An insight into the diagnostic and prognostic value of HOX A13's expression in non-muscle invasive bladder cancer.

BACKGROUND: Several studies have interrogated the molecular pathways and their interacting genes underlying bladder cancer (BCa) tumorigenesis, yet, the role of homeobox genes is still poorly understood. Specifically, HOXA13, which plays an important role as a major actor in the urogenital tract's development. METHODS: Immunohistochemical (IHC) staining was performed to inspect the differential expression of HOXA13 protein in non-muscle-invasive bladder cancer (NMIBC) and non-tumoral tissues. A semiquantitative scoring system was adopted to evaluate the IHC labeling. Correlation to clinical parameters was performed by descriptive statistics. Overall survival was estimated by the Kaplan-Meier method and Cox regression model. The functional HOX A13 protein association networks (PPI) were obtained using String 11.0 database. RESULTS: HOX A13 exhibited cytoplasmic and nuclear staining. Its expression levels were lower in high-grade NMIBC (HG NMIBC) compared to low-grade ones (LG NMIBC). The expression of HOX A13 was correlated to tumor grade (LG/HG) (p = 0.036) and stage (TA/T1) (p = 0.036). Nevertheless, its expression was not correlated to clinical parameters and was not able to predict the overall survival of patients with HG NMIBC. Finally, PPI analysis revealed that HOX A13 seems to be a part of a molecular network holding mainly PBX1, MEIS, ALDH1A2, HOX A10, and HOX A11. CONCLUSION: The deregulation of HOX A13 is not associated with the prognosis of BCa. It seems to be rather implicated in the early initiation of urothelial tumorigenesis and thus may serve as a diagnostic marker in patients with NMIBC. Further experimentations on larger validation sets are mandatory.

FGFR2 fusion proteins drive oncogenic transformation of mouse liver organoids towards cholangiocarcinoma.

BACKGROUND & AIMS: About 15% of intrahepatic cholangiocarcinomas (iCCAs) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), usually alongside mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing oncogenic FF activation. While FGFR-specific tyrosine kinase inhibitors (F-TKI) provide clinical benefit in FF+ iCCA, responses are partial and/or limited by resistance mechanisms, such as the V565F substitution in the FGFR2 gatekeeper residue. Improving on FF targeting in iCCA therefore remains a critical unmet need. Herein, we aimed to generate a murine model of FF-driven iCCA and use this to uncover actionable FF-associated dependencies. METHODS: Four iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by transplantation into immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies. RESULTS: Transplantation of FF-expressing liver organoids yielded tumors diagnosed as CCA based on histological, phenotypic and transcriptomic analyses. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation. Dual blockade of FF and the Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo. CONCLUSIONS: FF-driven iCCA pathogenesis was successfully modeled on a Tp53-/- murine background, revealing biological heterogeneity among structurally different FFs. Double blockade of FF-ERK signaling deserves consideration for precision-based approaches against human FF+ iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma (iCCA) is a rare cancer that is difficult to treat. A subtype of iCCA is caused by genomic alterations that generate oncogenic drivers known as FGFR2 fusions. Patients with FGFR2 fusions respond to FGFR inhibitors, but clinical responses are often of modest duration. We used animal and cellular models to show that FGFR2 fusions require the activity of a downstream effector named Mek1/2. We found that dual blockade of FGFR2 fusions and Mek1/2 was more effective than isolated inhibition of FGFR2 fusions, pointing to the potential clinical utility of dual FGFR2-MEK1/2 blockade in patients with iCCA.

The diagnostic applicability of A-type Lamin in non-muscle invasive bladder cancer.

PURPOSE: Lamin A is a major component of the nuclear lamina maintaining nuclear integrity, regulation of gene expression, cell proliferation, and apoptosis. Its deregulation in cancer has been recently reported to be associated with its prognosis. However, its clinical significance in non-muscle invasive bladder cancer (NMIBC) remains to be defined. MATERIAL/METHODS: Immunohistochemical staining and RT-qPCR were performed to screen the expression patterns of Lamin A/C protein and Lamin A mRNA respectively in 58 high and low grade NMIBC specimens. RESULTS: Lamin A/C protein was expressed only in the nucleus and less exhibited in NMIBC tissues compared to non-tumoral ones. On the other side, Lamin A mRNA was up-regulated in NMIBC compared to controls. Nevertheless, both expression patterns (protein and mRNA) were not correlated to clinical prognosis factors and were not able to predict the overall survival of patients with high-grade NMIBC. CONCLUSIONS: The deregulation of A-type Lamin is not associated with the prognosis of NMIBC, but it could serve as a diagnostic biomarker distinguishing NMIBC patients from healthy subjects suggesting its involvement as an initiator event of tumorigenesis in our cohort.

