Blood-based transcriptomic signature panel identification for cancer diagnosis: benchmarking of feature extraction methods.

Liquid biopsy has shown promise for cancer diagnosis due to its minimally invasive nature and the potential for novel biomarker discovery. However, the low concentration of relevant blood-based biosources and the heterogeneity of samples (i.e. the variability of relative abundance of molecules identified), pose major challenges to biomarker discovery. Moreover, the number of molecular measurements or features (e.g. transcript read counts) per sample could be in the order of several thousand, whereas the number of samples is often substantially lower, leading to the curse of dimensionality. These challenges, among others, elucidate the importance of a robust biomarker panel identification or feature extraction step wherein relevant molecular measurements are identified prior to classification for cancer detection. In this work, we performed a benchmarking study on 12 feature extraction methods using transcriptomic profiles derived from different blood-based biosources. The methods were assessed both in terms of their predictive performance and the robustness of the biomarker panels in diagnosing cancer or stratifying cancer subtypes. While performing the comparison, the feature extraction methods are categorized into feature subset selection methods and transformation methods. A transformation feature extraction method, namely partial least square discriminant analysis, was found to perform consistently superior in terms of classification performance. As part of the benchmarking study, a generic pipeline has been created and made available as an R package to ensure reproducibility of the results and allow for easy extension of this study to other datasets (https://github.com/VafaeeLab/bloodbased-pancancer-diagnosis).

Chemotherapeutic Potential of Chlorambucil-Platinum(IV) Prodrugs against Cisplatin-Resistant Colorectal Cancer Cells.

Chlorambucil-platinum(IV) prodrugs exhibit multi-mechanistic chemotherapeutic activity with promising anticancer potential. The platinum(II) precursors of the prodrugs have been previously found to induce changes in the microtubule cytoskeleton, specifically actin and tubulin of HT29 colon cells, while chlorambucil alkylates the DNA. These prodrugs demonstrate significant anticancer activity in 2D cell and 3D spheroid viability assays. A notable production of reactive oxygen species has been observed in HT29 cells 72 h post treatment with prodrugs of this type, while the mitochondrial membrane potential was substantially reduced. The cellular uptake of the chlorambucil-platinum(IV) prodrugs, assessed by ICP-MS, confirmed that active transport was the primary uptake mechanism, with platinum localisation identified primarily in the cytoskeletal fraction. Apoptosis and necrosis were observed at 72 h of treatment as demonstrated by Annexin V-FITC/PI assay using flow cytometry. Immunofluorescence measured via confocal microscopy showed significant changes in actin and tubulin intensity and in architecture. Western blot analysis of intrinsic and extrinsic pathway apoptotic markers, microtubule cytoskeleton markers, cell proliferation markers, as well as autophagy markers were studied post 72 h of treatment. The proteomic profile was also studied with a total of 1859 HT29 proteins quantified by mass spectroscopy, with several dysregulated proteins. Network analysis revealed dysregulation in transcription, MAPK markers, microtubule-associated proteins and mitochondrial transport dysfunction. This study confirms that chlorambucil-platinum(IV) prodrugs are candidates with promising anticancer potential that act as multi-mechanistic chemotherapeutics.

Pan-Cancer Analysis Reveals Functional Similarity of Three lncRNAs across Multiple Tumors.

Long non-coding RNAs (lncRNAs) are emerging as key regulators in many biological processes. The dysregulation of lncRNA expression has been associated with many diseases, including cancer. Mounting evidence suggests lncRNAs to be involved in cancer initiation, progression, and metastasis. Thus, understanding the functional implications of lncRNAs in tumorigenesis can aid in developing novel biomarkers and therapeutic targets. Rich cancer datasets, documenting genomic and transcriptomic alterations together with advancement in bioinformatics tools, have presented an opportunity to perform pan-cancer analyses across different cancer types. This study is aimed at conducting a pan-cancer analysis of lncRNAs by performing differential expression and functional analyses between tumor and non-neoplastic adjacent samples across eight cancer types. Among dysregulated lncRNAs, seven were shared across all cancer types. We focused on three lncRNAs, found to be consistently dysregulated among tumors. It has been observed that these three lncRNAs of interest are interacting with a wide range of genes across different tissues, yet enriching substantially similar biological processes, found to be implicated in cancer progression and proliferation.

The nuclear transporter importin 13 is critical for cell survival during embryonic stem cell differentiation.

