The role of autophagy in metal-induced urogenital carcinogenesis

Abstract

Environmental and/or occupational exposure to metals such as Arsenic (As), Cadmium (Cd), and Chromium (Cr) have been shown to induce carcinogenesis in various organs, including the urogenital system. However, the mechanisms responsible for metal-induced carcinogenesis remain elusive. We and others have shown that metals are potent inducers of autophagy, which has been suggested to be an adaptive stress response to allow metal-exposed cells to survive in hostile environments. Albeit few, recent experimental studies have shown that As and Cd promote tumorigenesis via autophagy and that inhibition of autophagic signaling suppressed metal-induced carcinogenesis. In light of the newly emerging role of autophagic involvement in metal-induced carcinogenesis, the present review focuses explicitly on the mechanistic role of autophagy and potential signaling pathways involved in As-, Cd-, and Cr-induced urogenital carcinogenesis.

1. Introduction:

Metal-induced carcinogenesis caused by environmental and/or occupational exposure to heavy metals is rapidly becoming a global health problem due to their increased use in industrial, agricultural, and pharmaceutical applications [1]. Unlike organic contaminants, metals cannot be broken down by microorganisms and tend to bioaccumulate in living organisms [2, 3]; this chronic exposure, in turn, has been linked to various malignancies in humans [1, 4–6]. Heavy metals such as Arsenic (As), Cadmium (Cd), and Chromium (Cr) are well-known carcinogens [2, 7, 8] that have been associated with an increased risk of urogenital cancers [3, 5, 9–11]. While the exact mechanism of heavy metal-induced carcinogenesis remains elusive, studies have shown that metals may drive carcinogenesis via several mechanisms, including by causing DNA damage, uncontrolled oxidative stress, impaired autophagy, and epigenetic modifications such as the silencing of DNA repair and tumor-suppressor genes [1, 9, 12]. In the present review, we focus specifically on the role of autophagy and discuss possible signaling pathways responsible for metal-induced urogenital carcinogenesis.

2. Molecular machinery of autophagy

Mammalian autophagy is an essential catabolic process involving the lysosomal degradation and recycling of cellular proteins and organelles. Essentially a survival mechanism, autophagy is dramatically upregulated in conditions of metabolic stress (such as nutrient deprivation and bioenergetics failure) to increase the availability of critical metabolic intermediates [13, 14]. On the other hand, autophagy has also been shown to accompany type II programmed cell death, as characterized by the appearance of large intracellular vesicles and the engagement of the autophagy machinery; however, its function as an active cell death mechanism remains controversial [15].

Three types of autophagy are known to occur in eukaryotic cells, namely, macroautophagy, microautophagy, and chaperone-mediated autophagy [16], with macroautophagy (referred to as autophagy) providing the basis for the majority of our knowledge of this process. Autophagy is initiated by forming an isolating membrane or phagophore, which gradually closes to form a double-membrane vesicle or autophagosome. The autophagosome then fuses with a lysosome to form an autolysosome, which enables its contents to be degraded and recycled. This multi-step process is tightly regulated by autophagy-related genes (Atg) and several downstream signaling pathways (Figure 1), which have been discussed below.

Figure 1:

Molecular pathways involved in the regulation of autophagy in metal-induced carcinogenesis. Metal induces uncontrolled oxidative stress and excessive ROS production, which triggers ER stress, activation of antioxidant response (p62/Nrf2), and DNA damage. Metal exposure also induces epigenetic changes that affect DNA repair pathways (ERCC1, XRCC), inflammation (NFκB), cell proliferation (Ras/MAPK/PI3K/mTORC1), and survival (Bcl-2). Cellular stress conditions like oxidative stress, hypoxia and ER stress activate autophagy to enable cells to adapt to unfavorable conditions.

2.1. Phagophore formation:

Autophagy is initiated by the activation of the Unc-51-like Autophagy Activating kinase 1/2 (ULK 1/2) complex (comprising ULK 1/2 kinases, Atg13, Atg101, and FIP200 proteins) [17]. In low nutrient conditions, the energy senor kinase of 5’ AMP-activated protein kinase (AMPK) senses alterations in intracellular AMP/ATP ratios and activates the ULK 1/2 complex by phosphorylating the serine residues (467, 555, and 638) of ULK-1 while simultaneously inhibiting the autophagy repressor mammalian Target of Rapamycin (mTOR) [18–22]. The phosphorylation of ULK1 triggers the translocation of a multi-protein complex comprising Beclin 1 (or Atg6), Activating molecule in Beclin 1-regulated autophagy (AMBRA), Atg14L, Vps15, and class III phosphoinositide 3-kinase (also known as vacuolar protein sorting 34 (Vps34)) from the cytoplasm to the nascent phagophore [23–25]. Beclin 1 plays a key role in the complex by forming an assembly platform.

