Rajib Shome1, Siddhartha Sankar Ghosh2,3. 1. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India. 2. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India. sghosh@iitg.ac.in. 3. Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India. sghosh@iitg.ac.in.
Abstract
PURPOSE: Due to a lack of effective targeted therapies, patients with metastatic triple-negative breast cancer (TNBC) have poor clinical outcomes. Epithelial to mesenchymal transition (EMT) is known to contribute to cancer progression, invasiveness and multidrug resistance (MDR). There is a strong correlation between various drug efflux mechanisms, cancer stem cells and tumor microenvironments, which in turn is synchronized by complex signaling crosstalk between EMT and MDR. We hypothesize that combining these regulatory connections with targeted combinatorial therapies may be an effective approach to annihilate the progression/metastasis of TNBC. METHODS: AlamarBlue assays were used to depict TNBC cell viability, whereas flow cytometry was used to detect apoptotic cell populations, reactive-oxygen species (ROS) levels as well as mitochondrial depolarization. qRT-PCR, Western blotting and confocal microscopy were used to provide molecular-level information of the genes and proteins involved. RESULTS: Our initial analyses showed that targeting EGFR by either erlotinib (EGFR inhibitor) or lapatinib (EGFR/HER-2 inhibitor) alone was ineffective against TNBC. Interestingly, we subsequently found that a low dose of lapatinib did act as a substrate rather than as an inhibitor facilitating EMT and MDR, leading to metastasis. Additional gene expression studies indicated that co-targeting the EGFR and Wnt/β-catenin pathways with lapatinib and XAV939 (a tankyrase inhibitor) promoted mesenchymal to epithelial transition (MET). Application of these inhibitors led to a 5.62-fold increase in the epithelial marker E-cadherin and a 3.33-fold decrease in the stemness marker EpCAM, with concomitant 1.5-fold and 3.22-fold reductions in the ABC transporters ABCB1 and ABCG2, respectively. These co-targeting effects resulted in overcoming EMT and MDR, which in turn was highlighted by reduced levels of pEGFR, pAKT, pMAPK, pSTAT-3, pGSK-3β and β-catenin. CONCLUSIONS: Our data indicate that the synergistic action of targeting both the EGFR and Wnt/β-catenin signaling pathways in TNBC cells may open up new avenues for combatting this disease.
PURPOSE: Due to a lack of effective targeted therapies, patients with metastatic triple-negative breast cancer (TNBC) have poor clinical outcomes. Epithelial to mesenchymal transition (EMT) is known to contribute to cancer progression, invasiveness and multidrug resistance (MDR). There is a strong correlation between various drug efflux mechanisms, cancer stem cells and tumor microenvironments, which in turn is synchronized by complex signaling crosstalk between EMT and MDR. We hypothesize that combining these regulatory connections with targeted combinatorial therapies may be an effective approach to annihilate the progression/metastasis of TNBC. METHODS: AlamarBlue assays were used to depict TNBC cell viability, whereas flow cytometry was used to detect apoptotic cell populations, reactive-oxygen species (ROS) levels as well as mitochondrial depolarization. qRT-PCR, Western blotting and confocal microscopy were used to provide molecular-level information of the genes and proteins involved. RESULTS: Our initial analyses showed that targeting EGFR by either erlotinib (EGFR inhibitor) or lapatinib (EGFR/HER-2 inhibitor) alone was ineffective against TNBC. Interestingly, we subsequently found that a low dose of lapatinib did act as a substrate rather than as an inhibitor facilitating EMT and MDR, leading to metastasis. Additional gene expression studies indicated that co-targeting the EGFR and Wnt/β-catenin pathways with lapatinib and XAV939 (a tankyrase inhibitor) promoted mesenchymal to epithelial transition (MET). Application of these inhibitors led to a 5.62-fold increase in the epithelial marker E-cadherin and a 3.33-fold decrease in the stemness marker EpCAM, with concomitant 1.5-fold and 3.22-fold reductions in the ABC transporters ABCB1 and ABCG2, respectively. These co-targeting effects resulted in overcoming EMT and MDR, which in turn was highlighted by reduced levels of pEGFR, pAKT, pMAPK, pSTAT-3, pGSK-3β and β-catenin. CONCLUSIONS: Our data indicate that the synergistic action of targeting both the EGFR and Wnt/β-catenin signaling pathways in TNBC cells may open up new avenues for combatting this disease.
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