Abstract PR4: Dual inhibition of TTK and HSPA9 functions to synergistically decrease viability in triple-negative breast cancer cell lines
Authors: Lavender Nicole A. , Chow Donald , Yin Holly , Berens Michael E. , Carpten John D. 
Published: May 2015

Abstract
Background: Triple-negative breast cancer (TNBC) is an aggressive subtype that is more likely to develop in African-American women and younger patients. Not only does TNBC have higher rates of metastasis, recurrence, and mortality; it lacks effective targeted treatments. We have found that compared to normal tissue, expression levels of the mitotic kinase TTK are elevated in metastatic TNBC tumors, particularly in African-American patients. We have demonstrated in TNBC cell lines that inhibiting TTK reduces cell viability, proliferation, and survival. Unfortunately, none of TTK inhibitors currently available are suitable for clinical use. Therefore, in an effort to generate an accessible treatment, we targeted a protein-protein interaction between TTK and the chaperone protein, HSPA9. This interaction results in super-activation of TTK and we hypothesized that disrupting this event may serve as an alternative and more effective method for targeting TTK as a TNBC chemotherapeutic.
Methods: We evaluated the effects of single and combined inhibition of TTK and HSPA9 on cell viability in eight TNBC cell lines. The cell lines used for this analysis were BT-20, BT-549, CAL-51, HCC38, MDA-MB-231, MDA-MB-436, MDA-MB-453, and MDA-MB-468. Cells were treated with the small molecular compounds AZ3146 and MKT-077 to inhibit TTK and HSPA9, respectively. The cells were first treated with the each agent individually to determine the dose response profile and IC50 values. Then, to achieve therapeutically relevant dose combinations, cells were treated with IC20 doses of AZ3146 and sub-nanomolar (&lt;IC10) doses of MKT-077. Changes in viability were measured after 96 hours of treatment.
Results: For the single agent treatments, the cells were more sensitive to MKT-077 alone than AZ3146. MKT-077 IC50 values ranged between 0.95 - 4.24μM, while AZ3146 values were 1.55 - 52.4μM. However, when used together at low doses, we observed synergistic effects in seven of the eight cell lines. With the exception of BT-20 cells, the combination of AZ3146 and MKT-077 resulted in significantly lower cell viability compared to treatment with either agent alone (p = 0.04 - 2.0x10-10). The combination was particularly effective in BT-549, MDA-MB-231, and MDA-MB-436 cells since they exhibited little sensitivity to AZ3146 when it was used as a single agent (IC50 ≥ 22.8μM). However, when each cell line was treated with an IC20 dose of AZ3146 and 0.05-100nM of MKT-077, we observed a statistically significant decrease in cell viability (p = 0.03 - 8.5x10-10) and drastically reduced IC50 values (combination IC50 ≤ 0.73nM).
Conclusions: Our findings show that simultaneously treating TNBC cells with low doses of TTK and HSPA9 inhibitors results in substantially greater reductions in viability. Although previous in vitro data demonstrate that TTK inhibitors cause selective cancer cell death, none of these agents are clinically available. Additionally, MKT-077 was shown to exhibit significant antitumor activity in a variety of in vitro and in vivo model systems. Unfortunately, it was found to cause reversible, but dose-dependent renal toxicity and deemed undesirable for patient use. However, we have shown that TNBC cells can be effectively treated with very low doses of MKT-077 when combined with a TTK inhibitor. More importantly, since these proteins are both overexpressed in tumor cells, our findings suggest that dual inhibition of TTK and HSPA9 may be a selective, safe, and highly effective chemotherapeutic strategy for TNBC.
This abstract is also presented as Poster B37.
Citation Format: Nicole A. Lavender, Donald Chow, Holly Yin, Michael E. Berens, John D. Carpten. Dual inhibition of TTK and HSPA9 functions to synergistically decrease viability in triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr PR4. doi:10.1158/1538-7755.DISP13-PR4