2.2. Elongation:

Two ubiquitin-like conjugation systems (Atg5-Atg12 and microtubule-associated protein 1A/1B-light chain 3 [LC3B]) and several soluble NSF attachment protein receptor (SNARE) proteins are required for the subsequent elongation of the phagophore membrane [26, 27]. The Atg5-Atg12 complex is first formed by the action of Atg7 (ubiquitin-E1-like enzyme) and Atg10 (ubiquitin-E2-like enzyme) and then noncovalently conjugated to Atg16 to form the Atg12-Atg5-Atg16 complex, which displays E3 ligase activity [28, 29]. This large complex then forms the autophagosome membrane by recruiting the second conjugation system, the LC3B [30, 31]. Prior to its recruitment, the LC3B protein is cleaved by Atg4 and then lapidated by the addition of a phosphatidyl ethanolamine group through the action of Atg7 and Atg3 [14, 30, 31]. Subsequent phagophore closure is regulated by members of the endosomal sorting complex required for transport (ESCRT), CHMP2A, and VPS4 [32]. Proteins such as p62 (sequestosome-1), NBR1, and NIX that harbor an LC3-interacting region (LIR) also facilitate the recognition of ubiquitylated proteins or specific organelle membranes to be selectively delivered as cargo to the autophagosomes [33].

2.3. Maturation:

The autophagic process is completed with the maturation stage, in which the autophagosomes fuse with the lysosomes to form autolysosomes, leading to their degradation by acid hydrolases. This final step is regulated by members of the SNARE family, including Qa-SNARE, syntaxin 17, Qbc-SNARE, and lysosomal R-SNARE [34]. Autophagy degradation products are then exported from lysosomes into the cytoplasm, where they are recycled for cellular metabolism and repair mechanisms [35].

3. Molecular mechanism of autophagy

Several signaling pathways are known to regulate autophagy in response to cellular and environmental cues. Depletion of nutrients is the most potent physiological inducer of autophagy, in which critical energy sensor pathways such as mTOR and AMPK have been shown to regulate starvation-induced autophagy [36–38]. Additionally, autophagy is induced by extra- and intracellular stresses such as hypoxia, endoplasmic reticulum (ER), and oxidative stress, enabling cells to adapt to or overcome unfavorable conditions [39].

3.1. mTOR pathway:

As an essential signaling molecule that regulates diverse cellular functions, including cell growth and proliferation, mTOR functions as a convergent point for multiple diverse extracellular stimuli (such as growth factors, amino acids, glucose, energy status) and upstream signaling cascades involved in the regulation of autophagy [40, 41]. The three main signaling pathways that regulate mTOR activity are the class I PtdIns3K-protein kinase B (PI3K/AKT), the RAS-proto-oncogene, and the liver kinase B1-AMPK (LKB1/AMPK) pathways [42]. Activation of mTOR negatively regulates autophagy via the action of its two distinct complexes: complex 1 (mTORC1) and 2 (mTORC2), with the former playing a direct role in inhibiting autophagy by phosphorylating the ULK 1/2 complex [19–22, 42, 43]. mTORC2, on the other hand, inhibits autophagy through the phosphorylation and activation of PKB/AKT [44, 45]. Activation of growth factor receptors in nutrient-rich environments stimulates the PI3K complex and small GTPase Ras, leading to the subsequent activation of PI3K/AKT/mTORC1 and Raf-1-MEK1/2-ERK1/2 pathways, respectively. Both PKB/AKT and ERK1/2 phosphorylate and inhibit the GTPase-activating protein complex TSC1/TSC2, resulting in the stabilization of Rheb-GTPase and the activation of mTORC1, which in turn inhibits autophagy [46–49]. In contrast, nutrient depletion triggers autophagy by activating the AMPK pathway, which inhibits mTORC1 activity, causing it to dissociate from the ULK 1/2 complex [19–21, 50].

