Longitudinal cancer monitoring is crucial to clinical implementation of precision medicine. There is growing evidence indicating important functions of extracellular vesicles (EVs) in tumor ...progression and metastasis, including matrix remodeling via transporting matrix metalloproteases (MMPs). However, the clinical relevance of EVs remains largely undetermined, partially owing to challenges in EV analysis. Distinct from existing technologies mostly focused on characterizing molecular constituents of EVs, here we report a nanoengineered lab-on-a-chip system that enables integrative functional and molecular phenotyping of tumor-associated EVs. A generalized, high-resolution colloidal inkjet printing method was developed to allow robust and scalable manufacturing of three-dimensional (3D) nanopatterned devices. With this nanochip platform, we demonstrated integrative analysis of the expression and proteolytic activity of MMP14 on EVs to detect in vitro cell invasiveness and monitor in vivo tumor metastasis, using cancer cell lines and mouse models. Analysis of clinical plasma specimen showed that our technology could be used for cancer detection including accurate classification of age-matched controls and patients with ductal carcinoma in situ, invasive ductal carcinoma, or locally metastatic breast cancer in a training cohort (
= 30, 96.7% accuracy) and an independent validation cohort (
= 70, 92.9% accuracy). With clinical validation, our technology could provide a useful liquid biopsy tool to improve cancer diagnostics and real-time surveillance of tumor evolution in patients to inform personalized therapy.
Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is upregulated in breast cancer, and elevated cytoplasmic ...HuR correlates with high-grade tumors and poor clinical outcome of breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors, and cytokines that support major tumor hallmarks including invasion and metastasis. Here, we report a HuR inhibitor KH-3, which potently suppresses breast cancer cell growth and invasion. Furthermore, KH-3 inhibits breast cancer experimental lung metastasis, improves mouse survival, and reduces orthotopic tumor growth. Mechanistically, we identify FOXQ1 as a direct target of HuR. KH-3 disrupts HuR-FOXQ1 mRNA interaction, leading to inhibition of breast cancer invasion. Our study suggests that inhibiting HuR is a promising therapeutic strategy for lethal metastatic breast cancer.
Abstract
Triple-negative breast cancer (TNBC) has a much lower 5-year relative survival rate (77%) than the overall breast cancer (91%). Chemotherapy remains the primary choice for the treatment of ...TNBC. However, patients often develop resistance to conventional chemotherapy after long-term exposure to the chemo-drugs, resulting in poorer prognosis and higher tumor reoccurrence compared to other subtypes of breast cancer. Therefore, understanding and overcoming drug resistance is critical for the successful treatment of TNBC. The Hu antigen R (HuR) or ELAVL1 (embryonic lethal, abnormal vision, Drosophila-like protein 1) plays an important role in chemotherapy resistance. The RNA-binding protein HuR is a posttranscriptional regulator, which can stabilize target mRNAs by binding to U- or AU-rich elements (ARE) mainly in 3’ untranslated region (UTR) of mRNAs and upregulate the translation of them. The encoded proteins are implicated in multiple cancer hallmarks, including chemoresistance. The overexpression of HuR, especially accumulated cytoplasmic expression, has been identified to be related to chemoresistance in many types of cancer. We hypothesize that inhibition of HuR function by disrupting its interaction with mRNAs can accelerate the decay of mRNAs and thus reduce the translation of proteins responsible for chemoresistance.Recently, we reported a small molecule HuR inhibitor, KH-3, which potently inhibit HuR function by disrupting the HuR-mRNA interaction. KH-3 can effectively suppress the growth and invasion of TNBC cells in vitro and in vivo. In this study, we aim to verify that HuR is a target for overcoming chemoresistance and evaluate that KH-3 as a HuR functional inhibitor can enhance the efficacy of chemotherapy for TNBC cells. To determine whether HuR inhibition can overcome acquired chemotherapy resistance of TNBCs, we generated MDA-MB-231 sub-cell lines with acquired resistance against docetaxel or doxorubicin. Our results show that inhibition of HuR by KH-3 could synergize chemotherapy for TNBC in vitro and in vivo. More interestingly, KH-3 treatment could re-sensitize resistant TNBC cells to chemo-drugs, indicating that HuR inhibition can overcome acquired chemoresistance. In the study of mechanism of actions, several pathways and HuR direct target mRNAs are found to be involved in acquired docetaxel and doxorubicin resistance. The detailed molecular mechanisms of how KH-3 sensitizes TNBC to chemotherapy is currently under investigation. This study provides a new strategy to overcome chemotherapy resistance and improve the overall survival rate of patients with TNBC.
