Cancer Letters 337 (2013) 35–40

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Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

Mini-review

Heat stress: A risk factor for skin carcinogenesis

0304-3835/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2013.05.039

⇑ Corresponding author. Address: Edith Cowan University Biomedical Science(SMS), School of Medical Sciences Faculty of Computing, Health and Science, 270Joondalup Drive, Joondalup WA 6027, Australia. Tel.: +61 8 6304 3640.

E-mail address: m.ziman@ecu.edu.au (M. Ziman).

Leslie Calapre a, Elin S. Gray a, Mel Ziman a,b,*

a School of Medical Science, Edith Cowan University, Perth, Western Australia, Australiab Department of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Australia

a r t i c l e i n f o

Article history:Received 22 April 2013Received in revised form 24 May 2013Accepted 27 May 2013

Keywords:Skin cancerHeat stressHeat shock proteinsHeat shock responseHSP72HSP90

a b s t r a c t

Recent evidence suggests that heat stress may also be a risk factor of skin carcinogenesis. Heat stresscauses activation of heat shock proteins (HSPs), chaperone proteins which prevent cells from undergoingapoptosis and ensuring their cellular function. However, HSPs recruitment may also have deleteriouseffects particularly if the cells rescued from apoptosis carry oncogenic mutations. We hypothesise thatexposures to both heat and UV induce skin cancer(s) by concomitant expression of HSPs and oncogenicmutant proteins. Here we review studies demonstrating that heat stress-activated heat shock proteinssuch as HSP72 and HSP90 can influence signalling pathways such as MAPK, JNK and p53, which are allinvolved in regulating cell proliferation, survival and apoptosis.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Increasing global temperature as a result of climate change is asignificant primary environmental issue likely to impact on futureclimates worldwide. In fact, the Intergovernmental Panel on Cli-mate Change (IPCC), a global organisation dedicated to monitoringclimate change, suggested that if global warming continues, theambient global temperature can be expected to rise between 2 -4.5 �C by 2100 [1]. This means that arid areas in Australia, whichhave average temperature ranges of 36–38 �C [2], may normaliseat 40–42 �C in the next few decades. This is alarming not only interms of global ecology but also because it could pose a significanthealth hazard. There is the possibility that exposure to such ex-tremes of temperature may result in increased incidences of can-cer, particularly of the skin.

The core body temperature of 37 �C sustains activity and tran-scription of genes that are vital for maintenance of cellular homeo-stasis as well as differentiation and proliferation [3–7]. However,as the body is constantly exposed to the environment, some cells,particularly those located in the skin, may be subjected to in-creased temperatures with deleterious effects [8]. Exposure totemperatures of 39 �C and above (heat shock), can cause morpho-logical damage to cells. For example, heat-exposed keratinocytes

have enlarged nuclei with dispersed chromatin changes [9]. Fur-thermore heat causes the enlargement of the cytoplasm of melano-cytes, as well as an increase in the number and size of theirdendritic processes [10].

Higher temperatures can trigger extensive denaturation, degra-dation and aggregation of critical intracellular proteins triggeringcell death pathways [4,11]. The deleterious consequences of in-creased temperatures are prevented, to some extent, by an adap-tive response that ensures cell survival in the presence of heat[12]. The survival pathway activated by heat is the heat shock re-sponse, a cascade of events that lead to induction of heat shockproteins (HSPs) which minimise acute cell damage (reviewed by[13]).

Several studies have documented the major pathways involvedin the heat stress response in various cells, including epidermalcells [4,14,15]. A major effect of the heat stress response on cellsis prevention of apoptosis [16]. As a decreased rate of apoptosis isalso a hallmark of cancer, it is possible that heat stress may be arisk factor for cancer, particularly of the skin, since epidermalcells can be constantly exposed to heat stress [17]. The fact thatregulators of the heat shock response, namely heat shock fac-tor-1 (HSF-1) and heat shock proteins (HSPs) [18], are commonlyfound upregulated in many cancer cells, including skin cancer,supports this notion. Furthermore, squamous cell carcinoma hasbeen known to arise from lesions of the heat-induced skin condi-tion called Erythema ab Igne (EAI) [19] which develops in theskin of people constantly exposed to intense heat such as bakersand glass blowers [10]. There is also an increased incidence of

36 L. Calapre et al. / Cancer Letters 337 (2013) 35–40

skin cancers among mine workers, constantly exposed to intensetemperatures [20]. Taken together, these facts support the pro-posal that heat may pose an as yet unidentified risk factor forskin cancer [21].

