Regulation of alphaB-crystallin gene expression by the transcription factor Ets1 in breast cancer.

Recent studies indicate that the small heat shock protein alphaB-crystallin is expressed in poor prognosis basal-like breast tumors and likely contributes to their aggressive phenotype. However, the mechanisms underlying the deregulated expression of alphaB-crystallin in basal-like tumors are poorly understood. Using a bioinformatics approach, we identified a putative DNA binding motif in the human alphaB-crystallin promoter for the proto-oncogene Ets1, a member of the ETS transcription factor family that bind to DNA at palindromic ETS-binding sites (EBS). Here we demonstrate that ectopic expression of Ets1 activates the alphaB-crystallin promoter by an EBS-dependent mechanism and increases alphaB-crystallin protein levels, while silencing Ets1 reduces alphaB-crystallin promoter activity and protein levels. Chromatin immunoprecipitation analyses showed that endogenous Ets1 binds to the alphaB-crystallin promoter in basal-like breast cancer cells in vivo. Interrogation of publically available gene expression data revealed that Ets1 is expressed in human basal-like breast tumors and is associated with poor survival. Collectively, our results point to a previously unrecognized link between the oncogenic transcription factor Ets1 and alphaB-crystallin in basal-like breast cancer.

Induction of the small heat shock protein alphaB-crystallin by genotoxic stress is mediated by p53 and p73.

The small heat shock protein alphaB-crystallin is a molecular chaperone that is induced by stress and protects cells by inhibiting protein aggregation and apoptosis. To identify novel transcriptional regulators of the alphaB-crystallin gene, we examined the alphaB-crystallin promoter for conserved transcription factor DNA-binding elements and identified a putative response element for the p53 tumor suppressor protein. Ectopic expression of wild-type p53 induced alphaB-crystallin mRNA and protein with delayed kinetics compared to p21. Additionally, the induction of alphaB-crystallin by genotoxic stress was inhibited by siRNAs targeting p53. Although the p53-dependent transactivation of an alphaB-crystallin promoter luciferase reporter required the putative p53RE, chromatin immunoprecipitation failed to detect p53 binding to the alphaB-crystallin promoter. These results suggested an indirect mechanism of transactivation involving p53 family members p63 or p73. DeltaNp73 was dramatically induced by p53 in a TAp73-dependent manner, and silencing p73 suppressed the transcriptional activation of alphaB-crystallin by p53. Moreover, ectopic expression of DeltaNp73alpha (but not other p73 isoforms) increased alphaB-crystallin mRNA levels in the absence of p53. Collectively, our results link the molecular chaperone alphaB-crystallin to the cellular genotoxic stress response via a novel mechanism of transcriptional regulation by p53 and p73.

The small heat shock protein HspB2 is a novel anti-apoptotic protein that inhibits apical caspase activation in the extrinsic apoptotic pathway.

Members of the conserved small heat shock protein (sHSP) family, such as αB-crystallin and Hsp27, are constitutively expressed in diverse malignancies and have been linked to several hallmark features of cancer including apoptosis resistance. In contrast, the sHSP HspB2/MKBP, which shares an intergenic promoter with αB-crystallin, was discovered as a chaperone of the myotonic dystrophy protein kinase and has not been previously implicated in apoptosis regulation. Here we describe a new function for HspB2 as a novel inhibitor of apical caspase activation in the extrinsic apoptotic pathway. Specifically, we demonstrate that HspB2 is expressed in a subset of human breast cancer cell lines and that ectopic expression of HspB2 in breast cancer cells confers resistance to apoptosis induced by both TRAIL and TNF-α. We also show that HspB2 inhibits the extrinsic apoptotic pathway by suppressing apical caspases-8 and 10 activation, thereby blocking downstream apoptotic events, such as Bid cleavage and caspase-3 activation. Consistent with these in vitro effects, HspB2 attenuates the anti-tumor activity of TRAIL in an orthotopic xenograft model of breast cancer. Collectively, our results reveal a novel function of HspB2 as an anti-apoptotic protein that negatively regulates apical caspase activation in the extrinsic apoptotic pathway.

