The Lysosome in Malignant Melanoma: Biology, Function and Therapeutic Applications

Lysosomes are membrane-bound vesicles that play roles in the degradation and recycling of cellular waste and homeostasis maintenance within cells. False alterations of lysosomal functions can lead to broad detrimental effects and cause various diseases, including cancers. Cancer cells that are rapidly proliferative and invasive are highly dependent on effective lysosomal function. Malignant melanoma is the most lethal form of skin cancer, with high metastasis characteristics, drug resistance, and aggressiveness. It is critical to understand the role of lysosomes in melanoma pathogenesis in order to improve the outcomes of melanoma patients. In this mini-review, we compile our current knowledge of lysosomes’ role in tumorigenesis, progression, therapy resistance, and the current treatment strategies related to lysosomes in melanoma.


Introduction
Lysosomes are membrane-enclosed organelles responsible for the disposal and recycling of worn out and damaged cellular macromolecules and organelles. Cells digest foreign materials by endocytosis and phagocytosis in lysosomes [1,2]. Lysosomal degradation products are recycled back to the cytosol via diffusion and specific transport channels or released to the extracellular space by exocytosis [1]. Moreover, lysosomes play essential roles in other cellular processes, including nutrient sensing and the control of energy metabolism [3].
The alteration of lysosomal functions can lead to broad detrimental effects, such as inflammation, apoptosis, failure to clear potentially toxic cellular waste, and the dysregulation of cellular signaling, causing a variety of diseases, including cancers [3]. It is expected that the expression and function of various lysosomal hydrolases are increased in human tumors, and they often correlate with a higher risk of recurrence and poor prognosis [1]. In addition, studies have shown that lysosomal enzymes play critical roles in cancer invasiveness, angiogenesis, and progression [1].
Malignant melanoma, a highly metastatic, drug-resistant, and aggressive malignancy, is the most lethal form among skin cancers [4]. Although it comprises only 5-10% of all skin cancers, malignant melanoma is responsible for more than 75% of all skin cancer-related deaths [4]. The incidence rates of melanoma have been augmenting rapidly over the past few decades. According to American Cancer Society's estimates, about 99,780 (57,180 in men and 42,600 in women) new melanomas will be diagnosed in the United States in 2022 [5]. Several risk factors are reported to be associated with cutaneous melanoma, including skin type (fair skin, freckling, and light hair), exposure to sun radiation, number of nevi (>50 moles), age (being older), gender (being male), immune status (having a weakened immune system), family history and former removed melanomas [6]. The early stages of melanoma are relatively easy to manage by surgical excision. However, melanoma is highly invasive and metastatic, and the prognosis for long-term survival is poor when the disease reaches advanced stages. Even though the use of targeted therapies and immunotherapies has improved the clinical outcomes, melanoma remains notoriously difficult to treat once it metastasizes to other sites, including brain, lungs, liver or bone [7].
Increasing efforts are currently focusing on novel biomarker identification for predicting the treatment response and prognosis of melanoma. Zhang et al. established a 14 cytolytic activity (CYT)-related-gene prognostic model, which can be used as a predictive biomarker and therapeutic target for cutaneous melanoma patients [8]. Bacchetti et al. reported that the expression levels of paraoxonase-2 (PON2) could be positively related to the tumor aggressiveness of skin cancers, including melanoma [9]. Campagna et al. demonstrated that nicotinamide N-methyltransferase (NNMT) knockdown led to a significant reduction in cell proliferation and migration of human melanoma cell lines and was correlated to enhanced chemosensitivity to dacarbazine, suggesting that NNMT could represent a molecular target for the effective treatment of melanoma [10]. Still, it is important to understand melanoma pathogenesis and factors that contribute to drug resistance to develop new therapeutic strategies.
In this review, we mainly focus on the role of lysosomes in melanoma tumorigenesis and progression, along with therapy resistance, including chemotherapies, targeted therapies, and immunotherapies. We also highlight the current treatment strategies related to lysosomes in melanoma.

