Cancer is one of the most dreaded diseases in humans and most common cause of death in twenty-first century. New cancer therapies are urgently required because of the existing pharmacological side effects of the conventional chemotherapy, radiation, or surgery. Newer modalities such as cancer vaccines and biological therapies are proving very helpful in the treatment of cancer along with the conventional therapies. The success of these novel cancer therapies is attributed to their lesser toxicity and the specific killing of the cancer cells. Bacterial therapy for cancer has been recognized a century ago. Live, attenuated, or genetically modified obligate or facultative anaerobic bacterial species exhibit the inherent property of colonizing the tumors and are capable of multiplying selectively inside the tumors, thereby inhibiting cancerous growths. The bacteria and their spores are used in the target specific therapies, delivering the prodrugs and the various proteins to the tumors. Albeit bacterial treatment of cancer is providing new perspective in the treatment of disease, the use of microorganisms to target tumors has certain confinements. The biosafety, genetic instability and the confounded interaction of the bacteria with treatment drugs, requires the more noteworthy consideration regarding the use of this novel treatment in the cancer treatment.

For suicide gene therapy, initially prodrug-converting enzymes (gene-directed enzyme-producing therapy, GDEPT) were employed to intracellularly metabolize non-toxic prodrugs into toxic compounds, leading to the effective suicidal killing of the transfected tumor cells. In this regard, the suicide gene therapy has demonstrated its potential for efficient tumor eradication. Numerous suicide genes of viral or bacterial origin were isolated, characterized, and extensively tested in vitro and in vivo, demonstrating their therapeutic potential even in clinical trials to treat cancers of different entities. Apart from this, growing efforts are made to generate more targeted and more effective suicide gene systems for cancer gene therapy. In this regard, bacterial toxins are an alternative to the classical GDEPT strategy, which add to the broad spectrum of different suicide approaches. In this context, lytic bacterial toxins, such as streptolysin O (SLO) or the claudin-targeted Clostridium perfringens enterotoxin (CPE) represent attractive new types of suicide oncoleaking genes. They permit as pore-forming proteins rapid and also selective toxicity toward a broad range of cancers. In this chapter, we describe the generation and use of SLO as well as of CPE-based gene therapies for the effective tumor cell eradication as promising, novel suicide gene approach particularly for treatment of therapy refractory tumors.

Multidrug resistance (MDR) to pharmaceutical active agents is a common clinical problem in patients suffering from cancer. MDR is often mediated by over expression of trans-membrane xenobiotic transport molecules belonging to the superfamily of ATP-binding cassette (ABC)-transporters. This protein family includes the classical MDR-associated transporter ABCB1 (MDR1/P-gp). Inhibition of ABC-transporters by low molecular weight compounds in cancer patients has been extensively investigated in clinical trials, but the results have been disappointing. Thus, in the last decades alternative experimental therapeutic strategies for overcoming MDR were under extensive investigation. These include gene therapeutic approaches applying antisense-, ribozyme-, RNA interference-, and CRISPR/Cas9-based techniques. Various delivery strategies were used to reverse MDR in different tumor models in vitro and in vivo. Results and conclusions of these gene therapeutic studies will be discussed.

mRNA cancer vaccines are a relatively new class of vaccines, which combine the potential of mRNA to encode for almost any protein with an excellent safety profile and a flexible production process. The most straightforward use of mRNA vaccines in oncologic settings is the immunization of patients with mRNA vaccines encoding tumor-associated antigens (TAAs). This is exemplified by the RNActive® technology, which induces balanced humoral and cellular immune responses in animal models and is currently evaluated in several clinical trials for oncologic indications. A second application of mRNA vaccines is the production of personalized vaccines. This is possible because mRNA vaccines are produced by a generic process, which can be used to quickly produce mRNA vaccines targeting patient-specific neoantigens that are identified by analyzing the tumor exome. Apart from being used directly to vaccinate patients, mRNAs can also be used in cellular therapies to transfect patient-derived cells in vitro and infuse the manipulated cells back into the patient. One such application is the transfection of patient-derived dendritic cells (DCs) with mRNAs encoding TAAs, which leads to the presentation of TAA-derived peptides on the DCs and an activation of antigen-specific T cells in vivo. A second application is the transfection of patient-derived T cells with mRNAs encoding chimeric antigen receptors, which allows the T cells to directly recognize a specific antigen expressed on the tumor. In this chapter, we will review preclinical and clinical data for the different approaches.

