Expressão dos genes C-MYC, HER-2 e receptores hormonais como preditores de resposta à quimioterapia neoadjuvante em câncer mamário

O câncer de mama é um dos tumores de maior incidência na mulher, e por isso, muitas pesquisas vêm sendo realizadas, desde a avaliação das características epidemiológicas, à dinâmica biocelular e o tratamento desta doença. Na avaliação de respostas ao tratamento, os fatores preditivos são marcadores que auxiliam na escolha da melhor droga a ser usada. Esta dissertação teve o objetivo de avaliar os genes de receptores de estrogênio e progesterona, HER-2 e C-MYC em tumores localmente avançados da mama, como fatores preditivos de resposta à quimioterapia neoadjuvante. Estudaram-se fragmentos da neoplasia maligna mamária de 50 pacientes com carcinoma ductal infiltrativo, com estadiamento clínico E-III e tratadas com quimioterapia neoadjuvante. Utilizaram-se as técnicas de imunohistoquímica e de hibridização in situ por fluorescência (FISH). A análise dos receptores hormonais não apresentou diferença estatisticamente significativa comparando as pacientes com resposta satisfatória à quimioterapia, das insatisfatórias; a análise do HER-2 apresentou significância apenas para as respostas satisfatórias, onde houve baixa amplificação deste gene. Em relação ao C-MYC observou-se uma diferença estatisticamente significativa comparando a alta amplificação deste gene a uma resposta insatisfatória à quimioterapia. O estudo concluiu que o gene C-MYC pode ser um importante marcador de predição nos tratamentos quimioterápicos neoadjuvantes usados em câncer mamário.

Expressão de HER-1 e HER-2 totais e fosforilados em queilite actínica e carcinoma epidermóide de lábio inferior

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Melatonin attenuates Her-2, p38 MAPK, p-AKT, and mTOR levels in ovarian carcinoma of ethanol-preferring rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene Expression Profile in Response to Doxorubicin-Rapamycin Combined Treatment of HER-2-Overexpressing Human Mammary Epithelial Cell Lines

HER-2-positive breast cancers frequently sustain elevated AKT/mTOR signaling, which has been associated with resistance to doxorubicin treatment. Here, we investigated whether rapamycin, an mTOR inhibitor, increased the sensitivity to doxorubicin therapy in two HER-2-overexpressing cell lines: C5.2, which was derived from the parental HB4a by transfection with HER-2 and SKBR3, which exhibits HER-2 amplification. The epithelial mammary cell line HB4a was also analyzed. The combined treatment using 20 nmol/L of rapamycin and 30 nmol/L of doxorubicin arrested HB4a and C5.2 cells in S to G(2)-M, whereas SKBR3 cells showed an increase in the G(0)-G(1) phase. Rapamycin increased the sensitivity to doxorubicin in HER-2-overexpressing cells by approximately 2-fold, suggesting that the combination displayed a more effective antiproliferative action. Gene expression profiling showed that these results might reflect alterations in genes involved in canonical pathways related to purine metabolism, oxidative phosphorylation, protein ubiquitination, and mitochondrial dysfunction. A set of 122 genes modulated by the combined treatment and specifically related to HER-2 overexpression was determined by finding genes commonly regulated in both C5.2 and SKBR3 that were not affected in HB4a cells. Network analysis of this particular set showed a smaller subgroup of genes in which coexpression pattern in HB4a cells was disrupted in C5.2 and SKBR3. Altogether, our data showed a subset of genes that might be more robust than individual markers in predicting the response of HER-2-overexpressing breast cancers to doxorubicin and rapamycin combination. Mol Cancer Ther; 11(2); 464-74. (C) 2011 AACR.

Prognostication of Soft Tissue Sarcomas Based on Chromosome 17q Gene and Protein Status: Evaluation of TOP2A, HER-2/neu, and Survivin

Topoisomerase 2 alpha (), HER-2/ and are genes that lie on chromosome 17 and correlate with the prognosis and prediction of target-driven therapy against tumors. In a previous study, we showed that TOP2A transcripts levels were significantly higher in soft tissue sarcomas (STS) than in benign tumors and desmoid-type fibromatoses (FM). Because these genes have been insufficiently examined in STS, we aimed to identify alterations in TOP2A and HER-2 expression by fluorescent in situ hybridization and immunohistochemistry, as well as that of survivin, and correlate them with clinicopathologic findings to assess their prognostic value. Eighteen FM and 244 STS were included. Fluorescent in situ hybridization and immunohistochemistry were performed on a tissue microarray. TOP2A and survivin were more highly expressed in sarcomas than in FM. TOP2A was an independent predictor of an unfavorable prognosis; it was combined with formerly established prognostic factors (primarily histologic grade and tumor size at diagnosis) to create a prognostic index that evaluated overall survival. Gene amplification/polysomy (13%) did not correlate with protein overexpression. Survivin and HER-2 expression were not associated with patient outcomes. These findings might become valuable in the management of patients with STS and possibly in the prospective evaluation of responses to new target-driven therapies.

