MILIP Binding to tRNAs Promotes Protein Synthesis to Drive Triple-Negative Breast Cancer.

Patients with triple-negative breast cancer (TNBC) have a poor prognosis due to the lack of effective molecular targets for therapeutic intervention. Here we found that the long noncoding RNA (lncRNA) MILIP supports TNBC cell survival, proliferation, and tumorigenicity by complexing with transfer RNAs (tRNA) to promote protein production, thus representing a potential therapeutic target in TNBC. MILIP was expressed at high levels in TNBC cells that commonly harbor loss-of-function mutations of the tumor suppressor p53, and MILIP silencing suppressed TNBC cell viability and xenograft growth, indicating that MILIP functions distinctively in TNBC beyond its established role in repressing p53 in other types of cancers. Mechanistic investigations revealed that MILIP interacted with eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) and formed an RNA-RNA duplex with the type II tRNAs tRNALeu and tRNASer through their variable loops, which facilitated the binding of eEF1α1 to these tRNAs. Disrupting the interaction between MILIP and eEF1α1 or tRNAs diminished protein synthesis and cell viability. Targeting MILIP inhibited TNBC growth and cooperated with the clinically available protein synthesis inhibitor omacetaxine mepesuccinate in vivo. Collectively, these results identify MILIP as an RNA translation elongation factor that promotes protein production in TNBC cells and reveal the therapeutic potential of targeting MILIP, alone and in combination with other types of protein synthesis inhibitors, for TNBC treatment. SIGNIFICANCE: LncRNA MILIP plays a key role in supporting protein production in TNBC by forming complexes with tRNAs and eEF1α1, which confers sensitivity to combined MILIP targeting and protein synthesis inhibitors.

Diagnostic value of serum COMP and ADAMTS7 for intervertebral disc degeneration.

OBJECTIVE: Intervertebral disc degeneration (IVDD) is a major cause of morbidity and disability. Our study aimed to investigate the potential of cartilage oligomeric matrix protein (COMP) and ADAMTS7 (A disintegrin and metalloproteinases with thrombospondin motifs 7) as biomarkers for IVDD together with their functional relationship. METHODS: IVD tissues and peripheral blood samples were collected from IVDD rabbit models over 1-4 weeks. Tissues and blood samples were also collected from clinical patients those were stratified into four equal groups according to Pfirrmann IVDD grading (I-V) with baseline data collected for each participant. COMP and ADAMTS7 expression were analyzed and biomarker characteristics were assessed using linear regression and receiver operating curve (ROC) analyses. RESULTS: COMP and ADAMTS7 expression increased in tissues and serum during IVDD progression. Serum COMP (sCOMP) and serum ADAMTS7 (sADAMTS7) levels increased in a time-dependent manner following IVD damage in the rabbit model while significant positive correlations were detected between sCOMP and sADAMTS7 and Pfirrmann grade in human subjects. ROC analysis showed that combining sCOMP and sADAMTS7 assay results produced an improved diagnostic measure for IVDD compared to individual sCOMP or sADAMTS7 tests. In vitro assays conducted on human cell isolates revealed that COMP prevented extracellular matrix degradation and antagonized ADAMTS7 expression although this protective role was uncoupled under microenvironmental conditions mimicking IVDD. CONCLUSIONS: Increases in circulating COMP and ADAMTS7 correlate with IVDD progression and may play regulatory roles. Assays for sCOMP and/or sADAMTS7 levels can discriminate between healthy subjects and IVDD patients, warranting further clinical assessment.

The long noncoding RNA HNF1A-AS1 with dual functions in the regulation of cytochrome P450 3A4.

