Synergistic antitumor immune response mediated by paclitaxel-conjugated nanohybrid oncolytic adenovirus with dendritic cell therapy.

Dendritic cell (DC)-based vaccines have emerged as a promising strategy in cancer immunotherapy due to low toxicity. However, the therapeutic efficacy of DC as a monotherapy is insufficient due to highly immunosuppressive tumor environment. To address these limitations of DC as immunotherapeutic agent, we have developed a polymeric nanocomplex incorporating (1) oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and (2) arginine-grafted bioreducible polymer with PEGylated paclitaxel (APP) to restore antitumor immune surveillance function in tumor milieu and potentiate immunostimulatory attributes of DC vaccine. Nanohybrid complex (oAd/APP) in combination with DC (oAd/APP+DC) induced superior expression level of antitumor cytokines (IL-12, GM-CSF, and interferon gamma) than either oAd/APP or DC monotherapy in tumor tissues, thus resulting in superior intratumoral infiltration of both endogenous and exogenous DCs. Furthermore, oAd/APP+DC treatment led superior migration of DC to secondary lymphoid organs, such as draining lymph nodes and spleen, in comparison with either monotherapy. Superior migration profile of DCs in oAd/APP+DC treatment group resulted in more prolific activation of tumor-specific T cells in these lymphoid organs and greater intratumoral infiltration of T cells. Additionally, oAd/APP+DC treatment led to lower subset of tumor infiltrating lymphocytes and splenocytes being immunosuppressive regulatory T cells than any other treatment groups. Collectively, oAd/APP+DC led to superior induction of antitumor immune response and amelioration of immunosuppressive tumor microenvironment to elicit potent tumor growth inhibition than either monotherapy.

Tumor microenvironment-modulating oncolytic adenovirus combined with GSK-3ß inhibitor enhances antitumor immune response against bladder cancer.

Bladder cancer is a common type of cancer around the world, and the majority of patients are diagnosed with non-muscle-invasive bladder cancer (NMIBC). Although low-risk NMIBC has a good prognosis, the disease recurrence rate and development of treatment-refractory disease remain high in intermediate- to high-risk NMIBC patients. To address these challenges for the treatment of NMIBC, a novel combination therapy composed of an oncolytic adenovirus (oAd) co-expressing interleukin (IL)-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and relaxin (RLX; HY-oAd) and a clinical-stage glycogen synthase kinase (GSK)-3ß inhibitor (9-ING-41; elraglusib) was investigated in the present report. Our findings demonstrate that HY-oAd and 9-ING-41 combination therapy (HY-oAd+9-ING-41) exerted superior inhibition of tumor growth compared with respective monotherapy in a syngeneic NMIBC tumor model. HY-oAd+9-ING-41 induced high-level tumor extracellular matrix (ECM) degradation and a more potent antitumor immune response than the respective monotherapy. In detail, HY-oAd+9-ING-41 induced superior accumulation of intratumoral T cells, prevention of immune cell exhaustion, and induction of tumor-specific adaptive immune response compared to either monotherapy. Collectively, these results demonstrate that the combination of HY-oAd and 9-ING-41 may be a promising approach to elicit a potent antitumor immune response against bladder cancer.

Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression.

Melanoma is a type of skin cancer that originates in melanin-producing melanocytes. It is considered a multifactorial disease caused by both genetic and environmental factors, such as UV radiation. Dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) phosphorylates many substrates involved in signaling pathways, cell survival, cell cycle control, differentiation, and neuronal development. However, little is known about the cellular function of DYRK3, one of the five members of the DYRK family. Interestingly, it was observed that the expression of DYRK3, as well as p62 (a multifunctional signaling protein), is highly enhanced in most melanoma cell lines. This study aimed to investigate whether DYRK3 interacts with p62, and how this affects melanoma progression, particularly in melanoma cell lines. We found that DYRK3 directly phosphorylates p62 at the Ser-207 and Thr-269 residue. Phosphorylation at Thr-269 of p62 by DYRK3 increased the interaction of p62 with tumor necrosis factor receptor-associated factor 6 (TRAF6), an already known activator of mammalian target of rapamycin complex 1 (mTORC1) in the mTOR-involved signaling pathways. Moreover, the phosphorylation of p62 at Thr-269 promoted the activation of mTORC1. We also found that DYRK3-mediated phosphorylation of p62 at Thr-269 enhanced the growth of melanoma cell lines and melanoma progression. Conversely, DYRK3 knockdown or blockade of p62-T269 phosphorylation inhibited melanoma growth, colony formation, and cell migration. In conclusion, we demonstrated that DYRK3 phosphorylates p62, positively modulating the p62-TRAF6-mTORC1 pathway in melanoma cells. This finding suggests that DYRK3 suppression may be a novel therapy for preventing melanoma progression by regulating the mTORC1 pathway.

