Citations

Abstract Despite a good response rate to front-line treatment, most of the patients with epithelial ovarian cancer (EOC) will eventually develop an incurable state of platinum (Pt)-resistant disease. Pt resistance is a multifactorial event driven by different molecular mechanisms such as increased ability to tolerate or repair DNA lesions, tendency to plasticity with acquisition of mesenchymal phenotype, and ability to modify/reprogram cellular metabolism. We recently identified in EOC relevant alterations in all of these factors, largely contributing to EOC aggressiveness. Focusing our attention on microRNAs (miRNAs), the master regulator of gene expression, we identified a cluster of miRNAs located on ChrXq27.3 (miR-506 family) whose downregulation is associated with advanced-stage EOC early relapse, and we found it to be the main contributor of a 35-miRNA based predictor (MiROvaR) of EOC risk of progression/relapse. We also contributed in defining altered choline metabolism in EOC that, through overexpression of choline kinase alpha (ChoK/CHKA) enzyme whose activity sustains tumor addiction to antioxidants, fosters EOC aggressiveness also by increasing chemoresistance. By in vitro and ex vivo studies performed in human EOC cell lines, non-tumoral immortalized ovarian cells and ex vivo EOC patient-derived ascitic tumor cells (PDAT), here we analyzed the role of miR-506 in regulating molecular network associated with drug resistance and evaluated the druggability of the cholinic phenotype, exploring its possible crosstalk with the immune system. We confirmed miR-506 to directly regulate the predicted target RAD17, a gene with a key role in guiding BRCA1 to DNA damage sites and stabilizing active signaling. Since an inactivation of DNA repair genes (BRCA1/2) is associated with better prognosis due to sensitization to DNA-damaging drugs, we investigated whether miR-506 can regulate DNA repair machinery by phenocopying/supporting the effects of BRCA1/2 mutation on Pt and PARP inhibitor sensitivity. We observed that miR-506 forced expression or RAD17 knockdown sensitized EOC cells to Pt and PARP inhibitors and, interestingly, showed a synthetic lethal effect with inhibitors of cell-cycle checkpoint kinases CHK1 and WEE1. Association of miR-506 cluster expression (RT-qPCR) with the BRCAness-phenotype is currently ongoing on a retrospective FFPE case material. We also recently link EOC cells plasticity, miRNAs expression, and Pt-sensitivity. Indeed, E-cadherin can exert a new and unexpected functional role in EOC cells by contributing to Pt susceptibility. We determined that overexpression of PLEKHA7 (Pleckstrin homology domain-containing family A member 7), a component of the zonula adherens in polarized epithelia and expressed at low levels in EOC biopsies, inhibits cell tumorigenicity and sensitizes EOC cells to Pt treatment. We will evaluate PLEKHA7 ability to regulate miRNAs driving tumor progression, including some of the miR-506 family. Concerning chemoresistance associated with metabolic rewiring, we showed that CHKA silencing altered the cellular oxidative stress redox status, decreasing glutathione content and raising reactive oxygen species (ROS) levels. Interestingly, the increase in intracellular ROS species improved EOC cells’ sensitivity to Pt/taxane treatments, contributing to cell death triggering. Moreover, upon CHKA silencing both in vitro and in PDAT cells, we observed an enhanced sensitization of cancer cells to TRAIL-mediated citotoxicity due to an augmented expression of pro-apoptotic/reduced expression of anti-apoptotic TRAIL receptor members, thus further increasing tumor cells’ weakness. Exploiting tumor’s reliance on these altered molecular and metabolic pathways is expected to be an appropriate strategy to improve efficacy of current treatment, possibly circumventing drug resistance. Funded by CARIPLO Foundation, AIRC and 5x1000 Institutional Research Funding. Citation Format: Marina Bagnoli, Roberta Nicoletti, Andrea Rizzo, Francesca Roggiani, Alessandro Satta, Mariangela Figini, Wei Zhang, Panagiotis Z. Anastasiadis, Egidio Iorio, Silvana Canevari, Antonella Tomassetti, Francesco Raspagliesi, Delia Mezzanzanica. An integrated molecular and metabolic approach for harnessing early relapse and chemoresistance in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A39.