Cytochrome P450 3A4 (CYP3A4) is the most important and abundant drug-metabolizing enzyme in the human liver. Inter-individual differences in the expression and activity of CYP3A4 affect clinical and precision medicine. Increasing evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in the regulation of CYP3A4 expression. Here, we showed that lncRNA hepatocyte nuclear factor 1 alpha-antisense 1 (HNF1A-AS1) exerted dual functions in regulating CYP3A4 expression in Huh7 and HepG2 cells. Mechanistically, HNF1A-AS1 served as an RNA scaffold to interact with both protein arginine methyltransferase 1 and pregnane X receptor (PXR), thereby facilitating their protein interactions and resulting in the transactivation of PXR and transcriptional alteration of CYP3A4 via histone modifications. Furthermore, HNF1A-AS1 bound to the HNF1A protein, a liver-specific transcription factor, thereby blocking its interaction with the E3 ubiquitin ligase tripartite motif containing 25, ultimately preventing HNF1A ubiquitination and protein degradation, further regulating the expression of CYP3A4. In summary, these results reveal the novel functions of HNF1A-AS1 as the transcriptional and post-translational regulator of CYP3A4; thus, HNF1A-AS1 may serve as a new indicator for establishing or predicting individual differences in CYP3A4 expression.

RNA m6A modifications regulate crosstalk between tumor metabolism and immunity.

In recent years, m6A modifications in RNA transcripts have arisen as a hot topic in cancer research. Indeed, a number of independent studies have elaborated that the m6A modification impacts the behavior of tumor cells and tumor-infiltrating immune cells, altering tumor cell metabolism along with the differentiation and functional activity of immune cells. This review elaborates on the links between RNA m6A modifications, tumor cell metabolism, and immune cell behavior, discussing this topic from the viewpoint of reciprocal regulation through "RNA m6A-tumor cell metabolism-immune cell behavior" and "RNA m6A-immune cell behavior-tumor cell metabolism" axes. In addition, we discuss the various factors affecting RNA m6A modifications in the tumor microenvironment, particularly the effects of hypoxia associated with cancer cell metabolism along with immune cell-secreted cytokines. Our analysis proposes the conclusion that RNA m6A modifications support widespread interactions between tumor metabolism and tumor immunity. With the current viewpoint that long-term cancer control must tackle cancer cell malignant behavior while strengthening anti-tumor immunity, the recognition of RNA m6A modifications as a key factor provides a new direction for the targeted therapy of tumors. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

FAT1 Gene Expression in Iranian Acute Lymphoid and Myeloid Leukemia Patients.

BACKGROUND: FAT atypical cadherin 1 (FAT1) is a member of the cadherin superfamily whose loss or gain is associated with the initiation and/or progression of different cancers. FAT1 overexpression has been reported in hematological malignancies. This research intended to investigate FAT1 gene expression in adult Iranian acute leukemia patients, compared to normal mobilized peripheral blood CD34+ cells. MATERIALS AND METHODS: The peripheral blast (peripheral blood mononuclear cells) cells of 22 acute myeloid leukemia (AML), 14 acute lymphoid leukemia (ALL) patients, and mobilized peripheral blood CD34+ cells of 12 healthy volunteer stem cell donors were collected. Then, quantitative real-time polymerase chain reaction (qPCR) was used to compare FAT1 gene expression. RESULTS: Overall, there were no significant differences in FAT1 expression between AML and ALL patients (p>0.2). Nonetheless, the mean expression level of FAT1 was significantly higher in leukemic patients (AML and ALL) than in normal CD34+ cells (p=0.029). Additionally, the FAT1 expression levels were significantly higher in both CD34+ and CD34- leukemic patients than in normal CD34+ cells (p=0.028). CONCLUSION: No significant differences were found between FAT1 expression in CD34+ and CD34- leukemic samples (p> 0.3). Thus, higher FAT1 expression was evident in ALL and AML leukemia cells but this appeared unrelated to CD34 expression. This suggests in a proportion of adult acute leukemia, FAT1 expression may prove to be a suitable target for therapeutic strategies.

Dexamethasone Induces Senescence-Associated Changes in Trabecular Meshwork Cells by Increasing ROS Levels Via the TGFß/Smad3-NOX4 Axis.

