Mechanisms of HER2/{\it neu\/} proto-oncogene overexpression in breast cancer cells

Overexpression and amplification of HER2/neu have been documented in many primary tumors, most notably in breast. Not only do approximately 30% of breast cancer patients carry tumors that overexpress the gene, but those that do generally have shorter overall and disease-free survival times than patients with tumors expressing low levels of HER2/neu. Thus, overexpression of HER2/neu plays an important role in the pathogenesis of breast cancer. We have examined the mechanisms that result in HER2/neu overexpression in breast cancer by using, as a model system, established breast cancer cell lines that express much higher levels of HER2/neu mRNA than normal breast tissue while maintaining a near normal HER2/neu gene copy number. Nuclear run-on experiments indicate that the breast cancer cell lines MDA-MB453, BT483, and BT474 have an increased HER2/neu gene transcription rate. By using HER2/neu promoter-CAT constructs, we have found that the enhanced HER2/neu transcription rate in MDA-MB453 cells is due to activation of the gene in trans, while the enhanced transcription rate in BT483 cells is due to activation of the gene in either trans or cis. In BT474 cells, transcriptional upregulation is primarily due to gene amplification. Since the levels of increased transcription are not as high as the levels of HER2/neu mRNA in any of these three lines, post-transcriptional deregulation that increases HER2/neu expression must also be functioning in these cells. The half-life of HER2/neu mRNA was measured and found to be equivalent in these lines as in a control. Thus, the post-transcriptional deregulation is not increased stability of the HER2/neu transcript.^ Much work has been performed in characterizing the altered trans-acting factor involved in increased HER2/neu transcription in MDA-MB453 cells. Using promoter deletion constructs linked to a reporter gene, the region responsive to this factor was localized in the rat neu promoter. When human HER2/neu promoter constructs were used, the homologous sequence in the human promoter was identified. Furthermore, a number of protein/DNA complexes are detected when these promoter regions are used in gel mobility shift assays. UV-crosslinking experiments indicate DNA-binding proteins of roughly 110 kDa, 70 kDa, and 35 kDa are capable of interacting with the human promoter element. ^

Modulation of BCL-X$\sb{\rm L}$ is a critical determinant of c-myc induced apoptosis

The fine balance between proliferation and apoptosis plays a primary role in carcinogenesis. Proto-oncogenes that induce both proliferation and apoptosis provide a powerful inbuilt system to inhibit clonal expansion of cells with high proliferation rates. This provides a restraint to the development of neoplasms. C-myc expressing cells undergo apoptosis in low serum by an unknown mechanism. Several lines of evidence suggested that c-myc induces apoptosis by a transcriptional mechanism. However, the target genes of this program have not been fully defined. Protein synthesis inhibitors induce apoptosis in c-myc over-expressing cells at high serum levels suggesting that inhibition of synthesis of a survival factor may induce apoptosis. We show that the expression of c-myc directly correlates with an increase in the level of a survival protein, bcl-$\rm x\sb{L},$ and a decrease in the pro-apoptotic protein, bax, at both the protein and mRNA level. Furthermore, a significant decrease of the bcl-$\rm x\sb{L}$ protein levels is observed under low serum conditions. In order to investigate the mechanism of regulation of bcl-$\rm x\sb{L}$ and bax by c-myc, the bcl-x and bax promoters were cloned, sequenced and shown to contain c-myc binding sites. The chloramephenicol acetyl transferase (CAT) reporter assay was used to demonstrate activation of the bcl-x promoter by increasing levels of c-myc when co-transfected in COS cells. The bax promoter was also shown to be transrepressed in c-myc expressing cells. The role of bcl-$\rm x\sb{L}$ in apoptosis regulation in c-myc cell lines in normal and low serum was then investigated. Cells lines expressing c-myc and bcl-$\rm x\sb{L}$ were generated and were shown to be resistant to apoptosis induction in low serum. Furthermore, cell lines expressing c-myc, anti-sense bcl-$\rm x\sb{L}$ and $\beta$-galactosidase demonstrated significantly enhanced rates of apoptosis in high serum compared to c-myc Rat 1a cells. These findings suggest that c-myc activates a survival program involving bcl-$\rm x\sb{L}$ upregulation and bax downregulation. However, this survival signal is reduced under low serum conditions by the relative downregulation of bcl-$\rm x\sb{L}$ allowing for apoptosis to proceed. These data also directly demonstrates that downregulation in the level of bcl-$\rm x\sb{L}$ associated with low serum conditions is a critical determinant of c-myc induced apoptosis. ^

