Abstract Background: Parasitic flatworms of the Schistosoma genus cause schistosomiasis, which affects over 230 million people. Schistosoma haematobium causes the urogenital form of schistosomiasis (UGS), which can lead to hematuria, fibrosis, and increased risk of secondary infections by bacteria or viruses. UGS is also linked to bladder cancer. To understand the bladder pathology during S. haematobium infection, our group previously developed a mouse model that involves the injection of S. haematobium eggs into the bladder wall. Using this model, we studied changes in epigenetics profile, as well as changes in gene and protein expression in the host bladder tissues. In the current study, we expand upon this work by examining the expression level of both host and parasite genes using RNA sequencing (RNA-seq) in the mouse bladder wall injection model of S. haematobium infection. Methods: We used a mouse model of S. haematobium infection in which parasite eggs or vehicle control were injected into the bladder walls of female BALB/c mice. RNA-seq was performed on the RNA isolated from the bladders four days after bladder wall injection. Results/Conclusions: RNA-seq analysis of egg- and vehicle control-injected bladders revealed the differential expression of 1025 mouse genes in the egg-injected bladders, including genes associated with cellular infiltration, immune cell chemotaxis, cytokine signaling, and inflammation We also observed the upregulation of immune checkpoint-related genes, which suggests that while the infection causes an inflammatory response, it also dampens the response to avoid excessive inflammation-related damage to the host. Identifying these changes in host signaling and immune responses improves our understanding of the infection and how it may contribute to the development of bladder cancer. Analysis of the differential gene expression of the parasite eggs between bladder-injected versus uninjected eggs revealed 119 S. haematobium genes associated with transcription, intracellular signaling, and metabolism. The analysis of the parasite genes also revealed fewer transcript reads compared to that found in the analysis of mouse genes, highlighting the challenges of studying parasite egg biology in the mouse model of S. haematobium infection. Author summary More than 230 million people worldwide are estimated to carry infection with parasites belonging to the Schistosoma genus, which cause morbidity associated with parasite egg deposition. Praziquantel, the drug of choice to treat the infection, does not prevent reinfection, and its decades-long history as the main treatment raises concerns for drug resistance. Of the schistosome species, Schistosoma haematobium causes urogenital disease and has a strong association with bladder cancer. The possibility for drug resistance and the gap in knowledge with respect to the mechanisms driving S. haematobium -related bladder cancer highlight the need to better understand the biology of the infection to aid in the development of new therapeutic strategies. In this study, we used a mouse model of S. haematobium infection that delivers parasite eggs directly to the host mouse bladder wall, and we examined the changes in the gene expression profile of the host and the parasite by RNA-sequencing. The results corroborated previous findings with respect to the host’s inflammatory responses against the parasite eggs, as well as revealed alterations in other immune response genes that deepen our understanding of the mechanisms involved in urogenital schistosomiasis pathogenesis.
Inherently unstable mRNAs contain AU-rich elements (AREs) in the 3′ untranslated regions. Expression of ARE-containing type I interferon transcripts is robustly induced upon viral infection and rapidly shut off thereafter. Their transient accumulation is partly mediated through posttranscriptional regulation. Here we show that mouse embryonic fibroblasts derived from knockout mice deficient in KH-type splicing regulatory protein (KSRP), an RNA-binding protein required for ARE-mediated mRNA decay, produce higher levels of Ifna and Ifnb mRNAs in response to viral infection as a result of decreased mRNA decay. Functional analysis showed that KSRP is required for the decay of Ifna4 and Ifnb mRNAs by interaction with AREs. The increased IFN expression renders Ksrp−/− cells refractory to herpes simplex virus type 1 and vesicular stomatitis virus infection. These findings support a role of a posttranscriptional mechanism in the control of type I IFN expression and highlight the function of KSRP in innate immunity by negatively regulating IFN production.