Uncovering the expression patterns and the clinical significance of miR-182, miR-205, miR-27a and miR-369 in patients with urinary bladder cancer.

BACKGROUND: Given the high recurrence and progression rates and the absence of reliable markers for early detection and prognosis prediction of patients with urothelial bladder cancer (BCa), the exploration of new biomarkers with high specificity is imperative. Mainly, microRNAs (miRNAs), which are involved in the initiation and the progression of BCa. Herein, the expression patterns of miR-182, miR-205, miR-27a and miR-369 were evaluated in patients with urothelial BCa. METHODS AND RESULTS: The expression levels of the miRNAs were investigated in 90 FFPE tissue samples (23 LG NMIBC, 44 HG NMIBC, 23 MIBC) and 10 non tumoral bladder tissues using TaqMan based RT-qPCR. Data analysis was performed using 2-ΔΔCT method. Correlation to clinical characteristics of the patients was performed using descriptive statistics and the receiver operating characteristic (ROC) curve was performed to evaluate the diagnostic value of all miRNAs. MiR-27a, miR-205 and miR-369 were down-regulated whereas miR-182 was up-regulated in patients compared to controls (p < 0.001). MiR-205 and miR-182 positively segregate between NMIBC and MIBC (p = 0.002 and p = 0.000 respectively) whereas the distribution of miR-27a's expression among these tumor groups was almost significant (p = 0.05) and that of miR-369's expression was irrelevant (p = 0.618). Interestingly, miR-182 was discriminative between LG NMIBC and HG NMIBC (p < 0.001) and Ta/T1 tumors (p = 0.000). Furthermore, high levels of miR-182 were potentially predictive of progression in NMIBC patients (p = 0.01). CONCLUSION: Collectively, a selection of miRNAs was found to be aberrantly expressed in BCa suggesting a potential diagnostic value in BCa. In addition, the clinical value of miR-182 and miR-205 as potential prognosis biomarkers was highlighted. Indeed, our data provide additional insights into cancer biology. Further functional or target studies are mandatory to strengthen these findings.

The advanced glycation end-product Nϵ -carboxymethyllysine promotes progression of pancreatic cancer: implications for diabetes-associated risk and its prevention.

Diabetes is an established risk factor for pancreatic cancer (PaC), together with obesity, a Western diet, and tobacco smoking. The common mechanistic link might be the accumulation of advanced glycation end-products (AGEs), which characterizes all of the above disease conditions and unhealthy habits. Surprisingly, however, the role of AGEs in PaC has not been examined yet, despite the evidence of a tumour-promoting role of receptor for advanced glycation end-products (RAGE), the receptor for AGEs. Here, we tested the hypothesis that AGEs promote PaC through RAGE activation. To this end, we investigated the effects of the AGE Nϵ -carboxymethyllysine (CML) in human pancreatic ductal adenocarcinoma (PDA) cell lines and in a mouse model of Kras-driven PaC interbred with a bioluminescent model of proliferation. Tumour growth was monitored in vivo by bioluminescence imaging and confirmed by histology. CML promoted PDA cell growth and RAGE expression, in a concentration-dependent and time-dependent manner, and activated downstream tumourigenic signalling pathways. These effects were counteracted by RAGE antagonist peptide (RAP). Exogenous AGE administration to PaC-prone mice induced RAGE upregulation in pancreatic intraepithelial neoplasias (PanINs) and markedly accelerated progression to invasive PaC. At 11 weeks of age (6 weeks of CML treatment), PaC was observed in eight of 11 (72.7%) CML-treated versus one of 11 (9.1%) vehicle-treated [control (Ctr)] mice. RAP delayed PanIN development in Ctr mice but failed to prevent PaC promotion in CML-treated mice, probably because of competition with soluble RAGE for binding to AGEs and/or compensatory upregulation of the RAGE homologue CD166/ activated leukocyte cell adhesion molecule, which also favoured tumour spread. These findings indicate that AGEs modulate the development and progression of PaC through receptor-mediated mechanisms, and might be responsible for the additional risk conferred by diabetes and other conditions characterized by increased AGE accumulation. Finally, our data suggest that an AGE reduction strategy, instead of RAGE inhibition, might be suitable for the risk management and prevention of PaC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Monitoring the Response of Hyperbilirubinemia in the Mouse Brain by In Vivo Bioluminescence Imaging.

