Immune-related lncRNA classification of head and neck squamous cell carcinoma

Background Long noncoding RNAs (lncRNAs) play a critical role in innate and adaptive immune responses. Thus, we aimed to identify ideal subtypes for head and neck squamous cell carcinoma (HNSCC) based on immune-related lncRNAs. Methods TCGA HNSCC cohort was divided into two datasets (training and validation dataset), and 960 previously characterized immune-related lncRNAs were extracted for non-negative matrix factorization analysis. We characterized our HNSCC subtypes based on biological behaviors, immune landscape and response to immunotherapy in both training and validation cohort. A lncRNA-signature was generated to predict our HNSCC subtypes, and essential lncRNAs involved in tumor microenvironment (TME) were identified. Results We developed and validated two HNSCC subtypes (C1 and C2) based on the 70 lncRNAs in the training and validation cohort. C2 subtype displayed good prognosis, high immune cell infiltration, immune-related genes expression and sensitivity to PD-1 blockade. C1 subtype was associated with high activity of mTORC1 signaling and glycolysis as well as high fraction of inactive immune cells. Finally, we generated a 31-lncRNA signature that could predict our above subtypes with high accurate. Additionally, TRG-AS1 was identified as the essential lncRNA involving TME formation. Knockdown of TRG-AS1 inhibited the expression of HLA-A, HLA-B, HLA-C, CXCL9, CXCL10 and CXCL11. High expression of TRG-AS1 indicated a favorable prognosis in HNSCC and anti-PD-L1 cohort (IMvigor210). Conclusions Our study establishes a novel HNSCC classification on the basis of 31-lncRNA, helping to identify beneficiaries for anti-PD-1 treatment. In addition, a critical lncRNA TRG-AS1 is identified as a new potential prognosis biomarker as well as therapeutic target.

circRNA LDLRAD3 Enhances the Malignant Behaviors of NSCLC Cells via the miR-20a-5p-SLC7A5 Axis Activating the mTORC1 Signaling Pathway

Circular RNA LDLRAD3 behaved as an oncogene in several malignancies, but its effects in NSCLC and the involvement of downstream molecules and activation of signaling pathways had not been fully reported. We planned to explore how LDLRAD3 facilitated the malignancy of NSCLC. QRT-PCR was performed to evaluate the expression levels of LDLRAD3, miR-20a-5p, and SLC7A5 in NSCLC tissues and cells. si-LDLRAD3 was transfected to A549 and H1299 cells to knock down intrinsic LDLRAD3 to determine its oncogenic roles. CCK-8 assay and transwell assay were executed to assess cell proliferative, migrative, and invasive abilities. Dual-luciferase reporter (DLR) assay was manipulated to verify the ENCORI-predicted relationships between LDLRAD3 and miR-20a-5p and between miR-20a-5p and SLC7A5. Western blot, immunofluorescent assay, and immunohistochemistry were applied to explore the expression levels of SLC7A5, and the levels of mTORC1 pathway-related proteins were evaluated using western blot. Rescue experiments were conducted by transfecting si-LDLRAD3, miR-20a-5p inhibitor, and si-SLC7A5 to explore the influence of the LDLRAD3-miR-20a-5p-SLC7A5 axis on the malignant behaviors of NSCLC cells. The expression levels of LDLRAD3 and SLC7A5 were boosted, whereas miR-20a-5p was impeded in NSCLC tissues and cell lines. Knockdown of LDLRAD3 weakened the proliferation, migration, and invasion of A549 and H1299 cells. LDLRAD3 was verified to sponge miR-20a-5p and miR-20a-5p targeted SLC7A5. LDLRAD3 activated the mTORC1 singling pathway via the miR-20a-5p-SLC7A5 axis to strengthen the malignant properties of A549 and H1299 cells. We concluded that LDLRAD3 exerted oncogenic effects via the miR-20a-5p-SLC7A5 axis to activate the mTORC1 signaling pathway in NSCLC. Our findings enlightened that LDLRAD3 could become a potential therapeutic target in the treatment and management of NSCLC.

