Autophagy in UV damage response

Abstract

UV radiation exposure from sunlight and artificial tanning beds is the major risk factor for the development of skin cancer and skin photoaging. UV-induced skin damage can trigger a cascade of DNA damage response signaling pathways, including cell cycle arrest, DNA repair, and, if damage is irreparable, apoptosis. Compensatory proliferation replaces the apoptotic cells to maintain skin barrier integrity. Disruption of these processes can be exploited to promote carcinogenesis by allowing the survival and proliferation of damaged cells. UV radiation also induces autophagy, a catabolic process that clears unwanted or damaged proteins, lipids, and organelles. The mechanisms by which autophagy is activated following UV exposure, and the functions of autophagy in UV response are only now being clarified. Here, we summarize the current understanding of the mechanisms governing autophagy regulation by UV, the roles of autophagy in regulating cellular response to UV-induced photodamage, and the implications of autophagy modulation in the treatment and prevention of photoaging and skin cancer.

Introduction

Exposure to ultraviolet (UV) radiation through sunlight and indoor tanning beds is the major risk factor for skin cancer development and skin photoaging. UV radiation is divided into three major types by wavelength: UVA (315-400 nm), UVB (280-315 nm), and UVC (100-280 nm). Of these, UVA is the most abundant, as it accounts for about 95% of solar UV radiation, and indoor tanning beds emit UVA at doses up 12-fold higher than the sun¹. UVB accounts for the remaining 5% of solar UV radiation, while UVC is filtered out by the ozone².

Absorption of UV radiation, and consequently the extent of skin damage, is dependent on a number of factors, including skin color/type, time of exposure, latitude, altitude, season, and wavelength³. UVA penetrates deep into the dermis⁴, while UVB reaches only the epidermis⁵.However, UVA is much less efficient in causing direct DNA damage than UVB, instead inducing oxidative DNA damage through reactive oxygen species (ROS)⁶. Both UVA and UVB have been shown to cause cell proliferation, growth arrest, and apoptosis, although these responses can be context-dependent. These effects of UVA and UVB on skin will be reviewed here. UVC, although it is the most mutagenic type of UV radiation⁷, will not be discussed here, as it is not a major relevant source of skin-damaging radiation.

Macroautophagy (hereafter autophagy) is an essential, homeostatic cellular process of “self-eating.” Through this process, cells clear unwanted or damaged proteins, lipids, and other cellular components, and in doing so regulate the availability of a number of cell signaling factors. Furthermore, autophagy-mediated recycling of cytoplasmic contents facilitates cell survival and adaptation during starvation, genotoxic stress, and oxidative stress in normal cells⁸. Autophagy can also provide nutrients to sustain high rates of proliferation in times of growth. Dysregulation of autophagy can therefore contribute to the development of a number of skin diseases, including skin cancer. UVA, UVB, and UVC have all been reported to induce autophagosome formation and upregulation of autophagy markers^9–11. However, given the varying effects of UV radiation, it is likely autophagy plays a number of context-dependent roles in UV response. Here, we will examine our current understanding of the regulation and function of autophagy in photodamage response, as well as the implications for UV-induced skincancer and aging.

Mechanisms of Autophagy

Autophagy Induction

Genotoxic stress can be induced by a number of pharmacological and environmental factors, including UV radiation. Genotoxic stress induces autophagy to mitigate the effects of DNA damage¹². Defects in autophagy are associated with increased DNA damage, gene amplification, and aneuploidy¹³. These effects are likely due to insufficient metabolic precursors in the absence of autophagy¹³. Genotoxic stress regulates autophagy at least in part through the stabilization of p53 (Figure 1, 2 and 3), which regulates the transcription of various autophagy and lysosomal genes¹⁴.

Figure 1. Mechanisms of Autophagy.

Autophagy is initiated upon activation of the AMPK signaling pathway. AMPK can be activated by p53 and Sesn2 in response to cellular stress. AMPK then activates TSC1/2 to inhibit mTORC1. mTORC1 is further negatively regulated by PTEN to activate autophagy. ULK1 is activated by inhibition of mTORC1, direct phosphorylation by AMPK, or p53-mediated transcriptional upregulation. ULK1 then forms a complex which activates Beclin1. Beclin1, which binds to Bcl-2 in normal conditions, dissociates from Bcl-2 and binds to UVRAG. UVRAG, a p53 target gene, promotes the formation of the Beclin1 complex to initiate phagophore nucleation. Two ubiquitin-like conjugation systems facilitate the lipidation of LC3-I to form LC3-II, and elongation of the burgeoning phagophore. E1-like activating enzyme Atg7 leads to the activation of E2-like enzyme Atg3 (System 1) or Atg10 (System 2), subsequently the activation of the E3-link enzyme complex Atg5-Atg12 conjugate with Atg16, and subsequently the generation of LC3-II. LC3-II binds to an adaptor protein or autophagy receptor, such as p62, which in turn binds to cargo with modifications such as polyubiquitination. The autophagosome encloses the cargo and subsequently fuses with a lysosome to form an autolysosome. Adaptor and cargo are degraded at the autolysosome and the resulting molecules are recycled to the cytoplasm.

