Oncogene (2013) 32, 1995–2004 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

REVIEW Cancer-associated lysosomal changes: friends or foes?

T Kallunki, OD Olsen and M Ja¨a¨ttela¨

Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal function. Accordingly, transformation and cancer progression are characterized by dramatic changes in lysosomal volume, composition and cellular distribution. Depending on one’s point of view, the cancer-associated changes in the lysosomal compartment can be regarded as friends or foes. Most of them are clearly transforming as they promote invasive growth, angiogenesis and drug resistance. The same changes can, however, strongly sensitize cells to lysosomal membrane permeabilization and thereby to -targeting anti-cancer drugs. In this review we compile our current knowledge on cancer-associated changes in lysosomal composition and discuss the consequences of these alterations to cancer progression and the possibilities they can bring to cancer therapy.

Oncogene (2013) 32, 1995–2004; doi:10.1038/onc.2012.292; published online 9 July 2012 Keywords: Lysosome; Cancer; metastasis; death

INTRODUCTION LYSOSOMAL BIOGENESIS AND COMPOSITION are acidic (pH 4.5–5) catabolic found in all Lysosome formation mammalian cells except for mature erythrocytes. They are Lysosomal biogenesis requires a complex integration of the responsible for the disposal and recycling of worn out and biosynthetic and endocytic pathways2,3,12 (Figure 2). The endoly- damaged cellular macromolecules and organelles as well as the sosomal system is a dynamic network of vesicles connecting the digestion of extracellular and foreign materials delivered to them plasma membrane to the lysosome. It starts from the budding of by endocytosis, autophagy and phagocytosis1–3 (Figure 1). The early from the plasma membrane and continues via a digestion of the cargo is brought about by residing in gradual maturation to late endosomes and finally to lysosomes. the lysosomal lumen, and the products are recycled back to the During this process that takes B30 min, lysosomes receive a via diffusion and specific transport channels or released to number of essential , including most of the lysosomal the extracellular space by exocytosis. hydrolases, either directly from the Golgi complex or via Malignant transformation introduces series of changes in exocytosis followed by endocytosis. Of these, the vacuolar H þ - lysosomes and their function (Figure 1). These include increased ATPase (V-ATPase) pump at the limiting membrane gradually lysosomal biogenesis and activity, changes in the lysosomal acidifies the maturing endosomes from pH around 6.2 in early membrane composition and enhanced secretion of lysosomal endosomes to a final pH around 4.5–5 in mature lysosomes.13,14 contents to the extracellular space.4–7 Accordingly, increases in The maturation process involves an inward budding of the the expression and function of various lysosomal hydrolases are limiting membrane resulting in numerous intraluminal vesicles.15 common in human tumors, and they often correlate with a higher The intraluminal vesicles function as important endosomal sorting risk of recurrence and poor prognosis.8–10 In addition to their stations and contain proteins (for example, endocytosed possible use as prognostic biomarkers, mounting evidence points receptors) and lipids destined to cycling back to the plasma to essential roles for extracellular lysosomal in cancer membrane or to the lysosomal degradation. invasiveness, angiogenesis and progression, making these enzymes attractive targets for cancer therapy.5,10 By contrast, intracellular release of lysosomal hydrolases can trigger apoptosis Synthesis and transport of lysosomal hydrolases or non-apoptotic lysosomal cell death providing a rational for Lysosomes contain over 50 hydrolases including proteinases, cancer therapies aiming at destabilization of the lysosomal 11 glycosidases, phosphatases, sulfatases, nucleases and lipases. membranes. Lysosomal hydrolases are synthesized in the rough endoplasmic Below, we review the fundaments of lysosomal biogenesis reticulum as proforms and processed in the Golgi complex, where and composition, and assemble our current knowledge on many of them are tagged with mannose-6-phosphate (M6P) cancer-associated changes in the expression and activity of residues and bind to specific M6P receptors (M6PRs) that target lysosomal components with focus on the therapeutic opportu- the complexes to clathrin-coated pits destined to early endo- nities they provide. We will focus here on those changes that are somes.3 Upon endosomal maturation, the hydrolases are released best studied and currently considered as putative therapeutic in late endosomes as a result of decreasing pH and they continue targets in cancer. their journey to the mature lysosomes, whose acidic pH provides

Cell Death and Metabolism and Centre for Genotoxic Stress Research, Danish Cancer Society Research Center, Copenhagen, Denmark. Correspondence: Professor M Ja¨a¨ttela¨, Cell Death and Metabolism and Centre for Genotoxic Stress Research, Danish Cancer Society Research Center, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. E-mail: [email protected] Received 24 April 2012; revised and accepted 1 June 2012; published online 9 July 2012 Cancer-associated lysosomal changes T Kallunki et al 1996

