Beta-Transducin Repeats-Containing Proteins as an Anticancer Target.

Beta-transducin repeat-containing proteins (ß-TrCPs) are E3-ubiquitin-ligase-recognizing substrates and regulate proteasomal degradation. The degradation of ß-TrCPs' substrates is tightly controlled by various external and internal signaling and confers diverse cellular processes, including cell cycle progression, apoptosis, and DNA damage response. In addition, ß-TrCPs function to regulate transcriptional activity and stabilize a set of substrates by distinct mechanisms. Despite the association of ß-TrCPs with tumorigenesis and tumor progression, studies on the mechanisms of the regulation of ß-TrCPs' activity have been limited. In this review, we studied publications on the regulation of ß-TrCPs themselves and analyzed the knowledge gaps to understand and modulate ß-TrCPs' activity in the future.

OSGIN1 is a novel TUBB3 regulator that promotes tumor progression and gefitinib resistance in non-small cell lung cancer.

BACKGROUND: Oxidative stress induced growth inhibitor 1 (OSGIN1) regulates cell death. The role and underlying molecular mechanism of OSGIN1 in non-small cell lung cancer (NSCLC) are uncharacterized. METHODS: OSGIN1 expression in NSCLC samples was detected using immunohistochemistry and Western blotting. Growth of NSCLC cells and gefitinib-resistant cells expressing OSGIN1 or TUBB3 knockdown was determined by MTT, soft agar, and foci formation assays. The effect of OSGIN1 knockdown on in vivo tumor growth was assessed using NSCLC patient-derived xenograft models and gefitinib-resistant patient-derived xenograft models. Potentially interacting protein partners of OSGIN1 were identified using IP-MS/MS, immunoprecipitation, PLA, and Western blotting assays. Microtubule dynamics were explored by tubulin polymerization assay and immunofluorescence. Differential expression of signaling molecules in OSGIN1 knockdown cells was investigated using phospho-proteomics, KEGG analysis, and Western blotting. RESULTS: We found that OSGIN1 is highly expressed in NSCLC tissues and is positively correlated with low survival rates and tumor size in lung cancer patients. OSGIN1 knockdown inhibited NSCLC cell growth and patient-derived NSCLC tumor growth in vivo. Knockdown of OSGIN1 strongly increased tubulin polymerization and re-established gefitinib sensitivity in vitro and in vivo. Additionally, knockdown of TUBB3 strongly inhibited NSCLC cell proliferation. Mechanistically, we found that OSGIN1 enhances DYRK1A-mediated TUBB3 phosphorylation, which is critical for inducing tubulin depolymerization. The results of phospho-proteomics and ontology analysis indicated that knockdown of OSGIN1 led to reduced propagation of the MKK3/6-p38 signaling axis. CONCLUSIONS: We propose that OSGIN1 modulates microtubule dynamics by enhancing DYRK1A-mediated phosphorylation of TUBB3 at serine 172. Moreover, elevated OSGIN1 expression promotes NSCLC tumor growth and gefitinib resistance through the MKK3/6-p38 signaling pathway. Our findings unveil a new mechanism of OSGIN1 and provide a promising therapeutic target for NSCLC treatment in the clinic.

Extracellular Vesicles as a New Therapeutic Entity.

Collectively, extracellular vesicles (EVs) refer to vesicular entities secreted by live cells, including microvesicles, exosomes, and apoptotic bodies [...].

TGFß4 alleviates the phenotype of Charcot-Marie-Tooth disease type 1A.