A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. Importance Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There’s a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

Hbxip (Lamtor5) is essential for embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1

Hbxip, also named Lamtor5, has been well characterized as a transcriptional coactivator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5. Using Hbxip knockout embryonic stem cells (ESCs), we showed that depletion of Hbxip compromises the self-renewal of ESCs, with reduced expression of pluripotency genes, reduced cell proliferation, and decreased colony forming capacity. In addition, Hbxip-/- ESCs are defective in differentiation, particularly ectodermal and mesodermal differentiation. Consistently, Hbxip-/- epiblast fails to differentiate properly, indicated by sustained expression of Oct4 in E8.5 Hbxip-/- epiblast. Mechanistically, in ESCs, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, display differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/-, and p18-/- mice, lacking subunits of the Ragulator complex, also share similar phenotypes, embryonic lethality and retarded growth around E7-8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

The endocrine and epigenetic impact of persistent cow milk consumption on prostate carcinogenesis

This review analyzes the potential impact of milk-induced signal transduction on the pathogenesis of prostate cancer (PCa). Articles in PubMed until November 2021 reporting on milk intake and PCa were reviewed. Epidemiological studies identified commercial cow milk consumption as a potential risk factor of PCa. The potential impact of cow milk consumption on the pathogenesis of PCa may already begin during fetal and pubertal prostate growth, critical windows with increased vulnerability. Milk is a promotor of growth and anabolism via activating insulin-like growth factor-1 (IGF-1)/phosphatidylinositol-3 kinase (PI3K)/AKT/mechanistic target of rapamycin complex 1 (mTORC1) signaling. Estrogens, major steroid hormone components of commercial milk of persistently pregnant dairy cows, activate IGF-1 and mTORC1. Milk-derived signaling synergizes with common driver mutations of the PI3K/AKT/mTORC1 signaling pathway that intersect with androgen receptor, MFG-E8, MAPK, RUNX2, MDM4, TP53, and WNT signaling, respectively. Potential exogenously induced drivers of PCa are milk-induced elevations of growth hormone, IGF-1, MFG-E8, estrogens, phytanic acid, and aflatoxins, as well as milk exosome-derived oncogenic microRNAs including miR-148a, miR-21, and miR-29b. Commercial cow milk intake, especially the consumption of pasteurized milk, which represents the closest replica of native milk, activates PI3K-AKT-mTORC1 signaling via cow milk’s endocrine and epigenetic modes of action. Vulnerable periods for adverse nutrigenomic impacts on prostate health appear to be the fetal and pubertal growth periods, potentially priming the initiation of PCa. Cow milk-mediated overactivation of PI3K-AKT-mTORC1 signaling synergizes with the most common genetic deviations in PCa, promoting PCa initiation, progression, and early recurrence.

mTORC1 Signaling Pathway Mediated SIRT6 Overexpression in TGF-β1-Induced Pulmonary Fibrosis

Fibroblast-to-myofibroblast transdifferentiation and myofibroblast hyperproliferation play a major role in Idiopathic pulmonary fibrosis (IPF). It was also reported that mTOR signaling pathway and SIRT6 have a critical role in pulmonary fibrosis. However, the mechanisms whether mTOR signaling pathway and SIRT6 affect the myofibroblasts differentiation in IPF remain unclear. The results show that SIRT6 is significantly upregulated by TGF-β1 with a time and concentration-dependent manner in MRC5 line and primary lung fibroblasts isolated from IPF patients. SIRT6 protein is also increased in IPF fibrotic lung tissues and bleomycin-challenged mice lung tissues. Also, the activity of mTOR signaling is activated in MRC5 and primary lung fibroblasts. Furthermore, the inhibitor of mTOR, rapamycin treatment significantly suppress mTORC1 pathway activity and SIRT6 protein expression. SIRT6 siRNA failed to mediate the activity of mTORC1 pathway and autophagy induction. Finally, deficiency of SIRT6 could promote TGF-β1 induced pro-fibrotic cytokines. In summary, the study have suggested that SIRT6 is a downstream of mTORC1 signaling pathway in the pulmonary fibrosis caused by TGF-β1-induced. Deficiency of SIRT6 mediated myofibroblasts differentiation through induced pro-fibrotic cytokines production but not induced-autophagy. It was indicated that manipulations of SIRT6 expression may provide a new therapeutic strategy to reverse the progression of pulmonary fibrosis.

