Search

Blood circulating microRNAs (c‐miRs) are potential biomarkers to trace aging and longevity trajectories to identify molecular targets for anti‐aging therapies. Based on a cross‐sectional study, a discovery phase was performed on 12 donors divided into four groups: young, old, healthy, and unhealthy centenarians. The identification of healthy and unhealthy phenotype was based on cognitive performance and capabilities to perform daily activities. Small RNA sequencing identified 79 differentially expressed c‐miRs when comparing young, old, healthy centenarians, and unhealthy centenarians. Two miRs, that is, miR‐19a‐3p and miR‐19b‐3p, were found increased at old age but decreased at extreme age, as confirmed by RT‐qPCR in 49 donors of validation phase. The significant decrease of those miR levels in healthy compared to unhealthy centenarians appears to be due to the presence of isomiRs, not detectable with RT‐qPCR, but only with a high‐resolution technique such as deep sequencing. Bioinformatically, three main common targets of miR‐19a/b‐3p were identified, that is, SMAD4, PTEN, and BCL2L11, converging into the FoxO signaling pathway, known to have a significant role in aging mechanisms. For the first time, this study shows the age‐related increase of plasma miR‐19a/b‐3p in old subjects but a decrease in centenarians. This decrease is more pronounced in healthy centenarians and was confirmed by the modified pattern of isomiRs comparing healthy and unhealthy centenarians. Thus, our study paves the way for functional studies using c‐miRs and isomiRs as additional parameter to track the onset of aging and age‐related diseases using new potential biomarkers., Small RNA deep sequencing on plasma‐derived RNA from 12 donors (young and old subjects, healthy, and unhealthy centenarians) was performed: 79 differentially expressed c‐miRs were identified. MiR‐19a/b‐3p increased at old age but decreased at extreme age, as confirmed by RT‐qPCR in 49 subjects. The decrease was more relevant in healthy centenarians, also confirmed by the modified pattern of isomiRs. Three common targets of miR‐19a/b‐3p were identified converging into the FoxO signaling pathway.

An understanding of the causative mechanisms of the harmful effects of cigarette smoke on the male reproductive system remains incomplete. Here, we investigated three different inhaled cigarette smoke doses over five different exposure durations to identify how the testis is affected. The effects of cigarette smoke exposure on testicular germ cells were characterized by morphological changes and a significant elevation in the number of apoptotic cells. Caspase 3 activation increased dramatically after cigarette smoke exposure, accompanied by significant time-dependent expression of the pro-apoptotic proteins Bak (B cell lymphoma/leukemia 2 [Bcl-2] homologous antagonist killer), Bcl2l11 (a BH3 domain-only protein related to Bcl-2), Apaf1 (Apoptotic protease-activating factor-1), and Caspase 9. Conversely, the abundance of anti-apoptotic Bcl2l2 decreased. Taken together, our findings suggest that extensive inhalation of cigarette smoke damages testicular germ cells through the induction of the mitochondrial apoptotic pathway through the Bcl-2 protein family.

BACKGROUND The authors' previous study demonstrated that the B-cell chronic lymphocytic leukemia/lymphoma (Bcl-2)-like 11 (BCL2L11) (Bim) deletion polymorphism was associated with poor clinical response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in patients with non–small cell lung cancer (NSCLC) with EGFR mutations. The objective of the current study was to investigate the impact of the Bim deletion polymorphism among patients with anaplastic lymphoma kinase (ALK)-positive or ROS proto-oncogene 1, receptor tyrosine kinase (ROS1)-positive NSCLC who were treated with crizotinib. METHODS A total of 55 patients with ALK-positive NSCLC and 14 patients with ROS1-positive NSCLC who were treated with crizotinib were enrolled into the current study. The Bim deletion polymorphism was analyzed by polymerase chain reaction. The clinical features of the Bim deletion polymorphism and its impact on the effect of crizotinib were investigated. RESULTS The Bim deletion polymorphism was present in 9 of 69 patients with ALK-positive or ROS1-positive NSCLC (13.0%). There were no differences noted with regard to clinicopathological features between patients with and without the Bim deletion polymorphism. Patients with the Bim deletion polymorphism had a significantly shorter progression-free survival (PFS) and lower objective response rate compared with those without (median PFS, 182 days vs 377 days [P = .008]) (objective response rate, 44.4% vs 81.7% [P =.041]) in all populations. The significant difference in PFS was observed in patients with ALK-positive NSCLC (83 days vs 305 days [P =.0304]) compared with those with ROS1-positive NSCLC (218 days vs not reached [P =.082]). Multivariate analysis indicated that the Bim deletion polymorphism was an independent predictive factor for patients with ALK-positive NSCLC who were treated with crizotinib (hazard ratio, 4.786 [P =.006]). CONCLUSIONS The Bim deletion polymorphism was found to be associated with poor clinical response to crizotinib in patients with ALK fusion-positive NSCLC. Cancer 2017;123:2927–35. © 2017 American Cancer Society.