The interferon-inducible murine p48 (ISGF3γ) gene is regulated by protooncogene c-myc

p48 protein is an integral component of the multimeric interferon (IFN)-regulated transcription factor, ISGF3. We have shown earlier that this gene is regulated by a novel IFN-γ-regulated element. In addition to the IFN-regulated element, a myc–max binding site is also present in this promoter. In this investigation we have studied the role of this site in the regulation of the p48 gene. In serum-induced quiescent cells Myc up-regulated the expression of p48 mRNA. We show that the protooncogene Myc regulates the expression of p48 through the element CACGTG. Mutations in this motif abolish Myc-inducibility of the reporter genes carrying p48 promoter elements. Purified Myc and Max proteins interact with the Myc-stimulated element of the p48 promoter. We also show that cells lacking p48 expression are highly susceptible to the cytocidal action of anticancer drugs. Taken together these data suggest that p48 may function as an anti-stress cell survival factor.

c-Myc enhances protein synthesis and cell size during B lymphocyte development

Members of the myc family of nuclear protooncogenes play roles in cell proliferation, differentiation, and apoptosis. Moreover, inappropriate expression of c-myc genes contributes to the development of many types of cancers, including B cell lymphomas in humans. Although Myc proteins have been shown to function as transcription factors, their immediate effects on the cell have not been well defined. Here we have utilized a murine model of lymphomagenesis (Eμ-myc mice) to show that constitutive expression of a c-myc transgene under control of the Ig heavy-chain enhancer (Eμ) results in an increase in cell size of normal pretransformed B lymphocytes at all stages of B cell development. Furthermore, we show that c-Myc-induced growth occurs independently of cell cycle phase and correlates with an increase in protein synthesis. These results suggest that Myc may normally function by coordinating expression of growth-related genes in response to mitogenic signals. Deregulated c-myc expression may predispose to cancer by enhancing cell growth to levels required for unrestrained cell division.

c-Myc target gene specificity is determined by a post-DNAbinding mechanism

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

YY1 and c-Myc associate in vivo in a manner that depends on c-Myc levels.

The c-Myc oncoprotein has previously been shown to associate with transcription regulator YY1 and to inhibit its activity. We show herein that endogenous c-Myc and YY1 associate in vivo and that changes in c-Myc levels, which accompany mitogenic stimulation or differentiation of cultured cells, affect the ratio of free to c-Myc-associated YY1. We have also investigated the mechanism by which association with c-Myc inhibits YY1's ability to regulate transcription. c-Myc does not block binding of YY1 to DNA. However, protein association studies suggest that c-Myc interferes with the ability of YY1 to contact basal transcription proteins TATA-binding protein and TFIIB.

Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element.

NM23-H2, a presumed regulator of tumor metastasis in humans, is a hexameric protein with both enzymatic (NDP kinase) and regulatory (transcriptional activation) activity. While the structure and catalytic mechanisms have been well characterized, the mode of DNA binding is not known. We examined this latter function in a site-directed mutational study and identified residues and domains essential for the recognition of a c-myc regulatory sequence. Three amino acids, Arg-34, Asn-69, and Lys-135, were found among 30 possibilities to be critical for DNA binding. Two of these, Asn-69 and Lys-135, are not conserved between NM23 variants differing in DNA-binding potential, suggesting that DNA recognition resides partly in nonconserved amino acids. All three DNA-binding defective mutant proteins are active enzymatically and appear to be stable hexamers, suggesting that they perform at the level of DNA recognition and that separate functional domains exist for enzyme catalysis and DNA binding. In the context of the known crystal structure of NM23-H2, the DNA-binding residues are located within distinct structural motifs in the monomer, which are exposed to the surface near the 2-fold axis of adjacent subunits in the hexamer. These findings are explained by a model in which NM23-H2 binds DNA with a combinatorial surface consisting of the "outer" face of the dimer. Chemical crosslinking data support a dimeric DNA-binding mode by NM23-H2.