ABSTRACT Bacteria that produce heat‐stable enterotoxins (STs), a leading cause of secretory diarrhea, are a major cause of morbidity and mortality worldwide. ST stimulates guanylyl cyclase C (GCC) and accumulation of intracellular cyclic GMP ([cGMP] i ), which opens the cystic fibrosis transmembrane conductance regulator (CFTR)‐related chloride channel, triggering intestinal secretion. Although the signaling cascade mediating ST‐induced diarrhea is well characterized, antisecretory therapy targeting this pathway has not been developed. 2‐ChloroATP (2ClATP) and its cell‐permeant precursor, 2‐chloroadenosine (2ClAdo), disrupt ST‐dependent signaling in intestinal cells. However, whether the ability to disrupt guanylyl cyclase signaling translates into effective antisecretory therapy remains untested. In this study, the efficacy of 2ClAdo to prevent ST‐induced water secretion by human intestinal cells was examined. In Caco‐2 human intestinal cells, ST increased [cGMP] i , induced a chloride current, and stimulated net basolateral‐to‐apical water secretion. This effect on chloride current and water secretion was mimicked by the cell‐permeant analog of cGMP, 8‐bromo‐cGMP. Treatment of Caco‐2 cells with 2ClAdo prevented ST‐induced increases in [cGMP] i , chloride current and water secretion. Inhibition of the downstream consequences of ST‐GCC interaction reflects proximal disruption of cGMP production because 8‐bromo‐cGMP stimulated chloride current and water secretion in 2ClAdo‐treated cells. Thus, this study demonstrates that disruption of guanylyl cyclase signaling is an effective strategy for antisecretory therapy and provides the basis for developing mechanism‐based treatments for enterotoxigenic diarrhea.—Zhang, W., Mannan, I., Schulz, S., Parkinson, S. J., Alekseev, A. E., Gomez, L. A., Terzic, A., Waldman, S. A. Interruption of transmembrane signaling as a novel antisecretory strategy to treat enterotoxigenic diarrhea. FASEB J. 13, 913–922 (1999)

Significance We have identified a lethal subtype of gastric cancer (GC) that is characterized by high levels of clonal heterogeneity and TP53 (tumor protein P53) mutation. We have also uncovered key novel mutations in the targetable NRG1 (neuregulin-1) and ERBB4 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 4) ligand-receptor pair and identified BRCA2 (breast cancer 2, early onset) mutations as new genetic markers to predict better survival for GC. Our study represents a novel approach for GC personalized medicine and identified novel clinical actionable therapies for GC therapy.

Key PointsMGUS patients have significantly increased cortical bone porosity and reduced bone strength relative to matched controls.

KCNJ13 encodes Kir7.1, an inwardly rectifying K+ channel that is expressed in multiple ion-transporting epithelia. A mutation in KCNJ13 resulting in an arginine-to-tryptophan change at residue 162 (R162W) of Kir7.1 was associated with snowflake vitreoretinal degeneration, an inherited autosomal-dominant disease characterized by vitreous degeneration and mild retinal degeneration. We used the Xenopus laevis oocyte expression system to assess the functional properties of the R162W (mutant) Kir7.1 channel and determine how wild-type (WT) Kir7.1 is affected by the presence of the mutant subunit. Recordings obtained via the two-electrode voltage-clamp technique revealed that injection of oocytes with mutant Kir7.1 cRNA resulted in currents and cation selectivity that were indistinguishable from those in water-injected oocytes, suggesting that the mutant protein does not form functional channels in the plasma membrane. Coinjection of oocytes with equal amounts of mutant and WT Kir7.1 cRNAs resulted in inward K+ and Rb+ currents with amplitudes that were ∼17% of those in oocytes injected with WT Kir7.1 cRNA alone, demonstrating a dominant-negative effect of the mutant subunit. Similar to oocytes injected with WT Kir7.1 cRNA alone, coinjected oocytes exhibited inwardly rectifying Rb+ currents that were more than seven times larger than K+ currents, indicating that mutant subunits did not alter Kir7.1 channel selectivity. Immunostaining of Xenopus oocytes or Madin-Darby canine kidney cells expressing mutant or WT Kir7.1 demonstrated distribution of both proteins primarily in the plasma membrane. Our data suggest that the R162W mutation suppresses Kir7.1 channel activity, possibly by negatively impacting gating by membrane phosphadidylinositol 4,5-bisphosphate.

