O splicing alternativo é o processo pelo qual diversos transcritos podem ser gerados a partir de um único gene, sendo de extrema importância para diversidade do repertório transcricional e proteico. Diferentes variantes de splicing são expressas entre os diferentes tecidos e estágios de desenvolvimento garantindo o funcionamento normal da célula, portanto, qualquer alteração neste padrão pode resultar no aparecimento de doenças. Neste contexto, o objetivo deste trabalho foi o estabelecimento de metodologias para identificação de variantes de splicing em câncer de mama sob influência do oncogene ERBB2, o qual é um marcador de mau prognóstico altamente expresso em cerca de 30% dos tumores de mama. Foram estabelecidas duas estratégias para construção de bibliotecas de cDNA. A construção de bibliotecas de cDNA enriquecidas para splicing alternativo, baseada na formação e captura de moléculas de heteroduplexes em combinação com a amplificação de RNAm, foi realizada a partir de RNA total de linhagem celular de mama e a partir de um grupo de cinco amostras tumorais, todas com alta expressão de ERBB2. Foram identificadas 79 possíveis variantes de splicing alternativo em câncer de mama, das quais 18 foram selecionadas para validação por RT-PCR. Foi obtida uma taxa de vallidação de 94% e foram identificadas duas novas variantes de splicing alternativo. A regulação da expressão mediada por ERBB2 de três variantes de splicing foi confirmada por duas metodologias distintas, eletroforese em chip e estratégia baseada na ligação de sondas específicas, que revelou desbalanço de expressão entre as variantes, demonstrando a influência do oncogene na regulação de variantes de splicing. A segunda abordagem utilizada, foi a construção de bibliotecas de cDNA para avaliação do transcriptoma total, utilizando sequênciamento de alto desempenho. Foram utilizados RNA total de duas linhagens celulares de mama que diferem apenas na expressão do gene ERBB2. Foram identificadas 2.865 novas variantes de splicing, das quais 20, que reportaram a identificação de um novo éxon, foram selecionadas para validação, com uma taxa de validação de 90%. Seis destas variantes apresentaram aumento de expressão na linhagem com alta expressão de ERBB2. Além disso, foi detectado um enriquecimento de algumas categorias de variantes na linhagem celular com alta expressão de ERBB2, reforçando a influência do oncogene na regulação do splicing alternativo, podendo resultar em variantes de splicing associadas a este grupo de câncer de mama, que podem ser candidatas a marcadores moleculares. / Alternative splicing is a process, by which many differente transcripts can be generated by one single gene, significantly expanding the transcriptional and proteomic diversity. Different splicing variants are generated among different transcripts and developmental stages, assuring normal cell function. Therefore, alterations in the splicing process can lead to diseases outcome. In this context, the aim of this study was the establishment of methodologies for the identification of alternative splicing in breast cancer influenced by ERBB2 oncogene, which is a poor prognostic molecular marker, highly expressed in 30% of human breast cancer. Two strategies were established for the construction of cDNA libraries. Alternative enriched splicing libraries, based on heteroduplex capture combined with mRNA amplification, were constructed from total RNA from a cell line and also from five tumor samples, all of them presenting high ERBB2 expression. Seventy nine putative splicing variants were identified and 18 of them were selected for RT-PCR validation. A high validation level was obtained (94%) and two novel alternative splicing variants were identified. ERBB2 mediated regulation was confirmed for three variants by two distinct methodologies, electrophoresis on a chip and probe specific ligation approach. The alteration in the expression balance of variants suggests the influence of the oncogene in the splicing pattern regulation. The second strategy was the construction of cDNA libraries for global transcriptome analysis based on deep sequencing. Total RNA from two mammary epithelial cell lines expressing different ERBB2 levels were used and 2,865 novel splicing variants were identified. Twenty novel events reporting the inclusion of novel exons were selected for RT-PCR validation with 90% validation rate. Six variants presented higher expression in the cell line with high levels of ERBB2. Moreover enrichment in splicing events was detected in the ERBB2 high expressing cell line, supporting the ERBB2 influence in alternative splicing regulation, possibly resulting in splicing variants associated to this subgroup of cancer that can be tested as molecular markers.

