Adenovirus Lacking E1b Efficiently Induces Cytopathic Effect in HPV-16-Positive Murine Cancer Cells via Virus Replication and Apoptosis.

Conditionally replicative adenoviruses (CRAds) replicate poorly in murine cancer cells; however, E1b-deleted CRAds may replicate effectively in HPV16-E6/E7-positive murine cancer cells (TC-1). The HPV16 E7 open reading frame encodes functions analogous to these deleted adenovirus E1 proteins. In this study, an E1b-deleted CRAd (Adhz60) was evaluated for its ability to replicate and induce oncolysis in TC-1 cells. Adhz60-mediated oncolysis was similar in TC-1 and HeLa cells. Productive viral replication was evident based on expression of E1A and hexon, production of infectious virus progeny, and Adhz60-induced apoptosis. The results suggest that TC-1 murine cancer cells allow Adhz60 replication and oncolysis.

Tumor specific liposomes improve detection of pancreatic adenocarcinoma in vivo using optoacoustic tomography.

BACKGROUND: Pancreatic cancer often goes undiagnosed until late stage disease due in part to suboptimal early detection. Our goal was to develop a Syndecan-1 tagged liposome containing fluorescent dye as an improved contrast agent for detection of pancreatic adenocarcinoma in vivo using multispectral optoacoustic tomography. RESULTS: The diagnostic capabilities and specificity to pancreatic adenocarcinoma of Syndecan-1 targeted liposomes were evaluated both in vitro and in vivo. Immunocytochemistry showed that liposomes preferentially bound to and released their contents into cells expressing high levels of insulin-like growth factor 1 receptor. We determined that the contents of the liposome were released into the cell as noted by the change in propidium iodide fluorescence from green to red based upon nucleic acid binding. In an orthotopic mouse model, the liposomes preferentially targeted the pancreatic tumor with little off-target binding in the liver and spleen. Peak accumulation of the liposomes in the tumor occurred at 8 h post-injection. Multispectral optoacoustic tomographic imaging was able to provide high-resolution 3D images of the tumor and liposome location. Ex vivo analysis showed that non-targeted liposomes accumulated in the liver, suggesting that specificity of the liposomes for pancreatic adenocarcinoma was due to the presence of the Syndecan-1 ligand. CONCLUSIONS: This study demonstrated that Syndecan-1 liposomes were able to release cargo into IGF1-R expressing tumor cells. The Syndecan-1 liposomes demonstrated tumor specificity in orthotopic pancreatic cancer as observed using multispectral optoacoustic tomography with reduced kidney and liver uptake. By targeting the liposome with Syndecan-1, this nanovehicle has potential as a targeted theranostic nanoparticle for both drug and contrast agent delivery to pancreatic tumors.

Correction: Garza-Morales et al. Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy In Vitro. Cancers 2018, 10, 144.

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Oncolytic Adenovirus Armed with a Novel Agonist of the CD137 Immune Checkpoint Stimulator Suppresses Tumor Growth.

Natural 4-1BBL (CD137L) is a cell membrane-bound protein critical to the expansion, effector function, and survival of CD8+ T cells. We reported the generation of an active soluble oligomeric construct, SA-4-1BBL, with demonstrated immunoprevention and immunotherapeutic efficacy in various mouse tumor models. Herein, we developed an oncolytic adenovirus (OAd) for the delivery and expression of SA-4-1BBL (OAdSA-4-1BBL) into solid tumors for immunotherapy. SA-4-1BBL protein expressed by this construct produced T-cell proliferation in vitro. OAdSA-4-1BBL decreased cell viability in two mouse lung cancer cell lines, TC-1 and CMT64, but not in the non-cancerous lung MM14.Lu cell line. OAdSA-4-1BBL induced programmed cell death types I and II (apoptosis and autophagy, respectively), and autophagy-mediated adenosine triphosphate (ATP) release was also detected. Intratumoral injection of OAdSA-4-1BBL efficiently expressed the SA-4-1BBL protein in the tumors, resulting in significant tumor suppression in a syngeneic subcutaneous TC-1 mouse lung cancer model. Tumor suppression was associated with a higher frequency of dendritic cells and an increased infiltration of cytotoxic CD8+ T and NK cells into the tumors. Our data suggest that OAdSA-4-1BBL may present an efficacious alternative therapeutic strategy against lung cancer as a standalone construct or in combination with other immunotherapeutic modalities, such as immune checkpoint inhibitors.

E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK.

E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.

Differential expression of microRNA between triple negative breast cancer patients of African American and European American descent.

