Abstract
Combinatorial targeted therapies are more effective in treating cancer by blocking by-pass mechanisms or inducing synthetic lethality. However, their clinical application is hampered by resistance and toxicity. To meet this important challenge, we developed and tested a novel concept of biomarker-guided sequential applications of various targeted therapies using ErbB2-overexpressing/PTEN-low, highly aggressive breast cancer as our model. Strikingly, sustained activation of ErbB2 and downstream pathways drives trastuzumab resistance in both PTEN-low/trastuzumab-resistant breast cancers from patients and mammary tumors with intratumoral heterogeneity from genetically-engineered mice. Although lapatinib initially inhibited trastuzumab-resistant mouse tumors, tumors by-passed the inhibition by activating the PI3K/mTOR signaling network as shown by the quantitative protein arrays. Interestingly, activation of the mTOR pathway was also observed in neoadjuvant lapatinib-treated patients manifesting lapatinib resistance. Trastuzumab + lapatinib resistance was effectively overcome by sequential application of a PI3K/mTOR dual kinase inhibitor (BEZ235) with no significant toxicity. However, our p-RTK array analysis demonstrated that BEZ235 treatment led to increased ErbB2 expression and phosphorylation in genetically-engineered mouse tumors and in 3-D, but not 2-D, culture, leading to BEZ235 resistance. Mechanistically, we identified ErbB2 protein stabilization and activation as a novel mechanism of BEZ235 resistance, which was reversed by subsequent treatment with lapatinib + BEZ235 combination. Remarkably, this sequential application of targeted therapies guided by biomarker changes in the tumors rapidly evolving resistance doubled the life-span of mice bearing exceedingly aggressive tumors. This fundamentally novel approach of using targeted therapies in a sequential order can effectively target and reprogram the signaling networks in cancers evolving resistance during treatment.
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Acknowledgements
We thank Yu laboratory members for helpful discussions on the manuscript. We thank Novartis for providing BEZ235. We also thank Dr Mark Greene (University of Pennsylvania) for generously providing the hybridoma for 7.16.4 mAb. This study is supported by NIH grants P30-CA 16672 (MDACC), PO1-CA099031 project 4 (DY), RO1-CA112567-07 (DY), Susan G Komen Breast Cancer Foundation Promise Grant KG091020 (DY), Cancer Prevention Research Institute of Texas Grant RP100726 (DY), Career Development Award of MD Anderson Cancer Center Breast SPORE (OS), NIH/NCI pre-doctoral fellowship F31CA165819 (SB) and NIH grant U54CA149169 (STW). D Yu is the Hubert L & Olive Stringer Distinguished Chair in Basic Science at MD Anderson Cancer Center.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Table S1
List of differentially expressed probes between PTEN low/trastuzumab resistant (red) versus PTEN normal/trastuzumab sensitive (blue) breast cancer patients. (XLSX 6366 kb)
Supplementary information, Table S2
List of genes used for IPA to determine canonical pathways differentially enriched between PTEN normal/trastuzumab resistant vs. PTEN high/trastuzumab sensitive breast cancer patients. (PDF 186 kb)
Supplementary information, Table S3
List of proteins with downregulated expression/phosphorylation in Ab7.16.4+lapatinib sensitive PTEN−/−/NIC transplants compared to control and resistant tumors. (PDF 219 kb)
Supplementary information, Table S4
List of antibodies used in western blotting (PDF 182 kb)
Supplementary information, Figure S1
PTEN loss leads to activation of ErbB2 and its downstream signaling in PTEN−/−/NIC mice. (PDF 297 kb)
Supplementary information, Figure S2
TUNEL staining of the tumor tissue obtained from PTEN−/−/NIC mice treated with different drugs or their combination for determination of apoptosis. (PDF 177 kb)
Supplementary information, Figure S3
Syngeneic tumor transplantation model recapitulates the both molecular and biological features of the original PTEN−/−/NIC mice. (PDF 393 kb)
Supplementary information, Figure S4
TUNEL staining of the tumor tissue obtained from PTEN−/−/NIC mice treated with vehicle, Ab+Lapa or sequential A+L/A+B for determination of apoptosis. (PDF 166 kb)
Supplementary information, Figure S5
BEZ235 does not stabilize ErbB2 in 2-D cell culture in BT474.Par or BT474.LapR cells and does not affect ErbB2 mRNA level in 3-D culture. (PDF 265 kb)
Supplementary information, Figure S6
BEZ235 treatment does not significantly alter c-MYC level in 3-D culture. (PDF 153 kb)
Supplementary information, Figure S7
PTEN loss is heterogeneous in Neu-driven, PTEN-loss mouse mammary tumors. (PDF 245 kb)
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Sahin, O., Wang, Q., Brady, S. et al. Biomarker-guided sequential targeted therapies to overcome therapy resistance in rapidly evolving highly aggressive mammary tumors. Cell Res 24, 542–559 (2014). https://doi.org/10.1038/cr.2014.37
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DOI: https://doi.org/10.1038/cr.2014.37
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