bims-lymeca Biomed News
on Lysosome metabolism in cancer
Issue of 2022‒01‒23
twenty-five papers selected by
Charilaos Filippakis
Harvard University

  1. Front Cell Dev Biol. 2021 ;9 814955
      Nucleophagy is an organelle-selective subtype of autophagy that targets nuclear material for degradation. The macroautophagic delivery of micronuclei to the vacuole, together with the nucleus-vacuole junction-dependent microautophagic degradation of nuclear material, were first observed in yeast. Nuclear pore complexes and ribosomal DNA are typically excluded during conventional macronucleophagy and micronucleophagy, indicating that degradation of nuclear cargo is tightly regulated. In mammals, similarly to other autophagy subtypes, nucleophagy is crucial for cellular differentiation and development, in addition to enabling cells to respond to various nuclear insults and cell cycle perturbations. A common denominator of all nucleophagic processes characterized in diverse organisms is the dependence on the core autophagic machinery. Here, we survey recent studies investigating the autophagic processing of nuclear components. We discuss nucleophagic events in the context of pathology, such as neurodegeneration, cancer, DNA damage, and ageing.
    Keywords:  ageing; autophagy; cancer; neurodegeneration; nucleophagy
  2. Science. 2021 Jun 25. pii: eabg0269. [Epub ahead of print]372(6549):
      Host cells initiate cell death programs to limit pathogen infection. Inhibition of transforming growth factor-β-activated kinase 1 (TAK1) by pathogenic Yersinia in macrophages triggers receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-dependent caspase-8 cleavage of gasdermin D (GSDMD) and inflammatory cell death (pyroptosis). A genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen to uncover mediators of caspase-8-dependent pyroptosis identified an unexpected role of the lysosomal FLCN-FNIP2-Rag-Ragulator supercomplex, which regulates metabolic signalling and the mechanistic target of rapamycin complex 1 (mTORC1). In response to Yersinia infection, FADD, RIPK1 and caspase-8 were recruited to Rag-Ragulator, causing RIPK1 phosphorylation and caspase-8 activation. Pyroptosis activation depended on Rag GTPase activity and lysosomal tethering of Rag-Ragulator, but not mTORC1. Thus, the lysosomal metabolic regulator Rag-Ragulator instructs the inflammatory response to Yersinia.
  3. ACS Omega. 2022 Jan 11. 7(1): 5-16
      Lysosomes are membranous compartments containing hydrolytic enzymes, where cellular degradation of proteins and enzymes among others occurs in a controlled manner. Lysosomal dysfunction results in various pathological situations, such as several lysosomal storage disorders, neurodegeneration, infectious diseases, cancers, and aging. In this review, we have discussed different strategies for synthesizing peptides/chimeric molecules, their lysosome-targeting ability, and their ability to treat several lysosomal associated diseases, including lysosomal storage diseases and cancers. We have also discussed the delivery of cargo molecules into the lysosome using lysosome-targeting ligand-decorated nanocarriers. The introduction of a protein-binding ligand along with a lysosome-targeting ligand to manufacture a chimeric architecture for cell-specific protein (extracellular and membrane protein) degradation ability has been discussed thoroughly. Finally, the future applications of these lysosome-targeting peptides, nanocarriers, and chimeric molecules have been pointed out.
  4. Stem Cell Reports. 2022 Jan 03. pii: S2213-6711(21)00652-4. [Epub ahead of print]
      Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.