Gene therapy is a prospective strategy to modulate gene expression level in specific cells to treat human inherited diseases, cancers, and acquired disorders. A subset of noncoding RNAs, microRNAs (miRNAs) and small interference RNAs (siRNAs), compose an important class of widely used effectors for gene therapy, especially in cancer treatment. Functioning through the RNA interference (RNAi) mechanism, miRNA and siRNA show potent ability in silencing oncogenic factors for cancer gene therapy. For a better understanding of this field, we reviewed the mechanism and biological function, the principles of design and synthesis, and the delivery strategies of noncoding RNAs with clinical potentials in cancer gene therapy.

Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an important goal in vector development was to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle (MC) DNA. The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform enabling a close-to-random profile of genomic integration. In combination, the MC platform greatly enhances SB transposition and transgene integration resulting in higher numbers of stably modified target cells. We have recently developed a strategy for MC-based SB transposition of chimeric antigen receptor (CAR) transgenes that enable improved transposition rates compared to conventional plasmids and rapid manufacturing of therapeutic CAR T cell doses (Monjezi et al. 2016). This advance enables manufacturing CAR T cells in a virus-free process that relies on SB-mediated transposition from MC DNA to accomplish gene-transfer. Advantages of this approach include a strong safety profile due to the nature of the MC itself and the genomic insertion pattern of MC-derived CAR transposons. In addition, stable transposition and high-level CAR transgene expression, as well as easy and reproducible handling, make MCs a preferred vector source for gene-transfer in advanced cellular and gene therapy. In this chapter, we will review our experience in MC-based CAR T cell engineering and discuss our recent advances in MC manufacturing to accelerate both pre-clinical and clinical implementation.

Advances in molecular technologies have led to the discovery of many disease-related genetic mutations as well as elucidation of aberrant gene and protein expression patterns in several human diseases, including cancer. This information has driven the development of novel therapeutic strategies, such as the utilization of small molecules to target specific cellular pathways and the use of retroviral vectors to retarget immune cells to recognize and eliminate tumor cells. Retroviral-mediated gene transfer has allowed efficient production of T cells engineered with chimeric antigen receptors (CARs), which have demonstrated marked success in the treatment of hematological malignancies. As a safety point, these modified cells can be outfitted with suicide genes. Customized gene editing tools, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated nucleases (CRISPR-Cas9), zinc-finger nucleases (ZFNs), or TAL-effector nucleases (TALENs), may also be combined with retroviral delivery to specifically delete oncogenes, inactivate oncogenic signaling pathways, or deliver wild-type genes. Additionally, the feasibility of retroviral gene transfer strategies to protect the hematopoietic stem cells (HSC) from the dose-limiting toxic effects of chemotherapy and radiotherapy was demonstrated. While some of these approaches have yet to be translated into clinical application, the potential implications for improved cellular replacement therapies to enhance and/or support the current treatment modalities are enormous.

Tumor suppressor gene (TSG) replacement therapy that involves various delivery systems is emerging as a promising antitumor strategy because malignant tumors develop through genetic alterations in TSGs. The most potent therapeutic TSG for tumor suppression is the multifunctional transcription factor p53 gene that regulates diverse cellular phenomena such as cell cycle arrest, senescence, apoptosis, and autophagy. Since the p53 gene is frequently inactivated by aberrant genetic regulation in human cancers, p53 replacement therapy is widely and frequently used as a potent antitumor strategy to restore wild-type p53 function in the p53-inactivated tumors. This chapter focuses on four types of p53 transfer systems: cationic liposome-DNA plasmid complexes, a replication-deficient adenovirus vector, a replication-competent adenovirus vector, and a protein transduction system. Moreover, we discuss recent advances in our understanding of the molecular basis of the p53-mediated cell death signaling pathway and therapeutic methods for enhancing tumor cell death and induction of bystander effects within tumor tissues in p53 replacement therapy. Exploration of the molecular mechanism underlying the p53-mediated tumor-suppressive network system and development of an effective strategy for enhancing p53-mediated cell death signaling pathways would lead to an improvement in the clinical outcome of patients with p53-inactivated cancers.

