Dr. Alain Nepveu, Goodman Cancer Research Centre
Dr. Alain Nepveu, Professor

Rosalind and Morris Goodman Cancer Research Centre

1160 Pine Avenue
Office Room 414; Lab Room 411
Montreal, Quebec H3A 1A3

Tel  (514) 398-5839
Lab (514) 398-5163
Fax  (514) 398-6769

alain.nepveu@mcgill.ca
Publications
Video
PowerPoint
 

Affiliation

Rosalind and Morris Goodman Cancer Research Centre
Department of Oncology, Biochemistry and Medicine
McGill University

Awards

1. James McGill Professor, McGill University, 2006

Research

Dr. Nepveu’s laboratory studies the regulation of transcription in mammalian cells and, in particular, the roles of transcription factors in DNA damage responses. We combine a vast array of molecular biology and functional genomic approaches together with tissue culture and mouse models to investigate how alterations in DNA repair and DNA damage responses contribute to the initiation and progression of cancer. We study how defects in DNA repair can contribute to tumor initiation and how certain cancer cells become dependent on specific DNA repair pathways. It is generally accepted that defects in DNA repair, whether transient or permanent, contribute to tumor development and progression. Yet, to replicate their DNA and proliferate, cancer cells need DNA repair mechanisms, perhaps even more so than normal cells. Moreover, efficient DNA repair mechanisms can enable cancer cells to resist radiotherapy and chemotherapy. We are entering a new era of cancer research in which patients will be stratified for appropriate therapy on the basis of their DNA repair status, rather than on the tissue of origin, and where combination treatments will include DNA repair inhibitors. The goal of our work is to use the acquired knowledge to exacerbate the sensitivity of cancer cells to radiotherapy and specific chemotherapeutic treatments.

In particular, we investigate the molecular and cellular functions of the Cut homeobox 1 (CUX1) gene, a haploinsufficient tumour suppressor gene that is often amplified and overexpressed in advanced cancer. We study which molecular functions of CUX1 protect against cancer and which molecular functions are associated with tumour progression. CUX1 codes for several protein isoforms. Some CUX1 isoforms function as transcriptional activators or repressors, depending on promoter context. Transcriptional activities of CUX1 promote cell cycle progression, DNA replication and DNA damage responses. Another CUX1 isoform which is very abundant functions as an accessory factor in DNA repair and stimulates the efficiency of base excision repair. While this activity in DNA repair protects normal cells against DNA damage and mutations, increased expression of this isoform in cancer cells contributes to their resistance to radiotherapy and chemotherapy. In contrast, knockdown of CUX1 expression sensitizes cancer cells to treatments. We are in the process of identifying drugs that inhibit the DNA repair functions of CUX1 with the aim of sensitizing cancer cells to treatments.

Goodman Cancer Research Centre, CUX 1, Alain Nepveu

Figure 1. Regulation and Biochemical Activities of p200 CUX1 and p110 CUX1
 


Goodman Cancer Research Centre, CUX 1-GFP, Alain Nepveu

Figure 2.  CUX1 Is Rapidly Recruited To Sites of DNA Damage Through Its Cut Repeat Domains
DLD-1 cells were transfected with the indicated expression vectors and submitted to 405 nM laser microirradiation. Pictures were taken from movies at the indicated times after irradiation.


Goodman Cancer Research Centre, Alain Nepveu, DNA Damage, Comets MEFs

Figure 3. Genetic inactivation of Cux1 reduces the DNA repair efficiency of MEFs 
Mouse embryo fibroblasts (MEFs) from Cux1+/+, Cux1+/- and Cux1-/- mice were exposed to 10 μm H2O2 for 20 min on ice, allowed to recover at 37°C for the indicated time. DNA damage before and after treatment was measured by comet assay at pH >13. Each bar represents the average of at least 30 comets. *: p<0.05, **: p<0.01, ***: p<0.001.


Goodman Cancer Research Centre, Alain Nepveu, DNA Damage, CUX 1

Figure 4. Knockdown of CUX1 Reduces DNA Repair Efficiency
MCF7 cells were transfected with CUX1-specific siRNA and then exposed to 2 Gy IR (left) or 10 μM H2O2 for 30 min (Right). At the indicated times, cells were collected and strand breaks quantified by Alkaline Single Cell Gel Electrophoresis. Comet tail moments were scored for at least 30 cells per conditions. Error bars represent standard error. * Indicates p Value <0.05, ** <0.01, *** <0.001 on a student's T test.


Goodman Cancer Research Centre, Alain Nepveu, Glycosylase and AP/Lyase

Figure 5. Cut Repeat Domains Stimulate the Glycosylase and AP/Lyase Activities of the 8-Oxo-Guanine DNA Glycosylase (OGG1) 
The oligonucleotide includes at its 5'end a fluorescent reporter, carboxytetramethyl rhodamine (TAMRA), and an 8-oxoguanine residue at position 6 in the sequence TAMRA-TCACC{{8-oxoG}}, while the other strand contains the Black Hole Quencher-2 (BHQ-2) at its 3'end, such that the quencher resides.

Following cleavage by OGG1, a short deoxyoligonucleotide fluorophore-labeled product is spontaneously released from the remaining DNA fragment possessing the quencher, causing the fluorophore emission to increase. 


Figure 6. CUX1 Transgenic Mice Develop Mammary Gland Tumors With Various Histopathologies
Immunohistochemical staining of an adenomyoepithelioma, a solid carcinoma, an adenosquamous carcinoma, and an adenoma for cytokeratin 6, 14 and 8/18.  Stainings are representative of three mice each for the solid carcinoma and the adenosquamous carcinoma and of one mouse each for the adenomyoepithelioma and the adenoma. H&E stainings are shown at lower magnification.


Goodman Cancer Research Centre, Alain Nepveu, DNA Damage, CUX 1, Tetraploid Cells

Figure 7. Tetraploid Cells Overexpressing CUX1 Survive and Proliferate But Loose Chromosomes Though Merotelic Kinetochore Attachments
Chromosome segregation during anaphase was studied by confocal microscopy. Cells were stained for γ-tubulin (green), α-tubulin (red) and DNA (DAPI, blue). Images are a composite of z-sections encompassing entire cells.


Goodman Cancer Research Centre, Alain Nepveu, DNA Damage, CUX 1-GFP, sub tetraploid

Figure 8. Tumor Cells from CUX1 Transgenic Mice Are Sub-Tetraploid
Chromosome number of cells from six mammary gland tumors arising in p110 or p75 CUX1 transgenic mice was determined from metaphase spreads. **: p < 0.0003 for both tumor sizes and tumor numbers.