The Roles of Lysosomes in Melanoma Progression
Hanahan and Weinberg proposed that cancers have several hallmarks, such as sustaining proliferative signaling, evading tumor suppressors, and promoting invasion and metastasis [11]. Lysosomes play critical roles in tumorigenesis and cancer progression. Rapidly proliferating cancer cells place heavy demands on the synthesis rates of new proteins, membrane lipids, DNA, and RNA [12]. In addition, cancer cells need to compete with extreme fluctuations in the poor vascularized tumor microenvironment with limited nutrient and oxygen levels [13]. Thus, nutrient-scavenging pathways associated with lysosomes help generate the nutrients and energy required by cancer cells [13]. Compared to healthy tissue, melanoma has higher level of reactive oxidative species (ROS), which can damage DNA, proteins and lipids, resulting in senescence and cytotoxicity [14,15]. Lysosomal autophagy helps melanoma cells escape senescence and promote adaptive survival by recycling damaged cellular organelles and proteins [16].

Lysosomes Mediate Melanoma Cell Proliferation, Survival and Death
In mammalian and yeast cells, the master growth regulator mTORC1 protein kinase is recruited and activated at the lysosome in response to nutrients [17]. In tumor development, cancer cells often experience stressed and nutrient-limited milieu. Through phagocytosis, endocytosis, and macropinocytosis, extracellular substances are delivered to lysosomes and undergo lysosomal degradation to generate nutrients [18]. In melanoma, it is reported that Ca 2+ channel mucolipin 1 (MCOLN1) is preferentially required for the survival and proliferation of melanoma cells by negatively regulating MAPK and mTORC1 signaling [19]. Additionally, macropinocytosis is upregulated in melanoma cells relative to normal melanocytes and is sustained by MCOLN1 [19].
The MiT/TFE transcription factor family, encoding MITF (micropthalmia-associated transcription factor), TFEB, TFE3, and TFEC, belongs to the MYC superfamily of ba-sic helix-loop-helix leucine zipper (bHLH-ZIP) proteins [20]. MITF is recognized as a master regulator of melanocytes required for the development, growth and survival of melanocytes and as a melanoma oncogene amplified in 30-40% of melanomas [21][22][23]. Additionally, it is observed that the MITF E318K mutation predisposes to familial and sporadic melanoma [24]. MITF in melanoma not only acts as a major oncogene, but also correlates with many lysosomal genes and generates late endosomes that are not functional in proteolysis [25]. MITF is also the transcriptional regulator of v-ATPase gene expression in melanocytes and melanomas [26,27].
Ploper et al. reported that, in melanoma cells, the nuclear accumulation and stabilization of MITF caused an expansion of the late endolysosome/multivesicular body (MVB) compartment and elevated expression of late endosomal proteins, including Rab7, LAMP1, and CD63. This increased endosome/MVB biogenesis enhanced Wnt signaling by increasing the sequestration of destruction complex components, such as GSK3 and Axin1 inside MVBs, generating a positive feedback loop and ultimately contributing to melanoma proliferation [22,25]. Based on this finding and the fact that MITF-M isoform, which is the type of isoform expressed in melanomas, lacks this N-terminal domain which is required for lysosomal localization and mTOR phosphorylation, it is proposed that overexpressing MITF-M binds promiscuously to CLEAR element lysosomal genes without being restrained by mTOR signaling and hence regulates endolysosomal biogenesis [25].
Similarly, Möller et al. demonstrated that MITF can bind to the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells [28]. In metastatic melanoma cell lines and tumors, MITF positively correlated with the expression of lysosomal and autophagosomal genes, and the knockdown of MITF resulted in starvation-induced autophagy degradation in both melanocytes and melanoma cells, presumably due to less autophagosomal formation [28]. On the other hand, the overexpression of MITF in melanoma cells elevated the expression of lysosomal and autophagosomal genes and induced autophagosome formation, but not sufficiently to induce autophagic flux [28], echoing the finding observed by Ploper et al. [25].
Furthermore, it is reported that an MITF-dependent melanoma patient-derived cell line, 501Mel, showed induction of RagD expression and increased mTORC1 activation, and the silencing of RagD was sufficient to greatly revert the hyperproliferative phenotype of this melanoma cell line [29]. Notably, markedly reduced xenograft tumor growth upon RagD silencing was observed in mice bearing 501Mel melanoma cells, indicating a key role of RagD in promoting tumor growth [29]. A significant correlation between MITF and RagD gene expression levels is demonstrated by the analysis of microarray data of melanoma metastatic patients and melanoma cell lines [29]. These findings support the idea that melanomas associated with MITF hyperactivation lead to constitutive RagD GTPase transcriptional induction and enhanced mTORC1 signaling, which fuels tumor growth [29].
On the other hand, melanoma cells with enhanced expression of LAMP-2C, a lysosomeassociated membrane protein, displayed elevated cell cycle arrest, increased the expression of the cell cycle regulators Chk1 and p21, and greater apoptosis and necrosis [30]. Reduced tumor growth was also observed in immune-compromised mice bearing melanoma cells with increased LAMP-2C expression, suggesting a potential role for LAMP-2C as a tumor suppressor in melanoma progression [30].