    Keywords:  autophagosome; autophagy; cancer stem cells; embryonal carcinoma stem cells; embryonic stem cells; induced pluripotent stem cells; lysosome
  5. Cancers (Basel). 2022 Jan 11. pii: 339. [Epub ahead of print]14(2):
      Glioblastoma (GBM) is a devastating disease and the most common primary brain malignancy of adults with a median survival barely exceeding one year. Recent findings suggest that the antipsychotic drug pimozide triggers an autophagy-dependent, lysosomal type of cell death in GBM cells with possible implications for GBM therapy. One oncoprotein that is often overactivated in these tumors and associated with a particularly dismal prognosis is Signal Transducer and Activator of Transcription 3 (STAT3). Here, we used isogenic human and murine GBM knockout cell lines, advanced fluorescence microscopy, transcriptomic analysis and FACS-based assessment of cell viability to show that STAT3 has an underappreciated, context-dependent role in drug-induced cell death. Specifically, we demonstrate that depletion of STAT3 significantly enhances cell survival after treatment with Pimozide, suggesting that STAT3 confers a particular vulnerability to GBM. Furthermore, we show that active STAT3 has no major influence on the early steps of the autophagy pathway, but exacerbates drug-induced lysosomal membrane permeabilization (LMP) and release of cathepsins into the cytosol. Collectively, our findings support the concept of exploiting the pro-death functions of autophagy and LMP for GBM therapy and to further determine whether STAT3 can be employed as a treatment predictor for highly apoptosis-resistant, but autophagy-proficient cancers.
    Keywords:  STAT3; autophagy; autophagy-dependent cell death; glioblastoma; lysosomal-dependent cell death; lysosome; pimozide
  6. FEBS J. 2022 Jan 21.
      Macroautophagy is a membrane-trafficking process that delivers cytoplasmic material to lysosomes for degradation. The process preserves cellular integrity by removing damaged cellular constituents and can promote cell survival by providing substrates for energy production during hiatuses of nutrient availability. The process is also highly responsive to other forms of cellular stress. For example, DNA damage can induce autophagy and this involves up-regulation of the Damage-Regulated Autophagy Modulator-1 (DRAM-1) by the tumor suppressor p53. DRAM-1 belongs to an evolutionarily-conserved protein family, which has five members in humans and we describe here the initial characterization of two members of this family, which we term DRAM-4 and DRAM-5 for DRAM-Related/Associated Member 4/5. We show that the genes encoding these proteins are not regulated by p53, but instead are induced by nutrient deprivation. Similar to other DRAM family proteins, however, DRAM-4 principally localizes to endosomes and DRAM-5 to the plasma membrane and both modulate autophagy flux when over-expressed. Deletion of DRAM-4 using CRISPR/Cas-9 also increased autophagy flux, but we found that DRAM-4 and DRAM-5 undergo compensatory regulation, such that deletion of DRAM-4 does not affect autophagy flux in the absence of DRAM-5. Similarly, deletion of DRAM-4 also promotes cell survival following growth of cells in the absence of amino acids, serum or glucose, but this effect is also impacted by the absence of DRAM-5. In summary, DRAM-4 and DRAM-5 are nutrient-responsive members of the DRAM family that exhibit interconnected roles in the regulation of autophagy and cell survival under nutrient-deprived conditions.
  7. Elife. 2022 Jan 18. pii: e69709. [Epub ahead of print]11
      Cell-surface receptors control how cells respond to their environment. Many cell-surface receptors recycle from endosomes to the plasma membrane via a recently discovered pathway, which includes sorting-nexin SNX17, Retriever, WASH and CCC complexes. Here, using mammalian cells, we discover that PIKfyve and its upstream PI3-kinase VPS34 positively regulate this pathway. VPS34 produces PI3P, which is the substrate for PIKfyve to generate PI3,5P2. We show that PIKfyve controls recycling of cargoes including integrins, receptors that control cell migration. Furthermore, endogenous PIKfyve colocalizes with SNX17, Retriever, WASH and CCC complexes on endosomes. Importantly, PIKfyve inhibition results displacement of Retriever and CCC from endosomes. In addition, we show that recruitment of SNX17 is an early step and requires VPS34. These discoveries suggest that VPS34 and PIKfyve coordinate an ordered pathway to regulate recycling from endosomes and suggest how PIKfyve functions in cell migration.