3.2. AMPK signaling:

Like the mTOR pathway, AMPK also functions as a sensor of cellular bioenergetics and is a well-known autophagy regulator [51, 52]. Studies have shown that at least three independent signaling pathways (LKB1, Ca (2+)/calmodulin-dependent kinase kinase-β [CaMKKβ], and transforming growth factor-β-activating kinase 1 [TAK1]) induce autophagy in an AMPK-dependent manner [53, 54]. AMPK, in turn, initiates autophagy directly by phosphorylating the ULK 1/2 complex and does so indirectly by suppressing mTORC1 activity [50]. Hypoxia is also a potent inducer of AMPK activation, an event independent of HIF activity and that can occur even in the absence of any detectable fluctuations of intracellular ATP levels [55–57]. AMPK’s role in carcinogenesis is controversial, as it can function as either a tumor suppressor or promoter in a context-dependent manner. On one hand, AMPK signaling has been shown to inhibit carcinogenesis by either triggering p53 and FOXO pathways and/or inhibiting AKT/mTOR and nuclear factor-κB (NFκB) pathways [58–60]. It has also been shown to support cancer cell survival by promoting autophagy by stabilizing cytosolic p27 levels or in response to starvation or hypoxic conditions [18, 61].

3.3. Other autophagy signaling pathways:

Autophagy is also regulated by other pathways such as Ras, a membrane-anchored protein, which once activated, stimulates diverse downstream effectors and cellular signaling networks, including class I PI3K/AKT/mTOR1, Raf-1/MEK/ERK, and Rac1/JNK pathways [62]. Ras seemingly plays complex but opposing roles in autophagy regulation. While Ras inhibits autophagy by activating the class I PI3K/AKT/mTOR1 pathway [47, 48], it also promotes autophagy via the activation of Raf-1-MEK1/2-ERK1/2 [49, 50] and Rac1/MKK7/JNK [54, 61] pathways. The former pathway inhibits Bcl-2 binding of Beclin 1, allowing for the formation of the class III PI3K complex, while the latter pathway promotes autophagy by upregulating Atg5/Atg7. Interestingly, Ras-driven tumors have been shown to induce autophagy as an adaptive response to oxidative and metabolic stress while promoting survival [63]. Thus, autophagy induced by Ras can affect tumor progression by modulating cell death, cell proliferation, mitochondrial integrity, and sensitivity to matrix detachment and metabolic stress. Another important pathway that regulates autophagy is the NFκB cascade, which plays a critical role in stress-related cellular inflammation and survival. Both NFκB and autophagy have been shown to regulate one another’s activity in a context-dependent manner. While NFκB can promote autophagy by inducing the expressions of various autophagy-associated proteins such as Beclin 1, it’s signaling can also suppress the expression of autophagic inducers like BNIP3, Jun‐N‐terminal kinase (JNK), and reactive oxygen species (ROS). In contrast, increased autophagy has been shown to inhibit NFκB signaling by degrading IKK subunits (detailed in [64, 65]). Similar to NFκB, the B-cell lymphoma 2 (Bcl-2) protein family also plays a dual role in autophagic regulation. While the oncogenic potential of the Bcl-2 family is attributed mainly to its inhibition of apoptotic signals [detailed in [66]), its emerging role as an autophagy regulator suggests that members such as Bcl-2/Bcl-X_L promote carcinogenesis by impairing autophagic function, leading to increased genomic instability and improved survival of cancer cells.

3.4. Oxidative stress:

Excessive ROS production in tissues or cells can induce oxidative stress and lead to oxidative damage such as DNA hydroxylation, protein degeneration, and tissue damage [67]. While the mitochondria are generally accepted as being the main sources of ROS, various intracellular stresses, including nutrition starvation, ER stress, and hypoxia can also induce oxidative stress and damage [68–72]. ROS induces autophagy through various signaling pathways including inhibiting PI3K-AKT-mTOR [67, 73] whilst activating AMPK [74, 75], mitogen activated-protein kinase (MAPK) [76], extracellular signal-regulated kinase (ERK) [77, 78], and JNK [79] signaling. Aside from eliminating ROS via autophagy, cells protect themselves from oxidative stress by promoting the transcription of antioxidant-defense genes. A key transcriptional factor regulating this response is nuclear factor erythroid 2-related factor 2 (Nrf2) [80, 81]. During oxidative stress, Nrf2 is released either through its dissociation from Kelch-like ECH-associated protein 1 (Keap1) or by the upregulation of SQSTM1 (the gene encoding p62). The dissociated Nrf2 then translocates to the nucleus, where it activates the expression of anti-oxidant genes and promotes cell survival [82, 83]. While p62 is degraded during the autophagic process [33], its accumulation in autophagy-impaired cells results in the constitutive activation of Nrf2 and increased production of antioxidant proteins to maintain the redox balance. Moreover, upregulation of p62 activates other pro-survival oncogenic pathways, including NF-κB and mTOR [84]. Oxidative stress and DNA damage have been suggested to play a central role in metal-induced carcinogenesis [85, 86].