Lysosome Alterations Promote Melanoma Invasiveness and Metastasis
Cysteine cathepsin proteases (herein referred to as cathepsins) are involved in almost all processes related to lysosomes, such as protein degradation, protein and lipid metabolism, autophagy, antigen presentation, growth factor receptor recycling, cellular stress signaling, and lysosome-mediated cell death [31]. In addition, these enzymes can be secreted via lysosomal exocytosis, resulting in the degradation of extracellular targets [31]. Accumulating evidence suggests that cathepsins critically contribute to tumor progression in a variety of cancers [31]. Cathepsins are crucial acid hydrolases within lysosomes and are the key effectors of protein catabolism and autophagy, which support the elevating metabolic needs of proliferating cancer cells [31][32][33]. Moreover, secreted cathepsins can modify the tumor microenvironment by degrading the extracellular matrix and contribute to tissue invasion and metastasis [31,[34][35][36].
In invasive human melanomas, cathepsins B and L were found to be upregulated and were correlated with metastasis. The inhibition of the cell-membrane-permeable cathepsins B and L suppressed the invasive growth of the melanoma cells [37]. Similarly, cathepsin K was strongly expressed in most primary melanomas and all cutaneous melanoma metastases. The inhibition of cathepsin K significantly reduced melanoma cell invasion and increased detection of internalized collagen in vitro [38]. Additionally, Alonso-Curbelo et al. reported that cathepsins can be misrouted within the cell via the lineage-specific wiring of the endolysosomal pathway [39].
Rab7, a GTPase, has a key role in lysosome biogenesis and the lysosomal-associated degradation of cytoplasmic vesicles. The knockdown of Rab7 promoted the secretion of multiple lysosomal cathepsins and matrix proteins, and subsequently increased melanoma invasion [39]. Moreover, Tripathi et al. investigated the signaling pathways leading to increased cathepsin secretion in melanoma cells and found that the nonreceptor tyrosine kinases Abl and Arg induced the secretion of cathepsins B and L by activating transcription factors, including Ets1, Sp1, and NF-κB/p65, which play critical roles in driving melanoma invasion and metastasis [40].
Furthermore, Johansson et al. revealed that the oncogene PRL3 bound and dephosphorylated RNA helicase DDX21 to restrain the transcription of MITF-regulated endolysosomal genes in the melanocyte stem cell [41]. In clinical melanoma samples, PRL3 expression was inversely correlated with endolysosomal vesicle gene expression, and high PRL3 expression was significantly associated with melanoma-specific death, making it a valuable predictive marker for metastatic melanoma-specific death in all stages [41]. Together, the abovementioned studies support the idea that the dysregulation of endolysosomal pathways has emerged as a hallmark of melanoma and a driver of metastasis (Table 1) [39,41,42].

