Chemoresistance in cancer is a complex and multifaceted phenomenon, with both inherent and acquired resistance pathways contributing to treatment failure (13). This complexity is further compounded by the heterogeneous nature of cancer, making it difficult to fully understand the mechanisms of chemoresistance (14). One significant factor in chemoresistance is the presence of cancer stem cells, which possess intrinsic resistance to chemotherapy (9). Despite these challenges, several strategies have been proposed to overcome chemoresistance, including the use of novel agents, combination treatments, and targeted therapies (9, 10). However, the effectiveness of these strategies in clinical settings remains a major hurdle (13). Our findings reveal that the effects of the nine chemotherapeutic drugs tested can be classified into three distinct groups based on their impact on canonical pathways. These groups are: Group I (Arabinosylcytosine, Methotrexate), Group II (Paclitaxel, Cyclophosphamide, Nocodazole), and Group III (5-fluorouracil, Etoposide, Doxorubicin, Cisplatin). We propose a combination therapy strategy that utilizes chemotherapeutic drugs from different groups. In the event of a relapse, the treatment should involve drugs from a different group than those used in the initial treatment. This approach needs further validation through in vitro and in vivo studies.
Continuous exposure to increasing concentrations of chemotherapeutic drugs leads to the emergence of drug-resistant cells, a process that reflects the selective pressures experienced by cancer cells in vivo (15). This approach leads to the enrichment of a drug-resistant cell population through genetic and epigenetic alterations (16). These alterations include the overexpression of drug efflux pumps, changes in drug metabolism, enhanced DNA repair capacity, and modifications in cell signaling pathways (15). The development of drug resistance is further complicated by the evolution of multifactorial mechanisms, such as gene amplification and increased enzyme activity (16). These mechanisms are associated with altered sequestration and efflux of chemotherapeutic drugs by multidrug-resistant cells (17). Despite the identification of these mechanisms, strategies to overcome drug resistance have largely failed due to the complexity of the resistance phenotype.
Several studies have reported the development of MCF7 resistant sublines through stepwise increases in different chemotherapeutic drugs. The establishment of MCF7 sublines, including MCF7/VP (Vincristine), MCF7/Pac (Paclitaxel), MCF7/TopoisomeraseII (Etoposide), MCF7/MTX (Methotrexate), MCF7/5-FU (5-fluorouracil), MCF7/Cis (Cisplatin), MCF7/Cyclo (Cyclophosphamide), and MCF7/AraC (Arabinosylcytosine), involves the selection and expansion of inherently resistant cells (18-21). These sublines display various characteristics of drug resistance, including altered gene expression patterns and increased expression of drug resistance-related proteins (18, 20-22). The resistance mechanisms include increased expression of multidrug resistance protein (MRP) and glutathione S-transferase P1-1 (GSTP1-1) (18), reduced drug sensitivity of topoisomerase II (22), enhanced energy-dependent drug efflux (20), and upregulation of MDR1 gene and detoxifying enzymes (21). These studies collectively highlight the complex and multifaceted nature of drug resistance in MCF7 cells.
The use of a singular cell line in cancer research offers several advantages, including the elimination of cellular heterogeneity, unifying genetic background, facilitating comparative studies, and ensuring reproducibility and consistency (23). However, the presence of tumor heterogeneity poses a challenge to the effectiveness of drug delivery and treatment methods. Therefore, while the use of a singular cell line can provide valuable insights, it is crucial to consider the limitations and utilize multiple cell lines. Thus, our proposed classification for chemotherapeutic drugs based on canonical pathway activation/inactivation should be validated using a diverse panel of breast cancer cell lines representing the heterogeneity of tumor cells.
Despite the success of established combination regimens, there exists an opportunity to optimize treatment strategies by exploring novel combinations and integrating additional highly effective oncology drugs. This rationale is grounded in understanding the shared and distinct signaling pathways activated and inactivated by various chemotherapeutic agents commonly used in breast cancer treatment. To address this, we systematically grouped nine chemotherapeutic agents based on their activation scores and intersections within various signaling pathways in our MCF7 chemoresistant sublines. The resulting three groups provide a framework for comparing newly assembled groups with traditional breast cancer regimens. This comparative analysis, considering indications, mechanisms of action, resistance, and relapse rates, aims to deepen our understanding of the molecular mechanisms governing MDR across these diverse drug classes. The ultimate goal is to provide a foundation for developing innovative regimens that integrate additional drugs with minimal overlap in signaling pathways and toxicities. This strategic approach has the potential to significantly enhance chemotherapy effectiveness and reduce relapse rates in the future (24). Based on the proposed classifications, it becomes evident that certain drugs share similar groupings concerning both their activation scores in the canonical pathways and their pharmacokinetics (24). Notably, 5-Fluorouracil and Arabinosylcytosine, classified as antimetabolites, function as inactivators in various signaling pathways. Similarly, Paclitaxel and Nocodazole, which primarily act as mitotic spindle inhibitors, show no difference in terms of their roles as activators or inactivators in the analyzed signaling pathways illustrated in Figure 1. Lastly, Etoposide, Doxorubicin, and Cisplatin—classified as genotoxic agents based on their chemotherapeutic mechanisms primarily serve as activators in a diverse array of signaling pathways detailed in Figure 1. These classifications hold particular significance in endeavors aimed at comprehending the formation of multidrug resistance and determining suitable chemotherapeutic agents in the event of relapse. By considering the activation/inhibition of canonical pathways and pharmacokinetic properties of these drugs, researchers can develop more effective combination therapies and predict potential drug resistance mechanisms. Thus, the grouping of anti-cancer drugs based on their canonical signaling pathways and pharmacokinetics offers a novel approach to understanding and optimizing chemotherapy regimens, ultimately leading to improved patient outcomes in breast cancer treatment.
