Combating the Epigenome: Elucidation of Mechanisms Underlying Chemoresistance and Enhancing Tumor Immunogenicity

Chemotherapy and radiation therapy remain the backbone of cancer treatments, and now cancer immunotherapy offers promising new approaches for the treatment of malignancies. One of the major obstacles for chemo-based therapies is acquired chemoresistance. We find Regulator of G-protein signaling protein (RGS10) is an important regulator of cell survival and chemoresistance in ovarian cancer. Our findings further indicate RGS10 transcript expression is suppressed by DNA hypermethylation and histone deacetylation during acquired chemoresistance in ovarian cancer. We identify two important epigenetic regulators, HDAC1 and DNMT1, which exhibit aberrant association with RGS10 promoters in chemoresistant ovarian cancer cells. Inhibition of DNMT1 or HDAC1 significantly increases RGS10 expression and cisplatin-mediated cell death. We further focus on modulation of death receptors in advanced colorectal cancer (CRC) cells. We use a combination treatment of irradiation and proteasome inhibition to further induce activation of tumor-specific immune responses. We investigate the effect of the 26S proteasome inhibitor bortezomib alone or in combination with radiotherapy, on the expression of death receptors in normal colon and in colorectal cancer cell lines. Our results indicate a combination of 26S proteasome inhibition and sub-lethal radiation significantly increases the sensitivity of carcinoma cells to apoptosis. Combination treatment up-regulates cell surface expression of DR4, DR5 and Fas by increasing their transcriptional activation. Thus, the combination treatment enhanced sensitivity to killing through FAS and TRAIL receptors by CD8+ T cells. We further characterized the mechanisms by which radiation controls CRC expression of death receptors. We have shown that sub-lethal irradiation increases expression of our target molecules by enhancing histone acetylation at promoter regions through decreasing binding of HDAC2 and HDAC3, and by DNA hypomethylation, via decreasing binding of DNMT1. In sum, our studies provide insight into the alteration of molecular pathways involved in cancer cell death and survival

Serum acidic mammalian kinase as a new laboratory test to define subclinical inflammation in Familial Mediterranean fever

Aim: To investigate the relationship between acidic mammalian chitinase (CHIA) level and autoinflammatory diseases, especially in Familial Mediterranean fever. Methods: We first analyzed CHIA expression, methylation in various autoinflammatory diseases, including, SLE (Systemic Lupus Erythematosus), RA (Rheumatoid Arthritis), SJS (Stevens-Johnson syndrome), SSc (Systemic Sclerosis) and T1D (Type 1 diabetes) patients, case-control and correlation between the MEFV and CHIA genes by using bioinformatics tools. We then measured serum CHIA level in ninety individuals; thirty FMF attacks, thirty FMF remissions and thirty healthy control groups. Statistical analysis was used to evaluate the interaction between clinical parameters and serum CHIA level. The potential of serum CHIA level was tested using AUC and ROC analysis. Results: According to our ADEx analysis, we observed high CHIA expression in SLE, RA and T1D patients than in the control group. Moreover, we detected that the methylation level decreased in each disease, especially in the cg17143643 and cg7497781 probes. We also observed a correlation between MEFV and CHIA in these autoinflammatory diseases. According to our ELISA results, we also showed an increased CHIA level in FMF attack and remission as compared to the control group in serum (p <0.001, p =0.007; resp). We further observed a relationship between CHIA level and patients with amyloidosis, attack per month, and neutrophil and WBC levels.  Conclusion: Our primary data suggest that CHIA is related to FMF pathogenesis and can be followed in the subclinical period of the disease

Combination Treatment with Sublethal Ionizing Radiation and the Proteasome Inhibitor, Bortezomib, Enhances Death-Receptor Mediated Apoptosis and Anti-Tumor Immune Attack

Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity

Interchanging Functionality Among Homologous Elongation Factors Using Signatures of Heterotachy

