DNA Methylome Changes of Muscle- and Neuronal-Related Processes Precede Bladder Cancer Invasiveness.

Bladder cancer (BC) is the ninth leading cause of cancer death with one of the highest recurrence rates among all cancers. One of the main risks for BC development is exposure to nitrosamines present in tobacco smoke or in other products. Aberrant epigenetic (DNA methylation) changes accompanied by deregulated gene expression are an important element of cancer pathogenesis. Therefore, we aimed to determine DNA methylation signatures and their impacts on gene expression in mice treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), a carcinogen similar to compounds found in tobacco smoke. Following BBN administration mice developed non-invasive or invasive bladder cancers. Surprisingly, muscle- and neuronal-related pathways emerged as the most affected in those tumors. Hypo- and hypermethylation changes were present within non-invasive BC, across CpGs mapping to the genes involved in muscle- and neuronal-related pathways, however, methylation differences were not sufficient to affect the expression of the majority of associated genes. Conversely, invasive tumors displayed hypermethylation changes that were linked with alterations in gene expression profiles. Together, these findings indicate that bladder cancer progression could be revealed through methylation profiling at the pre-invasive cancer stage that could assist monitoring of cancer patients and guide novel therapeutic approaches.

Activation of cGAS-STING Pathway Is Associated with MSI-H Stage IV Colorectal Cancer.

Colorectal cancer is the second most common cause of cancer-related mortality in adults. Understanding colorectal tumorigenesis at both the cellular and molecular levels is crucial for developing effective treatment options. Forty-one biopsy samples from patients with metastatic CRC (mCRC) were collected at Split University Hospital in Croatia. A total of 41 patients (21 with microsatellite unstable tumours and 20 with microsatellite stable tumours) were randomly included in the study. Immunolabelling of cGAS and STING in metastatic CRC was performed and further complemented by histological classification, tumour grade, and KRAS, NRAS, and BRAF mutational status of mCRC. In bivariate analysis, elevated expression of cGAS and STING was positively associated with MSI-H colon cancer (Fisher's exact test, both p = 0.0203). Combined expression analysis of cGAS and STING showed a significantly higher percentage of patients with mCRC MSI-H with a fully or partially activated cGAS-STING signalling pathway (chi-square test, p = 0.0050). After adjusting for age, sex, and STING expression, increased cGAS expression remained significantly associated with MSI-H colon cancer in a multiple logistic regression model (ß = 1.588, SE = ±0.799, p = 0.047). The cGAS-STING signalling axis represents a compelling new target for optimization of immune checkpoint inhibitor therapeutic approaches in patients with MSI-H stage IV CRC.

BBN-driven urinary bladder cancer mouse model.

Around 3% of new cancer diagnoses and 2% of all cancer deaths every year are caused by urinary bladder cancer (BC). This indicates a great need for intensive studying of BC by using different approaches including indispensable mice models. The most common preclinical mouse model of bladder carcinogenesis relies on the use of a nitrosamine compound, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) which causes high-grade, invasive tumors in the urinary bladder. BBN-induced bladder cancer in mice recapitulates the histology and manifests genetic alterations similar to human muscle-invasive bladder cancer. Here we present a detailed protocol for the induction of BC in mice which is based on the administration of 0.05%-0.1% BBN in drinking water. Six-to-eight-week-old mice are treated orally with BBN for 12weeks and tumors are expected 8weeks after the termination of BBN regimen. Histopathologic examination of the lesions should be routinely assessed after hematoxylin and eosin staining by an experienced pathologist and it can vary from urothelial dysplasia to invasive bladder cancer with glandular and squamous divergent differentiation, the incidence of which might depend on the mouse strain, gender, BBN concentration and the timeline of the protocol. Utilizing half of the urinary bladder tissue for the isolation and analysis of RNA, DNA and proteins provides a comprehensive insight into the biology of BC and reduces the number of mice per study. Finally, the successful use of the BC model can facilitate fundamental biomedical discoveries leading to novel diagnostic and therapeutic approaches with clinical benefits.

Vitamin A Rich Diet Diminishes Early Urothelial Carcinogenesis by Altering Retinoic Acid Signaling.