    Keywords:  cell biology
  8. Nucleic Acids Res. 2022 Jan 17. pii: gkab1296. [Epub ahead of print]
      Although there are several pathways to ensure that proteins are folded properly in the cell, little is known about the molecular mechanisms regulating histone folding and proteostasis. In this work, we identified that chaperone-mediated autophagy (CMA) is the main pathway involved in the degradation of newly synthesized histones H3 and H4. This degradation is finely regulated by the interplay between HSC70 and tNASP, two histone interacting proteins. tNASP stabilizes histone H3 levels by blocking the direct transport of histone H3 into lysosomes. We further demonstrate that CMA degrades unfolded histone H3. Thus, we reveal that CMA is the main degradation pathway involved in the quality control of histone biogenesis, evidencing an additional mechanism in the intricate network of histone cellular proteostasis.
  9. Clin Transl Oncol. 2022 Jan 17.
      Cancer is one of the leading causes of death, with a heavy socio-economical burden for countries. Despite the great advances that have been made in the treatment of cancer, chemotherapy is still the most common method of treatment. However, many side effects, including hepatotoxicity, renal toxicity, and cardiotoxicity, limit the efficacy of conventional chemotherapy. Over recent years, natural products have attracted attention as therapeutic agents against various diseases, such as cancer. Resveratrol (RES), a natural polyphenol occurring in grapes, nuts, wine, and berries, exhibited potential for preventing and treating various cancer types. RES also ameliorates chemotherapy-induced detrimental effects. Furthermore, RES could modulate apoptosis and autophagy as the main forms of cancer cell deaths by targeting various signaling pathways and up/downregulation of apoptotic and autophagic genes. This review will summarize the anti-cancer effects of RES and focus on the fundamental mechanisms and targets for modulating apoptosis and autophagy by RES.
    Keywords:  Apoptosis; Autophagy; Cancer; Cancer cell death; Resveratrol
  10. Cell Death Dis. 2022 Jan 17. 13(1): 59
      Histone deacetylase (HDAC) inhibitors and proteasome inhibitors have been approved by the FDA for the treatment of multiple myeloma and lymphoma, respectively, but have not achieved similar activity as single agents in solid tumors. Preclinical studies have demonstrated the activity of the combination of an HDAC inhibitor and a proteasome inhibitor in a variety of tumor models. However, the mechanisms underlying sensitivity and resistance to this combination are not well-understood. This study explores the role of autophagy in adaptive resistance to dual HDAC and proteasome inhibition. Studies focus on ovarian and endometrial gynecologic cancers, two diseases with high mortality and a need for novel treatment approaches. We found that nanomolar concentrations of the proteasome inhibitor ixazomib and HDAC inhibitor romidepsin synergistically induce cell death in the majority of gynecologic cancer cells and patient-derived organoid (PDO) models created using endometrial and ovarian patient tumor tissue. However, some models were not sensitive to this combination, and mechanistic studies implicated autophagy as the main mediator of cell survival in the context of dual HDAC and proteasome inhibition. Whereas the combination of ixazomib and romidepsin reduces autophagy in sensitive gynecologic cancer models, autophagy is induced following drug treatment of resistant cells. Pharmacologic or genetic inhibition of autophagy in resistant cells reverses drug resistance as evidenced by an enhanced anti-tumor response both in vitro and in vivo. Taken together, our findings demonstrate a role for autophagic-mediated cell survival in proteasome inhibitor and HDAC inhibitor-resistant gynecologic cancer cells. These data reveal a new approach to overcome drug resistance by inhibiting the autophagy pathway.