Bone marrow‐derived macrophages (BMMs) treated with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or macrophage colony‐stimulating factor (M‐CSF), differentiate into GM‐CSF‐induced mouse bone marrow‐derived macrophages (GM‐BMMs) or M‐CSF‐induced mouse bone marrow‐derived macrophages (M‐BMMs), which have an M1 or M2 profile, respectively. GM‐BMMs produce large amounts of proinflammatory cytokines and mediate resistance to pathogens, whereas M‐BMMs produce antiinflammatory cytokines that contribute to tissue repair and remodeling. M‐BMMs stimulated with lipopolysaccharide (LPS) are in an antiinflammatory state, with an IL‐12lowIL‐10high phenotype. However, the regulation of this process remains unclear. Klf10 belongs to the family of Krüppel‐like transcription factors and was initially described as a TGF‐β inducible early gene 1. IL‐12p40 is upregulated in LPS‐stimulated M‐BMMs from Klf10‐deficient mice, but downregulated during Klf10 overexpression. Klf11, another member of the Krüppel‐like factor family, can also repress the production of IL‐12p40. Furthermore, Klf10 binds to the CACCC element of the IL‐12p40 promoter and inhibits its transcription. We have therefore identified Klf10 as a transcription factor that regulates the expression of IL‐12p40 in M‐BMMs.

Abstract Purpose: Cell-based approaches were used to identify genetic markers predictive of patients' risk for poor response prior to chemotherapy. Experimental Design: We conducted genome-wide association studies (GWAS) to identify single-nucleotide polymorphisms (SNP) associated with cellular sensitivity to carboplatin through their effects on mRNA expression using International HapMap lymphoblastoid cell lines (LCL) and replicated them in additional LCLs. SNPs passing both stages of the cell-based study were tested for association with progression-free survival (PFS) in patients. Phase 1 validation was based on 377 ovarian cancer patients receiving at least four cycles of carboplatin and paclitaxel from the Australian Ovarian Cancer Study (AOCS). Positive associations were then assessed in phase 2 validation analysis of 1,326 patients from the Ovarian Cancer Association Consortium and The Cancer Genome Atlas. Results: In the initial GWAS, 342 SNPs were associated with carboplatin-induced cytotoxicity, of which 18 unique SNPs were retained after assessing their association with gene expression. One SNP (rs1649942) was replicated in an independent LCL set (Bonferroni adjusted P < 0.05). It was found to be significantly associated with decreased PFS in phase 1 AOCS patients (Pper-allele = 2 × 10−2), with a stronger effect in the subset of women with optimally debulked tumors (Pper-allele = 4 × 10−3). rs1649942 was also associated with poorer overall survival in women with optimally debulked tumors (Pper-allele = 9 × 10−3). However, this SNP was not significant in phase 2 validation analysis with patients from numerous cohorts. Conclusion: This study shows the potential of cell-based, genome-wide approaches to identify germline predictors of treatment outcome and highlights the need for extensive validation in patients to assess their clinical effect. Clin Cancer Res; 17(16); 5490–500. ©2011 AACR.

Abstract Purpose: Esophageal adenocarcinoma is a highly aggressive malignancy that frequently develops from Barrett's esophagus, a premalignant pathologic change occurring in the lower end of the esophagus. Identifying Barrett's esophagus patients at high risk of malignant transformation is essential to the prevention of esophageal adenocarcinoma. Although microRNA (miRNA) expression signatures have been associated with the etiology and prognosis of several types of cancers, their roles in the development of esophageal adenocarcinoma have not been extensively evaluated. Experimental Design: In this study, we analyzed the expression patterns of 470 human miRNAs using Agilent miRNA microarray in 32 disease/normal-paired tissues from 16 patients diagnosed with Barrett's esophagus of either low- or high-grade dysplasia, or esophageal adenocarcinoma. Results: Using unsupervised hierarchical clustering and class comparison analyses, we found that miRNA expression profiles in tissues of Barrett's esophagus with high-grade dysplasia were significantly different from their corresponding normal tissues. Similar findings were observed for esophageal adenocarcinoma, but not for Barrett's esophagus with low-grade dysplasia. The expression patterns of selected miRNAs were further validated using quantitative reverse transcription real-time PCR in an independent set of 75 pairs of disease/normal tissues. Finally, we identified several miRNAs that were involved in the progressions from low grade-dysplasia Barrett's esophagus to esophageal adenocarcinoma. Conclusions: We showed that miRNAs were involved in the development and progression of esophageal adenocarcinoma. The identified significant miRNAs that may become potential targets for early detection, chemoprevention, and treatment of esophageal cancer. (Clin Cancer Res 2009;15(18):5744–52)