The duplication of the peripheral myelin protein 22 (PMP22) gene causes a demyelinating type of neuropathy, commonly known as Charcot-Marie-Tooth disease type 1A (CMT1A). Development of effective drugs for CMT1A still remains as an unmet medical need. In the present study, we assessed the role of the transforming growth factor beta 4 (TGFß4)/Nodal axis in the pathogenesis of CMT1A. First, we identified PMP22 overexpression-induced Nodal expression in Schwann cells, which might be one of the downstream effectors in CMT1A. Administration of Nodal protein at the developmental stage of peripheral nerves induced the demyelinating phenotype in vivo. Second, we further isolated TGFß4 as an antagonist that could abolish Nodal-induced demyelination. Finally, we developed a recombinant TGFß4-fragment crystallizable (Fc) fusion protein, CX201, and demonstrated that its application had promyelinating efficacy in Schwann cells. CX201 administration improved the demyelinating phenotypes of CMT1A mouse models at both pre-symptomatic and post-symptomatic stages. These results suggest that the TGFß4/Nodal axis plays a crucial role in the pathogenesis of CMT1A and might be a potential therapeutic target for CMT1A.

Therapeutic Implications of the Drug Resistance Conferred by Extracellular Vesicles Derived from Triple-Negative Breast Cancer Cells.

Anticancer drug resistance is a significant impediment in current cancer treatment. Extracellular vesicles (EVs) derived from cancer cells were recently acknowledged as a critical mechanism of drug resistance, tumor progression, and metastasis. EVs are enveloped vesicles comprising a lipid bilayer that transfers various cargo, including proteins, nucleic acids, lipids, and metabolites, from an originating cell to a recipient cell. Investigating the mechanisms whereby EVs confer drug resistance is still in the early stages. In this review, I analyze the roles of EVs derived from triple-negative breast cancer cells (TNBC-EVs) in anticancer drug resistance and discuss strategies to overcome TNBC-EV-mediated drug resistance.

Inhibition of IκB Kinase Is a Potential Therapeutic Strategy to Circumvent Resistance to Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) remains as an intractable malignancy with limited therapeutic targets. High expression of epidermal growth factor receptor (EGFR) has been associated with a poor prognosis of TNBC; however, EGFR targeting has failed with unfavorable clinical outcomes. Here, we performed a combinatorial screening of fifty-five protein kinase inhibitors with the EGFR inhibitor gefitinib in the TNBC cell line MDA-MB-231 and identified the IκB kinase (IKK) inhibitor IKK16 as a sensitizer of gefitinib. Cell viability and clonogenic survival assays were performed to evaluate the antiproliferative effects of the gefitinib and IKK16 (Gefitinib + IKK16) combination in TNBC cell lines. Western blot analyses were also performed to reveal the potential mode of action of this combination. In addition, next-generation sequencing (NGS) analysis was performed in Gefitinib+IKK16-treated cells. The Gefitinib+IKK16 treatment synergistically reduced cell viability and colony formation of TNBC cell lines such as HS578T, MDA-MB-231, and MDA-MB-468. This combination downregulated p-STAT3, p-AKT, p-mTOR, p-GSK3ß, and p-RPS6. In addition, p-NF-κB and the total NF-κB were also regulated by this combination. Furthermore, NGS analysis revealed that NF-κB/RELA targets including CCL2, CXCL8, EDN1, IL-1ß, IL-6, and SERPINE1 were further reduced and several potential tumor suppressors, such as FABP3, FADS2, FDFT1, SEMA6A, and PCK2, were synergistically induced by the Gefitinib-+IKK16 treatment. Taken together, we identified the IKK/NF-κB pathway as a potential target in combination of EGFR inhibition for treating TNBC.

Doxycycline potentiates the anti-proliferation effects of gemcitabine in pancreatic cancer cells.

