Role of endolysosomes and pH in the pathogenesis and treatment of glioblastoma

Abstract

Background

Glioblastoma multiforme (GBM) is a Grade IV astrocytoma with an aggressive disease course and a uniformly poor prognosis. Pathologically, GBM is characterized by rapid development of primary tumors, diffuse infiltration into the brain parenchyma, and robust angiogenesis. The treatment options that are limited and largely ineffective include a combination of surgical resection, radiotherapy, and chemotherapy with the alkylating agent temozolomide.

Recent findings

Similar to many other forms of cancer, the extracellular environment near GBM tumors is acidified. Extracellular acidosis is particularly relevant to tumorgenesis, and the concept of tumor cell dormancy because of findings that decreased pH reduces proliferation, increases resistance to apoptosis and autophagy, promotes tumor cell invasion, increases angiogenesis, obscures immune surveillance, and promotes resistance to drug and radio‐treatment. Factors known to participate in the acidification process are nutrient starvation, oxidative stress, hypoxia, and high levels of anaerobic glycolysis that lead to increases in lactate. Also involved are endosomes and lysosomes (hereafter termed endolysosomes), acidic organelles with highly regulated stores of hydrogen (H⁺) ions. Endolysosomes contain more than 60 hydrolases as well as about 50 proteins that are known to affect the number, sizes, and distribution patterns of these organelles within cells. Recently, vacuolar ATPase (v‐ATPase), the main proton pump that is responsible for maintaining the acidic environment in endolysosomes, was identified as a novel therapeutic target for glioblastoma.

Conclusions

Thus, a greater understanding of the role of endolysosomes in regulating cellular and extracellular acidity could result in a better elucidation of GBM pathogenesis and new therapeutic strategies.

1. OVERVIEW

Glioblastoma multiforme (GBM) is a very aggressive and highly invasive primary brain cancer that continues to hold a very poor prognosis for the patient.1 Pathologically, GBM is characterized by rapid development of primary glioblastomas, diffuse infiltration of tumors into the brain parenchyma, and robust angiogenesis. Despite years of research, life expectancy for the patient from the time of diagnosis to death is generally less than 24 months. Treatment options are limited and largely ineffective; these include a combination of surgical resection, radiotherapy, and chemotherapy with the alkylating agent temozolomide alone and in combination with other adjuvant therapies.

Tumor cells in GBM are mainly of astrocytic lineage, and morphological and functional abnormalities have been noted among subcellular organelles including mitochondria, and endosomes and lysosomes (hereafter referred to as endolysosomes). GBM has at least one thing in common with many other forms of cancer; the extracellular tumor environment adjacent to the tumor is increasingly acidic. Extracellular acidosis is particularly relevant to tumorgenesis and the concept of tumor cell dormancy because of findings that decreased pH reduces proliferation, increases resistance to apoptosis and autophagy, promotes tumor cell invasion, increases angiogenesis, obscures immune surveillance, and promotes resistance to drug and radio‐treatment.2 This extracellular acidification is influenced by a number of associated factors including nutrient starvation, oxidative stress, hypoxia, high levels of anaerobic glycolysis, and increased levels of lactic acid. Accordingly, mitochondria have been implicated, and multiple structural and functional abnormalities of mitochondria have been identified in GBM tumor cells.3 Hydrogen (H⁺) ions that help influence extracellular pH can originate from endolysosomes, highly acidic organelles that possess a number of pumps and channels that control their H⁺ stores. Recently, vacuolar ATPase (v‐ATPase), the main proton pump responsible for maintaining the acidic environment in endolysosomes as well as in extracellular spaces, was identified as a novel therapeutic target for GBM.4 However, very little is known about the coordinated effort of normal and cancer cells to regulate the pH of endolysosomes, cytoplasm, and the extracellular milieu. Here, we will discuss possible contributions of pH to the pathological development of GBM and to its possible treatment.

2. PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL ROLES OF ENDOLYSOSOMES

The original description of lysosomes in 1955 was honored by the award of a Nobel Prize.5 The description of endosomes and the process of endocytosis by Brown and Goldstein in 1985 also resulted in the award of a Nobel Prize.6 Subsequently, much has been learned about the many physiological functions regulated by endolysosomes including plasma membrane repair, cell homeostasis, energy metabolism, nutrient‐dependent signal transduction, and immune responses.7, 8 The ability of endolysosomes to control a wide spectrum of cellular functions is complemented by the tight control of endolysosome biogenesis, and expression levels and activity of a number of resident acidic enzymes and proteins, findings that highlight the ability of endolysosomes to match their activity to the needs of the cell. Endolysosomes contain more than 60 hydrolases as well as about 50 proteins that help control the cellular and subcellular distribution patterns of endolysosomes as well as their numbers, their sizes, and their activity levels within cells.9 Pathologically, morphological and functional changes to endolysosomes have been described for a wide spectrum of diseases including cancer and neurodegenerative diseases.10

3. ACIDIC NATURE OF ENDOLYSOSOMES

A defining feature of endolysosomes is their acidic luminal pH, a feature that is regulated mainly by v‐ATPases linked to the flux of anionic and cationic counterions including Na⁺, K⁺, Ca²⁺, and Cl⁻, and the free energy of ATP hydrolysis that drives protons against their electrochemical gradient into the lumen of endolysosomes.7 This acidic environment is essential for the functionality of endolysosome‐resident hydrolases that participate in the degradation of macromolecules into such cellular building blocks as amino acids, monosaccharides, and free fatty acids. The acidic pH is also important for the fusing of autophagosomes with lysosomes to form autophagolysosomes that control the important self‐catabolic (intracellular components) function known as autophagy and for which another Nobel prize was awarded.11 H⁺ ions are central to the establishment of transmembrane proton gradients that drive ATP synthesis in mitochondria, but such gradients exist also in other organelles including endolysosomes, Golgi complex, and secretory granules as well as on the plasma membrane.12 The acidic nature of endolysosomes predates the discovery of lysosomes by about 60 years.13

4. ROLE OF ENDOLYSOSOMES IN REGULATING THE CYTOSOLIC AND EXTRACELLULAR ENVIRONMENTS OF CANCER TUMORS

The extracellular environment adjacent to tumors is generally referred to as the tumor microenvironment, and this environment is acidic even in GBM.14, 15 In GBM, four zones each with distinct cellular phenotypes and pH levels have been described: the “necrotic zone” that exhibits pH values ≤3.4, the surrounding “pseudo‐palisading cell zone” with pH values <5.5, the “cellular tumor zone” with pH values of 6.2 to 7.0, and finally the “leading edge zone” with normal pH values of 7.2 to 7.4.16

This extracellular acidification observed with cancer is influenced by a number of associated factors involving mitochondria including nutrient starvation, oxidative stress, hypoxia, high levels of anaerobic glycolysis, and increased levels of lactic acid. Extracellular acidification and these same associated factors have all been found to be abnormal in GBM tumor cells3 and increase the fate and progression of GBM.16 H⁺ ions released from endolysosomes can not only cause endolysosome de‐acidification but can also lead to acidification of the cytoplasm and the extracellular space. v‐ATPase, the main regulator of H⁺ ions in endolysosomes, has been identified as a novel therapeutic target for GBM,4 and organelle pH generally, and endolysosome pH specifically, affects oncogenic signaling in GBM. Further implicating the role of endolysosome pH in GBM are findings that the pH‐dependent CLIC‐1 channel is required for GBM stem cell proliferation.17

