Reporter gene-based optoacoustic imaging of E. coli targeted colon cancer in vivo.

Bacteria-mediated cancer-targeted therapy is a novel experimental strategy for the treatment of cancers. Bacteria can be engineered to overcome a major challenge of existing therapeutics by differentiating between malignant and healthy tissue. A prerequisite for further development and study of engineered bacteria is a suitable imaging concept which allows bacterial visualization in tissue and monitoring bacterial targeting and proliferation. Optoacoustics (OA) is an evolving technology allowing whole-tumor imaging and thereby direct observation of bacterial colonization in tumor regions. However, bacterial detection using OA is currently hampered by the lack of endogenous contrast or suitable transgene fluorescent labels. Here, we demonstrate improved visualization of cancer-targeting bacteria using OA imaging and E. coli engineered to express tyrosinase, which uses L-tyrosine as the substrate to produce the strong optoacoustic probe melanin in the tumor microenvironment. Tumors of animals injected with tyrosinase-expressing E. coli showed strong melanin signals, allowing to resolve bacterial growth in the tumor over time using multispectral OA tomography (MSOT). MSOT imaging of melanin accumulation in tumors was confirmed by melanin and E. coli staining. Our results demonstrate that using tyrosinase-expressing E. coli enables non-invasive, longitudinal monitoring of bacterial targeting and proliferation in cancer using MSOT.

The Role of microRNAs in Epithelial Ovarian Cancer Metastasis.

Epithelial ovarian cancer (EOC) is the deadliest gynecological cancer, and the major cause of death is mainly attributed to metastasis. MicroRNAs (miRNAs) are a group of small non-coding RNAs that exert important regulatory functions in many biological processes through their effects on regulating gene expression. In most cases, miRNAs interact with the 3' UTRs of target mRNAs to induce their degradation and suppress their translation. Aberrant expression of miRNAs has been detected in EOC tumors and/or the biological fluids of EOC patients. Such dysregulation occurs as the result of alterations in DNA copy numbers, epigenetic regulation, and miRNA biogenesis. Many studies have demonstrated that miRNAs can promote or suppress events related to EOC metastasis, such as cell migration, invasion, epithelial-to-mesenchymal transition, and interaction with the tumor microenvironment. In this review, we provide a brief overview of miRNA biogenesis and highlight some key events and regulations related to EOC metastasis. We summarize current knowledge on how miRNAs are dysregulated, focusing on those that have been reported to regulate metastasis. Furthermore, we discuss the role of miRNAs in promoting and inhibiting EOC metastasis. Finally, we point out some limitations of current findings and suggest future research directions in the field.

Imaging of tumor colonization by Escherichia coli using 18F-FDS PET.

Tumor-targeting bacteria have been actively investigated as a new therapeutic tool for solid tumors. However, in vivo imaging of tumor-targeting bacteria has not been fully established. 18F-fluorodeoxysorbitol (FDS) positron emission tomography (PET) is known to be capable of imaging Gram-negative Enterobacteriaceae infection. In the present study, we aimed to validate the use of 18F-FDS PET for visualization of the colonization and proliferation of tumor-targeting Escherichia coli (E. coli) MG1655 in mouse tumor models. Methods:E. coli (5 × 107 colony forming unit) were injected intravenously into BALB/c mice bearing mouse colon cancer (CT26). Before and 1, 3, and 5 days after the bacterial injection, PET imaging was performed following i.v. injection of approximately 7.4 MBq of 18F-FDS. Regions of interest were drawn in the engrafted tumor and normal organs including the heart, liver, lung, brain, muscle, and intestine. Semiquantitative analysis was performed using maximum standardized uptake value (SUVmax). Results:18F-FDS uptake was significantly higher in tumors colonized by live E. coli MG1655 than in uncolonized tumors (p < 0.001). The PET signals in the colonized tumors at 3 days after bacterial injection were 3.1-fold higher than those in the uncolonized tumors. Tumoral 18F-FDS uptake correlated very strongly with the number of E. coli in tumors (r = 0.823, p < 0.0001). Cross sectional analysis of autoradiography, bioluminescence, and pathology revealed that the 18F-FDS uptake sites in tumors matched the locations of E. coli MG1655. Conclusion: In conclusion, 18F-FDS PET is expected to be useful for the semiquantitative visualization of tumor-targeting bacteria when bacterial cancer therapy is performed using Gram-negative Enterobacteriaceae such as E. coli.

Wnt/ß-catenin signalling in ovarian cancer: Insights into its hyperactivation and function in tumorigenesis.