Gemcitabine is often recommended as a first-line treatment for patients with metastatic pancreatic cancer. However, gemcitabine resistance is a major challenge in the treatment of pancreatic ductal adenocarcinoma. Our group serendipitously identified the role of doxycycline as a potentiator of gemcitabine efficacy in pancreatic cancer cells. Doxycycline and gemcitabine co-treatment was significantly more cytotoxic to pancreatic cancer cells compared to gemcitabine alone. Interestingly, doxycycline only exerted synergistic effects when coupled with gemcitabine as opposed to other conventional chemotherapeutics including nucleoside analogs. The anti-clonogenic effects of gemcitabine on pancreatic cancer cells were also enhanced by doxycycline. According to cell cycle analyses, doxycycline prolonged gemcitabine-mediated S phase cell cycle arrest. Further, gene expression profiling analyses indicated that a small set of genes involved in cell cycle regulation were uniquely modulated by gemcitabine and doxycycline co-treatment compared to gemcitabine alone. Western blot analyses indicated that several cell cycle-related proteins, including cyclin D1, p21, and DNA damage inducible transcript 4 (DDIT4), were further modulated by doxycycline and gemcitabine co-treatment. Taken together, our findings indicate that doxycycline enhances the effects of gemcitabine on cell cycle progression, thus rendering pancreatic cancer cells more sensitive to gemcitabine. However, additional studies are required to assess the mechanisms of doxycycline and gemcitabine synergism, which might lead to novel treatment options for pancreatic cancer.

Potentiating Therapeutic Effects of Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subset of breast cancer with aggressive characteristics and few therapeutic options. The lack of an appropriate therapeutic target is a challenging issue in treating TNBC. Although a high level expression of epidermal growth factor receptor (EGFR) has been associated with a poor prognosis among patients with TNBC, targeted anti-EGFR therapies have demonstrated limited efficacy for TNBC treatment in both clinical and preclinical settings. However, with the advantage of a number of clinically approved EGFR inhibitors (EGFRis), combination strategies have been explored as a promising approach to overcome the intrinsic resistance of TNBC to EGFRis. In this review, we analyzed the literature on the combination of EGFRis with other molecularly targeted therapeutics or conventional chemotherapeutics to understand the current knowledge and to provide potential therapeutic options for TNBC treatment.

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles.

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50-200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex "work-in-progress" MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.

Dual Inhibition of AKT and MEK Pathways Potentiates the Anti-Cancer Effect of Gefitinib in Triple-Negative Breast Cancer Cells.

There is an unmet medical need for the development of new targeted therapeutic strategies for triple-negative breast cancer (TNBC). With drug combination screenings, we found that the triple combination of the protein kinase inhibitors (PKIs) of the epidermal growth factor receptor (EGFR), v-akt murine thymoma viral oncogene homolog (AKT), and MAPK/ERK kinase (MEK) is effective in inducing apoptosis in TNBC cells. A set of PKIs were first screened in combination with gefitinib in the TNBC cell line, MDA-MB-231. The AKT inhibitor, AT7867, was identified and further analyzed in two mesenchymal stem-like (MSL) subtype TNBC cells, MDA-MB-231 and HS578T. A combination of gefitinib and AT7867 reduced the proliferation and long-term survival of MSL TNBC cells. However, gefitinib and AT7867 induced the activation of the rat sarcoma (RAS)/ v-raf-1 murine leukemia viral oncogene homolog (RAF)/MEK/ extracellular signal-regulated kinase (ERK) pathway. To inhibit this pathway, MEK/ERK inhibitors were further screened in MDA-MB-231 cells in the presence of gefitinib and AT7867. As a result, we identified that the MEK inhibitor, PD-0325901, further enhanced the anti-proliferative and anti-clonogenic effects of gefitinib and AT7867 by inducing apoptosis. Our results suggest that the dual inhibition of the AKT and MEK pathways is a novel potential therapeutic strategy for targeting EGFR in TNBC cells.

Ribosomal Protein S6: A Potential Therapeutic Target against Cancer?