Her2-positiver Brustkrebs, der Subtyp des Mammakarzinoms, bei dem eine Überexpression des epidermalen Wachstumsfaktor-Rezeptors-2 (ErbB2/Her2) vorliegt, hat für die betroffenen Frauen eine besonders schlechte Prognose. Eine positive Korrelation zwischen der ErbB2-Expression in Brusttumoren und der Expression des Makrophagen-Migration-inhibierenden Faktors (MIF), einem inzwischen gut bekannten tumorfördernden Protein, konnte bereits gezeigt werden. Ferner konnte gezeigt werden, dass MIF durch das Hitzeschockprotein 90 (Hsp90) in einem Mausmodell des Her2-positiven Brustkrebses stabilisiert wird. Der zugrundeliegende Mechanismus war bisher unverstanden. In dieser Doktorarbeit konnte erstmalig demonstriert werden, dass Hitzeschockfaktor-1 (HSF-1), der Transkriptionsfaktor der stressinduzierten Hitzeschockproteine (HSP), einschließlich Hsp90, in ErbB2-überexprimierenden SK-BR-3-Brustkrebszellen konstitutiv durch ErbB2 aktiviert wird. Durch eine Behandlung mit dem ErbB2-Inhibitor CP724.714 konnte die aktivierende Serin326-Phosphorylierung von HSF-1 verhindert werden. Als Folge wird, wie durch Western-Blot-Analysen gezeigt, die HSP-Maschinerie inhibiert und tumorfördernde Hsp90-Klienten wie MIF, Akt, mutiertes p53, ErbB2 und HSF-1 destabilisiert. Außerdem konnte die unterbleibende HSF-1-Aktivierung durch quantitative PCR-Analysen und Immunfluoreszenzmikroskopie bestätigt werden. Die mechanistischen Untersuchungen konnten die hier erstmalig beschriebene ErbB2-Akt-HSF-1-Achse aufdecken, über die HSF-1 in SK-BR-3-Zellen reguliert wird. Ferner konnte eine ErbB2-Inhibition sogar die HSF-1-Aktivierung durch einen Hitzeschock unterbinden. Unsere Ergebnisse zeigen zum ersten Mal, dass die ErbB-2-Überexpression in SK-BR-3-Zellen eine konstitutive HSF-1-Aktivierung bewirkt, mit der Folge, dass tumorfördernde Hsp90-Klienten wie MIF, HSF-1 selbst, ErbB2 und mutiertes p53 stabilisiert werden. Die neu entdeckte ErbB2-Akt-HSF-1-Hsp90-Klienten-Achse legt möglicherweise neue Angriffspunkte für zusätzliche Pharmaka bei der Therapie Her2-positiven Brustkrebses offen.

MUC4 is a heterodimeric mucin glycoprotein expressed in the epithelia of tissues. Previous studies in our laboratory demonstrated that MUC4 protein expression is regulated by exogenous growth factors and that MUC4 is found in complex with the receptor tyrosine kinase ErbB2. MUC4 protein expression in airway epithelia was evaluated using molecular biology techniques. The impact of the protein on ErbB2 activation was evaluated post mechanical wounding of airway epithelia, and upon MUC4 RNA silencing. MUC4 levels were increased with exposure to the differentiating agent retinoic acid and decreased upon exposure to epidermal growth factor, a proliferative agent. In the absence of MUC4, ErbB2 phosphorylation was diminished. These results support the hypothesis that MUC4 expression is enhanced during differentiation of epithelia. Furthermore these findings provide evidence for an additional level of ErbB regulation in airway injury and subsequent epithelial wound healing.