B-cell acute lymphoblastic leukemia (B‑ALL) is an aggressive hematological malignancy and a leading cause of cancer-related mortality in children and young adults. The molecular mechanisms involved in the regulation of its gene expression has yet to be fully elucidated. In the present study, we performed large scale expression profiling of microRNA (miRNA) and transcription factor (TF) by Illumina deep‑sequencing and TF array technology, respectively, and identified 291 differentially expressed miRNAs and 201 differentially expressed TFs in adult B‑ALL samples relative to their controls. After integrating expression profile data with computational prediction of miRNA and TF targets from different databases, we construct a comprehensive miRNA‑TF regulatory network specifically for adult B‑ALL. Network function analysis revealed 25 significantly enriched pathways, four pathways are well‑known to be involved in B‑ALL, such as PI3K‑Akt signaling pathway, Jak‑STAT signaling pathway, Ras signaling pathway and cell cycle pathway. By analyzing the network topology, we identified 28 hub miRNAs and 19 hub TFs in the network, and found nine potential B‑ALL regulators among these hub nodes. We also constructed a Jak‑STAT signaling sub‑network for B‑ALL. Based on the sub‑network analysis and literature survey, we proposed a cellular model to discuss MYC/miR‑15a‑5p/FLT3 feed-forward loop (FFL) with Jak‑STAT signaling pathway in B‑ALL. These findings enhance our understanding of this disease at the molecular level, as well as provide putative therapeutic targets for B-ALL.

Aurora kinase B, playing a vital, important role in mitosis, is frequently detected to be overexpressed in many cancer cell lines and various tumor tissues, including prostatic carcinoma. Given the essential function of Aurora kinase B in mitosis and its association with tumorigenesis, it might be a drug target for prostatic carcinoma treatment. In our study, short hairpin RNA targeting Aurora kinase B was cloned into a pGPU6 plasmid vector and then transfected into human prostatic carcinoma cells. The expression level of Aurora kinase B was verified by reverse transcription-polymerase chain reaction and Western blot. At the same time, cell apoptosis was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, fluorescent staining, and flow cytometric analysis. Furthermore, prostate carcinoma cells were injected into mice to establish a tumor xenograft model. Previous studies have shown the effect of pGPU6-shAURKB plasmid on tumor growth in a prostate carcinoma xenogenic implantation model. From the study, we knew that the Aurora kinase B was significantly downregulated in prostate carcinoma cells, and cell apoptosis was also detected higher in treated groups than that in control groups. Moreover, in the prostate carcinoma xenogenic implantation model, compared with the control groups, the tumor growth was inhibited about 78.7% in the pGPU6-shAURKB plasmid-treated group, and cell apoptosis in the experimental group was notably higher than that in control groups. The average duration of tumor-bearing mice was prolonged to about 35 days. The results of experiment indicated that specific knockdown of Aurora kinase B led to prostate carcinoma cells apoptosis and inhibited tumor growth. Our data clearly confirmed that specific knockdown of Aurora kinase B expression by vector-based short hairpin RNA/liposome may be a potential new approach to treat human prostatic carcinoma.

Kaposiform hemangioendothelioma is a rare, intermediate, malignant tumor. The tumor's etiology remains unknown and there are no specific treatments.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite the anticancer capabilities of emodin observed in many cancers, including EOC, the underlying molecular mechanism remains to be elucidated. A crucial link has been discovered between the acquisition of metastatic traits and the epithelial-mesenchymal transition (EMT). The present study aimed to determine whether emodin could inhibit the EMT of EOC cells and explore the underlying mechanism. The CCK-8 assay and transwell assay showed that emodin effectively repressed the abilities of proliferation, invasion, and migration in A2780 and SK-OV-3 cells. The Western blot showed that emodin upregulated epithelial markers (E-cadherin and Claudin) while it downregulated mesenchymal markers (N-cadherin and Vimentin) and transcription factor (Slug) in a dose-dependent fashion. After transfection of siRNA-Slug, both Slug and N-cadherin were downregulated in EOC cells while E-cadherin was upregulated, which was intensified by emodin. Besides, emodin decreased the expression of ILK, p-GSK-3β, β-catenin, and Slug. Transfection of siRNA-ILK also achieved the same effects, which was further strengthened by following emodin treatment. Nevertheless, SB216763, an inhibitor of GSK-3β, could reverse the effects of emodin except for ILK expression. These findings suggest that emodin inhibited the EMT of EOC cells via ILK/GSK-3β/Slug signaling pathway.