Figure 2. Autophagy in UVB response.

UVB radiation causes direct DNA damage in the form of photoproducts, and autophagy is induced by UVB to promote photoproduct repair. UVB directly induces autophagy activators AMPK, UVRAG and p53. p53, upon stabilization by UVB, activates transcription of AMPK, Sesn2, TSC2, and UVRAG to activate autophagy. ATR is also induced by UVB-induced DNA damage and can activate AMPK signaling. Sesn2 interacts with AMPK, TSC1, and TSC2 to activate autophagy in response to genotoxic stress. Activation of autophagy by these factors leads to the degradation of p62 and thus the decrease in Twist1 stability, which is required for XPC upregulation following UVB, and recruitment of DDB2 to sites of UVB-induced DNA damage. Conversely, autophagy activator PTEN is inhibited in response to UVB and this impairs NER by downregulating XPC.

Figure 3. Autophagy in UVA response.

UVA-induced ROS production stimulates autophagy, and autophagy regulates the oxidative stress response following UVA. UVA stabilizes p53, which induces transcription of autophagy activators AMPK and Sesn2. Conversely, UVA suppresses PTEN expression, which may negatively impact autophagy induction following UVA. Autophagy clears oxidized proteins and lipids following UVA exposure, but suppresses Nrf2-mediated antioxidant response following UVA.

Oxidative stress induced by the formation of reactive oxygen species (ROS) similarly induces autophagy¹⁵ (Figure 3). ROS are highly reactive molecules, including radicals (singlet oxygen) and non-radicals such as hydrogen peroxide (H₂O₂). Low basal levels of ROS act as signaling molecules in healthy cells, but elevated ROS levels cause oxidative damage to proteins, lipids, and DNA, as is seen in the response to UVA¹⁶. Autophagy provides protection against oxidative stress by clearing the damaged proteins, lipids, and DNA, and restoring metabolic homeostasis¹⁷. The antioxidant response triggered by nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is linked to autophagy through a feedback loop with adaptor protein p62. p62 competes with Nrf2 to bind to Kelch-like ECH-associated protein 1 (KEAP1)^18,19, preventing KEAP1-mediated degradation of Nrf2. Increasing p62 levels, therefore, leads to the stabilization of Nrf2, and the degradation of KEAP1 by autophagy²⁰. Nrf2 in turn binds to the antioxidant response element (ARE) within the p62 promoter to activate p62 transcription¹⁸.

Another autophagy regulator is the microphthalmia-associated transcription factor (MITF) family member transcription factor EB (TFEB)^21–23. TFEB is a basic helix-loop-helix leucine zipper transcription factor, which binds to the Coordinated Lysosomal Expression and Regulation (CLEAR) binding site found in the promoter of many autophagy and lysosomal genes²³. TFEB overexpression enhances transcription of an autophagy and lysosomal gene program²¹, as well as the degradation of autophagy substrates, mitochondria, and lipid droplets²⁴. TFEB has been reported to activate the transcription of an autophagy program in response to starvation²¹.

TFEB activation is thought to be regulated primarily through phosphorylation. Under nutrient-rich conditions, TFEB is primarily cytosolic and inactive²⁵. Upon nutrient deprivation, TFEB rapidly translocates to the nucleus and is activated to induce transcription of autophagy genes²⁵. Phosphorylation at two sites, Ser211 and Ser142, determine the localization and activity of TFEB²⁶. Ser211, when phosphorylated, is a docking site for chaperone 14-3-3, and this interaction retains TFEB in the cytoplasm^25,27. Furthermore, to maintain its cytosolic localization and inactivation, TFEB is phosphorylated at Ser211 by mammalian target of rapamycin complex 1 (mTORC1), a known negative regulator of autophagy ²⁷.

mTORC1 is a key regulator of cell growth, proliferation, protein synthesis, and autophagy²⁸ (Figure 1). mTORC1 consists of the core components mLST8, serine/threonine kinase mTOR, and adaptor protein Raptor. mTORC1 is regulated in response to cellular stress by 5′-AMP-activated protein kinase (AMPK)²⁸ (Figure 1). AMPK and its activator LKB1 sense reductions in cellular ATP and induce autophagy to replenish ATP stores. AMPK activation leads to the phosphorylation and activation of tuberous sclerosis complexes 1 and 2 (TSC1/2). TSC2 is a GTPase-activating protein which acts on small G-protein Rheb to inhibit mTORC1, and mTORC1 inhibition by TSC2 induces autophagy²⁸ (Figure 1).

mTORC1 is also regulated by the phosphatidylinositol-3 kinase (PI3K)-protein kinase B (AKT) pathway, and its negative regulator phosphatase and tensin homolog (PTEN). PI3K phosphorylates PIP2 to form signaling intermediate PIP3, while PTEN dephosphorylates PIP3. PIP3 activates AKT, which in turn activates mTOR and phosphorylates Beclin1 to inhibit autophagy^29,30. PTEN has been shown to negatively regulate mTOR to induce autophagy in a variety of cell types^31,32, and is activated by ATM in response to DNA damage to induce autophagy³³.