Le cancer du sein est le cancer le plus fréquent chez la femme (Boyle and Ferlay, 2005; Ferlay et al., 2007). Même si les traitements sont de plus en plus efficaces, il est responsable denviron 130 000 décès en Europe. Environ 20-30% des cancers du sein surexprime le gène ERBB2. Cette surexpression confère à la cellule un profil tumoral très agressif, et résistant aux chimiothérapies conventionnelles. Létude des facteurs responsable de la surexpression du gène ERBB2 dans les cancers du sein est le thème principal de recherche du laboratoire doncologie moléculaire. Notre laboratoire, a étudié les mécanismes moléculaires responsables de la surexpression du gène ERBB2 dans les cancers du sein. Nous avons montré que la surexpression est la conséquence dune stimulation de la transcription et non de la stabilisation de lARN (Pasleau et al., 1993). Pour étudier la régulation de la transcription un fragment de 6 kb du promoteur du gène ERBB2 a été cloné et séquencé. Différentes régions régulatrices ont été identifiées (Grooteclaes et al., 1994). Plusieurs sites de liaison pour des facteurs AP-2 ont été identifiés (Delacroix et al., 2005; Grooteclaes et al., 1999; Vernimmen et al., 2003a). La fixation des facteurs au promoteur a été vérifiée par Chromatin Immuniprecipitation (ChIP assay) (Begon et al., 2005; Delacroix et al., 2005). Afin de mieux comprendre le fonctionnement des facteurs de transcription AP-2, nous avons cherché les protéines interagissant avec ce facteur et contribuant à la surexpression dERBB2 dans des lignées de cancer du sein. Précédemment, Dominique Begon a montré linteraction de la protéine YY1 avec le facteur de transcription AP-2α et leur implication sur le promoteur du gène ERBB2 (Begon et al., 2005). La première partie de mon travail a été de mettre en corrélation par immunohistochimie lexpression des protéines YY1 et AP-2α avec la surexpression dERBB2 dans des tumeurs primaires. Nous avons également voulu étudier leffet dune diminution des protéines YY1 et AP-2 sur lexpression dERBB2, à laide de siRNAs (Voir article 1 en annexe, (Allouche A and Nolens G. et al., 2008). La deuxième partie de ce travail a porté sur lidentification dautres protéines pouvant interagir avec les protéines AP-2. Après purification, les protéines Ku70 et Ku80 furent identifiées. Nous avons voulu étudier leffet de cette interaction sur lactivité des protéines AP-2 (α et γ) et sur lexpression dERBB2 (voir article 2 en annexe).

Tolfenamic acid (TA), betulinic acid (BA) and acetylsalicylic acid (aspirin) are anticancer drugs, and Sp1, Sp3, Sp4 transcription factors and growth factor 2 (EGFR2, HER2 / ErbB2) are important molecular markers in cancer cells. In this study the molecular mechanisms by which these anticancer drugs target downregulation of Sp1, Sp3, Sp4 and ErbB2 are investigated in breast cancer cells. TA inhibits growth of ErbB2-overexpressing BT474 and SKBR3 breast cancer cells by inhibiting ErbB2 expression. In cells treated with TA, ErbB2 mRNA expression and promoter activity were decreased, and this was due to decreased mRNA stability in BT474 cells. In both cell lines, TA also decreased expression of the YY1 and AP-2 transcription factors that are required for basal ErbB2 expression. These effects were accompanied by decreased ErbB2-dependent kinase activities, induction of p27, and decreased expression of cyclin D1. In addition, TA also inhibited tumor growth in BT474 cells orthotopic mouse model. However, Sp proteins were not the major target of TA in these breast cancer cells and this contrasts to results in pancreatic cancer cells where TA decreased expression of Sp proteins. BA inhibits growth of ErbB2 –overexpressing BT474 and MDA-MB-453 and induced apoptosis in these cells. BA induced proteasome-independent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, Sp4 and survivin, an Sp-regulated gene, and BA also decreased expression of ErbB2, ErbB2-regulated kinases and also YY1, a transcription factor that regulates ErbB2 expression in these cells. Knockdown of Sp1, Sp3, Sp4 and their combination by RNA interference was accompanied by decreased expression of ErbB2, YY1 and luciferase activity in cells transfected with a construct containing the GC-rich YY1 promoter linked to a luciferase reporter gene. BA-dependent repression of Sp1, Sp3, Sp4 and Sp regulated genes was due in part to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a) which constitutively inhibits ZBTB10 expression. The effects of BA on the miR-27a:zBTB10-Sp transcription factor axis were inhibited in cells cotreated with the cannabinoid 1 (CB1) and CB2 receptor antagonists AM251 and AM630 respectively. However, in vitro binding studies with ≤ 10 mM BA and a radiolabeled cannabinoid did not indicate direct competitive binding of BA to the CB1 and CB2 receptors, suggesting a possible role for other CB-like G protein-coupled receptors. Aspirin inhibits growth, induces apoptosis and decreases cell migration in BT474 and MDA-MB-453 breast cancer cells that overexpress the ErbB2 oncogene. Aspirin also downregulated ErbB2 expression in these cells and this was accompanied by inhibition of downstream kinases including phospho-Akt (p-Akt) and phospho-mitogen-activated protein kinase (p-MAPK). Aspirin also decreased expression of survivin, vascular endothelial growth factor (VEGF) and YY1 which regulates ErbB2 expression in these cell lines. Aspirin also downregulates Sp1, Sp3 and Sp4, and further investigation of the underlying mechanism of action showed that aspirin-induced downregulation of Sp transcription factors and Sp-regulated genes could be inhibited in part by proteasome inhibitior lactacystin and phosphatase inhibitors including sodium orthovanadate (SOV). These results were consistant with the induction of several phosphatases by aspirin in BT474 and MDA-MB-453 cells and these include mitogen-activated protein kinase phosphatase-5 (MKP-5) and MKP-1. Aspirin-induced downregulation of Sp1, Sp3 and Sp4 are reversed in cells transfected with an oligonucleotide (siMKP5) that knocks down MKP5 by RNA interference whereas siMKP-1 did not block Sp protein downregulation demonstrating for the first time a linkage between a drug-induced phosphatase (MKP-5) and Sp downregulation. These results suggest that TA, BA and aspirin represent novel and promising new anticancer drugs for cancer treatment by targeting Sp proteins and ErbB2 oncogene.