3.5. ER stress:

In mammalian cells, the ER is the key compartment that facilitates the folding of newly synthesized proteins and the initiation of vesicular movement of the membrane and proteins to the various organelles and the cell surface. A major ER stress pathway is the unfolded protein response (UPR), which is a potent stimulus of autophagy. ER stress promotes autophagic response via the induction Ca2+ as well as UPR downstream effectors RNA-dependent protein kinase-like ER kinase (PERK) and the inositol-requiring protein‐1α (IRE1α) pathways [51, 87, 88]. Ca2+ released from the ER lumen induces autophagy through activation of the CaMKKβ-AMPK pathways [53, 89] as well as by the death‐associated protein kinase (DAPK)-mediated dissociation of Beclin 1 from the Beclin 1/Bcl‐2 complex [90, 91]. Similarly, IRE1α also promotes autophagy by JNK-mediated phosphorylation of Bcl‐2, resulting in the dissociation of Beclin 1 from Bcl‐2 [92, 93]. PERK, on the other hand, drives autophagy via Activating Transcription Factor 4 (ATF4)’s transcriptional regulation of Atg12, while ATF4-mediated activation of C/EBP‐homologous protein (CHOP) transcriptionally induces Atg5 [88, 94]. ER-stress-induced autophagy can be either pro-survival or pro-death. While many studies indicate that autophagy has a cytoprotective pro‐survival function following ER stress [95, 96], prolonged stress can induce autophagy‐dependent cell death mechanisms and cell damage [97].

3.6. Hypoxia:

Upregulation of autophagy has been observed in hypoxic regions of tumors [43]. Accumulating data show that hypoxia induces autophagy in mammalian cells [98, 99] by both hypoxia-inducible factor-1 (HIF-1)-dependent and independent mechanisms (like AMPK-mTOR, UPR). Considering that hypoxia induces ER stress through UPR and that mitochondria have reduced function in oxidative phosphorylation in hypoxic conditions, the induction of autophagy could allow cells to eliminate portions of compacted ER and reduce mitochondrial mass in periods when no oxygen is available to accept free electrons from the respiratory chain [99]. Hypoxia also increases the transcription of essential autophagy genes LC3 and Atg5 through transcription factors ATF4 and CHOP, respectively, which are in turn regulated by PERK [100].

Carcinogenic metals have been shown to be potent inducers of autophagy; an event likely triggered as a self-defense mechanism against metal-induced oxidative stress [10, 101]. Moreover, cancer cells are known to take advantage of the autophagic process to survive hostile environments. Concurrently, we and others have demonstrated that heavy metals like As and Cd promote tumorigenesis via impaired autophagy [102–106], whereas the subsequent inhibition of autophagy suppressed metal-induced carcinogenesis [105]. Thus, considering the newly emerging role of autophagic response as a key event in metal-induced carcinogenesis, this review will discuss autophagy’s mechanistic role in As-, Cd-, and Cr-induced urogenital carcinogenesis.

4. Metal-induced prostate carcinogenesis

Prostate cancer (CaP) is the most commonly diagnosed cancer and the second leading cause of cancer-related death among men in western countries [107]. Despite identifying several risk factors for CaP, including age, diet, ethnicity, and genetic/family history [108], CaP pathogenesis remains unclear. Several epidemiological studies have suggested a possible link between heavy metal exposure and increased risk of CaP [109–117] (Figure 2)

Figure 2:

Induction of autophagy in metal-induced prostate carcinogenesis. Cd and As have been shown to mediate effects using the same molecular pathways. Both promote carcinogenesis by directly triggering proteins involved in cell proliferation (ERK/MAPK), and by increasing oxidative stress through epigenetic changes or disruption of antioxidant signaling. Increased ROS triggers ER stress and autophagic responses that lead to CaP. Cr also causes excessive ROS production and NFκB activation resulting in cell survival. Albeit limited, studies have shown that autophagy is a key event during the malignant transformation of metal-exposed prostate cells. Dotted lines represent plausible mechanisms involved.