AlphaB-crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer.

Recent gene profiling studies have identified a new breast cancer subtype, the basal-like group, which expresses genes characteristic of basal epithelial cells and is associated with poor clinical outcomes. However, the genes responsible for the aggressive behavior observed in this group are largely unknown. Here we report that the small heat shock protein alpha-basic-crystallin (alphaB-crystallin) was commonly expressed in basal-like tumors and predicted poor survival in breast cancer patients independently of other prognostic markers. We also demonstrate that overexpression of alphaB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, alphaB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing alphaB-crystallin by RNA interference inhibited these abnormalities. alphaB-Crystallin overexpression also induced EGF- and anchorage-independent growth, increased cell migration and invasion, and constitutively activated the MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by alphaB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing alphaB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that alphaB-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors.

Deconstructing the molecular portrait of basal-like breast cancer.

Gene-expression profiling has revealed several molecular subtypes of breast cancer, which differ in their pathobiology and clinical outcomes. Basal-like tumors are a newly recognized subtype of breast cancer, which express genes that are characteristic of basal epithelial cells, such as the basal cytokeratins, and are associated with poor relapse-free and overall survival. However, the genetic and epigenetic alterations that are responsible for the biologically aggressive phenotype of these estrogen receptor-negative and HER2/ErbB2-negative tumors are not well understood, thereby hindering efforts to develop targeted therapies. Here, we focus on new insights into the molecular pathogenesis of basal-like breast cancer and explore how these discoveries might impact the treatment of these poor-prognosis tumors.

A member of the GAGE family of tumor antigens is an anti-apoptotic gene that confers resistance to Fas/CD95/APO-1, Interferon-gamma, taxol and gamma-irradiation.

Attractive targets for cancer therapy are gene products whose inactivation is not detrimental in essential tissues. The GAGE family of Cancer/Testis Antigens is a group of appealing candidates for cancer therapy since they are expressed in a wide variety of human tumors and are silent in most adult tissues, with the exception of testis. Interestingly, expression of GAGE has been associated with poor prognosis in some cancers. Nevertheless, no function has been reported for any of the GAGE family members. Here we describe for the first time an anti-apoptotic activity exerted by GAGE. We have cloned GAGE-7C from HeLa cells and showed that it renders transfected cells resistant to apoptosis induced by Interferon-gamma (IFN-gamma) or by the death receptor Fas/CD95/APO-1. Similarly, transfection of GAGE-7/7B also confers resistance to Fas induced apoptosis. In the Fas pathway, the anti-apoptotic activity of GAGE-7C maps downstream of caspase-8 activation and upstream of poly (ADP-ribose) polymerase (PARP) cleavage. Furthermore, GAGE-7C renders the cells resistant to the therapeutic agents Taxol and gamma-irradiation. Following the various apoptotic stimuli, the surviving GAGE-7C transfectants actively proliferate and exhibit enhanced long term survival in colony formation assays. Overall, our data establishes a functional link between GAGE-7C and two aspects of human tumor progression; namely, resistance to Fas induced apoptosis and to chemo- and radio-therapy.

Rational drug design: a GAGE derived peptide kills tumor cells.

The current therapy of cancer fails to completely and exclusively target tumor cells. An ideal target for cancer therapy should be expressed exclusively in tumor cells and contribute to tumorigenesis. Targeting such a candidate gene would cause cell death only in tumor cells. A cancer testis antigen, GAGE, is considered such a target as it is expressed in numerous tumors but silent in normal tissues. Contribution of GAGE to tumorigenesis is evident from the fact that overexpression of GAGE results in rescuing the cells from cell death induced by interferon-gamma, Fas, Taxol and ionizing radiation. GAGE binds to Interferon Response Factor-1 (IRF1) and decreases its abundance, thus impacting cell death pathways mediated by interferon-gamma. To dissect the region responsible for GAGE activities, five peptides encompassing the whole sequence of GAGE protein were designed. A string of arginine residues enabled the peptides to enter the cells. Surprisingly, peptide#1 consisting of N-terminal end of GAGE protein was able to induce cell death in HeLa cells. Interestingly, GAGE null HEK293 cells and primary fibroblasts were less sensitive to the cell death induced by this peptide. Furthermore, GAGE expression, endogenous or ectopic, resulted in increased sensitization to cell death induced by this peptide suggesting its dominant active properties. The tumors that express GAGE as a diagnostic marker should be sensitive to this peptide while sparing GAGE null normal tissues suggesting potential in cancer therapeutics.