Nuclear transporter Importin (Imp, Ipo) 13 is known to transport various mammalian cargoes into/out of the nucleus, but its role in directing cell-fate is unclear. Here we examine the role of Imp13 in the maintenance of pluripotency and differentiation of embryonic stem cells (ESCs) for the first time, using an embryonic body (EB)-based model. When induced to differentiate, Ipo13-/- ESCs displayed slow proliferation, reduced EB size, and lower expression of the proliferation marker KI67, concomitant with an increase in the number of TUNEL+ nuclei compared to wildtype ESCs. At days 5 and 10 of differentiation, Ipo13-/- EBs also showed enhanced loss of the pluripotency transcript OCT3/4, and barely detectable clusters of OCT3/4 positive cells. Day 5 Ipo13-/- EBs further exhibited reduced levels of the mesodermal markers Brachyury and Mixl1, correlating with reduced numbers of haemoglobinised cells generated. Our findings suggest that Imp13 is critical to ESC survival as well as early post-gastrulation differentiation.

Human Group IIA Phospholipase A2-Three Decades on from Its Discovery.

Phospholipase A2 (PLA2) enzymes were first recognized as an enzyme activity class in 1961. The secreted (sPLA2) enzymes were the first of the five major classes of human PLA2s to be identified and now number nine catalytically-active structurally homologous proteins. The best-studied of these, group IIA sPLA2, has a clear role in the physiological response to infection and minor injury and acts as an amplifier of pathological inflammation. The enzyme has been a target for anti-inflammatory drug development in multiple disorders where chronic inflammation is a driver of pathology since its cloning in 1989. Despite intensive effort, no clinically approved medicines targeting the enzyme activity have yet been developed. This review catalogues the major discoveries in the human group IIA sPLA2 field, focusing on features of enzyme function that may explain this lack of success and discusses future research that may assist in realizing the potential benefit of targeting this enzyme. Functionally-selective inhibitors together with isoform-selective inhibitors are necessary to limit the apparent toxicity of previous drugs. There is also a need to define the relevance of the catalytic function of hGIIA to human inflammatory pathology relative to its recently-discovered catalysis-independent function.

Interactome of the inhibitory isoform of the nuclear transporter Importin 13.

Importin 13 (Imp13) is a bidirectional nuclear transporter of proteins involved in a range of important cellular processes, with an N-terminally truncated inhibitory isoform (tImp13) specifically expressed in testis. To gain insight into tImp13 function, we performed a yeast-2-hybrid screen from a human testis cDNA library, identifying for the first time a suite of interactors with roles in diverse cellular process. We validated the interaction of tImp13 with Eukaryotic translation initiation factor 4γ2 (EIF4G2) and High mobility group containing protein 20A (HMG20A), benchmarking that with glucocorticoid receptor (GR), a known Imp13 interactor expressed in testis. Coimmunoprecipitation assays indicated association of both tImp13 and Imp13 with EIF4G2, HMG20A and GR. Quantitative confocal microscopic analysis revealed the ability of tImp13 to inhibit the nuclear localisation of EIF4G2, HMG20A and GR, as well as that of Imp13 to act as a nuclear exporter for both EIF4G2 and HMG20A, and as a nuclear importer for GR. The physiological relevance of these results was highlighted by the cytoplasmic localisation of EIF4G2, HMG20A and GR in pachytene spermatocytes/round spermatids in the murine testis where tImp13 is present at high levels, in contrast to the nuclear localisation of HMG20A and GR in spermatogonia, where tImp13 is largely absent. Interestingly, Imp13, EIF4G2, HMG20A and GR were found together in the acrosome vesicle of murine epididymal spermatozoa. Collectively, our findings show, for the first time, that tImp13 may have a functional role in the mature spermatozoa, in addition to that in the meiotic germ cells of the testis.

Anticancer Effect of PtIIPHENSS, PtII5MESS, PtII56MESS and Their Platinum(IV)-Dihydroxy Derivatives against Triple-Negative Breast Cancer and Cisplatin-Resistant Colorectal Cancer.

