Inhibition of Lysosomal Function Mitigates Protective Mitophagy and Augments Ceramide Nanoliposome–Induced Cell Death in Head and Neck Squamous Cell Carcinoma

Abstract

Therapies for head and neck squamous cell carcinoma (HNSCC) are, at best, moderately effective, underscoring the need for new therapeutic strategies. Ceramide treatment leads to cell death as a consequence of mitochondrial damage by generating oxidative stress and causing mitochondrial permeability. However, HNSCC cells are able to resist cell death through mitochondria repair via mitophagy. Through the use of the C6-ceramide nanoliposome (CNL) to deliver therapeutic levels of bioactive ceramide, we demonstrate that the effects of CNL are mitigated in drug-resistant HNSCC via an autophagic/mitophagic response. We also demonstrate that inhibitors of lysosomal function, including chloroquine (CQ), significantly augment CNL-induced death in HNSCC cell lines. Mechanistically, the combination of CQ and CNL results in dysfunctional lysosomal processing of damaged mitochondria. We further demonstrate that exogenous addition of methyl pyruvate rescues cells from CNL + CQ–dependent cell death by restoring mitochondrial functionality via the reduction of CNL- and CQ-induced generation of reactive oxygen species and mitochondria permeability. Taken together, inhibition of late-stage protective autophagy/mitophagy augments the efficacy of CNL through preventing mitochondrial repair. Moreover, the combination of inhibitors of lysosomal function with CNL may provide an efficacious treatment modality for HNSCC.

Targeting Sphingolipids for Cancer Therapy

“…Preclinical assays with cell lines and xenografts show that CNLs potentiate the effect of chemotherapy ( 114 – 116 , 120 ); reduce tumor proliferation mediated by apoptosis (increased cleavage of PARP and caspases) ( 110 – 112 , 114 , 116 – 119 , 121 , 123 ), autophagy (increased LC3-II and Atg5 levels) ( 117 , 122 ), necrosis ( 106 ), necroptosis ( 109 ), anoikis ( 108 ), mitophagy (mitochondrial membrane permeabilization) ( 116 , 119 , 121 – 123 ), and cell cycle arrest (increased p53 expression) ( 116 , 119 ); increase ROS levels ( 110 ); inhibit lysosomal function ( 116 , 122 ); inhibit integrin affinity ( 105 , 107 ); and target CD44 receptor ( 108 ), survivin ( 111 ), PI3K ( 107 , 114 ), MAPK ( 105 , 114 ), mammalian target of rapamycin (mTOR) ( 112 , 121 ), Akt and Erk1/2 ( 110 , 115 ) signaling ( Figure 3 ). For example, Shaw et al.…”

“…For example, Shaw et al. indicated that the combination of C6-CNLs with chloroquine (an inhibitor of lysosomal function and therefore an autophagy inhibitor) significantly increases apoptosis in response to ceramide by avoiding the repair of mitochondrial damage ( 122 ).…”

The Role of Ceramide Metabolism and Signaling in the Regulation of Mitophagy and Cancer Therapy

“…As mentioned in Section 3.2, cancer may upregulate basal mitophagy in a protective manner to reduce reactive oxygen species and restore depolarized mitochondria during chemotherapeutic treatment. Disruption of cancer-induced protective mitophagy through the use of lysosomal inhibitors was found to sensitize drug-resistant HNSCC cells to C6-ceramide nanoliposome delivery of therapeutic ceramide [174]. Conversely, knocking down neutral ceramidase in mouse embryonic fibroblasts stimulated protective autophagy and ceramide generation to protect against drug-induced necroptosis [175].…”

A nano-enhanced vaccine for metastatic melanoma immunotherapy

“…Indeed, many vaccines benefit from lipid-based delivery. Of particular note are the recently authorized COVID-19 vaccines, which utilize lipid nanoparticles containing antigenencoding RNA [18,19] . Our team has been successful in the use of nanotechnologies, including nanoliposomes, for a host of different therapies [20][21][22][23][24][25] .…”

“…Of particular note are the recently authorized COVID-19 vaccines, which utilize lipid nanoparticles containing antigen-encoding RNA [ 18 , 19 ] . Our team has been successful in the use of nanotechnologies, including nanoliposomes, for a host of different therapies [ 20 - 25 ] . Nanoliposomes have the capability of altering the pharmacokinetic properties of antigens and immunomodulators/adjuvants, with a marked reduction in off-target toxicity, protection of the therapeutic from degradation, along with more specific targeting to antigen-presenting cells [ 26 ] .…”