Increased levels of unconjugated bilirubin are neurotoxic, but the mechanism leading to neurological damage has not been completely elucidated. Innovative strategies of investigation are needed to more precisely define this pathological process. By longitudinal in vivo bioluminescence imaging, we noninvasively visualized the brain response to hyperbilirubinemia in the MITO-Luc mouse, in which light emission is restricted to the regions of active cell proliferation. We assessed that acute hyperbilirubinemia promotes bioluminescence in the brain region, indicating an increment in the cell proliferation rate. Immunohistochemical detection in brain sections of cells positive for both luciferase and the microglial marker allograft inflammatory factor 1 suggests proliferation of microglial cells. In addition, we demonstrated that brain induction of bioluminescence was altered by pharmacological displacement of bilirubin from its albumin binding sites and by modulation of the blood-brain barrier permeability, all pivotal factors in the development of bilirubin-induced neurologic dysfunction. We also determined that treatment with minocycline, an antibiotic with anti-inflammatory and neuroprotective properties, or administration of bevacizumab, an anti-vascular endothelial growth factor antibody, blunts bilirubin-induced bioluminescence. Overall the study supports the use of the MITO-Luc mouse as a valuable tool for the rapid response monitoring of drugs aiming at preventing acute bilirubin-induced neurological dysfunction.

A nitric oxide-dependent cross-talk between class I and III histone deacetylases accelerates skin repair.

In a mouse model of skin repair we found that the class I-IIa histone deacetylase inhibitor trichostatin A accelerated tissue regeneration. Unexpectedly, this effect was suppressed by Sirtinol, a class III histone deacetylase (HDAC) (sirtuin)-selective inhibitor. The role of sirtuins (SIRTs) was then investigated by using resveratrol and a novel SIRT1-2-3 activator, the MC2562 compound we synthesized recently. Both resveratrol and MC2562 were effective in accelerating wound repair. The local administration of natural or synthetic SIRT activators, in fact, significantly accelerated skin regeneration by increasing keratinocyte proliferation. In vitro experiments revealed that the activation of SIRTs stimulated keratinocyte proliferation via endothelial NO synthase phosphorylation and NO production. In this condition, the class I member HDAC2 was found S-nitrosylated on cysteine, a post-transduction modification associated with loss of activity and DNA binding capacity. After deacetylase inhibitor or SIRT activator treatment, ChIP showed, in fact, a significant HDAC2 detachment from the promoter region of insulin growth factor I (IGF-I), fibroblast growth factor 10 (FGF-10), and Epithelial Growth Factor (EGF), which may be the final recipients and effectors of the SIRT-NO-HDAC signaling cascade. Consistently, the effect of SIRT activators was reduced in the presence of NG-nitro-L-arginine methyl ester (L-NAME), a general inhibitor of NO synthesis. In conclusion, the NO-dependent cross-talk among class III and I histone deacetylases suggests an unprecedented signaling pathway important for skin repair.

The cross-talk between the macro and micro-environment in precursor lesions of pancreatic cancer leads to new and promising circulating biomarkers.

Pancreatic cancer (PC) is a clinically challenging tumor to combat due to its advanced stage at diagnosis as well as its resistance to currently available therapies. The absence of early symptoms and known detectable biomarkers renders this disease incredibly difficult to detect/manage. Recent advances in the understanding of PC biology have highlighted the importance of cancer-immune cell interactions, not only in the tumor micro-environment but also in distant systemic sites, like the bone marrow, spleen and circulating immune cells, the so-called macro-environment. The response of the macro-environment is emerging as a determining factor in tumor development by contributing to the formation of an increasingly immunogenic micro-environment promoting tumor homeostasis and progression. We will summarize the key events associated with the feedback loop between the tumor immune micro-environment (TIME) and the tumor immune macroenvironment (TIMaE) in pancreatic precancerous lesions along with how it regulates disease development and progression. In addition, liquid biopsy biomarkers capable of diagnosing PC at an early stage of onset will also be discussed. A clearer understanding of the early crosstalk between micro-environment and macro-environment could contribute to identifying new molecular therapeutic targets and biomarkers, consequently improving early PC diagnosis and treatment.