  11. FEBS J. 2022 Jan 18.
      zVAD-fmk is a widely used pan-caspase inhibitor that blocks apoptosis but has undesirable side effects, including autophagy. In this issue, Needs et al. propose that zVAD-fmk induces autophagy by inhibiting the N-glycanase NGLY1 rather than caspases. NGLY1 is essential for the ERAD response and patients with inactivating mutations in NGLY1 present with neurodevelopmental defects and organ dysfunction. The ability of NGLY1 to inhibit basal levels of autophagy may contribute to this pathology. This study demonstrates possible crosstalk between protein turnover and autophagy while also underscoring the importance of specificity when using chemical tools to interrogate these pathways. Comment on
    Keywords:  NLGY1; apoptosis; autophagy; caspase; zVAD-fmk
  12. Biomaterials. 2022 Jan 06. pii: S0142-9612(22)00004-7. [Epub ahead of print]281 121365
      Magnetic nanoparticles (MNPs) are potential theranostic tools that are biodegraded through different endocytic pathways. However, little is known about the endolysosomal network through which MNPs transit and the influence of the surface coating in this process. Here, we studied the intracellular transit of two MNPs with identical iron oxide core size but with two distinct coatings: 3-aminopropyl-trietoxysilane (APS) and dimercaptosuccinic acid (DMSA). Using endolysosomal markers and a high throughput analysis of the associated proteome, we tracked the MNPs intracellularly in two different mouse cell lines, RAW264.7 (macrophages) and Pan02 (tumor cells). We did not detect differences in the MNP trafficking kinetics nor in the MNP-containing endolysosome phenotype in Pan02 cells. Nonetheless, DMSA-MNPs transited at slower rate than APS-MNPs in macrophages as measured by MNP accumulation in Rab7+ endolysosomes. Macrophage DMSA-MNP-containing endolysosomes had a higher percentage of lytic enzymes and catalytic proteins than their APS-MNP counterparts, concomitantly with a V-type ATPase enrichment, suggesting an acidic nature. Consequently, more autophagic vesicles are induced by DMSA-MNPs in macrophages, enhancing the expression of iron metabolism-related genes and proteins. Therefore, unlike Pan02 cells, the MNP coating appears to influence the intracellular trafficking rate and the endolysosome nature in macrophages. These results highlight how the MNP coating can determine the nanoparticle intracellular fate and biodegradation in a cell-type bias.
    Keywords:  Autophagy; Degradative proteome; Intracellular trafficking; Magnetic nanoparticles; Proteomics
  13. J Cell Biol. 2022 Mar 07. pii: e202106124. [Epub ahead of print]221(3):
      Membrane trafficking plays many roles in morphogenesis, from bulk membrane provision to targeted delivery of proteins and other cargos. In tracheal terminal cells of the Drosophila respiratory system, transport through late endosomes balances membrane delivery between the basal plasma membrane and the apical membrane, which forms a subcellular tube, but it has been unclear how the direction of growth of the subcellular tube with the overall cell growth is coordinated. We show here that endosomes also organize F-actin. Actin assembles around late endocytic vesicles in the growth cone of the cell, reaching from the tip of the subcellular tube to the leading filopodia of the basal membrane. Preventing nucleation of endosomal actin disturbs the directionality of tube growth, uncoupling it from the direction of cell elongation. Severing actin in this area affects tube integrity. Our findings show a new role for late endosomes in directing morphogenesis by organizing actin, in addition to their known role in membrane and protein trafficking.
  14. Cells. 2022 Jan 07. pii: 197. [Epub ahead of print]11(2):
      Solute-linked cotransporter, SLC4A11, a member of the bicarbonate transporter family, is an electrogenic H+ transporter activated by NH3 and alkaline pH. Although SLC4A11 does not transport bicarbonate, it shares many properties with other members of the SLC4 family. SLC4A11 mutations can lead to corneal endothelial dystrophy and hearing deficits that are recapitulated in SLC4A11 knock-out mice. SLC4A11, at the inner mitochondrial membrane, facilitates glutamine catabolism and suppresses the production of mitochondrial superoxide by providing ammonia-sensitive H+ uncoupling that reduces glutamine-driven mitochondrial membrane potential hyperpolarization. Mitochondrial oxidative stress in SLC4A11 KO also triggers dysfunctional autophagy and lysosomes, as well as ER stress. SLC4A11 expression is induced by oxidative stress through the transcription factor NRF2, the master regulator of antioxidant genes. Outside of the corneal endothelium, SLC4A11's function has been demonstrated in cochlear fibrocytes, salivary glands, and kidneys, but is largely unexplored overall. Increased SLC4A11 expression is a component of some "glutamine-addicted" cancers, and is possibly linked to cells and tissues that rely on glutamine catabolism.
