Nlp-dependent ER-to-Golgi transport.

The mechanism that maintains ER-to-Golgi vesicles formation and transport is complicated. As one of the adapters, Ninein-like protein (Nlp) participated in assembly and transporting of partial ER-to-Golgi vesicles that contained specific proteins, such as ß-Catenin and STING. Nlp acted as a platform to sustain the specificity and continuity of cargoes during COPII and COPI-coated vesicle transition and transportation through binding directly with SEC31A as well as Rab1B. Thus, we proposed an integrated transport model that particular adapter participated in specific cargo selection or transportation through cooperating with different membrane associated proteins to ensure the continuity of cargo trafficking. Deficiency of Nlp led to vesicle budding failure and accumulation of unprocessed proteins in ER, which further caused ER stress as well as Golgi fragmentation, and PERK-eIF2α pathway of UPR was activated to reduce the synthesis of universal proteins. In contrast, upregulation of Nlp resulted in Golgi fragmentation, which enhanced the cargo transport efficiency between ER and Golgi. Moreover, Nlp deficient mice were prone to spontaneous B cell lymphoma, since the developments and functions of lymphocytes significantly depended on secretory proteins through ER-to-Golgi vesicle trafficking, including IL-13, IL-17 and IL-21. Thus, perturbations of Nlp altered ER-to-Golgi communication and cellular homeostasis, and might contribute to the pathogenesis of B cell lymphoma.

CDK7-YAP-LDHD axis promotes D-lactate elimination and ferroptosis defense to support cancer stem cell-like properties.

Reprogrammed cellular metabolism is essential for maintaining cancer stem cells (CSCs) state. Here, we report that mitochondrial D-lactate catabolism is a necessary initiating oncogenic event during tumorigenesis of esophageal squamous cell carcinoma (ESCC). We discover that cyclin-dependent kinase 7 (CDK7) phosphorylates nuclear Yes-associated protein 1 (YAP) at S127 and S397 sites and enhances its transcription function, which promotes D-lactate dehydrogenase (LDHD) protein expression. Moreover, LDHD is enriched significantly in ESCC-CSCs rather than differentiated tumor cells and high LDHD status is connected with poor prognosis in ESCC patients. Mechanistically, the CDK7-YAP-LDHD axis helps ESCC-CSCs escape from ferroptosis induced by D-lactate and generates pyruvate to satisfy energetic demands for their elevated self-renewal potential. Hence, we conclude that esophageal CSCs adopt a D-lactate elimination and pyruvate accumulation mode dependent on CDK7-YAP-LDHD axis, which drives stemness-associated hallmarks of ESCC-CSCs. Reasonably, targeting metabolic checkpoints may serve as an effective strategy for ESCC therapy.

Nlp promotes autophagy through facilitating the interaction of Rab7 and FYCO1.

Autophagy is the main degradation pathway to eliminate long-lived and aggregated proteins, aged or malfunctioning organelles, which is essential for the intracellular homeostasis and prevention of malignant transformation. Although the processes of autophagosome biogenesis have been well illuminated, the mechanism of autophagosome transport remains largely unclear. In this study, we demonstrated that the ninein-like protein (Nlp), a well-characterized centrosomal associated protein, was able to modulate autophagosome transport and facilitate autophagy. During autophagy, Nlp colocalized with autophagosomes and physically interacted with autophagosome marker LC3, autophagosome sorting protein Rab7 and its downstream effector FYCO1. Interestingly, Nlp enhanced the interaction between Rab7 and FYCO1, thus accelerated autophagic flux and the formation of autophagolysosomes. Furthermore, compared to the wild-type mice, NLP deficient mice treated with chemical agent DMBA were prone to increased incidence of hepatomegaly and liver cancer, which were tight associated with the hepatic autophagic defect. Taken together, our findings provide a new insight for the first time that the well-known centrosomal protein Nlp is also a new regulator of autophagy, which promotes the interaction of Rab7 and FYCO1 and facilitates the formation of autophagolysosome.