Autophagy Initiation

The mTORC1/AMPK pathway regulates the initiation of autophagy (Figure 1). In nutrient rich conditions, mTORC1 binds, phosphorylates, and inactivates Unc-51-like kinase 1 (ULK1) and Atg13³⁴. AMPK can also bind and phosphorylate ULK1³⁵, blocking inhibition by mTOR and activating ULK1³⁶. ULK1 activation by AMPK or by mTOR inhibition allows ULK1 to phosphorylate Atg13 and focal adhesion kinase family-interacting protein of 200 kD (FIP200)³⁴. ULK1, Atg13, and FIP200 form a scaffold (called the ULK1 complex)³⁴, which localizes to the burgeoning phagophore and promotes the recruitment of other proteins essential for autophagy.

The ULK1 complex activates essential autophagy gene Beclin1^37,38, via ULK1-mediated phosphorylation³⁹ (Figure 1). Beclin1, which is bound to antiapoptotic protein Bcl-2 in normal conditions, dissociates⁴⁰ and binds to UVRAG³⁸. UVRAG promotes the formation of the Beclin1 complex, consisting of core components Beclin1, Vps34, and p150⁴¹. The Beclin1 complex can interact with other proteins to induce phagophore nucleation in response to a variety of stressors⁴².

Elongation

Upon initiation of phagophore nucleation by Beclin1 complex formation, two sequential ubiquitin-like conjugation systems are induced to facilitate phagophore elongation⁴³ (Figure 1). In the first, ubiquitin-like Atg12 is activated by the E1-like activating enzyme Atg7. Atg7 transfers Atg12 to the E2 conjugating enzyme Atg10, and Atg12 is irreversibly attached to Atg5. The Atg12-Atg5 conjugate binds to Atg16 and attaches to the phagophore⁴³. In the second system, LC3 is then processed by cysteine protease Atg4 to allow activation by Atg7. Atg7 transfers the activated LC3 (LC3-I) to E2 enzyme Atg3. Finally, a phosphatidylethanolamine (PE) lipid is attached to LC3-I by Atg12-Atg5 conjugate to form LC3-II. LC3-II is linked to the phagophore membrane by the attached PE. The amount of LC3-II ultimately controls the size of the autophagosome, and the amount of cargo included for degradation⁴³.

Degradation

LC3-II facilitates selective autophagy of long-lived proteins, protein aggregates, or damaged organelles by forming interactions with adaptor proteins (Figure 1). Selective autophagy adaptors, such as p62/SQSTM1, bind to cargo for degradation, and to LC3-II on the autophagosome membrane through the LC3-interacting region (LIR)⁴⁴. Upon autophagosome-lysosome fusion, both the adaptor and cargo are degraded. The products of autophagy are recycled to the cytosol to maintain essential cellular processes after starvation or stress.

Autophagy in UV-Induced DNA Damage Response

UV-Induced DNA Photodamage

UVB radiation is efficiently absorbed by DNA within the epidermis⁵ and damages DNA directly to form photoproducts. The most common UVB-induced photoproducts are cyclopyrimidine dimers (CPDs) and, to a lesser extent, pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs)⁴⁵. Of these, 6-4PPs are bulkier, but more efficiently repaired than CPDs^46–48. As a result of this ineffective repair as well as the abundance of damage, CPDs are responsible for most (∼80%) UVB-induced mutations^49,50.

The ability of UVA to induce photoproduct formation, and the extent to which this contributes to skin damage, remains controversial. UVA causes the formation of far fewer photoproducts than UVB^51,52, and was previously thought to be harmless. However, multiple studies have reported UVA-induced CPD formation, and in many cases, CPDs were the predominant lesion formed by UVA^51,53–56. UVA does not cause significant formation of 6-4PPs^51,53,54,57. UVA-induced CPDs are predominantly T-T dimers⁵⁸, and these dimers persist longer than UVB-induced CPDs⁵¹. The abundance of UVA-induced CPDs, and ineffective repair of these lesions may therefore form a significant source of UVA-induced skin damage⁵⁶.

UV radiation-induced DNA photodamage activates DNA damage repair, cell cycle arrest, and apoptotic pathways. Cell cycle arrest allows time for recognition and repair of photoproducts, but if DNA damage is extensive and irreparable, apoptosis is initiated⁵⁹. In surviving cells, proliferation is induced to replace dying cells and maintain tissue homeostasis⁶⁰. Disrupting signaling through these pathways can lead to cancerous expansion of damaged cells.