Oncolytic adenovirus as pancreatic cancer-targeted therapy: Where do we go from here?

Pancreatic cancer remains one of the deadliest cancers with extremely high mortality rate, and the number of cases is expected to steadily increase with time. Pancreatic cancer is refractory to conventional cancer treatment options, like chemotherapy and radiotherapy, and commercialized immunotherapeutics, owing to its immunosuppressive and desmoplastic phenotype. Due to these reasons, development of an innovative treatment option that can overcome these challenges posed by the pancreatic tumor microenvironment (TME) is in an urgent need. The present review aims to summarize the evolution of oncolytic adenovirus (oAd) engineering and usage as therapeutics (either monotherapy or combination therapy) over the last decade to overcome these hurdles to instigate a potent antitumor effect against desmoplastic and immunosuppressive pancreatic cancer.

Current clinical landscape of oncolytic viruses as novel cancer immunotherapeutic and recent preclinical advancements.

Oncolytic viruses (OVs) have been gaining attention in the pharmaceutical industry as a novel immunotherapeutic and therapeutic adjuvant due to their ability to induce and boost antitumor immunity through multiple mechanisms. First, intrinsic mechanisms of OVs that enable exploitation of the host immune system (e.g., evading immune detection) can nullify the immune escape mechanism of tumors. Second, many types of OVs have been shown to cause direct lysis of tumor cells, resulting in an induction of tumor-specific T cell response mediated by release of tumor-associated antigens and danger signal molecules. Third, armed OV-expressing immune stimulatory therapeutic genes could be highly expressed in tumor tissues to further improve antitumor immunity. Last, these OVs can inflame cold tumors and their microenvironment to be more immunologically favorable for other immunotherapeutics. Due to these unique characteristics, OVs have been tested as an adjuvant of choice in a variety of therapeutics. In light of these promising attributes of OVs in the immune-oncology field, the present review will examine OVs in clinical development and discuss various strategies that are being explored in preclinical stages for the next generation of OVs that are optimized for immunotherapy applications.

ErbB3-Targeting Oncolytic Adenovirus Causes Potent Tumor Suppression by Induction of Apoptosis in Cancer Cells.

Cancer is a multifactorial and deadly disease. Despite major advancements in cancer therapy in the last two decades, cancer incidence is on the rise and disease prognosis still remains poor. Furthermore, molecular mechanisms of cancer invasiveness, metastasis, and drug resistance remain largely elusive. Targeted cancer therapy involving the silencing of specific cancer-enriched proteins by small interfering RNA (siRNA) offers a powerful tool. However, its application in clinic is limited by the short half-life of siRNA and warrants the development of efficient and stable siRNA delivery systems. Oncolytic adenovirus-mediated therapy offers an attractive alternative to the chemical drugs that often suffer from innate and acquired drug resistance. In continuation to our reports on the development of oncolytic adenovirus-mediated delivery of shRNA, we report here the replication-incompetent (dAd/shErbB3) and replication-competent (oAd/shErbB3) oncolytic adenovirus systems that caused efficient and persistent targeting of ErbB3. We demonstrate that the E1A coded by oAd/shErbB, in contrast to dAd/shErbB, caused downregulation of ErbB2 and ErbB3, yielding stronger downregulation of the ErbB3-oncogenic signaling axis in in vitro models of lung and breast cancer. These results were validated by in vivo antitumor efficacy of dAd/shErbB3 and oAd/shErbB3.

Why is Mortalin a Potential Therapeutic Target for Cancer?