Defect of SLC38A3 promotes epithelial-mesenchymal transition and predicts poor prognosis in esophageal squamous cell carcinoma.

OBJECTIVE: Solute carrier family 38 (SLC38s) transporters play important roles in amino acid transportation and signaling transduction. However, their genetic alterations and biological roles in tumors are still largely unclear. This study aimed to elucidate the genetic signatures of SLC38s transporters and their implications in esophageal squamous cell carcinoma (ESCC). METHODS: Analyses on somatic mutation and copy number alterations (CNAs) of SLC38A3 were performed as described. Immunohistochemistry (IHC) assay and Western blot assay were used to detect the protein expression level. MTS assay, colony formation assay, transwell assay and wound healing assay were used to explore the malignant phenotypes of ESCC cells. Immunofluorescence assay was used to verify the colocalization of two indicated proteins and immunopreciptation assay was performed to confirm the interaction of proteins. RESULTS: Our findings revealed that SLC38s family was significantly disrupted in ESCC, with high frequent CNAs and few somatic mutations. SLC38A3 was the most frequent loss gene among them and was linked to poor survival and lymph node metastasis. The expression of SLC38A3 was lower in tumor tissues compared to that in normal tissues, which was also significantly associated with worse clinical outcome. Further experiments revealed that depletion of SLC38A3 could promote EMT in ESCC cell lines, and the interaction of SLC38A3 and SETDB1 might lead to the reduced transcription of Snail. Pharmacogenomic analyses demonstrated that fifteen inhibitors were showed significantly correlated with SLC38A3 expression. CONCLUSIONS: Our investigations have provided insights that SLC38A3 could act as a suppressor in EMT pathway and serve as a prognostic factor and predictor of differential drug sensitivities in ESCC.

PFKP is transcriptionally repressed by BRCA1/ZBRK1 and predicts prognosis in breast cancer.

OBJECTIVES: The present study aims to elucidate the underlying mechanism how PFKP is regulated by BRCA1 and the clinical significance of PFKP in breast cancer. METHODS: MEF-BRCA1△/△ and the wild type counterpart MEF-BRCA1+/+ cell lines were used to test the sensitivity of glucose depletion in culture medium. Glucose Assay Kit was used to quantify glucose levels in cultural supernatant and cell lysate. Real time PCR was used to measure the mRNA expression levels of genes. Western blot was used to detect protein levels. Chromatin immunoprecipitation was used to verify the bindings between transcription factors and DNA elements. Luciferase reporter assay was performed to determine the transcriptional activity. Histochemistry assay was performed on tissue microarray. RESULTS: We found that MEF-BRCA1△/△ cells consumed more glucose and were more vulnerable to glucose-deprived culture medium. The mRNA profiles and qPCR assay of MEF-BRCA1△/△ and MEF-BRCA1+/+ cells revealed that PFKP, the rate-limiting enzyme of glycolysis, was significantly upregulated in MEF-BRCA1△/△ cells. Consistently, the repressive effects of BRCA1 on PFKP were confirmed by overexpression or knockdown of BRCA1. Moreover, we also demonstrated that PFKP was suppressed by ZBRK1 as well, which was the co-repression partner of BRCA1. Mechanistically, we figured out that BRCA1 formed a transcriptional repression complex with ZBRK1 on the promoter of PFKP and consequently restrained its expression. Importantly, the expression levels of PFKP were demonstrated to associate with poor survival of patients with breast cancer. CONCLUSION: Our study provided a new insight into the dysregulation of glycolysis in breast cancer, which might be partially due to the deficiency of BRCA1/ZBRK1 axis and subsequently reversed the transcriptional repressive effect on PFKP. We also found that PFKP overexpressed in a subset of breast cancer patients and could serve as a prognostic factor, which represented a potential target for BC therapy.