HER-2, p53, p21 and hormonal receptors proteins expression as predictive factors of response and prognosis in locally advanced breast cancer treated with neoadjuvant docetaxel plus epirubicin combination

Abstract Background Neoadjuvant chemotherapy has been considered the standard care in locally advanced breast cancer. However, about 20% of the patients do not benefit from this clinical treatment and, predictive factors of response were not defined yet. This study was designed to evaluate the importance of biological markers to predict response and prognosis in stage II and III breast cancer patients treated with taxane and anthracycline combination as neoadjuvant setting. Methods Sixty patients received preoperative docetaxel (75 mg/m2) in combination with epirubicin (50 mg/m2) in i.v. infusion in D1 every 3 weeks after incisional biopsy. They received adjuvant chemotherapy with CMF or FEC, attaining axillary status following definitive breast surgery. Clinical and pathologic response rates were measured after preoperative therapy. We evaluated the response rate to neoadjuvant chemotherapy and the prognostic significance of clinicopathological and immunohistochemical parameters (ER, PR, p51, p21 and HER-2 protein expression). The median patient age was 50.5 years with a median follow up time 48 months after the time of diagnosis. Results Preoperative treatment achieved clinical response in 76.6% of patients and complete pathologic response in 5%. The clinical, pathological and immunohistochemical parameters were not able to predict response to therapy and, only HER2 protein overexpression was associated with a decrease in disease free and overall survival (P = 0.0007 and P = 0.003) as shown by multivariate analysis. Conclusion Immunohistochemical phenotypes were not able to predict response to neoadjuvant chemotherapy. Clinical response is inversely correlated with a risk of death in patients submitted to neoadjuvant chemotherapy and HER2 overexpression is the major prognostic factor in stage II and III breast cancer patients treated with a neoadjuvant docetaxel and epirubicin combination.

