Uncoupling of EGFR-RAS signaling and nuclear localization of YBX1 in colorectal cancer.

The transcription factor YBX1 can act as a mediator of signals transmitted via the EGFR-RAS-MAPK axis. YBX1 expression has been associated with tumor progression and prognosis in multiple types of cancer. Immunohistochemical studies have revealed dependency between YBX1 expression and individual EGFR family members. We analyzed YBX1 and EGFR family proteins in a colorectal cancer (CRC) cohort and provide functional analyses of YBX1 in the context of EGFR-RAS-MAPK signaling. Immunohistochemistry for YBX1 and EGFR family receptors with two antibodies for YBX1 and EGFR were performed and related to clinicopathological data. We employed Caco2 cells expressing an inducible KRASV12 gene to determine effects on localization and levels of YBX1. Mouse xenografts of Caco2-KRASV12 cells were used to determine YBX1 dynamics in a tissue context. The two different antibodies against YBX1 showed discordant immunohistochemical stainings in cell culture and clinical specimens. Expression of YBX1 and EGFR family members were not correlated in CRC. Analysis of Caco2 xenografts displayed again heterogeneity of YBX1 staining with both antibodies. Our results suggest that YBX1 is controlled via complex regulatory mechanisms involving tumor stroma interaction and signal transduction processes. Our study highlights that YBX1 antibodies have different specificities, advocating their use in a combined manner.

Nuclear YB-1 expression as a negative prognostic marker in nonsmall cell lung cancer.

The human Y-box binding protein, YB-1, is a multifunctional protein that regulates gene expression. Nuclear expression of YB-1 has been associated with chemoresistance and poor prognosis of tumour patients. Representative samples from autopsied material of primary tumours from 77 patients with NSCLC were investigated by immunohistochemistry for subcellular distribution of YB-1 and p53, in order to evaluate the prognostic role of nuclear expression of YB-1. Cytoplasmic YB-1 expression was found in all tumour samples, whereas nuclear expression was only observed in 48%. There was no correlation with histological classification, clinical parameters or tumour size, stage and metastasis status. However, patients with positive nuclear YB-1 expression in tumours showed reduced survival times when compared with patients without nuclear expression. Including information about the histology and mutational status for p53 increased the prognostic value of nuclear YB-1. Patients with nuclear YB-1 expression and p53 mutations had the worst prognosis (median survival 3 months), while best outcome was found in patients with no nuclear YB-1 and wildtype p53 (median survival 15 months). This suggests that the combined analysis of both markers allows a better identification of subgroups with varying prognosis. Nuclear expression of Y-box binding protien seems to be an independent prognostic marker.

Hyperthermia-induced nuclear translocation of transcription factor YB-1 leads to enhanced expression of multidrug resistance-related ABC transporters.

Genotoxic stress leads to nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of the multidrug resistance gene MDR1. Because hyperthermia is used for the treatment of colon cancer in combination with chemoradiotherapy, we investigated the influence of hyperthermia on YB-1 activity and the expression of multidrug resistance-related genes. Here we report that hyperthermia causes YB-1 translocation from the cytoplasm into the nucleus of human colon carcinoma cells HCT15 and HCT116. Nuclear translocation of YB-1 was associated with increased MDR1 and MRP1 gene activity, which is reflected in strong efflux pump activity. However, a combination of hyperthermia and drug treatment effectively reduced cell survival of the HCT15 and HCT116 cells. These results demonstrate that activation of MDR1 and MRP1 gene expression and increased efflux pump activity after hyperthermia were insufficient to cause an increase in drug resistance in colon cancer cell lines. The ability of hyperthermia to abrogate drug resistance in the presence of an increase in functional MDR proteins may provide an explanation for the efficacious results seen in the clinic in colon cancer patients treated with a combination of hyperthermia and chemotherapy.

Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation.

Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.

Hodgkin/Reed-Sternberg cells induce fibroblasts to secrete eotaxin, a potent chemoattractant for T cells and eosinophils.