Ribosomal protein S6 (RPS6) is a component of the 40S small ribosomal subunit and participates in the control of mRNA translation. Additionally, phospho (p)-RPS6 has been recognized as a surrogate marker for the activated PI3K/AKT/mTORC1 pathway, which occurs in many cancer types. However, downstream mechanisms regulated by RPS6 or p-RPS remains elusive, and the therapeutic implication of RPS6 is underappreciated despite an approximately half a century history of research on this protein. In addition, substantial evidence from RPS6 knockdown experiments suggests the potential role of RPS6 in maintaining cancer cell proliferation. This motivates us to investigate the current knowledge of RPS6 functions in cancer. In this review article, we reviewed the current information about the transcriptional regulation, upstream regulators, and extra-ribosomal roles of RPS6, with a focus on its involvement in cancer. We also discussed the therapeutic potential of RPS6 in cancer.

Reproducible Large-Scale Isolation of Exosomes from Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells and Their Application in Acute Kidney Injury.

Acute kidney injury (AKI) is a fatal medical episode caused by sudden kidney damage or failure, leading to the death of patients within a few hours or days. Previous studies demonstrated that exosomes derived from various mesenchymal stem/stromal cells (MSC-exosomes) have positive effects on renal injuries in multiple experimental animal models of kidney diseases including AKI. However, the mass production of exosomes is a challenge not only in preclinical studies with large animals but also for successful clinical applications. In this respect, tangential flow filtration (TFF) is suitable for good manufacturing practice (GMP)-compliant large-scale production of high-quality exosomes. Until now, no studies have been reported on the use of TFF, but rather ultracentrifugation has been almost exclusively used, to isolate exosomes for AKI therapeutic application in preclinical studies. Here, we demonstrated the reproducible large-scale production of exosomes derived from adipose tissue-derived MSC (ASC-exosomes) using TFF and the lifesaving effect of the ASC-exosomes in a lethal model of cisplatin-induced rat AKI. Our results suggest the possibility of large-scale stable production of ASC-exosomes without loss of function and their successful application in life-threatening diseases.

Toxicological evaluation of exosomes derived from human adipose tissue-derived mesenchymal stem/stromal cells.

Several studies report that the therapeutic mechanism of action of mesenchymal stem/stromal cells (MSCs) is mainly mediated by paracrine factors that are released from MSCs such as exosomes. Exosomes are nano-sized extracellular vesicles that are transferred to target cells for cell-to-cell communication. Although MSC-derived exosomes (MSC-exosomes) are suggested as novel cell-free therapeutics for various human diseases, evaluation studies for the safety and toxicity of MSC-exosomes are limited. The purpose of our study was to evaluate the toxicological profile, including skin sensitization, photosensitization, eye and skin irritation, and acute oral toxicity using exosomes derived from human adipose tissue-derived MSCs (ASC-exosomes) in accordance with the OECD guidelines and the principles of Good Laboratory Practice. The ASC-exosomes were classified as a potential non-sensitizer in the skin sensitization test, UN GHS no category in the eye irritation test, and as a skin non-irritant in the skin irritation test, and did not induce any toxicity in the phototoxicity test or in acute oral toxicity testing. Our findings are the first to suggest that ASC-exosomes are safe for use as a topical treatment, with no adverse effects in toxicological testing, and have potential application as a therapeutic agent, cosmetic ingredient, or for other biological uses.

Mesenchymal Stem/Stromal Cell-Derived Exosomes for Immunomodulatory Therapeutics and Skin Regeneration.

Exosomes are nano-sized vesicles that serve as mediators for cell-to-cell communication. With their unique nucleic acids, proteins, and lipids cargo compositions that reflect the characteristics of producer cells, exosomes can be utilized as cell-free therapeutics. Among exosomes derived from various cellular origins, mesenchymal stem cell-derived exosomes (MSC-exosomes) have gained great attention due to their immunomodulatory and regenerative functions. Indeed, many studies have shown anti-inflammatory, anti-aging and wound healing effects of MSC-exosomes in various in vitro and in vivo models. In addition, recent advances in the field of exosome biology have enabled development of specific guidelines and quality control methods, which will ultimately lead to clinical application of exosomes. This review highlights recent studies that investigate therapeutic potential of MSC-exosomes and relevant mode of actions for skin diseases, as well as quality control measures required for development of exosome-derived therapeutics.