Immune escape durch Überexpression von HER-2/neu

In Tumoren und Onkogen-transformierten Zellen finden sich häufig Defizienzen in der Expression von Komponenten der MHC Klasse I-Antigenprozessierung, die mit einer verminderten MHC Klasse I-Oberflächenexpression und einer reduzierten Sensitivität der Zellen gegenüber einer ZTL-vermittelten Lyse gekoppelt sein können. Da in den meisten Fällen die reduzierten Expressionsmuster über Zytokine revertiert werden können, werden verschiedene Regulationsmechanismen als Ursache für die Defizienzen postuliert. Auch in Zellen, die den „human epidermal growth factor receptor 2“ (HER-2/neu) überexprimieren, wurden derartige „Immune escape“-Mechanismen identifiziert. Aufgrund der Amplifikation und/oder Überexpression dieses Onkogens in Tumoren, die mit einer schnellen Progression der Erkrankung und einer schlechten Heilungsprognose assoziiert ist, wurden zahlreiche Therapien entwickelt, die auf einer Mobilisierung des Immunsystems gegenüber HER-2/neu oder dessen Blockade durch spezifische Antikörper abzielen. Die bisher jedoch nur unzureichenden Erfolge dieser Therapien könnten ihre Ursache in einer verminderten Immunogenität der HER-2/neu+-Zellen aufgrund von Defizienzen in der MHC Klasse I-Antigenprozessierung haben, weshalb die Untersuchung der molekularen Ursachen dieser Suppression für die Therapie von HER-2/neu+-Tumoren von besonderer Bedeutung ist. In dieser Arbeit wurde anhand eines in vitro-Systems ein HER-2/neu-vermittelter „Immune escape“-Phänotyp charakterisiert und die zugrunde liegenden molekularen Mechanismen untersucht. Hierzu wurden murine, HER-2/neu--NIH3T3-Zellen mit HER-2/neu-transfizierten NIH3T3-Zellen verglichen. Die Untersuchung zeigte, dass die Oberflächenexpression von MHC Klasse I-Antigenen bei einer HER-2/neu-Überexpression vermindert ist. Dies ist assoziiert mit reduzierten Expressionen von LMP2, LMP10, PA28a, PA28b, ERAAP, TAP1, TAP2, und Tapasin, einem blockiertem TAP-Transport und einer fehlenden Sensitivität gegenüber einer ZTL-vermittelten Lyse. Da die analysierten Defekte durch eine Stimulation mit IFN‑g wieder revertiert werden können, wird eine transkriptionelle oder translationelle Regulation der betroffenen Gene durch HER-2/neu postuliert. Aufgrund dieser Ergebnisse ist eine T-Zell-vermittelte Therapie von HER-2/neu+-Tumoren als kritisch anzusehen. Die Untersuchung der Promotoren von TAP1/LMP2, TAP2 und Tapasin ergab geringere und durch IFN‑g-induzierbare Promotoraktivitäten in den HER-2/neu+-Zellen im Vergleich zu den HER-2/neu—-Zellen. Mittels Mutagenese-PCR und Gelretardationsanalysen konnte die Bindung eines Komplexes an zwei E2F- und einer P300-Bindungsstelle im Tapasin-Promotor identifiziert werden, die für die HER-2/neu-vermittelte Hemmung der Tapasin-Promotor­aktivität essentiell ist. Eine Inaktivierung der E2F- und P300-Motve in den TAP1/LMP2- und TAP2-Promotoren hatte dagegen keinen Einfluss auf die HER-2/neu-vermittelte Blockade der Promotoraktivität. Ein Vergleich der Promotoraktivitäten der HER-2/neu+- mit Ras-transformierten Zellen ergab, dass die TAP1/LMP2- und TAP2-Promotoren in beiden Zellen supprimiert werden, während der Tapasin-Promotor bei Ras-Transformation nicht beein­trächtigt ist. Der Einsatz von Inhibitoren zeigte, dass die Suppression des Tapasin-Promotors vermutlich über die PLC-g-PKC-Kaskade erfolgt. Dagegen konnte mit Inhibitoren gegen MAPK und PI3Kinase kein vergleichbarer Effekt erzielt werden. Aufgrund dieser Daten wird postuliert, dass HER-2/neu über die Signalkaskade PLC-g–PKC–E2F/P300 die Tapasin-Promotoraktivität supprimiert, wohingegen noch bisher unbekannte Signalkaskaden von HER-2/neu und Ras zu einer Hemmung der TAP1/LMP2- und TAP2-Promotoraktivität führen. Da die Komplexbildung von E2F und P300 auch im Zellzyklus eine Rolle spielt, wird eine negative Korrelation zwischen Zell-Proliferation und MHC Klasse I-Antigenpräsentation postuliert, die Gegenstand künftiger Studien sein wird.

