Withaferin A activates TRIM16 for its anti‐cancer activity in melanoma

Although selective BRAF inhibitors and novel immunotherapies have improved short-term treatment responses in metastatic melanoma patients, acquired resistance to these therapeutics still represent a major challenge in clinical practice. In this study, we evaluated the efficacy of Withaferin A (WFA), derived from the medicinal plant Withania Somnifera, as a novel therapeutic agent for the treatment of melanoma. WFA showed selective toxicity to melanoma cells compared to non-malignant cells. WFA induced apoptosis, significantly reduced cell proliferation and inhibited migration of melanoma cells. We identified that repression of the tumour suppressor TRIM16 diminished WFA cytotoxicity, suggesting that TRIM16 was in part responsible for the cytotoxic effects of WFA in melanoma cells. Together our data indicates that WFA has potent cytopathic effects on melanoma cells through TRIM16, suggesting a potential therapeutic application of WFA in the disease

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

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

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

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)

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

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

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