The LRIG1 gene (leucine-rich repeats and immunoglobulin like domains-1) at chromosome 3p14 is a proposed tumour suppressor gene whose gene product negatively regulates various receptor tyrosine kinases. This function has been the basis for classifying LRIG1 as a potential tumour suppressor gene (TSG). The ERBB receptor family is important in malignant cellular functions such as proliferation, survival, adhesion, migration and differentiation. In breast cancer, amplification of the ERBB2 proto-oncogene is an important negative prognostic factor. The epidermal growth factor receptor (EGFR/ERBB1), is expressed in colorectal cancer and has been correlated to a worse prognosis. Until recently, immunohistochemical analysis of EGFR expression was used to select patients suitable for treatment with EGFR targeted antibodies. This thesis characterizes LRIG1 in breast and colorectal cancer to gain further knowledge of the gene and its expression. Also, the EGFR expression in metastases and the invasive margin of colorectal cancers was investigated to correlate changes to clinical factors. Breast cancer samples and matched normal tissues were evaluated for LRIG1 and the ERBB receptors at gene, RNA and protein levels. An increase in copy number of the LRIG1 gene was evident. Also, increased LRIG1 copy number was associated with high levels of ERBB2 mRNA. Another set of breast cancer tumours were analysed for LRIG1 by FISH analysis. The results were coherent with the previous results. To further analyze the correlation to ERBB2, tumours with LRIG1 increased copy number were analysed for ERBB2. The data showed that 89% of tumours with increased LRIG1 copy number were either ERBB2 amplified or had an increased copy number of ERBB2. To investigate LRIG1 and the EGFR in colorectal cancer, the gene and protein expression was analysed by several methods in tumours and corresponding normal tissues. There were no significant changes at gene level found, but at the protein level, both over- and under expression were seen. No evident correlation between LRIG1 and EGFR expression was detected. The ERBB receptor family expression in colorectal cancer tumours and corresponding metastases was investigated to explore if the expression was altered in the metastatic lesion. The results showed that the EGFR expression was lost in the corresponding metastases in 33% of the tumours and that the same percentage of tumours gained expression in the metastases. Co-expression of the ERBB family members was also analysed; there was a significant increase of ERBB3/ERBB4 co-expression in late stage tumours. EGFR expression at the invasive margin of colorectal cancers was analysed to clarify whether expression correlated to the patient’s prognosis. Significant correlation to survival and the presence of budding was seen. In conclusion, 34% of the breast cancer tumours studied had an increased copy number of LRIG1 with a significant co-incidental increase in ERBB2 copy number. This raises the question of a functional correlation between LRIG1 and ERBB2, a finding that might be of clinical importance. The studies of EGFR and the ERBB receptors in colorectal cancer reflect the heterogeneity of EGFR expression in tumours. In addition, these findings suggest that survival of the patients correlates to an increasing EGFR expression at the invasive margin.

