TAK1 Inhibitor NG25 Enhances Doxorubicin-mediated Apoptosis In Breast Cancer Cells

Breast cancer is a group of heterogeneous diseases based on gene profiles,biological behaviors,morphological features and clinical outcomes.It accounts for 22% of all female cancers and 26% in affluent countries,which makes it the most common carcinoma in women.It is estimated that about 6% of women develop invasive breast cancer before age 75 in affluent populations in North America,Europe and Australia.With the improvement in the combination of more effective local regional disease control and systemic treatment protocols,the mortality of breast cancer has been reduced dramatically.The successful cancer-specific targeted therapies involving the endocrine therapy on estrogen receptor(ER)-positive tumors and HER2-targeted therapy on HER2 over-express tumors are major contributors.However,the cytotoxic agents are still the mainstream medications on advance breast cancer,especially for triple negative tumors.Doxorubicin(Dox,Adriamycin)is one of the most commonly used chemotherapy drugs for treating breast cancer.Doxcontaining adjuvant chemotherapy is recommended as the first line anti-cancer drug in 2016 NCCN's breast cancer guidelines,especially for HER-2 positive and triple negative breast cancer patients.Dox is well known to cause dose-dependent and progressive cardiac damage,profoundly affecting its clinical use.Different formulas of Dox such as liposomal doxorubicin or in combination with cardio-protective Dexrazoxane have been used clinically to minimize the cardio toxicity.These render limited benefit.Identification of new ways to maximize the benefits of Dox and at the same time minimize cardiac damage is in need for better treatment outcome.TAK1 was first discovered in 1995 as a mitogen-activated protein kinase kinase kinase(MAP3K),which is activated by TGF-? and bone morphogenetic protein(BMP).Recent studies emphasized that TAK1 has a powerful pro-survival role through activating the Ik B kinase(IKK)-NF-k B pathway.In addition,the activated TAK1 can phosphorylate and activate MAPKKs leading to activation of MAPKs such as ERK,p38 and JNK.The activation of NF-k B and MAPKs induces downstream expression of inflammatory cytokines and anti-apoptotic proteins which can block apoptosis and promote cell proliferation.Furthermore,elevated NF-k B activity has been shown to contribute to the development of resistance to chemotherapy,endocrine therapy and radiation therapy.Due to the critical role of TAK1 in cancer development and drug resistance,targeting TAK1 has been suggested as a promising anti-cancer strategy and has been studied in pancreatic cancer,breast cancer,colon cancer and non-small cell lung carcinoma.Based on the knowledge that TAK1 mediates NF-?B activation in response to genotoxic stresses,5Z-7-oxozeaenol(5Z-O),an irreversible ATP-competitive TAK-1 inhibitor,has been used to enhance the sensitivity of neuroblastoma cells to Dox therapeutic treatment.Recently,5Z-O also has been shown inhibiting TAK1 activity and then suppressing downstream signaling pathways including p38 and I?B kinase(IKK)pathways in breast cancer cells.Furthermore,inhibition of TAK-1 activity by knocking out TAK1-aasociated molecule TAB1 significantly suppressed breast cancer growth and metastasis in vivo.Considering the aforementioned information,we hypothesize that TAK1 inhibitor would probably enhance Dox-mediated cytotoxicity in breast cancer cells.NG25 is a synthesized type II TAK1 inhibitor which binds to the ATP binding pocket of the target kinase.Comparing to 5Z-O,a compound isolated from fungi,NG25 has economic benefits.Recently,NG25 has been shown to be able to effectively inhibit TAK-1 activity in several studies.However,the potential therapeutic effect of NG25 on cancer treatment has not been tested.In this preclinical study,we tested the cytotoxic effect of NG25 and its effects on Dox treatment on breast cancer cells by using a panel of breast cancer cell lines including T-47 D,MCF7,HCC1954,MDA-MB-231,and BT-549(representing ER/PR+,HER2+,or triple negative,respectively).TAK1,as an intracellular hub molecule that regulates both NF-?B and MAPK signaling pathways,plays an important role in cell survival,promotion of metastasis and development of drug resistance.Previous studies suggest that TAK1 may be a promising target for cancer treatment.Inhibition of TAK1 by RNAi-silencing or an oral inhibitor LYTAK1 significantly enhanced the cytotoxic effect of oxaliplain and gemcitabine in pancreatic cancer.TAK1 is a treatment target for enhanced efficacy of topoisomerase inhibitors in breast cancer.Additionally,5-Z-O,a TAK1 inhibitor isolated from fungi enhanced the cell-killing activity of Dox