One of the key signaling mediators is p53, a transcription factor that plays a key role in balancing the pathways activated in response to DNA photodamage (Figure 2). UV-induced photoproduct formation leads to the stabilization of p53⁶¹. p53 then activates transcription of a complex program of cell cycle inhibitors, DNA damage response genes^62,63, and apoptotic genes^64,65. p53 induces the transcription of cell cycle inhibitor p21 to arrest the cell cycle at the G1 phase following UV exposure⁶⁶, and p53-mediated DNA damage response gene induction is required for the repair of UV-induced DNA damage⁶². The decision to induce pro-apoptotic signals by p53 is context dependent, and can be impacted by p53 expression prior to UV exposure⁶⁷, skin cell layer^68,69, length of time since UV exposure⁶⁷, and cell type⁶⁴.

DNA Damage Repair

UV-induced photoproducts are repaired by either transcription-coupled (TC-) or global genome (GG-) nucleotide excision repair (NER)^70–73. TC-NER allows the rapid recognition and repair of damage in transcriptionally active genes, while GG-NER repairs damage throughout the genome independent of gene transcription. TC-NER begins when RNA polymerase stalls at distorted DNA. Initiation of GG-NER requires recognition of distorted DNA by xeroderma pigmentosum complementation group C (XPC) or damage specific DNA binding protein 2 (DDB2, also called XPE)⁷⁰.Transcription of XPC and DDB2/XPE is induced by p53 following UVB irradiation⁶².

Upon recognition of DNA damage, both NER subpathways converge on a common repair pathway⁷⁰. Transcription factor II H (TFIIH), a dimer of helicases XPB and XPD, is recruited to the damage site and unwinds DNA around the damage site. XPA and replication protein A (RPA) bind to the damaged strand and undamaged strand, respectively. This binding allows the recruitment of endonucleases XPG and XPF-ERCC1. XPG and XPF-ERCC1 then excise the damaged DNA to create a single strand of DNA complementary to the damage site. Replication factor C (RFC) loads proliferating cell nuclear antigen (PCNA) onto the DNA strand and a DNA polymerase synthesizes a strand complementary to the damage site. Finally, DNA ligase seals the nicks to complete NER⁷⁰.

Cell cycle arrest following UV exposure is critical to provide ample time for DNA damage repair, and to prevent proliferation of damaged cells. UV-induced DNA damage activates the sensors ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) to trigger cell cycle arrest via p53 stabilization⁶⁵ (Figure 2). ATR recognizes single strand DNA breaks caused by UV, including RPA-bound ssDNA formed during NER. ATR then phosphorylates checkpoint kinase 1 (Chk1) to activate checkpoints at the G1, S, and G2/M phases⁷⁴. ATM, which recognizes double strand breaks (DSBs), phosphorylates Chk2 to delay the cell cycle⁷⁵. XPC and DDB2 have been shown to facilitate the recruitment of ATM and ATR to sites of DNA damage and promote the activation of cell cycle arrest pathways⁷⁶.

Regulation of autophagy by UV-induced DNA damage response

UVB exposure directly and rapidly activates AMPK⁹, UVRAG⁷⁷, and p53⁷⁸ to activate autophagy (Figure 2). Upon activation by UV, p53 induces transcription of autophagy activators AMPK, TSC2, Sestrin 1, and Sestrin 2^79–81. Sestrin 1 and 2 have been shown to interact with AMPK, TSC1, and TSC2 to inhibit mTOR signaling in response to genotoxic stress⁸⁰.

Regulation of UV-induced DNA damage response by autophagy

Genotoxic stress is a trigger for autophagy, and repair of UV-induced DNA damage is regulated by autophagy. Knockdown of key autophagy genes AMPK, Atg5, Atg7, Atg12, and Atg14 impairs the repair of UVB-induced DNA damage^82,83. Recent work has identified multiple pathways through which autophagy regulates UV-induced DNA damage repair.

First, we have shown that autophagy positively regulates the recognition of DNA damage by XPC and DDB2. Knockdown of essential autophagy gene Atg5 decreases XPC transcription following UVB radiation by a Twist1-dependent mechanism⁸³. Autophagy deficiency leads to the accumulation of Twist1, which in turn activates transcriptional repressor complex E2F4-RBL2 through AKT signaling. The E2F4-RBL2 complex represses XPC transcription in autophagy-defective cells, leading to an accumulation of DNA damage⁸³.

Recognition of UV-induced DNA damage by DDB2 is also dependent on autophagy. Autophagy deficiency impairs DDB2 recruitment to CPD sites following UVB exposure⁸³ (Figure 2). This defect was found to be caused by Twist1 binding to and inhibiting p300, a key factor in DDB2 recruitment⁸³. We have previously shown that Twist1 is stabilized by p62 in autophagy-deficient conditions⁸⁴, and autophagy-mediated degradation of Twist1 can facilitate DDB2 recruitment and CPD repair⁸³. Collectively, this work suggests a tumor suppressive role for autophagy in the promotion of DNA damage repair.

