Tumor-targeted exosomes for delivery of anticancer drugs.

Exosomes are small phospholipid bilayer vesicles that are naturally produced by all living cells, both prokaryotes and eukaryotes. The exosomes due to their unique size, reduced immunogenicity, and their ability to mimic synthetic liposomes in carrying various anticancer drugs have been tested as drug delivery vehicles for cancer treatment. An added advantage of developing exosomes as a drug carrier is the ease of manipulating their intraluminal content and their surface modification to achieve tumor-targeted drug delivery. In the past ten-years, there has been an exponential increase in the number of exosome-related studies in cancer. Preclinical studies demonstrate exosomes-mediated delivery of chemotherapeutics, biologicals and natural products produce potent anticancer activity both, in vitro and in vivo. In contrast, the number of exosome-based clinical trials are few due to challenges in the manufacturing and scalability related to large-scale production of exosomes and their storage and stability. Herein, we discuss recent advances in exosome-based drug delivery for cancer treatment in preclinical and clinical studies and conclude with challenges to be overcome for translating a larger number of exosome-based therapies into the clinic.

RNA binding proteins (RBPs) and their role in DNA damage and radiation response in cancer.

Traditionally majority of eukaryotic gene expression is influenced by transcriptional and post-transcriptional events. Alterations in the expression of proteins that act post-transcriptionally can affect cellular signaling and homeostasis. RNA binding proteins (RBPs) are a family of proteins that specifically bind to RNAs and are involved in post-transcriptional regulation of gene expression and important cellular processes such as cell differentiation and metabolism. Deregulation of RNA-RBP interactions and any changes in RBP expression or function can lead to various diseases including cancer. In cancer cells, RBPs play an important role in regulating the expression of tumor suppressors and oncoproteins involved in various cell-signaling pathways. Several RBPs such as HuR, AUF1, RBM38, LIN28, RBM24, tristetrapolin family and Musashi play critical roles in various types of cancers and their aberrant expression in cancer cells makes them an attractive therapeutic target for cancer treatment. In this review we provide an overview of i). RBPs involved in cancer progression and their mechanism of action ii). the role of RBPs, including HuR, in breast cancer progression and DNA damage response and iii). explore RBPs with emphasis on HuR as therapeutic target for breast cancer therapy.

EWI2 prevents EGFR from clustering and endocytosis to reduce tumor cell movement and proliferation.

EWI2 is a transmembrane immunoglobulin superfamily (IgSF) protein that physically associates with tetraspanins and integrins. It inhibits cancer cells by influencing the interactions among membrane molecules including the tetraspanins and integrins. The present study revealed that, upon EWI2 silencing or ablation, the elevated movement and proliferation of cancer cells in vitro and increased cancer metastatic potential and malignancy in vivo are associated with (i) increases in clustering, endocytosis, and then activation of EGFR and (ii) enhancement of Erk MAP kinase signaling. These changes in signaling make cancer cells (i) undergo partial epithelial-to-mesenchymal (EMT) for more tumor progression and (ii) proliferate faster for better tumor formation. Inhibition of EGFR or Erk kinase can abrogate the cancer cell phenotypes resulting from EWI2 removal. Thus, to inhibit cancer cells, EWI2 prevents EGFR from clustering and endocytosis to restrain its activation and signaling.

Organically derived exosomes as carriers of anticancer drugs and imaging agents for cancer treatment.

Extracellular vesicles (EVs), is the umbrella term used for different types of vesicles produced by the cells, among which exosomes form the largest group. Exosomes perform intercellular communication by carrying several biologics from donor or parental cells and delivering them to recipient cells. Their unique cargo-carrying capacity has recently been explored for use as delivery vehicles of anticancer drugs and imaging agents. Being naturally produced, exosomes have many advantages over synthetic lipid-based nanoparticles currently being used clinically to treat cancer and other diseases. The finding of the role of exosomes in human diseases has led to numerous preclinical and clinical studies exploring their use as an amenable drug delivery vehicle and a theranostic in cancer diagnosis and treatment. However, there are certain limitations associated with exosomes, with the most important being the selection of the biological source for producing highly biocompatible exosomes on a large scale. This review article explores the various sources from which therapeutically viable exosomes can be isolated for use as drug carriers for cancer treatment. The methods of exosome isolation and the process of loading them with cancer therapeutics and imaging agents are also discussed in the follow-up sections. Finally, the article concludes with future directions for exosome-based applications in cancer diagnosis and treatment.