Glaucoma is a serious complication of glucocorticoid (GC) therapy arising through elevations in intraocular pressure (IOP). Dexamethasone (DEX) is reported to contribute to elevated IOP through different effects on the trabecular meshwork but whether DEX contributes to glaucoma development through the induction of cellular senescence is still unclear. We explored the actions of DEX on transformed human trabecular meshwork cells (HTMCs) using RNA-seq and conducted bioinformatic analyses to determine the affected pathways. Among the 4,103 differentially expressed genes identified in transformed HTMCs treated with 400 nM DEX (2,036 upregulated and 2,067 downregulated genes, respectively), bioinformatic analyses revealed significant enrichment and potential interplay between the transforming growth factor beta (TGFß)41; signaling and cellular senescence pathways. DEX treatment induced senescence changes in primary and transformed HTMCs as indicated by increases in SA-ß-gal positivity, interleukin (IL)-6 secretion, and senescence-associated heterochromatin foci (SAHF) along with selective accumulation of senescence marker p15 and elevations in reactive oxygen species (ROS) levels. Notably, the DEX-induced senescence changes were rescued by treatment with the TGFß/Smad3 pathway inhibitor SIS3. Furthermore, we show that DEX increases cellular ROS levels via upregulation of NADPH oxidase 4 (NOX4) through activation of Smad3, and that SIS3 decreases ROS levels by downregulating NOX4. Instructively, inhibiting NOX4 with GLX351322 and scavenging ROS with NAC were both effective in preventing DEX-induced senescence changes. Similarly, we found in the mouse model that DEX-ac upregulated p15 and NOX4 expression in the trabecular meshwork, with cotreatment with GLX351322 alleviating elevations in IOP. We establish that DEX induces senescence changes in HTMCs by increasing ROS levels via the TGFß/Smad3/NOX4 axis, increasing IOP and contributing to glaucoma development.

Elevated Angiotensin-II Levels Contribute to the Pathogenesis of Open-Angle Glaucoma Via Inducing the Expression of Fibrosis-Related Genes in Trabecular Meshwork Cells Through a ROS/NOX4/SMAD3 Axis.

Glaucoma including primary open-angle glaucoma (POAG) results from elevations in intraocular pressure (IOP). An eye-localized renin-angiotensin system (RAS) has been implicated in IOP regulation, although its mechanism of action and contribution to glaucoma is poorly understood. Here, we detected significant increases in the levels of angiotensin II (ANGII) in aqueous humor samples from POAG patients. Moreover, we determined that the concentrations of ANGII were positively correlated with IOP, suggesting a role for elevated ANGII levels in eye pathogenesis. Functional investigations demonstrated that ANGII induces the expression of fibrosis-related genes of transformed and primary human trabecular meshwork cells (HTMCs) through the transcriptional upregulation of key fibrotic genes. Parallel experiments using a murine periocular conjunctival fornix injection model confirmed that ANGII induces the expression of fibrosis-related genes in trabecular meshwork (TM) cells in vivo along with increasing IOP. ANGII was revealed to function through increasing the levels of reactive oxygen species (ROS) via selectively upregulating NOX4, with NOX4 knockdown or inhibition with GLX351322 alleviating fibrotic changes induced by ANGII. We further show that ANGII activates Smad3, with both GLX351322 and an inhibitor of Smad3 (SIS3) decreasing the phosphorylation of Smad3 and dampening the ANGII-induced increases in fibrotic proteins. Moreover, NOX4 and Smad3 inhibitors also partially rescued the elevated IOP levels induced by ANGII. Our collective results therefore highlight ANGII as a biomarker and treatment target in POAG together with establishing a causal relationship between ANGII and up-regulation of the expression of fibrosis-related genes of TM cells via a NOX4/ROS axis in cooperation with TGFß/Smad3 signaling.

Exploring neurotransmitters and their receptors for breast cancer prevention and treatment.