GAGE, an antiapoptotic protein binds and modulates the expression of nucleophosmin/B23 and interferon regulatory factor 1.

The GAGE family of highly related tumor antigens is expressed in a variety of tumors. This albeit silent gene expression resulted in resistance of cells to various apoptotic agents such as Fas, interferon-gamma, Taxol, or gamma-radiation. We now report that GAGE overexpression in either HeLa (expressing endogenous GAGE) or HEK293 (devoid of GAGE expression) rendered those cells unsusceptible to cell death induced by IFN-gamma. We investigated the underlying mechanism of GAGE-induced cell survival upon treatment with IFN-gamma in this report. We showed that GAGE overexpression resulted in down-regulation of a key player of IFN-gamma-signaling pathway, interferon regulatory factor 1 (IRF1), and its target genes caspase-1 and caspase-7. An interaction between GAGE and IRF1 is detected in cells. Furthermore, GAGE interacted with a multifunctional protein nucleophosmin (NPM)/B23 and increased its abundance by stabilizing the protein. Increased level of NPM/B23 in conjunction with decreased level of IRF1 could aid GAGE-induced resistance to IFN-gamma. Our results suggest that GAGE could rescue cell death induced by IFN-gamma by altering the level of key players in cell death pathways. As GAGE is silent in most healthy tissues, targeting GAGE could result in therapeutic interventions in cancer therapy.

Caspase cleavage of HER-2 releases a Bad-like cell death effector.

Human epidermal growth factor receptor-2 (HER-2/ErbB2/neu), a receptor tyrosine kinase that is amplified/overexpressed in poor prognosis breast carcinomas, confers resistance to apoptosis by activating cell survival pathways. Here we demonstrate that the cytoplasmic tail of HER-2 is cleaved by caspases at Asp(1016)/Asp(1019) to release a approximately 47-kDa product, which is subsequently proteolyzed by caspases at Asp(1125) into an unstable 22-kDa fragment that is degraded by the proteasome and a predicted 25-kDa product. Both the 47- and 25-kDa products translocate to mitochondria, release cytochrome c by a Bcl-x(L)-suppressible mechanism, and induce caspase-dependent apoptosis. The 47- and 25-kDa HER-2 cleavage products share a functional BH3-like domain, which is required for cytochrome c release in cells and isolated mitochondria and for apoptosis induction. Caspase-cleaved HER-2 binds Bcl-x(L) and acts synergistically with truncated Bid to induce apoptosis, mimicking the actions of the BH3-only protein Bad. Moreover, the HER-2 cleavage products cooperate with Noxa to induce apoptosis in cells expressing both Bcl-x(L) and Mcl-1, confirming their Bad-like function. Collectively, our results indicate that caspases activate a previously unrecognized proapoptotic function of HER-2 by releasing a Bad-like cell death effector.

Genes contributing to prion pathogenesis.

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrP(C)) to a misfolded, pathogenic isoform (PrP(Sc)). Prion inoculation experiments in mice expressing homologous PrP(C) molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrP(C), our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

Caspase proteolysis of the integrin beta4 subunit disrupts hemidesmosome assembly, promotes apoptosis, and inhibits cell migration.