Numerous models of molecular evolution have been formulated to describe the forces that shape sequence divergence among homologous proteins. These models have greatly enhanced our understanding of evolutionary processes. Rarely are such models empirically tested in the laboratory, and even more rare, are such models exploited to generate novel molecules useful for synthetic biology. Here, we experimentally demonstrate that the heterotachy model of evolution captures signatures of functional divergence among homologous elongation factors (EFs) between bacterial EF-Tu and eukaryotic eEF1A. These EFs are GTPases that participate in protein translation by presenting aminoacylated-tRNAs to the ribosome. Upon release from the ribosome, the EFs are recharged by nucleotide exchange factors EF-Ts in bacteria or eEF1B in eukaryotes. The two nucleotide exchange factors perform analogous functions despite not being homologous proteins. The heterotachy model was used to identify a set of sites in eEF1A/EF-Tu associated with eEF1B binding in eukaryotes and another reciprocal set associated with EF-Ts binding in bacteria. Introduction of bacterial EF-Tu residues at these sites into eEF1A protein efficiently disrupted binding of cognate eEF1B as well as endowed eEF1A with the novel ability to bind bacterial EF-Ts. We further demonstrate that eEF1A variants, unlike yeast wild-type, can function in a reconstituted in vitro bacterial translation system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00239-013-9540-9) contains supplementary material, which is available to authorized users

Evolutionary synthetic biology: structure/function relationships within the protein translation system

Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life. Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins. In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.MSCommittee Chair: Gaucher, Eric; Committee Member: Hammer, Brian; Committee Member: Wartell, Roge

Inhibition of HDAC1 and DNMT1 modulate RGS10 expression and decrease ovarian cancer chemoresistance.

RGS10 is an important regulator of cell survival and chemoresistance in ovarian cancer. We recently showed that RGS10 transcript expression is suppressed during acquired chemoresistance in ovarian cancer. The suppression of RGS10 is due to DNA hypermethylation and histone deacetylation, two important mechanisms that contribute to silencing of tumor suppressor genes during cancer progression. Here, we fully investigate the molecular mechanisms of epigenetic silencing of RGS10 expression in chemoresistant A2780-AD ovarian cancer cells. We identify two important epigenetic regulators, HDAC1 and DNMT1, that exhibit aberrant association with RGS10 promoters in chemoresistant ovarian cancer cells. Knockdown of HDAC1 or DNMT1 expression, and pharmacological inhibition of DNMT or HDAC enzymatic activity, significantly increases RGS10 expression and cisplatin-mediated cell death. Finally, DNMT1 knock down also decreases HDAC1 binding to the RGS10 promoter in chemoresistant cells, suggesting HDAC1 recruitment to RGS10 promoters requires DNMT1 activity. Our results suggest that HDAC1 and DNMT1 contribute to the suppression of RGS10 during acquired chemoresistance and support inhibition of HDAC1 and DNMT1 as an adjuvant therapeutic approach to overcome ovarian cancer chemoresistance

Antioxidant and antiproliferative activities of the n-butanol extract of Centaurea maroccana Ball aerial parts

The aim of the present study is to evaluate the total phenolic contents, antioxidant and anti-proliferative activities of the n-BuOH extract of Centaurea maroccana (BECM). The total phenolic and flavonoid of the butanolic extract of the plant were assessed by using Folin-Ciocalteau and aluminium chloride colorimetric assays, respectively, and the quantitative estimation of total flavonoids and phenols revealed the richness of the extract in these compounds. Antioxidant activity was evaluated using standard lab colorimetric methods, while the anti-proliferative activity was evaluated using sulforhodamine B (SRB) assay against C6 (Rattus norvegicus brain glioma) and HT29 (Homo sapiens colorectal adenocarcinoma) cell lines. The n-butanolic extract of Centaurea maroccana showed a strong antioxidant activity through DPPH, ABTS•+, DMSO alkalin, Reducing power, β-Carotene-linoleic acid and CUPRAC assays. Furthermore, the anti-proliferative activity against C6 and HT29 of Centaurea maroccana exhibited a high effect by the decrease in viability of both cancer cell lines. Our results suggest a possible use of Centaurea maroccana as a source of natural antioxidant and chemo-preventive agents against cancer

Effects of HDAC inhibitor trichostatin A (TSA) and DNMT inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) on RGS10 transcript expression and cell viabilities.