4.1. Cadmium:

Cd is a toxic heavy metal whose presence in nature has become magnified due to human activities. This persistent environmental pollutant is ranked #7 on the 2017 Agency for Toxic Substances and Disease Registry Substance Priority List and has been listed as a significant public health concern by the World Health Organization [118, 119]. Cd is a confirmed carcinogen [120] with a biological half-life >20 years [2, 121, 122]. It is widely found in metal coatings, food, and cigarette smoke [123–129]. The average intake of Cd from food varies between 9 and 25 μg/day in US and Europe and between 19.7 and 35.4 μg/day in Asia [130]. Cigarettes contain approximately 1–2μg of Cd, with about 50% of the inhaled Cd being absorbed by the body [131]. The prostate is one of the organs with the highest levels of Cd deposition [132, 133], and several epidemiological studies have suggested a link between Cd exposure and increased CaP risk/mortality [111, 134–138]. Risk assessment studies suggest that the likelihood of developing CaP is 1.1–2.76 times greater in Cd-exposed cases than in non-exposed cases [135], suggesting Cd as a risk factor for CaP.

Several studies have shown that chronic exposure to low concentrations of Cd-induced malignant transformation of normal human prostate epithelial cells (RWPE-1) [139–141]. These malignant Cd transformed cells, termed Cd-induced Prostate Epithelial cells (CTPE), were, in turn, shown to form aggressive but poorly differentiated adenocarcinomas when inoculated into nude mice [142, 143]. However, the precise molecular mechanisms involved in Cd-mediated carcinogenesis have been only partially elucidated. The complex network of mechanisms involved in Cd-mediated prostate carcinogenesis includes the induction of oxidative stress [144, 145], DNA damage [146, 147], autophagy [105, 106, 148–150], inhibition of antioxidant enzymes such as superoxide dismutase and GSH peroxidase [151, 152], suppression of DNA repair [146, 147, 153], epigenetic changes resulting in hypermethylation of tumor suppressor and apoptosis gene activities [141, 154–156], and enhanced proliferation [157, 158].

Acute or chronic Cd exposure increases ROS levels [120, 159], which, if persistent, induces alterations to the redox signaling pathway, DNA mutations, and changes in methylation and chromatin remodeling patterns [160]. A finding supported by studies demonstrating global hypermethylation and silencing of tumor suppressor genes such as p16 promoter and DNA repair genes ERCC1 and XRCC1 in CTPE cells via the overexpression of DNA methyltransferase 3b (DNMT3b) [141, 161–163]. Moreover, the activation of several signaling cascades, including mitogen-activated PI3K/AKT, NFκB, and p53 inactivation, has been shown to be involved in Cd-induced CaP [139, 164–167]. More recently, Dasgupta et al., [168] reported that enhanced ERK/MAPK signaling is a significant event in the malignant transformation of Cd-induced prostate carcinogenesis.

Our lab group was the first to report that autophagy plays a significant role in Cd-induced prostate carcinogenesis. While attempting to delineate the mechanism of Cd-mediated malignant transformation of prostate epithelial cells, we performed a comparative analysis of global gene arrays of RWPE-1 and CTPE cells and discovered that autophagy-regulated genes (Plac8, LC3B, STX8, STX17, and Lamp-1), particularly placental-specific 8 (Plac8), a precursor of autophagosome-lysosome fusion, were differentially expressed in both cell types [149]. Further examination showed a concomitant increase of both pro-survival proteins (pAKTS473, p65, Bcl-2) and autophagosome/lysosomal proteins (Plac8, Lamp-1, STX8, STX17, and LC3B) in CTPE cells, suggesting that the induction of the pro-survival function of autophagy protected these cells from Cd-induced toxicity. Our finding of the oncogenic role of Plac8 is supported by other studies that have reported a positive correlation between its increased expression and the activation of the pro-survival function of autophagy in other cancers [169–171]. We also found that the inhibition of Plac8-mediated autophagy and pro-survival signaling of NFκB/Bcl2 suppressed CTPE growth [105]. Subsequent delineation of the upstream events responsible for Cd-induced autophagy revealed that Cd-induced ROS, which in turn triggered ER stress and subsequent phosphorylation of downstream stress transducers (PERK, eIF2-α, and ATF4) [106]. Together, these results suggest that induction of ROS and subsequent ER stress are responsible for defective autophagy in Cd-induced prostate carcinogenesis.

4.2. Arsenic:

A naturally occurring metalloid found in food, soil, and water, As inorganic compounds such as As trioxide, sodium arsenite, As trichloride, and arsenates (e.g., calcium arsenate) are classified as class 1 human carcinogen due to their high toxicity and ability to cause multiple cancers including urogenital cancers [172]. As is a known pollutant, and its concentrations ranging from 1 ng/m³ to 2–3.6 mg/L have been reported in groundwater worldwide [160]. Drinking water, crops irrigated with contaminated water, and food are the main sources of As exposure [160]. A causal link between As and CaP has been described in several epidemiological studies in human populations exposed to high concentrations of As (reported range between>10 ppb and 2500 ppb) in drinking water [109, 112, 173–175].