UVB rapidly induces autophagy through activation of AMPK to regulate DNA damage repair^9,82 (Figure 2). AMPK activation by UVB increases XPC protein levels and increases CPD repair. Knockdown of AMPK reduces CPD repair following UVB, but has no effect on 6-4PP repair⁸². This work further links autophagy to the positive regulation of UVB-induced DNA damage repair.

Autophagy activator UVRAG was initially identified in a genetic screen as a protein able to partially complement the UV sensitivity of xeroderma pigmentosum (XP) cells defective in GG-NER⁸⁵. Until recently, however the function of UVRAG in DNA damage response was unknown. UVRAG has recently been shown to be essential for both autophagy and GG-NER in response to UV⁸⁶. UVRAG was found to facilitate the recruitment of DDB1 and DDB2 to sites of UV-induced DNA damage by binding DDB1. Knockdown of UVRAG inhibits transfer of damaged DNA from DDB1 to XPC during NER initiation. However, UVRAG activates autophagy and GG-NER independently⁸⁶, suggesting UVRAG may act as a signaling hub for concurrent activation of DNA damage repair and autophagy (Figure 2).

Conversely, autophagy activator PTEN is inhibited by Sestrin 2⁸⁷ and in response to UVB⁸⁸ (Figure 2). PTEN downregulation impairs GG-NER by downregulating XPC transcription⁸⁹. It is unclear, however, whether downregulation of autophagy has a significant role in PTEN-regulated DNA damage repair in response to UVB.

Autophagy in Oxidative Stress Response

UV-Induced Oxidative Damage

The most established effect of UVA is causing oxidative damage to DNA, proteins, and lipids (Figure 3). UVA is absorbed by cellular photosensitizers, which transfer energy in either type I or type II photosensitization reactions⁹⁰. In type I reactions, energy is transferred directly to DNA through free radical formation, resulting in oxidative modifications. In type II reactions, photosensitizers transfer energy to molecular oxygen, creating singlet oxygen⁹¹. Singlet oxygen reacts preferentially with guanine and can cause a number of oxidative DNA modifications. Of these, 8-oxo-2′-deoxyguanosine (8-oxo-dG) is the most common oxidative lesion caused by UVA⁵⁵. 8-oxo-dG can be repaired through base excision repair by 8-oxo-guanine glycosylase (OGG1)-mediated excision⁹². However, defective repair of 8-oxo-dG lesions following UVA exposure can cause GC→TA transversion⁵⁵. UVB can also cause ROS production and oxidative damage⁹³, although this effect is likely secondary to the direct DNA damage caused by UVB.

UVA-induced oxidative stress damages lipids and proteins in addition to DNA. UVA induces the oxidation of phospholipids in keratinocytes^10,94, and these oxidized phospholipids form adducts with proteins^95,96. Accumulation of oxidized phospholipid-protein adducts is found in a number of degenerative diseases and also in photoaged skin⁹³.

Accumulation of proteins with oxidative modifications is also seen in the dermis after UVA exposure⁹³, likely due to reduced expression of antioxidant enzymes^93,97. Recently, UVA has been shown to target OGG1 for oxidation⁹⁸, and in doing so, compromises BER of oxidized DNA⁹⁸. Furthermore, UVA has been shown to cause oxidative damage to single-strand DNA binding factor RPA, impairing NER^16,99. This work suggests there are combinatorial effects of UVA and UVB in inducing and perpetuating DNA damage.

ROS generation in response to UVA triggers the antioxidant response pathway, beginning with the stabilization of antioxidant response factor Nrf2¹⁰⁰. ROS formation triggers the dissociation of Nrf2 from KEAP1, stabilizing Nrf2 and allowing its nuclear translocation¹⁰¹. Nuclear Nrf2 activates an antioxidant response program through binding to antioxidant response elements in gene promoters¹⁰¹. Nrf2 also activates the transcription of genes involved in DNA damage response, including 8-oxo-dG-excising enzyme OGG1^102,103.

Regulation of Autophagy by UV-Induced Oxidative Stress

The mechanisms by which UV-induced oxidative stress regulates autophagy have only recently been clarified. UVA induces autophagy¹⁰ (Figure 3), and treatment with the singlet oxygen quencher NaN₃ impairs the induction of autophagy by UVA¹⁰. Similarly, antioxidants block the induction of autophagy following UVB exposure¹⁰⁴. Collectively, this work suggests that UV-induced ROS production leads to the activation of autophagy.

UVA exposure leads to an increased number of oxidized phospholipids, oxysterols, and cholesterols in keratinocytes^10,105. One oxidized lipid formed by UVA exposure, 25-hydroxycholesterol (25-OH), is sufficient to induce autophagy in keratinocytes¹⁰⁵. This further implicates UV-induced oxidative damage in the activation of autophagy (Figure 3).

