Tribbles homolog 3 denotes a poor prognosis in breast cancer and is involved in hypoxia response

Hypoxia in solid tumors is associated with treatment resistance, resulting in poor prognosis. Tribbles homolog 3 (TRIB3) is induced during hypoxia and is involved in multiple cellular pathways involved in cell survival. Here, we investigated the role of TRIB3 in breast cancer. TRIB3 mRNA expression was measured in breast tumor tissue from 247 patients and correlated with clinicopathological parameters and clinical outcome. Furthermore, we studied TRIB3 expression regulation in cell lines, xenografts tissues and human breast cancer material using Reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Finally, the effect of small interfering RNA (siRNA) mediated TRIB3 knockdown on hypoxia tolerance was assessed. Breast cancer patients with low, intermediate or high TRIB3 expression exhibited a mean disease free survival (DFS) of 80 (95% confidence interval [CI] = 74 to 86), 74 (CI = 67 to 81), and 63 (CI = 55 to 71) months respectively (P = .002, Mantel-Cox log-rank). The prognostic value of TRIB3 was limited to those patients that had received radiotherapy as part of their primary treatment (n = 179, P = .005) and remained statistically significant after correction for other clinicopathological parameters (DFS, Hazard Ratio = 1.90, CI = 1.17 to 3.08, P = .009). In breast cell lines TRIB3 expression was induced by hypoxia, nutrient starvation, and endoplasmic reticulum stress in an hypoxia inducible factor 1 (HIF-1) independent manner. TRIB3 induction after hypoxia did not increase with decreasing oxygen levels. In breast tumor xenografts and human breast cancer tissues TRIB3 co-localized with the hypoxic cell marker pimonidazole. The induction of TRIB3 by hypoxia was shown to be regulated via the PERK/ATF4/CHOP pathway of the unfolded protein response and knockdown of TRIB3 resulted in a dose-dependent increase in hypoxia sensitivity. TRIB3 is independently associated with poor prognosis of breast cancer patients, possibly through its association with tumor cell hypoxi

Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells

© 2018 The Author(s). Background: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. Methods: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. Results: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by >25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. Conclusions: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Reciprocal functions of hepatocyte growth factor and transforming growth factor-β1 in the progression of renal diseases: A role for CD44?

Reciprocal functions of hepatocyte growth factor and transforming growth factor-β1 in the progression of renal diseases: A role for CD44? Progressive renal fibrosis occurs via common pathophysiologic mechanisms, regardless of the primary underlying disease. This cascade includes release of cytokines/chemokines and toxic molecules, interstitial inflammation, tubular cell damage, accumulation of myofibroblasts, and finally, fibrosis. Hepatocyte growth factor (HGF) and transforming growth factor-β1 (TGF-β1) are key molecules in this cascade that, in general, exert opposite actions. Hepatocyte growth factor promotes, to some extent, inflammation, protects tubular epithelial cells, blocks myofibroblast transition, and contributes to tissue remodeling. In contrast, TGF-β1 has powerful anti-inflammatory actions, promotes apoptosis, induces myofibroblast transition, and is a strong pro-fibrotic agent. The mechanisms which orchestrate the reciprocal actions of HGF and TGF-β1 are still largely unknown and are probably multiple. One of these mechanisms involves the selective up-regulation of CD44 in damaged kidney. The glomerular and tubular expression of CD44 closely correlates with the degree of renal damage, and CD44 has been shown to facilitate the action of both HGF and TGF-β1. Moreover, during chronic obstructive nephropathy CD44 knock-out mice display much more tubular damage but develop less fibrosis in the course of the renal disease. These histologic findings are associated with impairment of signaling pathways of both HGF and TGF-β1. The development of new therapeutic strategies aimed at preventing progression of renal diseases that are based on HGF and/or TGF-β1 may take in account the pivotal role of CD44 expression in the functions of both molecules