    Keywords:  MCT4; ammonia; corneal endothelial dystrophy; glutamine; lactate
  15. Cells. 2022 Jan 17. pii: 304. [Epub ahead of print]11(2):
      Endolysosomal cation channels are emerging as key players of endolysosomal function such as endolysosomal trafficking, fusion/fission, lysosomal pH regulation, autophagy, lysosomal exocytosis, and endocytosis. Diseases comprise lysosomal storage disorders (LSDs) and neurodegenerative diseases, metabolic diseases, pigmentation defects, cancer, immune disorders, autophagy related diseases, infectious diseases and many more. Involvement in lung diseases has not been a focus of attention so far but recent developments in the field suggest critical functions in lung physiology and pathophysiology. Thus, loss of TRPML3 was discovered to exacerbate emphysema formation and cigarette smoke induced COPD due to dysregulated matrix metalloproteinase 12 (MMP-12) levels in the extracellular matrix of the lung, a known risk factor for emphysema/COPD. While direct lung function measurements with the exception of TRPML3 are missing for other endolysosomal cation channels or channels expressed in lysosome related organelles (LRO) in the lung, links between those channels and important roles in lung physiology have been established such as the role of P2X4 in surfactant release from alveolar epithelial Type II cells. Other channels with demonstrated functions and disease relevance in the lung such as TRPM2, TRPV2, or TRPA1 may mediate their effects due to plasma membrane expression but evidence accumulates that these channels might also be expressed in endolysosomes, suggesting additional and/or dual roles of these channels in cell and intracellular membranes. We will discuss here the current knowledge on cation channels residing in endolysosomes or LROs with respect to their emerging roles in lung disease.
    Keywords:  BK; COPD; TRPA1; TRPM2; TRPML; TRPML3; TRPV2; asthma; cystic fibrosis; emphysema; lung injury
  16. Biomolecules. 2022 Jan 13. pii: 126. [Epub ahead of print]12(1):
      BACKGROUND: The ARF tumour suppressor plays a well-established role as a tumour suppressor, halting cell growth by both p53-dependent and independent pathways in several cellular stress response circuits. However, data collected in recent years challenged the traditional role of this protein as a tumour suppressor. Cancer cells expressing high ARF levels showed that its expression, far from being dispensable, is required to guarantee tumour cell survival. In particular, ARF can promote autophagy, a self-digestion pathway that helps cells cope with stressful growth conditions arising during both physiological and pathological processes.METHODS: We previously showed that ARF is regulated through the activation of the protein kinase C (PKC)-dependent pathway and that an ARF phospho-mimetic mutant on the threonine residue 8, ARF-T8D, sustains cell proliferation in HeLa cells. We now explored the role of ARF phosphorylation in both basal and starvation-induced autophagy by analysing autophagic flux in cells transfected with either WT and ARF phosphorylation mutants by immunoblot and immunofluorescence.
    RESULTS: Here, we show that endogenous ARF expression in HeLa cells is required for starvation-induced autophagy. Further, we provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells' inability to elicit autophagosomes formation upon T8D expression.
    CONCLUSIONS: Our results lead to the hypothesis that ARF phosphorylation could be a mechanism through which the protein promotes or counteracts autophagy. Several observations underline how autophagy could serve a dual role in cancer progression, either protecting healthy cells from damage or aiding cancerous cells to survive. Our results indicate that ARF phosphorylation controls protein's ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression.