Hodgkin's disease is histopathologically characterized by the relative scarcity of neoplastic Hodgkin and Reed-Sternberg cells and for yet unknown reasons by an abundant reactive background of T lymphocytes and often eosinophils. Eotaxin is a CC-chemokine attracting eosinophils and T helper 2 (Th2) cells in allergic inflammation. We now report that eotaxin is strongly expressed in fibroblasts of Hodgkin's disease tissues, whereas Hodgkin/Reed-Sternberg cells do not express this chemokine. In tissue culture, Hodgkin's disease tumor cells induce eotaxin expression in cocultured dermal fibroblasts in a concentration leading to a specific chemotactic response of a Th2 cell clone. Production of tumor necrosis factor-alpha (TNF-alpha) by Hodgkin/Reed-Sternberg cells appears to be responsible for this induction, because blocking of TNF-alpha by neutralizing antibodies prevented fibroblast eotaxin expression. Our data suggest that eotaxin is involved in the pathobiology of Hodgkin's disease by contributing to eosinophil and T-lymphocyte recruitment.

Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression.

Breast cancers are either primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur with a multidrug-resistant phenotype because of overexpression of the multidrug transporter P-glycoprotein. The MDR1 gene encoding P-glycoprotein may be transcriptionally regulated by a Y-box transcription factor. We now report that, in multidrug-resistant MCF-7 breast cancer cells, nuclear localization of YB-1 is associated with MDR-1 gene expression. In drug-sensitive MCF-7 cells, however, YB-1 was localized to the cytoplasm. Regulated overexpression of YB-1 in drug-sensitive diploid breast epithelial cells induced MDR-1 gene expression and multidrug resistance. In 27 out of 27 untreated primary breast cancers, YB-1 protein was expressed in the cytoplasm although it was undetectable in normal breast tissue of these patients. In a subgroup of tumors (9/27), however, YB-1 was also localized to the nucleus and, in these cases, high levels of P-glycoprotein were present. These results show that in a subset of untreated primary breast cancers, nuclear localization of YB-1 protein is associated with intrinsic multidrug resistance. Our data show that YB-1 has an important role in controlling MDR1 gene transcription and this finding provides a basis for the analysis of molecular mechanisms responsible for intrinsic multidrug resistance in human breast cancer.

Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells.

The pathogenesis and etiology of Hodgkin's disease, a common human malignant lymphoma, is still unresolved. As a unique characteristic, we have identified constitutive activation of the transcription factor nuclear factor (NF)-kappaB p50-RelA in Hodgkin/Reed-Sternberg (H/RS) cells, which discriminates these neoplastic cells from most cell types studied to date. In contrast to other lymphoid and nonlymphoid cell lines tested, proliferation of H/RS cells depended on activated NF-kappaB. Furthermore, constitutive NF-kappaB p50-RelA prevented Hodgkin's lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkin's lymphoma cells depleted of constitutive nuclear NF-kappaB revealed strongly impaired tumor growth in severe combined immunodeficient mice. Our findings identify NF-kappaB as an important component for understanding the pathogenesis of Hodgkin's disease and for developing new therapeutic strategies against it.

Induction of the death-promoting gene bax-alpha sensitizes cultured breast-cancer cells to drug-induced apoptosis.

Resistance to apoptosis plays an important role in malignancies that are refractory to chemotherapy treatment. Recently we have shown that the expression of bax-alpha, a death-promoting member of the bcl-2 family, is down-regulated in breast cancer and have provided evidence that low bax expression might contribute to the pathogenesis of breast cancer. In this study we were able to demonstrate the role of this gene in chemotherapy-induced apoptosis. We transfected bax-alpha into the breast-cancer cell lines R30C and MCF-7 under the control of an inducible tetracycline-dependent expression system. Induction of bax-alpha expression did not affect viability by itself but strongly increased chemosensitivity to epirubicin. We were able to demonstrate that this sensitization is due to apoptosis. These data might explain the recently published observation that reduced expression of bax is associated with poor response rates to chemotherapy in breast cancer.

Overexpression of the death-promoting gene bax-alpha which is downregulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice.

We have studied the expression of members of the bcl-2 family in human breast cancer. The expression pattern of these genes in breast cancer tissue samples was compared with the expression pattern in normal breast epithelium. No marked difference with regard to bcl-2 and bcl-xL expression was observed between normal breast epithelium and cancer tissue. In contrast, bax-alpha, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal breast epithelium, whereas only weak or no expression could be detected in 39 out of 40 cancer tissue samples examined so far. Of interest, downregulation of bax-alpha was found in different histological subtypes. Furthermore, we transfected bax-alpha into breast cancer cell lines under the control of a tetracycline-dependent expression system. We were able to demonstrate for the first time that induction of bax expression in breast cancer cell lines restores sensitivity towards both serum starvation and APO-I/Fas-triggered apoptosis and significantly reduces tumor growth in SCID mice. Therefore, we propose that dysregulation of apoptosis might contribute to the pathogenesis of breast cancer at least in part due to an imbalance between members of the bcl-2 gene family.