Lysosomes Mediate Chemoresistance
Lysosomes play important roles in resistance to therapies such as chemotherapy, targeted therapy, and immunotherapy. Prior to recent therapeutic advances, chemotherapy was the treatment of choice for advanced melanoma, although the success has been minimal due to chemoresistance [44]. It may, however, remain important in the palliative treatment of refractory, progressed, and relapsed melanoma [45]. The molecular basis of chemoresistance in melanoma is multifactorial, including the overexpression of drug efflux proteins, the alteration of enzyme activation, the deregulation of apoptosis, Ras mutation, epithelial to mesenchymal transition, and the deregulation of microRNA expression [46].
Lysosomes contribute to hydrophobic weak base chemotherapeutic drug resistance via lysosomal sequestration, based on passive cation trapping [47]. Hydrophobic weak base chemotherapeutics become entrapped in lysosomes due to their protonation in the acidic lumen of lysosomes. Hence, these sequestered drugs can neither reach their target sites nor exert their cytotoxic effects [47]. In addition to the passive accumulation of chemotherapeutics, lysosomes can also mediate active lysosomal drug sequestration via transporters localized in the lysosomal membrane [47].
Autophagy is also emerging as a key player in therapy resistance. Therapy-induced autophagy has two possible effects; it may either contribute to the anticancer effects of chemotherapeutics or facilitate chemotherapy resistance [48]. In melanoma, Ma et al. showed that patients bearing melanomas with a high autophagic index were less likely to respond to treatment (temozolomide and sorafenib), and autophagy inhibition using either hydroxychloroquine or inducible shRNA against Atg5 led to a significant augmentation of temozolomide-induced cell death [49]. On the other hand, Li et al. demonstrated that the blockade of BRAF inhibitor-induced autophagy-lysosome activation in melanoma xenografts results in chemoresistance, which is associated with elevated TGF-β levels and enhanced TGF-β signaling [50].

Lysosomes Promote Resistance to Targeted Therapies
Kinase inhibitors, particularly BRAF and MEK inhibitors, have become the first line of treatment for advanced BRAF-mutated melanoma, which is the most common mutation in cutaneous melanoma. Although the response of these targeted therapies is impressive, the duration is short lived, with acquired resistance after a median of 6-8 months [51]. It is known that therapy-induced autophagy promotes resistance to kinase inhibitors. Ma et al. investigated tumor biopsies from patients treated with BRAF inhibitors or combined BRAF/MEF inhibitors and found that tumors with resistance to BRAF inhibitors had increased levels of autophagy relative to the baseline [52]. In addition, patients with higher therapy-induced autophagy levels experienced significantly lower response rates to BRAF inhibitors [52]. The in vitro experiments further demonstrated that BRAF inhibitors induced cytoprotective autophagy by activating an ER stress response [52]. Interestingly, it is shown that heterozygous (but not homozygous) Atg5 loss compromised the response of BRAF inhibitors in melanoma-specific mouse models, raising caution about the incomplete blockade of this gene in clinical aspects, since this may cause an unexpected worsening of patient outcomes [53].