Abstract: New therapies for metastatic breast cancer patients are urgently needed. The long-term survival rates remain unacceptably low for patients with recurrent disease or disseminated metastases. In addition, existing therapies often cause a variety of debilitating side effects that severely impact quality of life. Oncolytic viruses constitute a developing therapeutic modality in which interest continues to build due to their ability to spare normal tissue while selectively destroying tumor cells. A number of different viruses have been used to develop oncolytic agents for breast cancer, including herpes simplex virus, adenovirus, vaccinia virus, measles virus, reovirus, and others. In general, clinical trials for several cancers have demonstrated excellent safety records and evidence of efficacy. However, the impressive tumor responses often observed in preclinical studies have yet to be realized in the clinic. In order for the promise of oncolytic virotherapy to be fully realized for breast cancer patients, effectiveness must be demonstrated in metastatic disease. This review provides a summary of oncolytic virotherapy strategies being developed to target metastatic breast cancer. Keywords: oncolytic virus, virotherapy, breast cancer, metastasis
Abstract The purpose of this study was to improve the efficacy of oncolytic herpes simplex virus (HSV) against metastatic breast cancer by combining treatment with histone deacetylase (HDAC) inhibitors. Metastatic breast cancer continues to be a significant clinical challenge as the survival rates remain below 25%, illustrating the dire need for newly developed therapeutic strategies. Oncolytic viruses selectively replicate in cancer cells but not in normal cells and are an exciting potential therapy being evaluated in patients with breast cancer. HSV is a well-studied oncolytic virus that has been employed in clinical trials for several cancers. These trials have demonstrated safety, but HSV has generally lacked sufficient potency to yield durable tumor responses. In part, this is due to low viral replication and persistence. For tumor-selective replication, oncolytic HSV (oHSV) typically lack the diploid viral γ134.5 gene, which encodes a multifunctional protein that also contributes to viral evasion of host interferon response. Because γ134.5-deleted HSVs are particularly sensitive to interferon, reducing this response may be an effective means of enhancing oncolytic HSV therapy while maintaining deficiency of replication in normal cells. HDAC inhibitors have been shown to reduce the expression of interferon-stimulated genes. Therefore, we hypothesized that oHSV replication and efficacy could be improved by treatment with particular HDAC inhibitors. We tested a panel of HDAC inhibitors with distinct inhibition profiles and selectivity towards different classes of HDACs. Cell viability assays were used to establish lethal dose 50 (LD50) values for each compound and each was tested for its ability to modulate the replication of a γ134.5-deleted oncolytic HSV. The inhibitors were tested at three different dose levels: low (non-lethal), middle (partially lethal), and high (lethal), and two different treatment schedules: prior to viral infection (pre-treatment) or immediately following viral infection (co-treatment). The cell lines selected for this study included the metastatic breast cancer line MDA-MB-231 that was previously shown to be oHSV replication-competent and the oHSV replication-resistant cell lines: immortalized but otherwise normal breast epithelial cell line MCF10A and metastatic murine mammary carcinoma 4T1. Although pre-treatment did not affect viral replication and co-treatment actually reduced it at a high multiplicity of infection (MOI), at a low MOI, significantly enhanced viral replication was observed with both pre- and co-treatment in MDA-MB-231 cells (magnitude of increase dependent on particular HDAC inhibitor), but not in MCF10A. Interestingly, the oHSV-resistant 4T1 cells were rendered permissive by HDAC-inhibitor treatment as significant increases in oHSV replication were noted. These studies demonstrate that HDAC inhibitors can be used to induce viral replication depending on the particular compound and MOI of the virus. Furthermore, these results suggest that HDAC inhibitors could prove useful in combination with oHSV as a novel treatment strategy for metastatic breast cancer. Citation Format: James J. Cody, James M. Markert, Douglas R. Hurst. Treatment of breast cancer cells with histone deacetylase inhibitors increases the replication of an oncolytic herpes simplex virus. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B54.
