Alterations in Endogenous Retinoids with Acute UVB Exposure and in the Progression of Cutaneous Squamous Cell Carcinoma

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Katherine Gressel

Graduate Program in Human Nutrition

The Ohio State University

2015

Master's Examination Committee:

Dr. Helen Everts, Advisor

Dr. Earl Harrison

Dr. Tatiana Oberyszyn

Copyrighted by

Katherine Gressel

2015

Abstract

Exposure to UVB light is the largest risk factor for the development of cutaneous squamous cell carcinoma (cSCC), one of the most common cancers in the United States.

UVB exposure causes many changes to occur in the skin such as epidermal thickening, hyperproliferation, and alterations to epidermal differentiation. Retinoids regulate the differentiation and proliferation of the epidermis. The purpose of this study is to examine how the expression of retinoid metabolism proteins in the epidermis is altered with acute

UVB exposure and in the progression of cSCC. There is limited knowledge of how UVB exposure may affect this pathway; our results will allow us to gain a more complete understanding of changes in the entire retinoid metabolism pathway. We examined the expression of key retinoic acid metabolism proteins in the epidermis and hair follicle remnants 48 hours after UVB exposure in SKH-1 mice using immunohistochemistry.

UVB exposure increased the expression of retinoid synthesis, degradation, and binding proteins. These changes in expression suggest that acute UVB exposure increases the synthesis of retinoic acid in the epidermis. Many of these proteins changed their localization in the epidermis, concentrating in the more differentiated cells of the stratum granulosum and stratum corneum. To determine if retinoic acid was playing a role in repair from UVB light, we examined the changes in proliferation and terminal differentiation markers, keratin 6 and loricrin respectively, with disulfiram treatments, ii which inhibit retinoic acid synthesis. We found that retinoic acid synthesis is necessary for normal UVB repair. To further explore the role endogenous retinoids play in the formation and progression of cSCC, we examined STRA6, DHRS9, and ALDH1A1 expression using IHC in all tumor stages of cSCC. We found that these proteins change expression throughout cSCC development and may be contributing to changes in retinoic acid synthesis. Changes in endogenous retinoid metabolism in the progression of cSCC may explain why some retinoid treatments have been effective in treating this cancer.

These findings may contribute to the development of more effective prevention and treatments for cSCC.

iii

Acknowledgments

I would like to thank Dr. Helen Everts for her funding, support, and guidance throughout this research project.

I would like to thank Dr. Earl Harrison and Dr. Tatiana Oberyszyn for serving on my thesis committee.

Vita

Education

2013-Present……..…………………………….M.S. Human Nutrition,

The Ohio State University

2012...... B.S. Molecular Genetics,

The Ohio State University

Experience

2013 to present ...... Graduate Teaching Associate,

Department of Human Nutrition,

The Ohio State University

Publications

Gressel, K. L., J. F. Duncan, T. M. Oberyszyn, K. M. La Perle, and H. B. Everts, 2015, Endogenous Retinoic Acid Required to Maintain the Epidermis Following Ultraviolet Light Exposure in SKH-1 Hairless Mice:Photochem Photobiol. doi: 10.1111/php.12441

