Dataset Information

REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival.

ABSTRACT: Rab escort protein 1 (REP1), a component of the Rab geranyl-geranyltransferase 2 complex, plays a role in Rab protein recruitment in proper vesicles during vesicle trafficking. In addition to having well-known tissue degenerative phenotypes in the REP1 mutant, REP1 is tightly associated with cancer development and contributes to cell growth and survival. However, the functional mechanism of REP1 in cancer progression is largely uninvestigated. Here, we show that REP1 plays a crucial role in regulating mammalian target of rapamycin (mTOR) signaling and its downstream pathways, as well as autophagy and macropinocytosis, which are essential for cancer cell survival during metabolic stresses including starvation. REP1 small interfering RNA (siRNA) treatment downregulates mTORC1 activity in growing media, but blocks autophagosome formation under nutrient-depleted conditions. In contrast to the mild decrease of lysosomal enzyme activity seen in REP1 depletion, in REP1 knockdown the subcellular localization of lysosomes is altered, and localization of REP1 itself is modulated by intracellular nutrient levels and mTOR activity. Furthermore, REP1 depletion increases macro pinocytosis which may be a feedback mechanism to compensate autophagy inhibition. Concomitant treatment with macropinocytosis inhibitor and REP1siRNAresults in more significant cell death than autophagy blockade with REP1 knockdown. Therefore, REP1-mediated autophagy and lysosomal degradation processes act as novel regulatory mechanisms to support cancer cell survival, which can be further investigated as a potential cancer-targeting pathway.

SUBMITTER: Choi J

PROVIDER: S-EPMC5618515 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival.

Choi Jungwon J Kim Hyena H Bae Young Ki YK Cheong Heesun H

International journal of molecular sciences 20170828 9

Rab escort protein 1 (REP1), a component of the Rab geranyl-geranyltransferase 2 complex, plays a role in Rab protein recruitment in proper vesicles during vesicle trafficking. In addition to having well-known tissue degenerative phenotypes in the REP1 mutant, REP1 is tightly associated with cancer development and contributes to cell growth and survival. However, the functional mechanism of REP1 in cancer progression is largely uninvestigated. Here, we show that REP1 plays a crucial role in regu ...[more]

PMID: 28846638

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Project description:Macroautophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. The process operates under basal conditions as a mechanism to turnover damaged or misfolded proteins and organelles. As a result, it has a major role in preserving cellular integrity and viability. In addition to this basal function, macroautophagy can also be modulated in response to various forms of cellular stress, and the rate and cargoes of macroautophagy can be tailored to facilitate appropriate cellular responses in particular situations. The macroautophagy machinery is regulated by a group of evolutionarily conserved autophagy-related (ATG) proteins and by several other autophagy regulators, which either have tissue-restricted expression or operate in specific contexts. We report here the characterization of a novel autophagy regulator that we have termed DRAM-3 due to its significant homology to damage-regulated autophagy modulator (DRAM-1). DRAM-3 is expressed in a broad spectrum of normal tissues and tumor cells, but different from DRAM-1, DRAM-3 is not induced by p53 or DNA-damaging agents. Immunofluorescence studies revealed that DRAM-3 localizes to lysosomes/autolysosomes, endosomes and the plasma membrane, but not the endoplasmic reticulum, phagophores, autophagosomes or Golgi, indicating significant overlap with DRAM-1 localization and with organelles associated with macroautophagy. In this regard, we further proceed to show that DRAM-3 expression causes accumulation of autophagosomes under basal conditions and enhances autophagic flux. Reciprocally, CRISPR/Cas9-mediated disruption of DRAM-3 impairs autophagic flux confirming that DRAM-3 is a modulator of macroautophagy. As macroautophagy can be cytoprotective under starvation conditions, we also tested whether DRAM-3 could promote survival on nutrient deprivation. This revealed that DRAM-3 can repress cell death and promote long-term clonogenic survival of cells grown in the absence of glucose. Interestingly, however, this effect is macroautophagy-independent. In summary, these findings constitute the primary characterization of DRAM-3 as a modulator of both macroautophagy and cell survival under starvation conditions.

Project description:Rationale: Post-translational modifications have emerged as vital players in alterations to tumor metabolism, including amino acid metabolic reprogramming. Jumonji domain-containing protein 2B (JMJD2B) enhances colorectal cancer (CRC) cell survival upon glucose deficiency. In the present study, we hypothesized that JMJD2B affects tumor cell amino acid metabolism in CRC and consequently promotes survival of CRC cells upon glucose deprivation. Methods: Non-target metabolic profiling was used to evaluate the roles of JMJD2B in CRC cell metabolism under glucose starvation. The roles of amino acid alterations induced by JMJD2B on CRC cell survival were determined by cell viability, immunoblotting, and clonogenic assays, and flow cytometry. The underlying mechanisms by which JMJD2B affected CRC cell metabolism were assessed using immunofluorescence staining, chromatin immunoprecipitation assays, electron microscopy in CRC cell lines, and using xenograft models. The correlation between JMJD2B and LC3B expression in human CRC specimens was assessed using immunohistochemistry. Results: Profound metabolic reprogramming was detected in JMJD2B knockdown CRC cells under glucose deficiency, especially those involving amino acid metabolites. Silencing of JMJD2B reduced the levels of certain amino acids that were induced by glucose deficiency. Among these amino acids, asparagine (Asn), phenylalanine (Phe), and histidine (His) promoted CRC cell survival under glucose starvation when JMJD2B was knocked down. Mechanistically, downregulation of JMJD2B inhibited autophagy in CRC cells through epigenetic regulation of microtubule associated protein 1 light chain 3 beta (LC3B), and subsequently decreased intracellular amino acid (Asn, Phe, His) levels under glucose deprivation, thus suppressing the survival of CRC cells. Using a nude mouse xenograft model, we verified that inhibiting JMJD2B could decrease the levels of amino acids (Asn, Phe, His). In addition, the inhibitory effects of JMJD2B-knockdown on tumor growth and amino acids level were rescued by overexpression of LC3B. Furthermore, we observed that the high expression of LC3B was more likely detected in tissuses with high expression of JMJD2B (P < 0.001) in 60 human CRC tissues. Conclusion: These results indicated that JMJD2B sustained the intracellular amino acids derived from autophagy in CRC cells upon glucose deficiency, partly through epigenetic regulation of LC3B, thus driving the malignancy of CRC.

Dataset Information

REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival.

Publications

REP1 Modulates Autophagy and Macropinocytosis to Enhance Cancer Cell Survival.

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