RTK activation Cell growth H+ RTK H+ ECM Endocytosis P degradation

Lysosomal H+ Angiogenesis Oncogene exocytosis activation Recycling Early / MVB Oncogene Degradation activation Lysosome

Late endosome Lysosomal Degradation leakage Old and damaged organelles and macromolecuels Cell death Autophagosome Plasma membrane Figure 1. Multiple lysosomal functions in cancer cells. The main function of the lysosomes is the degradation of cargo derived by autophagy and endocytosis. They can also fuse with the plasma membrane during cell injury, and have more specialized secretory functions (lysosomal exocytosis) in some cell types including for example, osteoclasts and invasive cancer cells. In the extracellular space, lysosomal hydrolases can promote cancer progression by multiple means (see text for details). Oncogene activation can lead to the destabilization of lysosomal membranes and increased sensitivity to the leakage of lysosomal hydrolases into the cytosol, where they can contribute to the demise of the cancer cell. Upon tyrosine kinase activation, endolysosomal compartment regulates the recycling of receptors and their ligands. ECM, ; MVB, multivesicular body; RTK, receptor tyrosine kinase.

them with optimal working conditions. Less characterized M6PR- so via positively charged adaptor proteins (for example, SAPs). independent lysosomal targeting pathways include transport of The suggested backbones of BMP synthesis include phos- b- by lysosomal integral membrane 2 phatidylglycerol generated in the endoplasmatic reticulum and (LIMP2 or LGP85), all the way from ER to lysosomes via the mitochondrial cardiolipin, but its exact biosynthetic pathway is still endosomal compartment,16 and sortilin-mediated targeting of unclear.1 acid sphingomyelinase (ASM) and sphingolipid activator proteins In addition to lysosomal integral 2 (also directly to lysosomes.17,18 known as SCARB2) and V-ATPase, lysosomal membranes contain a variety of membrane-associated proteins including lysosomal- Extralysosomal roles of lysosomal hydrolases associated membrane proteins 1 and 2 (LAMP1 and LAMP2), In addition to their central catabolic role, lysosomes can also LIMP1 (also known as CD63 or tetraspanin) as well as numerous less characterized membrane components and transport pro- mediate programmed cell death, repair and tissue 24 19–21 teins. A large fraction of lysosomal membrane proteins travel remodeling. All of these processes involve regulated release of lysosomal hydrolases via processes such as lysosomal from the trans-Golgi network to plasma membrane via the default secretory pathway and reach the lysosomal compartment through membrane permeabilization or lysosomal exocytosis/lysosomal 25 secretion. Although lysosomal membrane permeabilization endocytosis. Alternatively, they can travel directly from the trans-Golgi network to the endolysosomal membranes along releases lysosomal hydrolases into cytosol, lysosomal exocytosis 3 releases them into the extracellular space. multiple as yet poorly defined pathways. LAMP1 and LAMP2 contribute to about half of the lysosomal transmembrane proteins and they are essential for the maintenance of the structural Lysosomal membranes integrity of the lysosomal compartment.26,27 LAMPs are highly Lysosomal membranes can be divided into the external limiting homologous proteins with short cytoplasmic C-terminal tails and membrane and a number of intraluminal vesicles. Akin to other highly glycosylated luminal N-terminal domains. Their cellular membranes, their lipid composition is highly regulated glycosylated tails form a sugar coat called glycocalix to the inner with regard to lipid classes, saturation and chain length.22 They are surface of the limiting membrane that protects the membrane mainly made up of glycerophospholipids and sphingolipids and from degradation by lysosomal hydrolases. LAMPs also serve differ from other cellular membranes by having high amounts overlapping and distinct functions in lysosomal trafficking and of bis(monoacylglycero)phosphate (BMP), 4–17% of the total exocytosis, chaperone-mediated autophagy, autophagosome- lysosomal lipid depending on cell type and up to 70% of the lipid lysosome fusion and cholesterol transport.27–30 in the intraluminal vesicles.23 The major function of BMP is associated with its localization to the intraluminal vesicles, where it serves as a docking lipid for soluble lipases allowing them to Transcriptional control of lysosomal biogenesis reach their substrates in the membrane.1,22 Lipases containing a The understanding of the transcriptional control of lysosomal positively charged lipid-binding domain (for example, ASM) bind biogenesis has only just begun to emerge. Transcription factor EB directly to the negatively charged BMP, whereas most lipases do (TFEB), which positively regulates the expression of many