Drug delivery approaches for HuR-targeted therapy for lung cancer.

Lung cancer (LC) is often diagnosed at an advanced stage and conventional treatments for disease management have limitations associated with them. Novel therapeutic targets are thus avidly sought for the effective management of LC. RNA binding proteins (RBPs) have been convincingly established as key players in tumorigenesis, and their dysregulation is linked to multiple cancers, including LC. In this context, we review the role of Human antigen R (HuR), an RBP that is overexpressed in LC, and further associated with various aspects of LC tumor growth and response to therapy. Herein, we describe the role of HuR in LC progression and outline the evidences supporting various pharmacologic and biologic approaches for inhibiting HuR expression and function. These approaches, including use of small molecule inhibitors, siRNAs and shRNAs, have demonstrated favorable results in reducing tumor cell growth, invasion and migration, angiogenesis and metastasis. Hence, HuR has significant potential as a key therapeutic target in LC. Use of siRNA-based approaches, however, have certain limitations that prevent their maximal exploitation as cancer therapies. To address this, in the conclusion of this review, we provide a list of nanomedicine-based HuR targeting approaches currently being employed for siRNA and shRNA delivery, and provide a rationale for the immense potential therapeutic benefits offered by nanocarrier-based HuR targeting and its promise for treating patients with LC.

Therapeutic approaches targeting molecular signaling pathways common to diabetes, lung diseases and cancer.

Diabetes mellitus (DM), is the most common metabolic disease and is characterized by sustained hyperglycemia. Accumulating evidences supports a strong association between DM and numerous lung diseases including chronic obstructive pulmonary disease (COPD), fibrosis, and lung cancer (LC). The global incidence of DM-associated lung disorders is rising and several ongoing studies, including clinical trials, aim to elucidate the molecular mechanisms linking DM with lung disorders, in particular LC. Several potential mechanisms, including hyperglycemia, hyperinsulinemia, glycation, inflammation, and hypoxia, are cited as plausible links between DM and LC. In addition, studies also propose a connection between the use of anti-diabetic medications and reduction in the incidence of LC. However, the exact cause for DM associated lung diseases especially LC is not clear and is an area under intense investigation. Herein, we review the biological links reported between DM and lung disorders with an emphasis on LC. Furthermore, we report common signaling pathways (eg: TGF-ß, IL-6, HIF-1, PDGF) and miRNAs that are dysregulated in DM and LC and serve as molecular targets for therapy. Finally, we propose a nanomedicine based approach for delivering therapeutics (eg: IL-24 plasmid DNA, HuR siRNA) to disrupt signaling pathways common to DM and LC and thus potentially treat DM-associated LC. Finally, we conclude that the effective modulation of commonly regulated signaling pathways would help design novel therapeutic protocols for treating DM patients diagnosed with LC.

Interleukin (IL)-24: Reconfiguring the Tumor Microenvironment for Eliciting Antitumor Response.

Interleukin (IL)-24 is a member of the IL-10 family of cytokines. Due to its unique ability to function as both a tumor suppressor and cytokine, IL-24-based cancer therapy has been developed for treating a broad spectrum of human cancers. Majority of the studies reported to date have focused on establishing IL-24 as a cancer therapeutic by primarily focusing on tumor cell killing. However, the ability of IL-24 treatment on modulating the tumor microenvironment and immune response is underinvestigated. In this article, we summarize the biological and functional properties of IL-24 and the benefits of applying IL-24-based therapy for cancer.

Extracellular Vesicles in Oncology: from Immune Suppression to Immunotherapy.