Urinary bladder cancer is one of the leading malignancies worldwide, with the highest recurrence rates. A diet rich in vitamin A has proven to lower the risk of cancer, yet the molecular mechanisms underlying this effect are unknown. We found that vitamin A decreased urothelial atypia and apoptosis during early bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Vitamin A did not alter urothelial cell desquamation, differentiation, or proliferation rate. Genes like Wnt5a, involved in retinoic acid signaling, and transcription factors Pparg, Ppara, Rxra, and Hoxa5 were downregulated, while Sox9 and Stra6 were upregulated in early urothelial carcinogenesis. When a vitamin A rich diet was provided during BBN treatment, none of these genes was up- or downregulated; only Lrat and Neurod1 were upregulated. The lecithin retinol acyltransferase (LRAT) enzyme that produces all-trans retinyl esters was translocated from the cytoplasm to the nuclei in urothelial cells as a consequence of BBN treatment regardless of vitamin A rich diet. A vitamin A-rich diet altered retinoic acid signaling, decreased atypia and apoptosis of urothelial cells, and consequently diminished early urothelial carcinogenesis.

Targeting Stat3 signaling impairs the progression of bladder cancer in a mouse model.

Bladder cancer is the fourth most commonly diagnosed malignancy in men worldwide and has one of the highest recurrence rates of all cancers. This cancer type is unique because chronic inflammation caused by Schistosoma haematobium can cause bladder cancer, while inflammation induced by Bacillus Calmette Guerin is the therapeutic cornerstone for this cancer type. Activation of proinflammatory IL-6/Stat3 axis promotes the development of different cancers by acting on cancer cells as well as by modulating cancer microenvironment. Using a genetic and pharmacological approach in a mouse model, we demonstrated the importance of IL-6 and Stat3 signaling in bladder cancer. Our findings show that pharmacological inhibition of Stat3 with WP1066 effectively delays progression and invasiveness of bladder cancer in N-butyl-N-(4-hydroxybutyl) nitrosamine-induced mouse model. Moreover, either IL-6 blockade or Stat3 inhibition sensitized bladder cancer to anti-PD-L1 immune therapy. Taken together, our study demonstrates an important role of IL-6/Stat3 signaling in bladder cancer and creates a rationale for testing the therapeutic potential of Stat3 inhibitors in human MIBC both alone or in combination with anti-PD-L1 and anti-IL-6 therapy.

Microbiota Alters Urinary Bladder Weight and Gene Expression.

We studied the effect of microbiota on the transcriptome and weight of the urinary bladder by comparing germ-free (GF) and specific pathogen-free (SPF) housed mice. In total, 97 genes were differently expressed (fold change > ±2; false discovery rate (FDR) p-value < 0.01) between the groups, including genes regulating circadian rhythm (Per1, Per2 and Per3), extracellular matrix (Spo1, Spon2), and neuromuscular synaptic transmission (Slc18a3, Slc5a7, Chrnb4, Chrna3, Snap25). The highest increase in expression was observed for immunoglobulin genes (Igkv1-122, Igkv4-68) of unknown function, but surprisingly the absence of microbiota did not change the expression of the genes responsible for recognizing microbes and their products. We found that urinary bladder weight was approximately 25% lighter in GF mice (p = 0.09 for males, p = 0.005 for females) and in mice treated with broad spectrum of antibiotics (p = 0.0002). In conclusion, our data indicate that microbiota is an important determinant of urinary bladder physiology controlling its gene expression and size.

The dynamics of the inflammatory response during BBN-induced bladder carcinogenesis in mice.

BACKGROUND: Bladder cancer (BC) is the most common malignant disease of the urinary tract. Recurrent high grade non muscle invasive BC carries a serious risk for progression and subsequent metastases. The most common preclinical mouse model for bladder cancer relies on administration of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) to mice. BBN-induced tumors in mice recapitulate the histology of human BC and were characterized with an overexpression of markers typical for basal-like cancer subtype in addition to a high mutational burden with frequent mutations in Trp53, similar to human muscle invasive BC. METHODS: Bladder cancer was induced in C57BL/6J male mice by administering the BBN in the drinking water. A thorough histopathological analysis of bladder specimen during and post BBN treatment was performed at 2, 4, 16, 20 and 25 weeks. RNA sequencing and qPCR was performed to assess the levels of expression of immunologically relevant genes at 2 weeks and 20 weeks during and post BBN treatment. RESULTS: We characterized the dynamics of the inflammatory response in the BBN-induced BC in mice. The treatment with BBN had gradually induced a robust inflammation in the first 2 weeks of administration, however, the inflammatory response was progressively silenced in the following weeks of the treatment, until the progression of the primary carcinoma. Tumors at 20 weeks were characterized with a marked upregulation of IL18 when compared to premalignant inflammatory response at 2 weeks. In accordance with this, we observed an increase in expression of IFNγ-responsive genes coupled to a pronounced lymphocytic infiltrate during the early stages of malignant transformation in bladder. Similar to human basal-like BC, BBN-induced murine tumors displayed an upregulated expression of immunoinhibitory molecules such as CTLA-4, PD-L1, and IDO1 which can lead to cytotoxic resistance and tumor escape. CONCLUSIONS: Despite the recent advances in bladder cancer therapy which include the use of checkpoint inhibitors, the treatment options for patients with locally advanced and metastatic BC remain limited. BBN-induced BC in mice displays an immunological profile which shares similarities with human MIBC thus representing an optimal model for preclinical studies on immunomodulation in management of BC.