AATF/Che-1 RNA polymerase II binding protein overexpression reduces the anti-tumor NK-cell cytotoxicity through activating receptors modulation.

Introduction: AATF/Che-1 over-expression in different tumors is well known and its effect on tumorigenicity is mainly due to its central role demonstrated in the oncogenic pathways of solid tumors, where it controls proliferation and viability. The effect exerted by tumors overexpressing Che-1 on the immune response has not yet been investigated. Methods: Starting from ChIP-sequencing data we confirmed Che-1 enrichment on Nectin-1 promoter. Several co-cultures experiments between NK-cells and tumor cells transduced by lentiviral vectors carrying Che-1-interfering sequence, analyzed by flow-cytometry have allowed a detailed characterization of NK receptors and tumor ligands expression. Results: Here, we show that Che-1 is able to modulate the expression of Nectin-1 ligand at the transcriptional level, leading to the impairment of killing activity of NK-cells. Nectin-1 down-modulation induces a modification in NK-cell ligands expression able to interact with activating receptors and to stimulate NK-cell function. In addition, NK-cells from Che-1 transgenic mice, confirming a reduced expression of activating receptors, exhibit impaired activation and a preferential immature status. Discussion: The critical equilibrium between NK-cell ligand expression on tumor cells and the interaction with NK cell receptors is affected by Che-1 over-expression and partially restored by Che-1 interference. The evidence of a new role for Che-1 as regulator of anti-tumor immunity supports the necessity to develop approaches able to target this molecule which shows a dual tumorigenic function as cancer promoter and immune response modulator.

The Activation of PPARγ by (2Z,4E,6E)-2-methoxyocta-2,4,6-trienoic Acid Counteracts the Epithelial-Mesenchymal Transition Process in Skin Carcinogenesis.

Cutaneous squamous cell carcinoma (cSCC) is the most common UV-induced keratinocyte-derived cancer, and its progression is characterized by the epithelial-mesenchymal transition (EMT) process. We previously demonstrated that PPARγ activation by 2,4,6-octatrienoic acid (Octa) prevents cutaneous UV damage. We investigated the possible role of the PPARγ activators Octa and the new compound (2Z,4E,6E)-2-methoxyocta-2,4,6-trienoic acid (A02) in targeting keratinocyte-derived skin cancer. Like Octa, A02 exerted a protective effect against UVB-induced oxidative stress and DNA damage in NHKs. In the squamous cell carcinoma A431 cells, A02 inhibited cell proliferation and increased differentiation markers' expression. Moreover, Octa and even more A02 counteracted the TGF-ß1-dependent increase in mesenchymal markers, intracellular ROS, the activation of EMT-related signal transduction pathways, and cells' migratory capacity. Both compounds, especially A02, counterbalanced the TGF-ß1-induced cell membrane lipid remodeling and the release of bioactive lipids involved in EMT. In vivo experiments on a murine model useful to study cell proliferation in adult animals showed the reduction of areas characterized by active cell proliferation in response to A02 topical treatment. In conclusion, targeting PPARγ may be useful for the prevention and treatment of keratinocyte-derived skin cancer.

Bcl-2 family inhibitors sensitize human cancer models to therapy.

BH3 mimetics, targeting the Bcl-2 family anti-apoptotic proteins, represent a promising therapeutic opportunity in cancers. ABT-199, the first specific Bcl-2 inhibitor, was approved by FDA for the treatment of several hematological malignancies. We have recently discovered IS21, a novel pan BH3 mimetic with preclinical antitumor activity in several tumor types. Here, we evaluated the efficacy of IS21 and other BH3 mimetics, both as single agents and combined with the currently used antineoplastic agents in T-cell acute lymphoblastic leukemia, ovarian cancer, and melanoma. IS21 was found to be active in T-cell acute lymphoblastic leukemia, melanoma, lung, pancreatic, and ovarian cancer cell lines. Bcl-xL and Mcl-1 protein levels predicted IS21 sensitivity in melanoma and ovarian cancer, respectively. Exploring IS21 mechanism of action, we found that IS21 activity depends on the presence of BAX and BAK proteins: complexes between Bcl-2 and Bcl-xL proteins and their main binding partners were reduced after IS21 treatment. In combination experiments, BH3 mimetics sensitized leukemia cells to chemotherapy, ovarian cancer cells and melanoma models to PARP and MAPK inhibitors, respectively. We showed that this enhancing effect was related to the potentiation of the apoptotic pathway, both in hematologic and solid tumors. In conclusion, our data suggest the use of inhibitors of anti-apoptotic proteins as a therapeutic strategy to enhance the efficacy of anticancer treatment.