UVA has been shown to regulate the transcription of a number of genes involved in autophagy (Figure 3). UVA induces transcription of autophagy adaptor protein p62 in skin fibroblasts¹⁰⁶, as well as autophagy activators p53^107,108 and Sestrin2⁸¹. As described above, p53 and Sestrin 2 can induce autophagy through AMPK signaling. In contrast, UVA suppresses expression of the autophagy activator PTEN¹⁰⁹. PTEN activates autophagy through the PI3K pathway; however, it remains to be seen whether UVA-mediated PTEN downregulation impairs autophagy in UVA response.

Regulation of UV-Induced Oxidative Stress Response by Autophagy

The function of autophagy is critical for oxidative stress response¹¹⁰. However, little work has been done to establish the role of autophagy in the response to UVA-induced oxidative stress. UVA-induced oxidative stress triggers autophagy to clear oxidized lipids and proteins¹⁰ (Figure 3). Autophagy deficiency leads to the accumulation of oxidized phospholipids and protein aggregates following UVA exposure¹⁰. Furthermore, autophagy deficiency increases the Nrf2-dependent antioxidant response in keratinocytes, even prior to UVA exposure¹⁰. We have found that in melanocytes the autophagy activator Sestrin 2 reduces Nrf2 levels upon induction by UVA, and increases UVA-induced ROS production⁸¹. Autophagy induction also serves to inhibit Nrf2 stabilization by degrading p62¹⁰. This work indicates that autophagy plays a complex role in UVA-induced oxidative stress response, by clearing oxidized proteins and lipids, while minimizing antioxidant response in different cell types.

Autophagy in UV-Induced Cell Proliferation and Apoptosis

UV radiation exposure can induce apoptosis and proliferation⁶⁰, often within the same sun-damaged tissue^111,112. In normal skin, compensatory hyperproliferation is induced to replace the cells cleared by apoptosis and maintain homeostasis. The mechanisms governing UV-induced proliferation and apoptosis are tightly regulated to prevent cancerous expansion of cells damaged by UV.

UVA and UVB induce apoptosis of epidermal cells by increasing p53 and Bax expression, while decreasing expression of Bcl-2^112,113. Furthermore, UVA and UVB can induce apoptosis through p38 activation^9,114,115. UVA has been reported to downregulate PTEN and upregulate AKT signaling to protect against apoptosis¹¹⁶. Conversely, UVB-induced UV radiation resistance associated gene (UVRAG) expression suppresses apoptosis by sequestering Bax away from the mitochondria, where it induces apoptosis¹¹⁷. As UVRAG is a p53 target gene¹⁴, this would suggest that UVRAG serves as a possible negative feedback loop in the regulation of UV-induced apoptosis.

Another essential mediator of both UV-induced proliferation and apoptosis is cyclooxygenase-2 (COX-2). Both UVA and UVB upregulate COX-2^118–120, an inducible prostaglandin synthase which catalyzes the rate-limiting step in prostaglandin E₂ (PGE₂) synthesis. PGE₂ signals through autocrine and paracrine mechanisms to promote cell proliferation¹²¹ and suppress apoptosis¹²². COX-2 is induced in the skin of hairless mice following UV radiation to increase proliferation¹²³, and induction of COX-2 protects against UVB-induced apoptosis^119,123,124.

In addition, the mTOR signaling pathway regulates cell growth and proliferation in response to stress. Sestrin 2, a stress-inducible protein that is activated following UVB exposure⁸⁷, inhibits cell proliferation through the negative regulation of mTOR signaling⁸⁰. Similarly, AMPK, an inhibitor of mTOR signaling, suppresses cell proliferation in response to UVB⁸².

UV exposure induces apoptosis and cell proliferation to maintain tissue homeostasis. Research suggests that autophagy plays key roles in the regulation of apoptosis and proliferation, but the contributions of autophagy are likely context-dependent. Knockdown of the autophagy activators AMPK^9,81, Sestrin 2⁸⁷, Beclin1¹²⁵, Atg5^9,125, and UVRAG¹²⁶ sensitizes skin and skin cancer cells to UVB-induced apoptosis, suggesting a protective role of autophagy. However, other research suggests that autophagy or AMPK signaling promote UVB-induced apoptosis^104,127.

The antiapoptotic Beclin1-binding protein Bcl-2 is downregulated following UVB exposure¹¹², which may free Beclin1 to bind UVRAG and induce autophagy³⁸. UVB-induced UVRAG expression suppresses apoptosis by sequestering Bax¹²⁶, independent of its role in autophagy induction¹²⁶. Furthermore, UVRAG is key to suppressing proliferation after UVB, independent of its function in autophagy activation³⁸. As the domains of UVRAG required for activating autophagy, promoting proliferation, and suppressing apoptosis differ, it is possible that UVRAG can activate these processes simultaneously, therefore forming a critical hub in the response to UV radiation.