Studies have shown that chronic exposure to low doses of As induced malignant transformation of RWPE-1 cells [176, 177], and subsequent in vivo experiments found that these transformed cells formed aggressive carcinomas exhibiting characteristics commonly found in human prostatic cancers [178]. As has been shown to induce oxidative stress by increasing intracellular ROS levels [179] and inhibiting glutathione (GSH) antioxidant activity [180], resulting in oxidative DNA damage [181–184]. Moreover, epigenetic changes and altered signaling that play important roles in cell proliferation, differentiation, and transformation have been reported in As-mediated malignant transformation of prostate cells [141, 185–188]. Although the role of autophagy in As-mediated prostate carcinogenesis has not yet been studied, autophagy has been shown to confer therapeutic resistance and enhance the invasive ability of CaP cells [189]. Moreover, the use of autophagy inhibitors in combination with therapeutic agents was found to suppress the proliferation of resistant CaP cells and promote cell death [190–192]. Based on this evidence, it is clear that autophagy plays a significant role in CaP pathogenesis and that more studies are needed to explore its role in metal-induced prostate carcinogenesis.

4.3. Chromium:

Cr toxicity is caused primarily by its hexavalent form, Cr(VI), and is classified as a Group 1 carcinogen by the IARC. Cr(VI)’s environmental presence is almost entirely industrial in origin [1], and many epidemiological studies have reported its carcinogenic effect in several organs, including the prostate [193]. Cr(VI)-induced epigenetic changes, genotoxicity, and oxidative DNA damage have been implicated as mechanisms through which it mediates its carcinogenicity [7, 116, 194]. Cr(VI) exposure has also been shown to induce ROS production, which, in turn, has been shown to activate NFκB and AP-1 signaling pathways [186, 187, 195, 196]. In CaP, Cr(VI) exposure was reported to induce ROS and increase HIF-1α and VEGF levels via activation of the p38 MAP kinase pathway [197]. However, despite being well studied in other cancers such as that of the lung, Cr’s carcinogenic role in CaP remains underexplored. Considering that studies have shown the activity of many upstream effectors of autophagy, it is possible that Cr(VI) may mediate some of its carcinogenic effects via defective autophagy. However, more studies are needed in this area.

5. Metal-induced bladder carcinogenesis

Bladder cancer is the fourth most prevalent cancer among men; while it can occur in women, its overall incidence is atleast three to four times lower [107]. Among the heavy metals discussed in this review, As is considered the most potent bladder carcinogen, followed by Cd and hexavalent Cr. Despite metal-induced carcinogenesis being documented in several epidemiological studies, the exact mechanisms underlying metal-induced bladder carcinogenesis have yet to be explored. Mechanistic insights suggest that autophagy may play an important role in bladder carcinogenesis (Figure 3).

Figure 3:

Metal-induced autophagy-mediated bladder carcinogenesis. As exposure induces GSH mediated superoxide formation that affects several pathways, including Nrf2-Keap1 and ATF4-mediated UPR. Inhibition of mTORC1 or activation of ULK1 and Atg induced autophagy resulting in the malignant transformation of bladder cells. Cd exposure increased Nrf2 binding to the ARE promoter in p62, causing an increase in antioxidants and concomitant ROS inhibition ultimately leading to carcinogenesis of bladder epithelial cells. Similarly, Cr-exposure dysregulated the p62 mediated Nrf2-Keap1 pathway and promoted constitutive activation of Nrf2.

5.1. Arsenic:

Although As carcinogenicity is associated mainly with skin and lung cancers [198, 199], its accumulation in the bladder makes it a potent carcinogen for the onset of bladder carcinogenesis [200]. The trivalent form of As (iAs³⁺) is known to induce ROS (O2⁻, H₂O₂) [201] and, consequently, the Nrf2-Keap1 pathway. In fact, As exposure in UROtsa cells has been shown to activate Nrf2 signaling [202], with the subsequent dysregulation of the Nrf2-Keap1 axis being shown to induce malignant transformation of normal bladder epithelial cells and increased cell proliferation [203]. As exposure also induces UPR and the activation of ATF4 signaling. While oxidative stress has been implicated as the major driver of malignant cellular transformation [204], it should be noted that the activation of autophagy in response to persistent oxidative stress maybe how the transforming cells survive and proliferate in these stressed conditions. As-induced ROS activation has been shown to activate AMPK signaling [205] resulting in the dephosphorylation of mTORC1 and activation of ULK1-induced protective autophagy in bladder cancer cells [206]. As has also been shown to bind to cysteine 299 of AMPKα directly [207]. Key autophagic proteins such as Beclin 1 and Atg7 protein have also been shown to be predominately expressed in high-grade bladder cancers. Moreover, modest alterations of autophagic proteins such as Beclin 1, Atg-5, −7, −12 and LC3B have been shown in As-exposed UROtsa cells [208]. Taken together, these studies suggest that ROS-mediated autophagy activation may be a key event cell survival during As-induced malignant transformation of normal bladder epithelial cells.