Exosomes from Human Adipose Tissue-Derived Mesenchymal Stem Cells Promote Epidermal Barrier Repair by Inducing de Novo Synthesis of Ceramides in Atopic Dermatitis.

Atopic dermatitis (AD) is a multifactorial, heterogeneous disease associated with epidermal barrier disruption and intense systemic inflammation. Previously, we showed that exosomes derived from human adipose tissue-derived mesenchymal stem cells (ASC-exosomes) attenuate AD-like symptoms by reducing multiple inflammatory cytokine levels. Here, we investigated ASC-exosomes' effects on skin barrier restoration by analyzing protein and lipid contents. We found that subcutaneous injection of ASC-exosomes in an oxazolone-induced dermatitis model remarkably reduced trans-epidermal water loss, while enhancing stratum corneum (SC) hydration and markedly decreasing the levels of inflammatory cytokines such as IL-4, IL-5, IL-13, TNF-α, IFN-γ, IL-17, and TSLP, all in a dose-dependent manner. Interestingly, ASC-exosomes induced the production of ceramides and dihydroceramides. Electron microscopic analysis revealed enhanced epidermal lamellar bodies and formation of lamellar layer at the interface of the SC and stratum granulosum with ASC-exosomes treatment. Deep RNA sequencing analysis of skin lesions demonstrated that ASC-exosomes restores the expression of genes involved in skin barrier, lipid metabolism, cell cycle, and inflammatory response in the diseased area. Collectively, our results suggest that ASC-exosomes effectively restore epidermal barrier functions in AD by facilitating the de novo synthesis of ceramides, resulting in a promising cell-free therapeutic option for treating AD.

Advances in Analysis of Biodistribution of Exosomes by Molecular Imaging.

Exosomes are nano-sized membranous vesicles produced by nearly all types of cells. Since exosome-like vesicles are produced in an evolutionarily conserved manner for information and function transfer from the originating cells to recipient cells, an increasing number of studies have focused on their application as therapeutic agents, drug delivery vehicles, and diagnostic targets. Analysis of the in vivo distribution of exosomes is a prerequisite for the development of exosome-based therapeutics and drug delivery vehicles with accurate prediction of therapeutic dose and potential side effects. Various attempts to evaluate the biodistribution of exosomes obtained from different sources have been reported. In this review, we examined the current trends and the advantages and disadvantages of the methods used to determine the biodistribution of exosomes by molecular imaging. We also reviewed 29 publications to compare the methods employed to isolate, analyze, and label exosomes as well as to determine the biodistribution of labeled exosomes.

Exosomes derived from human adipose tissue-derived mesenchymal stem cells alleviate atopic dermatitis.

Exosomes are nano-sized vesicles (30-200 nm) constantly released by almost all cells. The ability of exosomes to travel between cells and deliver their cargo, which includes lipids, proteins, and nucleic acids, makes them an appealing cell-free therapy option to treat multiple diseases. Here, we investigated for the first time whether human adipose tissue-derived mesenchymal stem cell-derived exosomes (ASC-exosomes) can ameliorate atopic dermatitis (AD) in an in vivo mouse model. When injected either intravenously (IV) or subcutaneously (SC) into NC/Nga mice treated with house dust mite antigens, ASC-exosomes were found to reduce pathological symptoms such as clinical score, the levels of serum IgE, the number of eosinophils in blood, and the infiltration of mast cells, CD86+, and CD206+ cells in skin lesions. ASC-exosomes also significantly reduced mRNA expression of various inflammatory cytokines such as interleukin (IL)-4, IL-23, IL-31, and tumor necrosis factor-α (TNF-α) in AD skin lesions of Nc/Nga mice. Taken together, these results suggest that ASC-exosomes can be a novel promising cell-free therapeutic modality for AD treatment.