There are racial disparities in the outcome of triple negative breast cancer (TNBC) patients between women of African ancestry and women of European ancestry, even after accounting for lifestyle, socioeconomic and clinical factors. MicroRNA (miRNA) are non-coding molecules whose level of expression is associated with cancer suppression, proliferation and drug resistance; therefore, these have potential for biomarker applications in cancers including TNBC. Historically, miRNAs up-regulated in African American (AA) patients have received less attention than for patients of European ancestry. Using laser capture microdissection (LCM) to acquire ultrapure tumor cell samples, miRNA expression was evaluated in 15 AA and 15 European American (EA) TNBC patients. Tumor sections were evaluated using RNA extraction followed by miRNA analysis and profiling. Results were compared based on ethnicity and method of tissue fixation. miRNAs that showed high differential expression in AA TNBC patients compared to EA included: miR-19a, miR-192, miR-302a, miR-302b, miR-302c, miR-335, miR-520b, miR-520f and miR-645. LCM is a useful technique for isolation of tumor cells. We found a greater abundance of RNA in frozen samples compared to formalin fixed, paraffin embedded samples. miRNA appears to be a useful biomarker for TNBC to improve diagnosis and treatment.

Probiotics and Trained Immunity.

The characteristics of innate immunity have recently been investigated in depth in several research articles, and original findings suggest that innate immunity also has a memory capacity, which has been named "trained immunity". This notion has revolutionized our knowledge of the innate immune response. Thus, stimulation of trained immunity represents a therapeutic alternative that is worth exploring. In this context, probiotics, live microorganisms which when administered in adequate amounts confer a health benefit on the host, represent attractive candidates for the stimulation of trained immunity; however, although numerous studies have documented the beneficial proprieties of these microorganisms, their mechanisms of action are not yet fully understood. In this review, we propose to explore the putative connection between probiotics and stimulation of trained immunity.

The neutral red assay can be used to evaluate cell viability during autophagy or in an acidic microenvironment in vitro.

Harsh conditions within the tumor microenvironment, such as hypoxia and extracellular acidic pH (pHe), inactivate some chemotherapies, which results in limited or no cytotoxicity. Standard MTT, ATPlite and protease assays that are used to determine the potency of newly developed drugs often give erroneous results when applied under hypoxic or acidic conditions. Therefore, development of a cytotoxicity assay that does not yield false positive or false negative results under circumstances of both hypoxia and acidic pHe is needed. We evaluated currently used cell viability assays as well as neutral red staining to assess viability of ovarian and pancreatic cancer cells grown in an acidic pHe microenvironment after treatment with carboplatin, gemcitabine or chloroquine. We validated cell viability using western blotting of pro-caspase-9 and cleaved-caspase-9, and LC3-I and - II. Standard cell viability assays indicated cell viability accurately at pHe 7.4, but was not correlated with induction of apoptosis or autophagy at acidic pHe. By contrast, our modified neutral red assay detected cell viability accurately over a range of pHe as demonstrated by its correlation with induction of apoptosis and autophagy. Neutral red staining is effective for evaluating the effect of chemotherapeutic agents on cell viability under acidic pHe or hypoxic conditions.

An Oncolytic Adenovirus Encoding SA-4-1BBL Adjuvant Fused to HPV-16 E7 Antigen Produces a Specific Antitumor Effect in a Cancer Mouse Model.

Human papillomaviruses (HPVs) are responsible for about 25% of cancer cases worldwide. HPV-16 E7 antigen is a tumor-associated antigen (TAA) commonly expressed in HPV-induced tumors; however, it has low immunogenicity. The interaction of 4-1BBL with its receptor induces pleiotropic effects on innate, adaptive, and regulatory immunity and, if fused to TAAs in DNA vaccines, can improve the antitumor response; however, there is low transfection and antitumor efficiency. Oncolytic virotherapy is promising for antitumor gene therapy as it can be selectively replicated in tumor cells, inducing cell lysis, and furthermore, tumor cell debris can be taken in by immune cells to potentiate antitumor responses. In this study, we expressed the immunomodulatory molecule SA-4-1BBL fused to E7 on an oncolytic adenovirus (OAd) system. In vitro infection of TC-1 tumor cells and NIH-3T3 non-tumor cells with SA/E7/4-1BBL OAd demonstrated that only tumor cells are selectively destroyed. Moreover, protein expression is targeted to the endoplasmic reticulum in both cell lines when a signal peptide (SP) is added. Finally, in an HPV-induced cancer murine model, the therapeutic oncolytic activity of OAd can be detected, and this can be improved when fused to E7 and SP.