Glycosylation of the c-Myc transactivation domain.

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic posttranslational modification composed of a single monosaccharide, GlcNAc, glycosidically composed of a single monosaccharide, GlcNAc, glycosidically linked to the side-chain hydroxyl of serine or threonine residues. Although O-GlcNAc occurs on a myriad of nuclear and cytoplasmic proteins, only a few have thus far been identified. These O-GlcNAc-bearing proteins are also modified by phosphorylation and form reversible multimeric complexes. Here we present evidence for O-GlcNAc glycosylation of the oncoprotein c-Myc, a helix-loop-helix/leucine zipper phosphoprotein that heterodimerizes with Max and participates in the regulation of gene transcription in normal and neoplastic cells. O-GlcNAc modification of c-Myc is shown by three different methods: (i) demonstration of lectin binding to in vitro translated protein using a protein-protein interaction mobility-shift assay; (ii) glycosidase or glycosyltransferase treatment of in vitro translated protein analyzed by lectin affinity chromatography; and (iii) direct characterization of the sugar moieties on purified recombinant protein overexpressed in either insect cells or Chinese hamster ovary cells. Analyses of serial deletion mutants of c-Myc further suggest that the O-GlcNAc site(s) are located within or near the N-terminal transcription activation/malignant transformation domain, a region where mutations of c-Myc that are frequently found in Burkitt and AIDS-related lymphomas cluster.

Prognostic role of the LCS6 KRAS variant in locally advanced rectal cancer: results of the EXPERT-C trial.

Background: Lethal-7 (let-7) is a tumour suppressor miRNA which acts by down-regulating several oncogenes including KRAS. A single-nucleotide polymorphism (rs61764370, T > G base substitution) in the let-7 complementary site 6 (LCS-6) of KRAS mRNA has been shown to predict prognosis in early-stage colorectal cancer (CRC) and benefit from anti-epidermal growth factor receptor monoclonal antibodies in metastatic CRC. Patients and methods: We analysed rs61764370 in EXPERT-C, a randomised phase II trial of neoadjuvant CAPOX followed by chemoradiotherapy, surgery and adjuvant CAPOX plus or minus cetuximab in locally advanced rectal cancer. DNA was isolated from formalin-fixed paraffin-embedded tumour tissue and genotyped using a PCR-based commercially available assay. Kaplan–Meier method and Cox regression analysis were used to calculate survival estimates and compare treatment arms. Results: A total of 155/164 (94.5%) patients were successfully analysed, of whom 123 (79.4%) and 32 (20.6%) had the LCS-6 TT and LCS-6 TG genotype, respectively. Carriers of the G allele were found to have a statistically significantly higher rate of complete response (CR) after neoadjuvant therapy (28.1% versus 10.6%; P = 0.020) and a trend for better 5-year progression-free survival (PFS) [77.4% versus 64.5%: hazard ratio (HR) 0.56; P = 0.152] and overall survival (OS) rates (80.3% versus 71.9%: HR 0.59; P = 0.234). Both CR and survival outcomes were independent of the use of cetuximab. The negative prognostic effect associated with KRAS mutation appeared to be stronger in patients with the LCS-6 TT genotype (HR PFS 1.70, P = 0.078; HR OS 1.79, P = 0.082) compared with those with the LCS-6 TG genotype (HR PFS 1.33, P = 0.713; HR OS 1.01, P = 0.995). Conclusion: This analysis suggests that rs61764370 may be a biomarker of response to neoadjuvant treatment and an indicator of favourable outcome in locally advanced rectal cancer possibly by mitigating the poor prognosis of KRAS mutation. In this setting, however, this polymorphism does not appear to predict cetuximab benefit.