UVB activates the AMPK signaling pathway to induce autophagy^9,82 and suppress apoptosis^9,128. Autophagy induction by UVB limits p62 levels to prevent p62-mediated p38 activation and subsequent apoptosis⁹. UVB similarly induces autophagy activator Sestrin 2 to promote cell survival⁸⁷. This work suggests that autophagy is activated by UVB to promote cell survival, and likely contributes to UVB-induced tumorigenesis.

Furthermore, we have previously shown that autophagy activator AMPK regulates expression of XPC, and consequently UVB-induced DNA damage repair⁸². AMPK-mediated DNA damage repair suppressed cell proliferation in response to UVB⁸², suggesting a key role for AMPK in the regulation of autophagy, proliferation, and apoptosis.

PTEN, which has been shown to negatively regulate autophagy, is involved in the regulation of cell proliferation and survival following UV exposure. Both UVA and UVB suppress PTEN expression to promote survival of keratinocytes^109,116,129. Sestrin 2 promotes the suppression of PTEN and subsequent AKT activation to promote survival in response to UVB⁸⁷. PTEN suppression by UVA was accompanied by upregulation of AKT signaling and an increase in proliferation¹³⁰. PTEN is therefore central to the regulation of proliferation and apoptosis after UV, and has previously been linked to autophagy. It is unclear whether PTEN regulates UV-induced autophagy, however.

Autophagy in Photodamage-Induced Disease

UV and Skin Cancer

Approximately 90% of skin cancers are attributed to UV exposure^131,132. Skin cancer is the most common form of cancer, with 3.5 million cases diagnosed each year in the United States alone^133,134. Skin cancer causes more than 20,000 deaths in the US and treatment costs the US $8.1 billion annually¹³⁵. Worldwide, incidence of skin cancer is rapidly rising each year ^134,136, increasing the number of people for whom skin cancer will become a costly and potentially deadly disease.

Skin cancers are broadly classified into two major types by the cell type of origin: melanoma and non-melanoma skin cancer (NMSC). NMSC consists of two major types: squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). BCC is the most common type of skin cancer, representing 80% of skin cancer cases¹³⁷. BCC accounts for approximately 3,000 deaths each year in the US and can be disfiguring¹³⁷. SCC represents accounts for approximately 16% of skin cancers¹³⁷ and has potential to metastasize¹³⁸. An estimated 9,000 Americans die each year of SCC¹³⁸. Melanoma is the least common, but most aggressive form of skin cancer. Despite accounting for only 4% of skin cancer cases, melanoma is responsible for nearly half of all skin cancer deaths¹³⁹.

UVA was initially believed to be non-tumorigenic due to its poor ability to cause direct DNA damage. This indirectly promoted the development of UVB-specific sunscreens¹⁴⁰ and the use of high-powered UVA lamps in indoor tanning beds¹⁴¹. It has since been shown that UVA induces skin carcinogenesis in vivo^142–144 and indoor tanning, even intermittently, significantly increases skin cancer risk^145,146. ROS production is thought to be the primary mechanism by which UVA causes skin cancer. Melanocytes are particularly sensitive to UVA, as melanin can act as a photosensitizer for UVA and enhances ROS production following UVA exposure^56,147–149. Consequently, UVA radiation is believed to play a major causal role in 67% of melanoma cases¹⁵⁰.

UVB radiation has been thought to contribute mostly to NMSC formation by causing direct DNA photodamage. UVB signature TC→TT and CC→TT mutations are commonly found in the p53 gene of skin cancer patients¹⁵¹, suggesting the importance of p53-mediated DNA damage response in preventing skin carcinogenesis. Defects in UV-induced DNA damage repair greatly accelerate skin cancer development, as is seen in xeroderma pigmentosum (XP) patients. XP is an autosomal recessive disorder caused by defects in GG-NER, and XP patients have a 10,000-fold increased susceptibility to UV-induced NMSC¹⁵². XP patients develop NMSC 50 years younger than the general population.

Tumor Suppressive Role of Autophagy in Skin Cancer

Autophagy can be tumor suppressive or pro-tumorigenic, depending on context¹⁵³. Autophagy acts as a tumor suppressor by promoting ROS clearance¹⁵⁴, DNA repair^13,83,155, and degrading oncogene p62¹⁵⁶. Autophagy deficiency causes accumulation of targets bound for degradation, including p62¹⁰⁶, promotes ROS generation¹⁵, and causes genomic instability¹².

Degradation of adaptor protein p62 has been found to be an important tumor suppressive function of autophagy¹⁵⁶. In autophagy-deficient conditions or upon transcriptional upregulation by UVA¹⁰⁶, p62 accumulates and acts as a signaling hub by forming interactions with a number of pro-tumorigenic proteins. We have found that p62 binds and stabilizes Twist1, a transcription factor involved in epithelial-mesenchymal transition⁸⁴. In doing so, p62 promotes proliferation and migration of skin cancer cells in vitro⁸⁴. In a mouse model of SCC, the p62-Twist1 interaction promotes tumor growth and metastasis⁸⁴.

p62 similarly activates NF-κB signaling in a feed-forward loop. The interaction between p62 and TRAF6^157,158, as well as interactions with death domain serine/threonine kinase RIP¹⁵⁹ and atypical protein kinases¹⁶⁰, ultimately activates NF-κBsignaling. NF-κB signaling in turn upregulates p62 transcription¹⁶¹. NF-κB signaling is activated by p62 to promote Ras-mediated tumorigenesis¹⁵⁸.