    Keywords:  INK4a/ARF locus; LC3; autophagy; cancer; cytoskeleton
  17. Molecules. 2022 Jan 07. pii: 369. [Epub ahead of print]27(2):
      Pterostilbene is a dietary phytochemical that has been found to possess several biological activities, such as antioxidant and anti-inflammatory. Recent studies have shown that it exhibits the hallmark characteristics of an anticancer agent. The aim of the study was to investigate the anticancer activity of pterostilbene against HT-29 human colon cancer cells, focusing on its influence on cell growth, differentiation, and the ability of this stilbene to induce cell death. To clarify the mechanism of pterostilbene activity against colon cancer cells, changes in the expression of several genes and proteins that are directly related to cell proliferation, signal transduction pathways, apoptosis, and autophagy were also evaluated. Cell growth and proliferation of cells exposed to pterostilbene (5-100 µM) were determined by SRB and BRDU assays. Flow cytometric analyses were used for cell cycle progression. Further molecular investigations were performed using quantitative real-time RT-PCR. The expression of the signaling proteins studied was determined by the ELISA method. The results revealed that pterostilbene inhibited proliferation and induced the death of HT-29 colon cancer cells. Pterostilbene, depending on concentration, caused inhibition of proliferation, G1 cell arrest, and/or triggered apoptosis in HT-29 cells. These effects were mediated by the down-regulation of the STAT3 and AKT kinase pathways. It may be concluded that pterostilbene could be considered as a potential therapeutic option in the treatment of colon cancer in the future.
    Keywords:  AKT; STAT3; autophagy; colon cancer; pterostilbene apoptosis
  18. Cells. 2022 Jan 10. pii: 219. [Epub ahead of print]11(2):
      ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes.
    Keywords:  ATP7B; Golgi; TFEB; chemoresistance; ovarian cancer
  19. Cells. 2022 Jan 13. pii: 270. [Epub ahead of print]11(2):
      Lysosomes-that is, acidic organelles known for degradation/recycling-move through the cytoplasm alternating between bursts of active transport and short, diffusive motions or even pauses. While their mobility is essential for lysosomes' fusogenic and non-fusogenic interactions with target organelles, their movements have not been characterized in adequate detail. Here, large-scale statistical analysis of lysosomal movement trajectories reveals that lysosome trajectories in all examined cell types-both cancer and noncancerous ones-are superdiffusive and characterized by heavy-tailed distributions of run and flight lengths. Consideration of Akaike weights for various potential models (lognormal, power law, truncated power law, stretched exponential, and exponential) indicates that the experimental data are best described by the lognormal distribution, which, in turn, can be related to one of the space-search strategies particularly effective when "thorough" search needs to balance search for rare target(s) (organelles). In addition, automated, wavelet-based analysis allows for co-tracking the motions of lysosomes and the cargos they carry-particularly the nanoparticle aggregates known to cause selective lysosome disruption in cancerous cells. The methods we describe here could help study nanoparticle assemblies, viruses, and other objects transported inside various vesicle types, as well as coordinated movements of organelles/particles in the cytoplasm. Custom-written code that includes integrated workflow for our analyses is made available for academic use.
    Keywords:  cancer lysosomes; confocal reflection microscopy; continuous wavelet transform; lognormal distribution; lysosome transport; lysosome-nanoparticle co-movement; maximum-likelihood estimates; mixed-charge nanoparticles
  20. Cancer Res. 2022 Jan 19. pii: canres.2609.2021. [Epub ahead of print]
      Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intra-tumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single cell proteomic approaches. To overcome this barrier, we applied single cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment. Compared with primary tumors and circulating tumor cells (CTC), bone metastases but not lung and liver metastases exhibited elevated PI3K/mTOR signaling and overexpressed receptor tyrosine kinases (RTK) including c-Met protein. Suppression of c-MET impaired tumor growth in the bone. Intra-tumoral heterogeneity within tumor compartments also arose from highly proliferative EpCAM-high epithelial cells with increased PI3K and mTOR kinase activities co-existing with poorly proliferating EpCAM-low mesenchymal populations with reduced kinase activities; these findings were recapitulated in epithelial and mesenchymal CTC populations in metastatic prostate and breast cancer patients. Increased kinase activity in EpCAM-high cells rendered them more sensitive to PI3K/mTOR inhibition, and drug-resistant EpCAM-low populations with reduced kinase activity emerged over time. Taken together, single cell proteomics indicate that microenvironment- and cell state-dependent activation of kinase networks create heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors.