Tumors are a serious threat to human health. The oncolytic virus is a kind of tumor killer virus which can infect and lyse cancer cells and spread through the tumor, while leaving normal cells largely unharmed. Mathematical models can help us to understand the tumor-virus dynamics and find better treatment strategies. This paper gives a new mathematical model of tumor therapy with oncolytic virus and MEK inhibitor. Stable analysis was given. Because mitogen-activated protein kinase (MEK) can not only lead to greater oncolytic virus infection into cancer cells, but also limit the replication of the virus, in order to provide the best dosage of MEK inhibitors and balance the positive and negative effect of the inhibitors, we put forward an optimal control problem of the inhibitor. The optimal strategies are given by theory and simulation.

Human papillomavirus (HPV) infection is an important etiologic factor in cervical carcinogenesis. Various HPV DNA detection methods have been evaluated for clinicopathological level. For the specimens with normal cytological finding, discrepancies among the detection methods were frequently found and adequate interpretation can be difficult. 6,322 clinical specimens were submitted and evaluated for real-time PCR and Hybrid Capture 2 (HC2). 573 positive or "Not Detected but Amplified" (NDBA) specimens by real-time PCR were additionally tested using genetic analyzer. For the reliability of real-time PCR, 325 retests were performed. Optimal cut-off cycle threshold (CT ) value was evaluated also. 78.7% of submitted specimens showed normal or nonspecific cytological finding. The distributions of HPV types by real-time PCR were not different between positive and NDBA cases. For positive cases by fragment analysis, concordance rates with real-time PCR and HC2 were 94.2% and 84.2%. In NDBA cases, fragment analysis and real-time PCR showed identical results in 77.0% and HC2 revealed 27.6% of concordance with fragment analysis. Optimal cut-off CT value was different for HPV types. NDBA results in real-time PCR should be regarded as equivocal, not negative. The adjustment of cut-off CT value for HPV types will be helpful for the appropriate result interpretation.

Overcoming temozolomide (TMZ) resistance is a great challenge in glioblastoma (GBM) treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide and has a crucial role in cancer cell metabolism. In this study, we investigated whether FK866 and CHS828, two specific NAMPT inhibitors, could sensitize GBM cells to TMZ. Low doses of FK866 and CHS828 (5 nM and 10 nM, resp.) alone did not significantly decrease cell viability in U251-MG and T98 GBM cells. However, they significantly increased the antitumor action of TMZ in these cells. In U251-MG cells, administration of NAMPT inhibitors increased the TMZ (100 μM)-induced apoptosis and LDH release from GBM cells. NAMPT inhibitors remarkably enhanced the activities of caspase-1, caspase-3, and caspase-9. Moreover, NAMPT inhibitors increased reactive oxygen species (ROS) production and superoxide anion level but reduced the SOD activity and total antioxidative capacity in GBM cells. Treatment of NAMPT inhibitors increased phosphorylation of c-Jun and JNK. Administration of JNK inhibitor SP600125 or ROS scavenger tocopherol with TMZ and NAMPT inhibitors substantially attenuated the sensitization of NAMPT inhibitor on TMZ antitumor action. Our data indicate a potential value of NAMPT inhibitors in combined use with TMZ for GBM treatment.

Purpose Imatinib (IM) can safely be discontinued in patients with chronic myeloid leukemia (CML) who have had undetectable minimal residual disease (UMRD) for at least 2 years. We report the final results of the Stop Imatinib (STIM1) study with a long follow-up. Patients and Methods IM was prospectively discontinued in 100 patients with CML with UMRD sustained for at least 2 years. Molecular recurrence (MR) was defined as positivity of BCR-ABL transcript in a quantitative reverse transcriptase polymerase chain reaction assay confirmed by a second analysis point that indicated an increase of one log in relation to the first analysis point at two successive assessments or loss of major molecular response at one point. Results The median molecular follow-up after treatment discontinuation was 77 months (range, 9 to 95 months). Sixty-one patients lost UMRD after a median of 2.5 months (range, 1 to 22 months), and one patient died with UMRD at 10 months. Molecular recurrence-free survival was 43% (95% CI, 33% to 52%) at 6 months and 38% (95% CI, 29% to 47%) at 60 months. Treatment was restarted in 57 of 61 patients with MR, and 55 patients achieved a second UMRD with a median time of 4 months (range, 1 to 16 months). None of the patients experienced a CML progression. Analyses of the characteristics of the study population identified that the Sokal risk score and duration of IM treatment were significantly associated with the probability of MR. Conclusion With a median follow-up of more than 6 years after treatment discontinuation, the STIM1 study demonstrates that IM can safely be discontinued in patients with a sustained deep molecular response with no late MR.