While psychological factors have long been linked to breast cancer pathogenesis and outcomes, accumulating evidence is revealing how the nervous system contributes to breast cancer development, progression, and treatment resistance. Central to the psychological-neurological nexus are interactions between neurotransmitters and their receptors expressed on breast cancer cells and other types of cells in the tumor microenvironment, which activate various intracellular signaling pathways. Importantly, the manipulation of these interactions is emerging as a potential avenue for breast cancer prevention and treatment. However, an important caveat is that the same neurotransmitter can exert multiple and sometimes opposing effects. In addition, certain neurotransmitters can be produced and secreted by non-neuronal cells including breast cancer cells that similarly activate intracellular signaling upon binding to their receptors. In this review we dissect the evidence for the emerging paradigm linking neurotransmitters and their receptors with breast cancer. Foremost, we explore the intricacies of such neurotransmitter-receptor interactions, including those that impinge on other cellular components of the tumor microenvironment, such as endothelial cells and immune cells. Moreover, we discuss findings where clinical agents used to treat neurological and/or psychological disorders have exhibited preventive/therapeutic effects against breast cancer in either associative or pre-clinical studies. Further, we elaborate on the current progress to identify druggable components of the psychological-neurological nexus that can be exploited for the prevention and treatment of breast cancer as well as other tumor types. We also provide our perspectives regarding future challenges in this field where multidisciplinary cooperation is a paramount requirement.

Mitochondrial SENP2 regulates the assembly of SDH complex under metabolic stress.

Succinate dehydrogenase (SDH) is a heterotetrameric enzyme complex belonging to the mitochondrial respiratory chain and uniquely links the tricarboxylic acid (TCA) cycle with oxidative phosphorylation. Cancer-related SDH mutations promote succinate accumulation, which is regarded as an oncometabolite. Post-translational modifications of SDH complex components are known to regulate SDH activity, although the contribution of SUMOylation remains unclear. Here, we show that SDHA is SUMOylated by PIAS3 and deSUMOylated by SENP2, events dictating the assembly and activity of the SDH complex. Moreover, CBP acetylation of SENP2 negatively regulates its deSUMOylation activity. Under glutamine deprivation, CBP levels decrease, and the ensuing SENP2 activation and SDHA deSUMOylation serve to concurrently dampen the TCA cycle and electron transport chain (ETC) activity. Along with succinate accumulation, this mechanism avoids excessive reactive oxygen species (ROS) production to promote cancer cell survival. This study elucidates a major function of mitochondrial-localized SENP2 and expands our understanding of the role of SUMOylation in resolving metabolic stress.

LncRNA LIMp27 Regulates the DNA Damage Response through p27 in p53-Defective Cancer Cells.

P53 inactivation occurs in about 50% of human cancers, where p53-driven p21 activity is devoid and p27 becomes essential for the establishment of the G1/S checkpoint upon DNA damage. Here, this work shows that the E2F1-responsive lncRNA LIMp27 selectively represses p27 expression and contributes to proliferation, tumorigenicity, and treatment resistance in p53-defective colon adenocarcinoma (COAD) cells. LIMp27 competes with p27 mRNA for binding to cytoplasmically localized hnRNA0, which otherwise stabilizes p27 mRNA leading to cell cycle arrest at the G0/G1 phase. In response to DNA damage, LIMp27 is upregulated in both wild-type and p53-mutant COAD cells, whereas cytoplasmic hnRNPA0 is only increased in p53-mutant COAD cells due to translocation from the nucleus. Moreover, high LIMp27 expression is associated with poor survival of p53-mutant but not wild-type p53 COAD patients. These results uncover an lncRNA mechanism that promotes p53-defective cancer pathogenesis and suggest that LIMp27 may constitute a target for the treatment of such cancers.

Microbiome and metabolic features of tissues and feces reveal diagnostic biomarkers for colorectal cancer.