Myeloid Cell Leukemia-1 (Mcl-1) is a widely expressed anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. Mcl-1 promotes tumour cell survival and drug resistance and was a mechanism of resistance to first generation Bcl-2 family inhibitors. To determine the significance of Mcl-1 in breast cancer, we evaluated the regulation of Mcl-1 by signalling via the epidermal growth factor receptors (EGFRs). EGFR signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival of tumour cells. We aimed to determine whether Mcl-1 is a critical downstream effector of this pathway and therefore an important therapeutic target. We found that Mcl-1 protein and messenger RNA levels were rapidly induced upon stimulation of breast cancer cells with epidermal growth factor. This induction was blocked by inhibitors of the Ras/Raf/Mek/Erk signalling cascade and was dependent upon activation of the transcription factor Elk-1. We found Mcl-1 to be an essential survival protein, as targeted knock-down with small interfering RNA alone was sufficient to induce apoptosis. Mcl-1 may be critical for the survival advantage conferred by EGFR activation, as prevention of its up-regulation by Mek/Erk inhibitors significantly reduced the drug resistance conferred by EGF. Furthermore, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels in breast tumour samples. Therefore, we conclude that Mcl-1 is an important downstream effector of survival and drug resistance mediated by elevated EGF signalling, making it an important therapeutic target in breast cancer.

Myeloid Cell Leukemia-1 (Mcl-1) is a widely expressed anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. Mcl-1 promotes tumour cell survival and drug resistance and was a mechanism of resistance to first generation Bcl-2 family inhibitors. To determine the significance of Mcl-1 in breast cancer, we evaluated the regulation of Mcl-1 by signalling via the epidermal growth factor receptors (EGFRs). EGFR signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival of tumour cells. We aimed to determine whether Mcl-1 is a critical downstream effector of this pathway and therefore an important therapeutic target. We found that Mcl-1 protein and messenger RNA levels were rapidly induced upon stimulation of breast cancer cells with epidermal growth factor. This induction was blocked by inhibitors of the Ras/Raf/Mek/Erk signalling cascade and was dependent upon activation of the transcription factor Elk-1. We found Mcl-1 to be an essential survival protein, as targeted knock-down with small interfering RNA alone was sufficient to induce apoptosis. Mcl-1 may be critical for the survival advantage conferred by EGFR activation, as prevention of its up-regulation by Mek/Erk inhibitors significantly reduced the drug resistance conferred by EGF. Furthermore, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels in breast tumour samples. Therefore, we conclude that Mcl-1 is an important downstream effector of survival and drug resistance mediated by elevated EGF signalling, making it an important therapeutic target in breast cancer.

No description available.

A amplificação gênica mais freqüente em câncer de mama é a do oncogene ERBB-2, observada em aproximadamente 30% dos tumores de mama e que está relacionada com menor intervalo livre de doença e sobrevida total das pacientes com câncer de mama. O ERBB-2 ativa importantes vias de sinalização celular, incluindo as vias MAPK e PI3K. Utilizando PCR em tempo real analisou-se o efeito do EGF e da HRG na regulação da expressão dos genes ANP32B, MATR3, ATAD4, NDRG1, ACTN1, SPARC, TPM1 e CENPH, nas células HB4a, C5.2 e SKBr3, que expressam diferentes níveis de ERBB2. Avaliou-se também o perfil de expressão destes transcritos após a supressão do ERBB2 pela técnica de siRNA nas células C5.2. O tratamento com EGF modulou de forma diferente a expressão dos genes estudados nas células HB4a, C5.2 e SKBr3. Nas células HB4a e SKBr3 a HRG também regulou a expressão dos genes acima. Após a transfecção das células C5.2 com siERBB2 houve alteração na expressão dos genes ATAD4, NDRG1, ACTN1, SPARC, MATR3, CENPH e TPM1. / The more frequent genic amplification observed in breast cancer is that of the ERBB2 oncogene, which occurs in approximately 30% of the breast cancers, and is associated with lower disease-free interval and survival of all patients with breast cancer. The ERBB-2 protein activates important cell signaling pathways such as MAPK and PI3K. Using real time PCR, it was investigated the effect of EGF and HRG on ANP32B, MATR3, ATAD4, NDRG1, ACTN1, SPARC, TPM1 and CENPH transcripts regulation in the HB4a, C5.2 and SKBr3 cell, that express different levels of ERBB2. It was also evaluated the expression profile of these transcripts in the C5.2 cell line after the suppression of ERBB2 expression by the siRNA technique. The treatments with EGF modulate differently the expression of the analysed transcripts in HB4a, C5.2 and SKBr3 cells. In HB4a and SKBr3 cells the treatments with HRG also modulate the expression of the transcripts above. The C5.2 cells transfected with siERBB2 showed alteration in the expression of ATAD4, NDRG1, ACTN1, SPARC, MATR3, CENPH and TPM1 transcripts.