5. EFFECTS OF GBM ON THE MORPHOLOGY AND FUNCTION OF ENDOLYSOSOMES

During transformation of normal cells into cancer cells, lysosomes have been found to be increased in size. They are more fragile than normal lysosomes, their distribution patterns in cells are affected, and the levels and activity of enzymes are increased.18, 19, 20 Furthermore, lysosome exocytosis was described as a mediator of cancer progression.21 More specifically to astrocytomas, including GBM, with increased tumor grade, a greater percentage of cells stained positively for the lysosomal marker LAMP‐1 and staining intensity was increased.22 The LAMP‐1 positive cells in GBM co‐stain for both the astrocyte marker GFAP and the tumor stem cell marker CD133.22 In high‐grade gliomas, levels of LAMP1 transcript and immunopositive staining for LAMP‐1 protein were higher than those measured in normal brain.23 Furthermore, higher levels of the lysosomal markers cathepsin B and D, and lysosomal biogenesis transcription factor TFEB were found in GBM, and the higher levels of cathepsin L may decrease apoptosis of glioma cells.24 The relevance of the aspartic endopeptidase cathepsin D to GBM is supported by findings that cathepsin B and D levels were elevated in glioblastoma and other high‐grade astrocytomas. The level of the cathepsins correlated with the grade of the gliomas as well as the shortened survival times of the individuals with GBM, and treatment with anti‐cathepsin D antibodies inhibited brain tumor cell invasion.24, 25 Furthermore, increased tumor invasion and progression may be linked to acidification of extracellular microenvironments because most cathepsins are active at acidic pH.26

6. ROLE OF ENDOLYSOSOMES AND AUTOPHAGY IN GBM

GBM cancer cells are often resistant to cell death via apoptosis because they acquire mutations or epigenetic silencing of key proteins in the apoptotic machinery. However, lysosomal cell death pathways including autophagic cell death are still functional, and, because of this, investigators continue to be interested in promoting glioblastoma cell death using lysosomal membrane permeabilization strategies. Autophagy is enhanced in astrocytomas compared with normal tissue but did not correlate with tumor grade or patient survival thus suggesting that activation of autophagolysosome pathways is secondary to restricted nutrient supply and not with more severe insults as hypoxia or amino acid restriction.1 Moreover, v‐ATPase is highly expressed by high‐grade gliomas compared with normal brain and less aggressive grade II tumors.4 While there continues to be ongoing debate as to whether autophagy promotes or limits cancer growth, some have found that inhibition of autophagy reduced GBM development.27 Lysosomes contain high levels of hydrolytic enzymes particularly the cathepsins, and limited release of cathepsins can cause apoptotic cell death, whereas large‐scale lysosomal permeabilization can cause cytosolic acidification and necrosis.

7. POSSIBLE USE OF LYSOSOME DESTABILIZING DRUGS IN GBM

With GBM, patient survival is very short because the cancer is highly infiltrative thus preventing radical surgery, the limited effectiveness of chemotherapy and radiation, and the proposed existence of tumor stem cells. Increasingly, it has been suggested that lysosome destabilizing drugs might be used therapeutically against GBM.28 LAMP‐1 might be involved in lysosome‐mediated cell death, and knocking down expression levels of LAMP1 using siRNA strategies was shown to decrease cancer cell proliferation, migration, and invasion.29 Lysosome‐destabilizing drugs such as the sigma receptor agonist siramesine caused permeabilization of lysosome membranes, endolysosome de‐acidification, the release of cathepsins, and increased oxidative stress. Importantly, it was suggested that the changes in endolysosome pH preceded the endolysosome permeabilization, the leakage of cathepsins, and the cathepsin‐mediated increases in cell death of immortalized and transformed glioblastoma cells.22

Using an in vivo approach where U87MG tumors were implanted subcutaneously in mice, the weak base anti‐malarial drug chloroquine and other quinoline‐based clinically used anti‐malarial drugs were found to block authophagy, increase endoplasmic reticulum stress, and increase the chemosensitivity of glioma cells to the anti‐glioma drug temozolomide.30, 31 Chloroquine might also function as an adjuvant therapeutic against GBM by virtue of its ability to increase cellular oxidative stress.32 A mechanism by which chloroquine increases reactive oxygen species might be because ferrous iron is released by endolysosomes, is taken up by mitochondria, and leads to increased levels of reactive oxygen species in the cytosol and in mitochondria. Similar to chloroquine, the v‐ATPase inhibitor bafilomycin A1 was toxic to glioma primary cultures and was synergistic with temozolomide and the tetracyclic triterpene alcohol euphol in activating autophagy‐associated cell death.33 Additionally, bafilomycin A1 was found to decrease the viability of primary GBM neurospheres, decrease the viability of GBM organotypic cultures, and induce stem cell depletion and reduce the expression of stem cell factors.4 Further linking the lysosomotropic drugs chloroquine and bafilomycin to GBM therapeutics are findings that transforming growth factor (TGF)‐beta contributes to the pathogenesis of GBM and that these agents reduce levels of secreted TGFbeta.34, 35 Bafilomycin A1 also increased the cell death of CD133+ cancer cells with a stem cell‐like phenotype when treated with a humanized monoclonal antibody to vascular endothelial growth factor‐A (VEGF‐A), bevacizumab.36