2. Heat stress and the heat shock proteins

To ensure that cells are protected against environmental stress-ors, cells have developed protective mechanisms against cellularapoptosis. This protective mechanism is commonly referred to asthe heat shock response (Fig. 1) [22,23], and is primarily controlledat the transcription level by the transcription factor heat shock fac-tor-1 (HSF-1) [24]. HSF-1 is normally present in the cytoplasm as amonomer which has no DNA binding capability [25]. However,when cells are exposed to environmental stresses such as heat,monomeric HSFs combine to form trimers which translocate tothe nucleus. Once in the nucleus, these transcription factors areactivated by phosphorylation and bind to DNA heat shock ele-ments (HSEs) [26]. These HSEs are consensus sequences of DNA lo-cated in the upstream promoters of genes responsive to HSF-1[5,27] including HSPs [28].

The HSP proteins are highly conserved in different species andare expressed in all cells [29]. There are many types of heat shockproteins, but HSP72 and HSP90 are the key proteins involved in theheat shock response [14,30]. These peptides have important rolesin various cellular processes but their main function is to act as amolecular chaperone, i.e. they bind to other proteins and mediatetheir folding, transport and protein–protein interactions [8]. Inaddition, these HSPs have the ability to direct re-folding of dena-tured proteins, damaged as a result of heat shock, thus suppressingfurther damage [11]. As a result, cells are stabilised and protectedagainst heat-induced damage [22].

It is important to note that intense temperatures, particularlyabove 45 �C (hyperthermia), can lead to the impairment of HSPsand lead to cell death [31]. The exact mechanism by which hyper-thermia induces cell death is not clear [32]. However, the generalconsensus is that death occurs due to the irreversible, unfoldingof a significant number of proteins. Resultant protein denaturationleads to defective DNA replication, transcription and repair [33–

Fig. 1. The heat shock response (adapted from [38]). The heat shock response is activatranslocation of phosphorylated, trimeric heat shock factor (HSF) into the nucleus wheproteins called heat shock proteins (HSPs).

35], with improper processing of DNA fragments, which resultsin genomic instability and cell death [36].

By contrast at temperatures between 39 and 43 �C, HSPs protectcells from apoptosis [37], by inhibiting cell death-signalling path-ways, c-Jun NH2 terminal kinase (JNK) and p53 pathways [16,38].In fact, when HSP72 is overexpressed, the activity of death signal-ling pathways such as JNK and p53 are decreased [26]. Inhibition ofapoptosis may allow DNA damaged cells to survive and proliferatethus contributing to cancer initiation [39,40]. Furthermore, HSP90has been found to stabilise mutant B-RAF and N-RAS proteins,which normally are controllers of cell proliferation, however whenactivated by mutations they are constitutively activated leading tooncogenesis [41]. Thus, HSP90 would support their constant acti-vation and oncogenic activity. Interestingly, overexpression ofHSPs, particularly the major chaperone proteins HSP72 andHSP90, is common in most cancers including BCC, SCC and mela-noma [42–45].

2.1. HSPs and inhibition of cell death-inducing pathways andactivation of survival pathways

HSP72 (or HSP70) is part of a family of proteins with the molec-ular weight of 70 kDa [11]. It is one of the most abundant HSPs andaccounts for approximately 2% of all cellular proteins [4]. As men-tioned above, expression of HSP72 is very important for the regu-lation of the heat shock response, and these proteins cantransiently interact with various signalling pathways and nega-tively regulate their function in order to prevent apoptosis [46].The c-Jun N-terminal kinase (JNK) signalling cascade is one path-way inhibited by heat induced HSPs [47]. This pathway is respon-sible for activation of apoptosis in damaged cells [48].

JNK is a member of an evolutionarily conserved sub-family ofmitogen-activated protein (MAP) kinases. Normally, cellular dam-age activates JNK, which then induces cleavage of the pro-apopto-tic protein Bid and/or phosphorylation of anti-apoptotic proteinsBcl-2 and Bcl-xl, leading to the efflux of cytochrome c from themitochondria [49]. The efflux of cytochrome c activates caspase 9and caspase 3, which are members of the caspase family of prote-ases involved in the activation of cell apoptosis.

ted upon exposure to environmental and chemical stressors. It is initiated by there it binds to heat shock elements promoters leading to expression of chaperone

L. Calapre et al. / Cancer Letters 337 (2013) 35–40 37

Down-regulation of the JNK pathway is required for the devel-opment of many cancers including hepatocellular carcinoma, hu-man gastric cancer and skin cancer [50]. Loss of JNK is importantin skin tumour formation as it normally functions as a tumour-sup-pressor [51]. Studies using mouse models showed that JNK1-knockout mice are more susceptible to skin tumour formation[51,52].

HSP72 inhibits JNK-induced apoptosis (Fig. 2) [6,15,53], via acti-vation of JNK phosphatase, an enzyme that deactivates JNK pro-teins, by binding directly to their ATPase domain [47]. Inaddition, HSP72 can bind directly to Apaf-1, via its ATPase domain,which prevents the recruitment of procaspase-9 to the apopto-some and subsequent caspase-3 activation [54].

