This experiment investigated a possible candidate for cancer treatment utilizing a cell’s own function for programmed cell death. The purpose of this study was to determine if upregulation of the apoptotic gene HtrA2/Omi in breast cancer cells would lead to increased apoptosis in the cells. Previous literature had described upregulation of apoptotic pathways as a possible viable mechanism for cancer treatment. However, this study did not find significant results to support these claims.
Breast cancer, one of the most prevalent forms of cancer in the world, disproportionately affects women in the United States. On average, 13 percent of women in the United States will be diagnosed with breast cancer at some point during their lifetime (Breast Cancer Facts and Statistics 2023). Every year, 42,000 women die from breast cancer in the United States, with 240,000 more diagnosed with breast cancer (Basic Information About Breast Cancer 2023).
Cells undergo a highly regulated process of programmed cell death called apoptosis that allows for natural development and growth of the organism. Through apoptosis, organisms are able to destroy surplus, infected, and damaged cells. Cancerous tumors develop when the apoptosis function of a cell is not working properly, resulting in a malignant cell that can grow and divide uncontrollably into a tumor. As apoptosis pathways can be induced non-surgically, it is a highly effective method used to control or terminate malignant cancer cells. By utilizing the cell’s own mechanism for death, research for cancer treatment has identified apoptosis as a way to target malignant tumors (Pfeffer et al., 2018).
Research has shown that apoptosis is induced by overexpressing certain genes. HtrA2/Omi is a gene that induces apoptosis when overexpressed in the cell. When released from the mitochondria, HtrA2 inhibits the function of an apoptosis inhibitor, effectively inducing cell death (Suzuki et al., 2001). These data suggest that modulating and upregulating HtrA2 expression shows promising findings in enhancing apoptosis in breast cancer.
CRISPR-Cas9 is a type of cellular biotechnology which can be used to study the manipulation of genomes by either adding, deleting, or altering genetic material in specific locations. This tool can be used to overexpress the HtrA2 gene in order to induce cell death. The process of CRISPR-Cas9 involves using sgRNA (a single guide RNA) with an enzyme to act as a gene-editing tool and introduce mutations into a desired target sequence in the genome. In order to modulate the HtrA2 gene, this experiment will require CRISPRa, a variant of CRISPR that uses a protein (dCas9) and transcriptional effector. The sgRNA navigates to the genome locus, guiding the dCas9. The dCas9 is unable to make a cut, so the effector instead activates the desired downstream gene expression (“Chapter 2: CRISPRa,” n.d.). This experiment will use CRISPRa technology to upregulate the HtrA2/Omi gene, which will inhibit the X chromosome-linked inhibitor of apoptosis, inducing either caspase-dependent cell death or Caspase-3 independent cell death in MCF-7 cells.
The pilot study for this experiment was conducted to determine the optimal level of Lipofectamine – which is a reagent that can be used for an efficient transfection without causing the cells to undergo apoptosis. The Lipofectamine concentration was varied to identify the fold change it would create in the expression of the target gene, HtrA2/Omi. After statistical analyses, researchers found no statistically significant correlation between the HtrA2/Omi gene expression and the Lipofectamine concentration in this experiment.
Overall, the results from conducting quantitative PCR (qPCR), which shows how much of the HtrA2 was transfected, demonstrated extreme variance, indicating that there may have been errors that significantly affected these results. One possible error was that qPCR was conducted as cells were undergoing apoptosis, which would skew the results as mRNA is destroyed in cells as they die, leaving fragments behind. Another error observed throughout this experiment was high cell confluence (number of cells covering the adherent surface). Much of this experiment was conducted with cells at 100% or almost 100% confluence, which means it is possible that the concentrations of Lipofectamine that were predicted to cause efficient transfection did not work because the reagent could not enter the cells. Ultimately, it was found that a cell seeding concentration of 1*104 cells/mL worked best with regard to transformation, but the experiment still did not yield statistically significant results.
References
ATCC. (n.d.). MCF-7. ATCC. Retrieved November 17, 2021, from https://www.atcc.org/products/htb-22
Breast cancer facts and statistics, 2023. (n.d.). https://www.breastcancer.org/facts-statistics
Siegel, R. L., Miller, K. D., Fuchs, H., & Jemal, A. (2021). Cancer Statistics, 2021. CA: A Cancer Journal for Clinicians, 71(1), 7–33. https://doi.org/10.3322/caac.21654
Basic Information About Breast Cancer, 2023. https://www.cdc.gov/cancer/breast/basic_info
Pfeffer, C. M., & Singh, A. T. K. (2018). Apoptosis: A Target for Anticancer Therapy. International Journal of Molecular Sciences, 19(2), 448. https://doi.org/10.3390/ijms19020448
Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell. 2001 Sep;8(3):613-21. doi: 10.1016/s1097-2765(01)00341-0. PMID: 11583623.
Hu, Q., Myers, M., Fang, W., Yao, M., Brummer, G., Hawj, J., Smart, C., Berkland, C., & Cheng, N. (2019). Role of ALDH1A1 and HTRA2 expression in CCL2/CCR2-mediated breast cancer cell growth and invasion. Biology open, 8(7), bio040873. https://doi.org/10.1242/bio.040873
Camarillo, Ignacio G., et al. “4 – Low and High Voltage Electrochemotherapy for Breast Cancer:
An in Vitro Model Study.” ScienceDirect, Woodhead Publishing, 1 Jan. 2014. www.sciencedirect.com/science/article/abs/pii/B9781907568152500042.
Rouhimoghadam M, Safarian S, Carroll JS, Sheibani N, Bidkhori G. Tamoxifen-Induced Apoptosis of MCF-7 Cells via GPR30/PI3K/MAPKs Interactions: Verification by ODE Modeling and RNA Sequencing. Front Physiol. 2018 Jul 11;9:907. doi: 10.3389/fphys.2018.00907. PMID: 30050469; PMCID: PMC6050429.
Mooney, L. M., Al-Sakkaf, K. A., Brown, B. L., & Dobson, P. R. (2002). Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells. British journal of cancer, 87(8), 909–917. https://doi.org/10.1038/sj.bjc.6600541
Suzuki, Y., Takahashi-Niki, K., Akagi, T. et al. Mitochondrial protease Omi/HtrA2 enhances caspase activation through multiple pathways. Cell Death Differ 11, 208–216 (2004). https://doi.org/10.1038/sj.cdd.440134
Chapter 2: CRISPRa and CRISPRi. (n.d.). In A Comprehensive Guide on CRISPR Methods. https://www.synthego.com/guide/crispr-methods/crispri-