Fields of Study

Major Fields: Human Nutrition

Table of Contents Abstract ...... ii

Acknowledgments ...... iv

Vita ...... v

List of Figures ...... ix

List of Tables ...... xi

Introduction ...... 1

Chapter 1: Review of Literature ...... 3

1.1 Retinol Transport & Metabolism ...... 3

1.2 Retinoids in the Epidermis ...... 12

1.3 Ultraviolet Light ...... 13

1.4 Cell Death ...... 15

1.5 Cell Death and Cornification ...... 18

1.6 Repair from Ultraviolet Light Exposure ...... 19

1.7 Comparison of Epidermal Models ...... 20

1.8 Cutaneous Squamous Cell Carcinoma ...... 21 vi

1.9 SKH-1 Hairless Mouse Model ...... 27

Chapter 2: Endogenous Retinoic Acid Required to Maintain the Epidermis

Following Ultraviolet Light Exposure in SKH-1 Hairless Mice ...... 30

2.1 Introduction ...... 30

2.2 Materials and Methods...... 35

2.3 Results ...... 36

2.31 Altered expression of retinoid metabolism proteins in the upper epidermis and

hair follicle remnants 48 hours after UVB treatment ...... 36

2.32 Reduced retinoic acid synthesis damaged the upper epidermis after UVB

treatment ...... 38

2.4 Discussion ...... 42

Chapter 3: Alterations in Endogenous Retinoid Metabolism in the Progression of

Cutaneous Squamous Cell Carcinoma ...... 46

3.1 Introduction ...... 46

3.2 Materials and Methods...... 50

3.3 Results ...... 52

3.31 Retinoid Metabolism proteins change expression throughout the progression of

cSCC ...... 52

3.32 Retinoid metabolism proteins change expressions between no exposure, acute

UVB exposure, and in cSCC and surrounding epidermis...... 57 vii

3.4 Discussion ...... 59

Chapter 4: Epilogue ...... 64

Appendix: Tables ...... 67

References ...... 73

viii

List of Figures

Figure 1. Retinoid Compounds………………………………………………………...31

Figure 2. RA metabolism proteins increase expression in the upper epidermis following UVB treatment……………………………………………………….……...37

Figure 3. Reducing RA synthesis damages the epidermis……………………………40

Figure 4. LOR IHCL changes with disulfiram and UVB treatments……………….41

Figure 5. KRT6 IHCL changes with disulfiram and UVB treatments……...………41

Figure 6. LOR and KRT6 IHCL changes in the lesions and thin epidermis……….42

Figure 7. STRA6 IHCL changes during the progression of cSCC………...………...53

Figure 8. DHRS9 IHCL changes during the progression of cSCC………………….54

Figure 9. Focal and nuclear expression of DHRS9…………………………..……….55

Figure 10. ALDH1A1 IHCL changes during the progression of cSCC……………..56

Figure 11. Comparison of undifferentiated and differentiated epidermis and tumors with varying UVB exposure……………………………………………………………58

List of Tables

Table 1. Highest intensity IHCL for STRA6 in uninvolved skin and tumors for specific stages………………………………………...………………………………….67 Table 2. Representative intensity IHCL for STRA6 in uninvolved skin and tumors for specific stages………………………………………………………………………..68 Table 3. Highest intensity IHCL for STRA6 in uninvolved skin and tumors for general stages…………………………………………………………………………..68 Table 4. Representative intensity IHCL for STRA6 in uninvolved skin and tumors for general stages………………………………………………………………..……...69 Table 5. Highest intensity IHCL for DHRS9 in uninvolved skin and tumors for specific stages………………………………………...………………………………….69 Table 6. Representative intensity IHCL for DHRS9 in uninvolved skin and tumors for specific stages……………………………………………………………………..70 Table 7. Highest intensity IHCL for DHRS9 in uninvolved skin and tumors for general stages…………………………………………………………………………...71 Table 8. Representative intensity IHCL for DHRS9 in uninvolved skin and tumors for general stages………………………………………………………………..……....71 Table 9. Highest intensity IHCL for ALDH1A1 in uninvolved skin and tumors for specific stages………………………………………...………………………………….71 Table 10. Representative intensity IHCL for ALDH1A1 in uninvolved skin and tumors for specific stages……………………………………………………………...72 Table 11. Highest intensity IHCL for ALDH1A1in uninvolved skin and tumors for general stages……………………………………………………………………………72 Table 12. Representative intensity IHCL for ALDH1A1 in uninvolved skin and tumors for general stages……………………………………………………..……….72

Introduction

Exposure to ultraviolet B (UVB) light is the largest risk factor for the development of cutaneous squamous cell carcinoma, one of the most common cancers in the United States (Lansbury et al., 2013). UVB exposure causes many changes to occur in the skin such as epidermal thickening, hyperproliferation, and alterations to epidermal differentiation (Polefka et al., 2012). Retinoids regulate differentiation, proliferation, and apoptosis in the epidermis and are often used in the treatment of some skin disorders and cancers (Huang et al., 2014; Jones et al., 2004; Roos et al., 1998). Retinoid metabolism proteins have aberrant expression in many cancers, however we have limited knowledge on what changes may be occurring in endogenous retinoid metabolism after UVB exposure and in the progression of cutaneous squamous cell carcinoma (cSCC) (Guo and

Gudas, 1998). We hypothesize that endogenous retinoid metabolism is altered with acute and chronic UVB exposure and in the progression to cSCC. Given retinoids’ role in the regulation of cell proliferation and differentiation in the epidermis, we hypothesis that alterations in endogenous retinoids will influence the regulation of these processes, greatly influencing the repair from UVB and the formation and progression of cSCC.

Aim 1: Determine the expression and localization changes of key retinoid metabolism proteins after UVB exposure. The results of this study will give us insight into what changes may occur in endogenous retinoids following UVB exposure that could contribute to an increased understanding of cSCC prevention.

Aim 2: Determine how chronic UVB exposure and the progression of cutaneous squamous cell carcinoma (cSCC) alters the expression of retinoid metabolism proteins.