Epithelial ovarian cancer (EOC) is the deadliest female malignancy. The Wnt/ß-catenin pathway plays critical roles in regulating embryonic development and physiological processes. This pathway is tightly regulated to ensure its proper activity. In the absence of Wnt ligands, ß-catenin is degraded by a destruction complex. When the pathway is stimulated by a Wnt ligand, ß-catenin dissociates from the destruction complex and translocates into the nucleus where it interacts with TCF/LEF transcription factors to regulate target gene expression. Aberrant activation of this pathway, which leads to the hyperactivity of ß-catenin, has been reported in ovarian cancer. Specifically, mutations of CTNNB1, AXIN, or APC, have been observed in the endometrioid and mucinous subtypes of EOC. In addition, upregulation of the ligands, abnormal activation of the receptors or intracellular mediators, disruption of the ß-catenin destruction complex, inhibition of the association of ß-catenin/E-cadherin on the cell membrane, and aberrant promotion of the ß-catenin/TCF transcriptional activity, have all been reported in EOC, especially in the high grade serous subtype. Furthermore, several non-coding RNAs have been shown to regulate EOC development, in part, through the modulation of Wnt/ß-catenin signalling. The Wnt/ß-catenin pathway has been reported to promote cancer stem cell self-renewal, metastasis, and chemoresistance in all subtypes of EOC. Emerging evidence also suggests that the pathway induces ovarian tumor angiogenesis and immune evasion. Taken together, these studies demonstrate that the Wnt/ß-catenin pathway plays critical roles in EOC development and is a strong candidate for the development of targeted therapies.

Salmonella-Mediated Cancer Therapy: Roles and Potential.

The use of bacteria for cancer therapy, which was proposed many years ago, was not recognized as a potential therapeutic strategy until recently. Technological advances and updated knowledge have enabled the genetic engineering of bacteria for their safe and effective application in the treatment of cancer. The efficacy of radiotherapy depends mainly on tissue oxygen levels, and low oxygen concentrations in necrotic and hypoxic regions are a common cause of treatment failure. In addition, the distribution of a drug is important for the therapeutic effect of chemotherapy, and the poor vasculature in tumors impairs drug delivery, limiting the efficacy of a drug, especially in necrotic and hypoxic regions. Bacteria-mediated cancer therapy (BMCT) relies on facultative anaerobes that can survive in well or poorly oxygenated regions, and it therefore improves the therapeutic efficacy drug distribution throughout the tumor mass. Since the mid-1990s, the number of published bacterial therapy papers has increased rapidly, with a doubling time of 2.5 years in which the use of Salmonella increased significantly. BMCT is being reevaluated to overcome some of the drawbacks of conventional therapies. This review focuses on Salmonella-mediated cancer therapy as the most widely used type of BMCT.2.

RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic Efficacy of Attenuated Salmonella-mediated Cancer Therapy.

Bacteria-based anticancer therapies aim to overcome the limitations of current cancer therapy by actively targeting and efficiently removing cancer. To achieve this goal, new approaches that target and maintain bacterial drugs at sufficient concentrations during the therapeutic window are essential. Here, we examined the tumor tropism of attenuated Salmonella typhimurium displaying the RGD peptide sequence (ACDCRGDCFCG) on the external loop of outer membrane protein A (OmpA). RGD-displaying Salmonella strongly bound to cancer cells overexpressing αvß3, but weakly bound to αvß3-negative cancer cells, suggesting the feasibility of displaying a preferential homing peptide on the bacterial surface. In vivo studies revealed that RGD-displaying Salmonellae showed strong targeting efficiency, resulting in the regression in αvß3-overexpressing cancer xenografts, and prolonged survival of mouse models of human breast cancer (MDA-MB-231) and human melanoma (MDA-MB-435). Thus, surface engineering of Salmonellae to display RGD peptides increases both their targeting efficiency and therapeutic effect.

Activation of inflammasome by attenuated Salmonella typhimurium in bacteria-mediated cancer therapy.

Escherichia coli and attenuated Salmonella both naturally accumulate in a tumor mass, yet have distinct therapeutic efficacy: the E. coli K-12 strain (MG1655) cannot induce as significant a tumor suppression as attenuated Salmonella typhimurium, despite similar levels of accumulation in the tumor. To elucidate the mechanism of the robust antitumor effect of S. typhimurium, the cytokine profiles elicited by bacterial colonization in tumors were analyzed. C57BL/6 mice bearing MC38 tumors were injected with Salmonella or MG1655 in the tail vein. Tumors were collected 3 days post-infection and homogenized. Inflammasome-related signals were measured by real-time PCR, ELISA and western blot analysis. Only attenuated Salmonella triggered significant levels of the inflammatory cytokine IL-1ß in the tumor, whereas tumor growth was significantly suppressed. In addition, transcript levels of the core molecules of inflammasome signaling, IPAF, NLRP3 and P2X7, were significantly elevated only in Salmonella-treated tumors. Upon direct interaction between Salmonella and BMDM, BMDM expressed inflammasome-related proteins such as NLRP3, IPAF and caspase-1 p10, and secreted a significant amount of IL-1ß in supernatants. Coincubation assays with BMDM and Salmonella-treated MC38 cells (damaged cancer cells) revealed secretion of IL-1ß only when TLR4 and inflammasome were activated by both LPS and damaged cancer cells. ATP released from damaged cancer cells was also identified as a mechanism of NLRP3 activation. In conclusion, Salmonella activate the inflammasome pathway using damage signals released from cancer cells and through direct interaction with macrophages.