Exosomes are involved in cell-to-cell communication and play a crucial role in cellular physiology. The role of exosomes in cancer has been widely explored. Tumor cells have evolved and adapted to evade the immune response. The study of the immune system's modulations in favor of rogue tumor cells led to the development of a novel immunotherapeutic strategy targeting the immune checkpoint proteins (ICPs). In clinical settings, the response to ICP therapy has been inconsistent and is difficult to predict. Quantitating the targeted ICPs through immunohistochemistry is one approach, but is not pragmatic in a clinical setting and is often not sensitive. Examining the molecules present in bodily fluids to determine ICP treatment response, "liquid biopsy" is a convenient alternative. The term "liquid biopsy" refers to circulating tumor cells (CTCs), extracellular vesicles (EVs), non-coding (nc) RNA, circulating tumor DNA (ctDNA), circulating free DNA (cfDNA), etc. EVs includes exosomes, microvesicles, and oncosomes. Herein, we focus on exosomes isolated from bodily fluids and their use in liquid biopsy. Due to their unique ability to transfer bioactive molecules and perturb the physiology of recipient cells, exosomes have garnered attention for their immune modulation role and as a resource to identify molecules associated with liquid biopsy-based diagnostic methods. In this review, we examine the putative role of exosomes and their cargo in influencing the immune system. We discuss the immune and tumor cells present in the tumor microenvironment (TME), and the exosomes derived from these cells to understand how they participate in creating the immune-suppressive TME. Additionally, use of exosomes in liquid biopsy-based methods to measure the treatment response elicited by immunotherapy is discussed. Finally, we describe how exosomes have been used to develop immune therapies, especially cell-free vaccines, for cancer treatment.

Molecular Targeting of HuR Oncoprotein Suppresses MITF and Induces Apoptosis in Melanoma Cells.

BACKGROUND: Treatment of metastatic melanoma possesses challenges due to drug resistance and metastases. Recent advances in targeted therapy and immunotherapy have shown clinical benefits in melanoma patients with increased survival. However, a subset of patients who initially respond to targeted therapy relapse and succumb to the disease. Therefore, efforts to identify new therapeutic targets are underway. Due to its role in stabilizing several oncoproteins' mRNA, the human antigen R (HuR) has been shown as a promising molecular target for cancer therapy. However, little is known about its potential role in melanoma treatment. METHODS: In this study, we tested the impact of siRNA-mediated gene silencing of HuR in human melanoma (MeWo, A375) and normal melanocyte cells in vitro. Cells were treated with HuR siRNA encapsulated in a lipid nanoparticle (NP) either alone or in combination with MEK inhibitor (U0126) and subjected to cell viability, cell-cycle, apoptosis, Western blotting, and cell migration and invasion assays. Cells that were untreated or treated with control siRNA-NP (C-NP) were included as controls. RESULTS: HuR-NP treatment significantly reduced the expression of HuR and HuR-regulated oncoproteins, induced G1 cell cycle arrest, activated apoptosis signaling cascade, and mitigated melanoma cells' aggressiveness while sparing normal melanocytes. Furthermore, we demonstrated that HuR-NP treatment significantly reduced the expression of the microphthalmia-associated transcription factor (MITF) in both MeWo and MITF-overexpressing MeWo cells (p < 0.05). Finally, combining HuR-NP with U0126 resulted in synergistic antitumor activity against MeWo cells (p < 0.01). CONCLUSION: HuR-NP exhibited antitumor activity in melanoma cells independent of their oncogenic B-RAF mutational status. Additionally, combinatorial therapy incorporating MEK inhibitor holds promise in overriding MITF-mediated drug resistance in melanoma.

Regorafenib sensitizes human breast cancer cells to radiation by inhibiting multiple kinases and inducing DNA damage.