Microbiome and their metabolites are increasingly being recognized for their role in colorectal cancer (CRC) carcinogenesis. Towards revealing new CRC biomarkers, we compared 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS) metabolite analyses in 10 CRC (TCRC) and normal paired tissues (THC) along with 10 matched fecal samples (FCRC) and 10 healthy controls (FHC). The highest microbial phyla abundance from THC and TCRC were Firmicutes, while the dominant phyla from FHC and FCRC were Bacteroidetes, with 72 different microbial genera identified among four groups. No changes in Chao1 indices were detected between tissues or between fecal samples whereas non-metric multidimensional scaling (NMDS) analysis showed distinctive clusters among fecal samples but not tissues. LEfSe analyses indicated Caulobacterales and Brevundimonas were higher in THC than in TCRC, while Burkholderialese, Sutterellaceaed, Tannerellaceaea, and Bacteroidaceae were higher in FHC than in FCRC. Microbial association networks indicated some genera had substantially different correlations. Tissue and fecal analyses indicated lipids and lipid-like molecules were the most abundant metabolites detected in fecal samples. Moreover, partial least squares discriminant analysis (PLS-DA) based on metabolic profiles showed distinct clusters for CRC and normal samples with a total of 102 differential metabolites between THC and TCRC groups and 700 metabolites different between FHC and FCRC groups. However, only Myristic acid was detected amongst all four groups. Highly significant positive correlations were recorded between genus-level microbiome and metabolomics data in tissue and feces. And several metabolites were associated with paired microbes, suggesting a strong microbiota-metabolome coupling, indicating also that part of the CRC metabolomic signature was attributable to microbes. Suggesting utility as potential biomarkers, most such microbiome and metabolites showed directionally consistent changes in CRC patients. Nevertheless, further studies are needed to increase sample sizes towards verifying these findings.

Chloride intracellular channel 1 promotes esophageal squamous cell carcinoma proliferation via mTOR signalling.

OBJECTIVES: To investigate the clinical significance of Chloride Intracellular Channel 1 (CLIC1) expression in esophageal squamous cell carcinoma (ESCC) and its functional contribution and molecular mechanisms to the progression of ESCC. METHODS: CLIC1 expression was analyzed by immunohistochemistry (IHC) in a cohort of 86 ESCC tissue specimens and paired normal adjacent esophageal tissues. Associations between clinicopathological features of ESCC and CLIC1 expression were determined. In vitro analyses examined CLIC1 expression in the ESCC cell lines KYSE150 and TE1 using RT-PCR and Western blotting. The downstream pathways of CLIC1 were detected by lentiviral shRNA knockdown and subsequent proteomic analyses. CLIC1 siRNA knockdown was performed in ESCC cell lines KYSE150 and TE1 and the functional effects of CLIC1 on the growth and proliferation of ESCC cells were evaluated combined with cell viability and colony formation assays; the mTOR signaling pathway-related proteins were detected by Western blotting based on the previous proteomic data. RESULTS: CLIC1 expression was significantly increased in ex vivo ESCC tissues compared with corresponding normal tissues, and the up-regulation was associated with clinical tumor node metastasis (TNM) classifications. Knockdown of CLIC1 inhibited in vitro cell proliferation of ESCC cell lines KYSE150 and TE1. CLIC1 knockdown down-regulated the protein expression of p-mTOR and the downstream targets Rictor and p-4EBP1 in both KYSE150 and TE1 cell lines. And the CLIC1 knockdown induced inhibition of cell proliferation on ESCC cells could be rescued by mTOR overexpression. CONCLUSIONS: CLIC1 expression increases during esophageal carcinogenesis and it may functionally contribute to the progression of ESCC through growth promotion effects by promoting the mTOR and downstream signaling pathway. CLIC1 therefore constitutes a candidate molecular biomarker of ESCC.

The N-Myc-responsive lncRNA MILIP promotes DNA double-strand break repair through non-homologous end joining.