<p>Total RNA was isolated from untreated control cells and TSA or 5-Aza-dC treated cells. The relative expression of RGS10 mRNA was quantified by qRT-PCR and normalized to GAPDH transcript expression * p<0.05; ** p<0.005. <b>A</b>) HDAC inhibition increases RGS10 expression in chemoresistant A2780-AD cells. The cells were plated in a 10 cm² plate and incubated for 24 hours. The following day, cells were treated with 500 nM TSA and were incubated for an additional 48 hours. <b>B</b>) DNMT inhibitor 5-Aza-dC enhances RGS10 expression in chemoresistant cells. Two million A2780-AD cells were seeded in 10 cm² plates and were incubated for 24 hours. The following day, cells were treated with 20 µM 5-Aza-dC. Media and drug were refreshed every 24 hours. After indicated incubation time (3, 5, and 7 days), cells were harvested, mRNA was isolated, and cDNA was generated and quantified using qRT-PCR with specific primers and probe. <b>C</b>) A2780-AD cells were plated in 96-well plates and treated with 5 µM 5-Aza-dC for 5 days, 500 nM TSA for 36 hours, a combination of 5 µM 5-Aza-dC for 5 days and 500 nM TSA for the final 36 hours or DMSO. Gene expression was assessed using qRT-PCR as described, and normalized to RPL13A gene expression. The arrow indicates the expression level predicted by an additive effect of TSA and 5-Aza-dC. <b>D</b>) In a parallel experiment, A2780-AD cells were treated under the same conditions as 4C with or without 30 µM cisplatin for the final 12 hours. Cell survival was assessed using CellTiter-Blue fluorimetric viability assays. ***: p<0.001 comparing epigenetic drug to DMSO control in the absence of cisplatin. ###: p<0.001 comparing vehicle versus cisplatin treatment within epigenetic drug treatment groups. The dotted box indicates the cell viability predicted by an additive effect of TSA and 5-Aza-dC. <b>E</b>) A2780/AD cells (5000 cells/well) were plated in 96-well plate and transfected with negative control or RGS10 siRNA duplexes (Ambion Grand Island, NY) as per the manufacturer's protocol using Dharmafect1 transfection reagent (Dharmacon). Cells were dosed with a combination of 5 µM 5-Aza-dC for 3 days and 500 nM TSA for the last 36 h or DMSO. 30 µM cisplatin or vehicle was added for the last 12 h. Cell survival was assessed using CellTiter-Blue fluorimetric viability assays.</p

HDAC1 knockdown increases cisplatin stimulated apoptosis in chemoresistant cells.

<p>A2780-AD cells were treated with HDAC1 siRNA or control siRNA and were incubated for 48 hours. Following incubation, cells were treated with 50 µM cisplatin and incubated for addition 48 hours in RPMI 1640 media. An Annexin V: PE Apoptosis Detection Kit I (BD Pharmingen) was used for staining; results were quantified by Flow cytometry analysis and were analyzed using FlowJo software. Viable cells were negative for both annexin V-PE and 7-AAD; early apoptotic cells were positive for annexin V-PE and negative for 7-AAD, whereas late apoptotic dead cells were positive for both annexin V-PE and 7-AAD labeling. <b>A)</b> The relative increase of apoptotic cells in HDAC1- siRNA transfected A2780-AD cells which incorporated the Annexin V-PE and 7-AAD stains. <b>B)</b> Graph represent average of three independent experiments, with error bars denoting SEM *p<0.05. <b>C)</b> Western blot analysis represents efficiency of HDAC1 knockdown and RGS10 protein expression following HDAC1 knock down.</p