributes to the radioresistance of Fhit-/- cells. These results indicate an association of Fhit gene inactivation with increased survival after DNA damage, which is related to the over-active checkpoints regulated by the ATR/CHK1 pathway. These results also suggest the potential effects of Fhit-dependent DNA damage response on tumor progression.     

Reduced thermotolerance in aged cells results from a loss of an hsp72- mediated control of JNK signaling pathway

Aged organisms exhibit a greatly decreased ability to induce the major heat shock protein, Hsp72, in response to stresses, a phenomenon that can also be observed in cell cultures (Heydari AR, Takahashi R, Gutsmann A, You S and Richardson A (1994) Hsp70 and aging. Experientia 50: 1092–1098). Hsp72 was shown to protect cells from a variety of stresses. The protective function of Hsp72 has been commonly ascribed to its chaperoning ability. However, recently we showed that Hsp72 protects cells from heat shock by suppression of a stress-kinase JNK, an essential component of the heat-induced apoptotic pathway (Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S, Shifrin VI and Sherman MY (1997) Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 272: 18033–18037). Here we demonstrate that because of the diminished inducibility of Hsp72 in aged cells, Hsp72-mediated control of JNK signaling pathway is compromised. This results in increased rate of apoptotic cell death following heat shock. We show that forced expression of Hsp72 in aged cells from an adenovirus-based vector completely suppresses activation of JNK by heat shock and consequently protects from heat-induced apoptosis. We also demonstrate for the first time that it is possible to restore endogenous expression of Hsp72 in aged cells. This can be achieved by treatment with the proteasome inhibitor MG132. Induction of Hsp72 in aged cells under these conditions leads to suppression of JNK activation by a heat shock and restoration of thermotolerance manifested in a lower rate of apoptosis.     

Biological functions of mammalian Nit1, the counterpart of the invertebrate NitFhit Rosetta stone protein, a possible tumor suppressor

The "Rosetta Stone" hypothesis proposes that the existence of a fusion protein in some organisms predicts that the separate polypeptides function in the same biochemical pathway in other organisms and may physically interact. In Drosophila melanogaster and Caenorhabditis elegans, NitFhit protein is composed of two domains, a fragile histidine triad homolog and a bacterial and plant nitrilase homolog. We assessed the biological effects of mammalian Nit1 expression in comparison with Fhit and observed that: 1) Nit1 expression was observed in most normal tissues and overlapped partially with Fhit expression; 2) Nit1-deficient mouse kidney cells exhibited accelerated proliferation, resistance to DNA damage stress, and increased cyclin D1 expression; 3) cyclin D1 was up-regulated in Nit1 null mammary gland and skin; 4) Nit1 overexpression induced caspase-dependent apoptosis in vitro; and 5) Nit1 allele deficiency led to increased incidence of N-nitrosomethylbenzylamine-induced murine forestomach tumors. Thus, the biological effects of Nit1 expression are similar to Fhit effects. Adenoviruses carrying recombinant NIT1 and FHIT induced apoptosis in Fhit- and Nit1-deficient cells, respectively, suggesting that Nit1-Fhit interaction is not essential for function of either protein. The results suggest that Nit1 and Fhit share tumor suppressor signaling pathways, while localization of the NIT1 gene at a stable, rather than fragile, chromosome site explains the paucity of gene alterations and in frequent loss of expression of the NIT1 gene in human malignancies.     