Citation Format: Lanjing Wei, Qi Zhang, Gulhumay Gardashova, Cuncong Zhong, Jeffrey Aubé, Danny R. Welch, Xiaoqing Wu, Liang Xu. Targeting the RNA-binding protein HuR to overcome chemoresistance in triple-negative breast cancer abstract. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-20.
Abstract
Triple negative breast cancer (TNBC) has a lower 5-year survival rate and higher recurrence rate compared to other types of breast cancer, which is partially due to the acquired resistance ...to current treatment regimens. The RNA-binding protein Hu antigen R (HuR) is overexpressed in breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by promoting mRNA stability and translation of proteins implicated in proliferation, survival, angiogenesis, invasion, and metastasis. HuR also modulates sensitivity of breast cancer cells to chemotherapy. HuR knockout in TNBC cells with high HuR sensitizes them to chemotherapy while HuR overexpression induces chemo-resistance. Meanwhile, chemotherapy promotes cytoplasmic HuR accumulation and increases expression of HuR target encoding proteins in TNBC cells. Therefore, there is a positive feedback loop between HuR and chemo-resistance. These findings suggest that HuR plays a critical role in promoting a drug-resistance mechanism and HuR is a potential target for developing a novel therapy to overcome chemo-resistance in TNBC. RNA-binding proteins such as HuR had previously been considered to be “undruggable” due to the lack of a well-defined binding pocket for target RNAs. Nevertheless, using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that potently inhibit HuR-mRNA interaction with nM to sub-µM potency. Our top HuR inhibitor, KH-3, inhibits TNBC cell growth in vitro and in vivo. KH-3 also sensitizes TNBC cells to docetaxel and doxorubicin treatment in vitro. Furthermore, acquired docetaxel-resistant MDA-MB-231 cells and doxorubicin-resistant MDA-MB-231 cells display similar sensitivity to KH-3 to their parental cells. In the study of mechanism of action, several HuR direct targets are found to be involved in acquired docetaxel and doxorubicin resistance. KH-3 can disrupt the interaction of HuR with those target mRNAs. In animal efficacy studies, the combination of KH-3 and chemotherapy shows synergistic effect in both parental and acquired resistant cell xenograft models. Our data provide a proof-of-principle that HuR inhibition by KH-3 may be developed as a promising molecular therapy to overcome chemo-resistance of TNBC with high HuR.
Citation Format: Xiaoqing Wu, Lanjing Wei, Gulhumay Gardashova, Cuncong Zhong, Lan Lan, Qi Zhang, Dan A Dixon, Danny R Welch, Jeffrey Aubé, Liang Xu. Chemo-sensitization of triple negative breast cancer by targeting RNA-binding protein HuR abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-09.
Abstract
As most of treatment options do not work very well for metastatic cancer. Patients with metastatic cancer have a greatly lower survival rate compared with patients with local cancer. ...Metastasis remains a life-threat to cancer patients and an unmet medical need. The RBP Hu antigen R (HuR) is overexpressed in virtually all malignancies tested, including breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by promoting mRNA stability and translation of proteins implicated in proliferation, survival, angiogenesis, invasion, and metastasis. We found that silencing of HuR inhibited cell invasion in vitro in breast cancer. Using RIP-seq (ribonucleoprotein immunoprecipitation-sequencing), a transcription factor FOXQ1, which is recently revealed to implicate in breast cancer invasion and metastasis processes, is found to be a direct HuR target. Furthermore, exogenous introduction of FOXQ1 can rescue cell invasive ability inhibited by HuR knockout. Taken together, HuR-FOXQ1 signaling axis is a potential target for blocking breast cancer metastasis. RNA-binding proteins had previously been considered “undruggable” due to lack of a well-defined binding pocket for target RNAs. Using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that inhibit HuR-mRNA interaction at nM to sub-µM potency. Our lead compound, KH-3, potently inhibits breast cancer cell growth and decreases cell invasion in vitro similar to HuR knockout, as well as increasing the expression of epithelial marker E-cadherin. FOXQ1 overexpression abolishes the effect of KH-3 on blocking metastasis in breast cancer cells, demonstrating that the HuR inhibitor KH-3 inhibits cell metastasis by blocking FOXQ1 function. Moreover, KH-3 treatment disrupts HuR-FOXQ1 interaction in RNP-IP and FOXQ1 3′-UTR luciferase reporter assays. In vivo efficacy studies show that KH-3 not only exhibits potent antitumor efficacy in orthotopic xenograft models of breast cancer, but also efficiently blocks lung metastasis in experimental metastatic cancer model. In conclusion, we identified a potent and specific small molecule disrupter of HuR-FOXQ1 interaction for potential novel anti-metastatic therapy of breast cancer with HuR overexpression.