Transcriptomic Profiling Identifies Differentially Expressed Genes in Palbociclib-Resistant ER+ MCF7 Breast Cancer Cells.

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition in estrogen receptor-positive (ER+) breast cancer remains a significant clinical challenge. Efforts to uncover the mechanisms underlying resistance are needed to establish clinically actionable targets effective against resistant tumors. In this study, we sought to identify differentially expressed genes (DEGs) associated with acquired resistance to palbociclib in ER+ breast cancer. We performed next-generation transcriptomic RNA sequencing (RNA-seq) and pathway analysis in ER+ MCF7 palbociclib-sensitive (MCF7/pS) and MCF7 palbociclib-resistant (MCF7/pR) cells. We identified 2183 up-regulated and 1548 down-regulated transcripts in MCF7/pR compared to MCF7/pS cells. Functional analysis of the DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified several pathways associated with breast cancer, including 'cell cycle', 'DNA replication', 'DNA repair' and 'autophagy'. Additionally, Ingenuity Pathway Analysis (IPA) revealed that resistance to palbociclib is closely associated with deregulation of several key canonical and metabolic pathways. Further studies are needed to determine the utility of these DEGs and pathways as therapeutics targets against ER+ palbociclib-resistant breast cancer.

Targeting Melanoma Hypoxia with the Food-Grade Lactic Acid Bacterium Lactococcus Lactis.

Melanoma is the most aggressive form of skin cancer. Hypoxia is a feature of the tumor microenvironment that reduces efficacy of immuno- and chemotherapies, resulting in poor clinical outcomes. Lactococcus lactis is a facultative anaerobic gram-positive lactic acid bacterium (LAB) that is Generally Recognized as Safe (GRAS). Recently, the use of LAB as a delivery vehicle has emerged as an alternative strategy to deliver therapeutic molecules; therefore, we investigated whether L. lactis can target and localize within melanoma hypoxic niches. To simulate hypoxic conditions in vitro, melanoma cells A2058, A375 and MeWo were cultured in a chamber with a gas mixture of 5% CO2, 94% N2 and 1% O2. Among the cell lines tested, MeWo cells displayed greater survival rates when compared to A2058 and A375 cells. Co-cultures of L. lactis expressing GFP or mCherry and MeWo cells revealed that L. lactis efficiently express the transgenes under hypoxic conditions. Moreover, multispectral optoacoustic tomography (MSOT), and near infrared (NIR) imaging of tumor-bearing BALB/c mice revealed that the intravenous injection of either L. lactis expressing ß-galactosidase (ß-gal) or infrared fluorescent protein (IRFP713) results in the establishment of the recombinant bacteria within tumor hypoxic niches. Overall, our data suggest that L. lactis represents an alternative strategy to target and deliver therapeutic molecules into the tumor hypoxic microenvironment.

Actively Targeted Nanodelivery of Echinomycin Induces Autophagy-Mediated Death in Chemoresistant Pancreatic Cancer In Vivo.

Pancreatic cancer remains a recalcitrant neoplasm associated with chemoresistance and high fatality. Because it is frequently resistant to apoptosis, exploiting autophagic cell death could offer a new treatment approach. We repurpose echinomycin, an antibiotic encapsulated within a syndecan-1 actively targeted nanoparticle, for treatment of pancreatic cancer. Tumor-specific uptake, biodistribution, efficacy of nanodelivered echinomycin, and mechanism of cell death were assessed in aggressive, metastatic models of pancreatic cancer. In these autophagic-dependent pancreatic cancer models, echinomycin treatment resulted in autophagic cell death noted by high levels of LC3 among other autophagy markers, but without hallmarks of apoptosis, e.g., caspase activation and chromatin fragmentation, or necrosis, e.g., plasma membrane degradation and chromatin condensation/degrading. In vivo, biodistribution of syndecan-1-targeted nanoparticles indicated preferential S2VP10 or S2CP9 tumor uptake compared to the liver and kidney (S2VP10 p = 0.0016, p = 0.00004 and S2CP9 p = 0.0009, p = 0.0001). Actively targeted nanodelivered echinomycin resulted in significant survival increases compared to Gemzar (S2VP10 p = 0.0003, S2CP9 p = 0.0017) or echinomycin only (S2VP10 p = 0.0096, S2CP9 p = 0.0073). We demonstrate that actively targeted nanodelivery of echinomycin results in autophagic cell death in pancreatic and potentially other high-autophagy, apoptosis-resistant tumors. Collectively, these findings support syndecan-1-targeted delivery of echinomycin and dysregulation of autophagy to induce cell death in pancreatic cancer.