Oncogene (2013) 1995 – 2004 & 2013 Macmillan Publishers Limited Cancer-associated lysosomal changes T Kallunki et al 1997 Plasma membrane

Early endosome

Increasing vesicular acidity Sorting endosome

Late endosome/ Multivesicular body M6P

TGN Lysosome

LIMP Golgi BMP LAMP

Figure 2. The endocytic system. The endolysosomal system is a dynamic network of vesicles connecting the plasma membrane to the lysosome with decreasing pH during maturation. The maturation process involves an inward budding of the limiting membrane resulting in numerous intraluminal vesicles. The intraluminal vesicles function as important endosomal sorting stations and contain proteins and lipids destined to cycling back to the plasma membrane or to the lysosomal degradation. Lysosomes receive most of their hydrolyzes and membrane proteins either directly from the trans-Golgi network or via exocytosis followed by endocytosis. The highly glycosylated proteins at the limiting lysosomal membrane protect the membrane from degradation by lysosomal hydrolases.

encoding for lysosomal hydrolases and lysosomal membrane leak to the cytosol where they can activate either the proteins, has recently been identified as the major regulator of mitochondrial apoptosis pathway or trigger non-apoptotic cell lysosomal biogenesis.31,32 Interestingly, its activity is controlled in death, a mode of cell demise that can effectively kill even highly opposite ways by two cancer-associated kinases, mitogen- apoptosis resistant cancer cells.7,38 Below we highlight the cancer- activated protein kinase 1/extracellular signal-regulated kinases associated roles of individual cathepsins. (MAPK1/ERK2) and mammalian target of rapamycin complex 1. MAPK1/ERK2-mediated phosphorylation of TFEB blocks its nuclear D (encoded by CTSD) transport,33 whereas, activation of mammalian target of rapamycin Cathepsin D is an aspartate cathepsin and belongs to the complex 1 can lead to nuclear transport-inducing phosphorylation 39 32 superfamily of proteases. First reports of increased levels of of TFEB. These studies indicate that the biological function of TFEB cathepsin D in human cancer date back to the 1980s.40,41 Since is under complex regulation and that kinase-mediated control then plentiful studies have confirmed these findings in the of TFEB and other transcription factors regulating lysosomal 37,42 majority of solid cancers. Furthermore, positive correlations biogenesis have a major role in their cancer-associated changes. between cathepsin D expression and tumor size, tumor grade, metastasis, poor prognosis, chemoresistance and risk of recurrence have been reported.42,43 Accordingly, cathepsin D OPPOSITE ROLES OF CATHEPSINS IN TUMOR PROGRESSION; plays an essential role in the multiple steps of tumor progression by stimulating cancer cell proliferation and survival, fibroblast LOCALIZATION MATTERS 37,43 Cathepsins are powerful hydrolases that can be divided into three outgrowth and angiogenesis. Interestingly, these cancer- subgroups depending on the amino acid located in their catalytic promoting functions of cathepsin D appear to be largely independent of its proteolytic activity suggesting that the dyad: aspartate cathepsins (cathepsins D and E), cysteine protein or its unprocessed precursors have or growth cathepsins (cathepsins B, C, F, H, K, L, O, S, V/U/L2, W, X/Z/Y) 37,43 and serine cathepsins (cathepsins A and G).34 Akin to most other factor-like activities. Fittingly, pro-cathepsin D and its lysosomal hydrolases, they function optimally at pH around 4.5 or propeptide can induce the activation of the mitogen-activated lower. With respect to their secretion to the extracellular space protein kinase (MAPK-ERK) signaling pathway, cytokine secretion and expression of genes encoding for proteins supporting and leakage to the cytosol, it is important to note that many 42–44 cysteine cathepsins can also be active at higher pH, albeit often proliferation, metastasis and angiogenesis. The importance of the pro-form rather than the active form of cathepsin D in with changed functional parameters such as kinetics and substrate specificity.35 cancer progression challenges the development of cathepsin D Increased expression, activity and secretion of cathepsins lead targeting anti-cancer drugs since small molecule enzyme to enhanced tumor growth, invasion and angiogenesis.4,36,37 inhibitors are not likely to be effective. Accordingly, several cathepsins are overexpressed in variety of cancers, often both in the tumor cells and in tumor-associated Cathepsin B (encoded by CTSB) leukocytes, fibroblasts, osteoclasts, myoepithelial cells and Cathepsin B is a cysteine cathepsin of the papain superfamily, endothelial cells.4 The tumor promoting effects of cathepsins whose increased expression has been widely reported in most have been mainly assigned to their proteolytic activities outside cancer types, often both in cancer cells themselves as well as in the cell, whereas increased cysteine cathepsin activity in the tumor-associated macrophages and fibroblasts.4,45,46 In line with lumen of lysosomes enhances the of LAMP1 and the idea that cathepsin B enhances invasion and metastasis, it is LAMP2 and leads to the destabilization of lysosomal membranes often localized at the surface of tumor cells, and its expression is thereby sensitizing cells to various stresses and limiting cell predominantly increased in the cells at the invasive edge of the survival.26 Upon destabilization of lysosomal membranes, tumors. In general, increased cathepsin B levels detected by