and etoposide in neuroblastoma.Furthermore,TAK1 inhibition had pro-apoptotic activity in KRASdependent colon cancer.Recently,Micro RNA-26 b has been shown to suppress the activation of NF-?B signaling and to enhance the chemosensitivity of hepatocellular carcinoma cells by targeting TAK1 and TAB3.Consistent with these findings,we found that NG25,a synthesized TAK1 inhibitor,could sensitize the breast cancer cells to Dox mediated cytotoxicity through inhibiting the activation of NF-?B pathway.Herein,in this study,by using a panel of breast cancer cell lines,representing different molecular subtypes of breast cancer,we provide clear evidence that NG25 can enhance Dox-induced cytotoxicity in breast cancer cells through TAK1 inhibition.The role of NF-?B as a master transcriptional factor promoting cell-survival,increasing therapeutic resistance and enhancing metastasis ability of cancer cells has been well documented.NF-?B can be activated by multiple stimuli including genotoxic stresses.Dox,as a widely used chemotherapy reagent in different types of cancer,triggers the apoptosis of cancer cells by interfering the DNA topoisomerase ?? and creating DNA double-stand breaks which subsequently activates NF-?B signaling pathway in order to repair the damage via a stimulatory action on homologous repair,involving the targets ATM and the tumor suppressor gene,breast cancer susceptibility gene 2(BRCA2).In our studies,NG25 is shown to inhibit Dox-induced NF-?B activation and disturb the balance between pro-apoptotic and pro-survival pathways and thus it increased the efficacy of Dox chemotherapy in breast cancer cells.By detecting the level of I?B?,we showed that NG25 can inhibit TAK1-mediated NF-?B activation,which possibly is one of the major mechanisms of NG25 activity in the breast cancer cells.Besides that,we also detected that the inhibition of TAK1-mediated MAPK activation had partially been blocked by NG25,which may also contribute to the effect of this drug on breast cancer cells.Taken together,we conclude that inhibition of TAK1 activity by NG25 can enhance Dox-induced apoptosis by blocking NF-?B and MAPK signaling pathways.Integration of NG25 with Dox chemotherapy can reduce Dox dosage and thus minimize its side effects.As known,breast cancer is a heterogeneous disease,which can be classified into four major subtypes,including luminal A,luminal B,HER2 positive,and triple negative tumors.Each subtype has specific molecular changes,yet it is still possible to highlight common molecular features among those different tumor subtypes.Constitutively activation of NF-?B,a transcription factor that plays critical role in cell survival and proliferation,is one of those common features found in most breast cancer tumors.Furthermore,TAK1 has been shown to be constitutively activated in human breast cancer by detecting the phospho-TAK1 level on tumor tissues.Inhibition of TAK1 by RNAi knockdown of TAB1 significantly suppressed tumor growth and metastasis in vivo,suggesting that TAK1 is a potential therapeutic target for breast cancer.In our study,we found that NG25 alone has cytotoxic effect on all of the breast cancer cell lines in a dose-dependent manner,but with varied efficacy.To be noted,TAK1 has been shown to be constitutively activated in highly metastatic MDA-MB-231 breast cancer cells while its activity is very low in non-metastatic MCF-7 cells.Consistent with these results,we found that NG25 had a relatively stronger inhibitory effect on MDA-MB-231 cells compared with that on MCF-7 cells,suggesting that TAK1 may be a promising therapeutic target,especially for highly aggressive triple negative breast cancers.Targeting TAK1 as a strategy to overcome chemotherapy resistance has been reported in pancreatic cancer,neuroblastoma and hepatocellular carcinoma.It has also been tested to reverse radiotherapy resistance in breast cancer.In this study,we provide clear evidence that NG25 enhanced the Dox-mediated cytotoxic activity in different breast cancer cell lines.This activity seems universal regardless of the molecular subtypes of breast cancer cells.Therefore,targeting TAK1 may be an option to overcome the chemotherapy resistance.Taken together,we demonstrated that TAK1 inhibitor NG25 can suppress the Dox-induced NF-?B and MAPK signaling pathway activation,which subsequently sensitized breast cancer cells to Dox-mediated cytotoxic effect and this effect is observed in all major breast cancer cell subtypes.The data we presented here suggest that NG25,as a potent inhibitor of TAK1-mediated NF-?B activation,might be useful in enhancing the efficacy of Dox while reducing its side effects and then broaden its clinical use,deserve further investigation and clinical validation.