Breast cancer is now recognized as the most common malignancy worldwide among women, irrespective of age. Today’s standard of care and breast cancer management relies heavily on chemotherapy, hormone therapy, and targeted therapy. However, there is still much to be learned, with ongoing efforts to elucidate the mechanism of tumorigenesis, and continued studies addressing novel treatments and drugs, along with new combinations of existing treatments.
We’re delighted that many of these ongoing efforts incorporate the use of the Gibson Assembly® method as an efficient means of cloning. In honor of Breast Cancer Awareness Month, we would like to highlight some recent publications focused on furthering our understanding of breast cancer.
A paper from scientists at the University of Macau describes work to determine the role of an enhancer of zeste homolog 2 (EZH2) — which functions as an oncogene or tumor suppressor gene, depending on the context of the tumor type, and its interplay with early growth response 1 (EGR1) in breast cancer. Specifically, the authors aimed to determine whether EZH2 represses the expression of EGR1, which is known to be involved in regulating cell growth and apoptosis, and is highly expressed in normal breast tissues.
The group developed a CRISPR-mediated silencing approach fusing the catalytically inactive mutant Cas9 (dCas9) with EZH2 using seamless Gibson Assembly® cloning, successfully generating constructs that encode sgRNAs targeting the different subregions — R1, R2, and R3. In subsequent functional experiments using the breast cancer cell lines, MCF-7 and MDA-MB-231 cells, the dCas9/EZH2 fusion protein directed by all three sgRNAs repressed the expression of EGR1. Subsequent experiments allowed the group to hypothesize that “EZH2/PRC2 acts as a ‘brake’ for EGR1 expression by targeting the EGR1 silencer, and EZH2 overexpression dampens tumor-suppressive signals mediated by EGR1 to drive breast tumorigenesis.”
Although only 5 to 10% of breast cancer cases are inherited, recent estimates suggest that 55 to 65% of BRCA1 mutation carriers and approximately 45% of BRCA2 mutation carriers will develop breast cancer by age 70. It is well established that BRCA1 and BRCA2 proteins play a crucial role in maintaining genomic integrity through DNA repair by homologous recombination. Indeed, during the process of mitosis, BRCA2 is a target of phosphorylation by Polo-like kinase 1 (PLK1), both in its N-terminal region and in its central region, although the functional role of these phosphorylation events remains unclear. To address this, scientists from the Institut Curie in France elucidated how this phosphorylation contributes to mitosis control.
The team strategically used the Gibson Assembly® method to clone PLK1 cDNA into an expression vector, as well as to generate Polo-like binding domain (PBD) of PLK1 – amino acid 326 to amino acid 603 – which was amplified from the pTK24 plasmid (Addgene) and cloned into a pT7-His6-SUMO expression vector. Ultimately, the group demonstrated that PLK1 phosphorylates two conserved residues at S193 and T207, and that phosphorylated BRCA2-T207 is a bona fide docking site for PLK1. The authors report that “reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex, resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation, and aneuploidy.” This may shed light on the mechanism of aneuploidy observed in BRCA2-mutated tumors.
Without question, the appreciable growth in our understanding of breast cancer has led to remarkable progress in early detection, treatment, and prevention strategies. The increasing focus on personalized therapy holds much promise for ultimately overcoming this devastating disease. The entire Codex DNA team congratulates those researchers around the world, whose commitment and hard work will put us on the path to stamping out the worst outcomes of breast cancer.