Development of resistance to cisplatin, oxaliplatin and carboplatin remains a challenge for their use as chemotherapies, particularly in breast and colorectal cancer. Here, we compare the anticancer effect of novel complexes [Pt(1,10-phenanthroline)(1S,2S-diaminocyclohexane)](NO3)2 (PtIIPHENSS), [Pt(5-methyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)](NO3)2 (PtII5MESS) and [Pt(5,6-dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)](NO3)2 (PtII56MESS) and their platinum(IV)-dihydroxy derivatives with cisplatin. Complexes are greater than 11-fold more potent than cisplatin in both 2D and 3D cell line cultures with increased selectivity for cancer cells over genetically stable cells. ICP-MS studies showed cellular uptake occurred through an active transport mechanism with considerably altered platinum concentrations found in the cytoskeleton across all complexes after 24 h. Significant reactive oxygen species generation was observed, with reduced mitochondrial membrane potential at 72 h of treatment. Late apoptosis/necrosis was shown by Annexin V-FITC/PI flow cytometry assay, accompanied by increased sub-G0/G1 cells compared with untreated cells. An increase in S and G2+M cells was seen with all complexes. Treatment resulted in significant changes in actin and tubulin staining. Intrinsic and extrinsic apoptosis markers, MAPK/ERK and PI3K/AKT activation markers, together with autophagy markers showed significant activation of these pathways by Western blot. The proteomic profile investigated post-72 h of treatment identified 1597 MDA-MB-231 and 1859 HT29 proteins quantified by mass spectroscopy, with several differentially expressed proteins relative to no treatment. GO enrichment analysis revealed a statistically significant enrichment of RNA/DNA-associated proteins in both the cell lines and specific additional processes for individual drugs. This study shows that these novel agents function as multi-mechanistic chemotherapeutics, offering promising anticancer potential, and thereby supporting further research into their application as cancer therapeutics.

Anti-cancer potential of synergistic phytochemical combinations is influenced by the genetic profile of prostate cancer cell lines.

Introduction: Despite strong epidemiological evidence that dietary factors modulate cancer risk, cancer control through dietary intervention has been a largely intractable goal for over sixty years. The effect of tumour genotype on synergy is largely unexplored. Methods: The effect of seven dietary phytochemicals, quercetin (0-100 µM), curcumin (0-80 µM), genistein, indole-3-carbinol (I3C), equol, resveratrol and epigallocatechin gallate (EGCG) (each 0-200 µM), alone and in all paired combinations om cell viability of the androgen-responsive, pTEN-null (LNCaP), androgen-independent, pTEN-null (PC-3) or androgen-independent, pTEN-positive (DU145) prostate cancer (PCa) cell lines was determined using a high throughput alamarBlue® assay. Synergy, additivity and antagonism were modelled using Bliss additivism and highest single agent equations. Patterns of maximum synergy were identified by polygonogram analysis. Network pharmacology approaches were used to identify interactions with known PCa protein targets. Results: Synergy was observed with all combinations. In LNCaP and PC-3 cells, I3C mediated maximum synergy with five phytochemicals, while genistein was maximally synergistic with EGCG. In contrast, DU145 cells showed resveratrol-mediated maximum synergy with equol, EGCG and genistein, with I3C mediating maximum synergy with only quercetin and curcumin. Knockdown of pTEN expression in DU145 cells abrogated the synergistic effect of resveratrol without affecting the synergy profile of I3C and quercetin. Discussion: Our study identifies patterns of synergy that are dependent on tumour cell genotype and are independent of androgen signaling but are dependent on pTEN. Despite evident cell-type specificity in both maximally-synergistic combinations and the pathways that phytochemicals modulate, these combinations interact with similar prostate cancer protein targets. Here, we identify an approach that, when coupled with advanced data analysis methods, may suggest optimal dietary phytochemical combinations for individual consumption based on tumour molecular profile.Graphical abstract.

Harnessing Liquid Biopsies to Guide Immune Checkpoint Inhibitor Therapy.

Immunotherapy (IO), involving the use of immune checkpoint inhibition, achieves improved response-rates and significant disease-free survival for some cancer patients. Despite these beneficial effects, there is poor predictability of response and substantial rates of innate or acquired resistance, resulting in heterogeneous responses among patients. In addition, patients can develop life-threatening adverse events, and while these generally occur in patients that also show a tumor response, these outcomes are not always congruent. Therefore, predicting a response to IO is of paramount importance. Traditionally, tumor tissue analysis has been used for this purpose. However, minimally invasive liquid biopsies that monitor changes in blood or other bodily fluid markers are emerging as a promising cost-effective alternative. Traditional biomarkers have limitations mainly due to difficulty in repeatedly obtaining tumor tissue confounded also by the spatial and temporal heterogeneity of tumours. Liquid biopsy has the potential to circumvent tumor heterogeneity and to help identifying patients who may respond to IO, to monitor the treatment dynamically, as well as to unravel the mechanisms of relapse. We present here a review of the current status of molecular markers for the prediction and monitoring of IO response, focusing on the detection of these markers in liquid biopsies. With the emerging improvements in the field of liquid biopsy, this approach has the capacity to identify IO-eligible patients and provide clinically relevant information to assist with their ongoing disease management.

The BRCA-1 binding protein BRAP2 is a novel, negative regulator of nuclear import of viral proteins, dependent on phosphorylation flanking the nuclear localization signal.