Salmonella typhimurium Suppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1ß.

Although strains of attenuated Salmonella typhimurium and wild-type Escherichia coli show similar tumor-targeting capacities, only S. typhimurium significantly suppresses tumor growth in mice. The aim of the present study was to examine bacteria-mediated immune responses by conducting comparative analyses of the cytokine profiles and immune cell populations within tumor tissues colonized by E. coli or attenuated Salmonellae. CT26 tumor-bearing mice were treated with two different bacterial strains: S. typhimurium defective in ppGpp synthesis (ΔppGpp Salmonellae) or wild-type E. coli MG1655. Cytokine profiles and immune cell populations in tumor tissue colonized by these two bacterial strains were examined at two time points based on the pattern of tumor growth after ΔppGpp Salmonellae treatment: 1) when tumor growth was suppressed ('suppression stage') and 2) when they began to re-grow ('re-growing stage'). The levels of IL-1ß and TNF-α were markedly increased in tumors colonized by ΔppGpp Salmonellae. This increase was associated with tumor regression; the levels of both IL-1ß and TNF-α returned to normal level when the tumors started to re-grow. To identify the immune cells primarily responsible for Salmonellae-mediated tumor suppression, we examined the major cell types that produce IL-1ß and TNF-α. We found that macrophages and dendritic cells were the main producers of TNF-α and IL-1ß. Inhibiting IL-1ß production in Salmonellae-treated mice restored tumor growth, whereas tumor growth was suppressed for longer by local administration of recombinant IL-1ß or TNF-α in conjunction with Salmonella therapy. These findings suggested that IL-1ß and TNF-α play important roles in Salmonella-mediated cancer therapy. A better understanding of host immune responses in Salmonella therapy may increase the success of a given drug, particularly when various strategies are combined with bacteriotherapy.

Regulation of acetylation of histone deacetylase 2 by p300/CBP-associated factor/histone deacetylase 5 in the development of cardiac hypertrophy.

RATIONALE: Histone deacetylases (HDACs) are closely involved in cardiac reprogramming. Although the functional roles of class I and class IIa HDACs are well established, the significance of interclass crosstalk in the development of cardiac hypertrophy remains unclear. OBJECTIVE: Recently, we suggested that casein kinase 2α1-dependent phosphorylation of HDAC2 leads to enzymatic activation, which in turn induces cardiac hypertrophy. Here we report an alternative post-translational activation mechanism of HDAC2 that involves acetylation of HDAC2 mediated by p300/CBP-associated factor/HDAC5. METHODS AND RESULTS: Hdac2 was acetylated in response to hypertrophic stresses in both cardiomyocytes and a mouse model. Acetylation was reduced by a histone acetyltransferase inhibitor but was increased by a nonspecific HDAC inhibitor. The enzymatic activity of Hdac2 was positively correlated with its acetylation status. p300/CBP-associated factor bound to Hdac2 and induced acetylation. The HDAC2 K75 residue was responsible for hypertrophic stress-induced acetylation. The acetylation-resistant Hdac2 K75R showed a significant decrease in phosphorylation on S394, which led to the loss of intrinsic activity. Hdac5, one of class IIa HDACs, directly deacetylated Hdac2. Acetylation of Hdac2 was increased in Hdac5-null mice. When an acetylation-mimicking mutant of Hdac2 was infected into cardiomyocytes, the antihypertrophic effect of either nuclear tethering of Hdac5 with leptomycin B or Hdac5 overexpression was reduced. CONCLUSIONS: Taken together, our results suggest a novel mechanism by which the balance of HDAC2 acetylation is regulated by p300/CBP-associated factor and HDAC5 in the development of cardiac hypertrophy.

Effect of Salmonella treatment on an implanted tumor (CT26) in a mouse model.

The use of bacteria has contributed to recent advances in targeted cancer therapy especially for its tumor-specific accumulation and proliferation. In this study, we investigated the molecular events following bacterial therapy using an attenuated Salmonella Typhimurium defective in ppGpp synthesis (ΔppGpp), by analyzing those proteins differentially expressed in tumor tissues from treated and untreated mice. CT26 murine colon cancer cells were implanted in BALB/c mice and allowed to form tumors. The tumor-bearing mice were treated with the attenuated Salmonella Typhimurium. Tumor tissues were analyzed by 2D-PAGE. Fourteen differentially expressed proteins were identified by mass spectrometry. The analysis revealed that cytoskeletal components, including vimentin, drebrin-like protein, and tropomyosin-alpha 3, were decreased while serum proteins related to heme or iron metabolism, including transferrin, hemopexin, and haptoglobin were increased. Subsequent studies revealed that the decrease in cytoskeletal components occurred at the transcriptional level and that the increase in heme and iron metabolism proteins occurred in liver. Most interestingly, the same pattern of increased expression of transferrin, hemopexin, and haptoglobin was observed following radiotherapy at the dosage of 14 Gy.