Oncogenic Role of Autophagy in Skin Cancer

Autophagy can facilitate tumor development by promoting cell survival in times of genotoxic, oxidative, or metabolic stress, and by providing the macromolecules necessary to sustain a high rate of proliferation¹⁵³. We have proposed an oncogenic function for autophagy in the development of SCC, in which autophagy promotes survival of SCC cells with extensive DNA damage⁹. Future investigations will help understand the role of autophagy in the regulation of critical signaling pathways in tumorigenesis.

UV and Photoaging

Photoaging (also called extrinsic aging) is premature aging of the skin caused by environmental effects, primarily UV exposure. Photoaging differs from intrinsic (chronological) aging, which affects the skin in ways similar to other organs and can be superimposed on photoaging¹⁶². While intrinsically aged skin is thin and smooth, photoaging can cause a leathery thickening, sagging, and wrinkling of skin^163,164. Histologically, photoaged skin is characterized by a loss of dermal collagen¹⁶⁵, induction of matrix metalloproteinases (MMPs)¹⁶⁶, and accumulation of elastin¹⁶⁷.

Both UVA and UVB have been shown to cause photoaging by inducing ROS production and subsequent oxidative damage to DNA, lipids, and proteins¹⁶⁷. UVA-induced alterations to skin lipid composition¹⁰⁵ and phospholipid oxidation¹⁰ cause skin sagging characteristic of photoaging. Furthermore, UV exposure leads to the accumulation of oxidatively modified proteins and depletes antioxidant enzymes in photoaged skin^93,168.

Autophagy in Photoaging

UVA-induced ROS generation leads to the oxidation of phospholipids, and subsequent formation of oxidized phospholiplid-protein adducts¹⁰. Autophagy induced by UVA degrades these adducts¹⁰ to prevent damage caused by aggregation of heavily oxidized protein adducts¹⁶⁹. Aging-related decline in autophagic clearance¹⁷⁰ leads to the accumulation of oxidized phospholipid-protein adducts¹⁷¹, and oxidized protein aggregates¹⁷². Adduct accumulation contributes to skin photoaging¹⁷¹.

Implications for Autophagy in UV-Induced Disease Prevention and Treatment

Autophagy modulators are of clinical interest for the treatment and prevention of skin cancer and many other diseases. However, the context-dependent and often opposing functions of autophagy make it difficult to predict response to autophagy modulators in the clinic. Here, we examine the current understanding of autophagy modulation in UV-induced skin cancer.

We have reported that AMPK is activated by UVB to induce autophagy⁹, promote DNA repair⁸², impair cell proliferation⁸², and inhibit apoptosis^9,128 under stress conditions. AMPK activation is reduced in human skin cancer samples⁸², and therefore, targeting AMPK for activation in skin cancers could provide an opportunity to block tumor growth. Our data supports the use of AMPK activators metformin and AICAR to block the growth of UVB-induced skin tumors in vivo⁸².

Autophagy activator rapamycin reduces UV-induced skin tumor formation and progression^83,173. Rapamycin treatment increases XPC levels through Twist1 downregulation⁸³, and consequently reduces the number of UV-induced mutations in p53 in skin tumors¹⁷³. Inhibition of autophagy by Spautin-1 increases Twist1, decreases XPC, and increases tumor growth induced by UVB⁸³. Collectively, this work suggests that autophagy plays a tumor suppressive function in UVB response.

Conclusions

While much progress has been made in understanding the regulatory and functional role of autophagy in energy stress response, the role of autophagy in response to UVB and UVA radiation is only beginning to be understood. Autophagy regulates UV-induced apoptosis, DNA damage repair, oxidized lipid removal, and other oxidative damage response. The role of autophagy in UV response, skin cancer, and aging remains to be elucidated.

First, while autophagy likely prevents the accumulation of oxidative damage that leads to UV-induced photoaging, it remains unclear whether activation of autophagy would be effective at preventing photoaging. Moreover, there is currently no clear indication that autophagy modulation would be effective in the treatment or prevention of skin cancer. The contribution of autophagy to the promotion or suppression of tumor growth and metastasis is likely dependent on a multitude of factors, including cell type and disease state. Understanding the context-dependency of the oncogenic and tumor suppressive roles of autophagy in skin cancer requires further research into UV response and could provide clearer insight into autophagy modulation as a treatment option for skin cancer. These future findings will define the regulatory and functional role of autophagy in UV response and skin cancer and aging, and provide molecular basis for targeting autophagy to prevent and treat skin cancer and aging-associated diseases.