Lysosomes Contribute to Immunotherapy Resistance
Immunotherapies with CD8+ cytotoxic T cells as major cellular effectors appear to be a promising treatment option for patients with advanced melanoma, since they show durable response rates in selected patients [7]. However, primary and acquired resistance to immunotherapies is common [7]. The secretion of the pore-forming protein perforin and granzyme B by human cytotoxic T cells is the crucial mechanism to kill target cells, which occurs at the cytotoxic T cell/target cell lytic synapse [54,55]. It is reported that human melanoma cells can develop a rapid response to cytotoxic T cells at the lytic synapse by a secretory burst of lysosome/late endosomes, which leads to cathepsin-mediated perforin degradation and deficient granzyme B penetration [55]. The inhibition of this melanoma response by the depletion of SNAP-23-dependent lysosomal trafficking, pH perturbation, or impairment of lysosomal proteolytic activity restores susceptibility to cytotoxic T cell assault. This finding supports this potential mechanism contributing to melanoma cell immune resistance [55]. Moreover, dendritic cells are required to license the cytotoxic activity of CD8+ T cells in T-cell-based immunotherapy. Santana-Magal et al. found that tumor-infiltrating monocyte-derived dendritic cells undergo apoptosis due to their excessive phagocytosis of melanoma-secreted lysosomes as melanoma progresses. In their absence, cytotoxic T cells fail to lyse melanoma cells, suggesting that lysosomes play a role in limiting the effect of immunotherapy [56].

Targeting Lysosomes in Cancer Therapeutics
The contribution of lysosomes to cancer progression and therapy resistance provides the rationale for targeting lysosomes as a cancer therapeutic strategy. Lysosomes are critical for both anabolic growth pathways driven by mTORC1 and catabolic pathways, including macropinocytosis as well as autophagy; these pathways are all potential targets in cancer [57]. mTOR is crucial for regulating cell growth, proliferation, metabolism, and angiogenesis. mTOR inhibitors have been widely used against cancers in multiple preclinical studies and clinical trials [57,58]. Despite having both pro-and antitumor effects, autophagy is a potential target of inhibition because it can promote the growth of established tumors and mediate therapy resistance during cancer therapy [59].

Lysosomotropic Agents
Lysosome-targeting strategies mainly focus on lysosomotropic compounds, such as chloroquine, ammonium chloride, methylamine, and siramesine [60]. Because of their weak base characteristics, lysosomotropic compounds act by elevating lysosomal pH when accumulating in the lysosomal lumen and usually inducing lysosomal membrane permeabilization [60,61]. Additionally, as repurposing approved and abandoned nononcological drugs becomes an alternative approach to identifying anticancer therapeutics, antimalarials that potentially inhibit lysosomal functions have been considered as promising candidates for oncological repurposing and have been tested in preclinical and clinical cancer studies [62,63]. Hydroxychloroquine (HCQ), one such antimalarial, has been widely investigated in many clinical trials combining other anticancer therapies [64]. A phase 1 trial of HCQ and temsirolimus (TEM) demonstrated that TEM and HCQ have antitumor effects by inhibiting autophagy in patient with a safe profile [65]. Another clinical study showed that the combination of high-dose HCQ and dose-intense temozolomide is safe and tolerable. The treatment outcome with a prolonged stable disease and responses suggested antitumor activity in melanoma patients [17]. These two clinical studies warranted the combination of potent autophagy modulating molecules for improving melanoma patients' outcomes.
In melanoma, it has been shown that amodiaquine (AQ), a clinical 4-aminoquinoline antimalarial, can cause an autophagic-lysosomal and proliferative blockade in melanoma cells. It can also sensitize melanoma cells to either starvation-or chemotherapeutic agentinduced cell death [63]. Xie et al. also demonstrated that the antimalarial lysosomotropic agent HCQ synergized with TEM, which in turn led to the suppression of melanoma growth and induced cell death via apoptosis in both three-dimensional spheroid cultures and tumor xenografts [66]. In addition, McAfee et al. synthesized a bisaminoquinoline autophagy inhibitor Lys05, which is more potent than HCQ, and assessed its antitumor activity in vivo [67]. This result showed that mice bearing melanoma xenografts treated with Lys05 exhibited more significant tumor growth reduction than mice treated with PBS and HCQ [67]. The authors also demonstrated that Lys05 caused lysosomal dysfunction by deacidifying the lysosome, leading to the impairment of lysosomal enzymes and effective autophagy inhibition [67].