The protooncoprotein N-Myc, which is overexpressed in approximately 25% of neuroblastomas as the consequence of MYCN gene amplification, has long been postulated to regulate DNA double-strand break (DSB) repair in neuroblastoma cells, but experimental evidence of this function is presently scant. Here, we show that N-Myc transcriptionally activates the long noncoding RNA MILIP to promote nonhomologous end-joining (NHEJ) DNA repair through facilitating Ku70-Ku80 heterodimerization in neuroblastoma cells. High MILIP expression was associated with poor outcome and appeared as an independent prognostic factor in neuroblastoma patients. Knockdown of MILIP reduced neuroblastoma cell viability through the induction of apoptosis and inhibition of proliferation, retarded neuroblastoma xenograft growth, and sensitized neuroblastoma cells to DNA-damaging therapeutics. The effect of MILIP knockdown was associated with the accumulation of DNA DSBs in neuroblastoma cells largely due to decreased activity of the NHEJ DNA repair pathway. Mechanistical investigations revealed that binding of MILIP to Ku70 and Ku80 increased their heterodimerization, and this was required for MILIP-mediated promotion of NHEJ DNA repair. Disrupting the interaction between MILIP and Ku70 or Ku80 increased DNA DSBs and reduced cell viability with therapeutic potential revealed where targeting MILIP using Gapmers cooperated with the DNA-damaging drug cisplatin to inhibit neuroblastoma growth in vivo. Collectively, our findings identify MILIP as an N-Myc downstream effector critical for activation of the NHEJ DNA repair pathway in neuroblastoma cells, with practical implications of MILIP targeting, alone and in combination with DNA-damaging therapeutics, for neuroblastoma treatment.

circPRKAA1 activates a Ku80/Ku70/SREBP-1 axis driving de novo fatty acid synthesis in cancer cells.

Many metabolism-related genes undergo alternative splicing to generate circular RNAs, but their functions remain poorly understood. Here we report that circPRKAA1, a circular RNA (circRNA) derived from the α1 subunit of AMP-activated protein kinase (AMPK), fulfills a fundamental role in maintaining lipid homeostasis. circPRKAA1 expression facilitates fatty acid synthesis and promotes lipid storage through two coordinated functions. First, circPRKAA1 promotes a tetrameric complex between the Ku80/Ku70 heterodimer and the mature form of sterol regulatory element-binding protein 1 (mSREBP-1) to enhance the stability of mSREBP-1. Second, circPRKAA1 selectively binds to the promoters of the ACC1, ACLY, SCD1, and FASN genes to recruit mSREBP-1, upregulating their transcription and increasing fatty acid synthesis to promote cancer growth. circPRKAA1 biogenesis is negatively regulated by AMPK activity, with lower AMPK activation in hepatocellular carcinoma tissue frequently associated with elevated circPRKAA1 expression. This work identifies circPRKAA1 as an integral element of AMPK-regulated reprogramming of lipid metabolism in cancer cells.

LncRNA MILIP links YBX1 to translational activation of Snai1 and promotes metastasis in clear cell renal cell carcinoma.

BACKGROUND: Distant metastasis is the major cause of clear cell renal cell carcinoma (ccRCC)-associated mortality. However, molecular mechanisms involved in ccRCC metastasis remain to be fully understood. With the increasing appreciation of the role of long non-coding RNAs (lncRNAs) in cancer development, progression, and treatment resistance, the list of aberrantly expressed lncRNAs contributing to ccRCC pathogenesis is expanding rapidly. METHODS: Bioinformatics analysis was carried out to interrogate publicly available ccRCC datasets. In situ hybridization and qRT-PCR assays were used to test lncRNA expression in human ccRCC tissues and cell lines, respectively. Chromatin immunoprecipitation and luciferase reporter assays were used to examine transcriptional regulation of gene expression. Wound healing as well as transwell migration and invasion assays were employed to monitor ccRCC cell migration and invasion in vitro. ccRCC metastasis was also examined using mouse models in vivo. RNA pulldown and RNA immunoprecipitation were performed to test RNA-protein associations, whereas RNA-RNA interactions were tested using domain-specific chromatin isolation by RNA purification. RESULTS: MILIP expression was upregulated in metastatic compared with primary ccRCC tissues. The increased MILIP expression in metastatic ccRCC cells was driven by the transcription factor AP-2 gamma (TFAP2C). Knockdown of MILIP diminished the potential of ccRCC cell migration and invasion in vitro and reduced the formation of ccRCC metastatic lesions in vivo. The effect of MILIP on ccRCC cells was associated with alterations in the expression of epithelial-to-mesenchymal transition (EMT) hallmark genes. Mechanistically, MILIP formed an RNA-RNA duplex with the snail family transcriptional repressor 1 (Snai1) mRNA and bound to Y-box binding protein 1 (YBX1). This promoted the association between the YBX1 protein and the Snai1 mRNA, leading to increased translation of the latter. Snai1 in turn played an important role in MILIP-driven ccRCC metastasis. CONCLUSIONS: The TFAP2C-responsive lncRNA MILIP drives ccRCC metastasis. Targeting MILIP may thus represent a potential avenue for ccRCC treatment.