& 2013 Macmillan Publishers Limited Oncogene (2013) 1995 – 2004 Cancer-associated lysosomal changes T Kallunki et al 1998 immunohistochemistry correlate well with higher mRNA levels angiogenesis are somewhat contradicting.65 Supporting its role in pointing to a transcriptional upregulation of cathepsin B encoding angiogenesis, cathepsin L expression is significantly associated CTSB .47 Accordingly, transformation of fibroblast and/or with the neovasculature of astrocytic tumors,67 and cathepsin L epithelial cells by activated Ras, Src or ERBB2 oncogenes increases specific inhibitor NSITC displays potent antiangiogenic activity in the levels of CTSB/CtsB mRNAs, protein and proteolytic activity and chick chorioallantoic membrane and mouse matrigel models of promotes pericellular distribution of lysosomes.26,48–50 Moreover, angiogenesis in vitro.68 Nevertheless, in the RT2 mouse model of ErbB2-induced upregulation of cathepsins B (and L) renders non- pancreatic islet cell carcinogenesis Ctsl deletion has no effect in invasive breast cancer cells highly invasive in a three-dimensional the angiogenic switch even though it effectively impairs tumor Matrigel invasion model.50 growth and invasiveness.52 The ErbB2-induced expression of cathepsin B-encoding CTSB Cathepsin L has an additional tumorigenic activity in the 50 gene appears to be independent of TFEB, the major regulator of nucleus. An isoform of cathepsin L that lacks the signal peptide the transcription of lysosomal genes.31 Instead, ErbB2 activates a necessary for the lysosomal targeting migrates to the nucleus signaling network consisting of PAK4, cdc42bpb, PKCa and MAPK- where it cleaves histone H3 and transcription factors such as the ERK2 serine–threonine kinases that activates a myeloid zinc-finger CCAAT-displacement protein/cut homeobox (CDP/Cux), thereby transcription factor 1 (MZF1), which in turn binds to an ErbB2- altering cell cycle progression and patterns induced enhancer element in the first intron of CTSB gene and contributing to oncogenic transformation.69–72 In colorectal in vivo.50 Interestingly, MAPK-ERK2 activity, which is essential for cancer increased nuclear versus lysosomal localization of cathepsin the ErbB2-induced activation of myeloid zinc-finger transcription L correlates with advanced tumor stage and poor prognosis.73 As factor 1 and expression of CTSB, inhibits the nuclear import of for cathepsin B, specific inhibitors have also been developed for TFEB.51 It remains to be studied whether similar transcriptional cathepsin L.74,75 Of these, the most promising metastasis and/or switch occurs in response to other CTSB-activating oncogenes and invasion blocking compounds include CLIK-148 and FF-FMK.65 FF- whether the transcription of other lysosomal genes also switches FMK, which can also inhibit cathepsin B at higher concentrations, from TFEB-dependency to dependency on myeloid zinc-finger decreases the invasion of human oral squamous cell carcinoma transcription factor 1 or other oncogene-activated transcription cell lines in vitro,76 and oral administration of CLIK-148 inhibits factors. human melanoma A375 cell metastasis to bone but not to liver or The active role of cathepsin B in tumorigenesis is strongly muscle in a xenograft mouse model.77 Several additional studies supported by murine cancer models. In the RIP1-Tag2/RT2 (RT2) support the bone degradative function of cathepsin L, making it a pancreatic islet cell carcinogenesis model, the multiple pancreatic possible target molecule in the treatment of metastatic bone islet tumors driven by the expression of oncogenic SV40 T-antigen disease.78 in insulin producing b-cells express high levels of cathepsin B.52,53 The usefulness of cathepsin L as a therapeutic target for cancer Crossing of these mice to a Ctsb-deficient background results in treatment has, however, been questioned by studies showing that strongly impaired tumor formation, tumor cell proliferation, its deficiency or inactivation promotes tumor progression