Konditionale Expression von HER-2, H-Ras oder BXB-Raf1 in einem Maustumormodell

Es ist bekannt, dass die Überexpression eines einzigen Onkogens im Tumorgewebe einen maligneren Phänotyp zur Folge haben kann. Ein Beispiel hierfür ist die Rezeptortyrosinkinase HER-2. Besonders in Mamma- und Ovarialkarzinomen tritt häufig eine HER-2 Überexpression auf, die mit einer schlechteren Prognose für die Patientinnen einhergeht. Die HER-2 blockierende Therapie mit Trastuzumab (Herceptin®) konnte zu einer signifikanten Verbesserung der Überlebenszeit bei Patientinnen mit metastasierendem Mammakarzinom führen. Es ist deshalb von großem Interesse herauszufinden, ob ein Tumor durch gezielte Blockade eines bestimmten Onkogens sein tumorigenes Potential verlieren kann, und dadurch das Tumorwachstum zumindest zeitweise unterbunden wird. Die Frage ist also, ob ein Tumor reversibel sein kann, wenn die Expression seiner Onkogene blockiert wird. Frühere Arbeiten meiner Arbeitsgruppe haben gezeigt, dass Tumore, die konditional humanes HER-2 exprimierten, nach Ausschalten von HER-2 tatsächlich in Remission gingen, d.h. reversibel waren. Tumorgrößenabhängig konnte sogar eine vollständige Tumorremission beobachtet werden. Die vorliegende Arbeit soll nun helfen, die beobachtete Remission nach Ausschalten von HER-2 besser verstehen zu können. Von Interesse sind dabei vor allem die molekularen Mechanismen, die in dem Tumor nach Ausschalten der HER-2 Expression ablaufen. Die konditionale Expression von HER-2 wurde mit Hilfe des TET-OFF Systems in NIH3T3 Mausfibroblasten erreicht. Mit dieser Technik wurde ein Maustumormodell etabliert, das ermöglichte, die Veränderungen in den Tumoren nach Ausschalten von HER-2 zu untersuchen. Ein besonderes Augenmerk wurde dabei auf zwei der durch HER-2 vermittelten Signalwege gerichtet, den Ras-MAP Kinase Signalweg und die Aktivierung von Akt über die Phosphoinositol-3 Kinase. Beide wurden nach Ausschalten der HER-2 Expression deaktiviert. Um herausfinden zu können, welcher der beiden Wege eine wichtigere Rolle bei der Tumorremission spielt, wurden in der vorliegenden Arbeit zwei weitere Maustumormodelle zur konditionalen Expression von humanem H-Ras bzw. einer Form des humanen c-Raf-1 (BXB-Raf1) etabliert. Die Modelle funktionierten auf dieselbe Weise wie das HER-2 Maustumormodell und es wurden auch dieselben Faktoren untersucht. Ras und Raf sind Mitglieder des Ras-MAP Kinase Signalweges. Raf ist aber im Gegensatz zu HER-2 und Ras nicht in der Lage, Akt zu aktivieren. Durch Vergleich der Ergebnisse der drei Maustumormodelle war es deshalb möglich zu differenzieren, ob Einflüsse auf die Tumorentwicklung über denn Ras-MAP Kinase oder den PI3K/Akt Signalweg vermittelt wurden. Auch Ausschalten von H-Ras oder BXB-Raf1 führte zu einer raschen Tumorremission. Damit wurde erneut die Frage nach der Reversibilität eines Tumors beantwortet. Ob die Remission auf einer Induktion von Apoptose beruhte, konnte nicht endgültig geklärt werden, da es zwar nach Ausschalten von HER-2 zu einer Erhöhung der Apoptoserate kam, nicht jedoch nach Ausschalten von H-Ras oder BXB-Raf1. Aufgrund der vorhandenen Ergebnisse wird vermutet, dass es zu einer Störung des Gleichgewichtes zwischen proliferationsfördernden und apoptotischen Faktoren nach Ausschalten der Onkogene kam. Die in den Tumoren vorhandene Spontanapoptose könnte dann ausreichen, den Prozess der Tumorremission auszulösen. Die Untersuchungen haben gezeigt, dass ERK bzw. der Ras-MAP Kinase Signalweg die bedeutendere Rolle bei der Tumorremission spielte. Zum einen wurde dies belegt durch die Beobachtung, dass die Tumorverläufe von HER-2 und BXB-Raf1 nahezu identisch waren. Zum anderen kam es in allen drei Modellen zu einer Dephosphorylierung von ERK, die der Tumorremission vorausging. Akt schien dagegen keine Rolle zu spielen, da das Ausschalten der HER-2, H-Ras oder BXB-Raf1 Expression zu keiner einheitlichen Veränderung des Posphorylierungsgrades von Akt führte. Demnach ist die Blockade des Ras-MAP Kinase Signalweges, der hauptsächlich proliferationsfördernde Eigenschaften besitzt, wichtiger für die Tumorremission als die Blockade des PI3K/Akt Signalweges, der hauptsächlich anti-apoptotische Eigenschaften vermittelt.

Preclinical development of anti-cancer strategies against human HER-2

HER-2 is a 185 kDa transmembrane receptor tyrosine kinase that belongs to the EGFR family. HER-2 is overexpressed in nearly 25% of human breast cancers and women with this subtype of breast cancer have a worse prognosis and frequently develop metastases. The progressive high number of HER-2-positive breast cancer patients with metastatic spread in the brain (up to half of women) has been attributed to the reduction in mortality, the effectiveness of Trastuzumab in killing metastatic cells in other organs and to its incapability to cross the blood-brain barrier. Apart from full-length-HER-2, a splice variant of HER-2 lacking exon 16 (here referred to as D16) was identified in human HER-2-positive breast cancers. Here, the contribution of HER-2 and D16 to mammary carcinogenesis was investigated in a model transgenic for both genes (F1 model). A dominant role of D16, especially in early stages of tumorigenesis, was suggested and the coexistence of heterogeneous levels of HER-2 and D16 in F1 tumors revealed the undeniable value of F1 strain as preclinical model of HER-2-positive breast cancer, closer resembling the human situation in respect to previous models. The therapeutical efficacy of anti-HER-2 agents, targeting HER-2 receptor (Trastuzumab, Lapatinib, R-LM249) or signaling effectors (Dasatinib, UO126, NVP-BKM120), was investigated in models of local or advanced HER-2-positive breast cancer. In contrast with early studies, data herein collected suggested that the presence of D16 can predict a better response to Trastuzumab and other agents targeting HER-2 receptor or Src activity. Using a multiorgan HER-2-positive metastatic model, the efficacy of NVP-BKM120 (PI3K inhibitor) in blocking the growth of brain metastases and the oncolytic ability of R-LM249 (HER-2-retargeted HSV) to reach and destroy metastatic HER-2-positive cancer cells were shown. Finally, exploiting the definition of “oncoantigen” given to HER-2, the immunopreventive activity of two vaccines on HER-2-positive mammary tumorigenesis was demonstrated.

