Poster H8 - Mitotic perturbations increase micronuclei formation in breast cancer cells

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Winnie F. Muiru

Undergraduate student
Bryn Mawr College, The University of Pennsylvania
Bryn Mawr, Pennsylvania, United States

Co-Author(s)

AA

Alişya Anlaş, PhD

Postdoctoral Researcher
University of Pennsylvania
Philadelphia, Pennsylvania, United States

Primary Investigator(s)

Dennis Discher, PhD

Robert D. Bent Professor, Director Phys Sci Onc Ctr/Proj
Biophysical Eng'g Labs, University of Pennsylvania
Philadelphia, Pennsylvania, United States

Introduction: Genomic instability is a hallmark of cancer and contributes to its initiation and progression1,2. Among one of the most damaging errors is whole chromosome loss, and this aneuploid state is frequent in solid tumors compared to liquid ones1. Chromosomal instability (CIN) is typically a result of errors in DNA replication and mitosis3. The monopolar spindle (MPS1) kinase ensures bipolar attachment of chromosomes along the mitotic spindle4 and has been found to be misregulated in tumors, leading to aneuploidy4.

The phosphatase and tensin (PTEN) homolog gene is a major lipid phosphatase known for its traditional role in enzymatic activation of the PI3K/AKT signaling pathway and has recently been shown to be necessary for centromere function by interacting with key centromere protein CENP-C5. Loss of PTEN can cause CIN via formation of centromere fragments, with aneuploidy in human primary breast cancer5, 6. Cytoskeletal motor protein myosin IIA, an ATPase encoded by the myosin heavy chain 9 (MYH9) gene is the main motor protein involved in cytokinesis, and has been implicated in cytokinesis failure7. Whereas PTEN is a major tumor suppressor gene (TSG) that is frequently mutated in cancers, MYH9 is more minor and not considered a TSG.

Chromosome missegregation can lead to mispackaging of chromosomes in the form of a micronucleus. Characterization of micronuclei offers insight into understanding the mechanisms of chromosome loss in solid tumors. Here, we pharmacologically inhibit MPS1, PTEN and MYH9 in a human breast cancer cell line to observe CIN in the form of micronuclei formation.

Materials and

Methods: MCF7 breast cancer cells were treated with an MPS1 inhibitor, a PTEN inhibitor or MPS1 inhibitor blebbistatin (20 uM). Following a 24-hour treatment, the cells were fixed using formaldehyde, permeabilized and blocked, then stained for F-actin. Nuclei were counterstained using Hoechst.
Images were then acquired with fluorescence microscopy using a x20 or x40 magnification air objective to visualize micronuclei and mitotic cells, and quantification was performed using Fiji (Image J).

Results, Conclusions, and Discussions: Our data suggest that inhibiting the SAC leads to increased micronuclei (p <.001). Treatment of MCF7 cells with a PTEN inhibitor also led to an increase in micronuclei and mitotic cells (p <.05). Treatment with blebbistatin followed the same trend as with PTEN inhibition, though this result was not significant. PTEN is a putative TSG and there’s some evidence of MYH9 also being a TSG. Here, we see their inhibition causes an increase in mitotic cells along with increased micronuclei. When genes involved in mitosis are perturbed, increased micronuclei formation is observed in the case of MPS1 and PTEN inhibition. These results further support other findings in which MPS1 and PTEN are dysregulated in different tumor types4,5,6. Myosin IIA links with actin to form the contractile ring during cytokinesis and this motor protein’s failure to divide the cytoplasm can lead to multipolar spindle formation, aneuploidy, cell cycle arrest or cell death. Multipolar spindle formation can increase micronuclei since there are more attachment points of spindles to centromeres. Cell cycle arrest or cell death could also explain why there was not a significant increase in micronuclei in the blebbistatin treatment despite studies that show that loss of this gene is associated with increased tumorigenesis8.

Identification of micronuclei is one step in understanding the effects of CIN, though the exact mechanisms of micronuclei formation such as merotelic attachment or centrosome amplification need to be further investigated in this breast cancer adenocarcinoma line and others.

Chromosomal instability (CIN) in cancer cells, driven by errors in mitotic processes, is a critical factor in tumor progression and resistance to commonly-used therapeutics. Our research demonstrates that pharmacological inhibition of key mitotic regulators—MPS1, PTEN, and MYH9—increases micronuclei formation in breast cancer cells, revealing their roles in maintaining chromosomal integrity. By identifying specific genes involved in CIN, our study emphasizes the importance of developing targeted therapies that can selectively address these dysregulations and improve patient outcomes in breast cancer.

Acknowledgements (Optional):