Hsp60 regulation of tumor cell apoptosis

Molecular chaperones may promote cell survival, but how this process is regulated, especially in cancer, is not well understood. Using high throughput proteomics screening, we identified the cell cycle regulator and apoptosis inhibitor survivin as a novel protein associated with the molecular chaperone Hsp60. Acute ablation of Hsp60 by small interfering RNA destabilizes the mitochondrial pool of survivin, induces mitochondrial dysfunction, and activates caspase-dependent apoptosis. This response involves disruption of an Hsp60-p53 complex, which results in p53 stabilization, increased expression of pro-apoptotic Bax, and Bax-dependent apoptosis. In vivo, Hsp60 is abundantly expressed in primary human tumors, as compared with matched normal tissues, and small interfering RNA ablation of Hsp60 in normal cells is well tolerated and does not cause apoptosis. Therefore, Hsp60 orchestrates a broad cell survival program centered on stabilization of mitochondrial survivin and restraining of p53 function, and this process is selectively exploited in cancer. Hsp60 inhibitors may function as attractive anticancer agents by differentially inducing apoptosis in tumor cells.     

Small stress protein Hsp27 accumulation during dopamine-mediated differentiation of rat olfactory neurons counteracts apoptosis

The small stress protein Hsp27 is expressed during mammalian neural development. We have analyzed the role of this protein in immortalized rat olfactory neuroblasts. In the presence of dopamine a fraction of these cells differentiate into neurons while the remaining cells undergo apoptosis. We report here that the dopamine induced differentiation and apoptosis are associated with a transient and specific accumulation of Hsp27. Moreover, transfection experiments have shown that Hsp27 overexpression drastically decreases the fraction of cells undergoing apoptosis. In contrast, reduction of the endogenous level of Hsp27 led to abortion of differentiation and, therefore, drastically increased the number of apoptotic cells. Furthermore, in the normal cell population we show that Hsp27 accumulation takes place only in differentiating cells that were not undergoing apoptosis. We therefore conclude that Hsp27 may represent a key protein that controls the decision of olfactory precursor cells to undergo either differentiation or cell death.     

Enhancement of oxidative stress-induced apoptosis by Hsp105alpha in mouse embryonal F9 cells.

Hsp105alpha is one of the major mammalian heat shock proteins that belongs to the HSP105/110 family, and is expressed at especially high levels in the brain as compared with other tissues in mammals. Previously, we showed that Hsp105alpha prevents stress-induced apoptosis in neuronal PC12 cells, and is a novel anti-apoptotic neuroprotective factor in the mammalian brain. On the other hand, we have also demonstrated that Hsp105alpha is expressed transiently at high levels during mouse embryogenesis and is found not only in various tissues but also in apoptotic cells. In the present study, to elucidate the role of Hsp105alpha during mouse embryogenesis, we established mouse embryonal F9 cell lines that constitutively over-express Hsp105alpha. Over-expression of Hsp105alpha enhanced hydrogen peroxide-induced apoptosis by enhancing the activation of caspase-3, poly(ADP-ribose)polymerase cleavage, cytochrome c release and activation of p38 mitogen-activated protein kinase (p38). Furthermore, oxidative stress-induced apoptosis was suppressed by SB202190, a potent inhibitor of p38, in F9 cells. These findings indicated that the activation of p38 is an essential step for apoptosis in F9 cells and that Hsp105alpha enhances activation of p38, release of cytochrome c and caspase activation. Hsp105alpha may play important roles in organogenesis, during which marked apoptosis occurs, by enhancing apoptosis during mouse embryogenesis.     

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.     

Identification of alpha-tubulin as an hsp105alpha-binding protein by the yeast two-hybrid system

Hsp105alpha is a mammalian stress protein that belongs to the HSP105/110 family. Hsp105alpha prevents stress-induced apoptosis in neuronal cells and binds to Hsp70/Hsc70 and suppresses the Hsp70 chaperone activity in vitro. In this study, to further elucidate the function of Hsp105alpha, we searched for Hsp105alpha-binding proteins by screening a mouse FM3A cell cDNA library with full-length Hsp105alpha using the yeast two-hybrid system and obtained alpha-tubulin as an Hsp105alpha-binding protein. Hsp105alpha bound directly to alpha-tubulin both in vitro and in vivo. Indirect immunofluorescence analysis with anti-Hsp105 and anti-alpha-tubulin antibodies indicated that Hsp105alpha was colocalized with microtubules. Furthermore, the disorganization of microtubules induced by heat shock was prevented in Hsp105alpha-overexpressing COS-7 cells. These findings suggested that Hsp105alpha associates with alpha-tubulin and microtubules in cells and plays a role in protection of microtubules under conditions of stress.     