Bcl-2-like protein-10 increases aggressive features of melanoma cells.

Aim: B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated. Methods: Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis. Results: Results demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR. Conclusions: Overall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features.

Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis.

Phosphorylation of p53 at Ser 46 was shown to regulate p53 apoptotic activity. Here we demonstrate that homeodomain-interacting protein kinase-2 (HIPK2), a member of a novel family of nuclear serine/threonine kinases, binds to and activates p53 by directly phosphorylating it at Ser 46. HIPK2 localizes with p53 and PML-3 into the nuclear bodies and is activated after irradiation with ultraviolet. Antisense inhibition of HIPK2 expression reduces the ultraviolet-induced apoptosis. Furthermore, HIPK2 and p53 cooperate in the activation of p53-dependent transcription and apoptotic pathways. These data define a new functional interaction between p53 and HIPK2 that results in the targeted subcellular localization of p53 and initiation of apoptosis.

Reduction of Cell Proliferation by Acute C2H6O Exposure.

Endogenous acetaldehyde production from the metabolism of ingested alcohol exposes hematopoietic progenitor cells to increased genotoxic risk. To develop possible therapeutic strategies to prevent or reverse alcohol abuse effects, it would be critical to determine the temporal progression of acute ethanol toxicity on progenitor cell numbers and proliferative status. We followed the variation of the cell proliferation rate in bone marrow and spleen in response to acute ethanol intoxication in the MITO-Luc mouse, in which NF-Y-dependent cell proliferation can be assessed in vivo by non-invasive bioluminescent imaging. One week after ethanol administration, bioluminescent signals in bone marrow and spleen decreased below the level corresponding to physiological proliferation, and they progressively resumed to pre-treatment values in approximately 4 weeks. Boosting acetaldehyde catabolism by administration of an aldehyde dehydrogenase activity activator or administration of polyphenols with antioxidant activity partially restored bone marrow cells' physiological proliferation. These results indicate that in this mouse model, bioluminescent alteration reflects the reduction of the physiological proliferation rate of bone marrow progenitor cells due to the toxic effect of aldehydes generated by alcohol oxidation. In summary, this study presents a novel view of the impact of acute alcohol intake on bone marrow cell proliferation in vivo.

MITO-Luc/GFP zebrafish model to assess spatial and temporal evolution of cell proliferation in vivo.

We developed a novel reporter transgenic zebrafish model called MITO-Luc/GFP zebrafish in which GFP and luciferase expression are under the control of the master regulator of proliferation NF-Y. In MITO-Luc/GFP zebrafish it is possible to visualize cell proliferation in vivo by fluorescence and bioluminescence. In this animal model, GFP and luciferase expression occur in early living embryos, becoming tissue specific in juvenile and adult zebrafish. By in vitro and ex vivo experiments we demonstrate that luciferase activity in adult animals occurs in intestine, kidney and gonads, where detectable proliferating cells are located. Further, by time lapse experiments in live embryos, we observed a wave of GFP positive cells following fin clip. In adult zebrafish, in addition to a bright bioluminescence signal on the regenerating tail, an early unexpected signal coming from the kidney occurs indicating not only a fin cell proliferation, but also a systemic response to tissue damage. Finally, we observed that luciferase activity was inhibited by anti-proliferative interventions, i.e. 5FU, cell cycle inhibitors and X-Rays. In conclusion, MITO-Luc/GFP zebrafish is a novel animal model that may be crucial to assess the spatial and temporal evolution of cell proliferation in vivo.

[Corrigendum] TOOLBOX: Strep­Tagged nano­assemblies of antibody­drug­conjugates (ADC) for modular and conditional cancer drugging.