High-level nuclear NF-kappa B and Oct-2 is a common feature of cultured Hodgkin/Reed-Sternberg cells.

There is a considerable lack of understanding about the common molecular defects that form the basis for the occurrence of Hodgkin's lymphoma. Despite a number of molecular tools used thus far in immunophenotypic and karyotypic studies, it has not been possible to establish a single common trait among various Hodgkin (H)-cell lines or primary tumor cells that would allow classification into a particular hematopoietic lineage. With this study, we demonstrate that a characteristic expression pattern of transcription factors provides a unifying principle. Seven different cell lines derived from patients with Hodgkin's disease (HD), as well as primary H/Reed-Sternberg (RS) cells isolated from the pericardial fluid of a patient with HD, were compared with a number of hematopoietic and nonhematopoietic cell lines for the expression of Oct-2, a tissue-specific transcription factor normally restricted to B cells, and nuclear factor kappa B (NF-kappa B), an inducible transcription factor. Regardless of the heterogeneous phenotypes and genotypes of the H cell lines, which varied inconsistently between B-cell-, T-cell-, or monocyte-like properties, all H cells tested displayed expression of Oct-2 protein at levels comparable to those seen in B cells. Furthermore, all cell lines showed an abundant constitutive nuclear NF-kappa B activity. Interestingly, anaplastic large-cell lymphoma (ALCL) cell lines, which have many features in common with H/RS cells, were devoid constitutive nuclear NF-kappa B activity. Unlike the constitutive NF-kappa B activity known for B cells, which mainly consists of the p50 and c-Rel or RelB subunits, we demonstrate by antibody supershifting experiments that H cells contain constitutive nuclear p50 and p65, the dimeric NF-kappa B normally observed only for limited time intervals after stimulation with diverse inducers. Additionally, some H-cell lines also displayed nuclear c-Rel activity, whereas RelB or p52 were not detected as part of the constitutive activity. The expression pattern of Oct-2 and NF-kappa B appears to be a unifying and characteristic property of H cells and might explain the deregulated expression of various cytokines leading to the clinical and pathologic manifestations of HD.

Cell cycle regulation of nuclear factor p32 DNA-binding activity by novel phase-specific inhibitors.

The nuclear factor p92, originally discovered by its interaction with the human papillomavirus type 18 enhancer, is a cellular protein whose activity is restricted to S phase in human primary fibroblasts. The human papillomavirus type 18 p92 binding sequence confers enhancer activity on a heterologous promoter, suggesting that p92 acts as a transcription factor. We have identified a class of nuclear inhibitory proteins, I-92s, which noncovalently associate with p92 but not with other transcription factors such as AP1, E2F, or NF-kappaB. Different I-92s occur in G1, G2, and G0, while no I-92 is detectable in S phase. Phase-specific inhibitors, therefore, are responsible for the cell cycle dependence of p92 activity and provide a novel mechanism linking transcription factor regulation with the cell cycle.

Blocking the transcription factor E2F/DP by dominant-negative mutants in a normal breast epithelial cell line efficiently inhibits apoptosis and induces tumor growth in SCID mice.

The transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis.

Multiple octamer-binding proteins are targets for the cell cycle-regulated nuclear inhibitor I-92.