Exploring the Life of Edith Fetherston through her Clothing

This research is an examination of the life of Edith Fetherston through studying her clothing and comparing her clothes to fashion history. The authors of Survey of Historic Costume, Tortora and Eubank, state that: 'Dress serves as a means of communication,' and that 'historic dress provides readers with some context for the period in which costumes were worn'. Most importantly, they state that clothing is 'a glimpse of attitudes and values as they were expressed by individuals of [a] period.' (4). Studying clothing history is a way to understand the attitudes of the times; it has a relationship with the attitudes of the society in which it is or was worn. Studying garments can identify personalities, as well as professions, and economic status. My research is based on the wardrobe of Edith Fetherston between the early twenties and late sixties. This thesis begins by examining Edith herself, then it examines each decade and its major characteristics, and finally my research examines at least two garments from each of the five decades.

Relationships Past and Present: Examining the Impact of One's Relationship with His/her Opposite-sex Parent on His/her Attitudes Toward and Engagement in Hookup Culture

Hooking up has become a common and public practice on university campuses across the country. While much research has determined who is doing it, with whom they are doing it, and what they are hoping to get out of it, little work has been done to determine what personal factors motivate students to participate in the culture. A total of 407 current students were surveyed to assess the impact of one’s relationship with his/her opposite-sex parent on his/her attitudestoward and engagement in hookup culture on campus. Scores were assigned to the participants to divide them into categories of high and low attachment with their parent. It was hypothesizedthat heterosexual students who do not perceive themselves as having a strong, close, positive relationship with their opposite-sex parent would be more likely to engage in or attempt to engage in casual sexual behavior. This pattern was expected to be strongest for women on campus. Men and women differed in their reasons for hooking up, with whom they hook up, to what they attribute the behaviors of their peers, and what they hope to gain from their sexual interactions. Effects of parent-child relationships were significant only for women who reported hooking up because “others are doing it,” men’s agreement with the behavior of their peers, and women’s overall satisfaction with their hookups. Developmental, social, and evolutionary perspectives are employed to explain the results. University status was determined to be most telling of the extent to which a student is engaged in hookup culture.

Dephosphorylation of p-ERK1/2 in relation to tumor remission after HER-2 and Raf1 blocking therapy in a conditional mouse tumor model

Several studies have shown that HER-2/neu (erbB-2) blocking therapy strategies can cause tumor remission. However, the responsible molecular mechanisms are not yet known. Both ERK1/2 and Akt/PKB are critical for HER-2-mediated signal transduction. Therefore, we used a mouse tumor model that allows downregulation of HER-2 in tumor tissue by administration of anhydrotetracycline (ATc). Switching-off HER-2 caused a rapid tumor remission by more than 95% within 7 d of ATc administration compared to the volume before switching-off HER-2. Interestingly, HER-2 downregulation caused a dephosphorylation of p-ERK1/2 by more than 80% already before tumor remission occurred. Levels of total ERK protein were not influenced. In contrast, dephosphorylation of p-Akt occurred later, when the tumor was already in remission. These data suggest that in our HER-2 tumor model dephosphorylation of p-ERK1/2 may be more critical for tumor remission than dephosphorylation of p-Akt. To test this hypothesis we used a second mouse tumor model that allows ATc controlled expression of BXB-Raf1 because the latter constitutively signals to ERK1/2, but cannot activate Akt/PKB. As expected, downregulation of BXB-Raf1 in tumor tissue caused a strong dephosphorylation of p-ERK1/2, but did not decrease levels of p-Akt. Interestingly, tumor remission after switching-off BXB-Raf1 was similarly efficient as the effect of HER-2 downregulation, despite the lack of p-Akt dephosphorylation. In conclusion, two lines of evidence strongly suggest that dephosphorylation of p-ERK1/2 and not that of p-Akt is critical for the rapid tumor remission after downregulation of HER-2 or BXB-Raf1 in our tumor model: (i) dephosphorylation of p-ERK1/2 but not that of p-Akt precedes tumor remission after switching-off HER-2 and (ii) downregulation of BXB-Raf1 leads to a similarly efficient tumor remission as downregulation of HER-2, although no p-Akt dephosphorylation was observed after switching-off BXB-Raf1.