This pilot feasibility clinical trial evaluated the coadministration of vemurafenib, a small-molecule antagonist of BRAFV600 mutations, and tumor-infiltrating lymphocytes (TIL) for the treatment of metastatic melanoma.

Worldwide, cancers of the oral cavity and pharynx comprise the sixth most common malignancies. Histologically, more than 90% of oral cancers are squamous cell carcinoma (SCC). Epidemiologic data strongly support the role of exogenous factors such as tobacco, alcohol, and human papilloma virus infection as major causative agents. Avoidance of risk factors has only been partially successful, and survival rates have not improved despite advances in therapeutic approaches. Therefore, new or improved approaches to prevention and/or early detection are critical. Better understanding of the mechanisms of oral carcinogenesis can assist in the development of novel biomarkers for early detection and strategies for disease prevention. Toward this goal, several animal models for carcinogenesis in the oral cavity have been developed. Among these are xenograft, and transgenic animal models, and others employing the synthetic carcinogens such as 7,12-dimethylbenz[a]anthracene in hamster cheek pouch and 4-nitroquinoline-N-oxide in rats and mice. Additional animal models employing environmental carcinogens such as benzo[a]pyrene and N'-nitrosonornicotine have been reported. Each model has certain advantages and disadvantages. Models that (1) utilize environmental carcinogens, (2) reflect tumor heterogeneity, and (3) accurately represent the cellular and molecular changes involved in the initiation and progression of oral cancer in humans could provide a realistic platform. To achieve this goal, we introduced a novel nonsurgical mouse model to study oral carcinogenesis induced by dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and tobacco smoke constituent, and its diol epoxide metabolite (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene [(±)-anti-DB[a,l]PDE]. On the basis of a detailed comparison of oral cancer induced by DB[a,l]P with that induced by the other above-mentioned oral carcinogens with respect to dose, duration, species and strain, cellular and molecular targets, and relative carcinogenic potency, our animal model may offer a more realistic platform to study oral carcinogenesis. In this perspective, we also discuss our preclinical studies to demonstrate the potential of black raspberry extracts on the prevention of OSCC. Specifically, we were the first to demonstrate that black raspberry inhibited DB[a,l]P-DNA binding and of particular importance its capacity to enhance the repair of DB[a,l]P-induced bulky lesions in DNA. We believe that the information presented in this perspective will stimulate further research on the impact of environmental carcinogens in the development of oral cancer and may lead to novel strategies toward the control and prevention of this disease.

Nicotine is the primary addictive agent in tobacco, and P450 2A6 (gene name: CYP2A6) is the primary catalyst of nicotine metabolism. It was proposed more than 20 years ago that individuals who metabolize nicotine poorly would smoke less, either fewer cigarettes per day or less intensely per cigarette, compared to smokers who metabolize nicotine more efficiently. These poor metabolizers would then be less likely to develop lung cancer due to their lower exposure to the many carcinogens delivered with nicotine in each puff of smoke. Numerous studies have reported that smokers who carry reduced activity or null CYP2A6 alleles do smoke less. Yet only in Asian populations, both Japanese and Chinese, which have a high prevalence of genetic variants, has a link between CYP2A6, smoking dose, and lung cancer been established. In other ethnic groups, it has been challenging to confirm a direct link between P450 2A6-mediated nicotine metabolism and the risk of lung cancer. This challenge is due in part to the difficulty in accurately quantifying smoking dose and accurately predicting or measuring P450 2A6-mediated nicotine metabolism. Biomarkers of nicotine metabolism and smoking exposure, including the ratio of trans-3-hydroxycotine to cotinine, a measure of P450 2A6 activity and plasma cotinine, or urinary total nicotine equivalents (the sum of nicotine and six metabolites) as measures of exposure are useful for addressing this challenge. However, to take full advantage of these biomarkers in the study of ethnic/racial differences in the risk of lung cancer requires the complete characterization of nicotine metabolism across ethnic/racial groups. Variation in metabolism pathways, other than those catalyzed by P450 2A6, can impact biomarkers of both nicotine metabolism and dose. This is clearly important for smokers with low levels of UGT2B10-catalyzed nicotine and cotinine glucuronidation because the UGT2B10 genotype influences plasma cotinine levels. Cotinine is not glucuronidated in 15% of African American smokers (compared to 1% of Whites) due to the prevalence of a UGT2B10 splice variant. This variant contributes significantly to the higher plasma cotinine levels per cigarette in this group and may also influence the accuracy of the 3HCOT to cotinine ratio as a measure of P450 2A6 activity.