Cancer is one of the leading causes of death worldwide, accounting for nearly 10 million deaths in 2020. Therefore, cancer therapy is a priority research field to explore the biology of the disease and identify novel targets for the development of better treatment strategies. Mortalin is a member of the heat shock 70 kDa protein family. It is enriched in several types of cancer and contributes to carcinogenesis in various ways, including inactivation of the tumor suppressor p53, deregulation of apoptosis, induction of epithelial-mesenchymal transition, and enhancement of cancer stemness. It has been studied extensively as a therapeutic target for cancer treatment, and several types of anti-mortalin molecules have been discovered that effectively suppress the tumor cell growth. In this review, we 1) provide a comprehensive sketch of the role of mortalin in tumor biology; 2) discuss various anti-mortalin molecules, including natural compounds, synthetic small molecules, peptides, antibodies, and nucleic acids, that have shown potential for cancer treatment in laboratory studies; and 3) provide future perspectives in cancer treatment.

Bioreducible polymer-mediated delivery of oncolytic adenovirus can attenuate antiviral immune response and concurrently enhance the induction of antitumor immune response to effectively prevent metastasis.

Oncolytic virotherapy is a highly promising and novel treatment modality for cancer. Several clinical trials with oncolytic viruses have illustrated that the potent antitumor efficacy of these viruses may rely on the efficient induction of antitumor immune response. In contrast, antiviral immune response is attributed to adverse side defects and diminishing therapeutic efficacy. In the present report, we generated a nanohybrid complex incorporating immune stimulatory oncolytic adenovirus (oAd) co-expressing decorin (DCN) and interleukin (IL)-12 with a bioreducible nanomaterial composed of PEI-Arg-mPEG-S-S-mPEG-Arg-PEI blocks (PAPS), ultimately aiming to modulate both antitumor and antiviral immune responses to be favorable toward oncolytic virotherapy. The transduction efficacy of the PAPS-incorporated nanohybrid vector (Ad/PAPS) was significantly higher than that of a complex using our previously reported polymer PPSA (Ad/PPSA) regardless of the cellular coxsackievirus and adenovirus receptor expression level of cancer cells. oAd complexed with PAPS (oAd/PAPS) also elicited a more potent cancer cell killing effect, antitumor efficacy, and metastasis inhibition than naked oAd or oAd complexed with PPSA (oAd/PPSA) through a higher level of therapeutic transgenes (DCN and IL-12), viral replication, and more efficient infiltration of T cells into tumor tissues. Notably, oAd/PAPS induced the highest level of antitumor immune response while the antiviral immune response was mediated at a significantly lower level than those of naked oAd. Adaptive immune response against the virus was also significantly attenuated in the oAd/PAPS group. oAd/PAPS treatment also led to the highest level of antitumor central memory T cells and the lowest level of immunosuppressive regulatory T cells in the spleen. Collectively, our findings illustrate that oAd/PAPS can simultaneously regulate both antitumor and antiviral immune responses to be more favorable to oncolytic virotherapy, leading to improved gene expression, viral replication, and growth inhibition of both primary and metastatic tumors.

Immunotherapy by mesenchymal stromal cell delivery of oncolytic viruses for treating metastatic tumors.

Oncolytic viruses (OVs) have emerged as a very promising anti-cancer therapeutic strategy in the past decades. However, despite their pre-clinical promise, many OV clinical evaluations for cancer therapy have highlighted the continued need for their improved delivery and targeting. Mesenchymal stromal cells (MSCs) have emerged as excellent candidate vehicles for the delivery of OVs due to their tumor-homing properties and low immunogenicity. MSCs can enhance OV delivery by protecting viruses from rapid clearance following administration and also by more efficiently targeting tumor sites, consequently augmenting the therapeutic potential of OVs. MSCs can function as "biological factories," enabling OV amplification within these cells to promote tumor lysis following MSC-OV arrival at the tumor site. MSC-OVs can promote enhanced safety profiles and therapeutic effects relative to OVs alone. In this review we explore the general characteristics of MSCs as delivery tools for cancer therapeutic agents. Furthermore, we discuss the potential of OVs as immune therapeutics and highlight some of the promising applications stemming from combining MSCs to achieve enhanced delivery and anti-tumor effectiveness of OVs at different pre-clinical and clinical stages. We further provide potential pitfalls of the MSC-OV platform and the strategies under development for enhancing the efficacy of these emerging therapeutics.