The function of HSP72 in suppression of c-Jun N-terminal kinase activation can be dissociated from its role in prevention of protein damage.

Activation of the c-Jun N-terminal kinase (JNK) by a variety of stimuli is critical for regulation of many cellular processes including apoptosis. The major inducible heat shock protein Hsp72 has previously been demonstrated to inhibit activation of JNK in cells exposed to heat shock and other protein-damaging agents, thus suppressing apoptosis. Hsp72 can protect proteins from stress-induced damage. To test if this protective function of Hsp72 is involved in JNK suppression, we investigated whether Hsp72 can avert activation of JNK by stimuli that do not cause protein damage. We show that Hsp72 suppresses activation of JNK induced by non-protein-damaging stimuli, interleukin-1 and UV irradiation, as well as by constitutively active components of the JNK signaling cascade Cdc42 and MEKK1. Furthermore, Hsp72 strongly reduced activation of JNK by phosphatase inhibitors. We also demonstrate that an Hsp72 mutant that lacks the ATPase domain is still capable of JNK suppression, thus indicating that the protein refolding activity of Hsp72 is not critical for inhibition of JNK activation. Taken together these data suggest that Hsp72 plays a regulatory role in JNK signaling and that the function of Hsp72 in protein protection or refolding is not involved in JNK regulation.     

Inhibition of Hsp90 function delays and impairs recovery from heat shock

The induction of the heat shock response as well as its termination is autoregulated by heat shock protein activities. In this study we have investigated whether Hsp90 functional protein levels influence the characteristics and duration of the heat shock response. Treatment of cells with several benzoquinone ansamycin inhibitors of Hsp90 (geldanamycin, herbimycin A) activated a heat shock response in the absence of heat shock, as reported previously. Pretreatment of cells with the Hsp90 inhibitors significantly delayed the rate of restoration of normal protein synthesis following a brief heat shock. Concurrently, the rate of Hsp synthesis and accumulation was substantially increased and prolonged. The cessation of heat shock protein synthesis did not occur until the levels of Hsp70 were substantially elevated relative to its standard threshold for autoregulation. The elevated levels of HSPS 22-28 (the small HSPS) and Hsp70 are not able to promote thermotolerance when Hsp90 activity is repressed by ansamycins; rather a suppression of thermotolerance is observed. These results suggest that a multicomponent protein chaperone complex involving both Hsp90 and Hsp70 signals the cessation of heat shock protein synthesis, the restoration of normal translation, and likely the establishment of thermotolerance. Impaired function of either component is sufficient to alter the heat shock response.     

Gambogic acid inhibits Hsp90 and deregulates TNF-α/NF-κB in HeLa cells

Gambogic acid (GB) is an important anti-cancer drug candidate, but the target protein by which it exerts its anti-cancer effects has not been identified. This study is the first to show that GB inhibits heat shock protein 90 (Hsp90) and down-regulates TNF-α/NF-κB in HeLa cells. The effects of GB on Hsp90 were studied by characterizing its physical interactions with Hsp90 upon binding, the noncompetitive inhibition of Hsp90 ATPase activity, and the degradation of Hsp90 client proteins (i.e., Akt, IKK) in HeLa cells. GB seems to bind to the N-terminal ATP-binding domain of Hsp90. Additionally, GB suppresses the activation of TNF-α/NF-κB and decreases XIAP expression levels and the ratio of Bcl-2/Bax, which in turn induces HeLa cell apoptosis. Thus, GB represents a promising therapeutic agent for cancer; it may also be useful as a probe to increase understanding of the biological functions of Hsp90.