The antipsychotic drug thioridazine inhibits late stage autophagy by impairing fusion between autophagosomes and lysosomes, and thioridazine in combination with temozolomide resulted in significant reduction in growth of human intracranial GBM xenografts in vivo and prolonged the survival of tumor‐bearing mice.37 The improved effectiveness of thioridazine with temozolomide may have resulted from decreased autophagy and/or endolysosome de‐acidification because similar effects were observed with chloroquine and bafilomycin A1, both of which de‐acidify endolysosomes.37 These effects of thioridazine are consistent with its physicochemical properties of it being a base with a pKa of 9.5 and with its ability to accumulate in and become trapped by endolysosomes.38

Given the above evidence that endolysosome de‐acidification and/or inhibition of autophagy might be of therapeutic benefit against GBM, clinical trials have been conducted with chloroquine and analogs of chloroquine.39, 40 Although some clinical successes have been noted with chloroquine, a phase I/II clinical trial with the autophagy inhibitor hydroxychloroquine administered in combination with standard and adjuvant chemoradiotherapy failed to show a significant benefit in patients with newly diagnosed GBM.41 However, as was pointed out by Johannessen and colleagues,37 pharmacologic inhibition of autophagy was not achieved in the patients due to toxic side effects, and it was not determined the extent to which endolysosomes were de‐acidified with hydroxychloroquine.

However, not all reported findings are consistent with this large body of accumulated evidence that endolysosome de‐acidification causes cell death associated with GBM. The lysosome inhibitor NH₄Cl at a concentration that itself did not affect glioma cell migration did block the ability of cordycepin‐induced inhibition of focal adhesion protein expression and glioma cell migration.42 Thus, more work needs to be conducted with new inhibitors of autophagy and/or drugs that de‐acidify endolysosomes.

8. POSSIBLE USE OF NANOMATERIALS AS GBM THERAPEUTICS

Nanomaterials are increasingly being used as therapeutic vehicles for a wide variety of disorders including cancer. These agents largely enter cells by endocytosis and accumulate in endolysosomes.43, 44, 45, 46 In addition to serving as vectors for anti‐tumor agents, the nanomaterials themselves may serve to decrease tumor size and migration. Similar to bafilomycin A1 and chloroquine, we reported recently the ability of silica nanomaterials to accumulate in endolysosomes, de‐acidify endolysosomes, and at higher concentrations decrease neuronal viability.47 Further, the ability of de‐acidifying agents to increase levels of amyloid beta protein provides a level of caution in terms of their possible chronic use in aged individuals who might be at risk of developing Alzheimer's disease.47, 48 However, acidic nanomaterials might find more therapeutic use because they can be uptaken by endolysosomes, promote and/or restore endolysosome acidification, increase cathepsin activity, and increase lysosome hydrolysis and function.10

9. ROLE OF CHOLESTEROL AND LRP1 IN GBM THERAPEUTICS

Low density lipoprotein receptor 1 (LRP1) is a highly promiscuous receptor that is activated by a wide spectrum of ligands including low density lipoprotein (LDL) cholesterol48 and HIV‐1 Tat.49 LRP1 is highly expressed in glioblastoma U87MG cells, especially in endolysosomes,50 and promoted glioblastoma cell migration and invasion.51 We have shown that LDL cholesterol accumulated in de‐acidified endolysosomes and increased autophagy.48 Because of findings that bafilomycin A1 and chloroquine de‐acidify endolysosomes, induce autophagy, and decrease viability of glioblastoma cells it is tempting to speculate that agents that alter cholesterol trafficking might find use as therapeutics against GBM. Indeed, it was shown recently that the antifungal drug itraconazole decreased plasma membrane cholesterol content, increased the accumulation of cholesterol in endosomes and lysosomes, and decreased the proliferation of glioblastoma cells in vitro and in vivo.52 These actions of itraconazole were confirmed using another compound capable of increasing cholesterol accumulation in late endosomes and lysosomes: U18666A.52 Caffeine decreased glioblastoma tumor size,53 and this too might be linked to LRP1 and cholesterol trafficking because we reported the ability of caffeine to block LDL cholesterol internalization through an apparent adenosine A₃ receptor‐mediated mechanism.54