Challenges and progress toward tumor-targeted therapy by systemic delivery of polymer-complexed oncolytic adenoviruses.

Oncolytic adenovirus (oAd) elicits antitumor activity by preferential viral replication in cancer cells. However, poor systemic administrability or suboptimal intratumoral retainment of the virus remains a major challenge toward maximizing the antitumor activity of oAd in a clinical environment. To surmount these issues, a variety of non-immunogenic polymers has been used to modify the surface of oAds chemically or physically. Complexation of oAd with polymers can effectively evade the host immune response and reduces nonspecific liver sequestration. The tumor-specific delivery of these complexes can be further improved upon by inclusion of tumor-targeting moieties on the surface. Therefore, modification of the Ad surface using polymers is viewed as a potential strategy to enhance the delivery of Ad via systemic administration. This review aims to provide a comprehensive overview of polymer-complexed Ads, their progress, and future challenges in cancer treatment.

Polymeric Systems for Cancer Immunotherapy: A Review.

Immunotherapy holds enormous promise to create a new outlook of cancer therapy by eliminating tumors via activation of the immune system. In immunotherapy, polymeric systems play a significant role in improving antitumor efficacy and safety profile. Polymeric systems possess many favorable properties, including magnificent biocompatibility and biodegradability, structural and component diversity, easy and controllable fabrication, and high loading capacity for immune-related substances. These properties allow polymeric systems to perform multiple functions in immunotherapy, such as immune stimulants, modifying and activating T cells, delivery system for immune cargos, or as an artificial antigen-presenting cell. Among diverse immunotherapies, immune checkpoint inhibitors, chimeric antigen receptor (CAR) T cell, and oncolytic virus recently have been dramatically investigated for their remarkable success in clinical trials. In this report, we review the monotherapy status of immune checkpoint inhibitors, CAR-T cell, and oncolytic virus, and their current combination strategies with diverse polymeric systems.

A pH- and Bioreducible Cationic Copolymer with Amino Acids and Piperazines for Adenovirus Delivery.

Adenoviruses (Ads) are attractive nonviral vectors and show great potential in cancer gene therapy. However, inherent properties of Ads, including immunogenicity, nonspecific toxicity, and coxsackie and adenovirus receptor (CAR)-dependent cell uptake, limit their clinical use. To surmount these issues, we developed a pH- and glutathione-responsive poly(ethylene glycol)-poly(ꞵ-aminoester)-polyethyleneimine (PPA) for conjugation with Ad. The pH sensitivity of the PPA copolymer was elegantly tuned by substitution with different amino acids (arginine, histidine, and tryptophan), piperazines (Pip1, Pip2, and Pip3), and guanidine residues in the backbone of the PPA conjugate. PPA copolymer was further functionalized with short-chain cross-linker succinimidyl 3-(2-pyridyldithio)propionate) (SPDP) to obtain PPA-SPDP for facile conjugation with Ad. The PPA-conjugated Ad (PPA-Ad) conjugate was obtained by reacting PPA-SPDP conjugate with thiolated Ad (Ad-SH). Ad-SH was prepared by reacting Ad with 2-iminothiolane. The size distribution and zeta potential results of PPA-Ad conjugate showed an increasing trend with an increase in copolymer dose. From in vitro test, it was found that the transduction efficiency of PPA-Ad conjugate in CAR-positive cells (A549 and H460 cells) was remarkably increased at the acidic pH condition (pH 6.2) when compared with PPA-Ad conjugate incubated under the physiological condition (pH 7.4). Interestingly, the increase in transduction efficiency was evidenced in CAR-negative cells (MDA-MB-231 and T24 cells). These results demonstrated that biocompatible and biodegradable PPA copolymers can efficiently cover the surface of Ad and can increase the transduction efficiency, and hence PPA copolymers can be a useful nanomaterial for viral vector delivery in cancer therapy.

GM101 in Combination with Histone Deacetylase Inhibitor Enhances Anti-Tumor Effects in Desmoplastic Microenvironment.