mTORC1-Mediated Angiogenesis is Required for the Development of Rosacea

Although multiple evidences suggest that angiogenesis is associated with the pathophysiology of rosacea, its role is still in debate. Here, we showed that angiogenesis was enhanced in skin lesions of both rosacea patients and LL37-induced rosacea-like mice. Inhibition of angiogenesis alleviated LL37-induced rosacea-like features in mice. Mechanistically, we showed that mTORC1 was activated in the endothelial cells of the lesional skin from rosacea patients and LL37-induced rosacea-like mouse model. Inhibition of mTORC1 decreased angiogenesis and blocked the development of rosacea in mice. On the contrary, hyperactivation of mTORC1 increased angiogenesis and exacerbated rosacea-like phenotypes. Our in vitro results further demonstrated that inhibition of mTORC1 signaling significantly declined LL37-induced tube formation of human endothelial cells. Taken together, our findings revealed that mTORC1-mediated angiogenesis responding to LL37 might be essential for the development of rosacea and targeting angiogenesis might be a novel potential therapy.

The Effect of Cancer Cachexia Progression on the Feeding Regulation of Skeletal Muscle Protein Turnover

Cancer cachexia is defined as the unintentional loss of skeletal muscle mass with or without fat loss that cannot be reversed by conventional nutritional support. Cachexia occurs in ~20% of cancer patients. More specifically, 50% of lung cancer patients, the most common cancer worldwide, develop cachexia. Cachexia occurs most often in lung and gastrointestinal cancers, whereas breast and prostate have the lowest rate of cachexia. Cancer-induced cachexia disrupts skeletal muscle protein turnover (decreasing protein synthesis and increasing protein degradation). Skeletal muscle’s capacity for protein synthesis is highly sensitive to local and systemic stimuli that are controlled by mTORC1 and AMPK signaling. During cachexia, altered protein turnover is thought to occur through suppressed anabolic signaling via mTORC1, coinciding with the chronic activation of AMPK. While progress has been made in understanding some of the mechanisms underlying the suppressed anabolic signaling in cachectic muscle, gaps still remain in our understanding of muscle’s ability to respond to anabolic stimulus prior to cachexia development. The purpose of this study was to determine if cachexia progression disrupts the feeding regulation of AMPK signaling and if gp130 signaling and muscle contraction could regulate this process. Specific aim 1 examined the feeding regulation of skeletal muscle protein synthesis in pre-cachectic tumor bearing mice. Feeding increased muscle protein synthesis, while lowering AMPK signaling in pre-cachectic tumor bearing mice. Importantly, pre-cachectic tumor bearing mice have overall suppressed muscle protein synthesis independent of the fast or fed condition. Muscle specific AMPK loss was sufficient to improve the fasting suppression of muscle mTORC1 and protein synthesis in pre-cachectic tumor bearing mice. Specific aim 2 examined if muscle gp130 signaling regulates the feeding regulation of AMPK during cancer cachexia progression. Muscle gp130 loss lowered the fasting induction of AMPK in pre-cachectic tumor bearing mice without improving protein synthesis. Muscle gp130 loss did not alter the feeding regulation of muscle Akt/mTORC1 signaling and protein synthesis. Specific Aim 3 examined if an acute bout of muscle contractions could improve the muscle protein synthesis response to feeding during the progression of cachexia. Pre-cachectic tumor bearing mice exhibit suppressed protein synthesis in response low frequency electrical stimulation, and the inability to synergistically induce protein synthesis in response to feeding and contraction. In summary, pre-cachectic tumor bearing mice have lowered Akt/mTORC1 signaling and protein synthesis. Feeding can induce Akt/mTORC1 and protein synthesis and AMPK regulates the fasting suppression of protein synthesis in pre-cachectic tumor bearing mice. While gp130 loss reduces AMPK it is not sufficient to improve protein synthesis in pre-cachectic tumor bearing mice. The added protein synthesis response to feeding and contraction is blunted in pre-cachectic tumor bearing mice. These findings provide novel insight into the regulation of Akt/mTORC1 signaling and protein synthesis in response to feeding. Additionally, these studies highlight gp130’s regulation of AMPK prior to cachexia development, and the blunted anabolic muscle response to feeding and contraction in pre-cachectic tumor bearing mice. By understanding these intracellular signaling processes and perturbations prior to cachexia development, we will be able to elucidate potential therapeutic targets and treatment options to manipulate and prevent cancer cachexia.