in angiogenesis and invasiveness. The suggested molecular basis in mouse skin cancer models79–81 as well as in intestinal epithelia.82 this model includes secretion of cathepsin B from tumor cells and The molecular explanation for this tumor suppressive role of tumor-associated macrophages and direct cathepsin B-mediated cathepsin L may be its essential role in the negative regulation of cleavage of extracellular matrix components, E-cadherin and the recycling of growth factors and their receptors from the urokinase plasminogen activator propeptide, the latter leading to endocytic compartment back to the plasma membrane.83 the activation of a potent proteolytic cascade involving sequential activation of urokinase plasminogen activator, plasmin and Cathepsin K (encoded by CTSK) various matrix metalloproteases.52,54–57 Similarly, Ctsb deficiency Cysteine cathepsin K has high collagenolytic and osteolytic significantly delays the onset and growth of primary mammary activity. It is predominantly expressed in osteoclasts, where it is tumors and their lung metastases in murine mammary tumor the key protease responsible for bone resorption and bone virus-polyoma middle T antigen (PyMT) transgenic mice.58 Vice metabolism.84 Correspondingly, inactivating mutations in human versa, cathepsin B overexpression in PyMT mice promotes cathepsin K gene (CTSK) result in pycnodysostosis characterized by mammary tumor growth and metastasis.59 These data reveal short stature, skull deformities and skeletal abnormalities.85 extracellular cathepsin B as an attractive target for cancer therapy. Cathepsin K expression is activated in breast and prostate Supporting this notion, a non-cell permeable cathepsin B specific carcinomas with higher expression levels in bone metastases as inhibitor CA-074 has given promising results in murine models of compared with primary tumors.86–88 Cathepsin K inhibitors (CKI, metastatic melanoma and breast cancer.60,61 AFG495 and Odanacatib), which have been successfully used to Contrary to the strong tumor-promoting properties of cathepsin treat osteoporosis-associated bone loss,84,89 reduce breast cancer- B, increased cathepsin B (and L) activity in the lumen of the induced osteolysis and skeletal tumor burden in a mouse model of lysosomes may form an Achilles heal for the cancer cells via the skeletal metastasis.87 Notably Odanacatib is currently on phase III cleavage of LAMP1 and LAMP2, and subsequent destabilization of trial as a treatment to reduce the risk of breast cancer-induced lysosomal membranes, decreased stress tolerance and sensitiza- bone metastasis (http://www.cancer.gov/drugdictionary). In tion to drugs that activate the lysosomal cell death pathway.26,62 melanoma, cathepsin K expression correlates with advanced Thus, high cathepsin B expression in tumor cells may prove to be a metastatic disease,90 and pharmacological inhibition of useful biomarker for the responsiveness to lysosome-targeting cathepsin K with Boc-I reduces melanoma cell invasion through therapies. the Matrigel basement membrane matrix.90 Inhibition of cathepsin K in this model results in the accumulation of internalized Cathepsin L (encoded by CTSL1) in the lysosomal compartment, suggesting that cathepsin K is essential for the effective intracellular degradation of Cathepsin L is another ubiquitously expressed cysteine cathepsin phagocytosed matrix proteins also outside the bone. that is frequently overexpressed in various human tumors.46 It enhances migration and invasion by decreasing cell–cell adhesion and increasing extracellular matrix degradation.4,36 Its identified Other cysteine cathepsins substrates that include extracellular matrix proteins, such as The expression of cysteine cathepsins S, F, H, O, V (also known as laminin, fibronectin and as well as E-cadherin and L2 or U) and Z (also known as Y or X) is also increased in human proheparanase.52,63–66 Studies on the role of cathepsin L in cancers. However, relatively little is known about their regulation