Clinical and molecular characterization of feline mammary carcinomas overexpressing HER2 proto-oncogene (FMC-HER2+) : new strategies for effective diagnostic and cancer therapy

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas

Proscillaridin A effects on histone acetylation and C-MYC degradation in acute lymphoblastic leukemia

La leucémie lymphoblastique aiguë (LLA) représente environ 25% des cancers pédiatriques diagnostiqués chaque année. Dans 80 % des cas, une rémission complète est observée. Cependant, les patients résistants aux traitements ainsi que les patients en rechute présentent un mauvais pronostique. Les altérations épigénétiques sont des facteurs essentiels dans le développement et la progression de la maladie, ainsi qu’à la résistance aux traitements. Lors d’un criblage de médicaments approuvés par la FDA, nous avons découvert des molécules ayant des caractéristiques anticancéreux et épigénétiques. Pour évaluer l’activité de ces molécules, nous avons procédé à un criblage secondaire sur plusieurs lignées cellulaires leucémiques. Nous avons découvert qu’une de ces molécules, un glucoside cardiotonique appelé la proscillaridine A, avait une activité anticancéreuse spécifique pour des cellules leucémiques. Nous faisons donc l’hypothèse que la proscillaridine A pourrait avoir des effets épigénétiques et anticancéreux dans des modèles précliniques de LLA. Pour tester cette hypothèse, nous avons traité deux lignées cellulaires de LLA Nalm-6 (LLA pre-B) et Molt-4 (T-LLA) in vitro pendant 2 à 96 heures à des doses pertinentes sur le plan clinique. Nous avons alors pu observer une inhibition de croissance qui était dépendante de la dose administrée dans les deux lignées cellulaires, avec des valeurs de 50% d’inhibition de croissance (CI50) de 3.0 nM pour les Nalm-6 et de et 2.3 nM pour les Molt-4. De plus, nos études sur le cycle cellulaire par BrdU démontrent un arrêt en phase G2/M. Nous avons également détecté par immunobuvardage de type western des baisses significatives de l’acétylation de résidus de l’histone 3. Les niveaux d’expression des enzymes responsables de cette acétylation, les histones acétyltransférases CBP, P300 et TIP60 ainsi que de l’oncogène C-MYC étaient également diminuées. Par des analyses de séquençage de l’ARN, nous avons observé une augmentation de l’expression des gènes impliquées dans les processus d’apoptose et de différentiation cellulaire, ainsi qu’une diminution des gènes impliqués dans la prolifération cellulaire comme en particulier les gènes cibles de C-MYC. Ces résultats prometteurs suggèrent le potentiel prometteur de la proscillaridine A comme nouvelle thérapie pour les patients atteints de LLA.

Proscillaridin A effects on histone acetylation and C-MYC degradation in acute lymphoblastic leukemia