TRIMming down autophagy in breast cancer.

Macroautophagy/autophagy is a conserved lysosome-dependent metabolic recycling pathway. ULK1 plays an essential role in autophagy initiation through a complex formed with ATG13, RB1CC1/FIP200, and ATG101 in mammalian cells. However, while autophagy is triggered by nutrient starvation where it is essential for cell survival, such conditions lead to the rapid degradation of ULK1, indicating that autophagy must be tightly controlled. Nevertheless, the precise mechanisms regulating the ULK1 complex are still largely unknown. Here we reveal the critical roles played by two novel ULK1 complex binding proteins in autophagy regulation, TRIM27 and STK38L. We show that basal autophagy is maintained through TRIM27-mediated ubiquitination and proteasomal degradation of ULK1, whereas under starvation conditions, excessive autophagy is restrained by the combined actions of TRIM27 and STK38L. TRIM27 ubiquitinates and activates STK38L which in turn phosphorylates ULK1, delivering ULK1 in a permissive state for hyper-ubiquitination by TRIM27. Thus, TRIM27 and STK38L kinase act in concert as a rheostat to control ULK1 levels. We further demonstrate increased basal autophagy in <i>trim27</i> knockout mice and establish physiological relevance in the context of breast cancer. Our study highlights the STK38L-TRIM27-ULK1 axis as a potential treatment avenue to explore for activating autophagy in various disease states.

High nerve density in breast cancer is associated with poor patient outcome.

Active crosstalk between the nervous system and breast cancer cells has been experimentally demonstrated in vitro and in animal models. However, low frequencies of peripheral nerve presence in human breast cancers reported in previous studies (~30% of cases) potentially negate a major role of the nervous system in breast cancer development and progression. This study aimed to clarify the incidence of nerves within human breast cancers and to delineate associations with clinicopathological features. Immunohistochemical staining was conducted in formalin-fixed paraffin-embedded breast cancer tissue sections using antibodies against the pan-neuronal markers protein gene product 9.5 and growth-associated protein 43, and the sympathetic nerve-specific marker tyrosine hydroxylase. Nerve trunks and isolated nerve fibers were quantitated. The chi-squared test was used to determine the associations between nerve counts and clinicopathological parameters. The log-rank test was used to compare differences in patient progression-free survival (PFS) and overall survival (OS). The overall frequency of peripheral nerves in breast cancers was 85%, a markedly higher proportion than reported previously. Of note, most nerves present in breast cancers were of the sympathetic origin. While high density of nerve trunks or isolated nerve fibers was associated with poor PFS and OS of patients, high nerve trunk density appeared also to predict poor patient PFS independently of lymph node metastasis. Innervation of breast cancers is a common event correlated with poor patient outcomes. These findings support the notion that the nervous system plays an active role in breast cancer pathogenesis.

TRIM27 cooperates with STK38L to inhibit ULK1-mediated autophagy and promote tumorigenesis.