p92 is a novel sequence-specific octamer-binding factor interacting with the enhancer of human papillomavirus type 18. The nuclear inhibitor I-92 regulates the DNA binding activity of p92 during the cell cycle such that p92 DNA binding is restricted to S-phase. The sequence motif ++ 5'-AATTGCTTGCATAA, consisting of two partially overlapping octamer-related sequences, represents a recognition site for p92. It was the aim of this study to characterize the complexity of proteins interacting with the 5'-AATTGCTTGCATAA motif and to determine their regulation by I-92. UV cross-linking experiments showed that, besides p92, multiple novel proteins interact with the 5'-AATTGCTGCATAA motif. These novel proteins p84, p75, p73, p69, p61, p57, p49, and p46 specifically bind to this motif, although with different affinities. The inhibitor I-92 regulates, besides p92, the DNA-binding activities of p84, p75, p73, p69, and p57 but not of p61, p49, and p46. The association of I-92 with p92, p84, p75, p73, p69, and p57 was completely reversible after treatment with the detergent deoxycholate (DOC). Finally, we analyzed I-92 specificity and found that I-92 selectively inhibited DNA binding activities of partially purified octamer-binding proteins p84 and p92 whereas DNA binding of the POU factor Oct-1 was not regulated by I-92. Our results show that I-92 regulates multiple octamer-binding proteins and these findings provide an example how gene regulation could be linked to cell cycle regulation.

Expression of the bcl-2 gene family in normal and malignant breast tissue: low bax-alpha expression in tumor cells correlates with resistance towards apoptosis.

We have studied the expression of the apoptosis-regulating genes bcl-2, bcl-x, bax and APO-1/fas (CD95) in human breast cancer. The expression pattern of these genes in human breast-cancer tissues and breast-cancer-derived cell lines was compared to that seen in normal breast epithelium and breast epithelial cell lines. No difference with regard to bcl-2 and bcl-xL expression was observed between normal breast epithelium and tumor tissue or breast cancer and non-malignant epithelial cell lines. In contrast, bax-alpha, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal cell lines and breast tissue, whereas only weak or no expression could be detected in cancer-cell lines and malignant tissue. In contrast to malignant cell lines, which express low levels of bax-alpha, non-malignant epithelial cell lines displaying high amounts of bax-alpha were highly sensitive to induction of programmed cell death by both serum starvation and APO-1/fas (CD95) triggering. We therefore propose that dysregulation of apoptosis contributes to the pathogenesis of breast cancer, at least in part, due to an imbalance between anti-apoptosis genes (such as bcl-2/bcl-x) and apoptosis-promoting genes (bax).

Characterization of the transcriptional regulatory region of the human WT1 gene.

Specific control of the expression of the Wilms tumor gene WT1 is important for normal development of the kidney. In order to characterise the transcriptional control region of the WT1 gene we have isolated genomic clones spanning the upstream region, the first WT1 exon and the 5' end of the Wit1 gene. DNA sequencing revealed that the WT1 promoter lacks a TATA box or CCAAT motif and has a GC content of 71%. Four transcriptional start sites are clustered within a 32 bp region. GC-boxes are present at nucleotide positions -413, -160, +84 and +158. DNAase I protection assays with purified Sp1 protein revealed the existence of 11 different binding sites in the WT1 promoter. WT1 and Wit1 promoter activities were tested in COS-7 cells with luciferase reporter gene constructs either containing or lacking an SV40 enhancer. WT1 promoter activity was found in a fragment extending from 449 bp upstream to 201 bp downstream of the WT1 start site. It was 26 fold lower in the absence of the SV40 enhancer than in the presence. Cotransfection with a Sp1 expression vector stimulated both constructs 3-4 fold. Wit1 promoter activity was identified in a DNA fragment extending from 200 bp upstream of the putative Wit1 TATA box to 130 bp downstream. Several potential recognition sites for WT1/EGR, Pax-8, and GAGA-like transcription factors are present in the WT1 promoter.

Identification of a negative regulatory domain in the human papillomavirus type 18 promoter: interaction with the transcriptional repressor YY1.

The human papillomavirus type 18 (HPV-18) promoter contains a TPA responsive element (TRE) which confers TPA responsiveness on a heterologous promoter. In the context of the HPV-18 promoter, however, this AP-1 site is inactive. We have identified a negative regulatory domain in the HPV-18 promoter which represses the constitutive and TPA-induced AP-1 activity. This negative regulatory sequence has been mapped to 44 nucleotides (OL13). We identified this element as a transcriptional silencer based on its ability to interfere with transcriptional initiation. This HPV-18 silencer domain was narrowed down further to 23 nucleotides, the OL13B element, which bears similarity to three other silencer sequences, present in the mouse N-ras gene upstream regulatory region, the mouse albumin gene enhancer and the adeno-associated virus P5 promoter. The transcriptional repressor protein YY1, which negatively regulates the P5 promoter, binds to the HPV-18 silencer with high affinity. Mutation of the YY1 binding site leads to an enhanced activity of the HPV-18 promoter, strongly suggesting that YY1 plays an important role in controlling HPV-18 early gene expression.

