33 research outputs found
Inhibition of polarity pathways as a synthetic lethal approach to aid targeted therapy in preventing breast tumor relapse.
No full textBreast cancer is the second most common cancer worldwide, and the one of the leading causes of cancer death in women. Due to recent advances in therapeutic approaches and early detection programs the breast cancer survival rate has seen a dramatic improvement. Consequently, it is now breast cancer recurrence, not the primary tumor, which is the leading cause of breast cancer related death. These relapses are due to malignant cells that survive treatment. Therefore, further characterization of the treatment-resistant cells is needed in order to improve the therapeutic approaches.
The overexpression of oncogenes drives tumorigenesis. In human breast cancer the epidermal growth factor receptor type-2 (Her2 also called Neu) is overexpressed in 20% of breast cancer, whereas the oncogenic transcription factor c-Myc is over-expressed in 16% of breast cancers. We model breast cancer through the use of an inducible mouse model in which the oncogenes NEU and c-MYC are under the control of a doxycycline inducible promoter. By administering a diet supplemented with doxycycline we are able to induce tumorigenesis, follow primary tumor development, and additionally inactivate the oncogene expression through withdrawal of doxycycline from the mouse diet. In essence, this would mimic a perfect therapeutic setting in which we are able to completely eradicate expression of the oncogenes, yet, these mice eventually succumb to tumor recurrence in the majority of cases. Therefore the aim of my thesis is to eliminate or disrupt the treatment-resistant cells so that they are unable to give rise to the tumor relapses even after the oncogene withdrawal.
We have taken an in vitro approach to screen candidates, which would interfere with the re-formation of the mammary gland following oncogene inactivation. To do so, we cultured primary mouse mammary epithelial cells, taken from the transgenic mice, in an in vitro 3D organotypic culture system. The structures, which grow in vitro closely, resemble the polarized, lumen filled acini which mammary glands are comprised of.
In these cultures, we tested different inhibitors for molecules which we confirmed were activated within the tumors derived from the c-MYC/NEU mouse model. Specifically, we tested inhibitors for Focal Adhesion Kinase, Integrin Linked Kinase, for signaling pathways, which regulate survival and membrane polarity as well an inhibitory antibody for E-cadherin bridges, which play an important role in establishing cell to cell contacts and cellular epithelial polarity.
Initial results of the in vitro assay show a decrease in the number of cells surviving following treatment with inhibitors. In order to determine the characteristics of the cells surviving treatment and their tumorigenic potential, we injected them into the cleared mammary fat pad of immunodeficient mice.
In the meantime using inducing a second round of induction in vitro we have seen that the survivors show reduced ability to re-induce. We also performed immunofluorescence staining on the treated cells to investigate the effect of the inhibitors on the re-polarized acinar structures. The first indications from these experiments hint that introduction of inhibitors at the time of oncogene inactivation severely disrupts reconstitution of the polarized acinar mammary gland structure. A clear distribution, ie (basal/luminal) of the structural molecular components, ZO-1, E-cad was missing, suggesting an inhibition in the re-establishment of polarity in the surviving structures.
Further studies are necessary to conclude that these inhibitors are able to target the treatment-resistant cells, but our first promising results encourage this line of research
Identification of Mechanisms of Breast Cancer Metastasis Using Tissue Specific Virus Delivery
No full textIdentification of Mechanisms of Breast Cancer Metastasis Using Tissue Specific Virus Delivery
No full textTowards a holistic and mechanistic understanding of tumourigenesis via genetically engineered mouse models
No full textDiverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis
No full text
Defining the mechanisms of breast tumorigenesis in the context of chromosome instability and oncogene dependence
No full textTesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 26-11-2014Intra-tumor heterogeneity is a widely demonstrated characteristic of human malignancies
(Hanahan and Weinberg, 2011). Despite this, cells that became malignant because of oncogenic
driven mutations still rely on their sustained over-expression for survival (Weinstein
and Joe, 2006). The concept of oncogene dependence has been the base for modern targeted
therapy, which, unfortunately still cannot completely eradicate the disease.
Resistance to targeted therapy might be elicited by mechanisms that cause genomic instability,
which is a striking feature of both solid and haemapoietic human tumors (Duijf et al.,
2012). The importance of chromosome instability (CIN) is underscored by its association
with poor patient outcome in di erent cancer types, including breast (McGranahan et al.,
2012). Furthermore, CIN has been shown to facilitate tumor relapse when found in primary
tumors (Sotillo et al., 2010).
CIN can be elicited by several molecular mechanisms (Holland and Cleveland, 2012). Among
those, over-activation of the mitotic checkpoint - in particular through the up-regulation of
the Mad2 protein - has been frequently observed in a variety of human tumors, including
breast (Rhodes et al., 2007). Characterization of the molecular mechanisms responsible for
tumor relapse has become a major focus in cancer, and the relevance of CIN in patient's
prognosis and survival suggests that the CIN status could be exploited in the clinical setting
(Holland and Cleveland, 2012; Pfau and Amon, 2012).
The work presented here focuses on the study of breast cancer, which is worldwide one
of the leading causes of death in women. Interestingly, CIN scores are prognostic in certain
subgroups (Smid et al., 2011); among those, Her2 positive breast tumors, are characterized
by extremely poor outcome.
