Presentations

Abstract

Introduction: Breast cancer is one of the most common forms of gene mutation related cancers in women (Van Pham, 2015). Current studies have established that BRCA1 strongly inhibits ER-a activity in breast cancer cells and blocks estrogen (ES) stimulated gene expression and cell proliferation. The BRCA1 repression of ER-a activity is due to a physical interaction between BRCA1 and ER-a protein, and this interaction acts as a breaking system for estrogen, driving breast cancer development. Treatment efficacy of breast cancer is often low while potential for remission of the tumor is high. As a result, new strides have been made in identifying potential treatment and therapy options following diagnosis. It is suggested that breast cancer, both hereditary and sporadic tumorigenesis, is caused by dysregulation in the cancer stem cell renewal and differentiation pathways (Zeng et al., 2021). Though the composition of breast cancer is primarily bulk-tumor cells, a subpopulation of breast cancer stem cells (BCSCs) has been suggested to contribute to the resistance of tumors to treatment options such as chemotherapy and surgical removal (Scioli et al., 2019). BCSCs possess stem cell features, namely self-renewal capacity, which allow for regeneration of both bulk-tumor cells and cancer stem cells. Recent improvements in breast cancer treatment and therapy have enhanced the potential of targeting BCSCs. Our project focuses on this possible targeted therapy. Our lab previously developed a homing peptide that is composed of two binding domains: a high-affinity estrogen receptor binding domain intended to target breast cancer cells, and a low affinity doublecortin-like kinase 1 (DCLK-1) binding domain which is a universally identified cancer stem cell marker. The compound would have the potential to mimic BRCA1 functionality to suppress breast cancer proliferation as it pertains to gene transcription. Following development of the structure of the homing peptide, we asked the following question: what is its binding affinity against BCSCs?

Methods: We began our investigation by culturing a BCSC line derived from an estrogen receptor positive, progesterone receptor positive, and HER2 negative breast cancer cell line, MCF7. Following development of the BCSC line, we tested the binding of the developed homing peptide. P100 plates were coated with a polylysine solution. Polylysine is a positively charged molecule which, when coated onto tissue-culture surfaces, promotes the attachment and growth of cells. After the MCF7-derived BCSCs were cultured on the polylysine-coated plates reached confluency, they were trypsinized and mechanically disassociated. Cell cultures were seeded onto new plates with a 0.1 µM concentration of the homing peptide and incubated for 1 hour. Following incubation, cells were washed with a phosphate-buffer solution three times, then stained with DAPI, a fluorescent stain that binds strongly to DNA sequences in cell nuclei, and a CD133 fluorescent-antibody marker, a common cell surface molecule in stem cells. The homing peptide itself contained rhodamine, an additional staining molecule. The stained cells were observed via fluorescence microscopy.

Results: Following this procedure, the homing peptide was observed to successfully bind to MCF7-derived BCSCs. The homing peptide had an affinity for the MCF7-derived BCSCs at homing peptide concentrations as low as 0.1 µM. Stem cell properties of the derived cell line were confirmed through CD133 fluorescent staining.

Conclusion: Our work demonstrates the efficacy of this homing peptide to guide the novel homing peptide to MCF7 BCSCs, providing proof of concept that this combination could stand as a potential candidate breast cancer therapy, perhaps for those cancers specifically relapsed after surgery or first-line chemotherapy as a result of BCSC proliferation. However, more work is needed to establish any inhibitory or toxigenic effect the homing peptide may have on this cell line. Further work should also investigate the use of this homing peptide on native rat or human BCSCs to determine its pharmacokinetics, affinity, toxicity, and anti-tumor effects.