PURPOSE: Triple-negative breast cancer (TNBC) is the most challenging and aggressive subtype of breast cancer with limited treatment options because of tumor heterogeneity, lack of druggable targets and therapy resistance. TNBCs are characterized by overexpression of growth factor receptors such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet derived growth factor receptor (PDGFR) making them promising therapeutic targets. Regorafenib is an FDA approved oral multi-kinase inhibitor that blocks the activity of multiple protein kinases including those involved in the regulation of tumor angiogenesis [VEGFR1-3, TIE2], tumor microenvironment [PDGFR-ß, FGFR] and oncogenesis (KIT, RET, RAF-1, BRAF). In the current study, we examined the radiosensitizing effects of Regorafenib on TNBC cell lines and explored the mechanism by which Regorafenib enhances radiosensitivity. METHODS: MDA-MB-231 and SUM159PT (human TNBC cell lines) and MCF 10a (human mammary epithelial cell line) were treated with Regorafenib, ionizing radiation or a combination of both. Following treatment with Regorafenib and radiation we conducted clonogenic assay to determine radiosensitivity, immunoblot analysis to assess the effect on key signaling targets, tube formation to evaluate effect on angiogenesis and comet assay as well as western blot for γH2AX to assess DNA damage response (DDR). RESULTS: Regorafenib reduced cell proliferation and enhanced radiosensitivity of MDA-MB-231 and SUM159PT cell lines but had no effect on the MCF 10a cells. Clonogenic survival assays showed that the surviving fraction at 2 Gy for both MDA-MB-231 and SUM159PT was reduced from 66.4 ± 8.9 and 88.2 ± 1.7 in controls to 38.1 ± 4.9 and 75.1 ± 1.1 following a 24 hr pretreatment with 10 µM and 5 µM Regorafenib, respectively. A marked reduction in the expression of VEGFR, PDGFR, EGFR and the downstream target, ERK, was observed with Regorafenib treatment alone or in combination with radiation. We also observed a significant inhibition of VEGF-A production in the TNBC cell lines following treatment with Regorafenib. Further, the addition of conditioned medium from Regorafenib-treated tumor cells onto human umbilical vein endothelial cells (HUVEC) suppressed tube formation, indicating an inhibition of tumor angiogenesis. Regorafenib also decreased migration of TNBC cells and suppressed radiation-induced DNA damage repair in a time-dependent manner. CONCLUSIONS: Our findings demonstrate that Regorafenib enhanced radiosensitivity of breast cancer cells by inhibiting the expression of multiple receptor tyrosine kinases, VEGF-mediated angiogenesis and DNA damage response in TNBC. Therefore, combining Regorafenib with radiation and antiangiogenic agents will be beneficial and effective in controlling TNBC.

Exosomes as drug delivery vehicle and contributor of resistance to anticancer drugs.

Exosomes are small membranous vesicles implicated in intercellular signalling. Through their uncanny ability to carry and deliver donor cellular cargo (biomolecules) to target cells, they exert a profound effect on the regular functioning of healthy cells and play a significant role in pathogenesis and progression of several diseases, including cancer. The composition and number of endogenously circulating exosomes frequently vary, which is often reflective of the pathophysiological status of the cell. Applicability of exosomes derived from normal cells as a drug carrier with or without modifying their intraluminal and surface components are generally tested. Conversely, exosomes also are reported to contribute to resistance towards several anti-cancer therapies. Therefore, it is necessary to carefully evaluate the role of exosomes in cancer progression, resistance and the potential use of exosomes as a delivery vehicle of cancer therapeutics. In this review, we summarize the recent advancements in the exploitation of exosomes as a drug delivery vehicle. We also discuss the role of exosomes in conferring resistance to anti-cancer therapeutics. While this review is focused on cancer, the exosome-based drug delivery and resistance is also applicable to other human diseases.

Progress in extracellular vesicle biology and their application in cancer medicine.

Under the broader category of extracellular vesicles (EVs), exosomes are now well recognized for their contribution and potential for biomedical research. During the last ten years, numerous technologies for purification and characterization of EVs have been developed. This enhanced knowledge has resulted in the development of novel applications of EVs. This review is an attempt to capture the exponential growth observed in EV science in the last decade and discuss the future potential to improve our understanding of EVs, develop technologies to overcome current limitations, and advance their utility for human benefit, especially in cancer medicine. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Tumor-Targeted Dendrimer Nanoparticles for Combinatorial Delivery of siRNA and Chemotherapy for Cancer Treatment.

In current cancer therapy, the combined targeted delivery of treatments is an important method to enhance the therapeutic efficiency and reduce adverse side effects. Dendrimer-based nanoparticles have received considerable attention for multifunctional therapeutic delivery. In this chapter, we describe the methods for encapsulating the chemotherapeutic drug, cisplatin (CDDP), and human antigen R (HuR)-targeted siRNA into dendrimer nanoparticles for folate receptor-targeted delivery. We discuss the methodologies for physical and biological characterization of synthesized multifunctional (Den-PEI-CDDP-HuR-FA) nanoparticles in detail. Physical characterization includes size and charge determination, drug encapsulation and release kinetics, ligand conjugation, etc., and functional characterization involves testing of the nanoparticles for receptor-specific uptake and cytotoxicity on human lung cancer and normal cells. The protocol provided is geared to provide the readers an overview of developing multifunctional dendrimer-based nanoparticles. However, based on the individual's objective and the type of combinatorial drugs to deliver, the protocol may need modifications in achieving maximal efficacy.

IL-24 Inhibits Lung Cancer Growth by Suppressing GLI1 and Inducing DNA Damage.