  21. Dev Cell. 2022 Jan 11. pii: S1534-5807(21)01038-8. [Epub ahead of print]
      The endoplasmic reticulum (ER)-to-Golgi transport is critical to protein secretion and intracellular sorting. Here, we report a highly elongated tubular ER-Golgi intermediate compartment (t-ERGIC) that selectively expedites the ER-to-Golgi transport for soluble cargoes of the receptor SURF4. Lacking the canonical ERGIC marker ERGIC-53 yet positive for the small GTPases Rab1A/B, the t-ERGIC is further marked by its extraordinarily elongated and thinned shape. With its large surface-to-volume ratio, high intracellular traveling speeds, and ER-Golgi recycling capabilities, the t-ERGIC accelerates the trafficking of SURF4-bound cargoes. The biogenesis and cargo selectivity of t-ERGIC both depend on SURF4, which recognizes the N terminus of soluble cargoes and co-clusters with the selected cargoes to expand the ER-exit site. In the steady state, the t-ERGIC-mediated fast ER-to-Golgi transport is antagonized by the KDEL-mediated ER retrieval. Together, our results argue that specific cargo-receptor interactions give rise to distinct transport carriers that regulate the trafficking kinetics.
    Keywords:  ER-Golgi intermediate compartment; ER-exit site expansion; ER-to-Golgi transport; N-terminal selective binding; SURF4-KDELR antagonism; SURF4-cargo co-clustering; membrane vesicle trafficking; protein secretion kinetics; soluble cargo; tubular carrier
  22. Mol Pharm. 2022 Jan 18.
      Intracellular delivery of protein therapeutics remains a significant challenge limiting the majority of clinically available protein drugs to extracellular targets. Strategies to deliver proteins to subcellular compartments have traditionally relied on cell-penetrating peptides, which can drive enhanced internalization but exhibit unreliable activity and are rarely able to target specific cells, leading to off-target effects. Moreover, few design rules exist regarding the relative efficacy of various endosomal escape strategies in proteins. Accordingly, we developed a simple fusion modification approach to incorporate endosomolytic peptides onto epidermal growth factor receptor (EGFR)-targeted protein conjugates and performed a systematic comparison of the endosomal escape efficacy, mechanism of action, and capacity to maintain EGFR-targeting specificity of conjugates modified with four different endosomolytic sequences of varying modes of action (Aurein 1.2, GALA, HA2, and L17E). Use of the recently developed Gal8-YFP assay indicated that the fusion of each endosomolytic peptide led to enhanced endosomal disruption. Additionally, the incorporation of each endosomolytic peptide increased the half-life of the internalized protein and lowered lysosomal colocalization, further supporting the membrane-disruptive capacity. Despite this, only EGFR-targeted conjugates modified with Aurein 1.2 or GALA maintained EGFR specificity. These results thus demonstrated that the choice of endosomal escape moiety can substantially affect targeting capability, cytotoxicity, and bioactivity and provided important new insights into endosomolytic peptide selection for the design of targeted protein delivery systems.