Oncogene (2013) 1995 – 2004 & 2013 Macmillan Publishers Limited Cancer-associated lysosomal changes T Kallunki et al 1999 or function. Cathepsin S may function in angiogenesis by in response to nucleotides such as ATP, ADP and degrading anti-angiogenic, type IV collagen derived peptides adenosine with similar kinetics than cathepsin D.104 Moreover, and generating pro-angiogenic peptides by cleavage of laminin- heparanase is found in the body fluids of patients with metastatic 5.91,92 Accordingly, genetic inactivation of cathepsin S encoding cancer, indicating its secretion in vivo.105,106 CTSS gene in the RT2 mouse model of pancreatic cancer leads to Heparan and its derivatives, sulfated oligoshaccarides and impaired tumor formation and impaired angiogenesis.52 In the suramin as well as suramin analogs show potent antimetastatic same model system, genetic inactivation of CTSH (cathepsin H) activity in mouse models of metastatic cancers as well as in clinical also significantly impaired angiogenic switching of the pre- trials.107–110 PI-88, a heparanase inhibitor with potent malignant hyperplastic islets and resulted in a reduction in the antiangiogenic activity,110,111 is currently on phase III clinical trial subsequent number of tumors.93 Moreover, the tumor burden in as ‘the adjuvant treatment of subjects with hepatitis virus-related CTSH null RT2 mice is significantly reduced, in association with hepatocellular carcinoma after surgical resection’ (http://www. defects in the blood vasculature and increased apoptosis. Finally, cancer.gov/drugdictionary). Moreover, a novel heparanase and simultaneous depletion of cathepsins B and Z results in synergistic angiogenesis inhibitor, a mimetic PG545, has antitumor effects in the murine PyMT breast cancer model, shown promise in numerous pre-clinical murine cancer models suggesting that most, if not all, cysteine cathepsins may posess and is now on phase I clinical trial.112 When designing clinical trials tumor-promoting features.94 for heparanase inhibitors, it should be taken into account that contrary to most other cancer types, multiple large array data sets reveal a highly significant decrease in HPSE mRNA levels in colon Pharmacological targeting of cysteine cathepsins cancer (www.oncomine.org). More than one cathepsin is usually upregulated upon oncogene- driven transformation in vitro in pre-clinical murine cancer models as well as in human cancer. Their highly overlapping substrate Neu1 sialidase 1 (encoded by NEU1) specificities and functions in tumorigenesis suggest thus that a Sialidases (also known as ) cleave sialic acids from combined inhibition of their activities might be required for an gangliosides and . There are four mammalian efficient cancer therapy. Accordingly, a single genetic inactivation sialidases (Neu1, Neu2, Neu3 and Neu4), of which Neu1 is 113 of Ctsb or Ctsz in the PyMT oncogene-induced mammary predominantly localized in lysosomes. Neu1 activity is carcinomas delays only slightly the appearance of tumors and decreased in oncogene-transformed rat fibroblasts and mouse lung metastasis, whereas their combined inactivation results in a adenocarcinoma colon 26 cells with inversed correlation between 114 significant delay in tumor growth and reduction in the number Neu1 expression and metastatic potential. Similarly, its ectopic and size of metastases.94,95 In line with this, a cell-permeable expression and depletion in the HT-29 human colon cancer cells broad-spectrum cysteine cathepsin inhibitor JPM-OEt is much inhibits and enhances liver metastasis in a mouse xenograft 115 more effective than single depletions of Ctsb, Ctsl or Ctss in model, respectively. The metastasis suppressing function of reducing angiogenic switching in the murine RT2 pancreatic Neu1 has been suggested to be due to its sialyation of b4 integrin 115 carcinoma model.52,92 By contrast, JPM-OEt shows poor efficacy in resulting in decreased b4 phosphorylation and signaling. thePyMTmurinebreastcancermodel,96 and fails to reduce Furthermore, Neu1 is a negative regulator of lysosomal metastasis in the breast cancer bone metastasis model in which exocytosis, which is likely to contribute to its antimetastatic 29 thenoncell-permeableCA-074iseffective.61 These results may be activity. Excessive lysosomal exocytosis in Neu1-deficient cells simply due to differential bioavailability of the drugs in different can be inhibited by silencing of LAMP1, suggesting that tissues.96 Alternatively, different cysteine cathepsins may have oversialylated LAMP1 is a key mediator of excessive exocytosis 29 opposing roles in some cancers, or the ability of JPM-OEt to enter of lysosomal proteases in these cells. Increased sialyation of the cell and inhibit intracellular cathepsins may stabilize lysosomal LAMP1 and LAMP2 is found in some human cancers, and in colon 116 membranes and provide cancer cells with a survival advantage that cancer, it strongly correlates with the metastatic potential. counteracts the effects of extracellular inhibition of cathepsins. Because sialyated LAMPs are essential for the lysosomal 24 However, the available preclinical data strongly encourage the membrane stability, the excessive sialyation of LAMPs may continuing development of broad-spectrum cysteine cathepsin also protect lysosomal membranes from destabilization and inhibitors as cancer therapeutics, but clearly more basic research thereby enhance cancer cell survival. The fact that the on the anticancer effects of individual cathepsins and their inactivation of this enzyme promotes cancer progression makes localization is needed to find the optimal treatment modalities. sialidase a challenging target for cancer treatment.