5.2. Cadmium:

Epidemiologic studies suggest that Cd is also a potent carcinogen for bladder cancer [209–212]. Chronic exposure to low doses of Cd has been shown to cause UROtsa cells to acquire clonogenic abilities and develop tumors in mice models [213]. Moreover, another study demonstrated that Cd exposure induces autophagy proteins’ expression, including Beclin 1 in Cd-induced malignant transformed bladder epithelial cells [208]. Similar to As-induced carcinogenesis, Cd-induced oncogenic transformation increases ROS production in cells, which in turn causes the constitutive activation of Nrf2 [214]. Cd exposure enhances the binding of Nrf2 to the antioxidant response element (ARE) promoter regions of p62/Bcl-2/Bcl-X_L. Cd exposure similar to that of As, causes epigenetic alterations resulting in impaired DNA repair mechanisms and subsequent carcinogenesis. Cd-induced activation of transcription factors such as Metal Regulatory Transcription Factor 1, upstream regulator of autophagy, has also been implicated as another mechanism through which this metal may drive bladder carcinogenesis [215]. Although new information pertaining Cd-induced carcinogenicity is rapidly evolving, more comprehensive studies are still needed to elucidate the roles of the various molecular mechanisms especially those related to autophagic activation in Cd-mediated bladder carcinogenesis.

5.3. Chromium:

Epidemiological evidence suggests that occupational exposure to Cr causes bladder cancer [216–219]. Kutze et al., [220] in their study, found a significantly increased probability of lower urinary tract urothelial tumors in patients occupationally exposed to Cr/chromate. Similarly, another study reported significantly higher Cr concentration in the bladder cancer tissues (99.632 ng/g) as compared to non-cancer tissue (33.144ng/g) [216–218]. Chronic Cr(VI) exposure was shown to induce chromosomal damage in bladder cells, which in turn was suggested as a possible mechanism for Cr-induced bladder carcinogenesis [221]. Cr has been linked to the reduced expressions of AMPK and peroxisome proliferator-activated receptor γ coactivator 1α, which resulted in the disruption of mitochondrial dynamics through the dysregulation of the Nrf2 axis [222]. Cr exposure has also been shown to induce autophagy through the activation of ROS-AKT-mTOR pathway [223, 224]. In another study, Yang et al., showed that trivalent Cr, Cr(III), induced autophagy by activating sphingomyelin phosphodiesterase 2 (SMPD2). SMPD2 increases levels of ceramide and ceramide signals overlapped with LC3, suggesting that ceramide might play an important role in the formation of autophagosomes. The study concluded that Cr induces autophagy via structural aberration of the organelle membrane, in particular by the increase of lipid compositions in addition to autophagy-associated proteins [103]. Although studies have shown potential Cr-induced oncogenic mechanisms involved in its promotion of bladder carcinogenesis, there is still a dearth of knowledge regarding the role of autophagy in Cr-mediated bladder carcinogenesis that requires further elucidation.

6. Metal-induced kidney carcinogenesis

Kidney cancer is the sixth and eighth most common cancer among men and women, respectively [107]. The kidney has long been recognized as one of the organs most affected by high metal exposure.

6.1. Cadmium:

Approximately 50% of Cd concentrations in the body is accumulated in the kidney [225]. Because of Cd’s low molecular mass (~7Kd), it is filtered through to proximal tubules, which are the parts most affected by Cd-related toxicity [226]. The free and metallothionein-bound Cd (Cd-MT) is taken up by receptors for small protein ligands such as megalin and cubilin [227]. Once internalized, Cd has been shown to induce autophagy signaling by mTORC1 downregulation and upregulating Atg5, Beclin 1, and LC3 in duck renal tubular epithelial cells [228], cultured mesangial cells [229], rat proximal tubule [230], and NRK-52E cells [231]. Studies have shown that an increase in LC3B expression coupled with a concomitant decrease in the expression of tumor suppressor miR-204, placed a critical role in the progression of renal cell carcinoma (RCC) [232]. Furthermore, autophagy defects caused by decreased microtubule-associated protein family 1 signaling [233] and deregulation of Keap1-Nrf2 axis [234] play an important role in promoting tumor cell survival in RCC. Although these studies present a link between Cd-induced autophagy and survival, the molecular interaction between these pathways, especially in the context of renal cancer, remains unclear. Future studies are urgently needed to amplify the current knowledge regarding the role of autophagy in Cd-induced renal carcinogenesis