Citation Format: Xiaoqing Wu, Gulhumay Gardashova, Lan Lan, Yu Zhan, Jiajun Liu, Dan A. Dixon, Jeffrey Aubé, Danny R. Welch, Liang Xu. Blocking breast cancer metastasis by targeting HuR-FOXQ1 signaling axis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 867.
Abstract
The majority of breast cancer-related mortality is due to metastasis. Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein ...Hu antigen R (HuR) is overexpressed in breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors and cytokines that implicate major tumor hallmarks including invasion and metastasis. Knocking out HuR by CRISPR/CAS9 technology inhibits cell invasion in breast cancer cells. Therefore, HuR is an emerging target for breast cancer therapy, especially the lethal metastatic breast cancer. RNA-binding proteins had previously been considered “undruggable” due to lack of a well-defined binding pocket for target RNAs. Using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that inhibit HuR-mRNA interaction at nM to sub-µM potency. Our lead compound, KH-3, potently inhibits breast cancer cell growth and decreases cell invasion in vitro similar to HuR knockout, as well as increasing the expression of epithelial marker E-cadherin. In the study of mechanism of action, a transcription factor, FOXQ1, which is recently revealed to implicate in breast cancer invasion and metastasis processes, is found for the first time to be a direct mRNA target of HuR and one of the top genes that are reduced by KH-3 treatment. Exogenous introduction of FOXQ1 can rescue cell invasive capability impaired by HuR knockout and abolish the effect of KH-3 on inhibiting cell invasion in breast cancer cells. Moreover, KH-3 disrupts HuR-FOXQ1 interaction in RNP-IP, RNA pull down and FOXQ1 3′-UTR luciferase reporter assays. In vivo efficacy studies show that KH-3 not only exhibits potent antitumor efficacy in an orthotopic xenograft model of breast cancer, but also efficiently inhibits lung metastasis and improves mouse survival in an experimental metastasis model. Our data provide a proof-of-principle that HuR inhibition by KH-3 may be developed as a promising molecular therapy for inhibiting progression and metastasis of breast cancer with high HuR.
Citation Format: Xiaoqing Wu, Gulhumay Gardashova, Lan Lan, Shuang Han, Cuncong Zhong, Ragul Gowthaman, John Karanicolas, Dan A. Dixon, Danny R. Welch, Ling Li, Min Ji, Jeffrey Aubé, Liang Xu. Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1235.
Abstract
RNA-binding proteins (RBPs) are key regulators of cellular functions, especially in post-transcriptional regulations. Dysregulation of RBPs is implicated in many diseases including cancer. ...One of the RBPs that is overexpressed in a variety of human cancer is Musashi-2 (MSI2). Elevated MSI2 expression is associated with ectopic oncogenic pathways, including but not limited to NUMB/Notch, PTEN/mTOR, TGF-β/SMAD3, making MSI2 a promising therapeutic target for cancer. In the case of NUMB, MSI2 binds to and negatively regulates translation of NUMB, a negative regulator of Notch signaling. Protein structure is critical for drug discovery and structure-based rational design. However, so far there is no structure available for MSI2 protein. Recently, using nuclear magnetic resonance (NMR) we solved the first solution structure of MSI2 RNA-recognition Motif 1 (MSI2-RRM1) that forms the major long narrow RNA-binding pocket. Using a fluorescence polarization (FP) chemical libraries screening, we identified several groups of hit compounds that disrupt the binding of MSI2-NUMB RNA potently. These compounds induced apoptosis, inhibited cancer cell proliferation, invasion and metastasis in multiple tumor models tested in vitro and in vivo. They also interfered with cancer stem cell functions with reduced tumorsphere formation. Several lead compounds showed promising efficacy in animal tumor models of human breast, prostate and colon cancer. To investigate the specificity of the compounds towards other RBPs, the compounds were tested in biophysical binding assays such as FP, time resolved Fluorescence Resonance Energy Transfer assay (TR-FRET), NMR spectroscopy and cell-based assays. We compared the affinity of these MSI2 inhibitors towards other RBPs such as MSI1 and HuR. Using docking and molecular dynamics simulation, we analyzed the structure-activity relationship of these inhibitors towards different RBP targets. This work adds significant information to the structure of MSI2-RRM1 and the structural basis for designing more potent and specific inhibitors of RBPs.
Citation Format: Lan Lan, Minli Xing, Xiaoqing Wu, Philip Gao, Justin T. Douglas, Yu Zhan, Gulhumay Gardashova, Jiajun Liu, Robert P. Hanzlik, Jeffrey Aubé, Kristi L. Neufeld, Berl R. Oakley, Roberto N. De Guzman, Liang Xu. Dissecting the structural basis for inhibitors of RNA-binding proteins abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2863.
PDF