Aberrant expression of GLI1 is responsible for aggressive tumor behavior and survival due to its effects on the DNA damage response (DDR). We investigated whether interleukin (IL)-24, a tumor suppressor, inhibits GLI1 and the associated DDR pathway in human NSCLCs. IL-24 treatment reduces mRNA and protein expression of GLI1 in lung tumor cells, but not in normal cells. GLI1 reporter assay and mRNA studies demonstrated that IL-24 regulates GLI1 at the post-transcriptional level by favoring mRNA degradation. Associated with GLI1 inhibition was marked suppression of the ATM-mediated DDR pathway resulting in increased DNA damage, as evidenced by γ-H2AX foci and Comet assay. Furthermore, attenuation of GLI1-associated DDR by IL-24 increased caspase-3 and PARP activity, resulting in cancer cell apoptosis. GLI1 inhibition and overexpression confirmed that IL-24-mediated anti-tumor effects involved the GLI-dependent pathway. Finally, we observed that IL-24-mediated alteration in GLI1 is independent of the canonical hedgehog-signaling pathway. Our study provides evidence that IL-24 treatment induces DNA damage, and reduces GLI1 expression and offers an opportunity for testing IL-24-based therapy for inhibiting GLI1 in lung cancer.

HuR Reduces Radiation-Induced DNA Damage by Enhancing Expression of ARID1A.

Tumor suppressor ARID1A, a subunit of the chromatin remodeling complex SWI/SNF, regulates cell cycle progression, interacts with the tumor suppressor TP53, and prevents genomic instability. In addition, ARID1A has been shown to foster resistance to cancer therapy. By promoting non-homologous end joining (NHEJ), ARID1A enhances DNA repair. Consequently, ARID1A has been proposed as a promising therapeutic target to sensitize cancer cells to chemotherapy and radiation. Here, we report that ARID1A is regulated by human antigen R (HuR), an RNA-binding protein that is highly expressed in a wide range of cancers and enables resistance to chemotherapy and radiation. Our results indicate that HuR binds ARID1A mRNA, thereby increasing its stability in breast cancer cells. We further find that ARID1A expression suppresses the accumulation of DNA double-strand breaks (DSBs) caused by radiation and can rescue the loss of radioresistance triggered by HuR inhibition, suggesting that ARID1A plays an important role in HuR-driven resistance to radiation. Taken together, our work shows that HuR and ARID1A form an important regulatory axis in radiation resistance that can be targeted to improve radiotherapy in breast cancer patients.

Combinatorial Nanoparticle Delivery of siRNA and Antineoplastics for Lung Cancer Treatment.

Recent developments in nanotechnology, especially in drug delivery systems, are advanced by featuring novel multifunctional nanoparticles that promise safe, specific, and efficient therapeutic delivery for cancer treatment. Multifunctional nanoparticle-based drug delivery systems enable simultaneous delivery of multiple therapeutic agents for effective combination therapy for cancer. In this chapter, we provide detailed protocols for development and application of a multifunctional nanoparticle system for combinatorial delivery of a chemotherapeutic (cisplatin) and small interfering RNA (siRNA) for human antigen R (HuR) mRNA in cancer cells using a polyamidoamine (PAMAM) dendrimer platform. Protocols for nanoparticle functionalization with folic acid (FA) for targeted delivery of therapeutics toward folate receptor (FR)-overexpressing cancer cells are also described. Further, methods employed for physiochemical and functional characterization of the multifunctional nanoparticle system are discussed in detail. Using the methods described in this chapter, researchers would be able to develop PAMAM dendrimer-based multifunctional nanoparticles for targeted delivery of chemotherapeutics and siRNA combinations. We also provide an example showing the dendrimer-polyethyleneimine-cis-diamminedichloroplatinum-siRNA-folic acid (Den-PEI-CDDP-siRNA-FA) nanoparticle system developed was therapeutically effective toward non-small cell lung cancer (NSCLC) cell lines (H1299 and A549) while exhibiting reduced toxicity to normal lung fibroblast (MRC9) cells.

Exosomes as Theranostics for Lung Cancer.

