End of year Report

According to the Susan G Komen Breast Cancer Foundation there will be 268,600 new cases of invasive or metastasized breast cancer diagnosed among women and approximately 41,760 women will die as a result of their Breast Cancer (“Find Breast Cancer Statistics at Susan G. Komen”). These statistics alone show just how significant of an impact breast cancer can have. Furthermore, cancer is characterized as a disease caused by uncontrolled proliferation. The cell division cycle is what controls cell division and is broken into four phases: G1, where the cell determines whether or not it should divide, S phase, where the DNA is replicated, G2 where the cell prepares for division, and finally M-phase, or when the cell actually separates or divides.

One way the cell is prohibited from undergoing cell division is by the regulation of tumor suppressor proteins like the Retinoblastoma protein or Rb. Rb is inactive in almost all cancers allowing proliferation to occur. Its normal function is to bind directly to and inhibit the transcription factor E2F which otherwise would stimulate S phase gene expression and commit the cell to dividing. In the presence of growth factors Cyclin Dependent Kinase activity is stimulated, phosphorylating Rb on 15 amino acids releasing it from E2F causing the cell to progress through the cell cycle and ultimately proliferate. Therefore, we can view Rb almost like a switch, when phosphorylated it is “off” and allows proliferation to occur but when it is not phosphorylated it is “on” and suppresses cell proliferation.
This understanding of the Rb switch lead researchers to develop CDK4/6 inhibitors like Palbociclib and Abemacicilib which Dr. Krucher and I use in our research. Researchers developed these inhibitors because they understood that in breast cancer there is a mutation that leads to the upregulation of CDK4/6 consequently phosphorylating Rb leading to uncontrolled proliferation. It is important to emphasize that Rb is not mutated but the mutation that is present in the cancer type causes upregulation of CDK4/6 activity instead.
Unfortunately, although there are these wonderful treatment options available after about a year of taking these medications like Abemaciclib or Palbociclib the cancer cells develop resistance by activating the AKT Signaling pathway and even in the experiments carried out by Dr. Krucher and myself we observed AKT activation in response to the two cdk4/6 inhibitors mentioned in the various cell types like MDA-MB-231, MCF7, & T47D.

AKT is involved in many molecular processes but to name a few it is involved in increasing glucose uptake as well as committing that glucose to glycolysis which in turn increases cytosolic citrate which can then be cleaved by the enzyme ACLY (ATP-citrate lyase), and lead to the formation of acetyl-coA which is a precursor for lipid synthesis.
Why is this important? Well if the cell has an indefinite supply of a material that is a precursor for the synthesis of lipids it has the materials necessary for the development of a new cell membrane and will use those materials to develop new cell membranes and ultimately proliferate.
Based on this information we developed the hypothesis that targeting ACLY might reduce resistance caused by Palbociclib and so in our experiments, using different breast cancer cell types like MCF7,MDA-MB-231, & T47D, an early breast cancer cell type, a later or invasive breast cancer cell type and a breast cancer cell type where ACLY is highly active, respectively, we test the efficacy of an ACLY inhibitor, SB, in combination with Palbociclib. Our experiment in this series was as follows: Day 1: count and plate cells in a 96 well plate, Day 2: Add drugs, Day 6: determine cell number by Cell Titer Fluor Assay.

The Cell Titer Fluorescence Assay allows us to count the number of viable cells because the assay measures constitutive protease activity within live cells. The live-cell protease activity is restricted to these intact viable cells and is measured using a fluorogenic, cell permeant, peptide substrate known as GF-AFC. This substrate enters live cells and is cleaved by said protease activity generating a fluorescent signal allowing us to count the number of viable cells. In addition, this substrate will not work in the event that the cell membrane has lost its integrity allowing for the capability reading of measurement of only viable cell number (“CellTiter-Fluor™ Cell Viability Assay Technical Bulletin”).

So far our conclusions in this project are consistent with the notion that both treatments together are significantly better than either alone and after each experiment. Dr. Krucher and I learned how to adapt to any unprecedented hindrances, like not realizing T47D may need a higher concentration of SB than the other cell types as a result of its highly active ACL enzyme, however we learned from those experiments and used it as a learning experience for the following. In the future we intend on continuing these experiments using other ACL inhibitors like Bempo which is currently being used clinically to decrease cholesterol levels and in treating other lipid diseases. Finally, we hope to extend these observations in other cancer types such as pancreatic cancer.

References

“Find Breast Cancer Statistics at Susan G. Komen.” Susan G. Komen®, ww5.komen.org/BreastCancer/Statistics.html.

“CellTiter-Fluor™ Cell Viability Assay Technical Bulletin.” CellTiter-Fluor™ Cell Viability Assay Protocol, www.promega.com/resources/protocols/technical-bulletins/101/celltiter-fluor-cell-viability-assay-protocol/.

 

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