Oncolytic adenoviruses (oAds) have been evaluated in numerous clinical trials due to their promising attributes as cancer therapeutics. However, the therapeutic efficacy of oAds was limited due to variable coxsackie and adenovirus receptor (CAR) expression levels and the dense extracellular matrix (ECM) of heterogenic clinical tumors. To overcome these limitations, our present report investigated the therapeutic efficacy of combining GM101, an oAd with excellent tumor ECM degrading properties, and histone deacetylase inhibitor (HDACi). Four different HDACi (suberohydroxamic acid (SBHA), MS-275, trichostatin A (TSA), and valproic acid) candidates in combination with replication-incompetent and GFP-expressing Ad (dAd/GFP) revealed that SBHA and MS-275 exerted more potent enhancement in Ad transduction efficacy than TSA or valproic acid. Further characterization revealed that SBHA and MS-275 effectively upregulated CAR expression in cancer cells, improved the binding of Ad with cancer cell membranes, and led to dynamin 2- and clathrin-mediated endocytosis of Ad. The combination of GM101 with HDACi induced superior cancer cell killing effects compared to any of the monotherapies, without any additional cytotoxicity in normal cell lines. Further, GM101+SBHA and GM101+MS-275 induced more potent antitumor efficacy than any monotherapy in U343 xenograft tumor model. Potent antitumor efficacy was achieved via the combination of GM101 with HDACi, inducing necrotic and apoptotic cancer cell death, inhibiting cancer cell proliferation, degrading ECM in tumor tissue, and thus exerting the highest level of virus dispersion and accumulation. Collectively, these data demonstrate that the combination of GM101 and HDACi can enhance intratumoral dispersion and accumulation of oAd through multifaced mechanisms, making it a promising strategy to address the challenges toward successful clinical development of oAd.

Optimizing Active Tumor Targeting Biocompatible Polymers for Efficient Systemic Delivery of Adenovirus.

Adenovirus (Ad) has risen to be a promising alternative to conventional cancer therapy. However, systemic delivery of Ad, which is necessary for the treatment of metastatic cancer, remains a major challenge within the field, owing to poor tumor tropism and nonspecific hepatic tropism of the virus. To address this limitation of Ad, we have synthesized two variants of folic acid (FA)-conjugated methoxy poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]-L-glutamate (P5N2LG-FA and P5N5LG-FA) using 5 kDa poly(ethylene glycol) (PEG) with a different level of protonation (N2 < N5 in terms of charge), along with a P5N5LG control polymer without FA. Our findings demonstrate that P5N5LG, P5N2LG-FA, and P5N5LG-FA exert a lower level of cytotoxicity compared to 25 kDa polyethyleneimine. Furthermore, green fluorescent protein (GFP)-expressing Ad complexed with P5N2LG-FA and P5N5LG-FA (Ad/P5N2LG-FA and Ad/P5N5LG-FA, respectively) exerted superior transduction efficiency compared to naked Ad or Ad complexed with P5N5LG (Ad/P5N5LG) in folate receptor (FR)-overexpressing cancer cells (KB and MCF7). All three nanocomplexes (Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA) internalized into cancer cells through coxsackie adenovirus receptor-independent endocytic mechanism and the cell uptake was more efficient than naked Ad. Importantly, the cell uptake of the two FA functionalized nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) was dependent on the complementary interaction of FA-FR. Systemically administered Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA showed exponentially higher retainment of the virus in blood circulation up to 24 h post-administration compared with naked Ad. Both tumor-targeted nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) showed significantly higher intratumoral accumulation than naked Ad or Ad/P5N5LG via systemic administration. Both tumor-targeted nanocomplexes accumulated at a lower level in liver tissues compared to naked Ad. Notably, the nonspecific accumulation of Ad/P5N2LG-FA was significantly lower than Ad/P5N5LG-FA in several normal organs, while exhibiting a significantly higher intratumoral accumulation level, showing that careful optimization of polyplex surface charge is critical to successful tumor-targeted systemic delivery of Ad nanocomplexes.

The role of viral infectivity in oncolytic virotherapy outcomes: A mathematical study.