Extensive research in genetics and genomics has revealed that lung cancer is a physiologically complex and genetically heterogeneous disease. Although molecular targets that can yield favorable response have been identified, those targets cannot be exploited due to the lack of suitable drug carriers. Furthermore, lung cancer often is diagnosed at an advanced stage when the disease has metastasized. Conventional treatments are not effective for treating metastatic lung cancer. Targeted therapeutics while beneficial has challenges that include poor tumor-targeting, off-target effects, and development of resistance to therapy. Therefore, improved drug delivery systems that can deliver drugs specifically to tumor will produce improved treatment outcomes. Exosomes have a natural ability to carry functional biomolecules, such as small RNAs, DNAs, and proteins, in their lumen. This property makes exosomes attractive for use in drug delivery and molecular diagnosis. Moreover, exosomes can be attached to nanoparticles and used for high precision imaging. Exosomes are now considered an important component in liquid biopsy assessments, which are useful for detecting cancers, including lung cancer. Several studies are currently underway to develop methods of exploiting exosomes for use as efficient drug delivery vehicles and to develop novel diagnostic modalities. This chapter summarizes the current status of exosome studies with regard to their use as theranostics in lung cancer. Examples from other cancers have also been cited to illustrate the extensive applicability of exosomes to therapy and diagnosis.

Recent Advances in Nanoparticle-Based Cancer Drug and Gene Delivery.

Effective and safe delivery of anticancer agents is among the major challenges in cancer therapy. The majority of anticancer agents are toxic to normal cells, have poor bioavailability, and lack in vivo stability. Recent advancements in nanotechnology provide safe and efficient drug delivery systems for successful delivery of anticancer agents via nanoparticles. The physicochemical and functional properties of the nanoparticle vary for each of these anticancer agents, including chemotherapeutics, nucleic acid-based therapeutics, small molecule inhibitors, and photodynamic agents. The characteristics of the anticancer agents influence the design and development of nanoparticle carriers. This review focuses on strategies of nanoparticle-based drug delivery for various anticancer agents. Recent advancements in the field are also highlighted, with suitable examples from our own research efforts and from the literature.

Chemo-biologic combinatorial drug delivery using folate receptor-targeted dendrimer nanoparticles for lung cancer treatment.

Co-administration of functionally distinct anti-cancer agents has emerged as an efficient strategy in lung cancer treatment. However, a specially designed drug delivery system is required to co-encapsulate functionally different agents, such as a combination of siRNA and chemotherapy, for targeted delivery. We developed a folic acid (FA)-conjugated polyamidoamine dendrimer (Den)-based nanoparticle (NP) system for co-delivery of siRNA against HuR mRNA (HuR siRNA) and cis-diamine platinum (CDDP) to folate receptor-α (FRA) -overexpressing H1299 lung cancer cells. The co-delivery of HuR siRNA and CDDP using the FRA-targeted NP had a significantly greater therapeutic effect than did individual therapeutics. Further, the FRA-targeted NP exhibited improved cytotoxicity compared to non-targeted NP against lung cancer cells. Finally, the NP showed negligible toxicity towards normal MRC9 lung fibroblast cells. Thus, the present study demonstrates FRA-targeted Den nanoparticle system as a suitable carrier for targeted co-delivery of siRNA and chemotherapy agents in lung cancer cells.

YAP1 inhibition radiosensitizes triple negative breast cancer cells by targeting the DNA damage response and cell survival pathways.

The Hippo pathway is an evolutionarily conserved signaling pathway that regulates proliferation and apoptosis to control organ size during developmental growth. Yes-associated protein 1 (YAP1), the terminal effector of the Hippo pathway, is a transcriptional co-activator and a potent growth promoter that has emerged as a critical oncogene. Overexpression of YAP1 has been implicated in promoting resistance to chemo-, radiation and targeted therapy in various cancers. However, the role of YAP1 in radioresistance in triple-negative breast cancer (TNBC) is currently unknown. We evaluated the role of YAP1 in radioresistance in TNBC in vitro, using two approaches to inhibit YAP1: 1) genetic inhibition by YAP1 specific shRNA or siRNA, and 2) pharmacological inhibition by using the small molecule inhibitor, verteporfin that prevents YAP1 transcriptional activity. Our findings demonstrate that both genetic and pharmacological inhibition of YAP1 sensitizes TNBC cells to radiation by inhibiting the EGFR/PI3K/AKT signaling axis and causing an increased accumulation of DNA damage. Our results reveal that YAP1 activation exerts a protective role for TNBC cells in radiotherapy and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of TNBC.