Autophagy represents a fundamental mechanism for maintaining cell survival and tissue homeostasis in response to physiological and pathological stress. Autophagy initiation converges on the FIP200-ATG13-ULK1 complex wherein the serine/threonine kinase ULK1 plays a central role. Here, we reveal that the E3 ubiquitin ligase TRIM27 functions as a negative regulatory component of the FIP200-ATG13-ULK1 complex. TRIM27 directly polyubiquitinates ULK1 at K568 and K571 sites with K48-linked ubiquitin chains, with proteasomal turnover maintaining control over basal ULK1 levels. However, during starvation-induced autophagy, TRIM27 catalyzes non-degradative K6- and K11-linked ubiquitination of the serine/threonine kinase 38-like (STK38L) kinase. In turn, STK38L ubiquitination promotes its activation and phosphorylation of ULK1 at Ser495, rendering ULK1 in a permissive state for TRIM27-mediated hyper-ubiquitination of ULK1. This cooperative mechanism serves to restrain the amplitude and duration of autophagy. Further evidence from mouse models shows that basal autophagy levels are increased in Trim27 knockout mice and that Trim27 differentially regulates tumorigenesis and metastasis. Our study identifies a key role of STK38L-TRIM27-ULK1 signaling axis in negatively controlling autophagy with relevance established in human breast cancer.

Stub1 maintains proteostasis of master transcription factors in embryonic stem cells.

The pluripotency and differentiation states of embryonic stem cells (ESCs) are regulated by a set of core transcription factors, primarily Sox2, Oct4, and Nanog. Although their transcriptional regulation has been studied extensively, the contribution of posttranslational modifications in Sox2, Oct4, and Nanog are poorly understood. Here, using a CRISPR-Cas9 knockout library screen in murine ESCs, we identify the E3 ubiquitin ligase Stub1 as a negative regulator of pluripotency. Manipulation of Stub1 expression in murine ESCs shows that ectopic Stub1 expression significantly reduces the protein half-life of Sox2, Oct4, and Nanog. Mechanistic investigations reveal Stub1 catalyzes the polyubiquitination and 26S proteasomal degradation of Sox2 and Nanog through K48-linked ubiquitin chains and Oct4 via K63 linkage. Stub1 deficiency positively enhances somatic cell reprogramming and delays differentiation, whereas its enforced expression triggers ESC differentiation. The discovery of Stub1 as an integral pluripotency regulator strengthens our understanding of ESC regulation beyond conventional transcriptional control mechanisms.

Verification and Validation of a Four-Gene Panel as a Prognostic Indicator in Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is a highly aggressive subtype with a high rate of metastasis, early distant recurrence and resistance to therapy leading to worse survival than other breast cancer subtypes. There are no well-established biomarkers that can determine women who will do better and those who are likely to have poorer outcomes with TNBC, nor are there targeted therapies. Thus, the identification of prognostic and/or predictive biomarkers will enable tailored therapies based on their likelihood of disease outcomes and may prevent over- and under-diagnosis. Previous studies from our laboratory have identified four genes (ANP32E, DSC2, ANKRD30A and IL6ST/gp130) that are specific to TNBC and were associated with lymph node metastasis (LNmets), the earliest indicator of tumor progression via distal spread. This study aimed to validate these findings using absolute quantitation by digital droplet PCR (ddPCR) and to determine relationships with clinicopathological features and survival. Our analysis confirmed all four genes displayed significant expression differences between TNBC cases and non-TNBC cases. Moreover, low IL6ST expression was significantly associated with grade 3 disease, hormone receptor negativity and earlier age at diagnosis; low ANKRD30A expression was associated with tumor size; and high ANP32E expression was significantly associated with grade and the number of positive lymph nodes. Individually, three of the four genes were associated with relapse-free survival in TNBC and in combination, all four genes were significantly associated with TNBC survival, but not in hormone receptor-positive cases. Collectively our results suggest that the four genes may have utility in TNBC prognostication.