A model capturing the dynamics between virus and tumour cells in the context of oncolytic virotherapy is presented and analysed. The ability of the virus to be internalised by uninfected cells is described by an infectivity parameter, which is inferred from available experimental data. The parameter is also able to describe the effects of changes in the tumour environment that affect viral uptake from tumour cells. Results show that when a virus is inoculated inside a growing tumour, strategies for enhancing infectivity do not lead to a complete eradication of the tumour. Within typical times of experiments and treatments, we observe the onset of oscillations, which always prevent a full destruction of the tumour mass. These findings are in good agreement with available laboratory results. Further analysis shows why a fully successful therapy cannot exist for the proposed model and that care must be taken when designing and engineering viral vectors with enhanced features. In particular, bifurcation analysis reveals that creating longer lasting virus particles or using strategies for reducing infected cell lifespan can cause unexpected and unwanted surges in the overall tumour load over time. Our findings suggest that virotherapy alone seems unlikely to be effective in clinical settings unless adjuvant strategies are included.

CRISPR-Cas12a with an oAd Induces Precise and Cancer-Specific Genomic Reprogramming of EGFR and Efficient Tumor Regression.

CRISPR-Cas12a represents a class 2/type V CRISPR RNA-guided endonuclease, holding promise as a precise genome-editing tool in vitro and in vivo. For efficient delivery of the CRISPR-Cas system into cancer, oncolytic adenovirus (oAd) has been recognized as a promising alternative vehicle to conventional cancer therapy, owing to its cancer specificity; however, to our knowledge, it has not been used for genome editing. In this study, we show that CRISPR-Cas12a mediated by oAd disrupts the oncogenic signaling pathway with excellent cancer specificity. The intratumoral delivery of a single oAd co-expressing a Cas12a and a CRISPR RNA (crRNA) targeting the epidermal growth factor receptor (EGFR) gene (oAd/Cas12a/crEGFR) induces efficient and precise editing of the targeted EGFR gene in a cancer-specific manner, without detectable off-target nuclease activity. Importantly, oAd/Cas12a/crEGFR elicits a potent antitumor effect via robust induction of apoptosis and inhibition of tumor cell proliferation, ultimately leading to complete tumor regression in a subset of treated mice. Collectively, in this study we show precise genomic reprogramming via a single oAd vector-mediated CRISPR-Cas system and the feasibility of such system as an alternative cancer therapy.

Mesenchymal stem cells used as carrier cells of oncolytic adenovirus results in enhanced oncolytic virotherapy.

Mesenchymal stem cells (MSCs) loaded with oncolytic viruses are presently being investigated as a new modality of advanced/metastatic tumors treatment and enhancement of virotherapy. MSCs can, however, either promote or suppress tumor growth. To address the critical question of how MSCs loaded with oncolytic viruses affect virotherapy outcomes and tumor growth patterns in a tumor microenvironment, we developed and analyzed an integrated mathematical-experimental model. We used the model to describe both the growth dynamics in our experiments of firefly luciferase-expressing Hep3B tumor xenografts and the effects of the immune response during the MSCs-based virotherapy. We further employed it to explore the conceptual clinical feasibility, particularly, in evaluating the relative significance of potential immune promotive/suppressive mechanisms induced by MSCs loaded with oncolytic viruses. We were able to delineate conditions which may significantly contribute to the success or failure of MSC-based virotherapy as well as generate new hypotheses. In fact, one of the most impactful outcomes shown by this investigation, not inferred from the experiments alone, was the initially counter-intuitive fact that using tumor-promoting MSCs as carriers is not only helpful but necessary in achieving tumor control. Considering the fact that it is still currently a controversial debate whether MSCs exert a pro- or anti-tumor action, mathematical models such as this one help to quantitatively predict the consequences of using MSCs for delivering virotherapeutic agents in vivo. Taken together, our results show that MSC-mediated systemic delivery of oncolytic viruses is a promising strategy for achieving synergistic anti-tumor efficacy with improved safety profiles.

Mesenchymal Stem Cell-Mediated Delivery of an Oncolytic Adenovirus Enhances Antitumor Efficacy in Hepatocellular Carcinoma.

