Akademik Veri Yönetim Sistemi
7th International Current Issues Congress on Medicine, Nursing, Midwifery, and Health Sciences, Sakarya, Türkiye, 27 - 29 Haziran 2025, cilt.1, ss.380-392, (Tam Metin Bildiri)
Introduction: According to the World Health Organization data from March 2023, breast cancer is
the most common cancer worldwide and the leading cause of cancer-related deaths among women. A
projected 47% increase in the number of cases by 2040 highlights the urgency for new and more
effective treatment strategies. For Luminal-type breast cancer (ER+ and/or PR+, HER2-), which is the
most common subtype (70%), there is a need for new drugs and therapeutic approaches due to variable
responses to therapy and the development of resistance.
The Hippo signaling pathway is a fundamental pathway that regulates many biological processes,
including cellular proliferation, survival, and tissue homeostasis. "Yes-associated protein 1" (YAP1) is
the main effector protein of the Hippo pathway and is closely associated with breast cancer formation,
progression, metastasis, and drug resistance. The nuclear accumulation of active YAP1 results in cell
hyperproliferation and cancerous transformation. Previous studies have shown that the YAP1 inhibitor
CA3 has an anticancer effect in various cancer types, particularly in breast cancer.
One of the hallmarks of cancer cells is the dysregulation of the cell cycle, leading to uncontrolled
proliferation. The hyperactivation of the Cyclin D-cyclin-dependent kinase 4/6 (CDK4/6) complex
plays a role in many types of cancer. The CDK4/6 inhibitors (CDKis) Palbociclib, Ribociclib, and
Abemaciclib (Ab) have shown impressive results in prolonging progression-free and overall survival
in patients with HR+, HER2- metastatic or advanced-stage breast cancer. However, side effects and
the development of secondary resistance necessitate the exploration of safer and more effective new
treatment combinations.
Considering the impact of both the cell cycle and the Hippo signaling pathway in breast cancer
development, it was hypothesized that targeting these two pathways could control cancer cell viability
and proliferation. Within the scope of this project, the anticancer effect of the YAP1 inhibitor CA3 and
its combined effect with the FDA-approved CDK4/6 inhibitor Abemaciclib were investigated in
Luminal A-type breast cancer cells (MCF7).
Materials and Methods: Cell viability in MCF7 cells was determined using the MTT assay.
Following 72-hour single-agent applications of CA3 (20 mM stock) and Abemaciclib (Ab) (10 mM
stock), percentage viability values were determined by the MTT test, and EC50 values were calculated
using the Dr. Fit program. For combined application experiments, 7 different concentrations of CA3
(0.005-2 μM), which inhibited viability by a maximum of 60%, and 5 different concentrations of Ab
(0.006-0.64 μM), which inhibited viability by a maximum of 45%, were selected for MCF7 cells. The
obtained viability data were analyzed for synergy/antagonism using the Combenefit program
according to the Bliss independence, Highest Single Agent (HSA), and Loewe additivity models.
Furthermore, the effect of different fixed concentrations of one compound on the dose-response curves
of the other was visualized to evaluate potential shifts. Statistical analyses were performed in
GraphPad Prism 8 (one-way ANOVA, Dunnett's test, P<0.05). Results: In the viability analyses conducted on MCF7 cells, the EC50 concentration of CA3 was
determined to be 1.02 μM according to the classical Hill equation based on a monophasic model. The
effect of Ab was found to be consistent with a biphasic model, with an EC50 concentration of 4.18
μM. Synergy analyses of the combined applications revealed distinct yet significant interactions
depending on the model used. The HSA and Loewe models showed strong and statistically significant
synergistic effects, particularly at high Ab concentrations (e.g., 2 μM) and mid-to-high CA3
concentrations (0.8 μM, 0.9 μM, 1 μM), with synergy scores reaching up to +23 for 0.8 μM CA3 and
2 μM Ab in the HSA model. These synergistic effects were also supported by dose-response shift
graphs for both compounds. Notably, the leftward and downward shift of CA3's dose-response curve
in the presence of Ab indicated an increase in CA3's potency, demonstrating greater efficacy even at
lower concentrations. Similarly, shifts suggestive of synergy were observed in Ab's dose-response
curve in the presence of CA3. Conversely, the Bliss independence model revealed pronounced
antagonistic interactions at high CA3 concentrations (1.2 μM and 1.5 μM) and mid-to-high
Abemaciclib concentrations (0.176 μM - 0.64 μM) (e.g., an antagonism score of -11 for 1.2 μM CA3
and 0.176 μM Ab).Conclusion and Discussion: This study demonstrates that the combination of the YAP1 inhibitor
CA3 and the CDK4/6 inhibitor Ab can exhibit both synergistic and antagonistic interactions in
Luminal A breast cancer cells. The strong synergistic regions, particularly identified by the HSA and
Loewe models, suggest that this combination holds the potential to enhance therapeutic efficacy and
possibly minimize side effects by reducing dosages in future breast cancer treatments. This supports
the approach that targeting multiple signaling pathways (Hippo and cell cycle) may be superior to
single-agent therapy. However, the antagonistic findings revealed by the Bliss model underscore the
complexity of drug interactions and the critical importance of concentration selection. Therefore, therisk that certain combinations might reduce efficacy should be considered. The fact that different
synergy models provide different interpretations indicates that a single reference model may not be
sufficient in combination studies and that results must be carefully evaluated in a biological context.
In summary, the Ab and CA3 combination has promising synergistic potential in Luminal A breast
cancer. Nevertheless, meticulous investigation of optimal dosage ratios is required to identify the most
synergistic and least toxic combinations for the transition to preclinical and clinical studies. These
findings provide crucial information for the rational design of combination therapies in drug discovery
and development processes.