This study describes for the first time the ability of the novel BRCA1-binding protein 2 (BRAP2) to inhibit the nuclear import of specific viral proteins dependent on phosphorylation. Ectopic expression of BRAP2 in transfected African green monkey kidney COS-7 cells was found to significantly reduce nuclear localization signal (NLS)-dependent nuclear accumulation of either simian virus SV40 large-tumor antigen (T-ag) or human cytomegalovirus DNA polymerase processivity factor ppUL44; this was also observed in HL-60 human promyelocytic leukemia cells on induction of BRAP2 expression by vitamin D3 treatment. BRAP2 inhibition of nuclear accumulation was dependent on phosphorylation sites flanking the respective NLSs, where substitution of the cyclin-dependent kinase site T124 of T-ag with Ala or Asp prevented or enhanced BRAP2 inhibition of nuclear import, respectively. Substitution of T427 within the NLS of ppUL44 gave similar results, whereas no effect of BRAP2 was observed on nuclear targeting of other viral proteins, such as herpes simplex virus-1 pUL30, which lacks a phosphorylation site near its NLS, and the human immunodeficiency virus-1 Tat protein. Pulldowns/AlphaScreen assays indicated direct, high-affinity binding of BRAP2(442-592) to T-ag(111-135), strictly dependent on negative charge at T124 and the NLS. All results are consistent with BRAP2 being a novel, phosphorylation-regulated negative regulator of nuclear import, with potential as an antiviral agent.

An update on novel approaches for diagnosis and treatment of SARS-CoV-2 infection.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) has made a serious public health and economic crisis worldwide which united global efforts to develop rapid, precise, and cost-efficient diagnostics, vaccines, and therapeutics. Numerous multi-disciplinary studies and techniques have been designed to investigate and develop various approaches to help frontline health workers, policymakers, and populations to overcome the disease. While these techniques have been reviewed within individual disciplines, it is now timely to provide a cross-disciplinary overview of novel diagnostic and therapeutic approaches summarizing complementary efforts across multiple fields of research and technology. Accordingly, we reviewed and summarized various advanced novel approaches used for diagnosis and treatment of COVID-19 to help researchers across diverse disciplines on their prioritization of resources for research and development and to give them better a picture of the latest techniques. These include artificial intelligence, nano-based, CRISPR-based, and mass spectrometry technologies as well as neutralizing factors and traditional medicines. We also reviewed new approaches for vaccine development and developed a dashboard to provide frequent updates on the current and future approved vaccines.

Integrative resource for network-based investigation of COVID-19 combinatorial drug repositioning and mechanism of action.

An effective monotherapy to target the complex and multifactorial pathology of SARS-CoV-2 infection poses a challenge to drug repositioning, which can be improved by combination therapy. We developed an online network pharmacology-based drug repositioning platform, COVID-CDR (http://vafaeelab.com/COVID19repositioning.html), that enables a visual and quantitative investigation of the interplay between the primary drug targets and the SARS-CoV-2-host interactome in the human protein-protein interaction network. COVID-CDR prioritizes drug combinations with potential to act synergistically through different, yet potentially complementary, pathways. It provides the options for understanding multi-evidence drug-pair similarity scores along with several other relevant information on individual drugs or drug pairs. Overall, COVID-CDR is a first-of-its-kind online platform that provides a systematic approach for pre-clinical in silico investigation of combination therapies for treating COVID-19 at the fingertips of the clinicians and researchers.

Interactome of the negative regulator of nuclear import BRCA1-binding protein 2.

Although the negative regulator of nuclear import (NRNI) BRCA1 binding protein 2 (BRAP2) is highly expressed in testis, its role is largely unknown. Here we address this question by documenting the BRAP2 interactome from human testis, using the yeast 2-hybrid system to identify BRAP2-interacting proteins with roles in diverse cellular processes, including regulation of the actin cytoskeleton, ubiquitinylation, cell cycle/apoptosis and transcription. Interaction with BRAP2 in adult mouse testis with three of these, PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), A-Kinase anchor protein (AKAP3) and DNA methyl transferase 1 (DNMT1), was confirmed by coimmunoprecipitation assays. BRAP2's ability to inhibit PHLPP1 and DNMT1 nuclear localisation was also confirmed by quantitative confocal microscopy. Importantly, the physiological relevance thereof was implied by the cytoplasmic localisation of PHLPP1, AKAP3 and DNMT1 in pachytene spermatocytes/round spermatids where BRAP2 is present at high levels, and nuclear localisation of PHLPP1 and DNMT1 in spermatogonia concomitant with lower levels of BRAP2. Interestingly, BRAP2 was also present in murine spermatozoa, in part colocalised with AKAP3. Together the results indicate for the first time that BRAP2 may play an important NRNI role in germ cells of the testis, with an additional, scaffold/structural role in mature spermatozoa.