Oncolytic virotherapy is a promising alternative to conventional treatment, yet systemic delivery of these viruses to tumors remains a major challenge. In this regard, mesenchymal stem cells (MSC) with well-established tumor-homing property could serve as a promising systemic delivery tool. We showed that MSCs could be effectively infected by hepatocellular carcinoma (HCC)-targeted oncolytic adenovirus (HCC-oAd) through modification of the virus' fiber domain and that the virus replicated efficiently in the cell carrier. HCC-targeting oAd loaded in MSCs (HCC-oAd/MSC) effectively lysed HCC cells in vitro under both normoxic and hypoxic conditions as a result of the hypoxia responsiveness of HCC-oAd. Importantly, systemically administered HCC-oAd/MSC, which were initially infected with a low viral dose, homed to HCC tumors and resulted in a high level of virion accumulation in the tumors, ultimately leading to potent tumor growth inhibition. Furthermore, viral dose reduction and tumor localization of HCC-oAd/MSC prevented the induction of hepatotoxicity by attenuating HCC-oAd hepatic accumulation. Taken together, these results demonstrate that MSC-mediated systemic delivery of oAd is a promising strategy for achieving synergistic antitumor efficacy with improved safety profiles. SIGNIFICANCE: Mesenchymal stem cells enable delivery of an oncolytic adenovirus specifically to the tumor without posing any risk associated with systemic administration of naked virions to the host.

CARF enrichment promotes epithelial-mesenchymal transition via Wnt/ß-catenin signaling: its clinical relevance and potential as a therapeutic target.

CARF (Collaborator of ARF)/CDKN2AIP was discovered as a novel ARF-binding protein. It has been established as an essential cell survival, p53-, and cell proliferation-regulatory protein. Although a moderate upregulation of CARF caused growth arrest and senescence, its excessively enriched levels were shown to facilitate aggressive proliferation and malignant transformation of cancer cells. Here, we examined the relevance of CARF levels in clinical tumors and found its amplification (both at gene and transcript levels) in a variety of invasive and metastatic malignancies. Consistent with the clinical readouts, enrichment of CARF in cancer cells promoted epithelial-mesenchymal transition (EMT). Cancer database and molecular analyses revealed that it activates Wnt/ß-catenin signaling axis, as evident by enhanced nuclear localization and function of ß-catenin marked by increased level of SNAIL1, SNAIL2, ZEB1, and TWIST1 and its downstream gene targets. Of note, targeted knockdown of CARF led to decrease in nuclear ß-catenin and its key downstream effectors, involved in EMT progression. Consistent with this, CARF targeting in vivo either by naked siRNA or CARF shRNA harboring adeno-oncolytic virus caused suppression of tumor progression and lung metastasis. Taken together, we report clinical and therapeutic relevance of CARF in EMT and cancer invasiveness/metastasis, and propose it as a potent therapeutic target of aggressive cancers.

Hepatocellular carcinoma-targeting oncolytic adenovirus overcomes hypoxic tumor microenvironment and effectively disperses through both central and peripheral tumor regions.

Cancer-specific promoter driven replication of oncolytic adenovirus (Ad) is cancer-specific, but shows low transcriptional activity. Thus, we generated several chimeric α-fetoprotein (AFP) promoter variants, containing reconstituted enhancer and silencer regions, to preferentially drive Ad replication in hepatocellular carcinoma (HCC). Modified AFP promoter, containing 2 enhancer A regions and a single enhancer B region (a2bm), showed strong and HCC-specific transcription. In AFP-positive HCCs, gene expression was 43- to 456-fold higher than those of control AFP promoter lacking enhancers. a2bm promoter was further modified by inserting multiple hypoxia-responsive elements (HRE) to generate Ha2bm promoter, which showed stronger transcriptional activity than a2bm promoter under hypoxic conditions. Ha2bm promoter-regulated oncolytic Ad (Ha2bm-d19) showed a stronger antitumor and proapoptotic effect than did a2bm promoter-regulated oncolytic Ad (a2bm-d19) in HCC xenograft tumors. Systemically administered Ha2bm-d19 caused no observable hepatotoxicity, whereas control replication-competent Ad, lacking cancer specificity (d19), induced significant hepatic damage. Ha2bm-d19 caused significantly lower expression of interleukin-6 than d19, showing that HCC-targeted delivery of Ad attenuates induction of the innate immune response against Ad. This chimeric AFP promoter enabled Ad to overcome the hypoxic tumor microenvironment and target HCC with high specificity, rendering it a promising candidate for the treatment of aggressive HCCs.