Following the publication of the above article, the authors have requested a change in the authorship on the paper, and the revised list of authors is presented above; essentially, the ninth intended author, Giuseppe Salvo (G.S.), was inadverently omitted from the author list. G.S. contributed towards the T design and the preparation of the tagged ScFv. Therefore, the revised authors' names and affiliations, as they should have been presented in the original version of this paper, are as follows: Elisa Tremante1, Leonardo Sibilio2,7, Fabio Centola2,8, Nadine Knutti3,9, Gerd Holzapfel4, Isabella Manni5, Matteo Allegretti1, Paolo Lombardi6, Giuseppe Salvo2,10, Loredana Cecchetelli2, Karlheinz Friedrich3, Joachim BertraM4 and Patrizio Giacomini1. 1Oncogenomics and Epigenetics, IRCCS Regina Elena National Cancer Institute, Rome; 2Ibi Lorenzini, Aprilia, Italy; 3University Hospital Jena, Institute of Biochemistry II, Jena; 4IBA GmbH, Göttingen, Germany; 5SAFU, IRCCS Regina Elena National Cancer Institute, Rome; 6NaxosPharma, Novate Milanese, Milan, Italy. Correspondence to: Dr Patrizio Giacomini. Oncogenomics and Epigenetics, IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy. E­mail: patrizio.giacomini@ifo.gov.it. Present address: 7Menarini Biotech, Pomezia, Rome, Italy. Present address: 8Merck Serono Spa, Global Analytical Department, Guidonia Montecelio, Rome, Italy. Present address: 9University Hospital Jena Institute for Clinical Chemistry and Laboratory Diagnostics, Jena, Germany. Present address: 10External Quality Assurance (ExM), MSD Italia S.r.l., Via Vitorchiano 151, 00189 Rome.Italy. Furthermore, the Authors' contributions section should be amended to read as follows: Authors' contributions: ET and MA tested the TOOLBOX concept and performed the flow cytometry experiments. LS, FC, GS and LC designed and prepared the tagged ScFv. NK and KF designed and prepared the GFP promoter­reporter construct. GH and JB designed and manufactured Strep­Tactins. ET and IM performed animal studies. PL designed and manufactured NAX and NAXT compounds. PG conceptualized TOOLBOX and wrote the manuscript with the contribution of all authors. All authors have approved the final version of the manuscript. All the authors agree with the inclusion of Giuseppe Salvo as an author on this paper, and are grateful to the Editor for allowing them the opportunity to publish this Corrigendum. Furthermore, they apologize to the readership of the Journal for any inconvenience caused. [the original article was published in Oncology Reports 45: 77, 2021; DOI: 10.3892/or.2021.8028].

TOOLBOX: Strep­Tagged nano­assemblies of antibody­drug­conjugates (ADC) for modular and conditional cancer drugging

Herein, we describe TOOLBOX, a 3­step modular nano­assembly targeting system that permits the combinatorial exchange of antibody specificities and toxic payloads, introducing modularity in antibody­drug conjugate (ADC) manufacturing. TOOLBOX integrates 3 building blocks: i) a recombinant antibody fragment (that in the selected setting targets the proto­oncogene ERBB2) genetically fused to an 8 amino acid Strep­Tag®; ii) a multivalent protein adapter, called Strep­Tactin®; iii) two anticancer agents, e.g. DNA nanobinders and the maytansinoid DM1, both carrying a chemically attached Strep­Tag that reversibly turns them into inactive prodrugs. Stoichiometrically optimized complexes of Strep­Tagged antibody fragments and drugs, bridged by Strep­Tactin, were specifically uptaken by breast cancer cells expressing ERBB2, and this unexpectedly resulted in conditional prodrug reactivation. A promoter­reporter system showed that TOOLBOX inhibited downstream ERBB2 signaling not only in ERBB2­overexpressing/­amplified SK­BR­3 cells grown in vitro, but also in ERBB2­low/non­amplified BRC230 triple­negative breast carcinoma cells xenotransplanted in nude mice. Thus, TOOLBOX is a modular ADC­like nano­assembly platform for precision oncology.

In Vivo Imaging of Thyroid Cancer with 99mTc-TR1401 and 99mTc-TR1402: A Comparison Study in Dogs.