Adenovirus E1B55K region is required to enhance cyclin E expression for efficient viral DNA replication.

Adenoviruses (Ads) with E1B55K mutations can selectively replicate in and destroy cancer cells. However, the mechanism of Ad-selective replication in tumor cells is not well characterized. We have shown previously that expression of several cell cycle-regulating genes is markedly affected by the Ad E1b gene in WI-38 human lung fibroblast cells (X. Rao, et al., Virology 350:418-428, 2006). In the current study, we show that the Ad E1B55K region is required to enhance cyclin E expression and that the failure to induce cyclin E overexpression due to E1B55K mutations prevents viral DNA from undergoing efficient replication in WI-38 cells, especially when the cells are arrested in the G(0) phase of the cell cycle by serum starvation. In contrast, cyclin E induction is less dependent on the function encoded in the E1B55K region in A549 and other cancer cells that are permissive for replication of E1B55K-mutated viruses, whether the cells are in the S phase or G(0) phase. The small interfering RNA that specifically inhibits cyclin E expression partially decreased viral replication. Our study provides evidence suggesting that E1B55K may be involved in cell cycle regulation that is important for efficient viral DNA replication and that cyclin E overexpression in cancer cells may be associated with the oncolytic replication of E1B55K-mutated viruses.

Targeting Palbociclib-Resistant Estrogen Receptor-Positive Breast Cancer Cells via Oncolytic Virotherapy.

While clinical responses to palbociclib have been promising, metastatic breast cancer remains incurable due to the development of resistance. We generated estrogen receptor-positive (ER+) and ER-negative (ER-) cell line models and determined their permissiveness and cellular responses to an oncolytic adenovirus (OAd) known as Ad5/3-delta24. Analysis of ER+ and ER- palbociclib-resistant cells revealed two clearly distinguishable responses to the OAd. While ER+ palbociclib-resistant cells displayed a hypersensitive phenotype to the effects of the OAd, ER- palbociclib-resistant cells showed a resistant phenotype to the OAd. Hypersensitivity to the OAd in ER+ palbociclib-resistant cells correlated with a decrease in type I interferon (IFN) signaling, an increase in viral entry receptor expression, and an increase in cyclin E expression. OAd resistance in ER- palbociclib-resistant cells correlated with an increase in type I IFN signaling and a marked decrease in viral entry receptor. Using the OAd as monotherapy caused significant cytotoxicity to both ER+ and ER- palbociclib-sensitive cell lines. However, the addition of palbociclib increased the oncolytic activity of the OAd only in ER+ palbociclib-sensitive cells. Our studies provide a mechanistic base for a novel anti-cancer regimen composed of an OAd in combination with palbociclib for the treatment of ER+ breast cancer.

A Novel Form of 4-1BBL Prevents Cancer Development via Nonspecific Activation of CD4+ T and Natural Killer Cells.

Costimulation through 4-1BB (CD137) receptor generates robust CD8+ T-effector and memory responses. The only known ligand, 4-1BBL, is a trimeric transmembrane protein that has no costimulatory activity as a soluble molecule. Thus, agonistic antibodies to the receptor have been used for cancer immunotherapy in preclinical models and are currently being evaluated in the clinic. Here, we report that treatment with an oligomeric form of the ligand, SA-4-1BBL, as a single agent is able to protect mice against subsequent tumor challenge irrespective of the tumor type. Protection was long-lasting (>8 weeks) and a bona fide property of SA-4-1BBL, as treatment with an agonistic antibody to the 4-1BB receptor was ineffective in generating immune protection against tumor challenge. Mechanistically, SA-4-1BBL significantly expanded IFNγ-expressing, preexisting memory-like CD44+CD4+ T cells and NK cells in naïve mice as compared with the agonistic antibody. In vivo blockade of IFNγ or depletion of CD4+ T or NK cells, but not CD8+ T or B cells, abrogated the immunopreventive effects of SA-4-1BBL against cancer. SA-4-1BBL as a single agent also exhibited robust efficacy in controlling postsurgical recurrences. This work highlights unexpected features of SA-4-1BBL as a novel immunomodulator with implications for cancer immunoprevention and therapy. SIGNIFICANCE: This study demonstrates the unique and unexpected immunomodulatory features of SA-4-1BBL that bridge innate and adaptive immune responses with both preventive and therapeutic efficacy against cancer.