La leucémie lymphoblastique aiguë (LLA) représente environ 25% des cancers pédiatriques diagnostiqués chaque année. Dans 80 % des cas, une rémission complète est observée. Cependant, les patients résistants aux traitements ainsi que les patients en rechute présentent un mauvais pronostique. Les altérations épigénétiques sont des facteurs essentiels dans le développement et la progression de la maladie, ainsi qu’à la résistance aux traitements. Lors d’un criblage de médicaments approuvés par la FDA, nous avons découvert des molécules ayant des caractéristiques anticancéreux et épigénétiques. Pour évaluer l’activité de ces molécules, nous avons procédé à un criblage secondaire sur plusieurs lignées cellulaires leucémiques. Nous avons découvert qu’une de ces molécules, un glucoside cardiotonique appelé la proscillaridine A, avait une activité anticancéreuse spécifique pour des cellules leucémiques. Nous faisons donc l’hypothèse que la proscillaridine A pourrait avoir des effets épigénétiques et anticancéreux dans des modèles précliniques de LLA. Pour tester cette hypothèse, nous avons traité deux lignées cellulaires de LLA Nalm-6 (LLA pre-B) et Molt-4 (T-LLA) in vitro pendant 2 à 96 heures à des doses pertinentes sur le plan clinique. Nous avons alors pu observer une inhibition de croissance qui était dépendante de la dose administrée dans les deux lignées cellulaires, avec des valeurs de 50% d’inhibition de croissance (CI50) de 3.0 nM pour les Nalm-6 et de et 2.3 nM pour les Molt-4. De plus, nos études sur le cycle cellulaire par BrdU démontrent un arrêt en phase G2/M. Nous avons également détecté par immunobuvardage de type western des baisses significatives de l’acétylation de résidus de l’histone 3. Les niveaux d’expression des enzymes responsables de cette acétylation, les histones acétyltransférases CBP, P300 et TIP60 ainsi que de l’oncogène C-MYC étaient également diminuées. Par des analyses de séquençage de l’ARN, nous avons observé une augmentation de l’expression des gènes impliquées dans les processus d’apoptose et de différentiation cellulaire, ainsi qu’une diminution des gènes impliqués dans la prolifération cellulaire comme en particulier les gènes cibles de C-MYC. Ces résultats prometteurs suggèrent le potentiel prometteur de la proscillaridine A comme nouvelle thérapie pour les patients atteints de LLA.

Caractérisation structurale de Miz-1 dans le cadre de la répression génique causée par le complexe c-Myc/Miz-1

Résumé : c-Myc est un facteur de transcription (FT) dont les niveaux cellulaires sont dérégulés dans la majorité des cancers chez l’homme. En hétérodimère avec son partenaire obligatoire Max, c-Myc lie préférentiellement les séquences E-Box (CACGTG) et cause l’expression de gènes impliqués dans la biosynthèse des protéines et des ARNs, dans le métabolisme et dans la prolifération cellulaire. Il est maintenant bien connu que c-Myc exerce aussi son potentiel mitogène en liant et inhibant différents FTs impliqués dans l’expression de gènes cytostatiques. Entre autres, c-Myc est en mesure d’inhiber Miz-1, un FT comportant 13 doigts de zinc de type Cys2-His2 (ZFs) impliqué dans l’expression de plusieurs gènes régulateurs du cycle cellulaire comprenant les inhibiteurs de CDK p15[indice supérieur INK4], p21[indice supérieur CIP1] et p57[indice supérieur KIP2]. Plus récemment, il fut démontré qu’en contrepartie, Miz-1 est aussi en mesure de renverser les fonctions activatrices de c-Myc et de prévenir la prolifération de cellules cancéreuses dépendantes de c-Myc. Ces différentes observations ont mené à la suggestion de l’hypothèse intéressante que la balance des niveaux de Miz-1 et c-Myc pourrait dicter le destin de la cellule et a permis d’établir Miz-1 comme nouvelle cible potentielle pour le développement d’agents anti-cancéreux. Malgré le fait que ces deux protéines semblent centrales à la régulation du cycle cellulaire, les mécanismes moléculaires leur permettant de s’inhiber mutuellement ainsi que les déterminants moléculaires permettant leur association spécifique demeurent assez peu documentés pour le moment. De plus, la biologie structurale de Miz-1 demeure à être explorée puisque qu’aucune structure de ses 13 ZFs, essentiels à sa liaison à l’ADN, n’a été déterminée pour l’instant. Les travaux réalisés dans le cadre cette thèse visent la caractérisation structurale et biophysique de Miz-1 dans le contexte de la répression génique causée par le complexe c-Myc/Miz-1. Nous présentons des résultats d’éxpériences in vitro démontrant que Miz-1 interagit avec c-Myc via un domaine contenu entre ses ZFs 12 et 13. De plus, nous démontrons que Miz-1 et Max sont en compétition pour la liaison de c-Myc. Ces résultats suggèrent pour la permière fois que Miz-1 inhibe les activités de c-Myc en prévenant son interaction avec son partenaire obligatoire Max. De plus, ils laissent présager que que Miz-1 pourrait servir de référence pour le développement d’inhibiteurs peptidiques de c-Myc. Finalement, nous avons réalisé la caractérisation structurale et dynamique des ZFs 1 à 4 et 8 à 10 de Miz-1 et avons évalué leur potentiel de liaison à l’ADN. Les résultats obtenus, couplés à des analyses bio-informatiques, nous permettent de suggérer un modèle détaillé pour la liaison spécifique de Miz-1 à son ADN consensus récemment identifié.

