No abstract

Breast cancer (BC) is a heterogeneous disease and it is one of the leading causes of mortality worldwide in women each year. However, in the United States, there is a disparity in survival between African American (AA) and Caucasian women with AA women having a higher rate of mortality. BC is typically classified based on the expression of the following receptors: Estrogen receptor α (ERα), Progesterone receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER2). Conventional treatment for BC relies on the expression of ERα for anti‐estrogenic therapy and HER2 for HER2 inhibition. Triple negative breast cancer (TNBC) is a form of breast cancer which lacks expression of ERα, PR and HER2 and affects minority population disproportionately. Potential mechanisms for the loss of ERα expression include hyperactivation of MAPK pathways due to EGFR or HER2 overexpression, estrogen withdrawal, hypoxia and methylation of the CpG island, cytosine‐guanine abundant area, on the ER gene promoter region. Due to lack of targetted treatment for TNBC, alternative treatment options are essential to increase patient survivability. 2, 3‐ dichloro‐5, 8‐dimethoxy‐1, 4‐naphthoquinone (Z285), a 1,4 napthoquinone analog, has demonstrated cytotoxic effects in estrogen positive and estrogen negative cell lines. Previous in silico data showed that this compound may function as an MAPK allosteric inhibitor. MCF7 cells were used as an ERα positive control and two TNBC cell lines, HCC1806, from a AA woman, and MDA MB 231, from a Caucasian woman, were used. Cell viability studies were carried out on cells treated with Z285 alone (0.5–10μM), 4 Hydroxy‐Tamoxifen (4OH‐Tam) alone (3–30μM) for 24,72 and 120hrs. In combination studies, the cells were pretreated with Z285 (2 or 5μM) for 6 hrs followed by 4OH‐Tam (1–15μM) for 24, 72 and 120hrs. These results showed a dose and time‐dependent response with individual treatment. Combinatory studies showed significant decreases in cell viability when compared to individual studies. Cells were treated with 5 μM Z285 and mRNA was collected for RNA microarray analysis to determine changes in mRNA expression profile. These results showed modulation of several genes involved in the MAPK pathway and suggest a mechanism of increased sensitivity of the TNBC cells to 4OH‐Tamoxifen. Therefore, this novel compound which targets the MAPK pathway may offer a model for treating TNBC breast cancers by reactivating ERα expression in these cancers and thus rendering them susceptible to anti‐estrogen therapy.Support or Funding InformationCharles and Mary Latham FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Triple negative breast cancer (TNBC) is a subtype of breast cancer (BC) that makes up 10–15% of BC diagnosis. This form of the disease disproportionately affects African American (AA) women as compared to women of other ethnicities. TNBC is classified by lack of expression of three receptors: Estrogen alpha (ERα), progesterone (PR) and human epidermal growth factor 2 (HER2). These receptors are used as therapeutic targets for BC that express them. As specific targets are not present, surgery, radiation and chemotherapy have been the mainstay of therapy for TNBC. Recently, newer drugs have become available based on specific genetic and epigenetic mutations like BRCA1, however more treatment options need to be produced to reduce the mortality rate in this disease. A novel compound, 2, 3‐dichloro‐5, 8‐dimethoxy‐1, 4‐naphthoquinone (Z285) belongs to the class of naphthoquinones that demonstrate activities as anticancer, antimalarial, and antibacterial compounds. These compounds have also been shown to cause an increase in reactive oxygen species (ROS) production as well as affect signaling pathways associated with epidermal growth factor receptor (EGFR). Moreover, it has been reported that treatment with tamoxifen can cause cell death. This is based on the accumulation of the drug and its active metabolites within cells that may cause an increase in the ROS production ultimately leading to cellular death, even in ERα negative cell lines. In the current study, three triple negative cell lines, HCC1806, MDA MB 231 and HS578T were used. These cells were treated with Z285 and 4‐hydroxy tamoxifen (4OH‐Tam) for 24 or 72hr with 1, 2, 4, 8, 16μM and 3, 6, 12, 24, 48μM respectively. Synergestic activity was demonstrated using SynergyFinder as well as the Chou‐Talalay method (CompuSyn). In other experiments, cells were treated with the IC50 of Z285 which was established in prior experiments. mRNA and protein were isolated between 6 and 24hr. Results showed alterations of several mRNA in response to these treatments. In addition, there were modifications to Nrf2 protein concentration, a transcription factor associated with oxidative stress. Therefore, these results indicate that combination treatment of Z285 and 4OH‐Tam induce cell death at lower concentrations. Thus this novel compound which causes an increase in ROS in these TNBC cells, may render them more susceptible to tamoxifen therapy. Support or Funding Information Charles and Mary Latham Fund

Triple-negative breast cancer (TNBC) is classified as a form of breast cancer in which cells do not express estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 (HER2). This type of cancer affects African American women disproportionately to Caucasian women. The absence of these receptors make treatment of TNBC difficult because hormonal therapy, i.e., estrogen antagonist, progesterone antagonist and anti-HER2, is ineffective. TNBC frequently presents with elevated growth factor receptor expression and/or signaling with a resultant increase in mitogen activated kinase (MAPK) signaling, thus causing repression of ERα expression in a reversible manner. Based on these observations, the hypothesis is that the attenuation of the MAPK pathway by direct inhibition of hyperactivated MAPK will result in re-expression and functionality of ERα using 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone (Z285), making cells susceptible to conventional antiestrogen therapy. Experiments were performed using TNBC cell lines (MDA MB 231 and HCC1806) derived from Caucasian and African American women, respectively, in concentration- and time-dependent assays. Cells were treated with Z285 alone and in combination with 4 hydroxy tamoxifen (4OH-Tam) and showed an increase in cell death using cell viability assay. Results show a stark difference in the effect of the two drugs on the two cell lines when given alone. From 24-120 hours the Z285 had minimal effect on both MDA-MB 231 and HCC 1806 cell lines with IC-50 above the highest concentrations given; however, the 4-OH Tam caused significant cell death, particularly at 72 to 120 hours with IC50 values of 6 μM. The combination study of pretreating the cells of 2 μM and 5 μM Z285 followed by 4 OH-Tam showed a significant decrease in the cell viability in the HCC 1806 cells with IC50s of 7 μM at 24 hours where the MDA-MB 231 cells were more resistant at 24 hours. The 72-120 hours showed significant concentration- and time-dependent decreases in cell viability for both cell types. Particularly, the HCC 1806 were more sensitive to the effects of 4-OH Tam with IC50 at 1 μM compared to MDA-MB 231 IC50 of 5 μM at 120 hours at both 2 μM and 5 μM pretreatment. Taken together, these drugs may work synergistically to promote cell death, thus providing a new potential avenue for therapy. Therefore, patients suffering from TNBC could benefit from combinational treatment with Z285 or its analogs and conventional chemo/hormonal therapy. Citation Format: Anastasia G.J. Robinson, Ridge Wells, Samantha J. Wilkinson, Robert L. Copeland. Comparative analysis of cell viability of two triple-negative breast cancer cell lines using 2,3-dichloro-5,8-dimethoxy-1,4-naphthoquinone and 4 hydroxy-tamoxifen [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B030.