GLYCOSIDASES REGULATE ANGIOGENESIS AND METASTASIS LYSOSOMAL SPHINGOLIPID CATABOLISM CONTROLS LYSOSOMAL INTEGRITY AND CELL SURVIVAL Heparanase (encoded by HPSE) Sphingolipids are sphingosine-based lipids, whose ‘bioactive’ Heparanase is an endo-b-D-glucuronidase and the predominant properties have made them interesting for cancer research.117,118 mammalian enzyme-degrading heparan sulfate. Heparanase is They modulate diverse physiological and pathological processes processed in late endosomes and lysosomes, and localizes including cell growth, cell death, autophagy and angiogenesis. primarily to these organelles as well as to the extracellular matrix. Sphingomyelinases and ceramidases are key enzymes of sphingo- Heparanase-mediated cleavage of heparan sulfate side chains lipid catabolism, as they regulate the formation and degradation of from the heparane sulfate leads to the disassembly ceramide, one of the most intensely studied classes of sphingolipids of the extracellular matrix and is associated with tissue remodel- with pro-apoptotic and growth inhibitory activities.119 Human cells ing, cell movement, cancer progression, metastasis and angiogen- express several forms of these enzymes that possess varying pH esis.97 Heparan sulfate binds and sequesters several growth optima. Here, we concentrate only on the lysosomal forms that act in factors, and into the extracellular matrix low pH, that is, ASM and acid ceramidase. and cell surface, and its cleavage will therefore increase their release and tissue availability.98,99 Heparanase expression is enhanced in numerous human cancers,6,100,101 and its activity Acid sphingomyelinase (encoded by SMPD1) positively correlates with the metastatic potential of tumor cells, The intraluminal lysosomal vesicles are the major sites for the attributes to the remodeling of the extracellular matrix degradation of sphingomyelin by ASM. ASM is a water-soluble and promotes neovascularization.102,103 Cancer cells secrete with positively charged regions that facilitate its