The deregulation of programmed cell death, apoptosis, is a major contributor to the development of diseases, including cancer or autoimmunity, and can impair the response of transformed cells to therapy. The Bcl-2 family of proteins includes critical regulators of the intrinsic apoptosis pathway. BH3 (Bcl-2 homologous 3)-only proteins are proapoptotic members that share with each other and the wider Bcl-2 family only the ‘BH3 domain’. This short stretch of about 16 amino acids is necessary for protein–protein interaction that is critical for their apoptosis-inducing capacity. BH3-only proteins promote cell death by regulating activation of Bax and Bak, in a cell-type and stimulus-specific manner. Studies of gene-targeted mice that lack two or more BH3-only proteins have helped to unravel the overlapping functions of these apoptosis initiators. With the dawn of CRISPR/Cas9 gene-editing technologies, this strategy was taken even further, opening up allegedly answered questions again, at least at the cellular level. BH3 mimetics were recently approved for the treatment of patients suffering from cancer and it can be anticipated that these drugs make second use to treat autoimmunity and ageing-related disorders in the future. Key Concepts The ‘Bcl-2-regulated’ (also called ‘intrinsic’ or ‘mitochondrial’) apoptotic pathway is triggered by developmental cues or a broad range of cell stressors (e.g. growth factor deprivation and γ-irradiation) and is regulated by the interplay of the pro- and antiapoptotic members of the Bcl-2 family of proteins controlling integrity of the outer mitochondrial membrane (OMM). The survival versus death fate of a cell is decided not only by the relative levels of pro- and antiapoptotic Bcl-2 family members, but also depends on their ‘activation status’, frequently imposed by different posttranslational modifications on these proteins. Abnormal cell survival during establishment and maintenance of immune-(self)-tolerance as well as defects in apoptosis signalling at the end of an immune response may lead to neoplastic transformation and tumourigenesis. The Bcl-2 family consists of three subgroups of proteins that can be differentiated based on amino acid sequence, 3D structure and function. Bcl-2, Bcl-xL, Bcl-w, Bcl-B, Mcl-1 and A1 are essential for cell survival, with cell-type-specific expression, while Bcl-B is found active only in humans. The two other, proapoptotic, subfamilies encompass the multi-BH domain Bcl-2 family members Bax, Bak and Bok as well as the BH3-only protein subfamily members Bad, Bid, Bik/Nbk/Blk, Hrk/DP5, Bim/Bod/Bcl2L11, Noxa/Pmaip, Bmf and Puma/Bbc3. The BH3-only proteins are critical activators of the effector phase of the intrinsic death pathway, while Bax and Bak act as executers of apoptosis by initiating mitochondrial outer membrane permeabilisation (MOMP). The role of Bok in this group remains to be clarified in full. Both Puma and Noxa, apoptosis initiators that can be transcriptionally activated by p53 in response to DNA damage or oncogenic stress, can act as tumour suppressors in their own right, particularly in the context of an oncogenic lesion that subverts cell cycle control. Yet, in most circumstances tested, Puma appears more effective, suggesting roles for Noxa outside the p53 response. Bid functions as the link between the ‘death receptor’ and the ‘Bcl-2-regulated’ apoptotic pathways by causing an amplification of the caspase cascade that leads to cell destruction. Remarkably, Bid is critical for Fas ‘death receptor’-induced apoptosis in certain cell types, such as hepatocytes, but dispensable in others, including lymphoid cells. Additional roles of Bid, downstream of other proteases, have been suggested. Bim, the most studied BH3-only protein, is critical for many physiologic and pathologic cell death processes and is a principal regulator of homeostasis in the lymphoid and myeloid compartment. Bim is crucial for the negative selection of autoreactive immature T- and B-lymphoid cells and for growth factor deprivation-induced apoptosis of many cell types. Loss of BIM in certain human cancers substantiates its role as a tumour suppressor and ample data supports a key role in anticancer therapy. Additional BH3-only proteins such as Bmf, Bad, Hrk and Bik are less well studied and poorly understood. Available data suggests that they support the key effectors Bim, Puma or Bid in potentiating cell killing in a cell-type and stimulus-dependent manner. Functions outside canonical apoptosis signalling remain plausible. BH3-only proteins have a crucial function in chemotherapeutic drug-induced killing of tumour cells and their loss is frequently associated with resistance to anticancer therapy. Some proteins are equally important in killing autoreactive immune cells or can become aberrantly activated in chronic inflammatory or degenerative disorders, contributing to diverse pathologies. Mimicking BH3-only proteins represents a promising strategy for enhancing the effects of conventional anticancer therapy and for treating autoimmune diseases, whereas the blockade of these proteins may be beneficial in the management of certain degenerative diseases that are characterised by abnormal killing of cells that should be kept alive. Keywords: BH3-only; apoptosis; Bcl-2; mitochondria; BH3-mimetics