Tissue restricted expression and chromosomal localization of the YB-1 gene encoding a 42 kD nuclear CCAAT binding protein.

YB-1 cDNA clones were isolated by binding site screening of a Hela expression library using a human papillomavirus type 18 enhancer oligonucleotide. YB-1 belongs to a family of transcription factors which bind to recognition sequences containing a core CCAAT element. YB-1 bound to its single stranded recognition sequence on the sense strand but not to the anti-sense strand. A synthetic peptide antiserum derived from the predicted YB-1 amino acid sequence identified a 42 kD nuclear protein in immunoblots. A protein with the same size was detected by binding site blotting experiments using the HPV18 enhancer oligonucleotide which bound YB-1. YB-1 gene expression was restricted in tissues from a human 24 week old fetus. High levels of YB-1 mRNA were present in heart, muscle, liver, lung, adrenal gland and the brain, in contrast, low amounts of YB-1 mRNA were found in thymus, kidney, bone marrow and spleen. In pancreas, bladder, stomach and testis YB-1 mRNA could not be detected by Northern hybridization. Finally, we have identified four YB-1 related loci in the mouse genome and have mapped these loci to four different mouse chromosomes by interspecific backcross analysis.

A novel nuclear inhibitor I-92 regulates DNA binding activity of octamer binding protein p92 during the cell cycle.

Nuclear DNA binding protein p92 is a sequence specific octamer binding protein with identical molecular weight as the ubiquitous octamer binding protein Oct-1. It binds to octamer related sequences from the enhancer of human papillomavirus type 18. The activity and intracellular distribution of p92 is regulated by extracellular signals. In serum starved Hela-fibroblast hybrid cells p92 is localized to the cytosol. Serum stimulation leads to nuclear import of p92. In fractions of asynchronously growing cells, which were separated according to cell cycle phases into G1, S, and G2 populations by centrifugal elutriation, p92 DNA binding is confined to S phase. In binding site blots however, p92 DNA binding activity is also present in G1 and G2. In G1 and G2 DNA binding activity of p92 is masked by a novel nuclear inhibitor I-92. The cyclic association of p92 with its inhibitor I-92 provides a new mechanism of regulating S phase dependent activity of a sequence specific DNA binding protein.

Delineation of human papillomavirus type 18 enhancer binding proteins: the intracellular distribution of a novel octamer binding protein p92 is cell cycle regulated.

The enhancer of human papillomavirus type 18 consists of two functionally redundant domains, one is partially conserved between HPV18 and HPV16, both mediate strong transcriptional enhancement. In contrast, short fragments of the enhancer mediate low transcriptional enhancement, suggesting that there is functional cooperation between HPV enhancer binding factors. Previously interactions of the enhancer with NF-1, AP1 and steroid receptors were shown by EMSA. Here we show by binding site blotting, that four novel sequence specific proteins p110, p92, p42 and p40 bind to the enhancer. Nuclear proteins p110 and p92 bind at repeated sites in the enhancer, proteins p42 and p40 only at one site. Recognition sequences for p110 and p92 were identified in a TTGCTTGCATAA sequence motif and consist of an overlapping p110 and p92 recognition site. The specific interaction of p110 with G residues of this 12 nucleotide long sequence was demonstrated by a mutant recognition site. Single recognition sites for p42 and p40 were localized in the enhancer by the use of overlapping oligonucleotides. In addition, electrophoretic mobility shift analysis identified Oct-1 and AP2 interactions with the enhancer. The AP2 binding site was mapped to a AGGCACATATT motif. The p92 protein binds to enhancer oligonucleotides, containing at least one copy of Oct-1 like recognition sequences, these oligonucleotides also bind synthetic Oct-1 protein. During serum starvation or at high saturation density, p92 moves from the nucleus into the cytoplasm. Immunoblots of cytoplasmic extracts with anti-Oct-1 antisera showed, that p92 is a novel octamer binding factor, which is not immunologically related to the Oct-1 protein. The intracellular p92 distribution is regulated at the G0/G1 boundary of the cell cycle, by nucleo-cytoplasmic translocation.