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/.

 

Blog Post #3

Thus far in our research, Dr. Krucher and I have made progress with determining whether or not certain resistance pathways in breast cancer cells remain inactive in response to reducing PNUTS levels or PNUTS Knockdown. One of the resistant pathways we have discovered as remaining inactive when PNUTS levels were reduced is AKT. In order to come to these results I would perform an experiment utilizing a series of laboratory techniques including but not limited to performing cell lysis protocols, protein concentration assays, western blotting, and running gel electrophoresis.

One of the challenges that I had with regard to our research was figuring out why some of the samples I had made were not easily visible or clean when it came time to view the Western blots via the Bio-Rad Imager. This problem lead to communication between Dr. Krucher and I in an attempt to determine why this was happening in my samples. After careful consideration and discussion with Dr. Krucher we came to the conclusion that the problem may have been a result of not always keeping the cell pellets on ice before performing the lysis protocol. This would potentially allow proteases within the cells to kill off the proteins that we want to isolate which would have accounted for the problems I had viewing the blots in the Bio-Rad Imager.

Through this project I have learned something new with each passing day. From learning laboratory techniques, to better understanding Cancer cells and learning more about the pathways that lead to their resistance, this research has allowed me to learn immensely and I am looking forward to continuing to learn throughout the duration of my time doing research with Dr. Krucher.

Blog #2: Investigation of Combination Treatments in Breast Cancer

The goal of the research I have been doing this year is to activate the tumor suppressor Rb, in breast cancer cells. To do that we activate an enzyme called PP1, which activates Rb, by a gene silencing technique called PNUTS knockdown. So far in this research I have learned many of the techniques necessary for setting up PNUTS Knockdown. I have learned how to efficiently count and plate MCF7 breast cancer cells, as well as learned how to trypsinize cells off of plates in order to remove the cancer cells and centrifuge them to collect them.

Throughout the semester I have also learned more about the intricacies of the PNUTS KD For example, I learned that using specific antibodies we tag the proteins that are on the nitrocellulose paper and after viewing them via specific computer software (Bio-rad image software) we determine whether or not PNUTS was knocked down by comparing a portion of cancer cells that didn’t receive any treatment (controls) that knocked down PNUTS to a portion of cancer cells that did have PNUTS knocked down and if the software shows the band where PNUTS is to be lighter or less prominent then we know the knockdown was a success. Furthermore, we also look to see if the band where Actin is, is more prominent or less prominent or the same. Ideally it is the same, meaning the same amount of protein was loaded in each cell. Therefore, expression of actin serves as our loading-control.

So far we don’t have any confirmed data because the results we do have need to be replicated several times to have confidence in the accuracy of the results. However, we do have general results. For example, so far the experiments that have shown promising results regard the phosphorylation of AKT detected by the antibody AKT-473. The way we know this antibody has shown promising results is by viewing the band on the nitrocellulose paper as mentioned before and determining whether or not the band is more prominent with the treatment or less prominent and in the experiments we have carried out so far the bands for this antibody have been less prominent after the PNUTS KD and this experiment will be carried out again a number of times to ensure the results are accurate.

 

UGR Blog Post 1: Investigation of Combination Treatments in Breast Cancer in Attempt to Overcome Drug Resistance

The title of the research project that Dr. Krucher and I are working on is Investigation of Combination Treatments in Breast Cancer in Attempt to Overcome Drug Resistance. The reason we are investigating combination treatments is because in breast cancer, like in many other cancers, the cancer has the ability to develop resistance to the drugs that are used to attack and kill the cancer cells. This means that after a period of time the drug that was used to treat the cancer is rendered ineffective due to the cancers ability to activate specific resistance pathways and continue proliferating. Therefore, through this research project Dr. Krucher and I plan on determining whether or not combination treatments would be a more effective means at combating this resistance in cancer. From this project I personally expect to learn in a variety of different ways. For example, I plan on learning new laboratory skills and techniques or developing the techniques that I have been taught so far like running a Gel Electrophoresis or Culturing cells or performing Protein Concentration Assays. Furthermore, I also plan on learning how to properly go about conducting research in a laboratory setting.

Our target in this research is the Retinoblastoma (Rb) protein, which is inactivated in almost all cancer types. In cancer cells Rb is highly phosphorylated which inactivates its ability to suppress unwarranted proliferation. The way that Dr. Krucher and I plan on answering these questions is by utilizing a method known as PNUTS (Phosphatase Nuclear Targeting Subunit) knockdown where we reduce PNUTS, a PP1 interacting protein, in cancer cells. PNUTS depletion leads to an activation of PP1 activity which leads to dephosphorylation of the Rb protein, and its activation. We will determine whether PNUTS depletion is a method to suppress cancer drug resistance. We plan on answering this question by continuing to utilize Gel Electrophoresis in order to separate proteins and Western Blot them in order to image them and determine whether or not PNUTS was knocked down. In future experiments we are interested to see if PNUTS Knockdown coupled with other drugs will aid in combating any resistance pathways that certain cancer cells develop.