v-ATPase Inhibits Melanoma Survival and Metastasis
v-ATPase inhibition is another potential strategy for cancer therapeutics because cancer cells are more reliant on v-ATPase for survival than nontransformed cells [68]. In melanoma, Pan et al. demonstrated that cleistanthin A (CA), a natural compound which has the ability to inhibit v-ATPase activity and to neutralize the pH of lysosomes, inhibited the invasion and migration of human melanoma A375 cells in vitro by downregulating the expression of matrix metallopeptidase (MMP)-2 and -9 [69]. In line with this, another in vitro study also showed that concanamycin, a very specific inhibitor of v-ATPases, significantly suppressed the migration and invasion of murine melanoma B16F10 cells, but not nontumor melanocyte Melan-A cells [70]. Additionally, Martins et al. reported that the inhibition of v-ATPases by Myrtenal disrupted the electrochemical H+ gradient across the tumor cell membranes, leading to reprogrammed cell death and decreased tumor cell migration and invasion in vitro using murine as well as human melanoma cell lines [71]. Myrtenal also significantly suppressed metastasis induced by B16F10 murine melanoma in vivo, supporting v-ATPase as a molecular target to inhibit cancer progression [71]. Furthermore, the inhibition of v-ATPase by esomeprazole, a proton pump inhibitor, inhibited proliferation of human melanoma cells in vitro and reduced the tumor growth in vivo as well as increased survival of mice bearing human melanoma [72]. Moreover, Nishisho et al. identified a particular v-ATPase subunit, a3 isoform, as a potential therapeutic target by showing that knockdown of a3 v-ATPase in B16F10 murine melanoma model significantly reduced invasiveness and metastases to lung and bone [73].

Cathepsins Inhibition Deactivates Melanoma Invasiveness and Metastasis
Cathepsins have been correlated with cancer metastasis by facilitating cell migration and invasiveness due to their proteolytic activity. Hence, targeting cathepsins has potential to be a therapeutic strategy [74][75][76]. It is reported that inhibition of cathepsin B by either a specific chemical inhibitor, CA-074, or specific anti-cathepsin B antibodies significantly prevented the in vitro invasiveness of metastatic melanoma cell lines [76]. Accordingly, CA-074 dramatically reduced human melanoma growth and lung metastases in murine xenograft models [76]. Similarly, Liu et al. demonstrated that cepharanthine (CEP), a natural alkaloid with cathepsin B inhibitory function, inhibited human primary cutaneous melanoma cell viability and proliferation in vitro. It also decreased the tumor growth in cutaneous melanoma mouse models by the topical application or intra-tumoral injection of CEP [77]. In addition, the incubation of primary cutaneous melanoma cells with CEP showed not only decreased cathepsin B expression, but also decreased autophagy-related protein, LC3-I and LC3-II, expression in a dose-dependent manner, while antioncogene p53, p21Cip1p, and p16Inka expression was upregulated [77].

Conclusions
The lysosome has emerged as a key regulatory hub in physiological homeostasis. Increasing evidence has also demonstrated its critical role in disease pathogenesis. Lysosomes are involved in many intracellular processes, including autophagy, and they have been shown to have a dual role in cancer progression and drug resistance. Therefore, to develop effective cancer therapeutics by targeting lysosomes, lysosomal activation and inhibition should be investigated cautiously. In addition, many lysosomotropic agents showing promising results are FDA approved and can translate to the clinic. A combination of lysosomotropic compounds with other therapies might improve the clinical outcomes of melanoma patients. However, more studies are needed to identify further lysosomotropic agents' specific roles in lysosome biogenesis and metabolism. Similarly, it is important to determine the consequences of v-ATPase and cathepsin inhibition, although a variety of studies have demonstrated their potential as therapeutic targets. The purpose of this mini-review is to highlight the role of lysosomes in melanoma progression and therapy resistance and to encourage the development of lysosome-targeted therapeutics, which hold hope for improving clinical outcomes of melanoma patients (Figure 1).  Funding: This research received no external funding.
Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.