    Keywords:  EGFR; endosomolytic peptides; intracellular protein delivery; targeted delivery
  23. J Exp Clin Cancer Res. 2022 Jan 19. 41(1): 28
      BACKGROUND: Estrogen-related receptor α (ERRα) has been reported to play a critical role in endometrial cancer (EC) progression. However, the underlying mechanism of ERRα-mediated lipid reprogramming in EC remains elusive. The transcription factor EB (TFEB)-ERRα axis induces lipid reprogramming to promote progression of EC was explored in this study.METHODS: TFEB and ERRα were analyzed and validated by RNA-sequencing data from the Cancer Genome Atlas (TCGA). The TFEB-ERRα axis was assessed by dual-luciferase reporter and chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR). The mechanism was investigated using loss-of-function and gain-of-function assays in vitro. Lipidomics and proteomics were performed to identify the TFEB-ERRα-related lipid metabolism pathway. Pseudopods were observed by scanning electron microscope. Furthermore, immunohistochemistry and lipidomics were performed in clinical tissue samples to validate the ERRα-related lipids.
    RESULTS: TFEB and ERRα were highly expressed in EC patients and correlated to EC progression. ERRα is the direct target of TFEB to mediate EC lipid metabolism. TFEB-ERRα axis mainly affected glycerophospholipids (GPs) and significantly elevated the ratio of phosphatidylcholine (PC)/sphingomyelin (SM), which indicated the enhanced membrane fluidity. TFEB-ERRα axis induced the mitochondria specific phosphatidylglycerol (PG) (18:1/22:6) + H increasing. The lipid reprogramming was mainly related to mitochondrial function though combining lipidomics and proteomics. The maximum oxygen consumption rate (OCR), ATP and lipid-related genes acc, fasn, and acadm were found to be positively correlated with TFEB/ERRα. TFEB-ERRα axis enhanced generation of pseudopodia to increase the invasiveness. Mechanistically, our functional assays indicated that TFEB promoted EC cell migration in an ERRα-dependent manner via EMT signaling. Consistent with the in vitro, higher PC (18:1/18:2) + HCOO was found in EC patients, and those with higher TFEB/ERRα had deeper myometrial invasion and lower serum HDL levels. Importantly, PC (18:1/18:2) + HCOO was an independent risk factor positively related to ERRα for lymph node metastasis.
    CONCLUSION: Lipid reprogramming induced by the TFEB-ERRα axis increases unsaturated fatty acid (UFA)-containing PCs, PG, PC/SM and pseudopodia, which enhance membrane fluidity via EMT signaling to promote EC progression. PG (18:1/22:6) + H induced by TFEB-ERRα axis was involved in tumorigenesis and PC (18:1/18:2) + HCOO was the ERRα-dependent lipid to mediate EC metastasis.
    Keywords:  EMT signaling; ERRα; Endometrial cancer; Lipid reprogramming; Mitochondrial stress; TFEB
  24. Front Plant Sci. 2021 ;12 784780
      CRISPR/Cas9-mediated genome editing has been demonstrated in the model diatom P. tricornutum, yet the currently available genetic tools do not combine the various advantageous features into a single, easy-to-assemble, modular construct that would allow the multiplexed targeting and creation of marker-free genome-edited lines. In this report, we describe the construction of the first modular two-component transcriptional unit system expressing SpCas9 from a diatom episome, assembled using the Universal Loop plasmid kit for Golden Gate assembly. We compared the editing efficiency of two constructs with orthogonal promoter-terminator combinations targeting the StLDP gene, encoding the major lipid droplet protein of P. tricornutum. Multiplexed targeting of the StLDP gene was confirmed via PCR screening, and lines with homozygous deletions were isolated from primary exconjugants. An editing efficiency ranging from 6.7 to 13.8% was observed in the better performing construct. Selected gene-edited lines displayed growth impairment, altered morphology, and the formation of lipid droplets during nutrient-replete growth. Under nitrogen deprivation, oversized lipid droplets were observed; the recovery of cell proliferation and degradation of lipid droplets were impaired after nitrogen replenishment. The results are consistent with the key role played by StLDP in the regulation of lipid droplet size and lipid homeostasis.
    Keywords:  CRISPR; Phaeodactylum tricornutum; RNA polymerase II promoter; Stramenopile-type lipid droplet protein; lipid droplet; multiplexed genome editing; two-component transcriptional unit