Abstract Autophagy is a catabolic process that is tightly regulated during normal cell growth, development, and homeostasis. Although it may be death inducing, it is also an important survival mechanism for cells in stressful environments, including hypoxia, nutrient deprivation, chemical or physical pressure, or detachment from colonies. Considering the stress associated with the process of metastasis, we hypothesized that autophagy may play a role in the ability of breast cancer cells to survive and metastasize. To test this, we first compared cell proliferation after autophagic induction between metastatic breast cancer cell lines (MDA-MB-231 and -435), a non-metastatic breast cancer cell line (MDA-MB-436), and a ‘normal’ breast epithelial cell line (MCF10A). Both metastatic cell lines continued to proliferate following induction of autophagy; however, the non-metastatic and normal cell lines were growth-inhibited. Autophagy was also more rapidly induced by rapamycin in the metastatic cell lines as demonstrated by an increase in LC3II expression. To test the different survival capabilities between metastatic and normal cells, we selected cells through five rounds of starvation with Earle's balanced salt solution (EBSS) and compared their ability to induce autophagy. Although both the normal and metastatic cells maintained their ability to proliferate under normal conditions, the MDA-MB-231 cells that survived starvation (231-EB5) proliferated more rapidly than the parental population following induction of autophagy. In contrast, both MCF10A and MCF10A-EB5 cells were growth-inhibited. The 231-EB5 cells induced autophagy more rapidly than the parental population as demonstrated by increased expression of LC3II (analyzed by immunoblot) and increased LC3 puncta (analyzed by immunofluorescence). Migration of parental 231 cells was inhibited by rapamycin; however, there was no change in the ability of 231-EB5 cells to migrate after treatment with rapamycin. Growth in three dimensions also was significantly altered. MCF10A-EB5 cells were more extended compared to the spherical growth of the parental MCF10A cells. Protrusions emitted from the 231-EB5 cells were more rounded compared to the extended spikes of the parental 231 cells. Altogether, these results demonstrate that select populations derived from metastatic breast cancer cells have different abilities to survive, proliferate, and migrate following induction of autophagy, suggesting that autophagy may be a key mechanism for tumor progression and metastasis. Citation Format: Yi Li, Monica J. Lewis, Jianzhong Liu, James J. Cody, Douglas R. Hurst. Metastatic breast cancer cell phenotype is regulated by autophagy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1678. doi:10.1158/1538-7445.AM2013-1678
Abstract Introduction Recurrent urinary tract infections have been linked to increased risk of bladder cancer, suggesting a potential role of the urinary microbiome in bladder cancer pathogenesis. Objective Compare the urinary microbiomes in mice with and without bladder. Methods Longitudinal study of mice exposed to a dilute bladder-specific carcinogen (0.05% n-butyl-n-(4-hydroxybutyl) nitrosamine, BBN mice, n=10), and control mice (n=10). Urine was sampled monthly from individual mice for 4 months. Microbial DNA was extracted from the urine, and the V4 region of the 16S rRNA gene sequenced. Animals were sacrificed and their bladders harvested for histopathology. Bladder sections were graded by a blinded pathologist. The composition and diversity of the urinary microbiome were compared between the BBN and control mice. Metabolic pathway analysis was completed using PICRUST. Results Bladder histology in the BBN group showed normal tissue with inflammation (BBN-normal, n=5), precancerous pathologies, (BBN-precancerous, n=3), and invasive cancer (BBN-cancer, n=2). Alpha diversity did not differ between the mice exposed to BBN and the control mice at any timepoint. There were no differences in the urinary microbiomes between the BBN and control mice at baseline. At month 4, mice exposed to BBN had higher proportion of both Gardnerella and Bifidobacterium compared to control mice. There were no differences in proportions of specific bacteria between either the BBN-precancer or BBN-cancer and controls at month 4. However, the BBN-normal mice had higher proportions of Gardnerella , Haemophilus , Bifidobacterium , and Ureaplasma Actinobaculum , and lower proportions of Actinomyces , compared to control mice at month 4. Functional pathway analysis demonstrated increases in genes related to purine metabolism, phosphotransferase systems, peptidases, protein folding, and bacterial toxins in the BBN-mice compared to control mice at month 4. Conclusion Mice exposed to 4 months of BBN, a bladder-specific carcinogen, have distinct urine microbial profiles compared to control mice.