Background Drug resistance significantly weakens the efficacy of cancer treatment, and the BIM (also known as the BCL2L11 gene) deletion polymorphism has been identified as a potential biomarker for drug resistance. In this retrospective study, we included a total of 290 patients with advanced non-small cell lung cancer (NSCLC) who received treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and chemotherapy. Methods The BIM deletion polymorphism of each patient was detected by polymerase chain reaction. EGFR mutations were detected by denaturing high-performance liquid chromatography methods and the amplification refractory mutation system. Results The BIM deletion polymorphism was detected in 45/290 (15.5%) Chinese NSCLC patients. No associations were observed between the BIM deletion and clinic-pathologic characteristics of patients. The BIM deletion polymorphism was predictive of shorter progression-free survival in Chinese patients with EGFR-mutant adenocarcinoma and who were treated with EGFR-TKIs (7.30 vs. 9.53 months, P = 0.034). Additionally, we found that the BIM deletion polymorphism was an effective predictor of short progression-free survival in individuals with EGFR-mutant NSCLC and treated with chemotherapy containing pemetrexed (3.32 vs. 5.30, P = 0.012) or second-/beyond-line chemotherapy containing taxanes (1.53 vs. 2.61 months, P = 0.025). The BIM deletion was not correlated with overall survival. Conclusion The BIM deletion polymorphism occurs in 15.5% of Chinese NSCLC patients, and is a biomarker for resistance to TKIs and chemotherapy. However, BIM deletion was not a decisive factor in overall survival.

Methylmalonic acidemia (MMA) is an autosomal-recessive inborn metabolic disorder that results from a deficiency in methylmalonyl-coenzyme A mutase or its cofactor, adenosylcobalamin. Currently, neurological manifestations in MMA are thought to be associated with neural apoptosis. BCL2L11, which is a proapoptotic Bcl-2 family member, is resident in the outer mitochondrial membrane, where this protein acts as a central regulator of the intrinsic apoptotic cascade and mediates excitotoxic apoptosis. MicroRNAs (miRNAs) are a class of non-coding RNAs that function as endogenous triggers of the RNA interference pathway. Currently, little is known regarding the role of miRNA in MMA. In our previous study, we preliminarily found that the expression of miR-9 was significantly down-regulated in MMA patient plasma and sensitively changed after VitB12 treatment, which may act as a potential "competitor" of gas chromatography-mass spectrometry for the diagnosis of MMA. In the present study, we first confirmed that miR-9 inhibited BCL2L11 expression by directly targeting its 3'-untranslated region, and the up-regulation of miR-9 reduced neural apoptosis induced by methylmalonate via targeting BCL2L11. Taken together, our results suggested that miR-9 might act as a monitor of changes in MMA and might provide new insights into a therapeutic entry point for treating MMA.

The most frequently occurring cancer in women, and the second leading cause of cancer death among women, is breast cancer. Cancer results from cellular mutations that enhance proliferation and decrease programmed cell death (apoptosis). Secreted frizzled-related proteins (SFRPs) are a family of proteins known for their ability to negatively modulate the Wnt signalling cascade. SFRP1 expression is lost in a multitude of cancers, including breast cancer, and SFRP1 down regulation reduces apoptosis in vitro but the mechanisms remain unclear, as also the effect of Sfrp1 deficiency on apoptosis on mammary epithelial cells in vivo. Our data show that mammary glands from Sfrp1(-/-) mice express significantly less Bcl2l11 (Bim) and Bax mRNA in response to DNA damage. The effect of Sfrp1 loss in reducing γ-irradiation induced apoptosis was examined by TUNEL staining and cleaved-caspase-3 immunostaining. The findings show that Sfrp1(-/-) mice have less DNA fragmentation, whilst caspase-3 expression is decreased, and that p53 expression is generally diminished. Recombinant SFRP1 could replace endogenous expression and elevate the levels of pro-apoptotic and p53-mediated gene expression (Bcl2l, Bax, Cdkn1a and Bbc3) in mammary epithelial cells derived from Sfrp1(-/-) mice. Thus Sfrp1 plays an important role in mediating mammary epithelial apoptotic response to DNA damage in vivo. The role SFRP1 plays in p53 target gene expression was also noted, which suggests that this pathway may be worth exploiting for novel therapies.

