Oncogenes y Antioncogenes

Dr. Daniel R. CIOCCA IMBECU (Unidad Ejecutora del CONICET) Fund. Argentina Investigacin Cncer MENDOZA

Oncognesis Mamaria
INICIADORES - Alteracin gentica Mutacin hereditaria (BRCA1, etc..) o Adquirida (radiaciones, metabolismo endgeno, qumicos, frmacos, virus, hormonas) oncogenes-antioncogenes

PROMOTORES Expanden alt. gentica Hormonas FACILITADORES Disminuyen defensas Inmunodepresores (stress, frmacos, enfermedades, hormonas)

Oncogenes

Proto-oncogenes: . Multiplicacin celular . Diferenciacin . Muerte/sobrevida

Alteracin dominante . Mutaciones puntuales . Amplificacin (mltiples copias de ADN) . Translocacin (rearreglos estructurales)

Oncogenes

Daos endgenos

Daos exgenos
Radiaciones: ionizante y UV

Durante la replicacin del ADN: incorporacin de U Desaminacin de A, G o C

Hidrlisis de uniones N-glucoslicas: despurinacin/despirimidacin Oxidacin de bases: ERO

Carcingenos qumicos: benzopireno, hidrocarburos aromticos policclicos

Drogas antineoplsicas

Ph cromosome, CML

t(9;22)(q34;q11)
Cromosomas involucrados

Abl codifica tirosina quinasa que cuando se fusiona con Bcr es constitutivamente activa

Bandas afectadas por los puntos de ruptura

Genes Supresores Tumorales (Antioncogenes): . Multiplicacin celular . Reparacin daos al ADN . Muerte/sobrevida

Mutaciones recesivas Metilaciones

Prdida del Antioncogen Cascada de Defectos del Genoma/Proteoma

Rb-Mediated Regulation of Cyclin E Transcription

HDAC-1 Rb E2F Pol

II

HDAC-1

CDK Cyclin

HDAC-1 T-Ag Rb T-Ag

Rb

HPV-16 E7

E2F Pol

II
CDK 2 Cyclin E

Rayos X Radicales libres Agentes alquilantes Reacciones espontneas

Luz UV Hidrocarburos aromticos policclicos

Rayos X Antineoplsicos (Cisplatino)

Errores de replicacin

Sndromes con reparacin del ADN defectuosa

C T

G T U G T C T G G A G

Uracilo Sitio absico 8-oxoguanina Rupturas de simple cadena

(6-4)PP Aductos DCP

Uniones cruzadas Rupturas de doble cadena

Bases mal apareadas Inserciones Deleciones

HSP in DNA repair Nadin & Ciocca In press

BER

NER

HR, EJ

MMR

Sndrome Xeroderma Pigmentosum (XP)

Mecanismo afectado NER (+/- TCR)

gen/protena afectada XPC/XPC XPA/XPA XPD/XPD XPG/XPG XPF/XPF hMLH1/hMSH2 hMSH6/hPMS1/hPMS2 BRCA1/BRCA2 ATM

Inestabilidad genmica Mutaciones puntuales

Relacin con cncer de piel inducido por UV o por exposicin a agentes qumicos Cncer colorectal Cncer de mama (ovario) Linfomas

HNPCC BRCA1/BRCA2 Ataxia Telangiectasia

MMR HR DSB repair

Mutaciones puntuales Aberraciones cromosmicas Aberraciones cromosmicas

Genomic analysis of a spontaneous model of breast cancer metastasis to bone revels a role for the extracellular matrix Eckhardt BL et al (Robin Anderson). Peter MacCallum Cancer Center, Australia

Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer BS Parker, DR Ciocca, et al Journal of Pathology 214:337-346 (2008).
Cysteine cathepsin inhibitor Stefin A as a gene differentially expressed in primary and metastatic mammary tumours. In primary tumours, Stefin A expression correlated inversely with metastatic potential in 4T1-derived lines and was not detected in tumour cells in culture, indicating induction only within the tumour microenvironment. Enforced expression of Stefin A in the highly metastatic 4T1.2 cell line significantly reduced spontaneous bone metastasis following orthotopic injection into the mammary gland. There have been no reports of Stefin A expression in metastases in vivo and the relative expression of Stefin A and the cysteine cathepsins, including their cellular localization, has not been studied in bone metastasis. In this report, we document the expression and localization of Stefin A in mouse and human breast tumours and provide functional evidence for its potential role as a prognostic/therapeutic in advanced breast cancer.

Stefin A reduces spontaneous metastasis to bone. Stefin A1-containing or BV-retroviral vectors were used to infect 4T1.2neo1 tumour cells.

RNA isolated from primary tumours was reverse-transcribed and real-time RTPCR was used to detect Stefin A1, A2 and A3 expression relative to GAPDH. RTA, relative transcript abundance. p < 0.05 (4T1 to 4T1.2/ 4T1.35)

A.- Real-time RTPCR and western blot detection of Stefin A1 RNA and protein expression (flag tag protein) in pooled and single cell clones. B.- RTQPCR detection of tumour burden in spine (using neomycin-tagged 4T1.2 cells) in mice injected with 4T1.2 BV or 4T1.2 StfA1. RTB is the relative tumour burden (left) and plasma calcium serum concentration (mg/dl) in tumour-bearing and non-tumour-bearing mice (right).

PEFF sections of reduction mammoplasty tissue, primary breast tumours and metastases in lung and bone were stained with mouse anti-human Stefin A or 1B5 hybridoma supernatant control and visualized with DAB.

KaplanMeier analysis comparing disease-free survival (DFS) (B) and death due to cancer (DDC) (C)

Co-expression of Stefin A and cathepsin B in lung and bone metastases. Stefin A fluorescence is visualized in red and cathepsin B in green (E), and these are shown both separately and as a merged image. Sections were additionally stained with PI (coloured blue) to visualize the nuclei (F).

Final model selected using Akaike information criteria. The model was initially fitted with all variables (Table 1), including Stefin A staining.

CONCLUSIONS In a multivariate disease-free survival analysis (Cox proportional hazards model), Stefin A expression remained a significant independent prognostic factor in patients with invasive ductal carcinoma (p = 0.0014), along with grade and progesterone receptor (PR) status. We propose that Stefin A, as a cysteine cathepsin inhibitor, may be a marker of increased cathepsin activity in metastases. Using immunohistology, the cathepsin inhibitor was detected co-expressed with cathepsin B in lung and bone metastases in both the murine model and human tissues. We conclude that Stefin A expression reduces distant metastasis in breast cancer and propose that this may be due to the inhibition of cysteine cathepsins, such as cathepsin B.

Multi-step mammary tumor progression C3(1) SV40 Tag Transgenic Model

NL

Dysplasia

DCIS

Inv Ca

Met Ca

1 month

6 months
(100% Incidence)

p53 + Rb Inactivation

Maroulakou et al. Proc. Natl. Acad. Sci. USA 91:11236, 1994

Categorizacin de los Eventos Moleculares en Cncer de Mama

Porteros: BRCA, p53, Rb

Dao al ADN, alteracin ciclo celular Mayor estmulo para crecimiento Falla apoptosis, y otras molc., angiognesis

Espordicos: HER-2/neu, c-myc, ras

Otros: Bcl-2, Hsps, ciclinas, VEGF

Carga gentica

Daos innatos al ADN Daos adquiridos Fallas controles de proliferacin Fallas controles de apoptosis

Cncer
Declinacin de la inmunidad Cambios hormonales Disminucin mecanismos reparativos Acumulacin de daos al genoma/proteoma

Edad