The urinary microbiome associated with bladder cancer.

Recent findings suggest that human microbiome can influence the development of cancer, but the role of microorganisms in bladder cancer pathogenesis has not been explored yet. The aim of this study was to characterize and compare the urinary microbiome of bladder cancer patients with those of healthy controls. Bacterial communities present in urine specimens collected from 12 male patients diagnosed with bladder cancer, and from 11 healthy, age-matched individuals were analysed using 16S sequencing. Our results show that the most abundant phylum in both groups was Firmicutes, followed by Actinobacteria, Bacteroidetes and Proteobacteria. While microbial diversity and overall microbiome composition were not significantly different between groups, we could identify operational taxonomic units (OTUs) that were more abundant in either group. Among those that were significantly enriched in the bladder cancer group, we identified an OTU belonging to genus Fusobacterium, a possible protumorigenic pathogen. In an independent sample of 42 bladder cancer tissues, 11 had Fusobacterium nucleatum sequences detected by PCR. Three OTUs from genera Veillonella, Streptococcus and Corynebacterium were more abundant in healthy urines. However, due to the limited number of participants additional studies are needed to determine if urinary microbiome is associated with bladder cancer.

SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks.

Ruijs-Aalfs syndrome is a segmental progeroid syndrome resulting from mutations in the SPRTN gene. Cells derived from patients with SPRTN mutations elicit genomic instability and people afflicted with this syndrome developed hepatocellular carcinoma. Here we describe the molecular mechanism by which SPRTN contributes to genome stability and normal cellular homeostasis. We show that SPRTN is a DNA-dependent mammalian protease required for resolving cytotoxic DNA-protein crosslinks (DPCs)- a function that had only been attributed to the metalloprotease Wss1 in budding yeast. We provide genetic evidence that SPRTN and Wss1 function distinctly in vivo to resolve DPCs. Upon DNA and ubiquitin binding, SPRTN can elicit proteolytic activity; cleaving DPC substrates and itself. SPRTN null cells or cells derived from patients with Ruijs-Aalfs syndrome are impaired in the resolution of covalent DPCs in vivo. Collectively, SPRTN is a mammalian protease required for resolving DNA-protein crosslinks in vivo whose function is compromised in Ruijs-Aalfs syndrome patients.

The impact of IL-6 and IL-28B gene polymorphisms on treatment outcome of chronic hepatitis C infection among intravenous drug users in Croatia.

BACKGROUND: Several genes and their single nucleotide polymorphisms (SNPs) are associated with either spontaneous resolution of hepatitis C infection or better treatment-induced viral clearance. We tested a cohort of intravenous drug users (IVDU) diagnosed with chronic hepatitis C virus (HCV) for treatment response and its association with the SNPs in the interleukin-6 (rs1800795-IL6) and the interleukin-28B (rs12979860-IL28B) genes. METHODS: The study included 110 Croatian IVDU positive for anti-HCV antibody. Genotyping was performed by polymerase chain reaction (PCR) based approach. Patients were treated by standard pegylated-interferon/ribavirin and followed throughout a period of four years, during which sustained virological response (SVR) was determined. All data were analysed with statistical package SPSS 19.0 (IBM Corp, Armonk, NY, USA) and PLINK v1.07 software. RESULTS: Patients showed a significantly better response to treatment according to the number of copies of the C allele carried at rs1800795-IL6 (P = 0.034). All but one of the patients with CC genotype achieved SVR (93%), whereas the response rate of patients with GG genotype was 64%. The association of rs1800795-IL6 with SVR status remained significant after further adjustment for patients' age, fibrosis staging, and viral genotype (OR 2.15, 95% CI 1.16-4.68, P = 0.019). Distributions of allele frequencies at the locus rs12979860-IL28B among the study cohort and the underlying general population were suggestive of a protective effect of CC genotype in acquiring chronic hepatitis C in the Croatian IVDU population. DISCUSSION: The rs1800795-IL6 polymorphism is associated with positive response to treatment in IVDU patients positive for HCV infection. A protective role of rs12979860-IL28B CC genotype in acquiring chronic hepatitis C is suggested for Croatian IVDU population.

Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features.

Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.

Carpal tunnel syndrome is associated with high fibrinogen and fibrinogen deposits.

BACKGROUND: Idiopathic carpal tunnel syndrome (ICTS) is a common entrapment neuropathy. Some cases of ICTS are linked to mutations of the transthyretin gene, whereas others are associated with systemic amyloidosis. The majority of ICTS cases are of unknown etiology. OBJECTIVE: To study molecular mechanisms of ICTS development. METHODS: A total of 71 ICTS patients and 68 control subjects were included in the study. The fibrinogen level was determined before surgery and its deposition in the transversal carpal ligament (TCL) was detected by immunohistochemistry, Western blot, and mass spectrometry. Fibrinogen interaction with other proteins was studied by immunoprecipitation assay. RESULTS: Plasma levels of the proinflammatory and hemostatic protein fibrinogen are elevated in ICTS patients. Other measured systemic inflammatory markers were not affected, and local inflammatory responses in TCL were absent. ICTS patients have shorter bleeding times, probably because of the elevated plasma levels of fibrinogen. Polymorphisms of the fibrinogen B promoter region were previously associated with increased plasma fibrinogen, but this association was not observed among patients with ICTS. Interestingly, we detected fibrinogen deposits in the TCL, whereas transcriptional activity of the fibrinogen genes was low. Amyloidogenic proteins, including transthyretin and α-synuclein, were also found in the TCL, whereas their local transcriptional activity was rather high. Finally, we demonstrated that fibrinogen interacts with transthyretin and α-synuclein in TCL lysates. CONCLUSION: Our data indicate that fibrinogen and other aggregation-prone proteins have potentially important roles in the pathogenesis of ICTS.

High NF-κB and STAT3 activity in human urothelial carcinoma: a pilot study.

PURPOSE: Given that the tumor-promoting inflammation has been previously established in squamous cell carcinoma of the bladder but its contribution to development of urothelial carcinoma (UC) still remains elusive, our aim was to study changes in expression and activity of inflammation-mediating NF-κB and STAT3 transcription factors in human urothelial bladder carcinoma as well as expression of their target genes cyclin D1, VEGFA and TGFß1. METHODS: Gene expression of STAT3, NF-κB, TGFß1, cyclin D1 and VEGFA was measured by quantitative real-time polymerase chain reaction in both tumor and healthy bladder tissue from 36 patients with UC of the bladder. Activation of STAT3 and NF-κB was assessed with immunohistochemistry and immunoblot. RESULTS: Urothelial bladder carcinoma displayed elevated expression as well as activation of NF-κB (P = 5.38e-10) and STAT3 (P = 0.002) transcription factors. Furthermore, elevated level of expression was observed for cyclin D1, VEGFA and TGFß1 (P = 9.71e-09, P = 9.71e-09, P = 5.38e-10). Preliminary statistical analysis indicated that the level of upregulation of STAT3 or NF-κB was probably not dependent upon the grade (P = 0.984 and 0.803, respectively) and invasiveness of the tumor (0.399 and 0.949), nor to the gender (0.780 and 0.536) and age (0.660 and 0.816) of the patients. CONCLUSIONS: NF-κB and STAT3 signaling pathways, as main inflammatory mediators, are found to be activated in urothelial bladder carcinoma indicating that chronic inflammatory processes are accompanying development of this tumor type. Future studies will have to determine possible causative role of inflammatory processes in development of urothelial bladder carcinomas.

Association of Kaposi's sarcoma-associated herpesvirus (KSHV) with bladder cancer in Croatian patients.

As the seventh most common human malignancy, bladder cancer represents a global health problem. In addition to well-recognized risk factors such as smoking and exposure to chemicals, various infectious agents have been implicated as cofactors in the pathogenesis of urothelial malignancies. The aim of the present study was to assess the possible association of viral infection and bladder cancer in Croatian patients. Biopsy specimens were collected from a total of 55 patients diagnosed with different stages of bladder cancer. Initial screening of DNA extracts for the presence of viruses on Lawrence Livermore Microbial Detection Array revealed Kaposi's sarcoma-associated herpesvirus (KSHV) in each of three randomly chosen biopsy specimens. The prevalence of infection with KSHV among study population was then examined by KSHV-specific polymerase chain reaction (PCR) and immunoblotting. By nested PCR, KSHV DNA was detected in 55% of patients. KSHV, also known as human herpesvirus 8, is an infectious agent known to cause cancer. Its oncogenic potential is primarily recognized from its role in Kaposi's sarcoma, but it has also been involved in pathogenesis of two lymphoproliferative disorders. A high prevalence of KSHV infection in our study indicates that KSHV may play a role in tumorigenesis of bladder cancer and warrants further studies.

Ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates.

Aggregation of misfolded proteins and the associated loss of neurons are considered a hallmark of numerous neurodegenerative diseases. Optineurin is present in protein inclusions observed in various neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Creutzfeld-Jacob disease and Pick's disease. Optineurin deletion mutations have also been described in ALS patients. However, the role of optineurin in mechanisms of protein aggregation remains unclear. In this report, we demonstrate that optineurin recognizes various protein aggregates via its C-terminal coiled-coil domain in a ubiquitin-independent manner. We also show that optineurin depletion significantly increases protein aggregation in HeLa cells and that morpholino-silencing of the optineurin ortholog in zebrafish causes the motor axonopathy phenotype similar to a zebrafish model of ALS. A more severe phenotype is observed when optineurin is depleted in zebrafish carrying ALS mutations. Furthermore, TANK1 binding kinase 1 (TBK1) is colocalized with optineurin on protein aggregates and is important in clearance of protein aggregates through the autophagy-lysosome pathway. TBK1 phosphorylates optineurin at serine 177 and regulates its ability to interact with autophagy modifiers. This study provides evidence for a ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates as well as additional relevance for TBK1 as an upstream regulator of the autophagic pathway.

Histamine N-methyltransferase Thr105Ile polymorphism is associated with Parkinson's disease.

Histamine is a central neurotransmitter degraded by histamine-N-methyltransferase (HNMT). Several abnormalities in the histaminergic system were found in patients with Parkinson's disease (PD), thus we tested the possible association of a Thr105Ile functional polymorphism in HNMT with PD. A total of 913 patients with PD and 958 controls were genotyped using a TaqMan RT-PCR Genotyping Assay (Foster City, California, USA). Lower frequency of HNMT Ile105 allele that is associated with decreased enzymatic activity was found in patients compared with controls (χ(2) = 11.65; p = 0.0006). We performed meta-analysis to confirm the association of Thr105Ile functional polymorphism with PD. Our results indicate that lower HNMT activity plays a role in the pathogenesis of PD.

SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis.

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

No association between histamine N-methyltransferase functional polymorphism Thr105Ile and Alzheimer's disease.

Several abnormalities, including lower histamine levels in brain, elevated serum histamine and degeneration of histaminergic neurons in tuberomammillary nucleus, were described in the histaminergic system of patients with Alzheimer's disease (AD). Histamine is a central neurotransmitter with several functions in brain including regulation of memory, cognition, locomotion, and is degraded in part by histamine N-methyltransferase (HNMT). A common Thr105Ile polymorphism within HNMT gene results in decreased enzyme activity. The Thr105Ile polymorphism was associated with Parkinson's disease, essential tremor, attention-deficit hyperactivity disorder (ADHD), asthma and alcoholism, thus we tested possible association of HNMT functional polymorphism with AD. We have tested 256 AD cases and 1190 healthy controls of Croatian origin. Thr105Ile polymorphism was determined by TaqMan RT-PCR Genotyping Assay and EcoRV digestion. Prevalence of functional HNMT polymorphism among all tested groups was similar and frequency of less active Ile105 variant was 11.5% among AD patients and 13.4% for healthy controls (p=0.26, X(2)=1.25). Our results indicate lack of the association of HNMT Thr105Ile functional polymorphism with Alzheimer's disease.

Inflammation and colon cancer.

The connection between inflammation and tumorigenesis is well-established and in the last decade has received a great deal of supporting evidence from genetic, pharmacological, and epidemiological data. Inflammatory bowel disease is an important risk factor for the development of colon cancer. Inflammation is also likely to be involved with other forms of sporadic as well as heritable colon cancer. The molecular mechanisms by which inflammation promotes cancer development are still being uncovered and could differ between colitis-associated and other forms of colorectal cancer. Recent work has elucidated the role of distinct immune cells, cytokines, and other immune mediators in virtually all steps of colon tumorigenesis, including initiation, promotion, progression, and metastasis. These mechanisms, as well as new approaches to prevention and therapy, are discussed in this review.

Nix is a selective autophagy receptor for mitochondrial clearance.

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.