Differentiated thyroid cancer (DTC) cells may lose NIS expression and iodine uptake, but usually express TSH receptors (TSHR). Therefore, the aim of our study was to compare two radiolabeled superagonist TSH analogues for DTC imaging. These analogues (namely TR1401 and TR1402) have a higher TSHR binding affinity than recombinant human TSH (Thyrogen®). Radiolabeling was performed with technetium-99m using an indirect method via HYNIC conjugation and was followed by in vitro quality controls and binding assay on TSHR-positive cell lines (ML-1). An in vitro binding assay was also performed and compared with radiolabeled human recombinant TSH. In vivo imaging was performed in four dogs with spontaneous follicular thyroid carcinoma with solid poorly differentiated areas with 99mTc-TR1401 SPECT/CT, 99mTc-TR1402 SPECT/CT, and [18F]FDG PET/CT on different days within 2 weeks. TR1401 and TR1402 were labeled with high specific activity (8.3 ± 1.2 MBq/µg) and retention of their biological activity and structural integrity. Both agonists were able to efficiently bind TSHR receptors expressed by cell lines with dissociation constants (Kd) of 2.7 nM for 99mTc-TR1401 and 0.5 nM for 99mTc-TR1402 compared with 99mTc-Thyrogen (Kd = 8.4 nM). In tumor-targeting experiments, a focal uptake was observed in dogs with spontaneous intraglandular thyroid carcinoma, in which TSHR expression was confirmed by immunohistochemistry. 99mTc-TR1402 provided higher T/B than 99mTc-TR1401 and [18F]FDG (12.9 ± 1.3, 10.2 ± 0.7, and 3.8 ± 0.6, respectively; all p < 0.001). Given these results, 99mTc-TR1402 appears to be a useful tool for in vivo imaging of thyroid cancer.

The RNA-binding protein MEX3A is a prognostic factor and regulator of resistance to gemcitabine in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer. Most patients present with advanced disease at diagnosis, which only permits palliative chemotherapeutic treatments. RNA dysregulation is a hallmark of most human cancers, including PDAC. To test the impact of RNA processing dysregulation on PDAC pathology, we performed a bioinformatics analysis to identify RNA-binding proteins (RBPs) associated with prognosis. Among the 12 RBPs associated with progression-free survival, we focused on MEX3A because it was recently shown to mark an intestinal stem cell population that is refractory to chemotherapeutic treatments, a typical feature of PDAC. Increased expression of MEX3A was correlated with higher disease stage in PDAC patients and with tumor development in a mouse model of PDAC. Depletion of MEX3A in PDAC cells enhanced sensitivity to chemotherapeutic treatment with gemcitabine, whereas its expression was increased in PDAC cells selected upon chronic exposure to the drug. RNA-sequencing analyses highlighted hundreds of genes whose expression is sensitive to MEX3A expression, with significant enrichment in cell cycle genes. MEX3A binds to its target mRNAs, like cyclin-dependent kinase 6 (CDK6), and promotes their stability. Accordingly, knockdown of MEX3A caused a significant reduction in PDAC cell proliferation and in progression to the S phase of the cell cycle. These findings uncover a novel role for MEX3A in the acquisition and maintenance of chemoresistance by PDAC cells, suggesting that it may represent a novel therapeutic target for PDAC.

Glabrescione B delivery by self-assembling micelles efficiently inhibits tumor growth in preclinical models of Hedgehog-dependent medulloblastoma.

Aberrant activation of the Hedgehog (Hh) pathway leads to the development of several tumors, including medulloblastoma (MB), the most common pediatric brain malignancy. Hh inhibitors acting on GLI1, the final effector of Hh signaling, offer a valuable opportunity to overcome the pitfalls of the existing therapies to treat Hh-driven cancers. In this study, the toxicity, delivery, biodistribution, and anticancer efficacy of Glabrescione B (GlaB), a selective GLI1 inhibitor, were investigated in preclinical models of Hh-dependent MB. To overcome its poor water solubility, GlaB was formulated with a self-assembling amphiphilic polymer forming micelles, called mPEG5kDa-cholane. mPEG5kDa-cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG5kDa-cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG5kDa-cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG5kDa-cholane micelles was delivered through the blood-brain barrier and drastically inhibited tumor growth in both allograft and orthotopic models of Hh-dependent MB. Our findings reveal that mPEG5kDa-cholane/GlaB is a good candidate for the treatment of Hh-driven tumors and provide relevant implications for the translation of GlaB into clinical practice.