Expression pattern of Oct-4, Sox2, and c-Myc in the primary culture of human dental pulp derived cells

Dental pulp cells (DPCs) have shown promising potential in dental tissue repair and regeneration. However, during in vitro culture, these cells undergo replicative senescence and result in significant alteration in cell proliferation and differentiation. Recently, the transcription factors of Oct-4, Sox2, c-Myc, and Klf4 have been reported to play a regulatory role in the stem cell self-renewal process, namely cell reprogramming. Therefore, it is interesting to know whether the replicative senescence during the culture of dental pulp cells is related to the diminishing of the expression of these transcription factors. In this study, we investigated the expression of the reprogramming markers Oct-4, Sox2, and c-Myc in the in vitro explant cultured dental pulp tissues and explant cultured dental pulp cells (DPCs) at various passages by immunofluorescence staining and real-time polymerase chain reaction analysis. Our results demonstrated that Oct-4, Sox2, and c-Myc translocated from nucleus in the first 2 passages to cytoplasm after the third passage in explant cultured DPCs. The mRNA expression of Oct-4, Sox2, and c-Myc elevated significantly over the first 2 passages, peaked at second passage (P < .05), and then decreased along the number of passages afterwards (P < .05). For the first time we demonstrated that the expression of reprogramming markers Oct-4, Sox2, and c-Myc was detectable in the early passaged DPCs, and the sequential loss of these markers in the nucleus during DPC cultures might be related to the cell fate of dental pulp derived cells during the long-term in vitro cultivation under current culture conditions.

Effect of bone morphogenetic protein-4 (BMP-4) on the expression of Sox2, Oct-4 and c-Myc in human periodontal ligament cells during long-term culture

Recent studies demonstrated endogenous expression level of Sox2, Oct-4 and c-Myc is correlated with the pluripotency and successful induction of induced pluripotent stem cells (iPSCs). Periondontal ligament cells (PDLCs)have multi-lineage diferentiation capability and ability to maintain undifferentiated stage, which makes PDLCs a suitable cell source for tissue repair and regeneration. To elucidate the effect of in vitro culture condition on the stemness potential of PDLCs, we explored the cell growth, proliferation, cell cycle, and the expression of Sox2, Oct-4 and c-Myc in PDLCs from passage 1 to 7 with or without the addition of recombinant human BMP4(rhBMP4). Our results revealed that BMP-4 promoted cell growth and proliferation, arrested PDLCs in S phase of cell cycle and upregulated PI value. It was revealed that without the addition of rhBMP4, the expression of Sox2, Oct-4 and c-Myc in PDLCs only maintained nucleus location until passage 3, then lost nucleus location subsequently. The mRNA expression in PDLCs further confirmed that the level of Sox2 and Oct-4 peaked at passage 3, then decreased afterwards, whereas c-Myc maintained consistently upregulation along passages. after the treatment with rhBMP4, the expression of Sox2, Oct-4 and c-Myc in PDLCs maintained nucleus location even at passage 7 and the mRNA expression of Sox2 and Oct-4 significantly upregulated at passage 5 and 7. These results demonstrated that addition of rhBMP-4 in the culture media could improve the current culture condition for PDLCs to maintain in an undifferentiated stage.