Given the lack of available CIN mouse models of breast tumorigenesis, we generated new
mouse models that would allow us to:
Faithfully model human disease by introducing CIN into established mouse models of
breast cancer,
Study the role of CIN in breast tumor initiation, progression and relapse,
Understand the molecular mechanisms of cancer relapse in the face of CIN.
The study of these mouse models lead to the following conclusions:
In the Her2 and c-MYC oncogenic backgrounds, Mad2 over-expression plays a dual
opposing role in breast tumorigenesis: tumor suppressing in primary tumor formation
while accelerating tumor recurrence,
Mad2 over-expression promotes tumor heterogeneity,
Mad2 induced heterogeneity in the primary tumor gives the possibility to choose among
di erent mechanisms of relapse.Una de las principales características del cáncer en humanos es la heterogeneidad intratumoral
(Hanahan and Weinberg, 2011). Las células que adquieren características tomorog
énicas debido a determinadas mutaciones necesitan mantener la sobreexpresión de
oncogenes para su supervivencia (Weinstein and Joe, 2006). La dependencia oncogénica ha
sido la base de la terapia dirigida que, desafortunadamente aún no ha sido capaz de erradicar
la enfermedad. Esta resistencia a la terapia puede explicarse a través de mecanismos que
causan inestabilidad cromosómica (CIN). Dicha CIN adquiere mayor importancia si consideramos
su asociación con la mala prognosis de los pacientes. (McGranahan et al., 2012).
Se ha demostrado que la CIN presente en tumores primarios es capaz de promover recidivas
(Sotillo et al., 2010). Varios mecanismos moleculares pueden dar lugar a CIN (Holland and
Cleveland, 2012). Entre ellos, la sobreactivación del punto de control mitótico en particular
a través de la sobreexpresión de Mad2- se ha encontrado en una gran variedad de tumores
humanos, entre ellos el cáncer de mama (Rhodes et al., 2007). La relevancia de la CIN en
la prognosis y supervivencia de los pacientes sugiere que podría ser explotada para el diseño
de nuevas terapias en clínica (Holland and Cleveland, 2012; Pfau and Amon, 2012). Este
trabajo se centra en el estudio del cáncer de mama. Los niveles de CIN son indicadores de
la prognosis de determinados subgrupos (Smid et al., 2011), siendo extremadamente grave
en los tumores de mama positivos para Her2. Dada la falta de modelos para estudiar la CIN
en tumorogenesis de mama, hemos generado una batería de modelos que nos han permitido:
Modelar la tumorogenesis humana mediante la introducción de inestabilidad cromosómica
en modelos murinos ya establecidos para el estudio del cáncer de mama,
El estudio del papel que la CIN cumple en la iniciación, progresión y aparición de
recidivas tumorales,
Entender los mecanismos moleculares de las recidivas tumorales en presencia de inestabilidad
cromosómica.
El estudio con los modelos animales presentados en este estudio llevaron a las siguientes
conclusiones:
en escenarios de sobreexpresión de los oncogenes Her2 y c-MYC durante la tumorogé-
nesis de mama, la sobreexpresión de Mad2 juega un papel dual: actúa como supresor
en la formación de tumores primarios pero actúa como promotor en la aparición de
recidivas tumorales,
La sobreexpresión de Mad2 promueve la heterogeneidad tumoral,
La heterogeneidad de los tumores primarios debida a la sobreexpresión de Mad2, otorga
la posibilidad de elegir entre diversos mecanismos promotores de recidivas tumorales
Subunit Composition and Quantitative Importance of Hetero-oligomeric Receptors: GABA<sub>A</sub>Receptors Containing α<sub>6</sub>Subunits
No full textIn cerebellum, GABAAreceptors containing α6subunits are expressed exclusively in granule cells. The number of α6receptor subtypes formed in these cells and their subunit composition presently are not known. Immunoaffinity chromatography on α6subunit-specific antibodies indicated that 45% of GABAAreceptors in cerebellar extracts contained α6subunits. Western blot analysis demonstrated that α1, β1, β2, β3, γ2, and δ subunits co-purified with α6subunits, suggesting the existence of multiple α6receptor subtypes. These subtypes were identified using a new method based on the one-by-one immunochromatographic elimination of receptors containing the co-purifying subunits in parallel or subsequent experiments. By quantification and Western blot analysis of α6receptors remaining in the extract, the proportion of α6receptors containing the eliminated subunit could be calculated and the subunit composition of the remaining receptors could be determined. Results obtained indicated that α6receptors in cerebellum are composed predominantly of α6βxγ2(32%), α1α6βxγ2(37%), α6βxδ (14%), or α1α6βxδ (15%) subunits. Other experiments indicated that 10%, 51%, or 21% of α6receptors contained homogeneous β1, β2, or β3subunits, respectively, whereas two different β subunits were present in 18% of all α6receptors. The method presented can be used to resolve the total number, subunit composition, and abundancy of GABAAreceptor subtypes in the brain and can also be applied to the investigation of other hetero-oligomeric receptors.</jats:p