HSP72 also maintains the activity of the cell survival kinase Akt,modulating its expression and function through phosphorylationof its unphosphorylated C-terminus [55], causing rephosphoryla-tion and activation [23]. Phosphorylated Akt positively regulatescellular proliferation [56,57]. In cancers, the aberrant activationof Akt disables pro-apoptosis proteins BAD and caspase and acti-vates pro-survival transcription factors such as nuclear factor-jB,resulting in cell survival and uncontrolled cell proliferation[6,58]. Akt inhibition of pro-apoptosis and activation of survivalmechanisms as a result of heat shock suggest that heat may allowcells, which carry a mutation(s) as a result of exposure to environ-mental stressors such as UV radiation, to escape death andproliferate.

It is important to note that Akt and JNK activities are down-reg-ulated by tumour suppressor phosphatase and tensin homologue(PTEN) [59]. PTEN mutations are present in many cancers includingin 10–27% of melanoma [57,60]. Studies have found that that lossof PTEN is sufficient to increase both JNK and Akt activity [61–63].Thus, even though loss of PTEN enhances both normal functions ofJNK and Akt, i.e. apoptosis and proliferation respectively, the pres-ence of HSP72 can counteract the effects of PTEN on JNK, suppress-ing its pro-apoptotic activity and promoting cell survival.Therefore, loss of PTEN in the presence of HSP72 may synergisti-cally enhance pro-survival and pro-proliferation activities.

Aside from HSP72, HSP90 is also found to modulate proteins inthe MAPK pathway, namely RAF and RAS, which regulate the pro-liferation and differentiation of keratinocytes and melanocytes(Fig. 3) [64,65]. HSP90, a 90 kDa protein activated as part of theheat shock response, has a wide range of ‘client’ proteins, includingN-RAS and B-RAF proteins which depend on HSP90 for conforma-tional maturation and folding [41,66]. However, studies have foundthat HSP90 is also responsible for stabilising mutant B-RAF and N-

Fig. 2. The heat shock proteins and JNK pathway (adapted from: [6]). Heat shockresponse has been found to inhibit apoptosis via inhibition of the JNK signallingpathway. HSP72 can induce activation of JNK phosphatase, which degrades the JNKproteins, and/or directly bind to Apaf-1to prevent binding of caspase-9 to derailapoptosis.

RAS proteins as these are found to be depleted and/or degraded inresponse to HSP90 inhibitors [67] N-RAS and B-RAF are constitu-tively activated when mutated and are crucial for the proliferationof cells in several form of cancer, particularly [57,61,68].

2.2. Induction of skin cancer by heat stress and UVR: mediated byHSP72 and p53 signalling pathways

In addition to its role in the JNK pathway, HSP72 is also involvedin regulation of the key p53 signalling pathways that regulateapoptosis. The TP53 gene encodes the p53 transcription factor, acrucial tumour-suppressor [69]. p53 is involved in maintaininggenomic stability, and can enforce a G1 cell cycle arrest or induceapoptosis in response to DNA damage [70]. Cell cycle arrest at theG1 phase is very important as it allows for cellular repair pathwaysto remove damage before DNA synthesis and cell mitosis can begin[71,72]. Conversely, p53 induced apoptosis helps eliminate poten-tial tumour progenitor cells. The p53 protein is activated by phos-phorylation at one or more serine residues at the N- and C-termini[73,74]. Once activated, the protein forms a multi-protein complexin the cytoplasm then translocates into the nucleus where it bindsto the enhancer or promoter regions of downstream gene targets,such as BAX and PUMA, to regulate their transcription [69,75].PUMA (p53-upregulated modulator of apoptosis) and BAX (Bcl2-associated X protein) are pro-apoptosis genes located in the mito-chondria and cytoplasm respectively [76]. Upon binding of p53,BAX and PUMA trigger the release of cytochrome c from the inter-membrane space of the mitochondria, which then activates cas-pase 3 and 9 to induce cell death (Fig. 4) [77,78].

Normally, low levels of p53 protein present in cells act as amolecular sensor of DNA damage induced by environmentalstressors, particularly UV radiation [73]. The action taken by p53is dependent on the level of DNA damage in cells [79]. If minorDNA damage is detected, p53 protein expression increases andaccumulates to inhibit cell division and promote repair before cellreplication begins [70]. If damage is excessive, the p53 protein ac-tively induces transcription of genes that may direct cells to under-go apoptosis [80,81]. As long as the TP53 gene is free frommutations or otherwise active, this system is maintained. However,

Fig. 3. The MAPK signalling pathway (adapted from: [91]). The MAPK pathway isimportant for regulating cell proliferation and apoptosis. N-RAS and B-RAF, twomain kinases of the pathway, are found to be client proteins of HSP90. Whenmutated, these proteins are stabilised by HSP90.