mTORC1 Signaling Pathway Mediates Chronic Stress-Induced Synapse Loss in the Hippocampus

Background: The mechanistic target of rapamycin complex 1 (mTORC1) signaling has served as a promising target for therapeutic intervention of major depressive disorder (MDD), but the mTORC1 signaling underlying MDD has not been well elucidated. In the present study, we investigated whether mTORC1 signaling pathway mediates synapse loss induced by chronic stress in the hippocampus.Methods: Chronic restraint stress-induced depression-like behaviors were tested by behavior tests (sucrose preference test, forced swim test and tail suspension test). Synaptic proteins and alternations of phosphorylation levels of mTORC1 signaling-associated molecules were measured using Western blotting. In addition, mRNA changes of immediate early genes (IEGs) and glutamate receptors were measured by RT-PCR. Rapamycin was used to explore the role of mTORC1 signaling in the antidepressant effects of fluoxetine.Results: After successfully establishing the chronic restraint stress paradigm, we observed that the mRNA levels of some IEGs were significantly changed, indicating the activation of neurons and protein synthesis alterations. Then, there was a significant downregulation of glutamate receptors and postsynaptic density protein 95 at protein and mRNA levels. Additionally, synaptic fractionation assay revealed that chronic stress induced synapse loss in the dorsal and ventral hippocampus. Furthermore, these effects were associated with the mTORC1 signaling pathway-mediated protein synthesis, and subsequently the phosphorylation of associated downstream signaling targets was reduced after chronic stress. Finally, we found that intracerebroventricular infusion of rapamycin simulated depression-like behavior and also blocked the antidepressant effects of fluoxetine.Conclusion: Overall, our study suggests that mTORC1 signaling pathway plays a critical role in mediating synapse loss induced by chronic stress, and has part in the behavioral effects of antidepressant treatment.

Activation of Downstream mTORC1 Target Ribosomal Protein S6 Kinase (S6K) Can Be Found in a Subgroup of Dutch Patients with Granulomatous Pulmonary Disease

Mechanistic target of rapamycin complex 1 (mTORC1) has been linked to different diseases. The mTORC1 signaling pathway is suggested to play a role in the granuloma formation of sarcoidosis. Recent studies demonstrated conflicting data on mTORC1 activation in patients with sarcoidosis by measuring activation of its downstream target S6 kinase (S6K) with either 33% or 100% of patients. Therefore, the aim of our study was to reevaluate the percentage of S6K activation in sarcoidosis patients in a Dutch cohort. To investigate whether this activation is specific for sarcoid granulomas, we also included Dutch patients with other granulomatous diseases of the lung. The activation of the S6K signaling pathway was evaluated by immunohistochemical staining of its downstream effector phospho-S6 in tissue sections. Active S6K signaling was detected in 32 (43%) of the sarcoidosis patients. Twelve (31%) of the patients with another granulomatous disorder also showed activated S6K signaling, demonstrating that the mTORC1 pathway may be activated in a range for different granulomatous diseases (p = 0.628). Activation of S6K can only be found in a subgroup of patients with sarcoidosis, as well as in patients with other granulomatous pulmonary diseases, such as hypersensitivity pneumonitis or vasculitis. No association between different clinical phenotypes and S6K activation can be found in sarcoidosis.