The goal of this study was to identify genes related to the metastasis of clear cell renal cell carcinoma (CRCC). We analyzed copy number alterations in renal tissue specimens of patients with CRCC patients with or without metastasis by using high-resolution single-nucleotide polymorphism (SNP) arrays and then integrated these data with gene expression profiling data obtained using oligonucleotide microarrays. The expression levels of target genes were determined by quantitative real-time RT-PCR (qRT-PCR) with an independent tumor set. An analysis of specimens from 23 CRCC cases with SNP arrays revealed that hemizygous deletions at 10q and 13q were found only in cases of metastatic disease. We found the homozygous deletion of TCF7L2 at 10q25.2 in an aggressive case that had hemizygous deletions at 10q. In addition, a qRT-PCR analysis of TCF7L2 mRNA levels in tumor samples revealed significantly lower levels in patients with metastasis when compared with those without metastasis. FOXO1 was identified as a down-regulated gene in the minimal overlapping region of the 13q hemizygous deletion in CRCC. Decreased FOXO1 expression was significantly correlated with metastasis and poor survival outcome. Knockdown of FOXO1 inhibited apoptosis after doxorubicin treatment in CRCC cells and reduced the expression of downstream genes involved in cell proliferation (CDKN1B) and survival (BCL2L11). Lower levels of FOXO1 expression were associated with decreased expression of CDKN1B and BCL2L11 in CRCC specimens. We conclude that FOXO1 and TCF7L2 are involved in metastasis and that molecules in these signaling pathways may be targets for diagnostic procedures and therapies for CRCC.

The deregulation of programmed cell death, apoptosis, is a major contributor to the development of cancer and can impair the response of tumour cells to anticancer therapy. The Bcl-2 family of proteins are critical regulators of apoptosis. BH3 (Bcl-2 homologous 3)-only proteins are pro-apoptotic members that share with each other and the wider Bcl-2 family only the BH3 domain that is critical for their killing capacity. These BH3-only proteins promote cell death by directly or indirectly activating Bax and Bak, in a cell-type and death stimulus-specific manner. Studies of gene-targeted mice that lack two or more BH3-only proteins are beginning to unravel the overlapping functions of these apoptosis initiators. The activation or mimicking of these proteins is expected to improve the treatment of patients suffering from cancer or autoimmune diseases. Key concepts The ‘Bcl-2-regulated’ (also called ‘intrinsic’ or ‘mitochondrial’) apoptotic pathway is triggered by developmental cues or a broad range of cell stressors (e.g. growth factor deprivation, γ-irradiation) and is regulated by the interplay of the pro- and antiapoptotic members of the Bcl-2 family of proteins. The survival versus death fate of a cell is decided by the relative levels of pro- and antiapoptotic Bcl-2 family members. During tumourigenesis defects in apoptosis signalling allow abnormal survival of cells undergoing neoplastic transformation and thereby facilitating sustained cell growth and accumulation of further oncogenic mutations. The Bcl-2 family consists of three subgroups of proteins that can be differentiated on the basis of amino acid sequence, 3D structure and function. The pro-survival members, Bcl-2, Bcl-xL, Bcl-w (Bcl-B), Mcl-1 and A1, are essential for cell survival, with cell-type specific expression. Two subfamilies encompass the apoptosis-promoting Bcl-2 family members: the multi-BH domain pro-apoptotic subfamily members Bax, Bak and Bok, and the BH3-only subfamily members Bad, Bid, Bik/Nbk/Blk, Hrk/DP5, Bim/Bod/Bcl2L11, Noxa, Bmf and Puma/Bbc3. Bax, Bak (and Bok), are the critical activators of the effector phase of the intrinsic death pathway. Both Puma and Noxa, apoptosis initiators that are transcriptionally activated by p53 in response to DNA damage or oncogenic stress, can function as tumour suppressors in their own right, particularly in the context of an oncogenic lesion that subverts cell cycle control. It is important to bear in mind that apoptosis initiation constitutes only one of several critical mechanisms by which p53 suppresses tumour development. Bid functions as the link between the ‘death receptor’ and the ‘Bcl-2-regulated’ apoptotic pathways by causing an amplification of the caspase cascade that leads to cell destruction. Remarkably, Bid is critical for Fas ‘death receptor’-induced apoptosis in certain cell types, such as hepatocytes, but dispensable in others, including lymphoid cells. Bim is critical for many physiologic and pathologic cell death processes and is a principal regulator of homeostasis in the lymphoid and myeloid compartment. Bim is crucial for the negative selection of autoreactive immature T- and B-lymphoid cells, for growth factor deprivation-induced apoptosis of many cell types and loss of BIM in certain human cancers substantiates its role as a tumour suppressor. BH3-only proteins have a crucial function in chemotherapeutic drug-induced killing of tumour cells and their loss is frequently associated with resistance to anticancer therapy. Mimicking BH3-only proteins represents a promising strategy for enhancing the effects of conventional anticancer therapy and for treating autoimmune diseases whereas the blockade of these proteins may be beneficial in the management of certain degenerative diseases that are characterized by abnormal killing of cells that should be kept alive. Keywords: BH3-only; apoptosis; Bcl-2