& 2013 Macmillan Publishers Limited Oncogene (2013) 1995 – 2004 Cancer-associated lysosomal changes T Kallunki et al 2000 binding to negatively charged lipids such as BMP, a binding which accumulate in acidic lysosomes where they interfere with the greatly increases ASM activity by bringing the enzyme to the electrostatic attraction between ASM and its docking lipid BMP on proximity of its neutral substrate in the intraluminal lysosomal the surface of intralumenal vesicles promoting proteolysis of the vesicles.120 Furthermore, the association of ASM with membranes enzyme.1 These drugs are, however, not specific for ASM, but protects it from proteolytic degradation by lysosomal inhibit also acid ceramidase and lysosomal phospholipases.127 proteases.121 Notably, Desipramine, a well-established inhibitor of ASM, Sphingolipids and their metabolites regulate cell survival with destabilizes lysosomal membranes in a manner similar to the diverse mechanisms that are not fully understood. The pro- RNAi-mediated depletion of Smpd1.125 Furthermore, most, if not apoptotic activity of ASM is largely attributed to the localization of all, cationic amphiphilic drugs are capable of reverting multidrug the enzyme to the plasma membrane in response to various resistant phenotype in cancer cells.11,136 It is, however, unclear stresses.122,123 The subsequent generation of ceramide at the whether the disturbance of sphingolipid metabolism contributes plasma membrane contributes to the formation of ordered to this effect, or whether it is mediated via the cationic membrane domains that can enhance apoptosis induction by neutralization of lysosomal pH or other yet undefined activities death receptors.122–124 By contrast, lysosomal ASM may have an of these drugs. Taken together, our knowledge of the actual roles opposite effect on cell survival as its activity is essential for the of ASM and acid ceramidase in tumor development and maintenance of the lysosomal membrane integrity.125 Notably, progression are sparse and calls for the development of more ASM may also contribute to cancer progression by a mechanism specific enzyme inhibitors suitable for the use in in vivo models of involving ceramide-mediated activation of pre-pro cathepsin D to cancer. enzymatically active isoforms of cathepsin D.126 Owing to the well-established pro-apoptotic properties of ceramide, ASM has been mainly considered as a negative V-ATPASE REGULATES ALL ASPECTS OF LYSOSOMAL regulator of cancer progression.119,127 Accordingly, our database The maintenance of highly acidic pH in the lysosomal lumen is search revealed a highly significant cancer-associated reduction in essential for the proper function of the lysosomes. The pH ASM, encoding SMPD1 mRNA levels in multiple studies of gradient between cytosol and lysosomal lumen is generated gastrointestinal, hepatocellular, salivary gland, renal and head by the action of a V-type ATPase that uses the free energy of and neck carcinomas, whereas as a modest but significant ATP hydrolysis to drive protons against their electrochemical increase in SMPD1 expression was found only in single studies gradient into the lysosome.137 This 14-subunit protein complex of hairy cell leukemia and smoldering myeloma (www.oncomine. is composed of a soluble ATP-hydrolyzing (V1) and a org). The decreased SMPD1 expression in several cancers may, transmembrane H þ -translocating (V0) subcomplexes. The however, contribute to the increased sensitivity of transformed V-ATPase activity does not only ensure lysosomal digestive cells to lysosomal cell death pathways. On the other hand, the functions but is also essential for the lysosomal calcium SMPD1 mRNA levels do not necessarily reflect the levels of ASM homeostasis as well as lysosomal trafficking and fusion protein whose binding to BMP, stability and activity can be events.137,138 Thus, the inhibition of V-ATPase activity impairs enhanced by heat shock protein 70,125 which is commonly endocytosis and autophagy and may even disrupt lysosomal relocated to the plasma membrane and thereby also to the membrane integrity. endolysomal compartment of cancer cells.128,129 In spite of the cancer-associated increase in acid production, the entire V-ATPase complex does not seem to be overexpressed in Acid ceramidase (encoded by ASAH1) neoplastic cells. Instead, the increased expression of specific subunits may contribute to altered activity of the pump. For Lysosomal ceramide is further degraded by acid ceramidase to example, overexpression of the gene encoding for the V1 subunit sphingosine that diffuses rapidly out of the lysosomes and can be C(ATP6V1C1) in oral squamous carcinoma cells has been converted to sphingosine-1-phosphate.130,131 The inhibition of suggested to enhance the pump activity by promoting the acid ceramidase has received much attention in anticancer assembly of V0 and V1 subcomplexes.139 On the other hand, therapy due to the cell death and cell survival promoting cisplatin-resistant cell lines have been reported to upregulate the attributes of ceramide and sphingosine-1-phosphate, respec- V0 subunit-c (ATP6V0C),140 and changes in the V0 subunit-a tively.127 Furthermore, a recent study identified acid ceramidase as isoform expression pattern affect the of the a positive regulator of prostate cancer invasion via a mechanism V-ATPase, the a3 isoform favoring the localization at the plasma involving sphingosine 1-phosphate-mediated upregulation of CTSB membrane.141 The plasma membrane V-ATPase acidifies the expression.132 extracellular space thereby easing the action of cathepsins on The expression of ASAH1 has been reported to be upregulated extracellular matrix and enhancing cancer cell invasion.142 in prostate cancer,133 and downregulated in thyroid cancer.134 Accordingly, depletion of a3 isoform in highly invasive breast Additionally, our database search reveals significantly increased cancer or melanoma cells results in decreased cytosolic pH and expression of ASAH1 in centroblastic, hairy cell and mantle cell reduced invasiveness.143,144 lympomas as well as in glioblastoma, and decreased expression Even though the specific targeting of the differently assembled especially in leukemias, lung cancers and sarcomas V-ATPase pumps is not possible with the tools available today, the (www.oncomine.org). In spite of the strong experimental existing V-ATPase inhibitors may prove useful in cancer therapy. evidence linking acid ceramidase activity to increased cell Several different types of V-ATPase inhibitors exist and they can survival, convincing in vivo evidence supporting its pro- induce programmed cell death and cell cycle arrest in various tumorigenic effect is still lacking. By contrast, the ASAH cancer cells, inhibit invasion, revert multidrug-resistant phenotype expression correlates with a better prognosis in ER-positive and some of them have even been shown to decrease xenograft breast cancer.135 Thus, more in vivo studies are clearly needed in tumor growth in vivo.13,145,146 Of these, special attention has been order to evaluate acid ceramidase as a putative cancer target. given to benzolactone enamides that can possibly even discriminate between different V-ATPases and thus are believed Pharmacological inhibition of sphingomyelin catabolism to serve as good basic compounds for the development of Several clinically relevant drugs, most notably the large group of V-ATPase targeting antitumor agents.147,148 Interestingly, in a cationic amphiphilic drugs (tricyclic antidepressants, malaria synthetic lethal screen, benzolactone enamide RTA203 and taxol drugs, antihistamines, and so on), induce a functional loss of synergistically killed H1155 cancer cells at doses where the single ASM activity.121 As basic substances, cationic amphiphilic drugs compounds only had minor effects,149 supporting the idea of

Oncogene (2013) 1995 – 2004 & 2013 Macmillan Publishers Limited Cancer-associated lysosomal changes T Kallunki et al 2001

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