Resumen de Identificación de genes candidatos involucrados en la progresión y la patogénesis del cáncer de mama
Breast cancer is a complex and heterogeneous disease and one of the most frequent causes of cancer-related deaths in developed countries. DNA copy number alterations occur frequently in breast tumours. Regions of DNA amplification are of major interest since often contain oncogenes whose increased expression confers tumour cells a selective advantage and contributes to the carcinogenesis process. In our laboratory prior analysis of familiar and sporadic breast tumours by using array-based Comparative Genomic Hybridization (aCGH) resulted in the identification of an amplification event at the 13q34 region with an overall frequency of 4.5% that increased to 8.1% in BRCA1-associated tumours and to 20% in basal-like tumours. A minimal common region of amplification that spanned 1.8Mb and contained 26 genes was defined.
With this antecedents and by using quantitative Real-Time PCR and immunohistochemistry we identified that, among the genes in the region, CUL4A and TFDP1 presented significant gene and protein overexpression when amplified, which supports their role as likely driver oncogenes within the 13q34 amplification.
We next focused our efforts on elucidating the possible implication of the CUL4A E3 ubiquitin ligase in the initiation and/or progression of breast cancer. By lentiviral infection we induced the stable up-regulation of CUL4A in human the 184B5 immortalized non-transformed mammary epithelial cells. We also stably silenced CUL4A in MDA-MB-157 and HCC1937, two basal-like breast cancer cell lines that amplify and/or overexpress CUL4A, the latter derived from the tumour of a BRCA1-mutation carrier. With these modified cell lines we performed in vitro functional assays that comprised cell viability experiments, S-phase BrdU incorporation assessment, anchorage-dependent and independent colony formation analysis or apoptosis evaluation. We eventually tested in vivo CUL4A-related changes in the tumorigenicity of our cell line models using xenografts in nude mice.
We observed that CUL4A overexpression in the 184B5 cell line increased cell viability and proliferation rate in association with a higher percentage of cells entering in the S-phase, which included a population of cycling polyploid cells; also cells showed enhanced anchorage-dependent and independent colony formation ability. We found evidences for cooperation between CUL4A and H-Ras-V12. Despite these in vitro transforming-related features, overexpression of CUL4A was not enough to generate 184B5-derived tumours in xenografted mice. Conversely, CUL4A-silencing in the CUL4A-overexpressing MDA-MB-157 and HCC1937 breast cancer cell lines resulted in diminished cell viability and proliferation accompanied by a decrease of cells entering the S-phase of cell cycle.
Also, CUL4A-silencing induced a decreased in the colony-forming ability of tumoural cells. In addition, decreased expression of CUL4A resulted in lower growth rate of tumours generated by both cells lines when xenografted in nude mice.
In order investigate the mechanisms by which CUL4A induces the observed phenotypes we analyzed some proteins known to be targeted for proteosomal degradation by CUL4A such as p21, p27 and p53. Following protein synthesis inhibition using cicloheximide we did not observed modifications in the degradation rate of p21 and p27 when overexpressing CUL4A; however, p53 levels were lower in the 184B5 cells that exogenously expressed CUL4A than in control cells. In contrast, CUL4A silencing in the HCC1937 cells did produce a reduced degradation rate and accumulation of p21 and p27 cyclin-dependent kinase inhibitors. Reduction and accumulation of these proteins involved in blocking cell cycle progression, might explain the increased and decreased cell viability and proliferation associated to the overexpresion and inhibition of CUL4A, respectively.
We also aimed to defined novel CUL4A mediators and by using a proteomic approach we performed differential protein expression profiling in our cell line models. Using this strategy we identified the Metallothionein-2 (MT2A), which is highly expressed in primary breast tumours, as a novel putative modulator of the CUL4A oncogenic role in breast cancer.
In summary, our results support the role of CUL4A as a driver oncogene at the 13q34 amplification. Our evidences suggest a role of CUL4A in modulating breast cancer aggressiveness and progression rather than a major involvement in the initial steps of the oncogenic process. We demonstrated that in breast cancer, as described for other tumour types, p21, p27 and/or p53 would mediate CUL4A oncogenic effect and in addition we defined novel candidate proteins that might help to further elucidate CUL4A oncogenic properties.