Caspases are a conserved family of cell death proteases that cleave intracellular substrates at Asp residues to modify their function and promote apoptosis. In this report we identify the integrin beta4 subunit as a novel caspase substrate using an expression cloning strategy. Together with its alpha6 partner, alpha6beta4 integrin anchors epithelial cells to the basement membrane at specialized adhesive structures known as hemidesmosomes and plays a critical role in diverse epithelial cell functions including cell survival and migration. We show that integrin beta4 is cleaved by caspase-3 and -7 at a conserved Asp residue (Asp(1109)) in vitro and in epithelial cells undergoing apoptosis, resulting in the removal of most of its cytoplasmic tail. Caspase cleavage of integrin beta4 produces two products, 1) a carboxyl-terminal product that is unstable and rapidly degraded by the proteasome and 2) an amino-terminal cleavage product (amino acids 1-1109) that is unable to assemble into mature hemidesmosomes. We also demonstrate that caspase cleavage of integrin beta4 sensitizes epithelial cells to apoptosis and inhibits cell migration. Taken together, we have identified a previously unrecognized proteolytic truncation of integrin beta4 generated by caspases that disrupts key structural and functional properties of epithelial cells and promotes apoptosis.

Cell Death Inhibiting RNA (CDIR) modulates IFN-gamma-stimulated sensitization to Fas/CD95/Apo-1 and TRAIL/Apo-2L-induced apoptosis.

We have previously demonstrated that overexpression of Cell Death Inhibiting RNA (CDIR), a portion of the 3'untranslated region (UTR) of KIAA0425, inhibits Interferon-gamma (IFN-gamma) induced apoptosis in HeLa cells (Shchors et al., J Biol Chem 2002; 277:47061-72). IFN-gamma is known to sensitize cells to killing induced by the death receptor ligands such as Fas/APO-1/CD95 and TNF-related apoptosis-inducing ligand (TRAIL/Apo-2L). Here we report that while CDIR does not alter the response of cells to Fas or TRAIL, it significantly modulates IFN-gamma-induced sensitization of HeLa cells to these death-inducing ligands. Interestingly, while CDIR abrogates the IFN-gamma-modulated sensitization to Fas, it enhances the sensitization to TRAIL. Expression of CDIR did not alter initial steps of IFN-gamma signaling including induction of Signal Transducer and Activator-1 (Stat1), caspase-1 or Interferon Regulatory Factor-1 (IRF1) transcription. In contrast, although expression of CDIR does not affect the protein level of caspase-1 or STAT1, it does significantly reduce the level of IRF1 protein. Thus, CDIR mediates IFN-gamma-induced apoptosis, at least in part, by reducing the level of the pro-apoptotic tumor suppressor gene IRF1 via a post-transcriptional mechanism. Since tumor cells are often less sensitive to Fas and more sensitive to TRAIL than normal cells, we suggest that CDIR or CDIR-like activity could contribute to such a phenotype of tumor cells.

Cell death inhibiting RNA (CDIR) derived from a 3'-untranslated region binds AUF1 and heat shock protein 27.

Regulators of programmed cell death were previously identified using a technical knockout genetic screen. Among the elements that inhibited interferon-gamma-induced apoptosis of HeLa cells was a 441-nucleotide fragment derived from the 3'-untranslated region (UTR) of KIAA0425, a gene of unknown function. This fragment was termed cell death inhibiting RNA (CDIR). Deletion and mutation analyses of CDIR were employed to identify the features required for its anti-apoptotic activity. Single nucleotide alterations within either copy of the duplicated U-rich motif found in the CDIR sequence abolished the anti-apoptotic activity of CDIR and altered its in vitro association with a protein complex. Further analysis of the CDIR-binding complex indicated that it contained heat shock protein 27 (Hsp27) and the regulator of mRNA turnover AUF1 (heterogeneous nuclear ribonucleoprotein D). In addition, recombinant AUF1 bound directly to CDIR. Furthermore, expression of another AUF1-binding RNA element, derived from the 3'-UTR of c-myc, inhibited apoptosis. We also demonstrate that the level and the stability of p21(waf1/Cip1/sdi1) mRNA, a target of AUF1 with anti-apoptotic activity, were increased in CDIR-transfected cells. The level of mRNA and protein of Bcl-2, another anti-apoptotic gene, containing an AUF1 binding site in its 3'-UTR was also increased in CDIR-transfected cells. Our data suggest that AUF1 regulates apoptosis by altering mRNA turnover. We propose that CDIR inhibits apoptosis by acting as a competitive inhibitor of AUF1, preventing AUF1 from binding to its targets.