6.2. Arsenic:

As has been associated with causing structural damage and renal dysfunction, resulting in nephrotoxicity and subsequent carcinogenesis [235–237]. Although kidney cancer generally occurs in older individuals, studies have shown that exposure to inorganic As increases the incidence of renal cancer among young adults [238]. Moreover, increased oxidative stress has been linked to elevated urine As levels and decreased renal function [239]. As exposure has been shown to induce significant ROS production and DNA damage, and autophagy in the kidney [240]. Studies have shown that As exposure induced p62 expression, which activates LC3B in the rat kidney, and the accumulation of autophagosome disrupted kidney ultrastructure [241]. While chronic As exposure has been shown to induce kidney carcinogenesis through sustained oxidative stress and autophagy signaling, more experimental studies are warranted to better establish the molecular mechanisms underlaying As-induced carcinogenesis.

6.3. Chromium:

Cr exposure has been strongly associated with increasing the mortality risk among patients with kidney cancer [242]. Decreased activity of glutathione reductase in the kidneys of rats exposed to Cr, has been suggested to be the result of Cr-induced oxidative stress. Moreover, Cr-transformed cells show constitutive expression of HIF-1α, which is linked to cellular survival by inducing mitophagy in clear cell renal cell carcinoma [243]. Studies have demonstrated that ROS and autophagy in Cr-exposed cells are closely linked and could play an important role in Cr-mediated malignant transformation of kidney cells. Considering that Cr induces activation of HIF-1α, Nrf2, and p62 pathways [10], which each play significant roles in autophagy activation, more studies are needed to delineate autophagy’s role in Cr-induced renal carcinogenesis.

7. Conclusions and future perspectives

Autophagy is considered to function as a double-edged sword in cancer cells due to its elusive switch from tumor suppressor to tumor promoter. Because cancer cells are known to exploit autophagy as an adaptive stress response to survive hostile environments, the recent decade has seen a resurgence of interest in developing autophagy inhibitors as a therapeutic strategy to combat refractory cancers. On the other hand, despite evidence from various experimental and epidemiological studies, the occurrence of metal-induced carcinogenesis is still a controversial topic among researchers. A factor that has greatly hampered progress in understanding the etiology of metal-induced carcinogenesis has been the lack of established in vivo models of the same, despite the availability of established in vitro models. Reasons for this include but are not limited to physiological and anatomical differences between humans and mice, making it hard to accurately mimic the carcinogenic effect of metals. Moreover, in real-world settings, humans are exposed to different concentrations of multiple metals simultaneously via various routes (drinking water, diet, smoking, and occupational), suggesting that metal-induced carcinogenesis may be a result of multiple hits. However, this aspect has yet to be mimicked in mice models where metal exposure is limited to one carcinogen and one exposure route. Moreover, the role of autophagy in metal-induced carcinogenesis is limited in urogenital cancers, though considering that the majority of experimental studies have shown a strong role of autophagy, it is essential to explore this avenue in the quest for therapeutic targets. Aside from establishing in vivo models that enable researchers to observe the carcinogenic effects of multiple metals through multiple exposure routes, there is a need to determine metal carcinogenesis using established autophagy mouse models.

Moving forward, there is a need to extend the current knowledge of metal-induced carcinogenesis from in vitro settings to in vivo settings, and compare these results to results acquired from human clinical specimens and epidemiological studies. Combining evidence from these fronts will enable us to acquire a complete picture of the etiology and pathology of metal-induced carcinogenesis.

Abbreviations:

Atg

Autophagy related genes

ATF4

Activating transcription factor 4

Cd-MT

metallothionein-bound Cd

CTPE

Cd-induced prostate epithelial cells

HIF-1

Hypoxia inducible factor 1

JNK

c-Jun N-terminal kinase

Keap1

Kelch-like ECH-associated protein

LC3B

microtubule associated protein 1A/1B light chain 3

MTF1

Metal Regulatory Transcription Factor 1

Nrf2

Nuclear factor erythroid 2- related factor 2

p62

Sequestosome-1

ROS

Reactive oxygen species

SNAREs

Soluble NSF attachment protein receptors

ULK1/2

Unc-51like autophagy activating kinase 1/2

UPR

Unfolded protein response