10. POSSIBLE ROLE OF INTERORGANELLAR SIGNALING IN GBM

It is becoming increasingly clear that endolysosomes physically and functionally interact with other intracellular organelles including mitochondria, peroxisomes, endoplasmic reticulum, and the plasma membrane. Such recognition has led to a resurgent interest in cell biology and an increased appreciation for the physiological and pathophysiological significance of the dynamic physical and chemical communications occurring between intracellular organelles including those regulated by pH. The ability of endolysosomes to change directional movements in cells almost certainly affects their ability to interact with other organelles and to regulate important physiological function including lysosome acidification, cell motility, and nutrient homeostasis. Others and we have shown that compounds that acidify endolysosomes such as the mucolipin synthetic agonist ML‐SA1 or the endogenous agonist of TRPML‐1 phosphatidlyinositol‐3,5‐bisphosphate (PI(3,5)P₂) cause endolysosomes to exhibit a mainly perinuclear pattern while compounds that de‐acidify endolysosomes cause these organelles to exhibit a larger profile and movement toward the plasma membrane.55 Given the above discussed roles of mitochondria and endolysosomes in GBM, it is likely that interorganellar signaling mechanisms are involved and might be targeted therapeutically.

Possibly the first physical interactions to be described between organelles were between mitochondria and endoplasmic reticulum some 60 years ago, and it was ~30 years ago that the functional significance of mitochondria‐associated membranes that link mitochondria to endoplasmic reticulum was described.56 Research on physical and functional interactions between organelles has increasingly escalated, and these organellar interactions have now been implicated in the pathogenesis of multiple diseases including cancer and neurodegenerative diseases.55, 57, 58, 59 As it relates to endolysosomes, it is now known that there are extensive physical interactions between endolysosomes and mitochondria and that these interorganellar communications participate in lipid and metabolite exchange as well as mitochondrial quality control.60 Conversely, mitochondrial dysfunction has been found to negatively affect lysosome structure and function through reactive oxygen species‐dependent mechanisms.61 Extensive membrane contact sites exist between lysosomes and endoplasmic reticulum, these contact sites are evolutionarily conserved, and the calcium released from lysosomes was sufficient to stimulate endoplasmic reticulum‐dependent calcium‐induced calcium release.62, 63, 64 Furthermore, endolysosomes maintain their thousand‐fold calcium concentration gradient in part by refilling endolysosome stores of calcium from IP₃‐regulated stores of calcium in endoplasmic reticulum.65 Because of the importance of store‐operated calcium entry in cancer,66 much more work is warranted into how interorganellar signaling regulates the pathogenesis of cancers including GBM and how such mechanisms might be manipulated for therapeutic benefit.

11. SUMMARY

Endolysosomes are acidic organelles that have been implicated in the pathogenesis of GBM. Further, possible therapeutic agents that affect endolysosomes and associated autophagolysosomes continue to be tested because glioblastoma cells die not from apoptosis but rather from autophagy. Central to pathological features of endolysosomes in U87MG glioblastoma cells is the regulation of pH. The findings of others and us suggest strongly that greater attention be paid to understanding better the effects of GBM therapeutic strategies on endolysosomes and levels of H+ ions in acidic organelles, cytoplasm, and extracellular environments.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AUTHORS' CONTRIBUTIONS

All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conceptualization, J.D.G., J.E.O.; Methodology, X.C., G.D., P.H., M.L.S.; Writing—Original Draft, J.D.G., P.H.; Writing—Review & Editing, G.D., J.E.O., M.L.S.; Funding Acquisition, J.D.G., X.C., J.E.O.

Halcrow P, Datta G, Ohm JE, Soliman ML, Chen X, Geiger JD. Role of endolysosomes and pH in the pathogenesis and treatment of glioblastoma. Cancer Reports. 2019;2:e1177. 10.1002/cnr2.1177