The strategy of combination therapy, involving the synergistic combination of multiple anti-cancer agents, has emerged as a promising avenue to enhance the efficacy of breast cancer treatment compared to monotherapy (25). This approach seeks to overcome the limitations associated with single-agent therapies by mitigating resistance, inhibiting metastasis, and reducing the number of mitotically active cells. Clinical trials consistently highlight the effectiveness of combination chemotherapy regimens in improving patient outcomes in breast cancer. A notable example is the cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) combination adjuvant chemotherapy regimen pioneered by the Istituto Nazionale Tumori in Milan. This regimen demonstrates improved overall survival among breast cancer patients, with no significant difference in relapse rates between pre-menopausal and post-menopausal women (12, 13). The US Breast Cancer Intergroup reported the efficacy of the CMF regimen in reducing recurrence and enhancing survival, particularly in patients with axillary node-negative disease (12, 13). Supporting the efficacy of combination therapy, the NSABP B-20 trial demonstrated that adding tamoxifen to the CMF regimen significantly decreased the relapse rate in patients with ER-positive, lymph node-negative disease. This effect aligns with the findings of the EBCTCG meta-analysis, indicating that CMF contributed to a 30% reduction in the recurrence rate over a 10-year period (26). Our proposed classification system, which assigns cyclophosphamide to group II, methotrexate to group I, and 5-fluorouracil to group III, further supports the potential of this categorization approach.
In addition to CMF, other combination regimens exhibit promise in breast cancer treatment. The AC combination therapy consisting of doxorubicin (group III) and cyclophosphamide (group II), administered every three weeks for four cycles, proves to be an effective and shorter alternative to the CMF regimen for patients with node-negative disease (27). The FAC regimen, comprising 5-fluorouracil (group III), doxorubicin (group III), and cyclophosphamide (group II), demonstrates superior efficacy compared to AC and CMF in postmenopausal women with hormone-receptor-positive, node-positive breast cancer (28, 29). More patient-customized regimens, such as doxorubicin (group III), cyclophosphamide (group II), followed by paclitaxel (group II) (AC-T), have been developed by Memorial Sloan Kettering Cancer Center for the treatment of aggressive early-stage breast cancer in young patients (30-32).
The efficacy of breast cancer chemotherapeutic regimens varies in their ability to prevent relapse and prolong disease-free survival (DFS) among patients. Bang et al. (2000) compared six cycles of AC to six cycles of oral CMF and found a DFS of 64% for AC-treated patients compared to 78% for CMF-treated patients (33). Another study by CALGB 9344 compared four cycles of AC to four cycles of AC followed by paclitaxel (AC-P) and reported a DFS rate of 65% for AC alone versus 70% for AC-P at five years (34). Additionally, Martin et al. (2003) compared six cycles of FAC to six cycles of CMF and found a DFS of 58% for FAC compared to 50% for CMF at five years (35). Finally, a study by MD Anderson Cancer Center compared four cycles of paclitaxel followed by four cycles of FAC to four cycles of FAC alone and reported a DFS of 86% for paclitaxel followed by FAC (pac/FAC) and 83% for FAC alone at 48 months (36). These studies demonstrate that contemporary regimens incorporating anthracyclines, such as doxorubicin, appear to be more effective in improving overall survival than CMF. However, they are associated with increased long-term toxicities, particularly an increased risk of acute myeloid leukemia (AML), compared to CMF. Therefore, it is crucial to establish an individualized selection process for the treatment of breast cancer patients. CMF may remain suitable for those with a low risk of recurrence, whereas patients at a high risk of recurrence may benefit from more effective treatments, such as regimens including anthracyclines (37). To further advance evidence-based regimen selection, it is essential to understand how these drugs interact in terms of their activated and inactivated biochemical pathways and how they overcome relapse. This knowledge can guide the development of more personalized and effective treatment strategies for breast cancer patients.
The increasing prevalence of MDR in cancer treatment has prompted the exploration of alternative strategies to improve patient outcomes. Our approach is to utilize drugs with minimally overlapping signaling pathways and different pharmacokinetic profiles to minimize the risk of MDR development. This strategy is particularly relevant in the context of relapse treatment, where cancer cells have already demonstrated resistance to initial treatment regimens. A notable study conducted in 1979 examined the efficacy of methotrexate (group I) and vincristine in patients with advanced breast cancer who had failed treatment with FAC (5-fluorouracil (group III), doxorubicin (group III), and cyclophosphamide (group II)) or experienced relapse. Among the 17 patients enrolled, four achieved partial remission lasting six months, with a median survival of ten months (38). Additionally, seven patients exhibited stable disease with minimal toxicity. This study is particularly intriguing in light of the distinct pharmacological profiles of methotrexate and vincristine compared to FAC drugs. As suggested by this study, methotrexate belongs to a different group based on its general function within the listed signaling pathways. Unfortunately, we did not examine vincristine canonical pathways in this study. These findings suggest that employing drugs with minimal or no overlapping signaling pathways may hold promise in reducing the likelihood of MDR development, particularly in the context of relapse treatment. However, further research, including additional data on multiple breast cancer cell lines and clinical trials, is warranted to fully elucidate the potential benefits of this approach.