Extracellular Vesicles as Transmitters of Hypoxia Tolerance in Solid Cancers

Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after treatment. This is mainly due to increased resistance of hypoxic cells to radio- and chemotherapy and the association of hypoxic cells with increased metastasis development. It is therefore not surprising that an increased hypoxic tumour fraction is associated with poor patient survival. The extent of hypoxia within a tumour is influenced by the tolerance of individual tumor cells to hypoxia, a feature that differs considerably between tumors. High numbers of hypoxic cells may, therefore, be a direct consequence of enhanced cellular capability inactivation of hypoxia tolerance mechanisms. These include HIF-1α signaling, the unfolded protein response (UPR) and autophagy to prevent hypoxia-induced cell death. Recent evidence shows hypoxia tolerance can be modulated by distant cells that have experienced episodes of hypoxia and is mediated by the systemic release of factors, such as extracellular vesicles (EV). In this review, the evidence for transfer of a hypoxia tolerance phenotype between tumour cells via EV is discussed. In particular, proteins, mRNA and microRNA enriched in EV, derived from hypoxic cells, that impact HIF-1α-, UPR-, angiogenesis- and autophagy signalling cascades are listed

Autophagy-Dependent Secretion: Contribution to Tumor Progression

Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular homeostasis. During autophagy, cytoplasmic content is recognized and packed in autophagic vacuoles, or autophagosomes, and targeted for degradation. However, during the last years, it has become evident that the role of autophagy is not restricted to degradation alone but also mediates unconventional forms of secretion. Furthermore, cells with defects in autophagy apparently are able to reroute their cargo, like mitochondria, to the extracellular environment; effects that contribute to an array of pathologies. In this review we discuss the current knowledge of the physiological roles of autophagy-dependent secretion, i.e. the effect on inflammation and insulin/ hormone secretion. Finally, we focus on the effects of autophagy-dependent secretion on the tumour microenvironment and tumour progression. The autophagy mediated secreted factors may stimulate cellular proliferation via auto- and paracrine signaling. The autophagy mediated release of immune modulating proteins change the immunosuppresive tumor microenvironment and may promote an invasive phenotype. These effects may be either direct or indirect through facilitating formation of the mobilized vesicle, aid in anterograde trafficking or alterations in homeostasis and/or autonomous cell signaling

Tumors Responsive to Autophagy-Inhibition:Identification and Biomarkers

Recent advances in cancer treatment modalities reveal the limitations of the prevalent “one-size-fits-all” therapies and emphasize the necessity to develop personalized approaches. In this perspective, identification of predictive biomarkers and intrinsic vulnerabilities are an important advancement for further therapeutic strategies. Autophagy is an important lysosomal degradation and recycling pathway that provides energy and macromolecular precursors to maintain cellular homeostasis. Although all cells require autophagy, several genetic and/or cellular changes elevate the dependence of cancer cells on autophagy for their survival and indicates that autophagy inhibition in these tumors could provide a favorable addition to current therapies. In this context, we review the current literature on tumor (sub)types with elevated dependence on autophagy for their survival and highlight an exploitable vulnerability. We provide an inventory of microenvironmental factors, genetic alterations and therapies that may be exploited with autophagy-targeted approaches to improve efficacy of conventional anti-tumor therapies

The Immunomodulatory Role of Hypoxic Tumor-Derived Extracellular Vesicles

Tumor-associated immune cells frequently display tumor-supportive phenotypes. These phenotypes, induced by the tumor microenvironment (TME), are described for both the adaptive and the innate arms of the immune system. Furthermore, they occur at all stages of immune cell development, up to effector function. One major factor that contributes to the immunosuppressive nature of the TME is hypoxia. In addition to directly inhibiting immune cell function, hypoxia affects intercellular crosstalk between tumor cells and immune cells. Extracellular vesicles (EVs) play an important role in this intercellular crosstalk, and changes in both the number and content of hypoxic cancer-cell-derived EVs are linked to the transfer of hypoxia tolerance. Here, we review the current knowledge about the role of these hypoxic cancer-cell-derived EVs in immunosuppression. In addition, we provide an overview of hypoxia-induced factors (i.e., miRNA and proteins) in tumor-derived EVs, and their role in immunomodulation