Inhibition of RPTOR overcomes resistance to EGFR inhibition in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) cells frequently exhibit activated growth factor signaling and resistance to inhibitors for epidermal growth factor receptor (EGFR), despite the overexpression of EGFR protein, and this is associated with a malignant behavior and a poor prognosis. In this study, to elucidate the underlying mechanisms of resistance to EGFR inhibitor and identify inhibitors that exert a synergistic effect with EGFR inhibition, we examined the inhibitory effects of selected protein kinase inhibitors (PKIs) in combination with gefitinib on the viability of a mesenchymal stem-like (MSL) subtype TNBC cell line. MK­2206, an AKT inhibitor, and a group of mammalian target of rapamycin (mTOR) inhibitors were found to exert synergistic lethal effects in combination with gefitinib in MDA­MB­231 cells. The combination of gefitinib/MK­2206 exerted a prominent synergistic lethal effect in an MTT cell viability assay and a growth inhibitory effect in a long-term colony-forming assay in 2 MSL subtype TNBC cell lines (MDA­MB­231 and HS578T) and one basal-like (BL) subtype TNBC cell line (MDA­MB-468). Gefitinib/MK­2206 treatment synergistically decreased the mTOR signaling target substrates along with the downregulation of ribosomal protein S6 (RPS6), a marker of cell proliferation and target substrate of the AKT-mTOR signaling pathway. In addition, gefitinib markedly reduced the viability of MDA­MD­231 and HS578T cells when regulatory-associated protein of mTOR (RPTOR) was suppressed by siRNA-based knockdown (KD). These results thus suggest that RPTOR mediates, at least partially, the resistance to EGFR inhibition in TNBC cells. Therefore, targeting the mTOR complex 1 (mTORC1) pathway may be a potential strategy for the treatment of EGFR-resistant TNBC.

Disruption of STAT3-DNMT1 interaction by SH-I-14 induces re-expression of tumor suppressor genes and inhibits growth of triple-negative breast tumor.

Epigenetic regulation of gene expression is an emerging target to treat several human diseases including cancers. In cancers, expressions of many tumor suppressor genes are suppressed by hyper-methylation in their regulatory regions. Herein, we describe a novel carbazole SH-I-14 that decreased the level of the acetyl-STAT3 at the K685 residue. Mutation analysis revealed that SH-I-14 disrupted STAT3-DNMT1 interaction by removing acetyl group from K685 of STAT3. Finally, the inhibition of STAT3-DNMT1 interaction by SH-I-14 resulted in re-expression of tumor suppressor genes such as VHL and PDLIM4 through de-methylation of their promoter regions. In addition, SH-I-14 showed anti-proliferative effect in triple-negative breast cancer (TNBC) cell lines in vitro and anti-tumor effect in a mouse xenograft model of MDA-MB-231 tumor. Taken together, our results suggest that targeting acetyl-STAT3 (K685) provides potential therapeutic opportunity to treat a subset of human cancers.

Application of a non-hazardous vital dye for cell counting with automated cell counters.

Recent advances in automated cell counters enable us to count cells more easily with consistency. However, the wide use of the traditional vital dye trypan blue (TB) raises environmental and health concerns due to its potential teratogenic effects. To avoid this chemical hazard, it is of importance to introduce an alternative non-hazardous vital dye that is compatible with automated cell counters. Erythrosin B (EB) is a vital dye that is impermeable to biological membranes and is used as a food additive. Similarly to TB, EB stains only nonviable cells with disintegrated membranes. However, EB is less popular than TB and is seldom used with automated cell counters. We found that cell counting accuracy with EB was comparable to that with TB. EB was found to be an effective dye for accurate counting of cells with different viabilities across three different automated cell counters. In contrast to TB, EB was less toxic to cultured HL-60 cells during the cell counting process. These results indicate that replacing TB with EB for use with automated cell counters will significantly reduce the hazardous risk while producing comparable results.