Fig. 4. The p53 signalling pathway (adapted from: [80]. An important regulator ofapoptosis, p53 activates PUMA and BAX to recruit pro-apoptotic proteins caspase-3and caspase-9 after UV exposure in response to cellular DNA damage.

38 L. Calapre et al. / Cancer Letters 337 (2013) 35–40

once the gene accumulates mutations, the system is disrupted, anddamaged cells may escape surveillance [82–84].

Due to its active role in regulating cell proliferation and apopto-sis in normal cells, alteration of TP53 is an important genetic eventleading to initiation and/or progression of many cancers includingBCC, SCC and melanoma [17,83–85]. UV radiation is considered themain mutagen of the TP53 gene (Fig. 4), with UV signature muta-tions C–T or CC–TT base transitions are frequently found in mela-noma as well as in 50–58% of non-melanoma skin cancers [73].Mutations in the TP53 gene cause an increase in the presence ofp53 monomers, which in turn leads to the increased presence ofmurine of double minute 2 (MDM2) [69]. MDM2 is a ubiquitin li-gase that can form a complex with p53, by binding to its amino ter-minal sequence, targeting it for proteosomal degradation [17]. Asthe mutant p53 gets phosphorylated the presence of unboundMDM2 is increased, which incapacitates the p53 signalling path-way via negative feedback [86]. This results in uncontrolled cellproliferation.

It is important to note that HSP72 and HSP90 can affect p53protein expression. Though the exact mechanism of the disruptive

Fig. 5. Hypothesised model for the rol

effect of HSP72 on the p53 pathway is not fully understood, it hasbeen shown that HSP72 can form stable complexes with mutantp53, which is important in stabilising and localising this protein[12,46]. In their study, Wiech and colleagues [87] confirmed thatHSP72 stabilises the mutant p53 protein and increasing its half-lifeby disrupting MDM2-dependent ubiquitination and degradation.Structural analysis of the interaction of HSP90 and p53 showedthat the C-terminal of HSP90 binds directly to the DNA-binding do-main of p53 where most of the cancer-causing mutations are foundto occur [88–90]. Taken together, the evidence supports ourhypothesis that combined exposure to UV radiation and heat couldlead to the formation of skin cancer.

In summary, exposure to intense heat may aid in keratinocyteand melanocyte transformation via mechanisms proposed inFig. 5. Basically, precursor cells would be characterised by abnor-mal expression of HSP72, resulting in low JNK protein activity. Fur-ther exposure to heat stress may induce overexpression of HSP72resulting in complete inhibition or minimal activity of JNK protein.There will also be overly high Akt activity, due to constant overex-pression of HSP72. These changes may cause deregulated prolifer-ation of cancer precursor cells.

Exposure to UV may induce mutations in p53. The resultantmutant p53 protein, together with abnormally high HSP72 andHSP90 activity (with subsequent heat exposure), may stabilise tu-mour progenitor cells, allowing them to proliferate leading to for-mation of melanoma, SCC or BCC. Mutations in N-RAS or B-RAF arenot sufficient to induce melanoma formation, but lead to formationof benign naevi. It is possible that constant activity of HSP90 as aresult of over-exposure to heat may promote survival of cells con-taining these mutant proteins, allowing additional mutations inp53 or PTEN to develop, thus facilitating transformation of precur-sor cells into cancer cells.

3. Conclusion

In conclusion, the increasing incidence of people diagnosedwith various forms of skin cancer annually makes skin cancersone of the major health concerns across the globe. The lack of curefor metastatic forms of melanoma and SCC makes it imperative todetermine all possible risk factors. So far, studies have confirmedthe role of UV radiation in skin cancer formation. The knowledge

e of heat in skin cancer induction.

L. Calapre et al. / Cancer Letters 337 (2013) 35–40 39

gained from studying UV radiation has seen the development ofpreventative strategies, which if utilised correctly could slow theever increasing incidence of skin cancer. The lack of informationon heat, as a risk factor for skin cancer, is potentially harmful. Asnoted above, heat stress has a dramatic effect on cells and caninfluence the activity of signal transduction pathways, particularlythose that are important in regulating keratinocyte and melano-cyte proliferation, survival and apoptosis. Further research is war-ranted to determine the role of heat in skin cancer formation, aloneor in synergism with UV radiation. Knowledge of this can be uti-lised to decrease risk exposures particularly for people exposedto combinations of these environmental hazards in workplacessuch as the mining, construction and petroleum industries.

Conflict of interest

We certify that there is no conflict of interest with any organi-zation, financial or otherwise, regarding the material discussed inthe manuscript.

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