A DNA Vaccine Encoding SA-4-1BBL Fused to HPV-16 E7 Antigen Has Prophylactic and Therapeutic Efficacy in a Cervical Cancer Mouse Model.

The SA-4-1BBL, an oligomeric novel form of the natural ligand for the 4-1BB co-stimulatory receptor of the tumor necrosis factor (TNF) superfamily, as a recombinant protein has potent pleiotropic effects on cells of innate, adaptive, and regulatory immunity with demonstrated therapeutic efficacy in several tumor models. However, the production of soluble form of SA-4-1BBL protein and quality control is time and resource intensive and face various issues pertinent to clinical development of biologics. The present study sought to take advantage of the simplicity and translatability of DNA-based vaccines for the production and delivery of SA-4-1BBL for cancer immune prevention and therapy. A chimeric HPV-16 E7 DNA vaccine (SP-SA-E7-4-1BBL) was constructed that contains the signal peptide (SP) of calreticulin (CRT), streptavidin (SA) domain of SA-4-1BBL, HPV-16 E7 double mutant gene, and the extracellular domain of mouse 4-1BBL. Immunization by gene gun with SP-SA-E7-4-1BBL induced greater prophylactic as well as therapeutic effects in C57BL/6 mice against TC-1 tumor model compared with immunization with E7wt, SP-SA-4-1BBL or reference-positive control CRT-E7wt. The therapeutic efficacy of the DNA vaccine was associated with increased frequency of E7-specific T cells producing interferon (IFN)-γ. Overall, our data suggest that this DNA-based vaccine strategy might represent a translational approach because it provides a simpler and versatile alternative to a subunit vaccine based on SA-4-1BBL and E7 proteins.

Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by Lactococcus lactis.

BACKGROUND: Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10. RESULTS: The IP-10 coding region was isolated from human cDNA and cloned into an L. lactis expression plasmid under the regulation of the pNis promoter. By fusion to the usp45 secretion signal, IP-10 was addressed out of the cell. Western blot analysis demonstrated that recombinant strains of L. lactis secrete IP-10 into the culture medium. Neither degradation nor incomplete forms of IP-10 were detected in the cell or supernatant fractions of L. lactis. In addition, we demonstrated that the NICE (nisin-controlled gene expression) system was able to express IP-10 "de novo" even two hours after nisin removal. This human IP-10 protein secreted by L. lactis was biological active as demonstrated by Chemotaxis assay over human CD3+T lymphocytes. CONCLUSION: Expression and secretion of mature IP-10 was efficiently achieved by L. lactis forming an effective system to produce IP-10. This recombinant IP-10 is biologically active as demonstrated by its ability to chemoattract human CD3+ T lymphocytes. This strain of recombinant L. lactis represents a potentially useful tool to be used as a live vaccine in vivo.

The role of JNK phosphorylation as a molecular target to enhance adenovirus replication, oncolysis and cancer therapeutic efficacy.

Oncolytic adenoviruses (Ads) are cancer selective tumoricidal agents; however their mechanism of Ad-mediated cancer cell lysis, or oncolysis, remains undefined. This report focuses upon the autophagy mediator c-JUN n-terminal kinase (JNK) and its effects upon Ad oncolysis and replication. Previously, E1b-deleted Ads have been used to treat several hundred cancer patients with limited clinical efficacy. We hypothesize that by studying the potential interactions between E1b and JNK, mechanisms to improve oncolytic Ad design and cancer therapeutic efficacy may be elucidated. To test this hypothesis, E1b was selectively deleted from the Ad genome. These studies indicated that Ads encoding E1b induced JNK phosphorylation predominately occurred via E1b-19K. The expression of another crucial Ad gene E1a was then overexpressed by the CMV promoter via the replication competent Ad vector Adhz69; these data indicated that E1A also induced JNK phosphorylation. To assess the effects of host cell JNK expression upon Ad oncolysis and replication, siRNA targeting JNK1 and JNK2 (JNK1/2) were utilized. The oncolysis and replication of the E1b-19K wild-type Ads Ad5 and Adhz63 were significantly attenuated following JNK1/2 siRNA transfection. However the oncolytic effects and replication of the E1b-19K deleted Ad Adhz60 were not altered by JNK1/2 siRNA transfection, further implicating the crucial role of E1b-19K for Ad oncolysis and replication via JNK phosphorylation. This study has demonstrated for the first time that JNK is an intriguing molecular marker associated with enhanced Ad virotherapy efficacy, influencing future Ad vector design.