Examining how the retinoid pathway changes as cSCC progresses can increase our understanding of the complex genetic changes that occur with this cancer and improve treatment and prevention approaches.

We expect that both acute and chronic UVB alters retinoid metabolism, thereby affecting the regulation of proliferation and differentiation in the epidermis. These processes are important in both repair from UVB and have altered regulations in cancer.

Determining what changes are occurring will improve our understanding of UVB repair and cSCC formation and may help the development of more effective preventions and treatments.

Chapter 1: Review of Literature

1.1 Retinol Transport & Metabolism

After eating, vitamin A is absorbed as retinol bound to retinol binding protein 4

(RBP4) into the blood and as retinyl esters in chylomicrons (Blomhoff et al., 1990).

Retinol is stored mainly in the liver (Blomhoff et al., 1990) and its release into the blood is highly regulated (Blomhoff et al., 1991). RBP4 transports and delivers retinol to target tissues (Blaner, 2007; Blomhoff et al., 1990). This is the main source of retinoids in cells, as opposed to from chlyomicrons (Blaner, 2007). Holo-RBP4 associates with - transthyretin (TTR) in the blood (Blomhoff and Blomhoff, 2006). There are mixed findings in the literature on whether STRA6 can take up retinol from RBP4-TTR complex or whether TTR must dissociate before RBP4 can interact with stimulated by retinoic acid 6 (STRA6) (Berry et al., 2012a; Kawaguchi et al., 2007).

STRA6 is an intermembrane protein that catalyzes the transport of retinol into cells that express it, including the epidermis (Kawaguchi et al., 2007). STRA6 has been shown to interact with both holo- and apo-RBP4 and is argued by some, but not all, researchers to be involved in both the influx and efflux of retinol from the cell

(Kawaguchi et al., 2012; Sun and Kawaguchi, 2011). STRA6 increases the uptake of

3 retinol from RBP4 and is specific to RBP4 (Kawaguchi et al., 2007). Retinol efflux will only occur when there is a significantly higher ratio of apo- to holo-RBP present in the extracellular fluid (Kawaguchi et al., 2012). However, when apo-RBP is at the same or lower concentration as holo-RBP, as in the blood, retinol influx into the cell predominates (Kawaguchi et al., 2012). Cellular retinol binding protein 1 (CRBP1) directly accepts retinol from STRA6 and binds STRA6 at its CRBP1 Binding Loop

(CBL) and the C-terminus domain to take up retinol into the cell (Berry et al., 2012b).

CRBP1 enhances retinol intake and is required for uptake via STRA6 (Berry et al.,

2012b).

STRA6 also functions in a janus kinase 2/Signal Transducer and Activator of

Transcription 5 (JAK2/STAT5) cytokine signaling cascade (Berry et al., 2012b). This signaling and retinol uptake are sequential (Berry et al., 2012b). Apo-CRBP1 and JAK2 are bound to STRA6 prior to STRA6-facilitated retinol uptake to apo-CRBP1. JAK2 is activated and in turn STRA6 is phosphorylated by JAK2 at its Y643 residue (Berry et al.,

2012b). Once STRA6 is phosphorylated, it releases its C-terminal domain, allowing greater access of retinol to apo-CRBP1 and increases retinol influx (Berry et al., 2012b).

Phosphorylated STRA6 then initiates STAT5 signaling cascade by recruiting STAT5, which then gets activated by JAK2 (Berry et al., 2012b). JAK2/STAT5 signaling promotes cell survival through the activation of Phosphoinositide 3-kinase (Pi3K) and

RAC-Alpha Serine/Threonine-Protein Kinas (Akt1) (Creamer et al., 2010; Schmidt et al.,

2014). Continual signaling through this pathway can promote tumor growth (Schmidt et al., 2014).

STRA6 localizes to the blood brain barriers and basolateral membranes, making it easily accessible to RBP in the blood (Kawaguchi et al., 2007). Large concentrations of

STRA6 have been found in the brain, spleen, the RPE in the eye, kidney, female genital tract, testis, and adipose tissue (Bouillet et al., 1997; Sima et al., 2011). STRA6 is expressed in the human epidermis and its expression is upregulated by retinoic acid

(Skazik et al., 2014).

Mutations in STRA6 in humans are classified as Matthew Wood/Spear syndrome/

Pulmonary hypoplasia-diaphragmatic hernia-anophthalmia-cardiac defect (PDAC) syndrome and result in defects of the eyes, lungs, diaphragm, and heart (Chassaing et al.,

2009). STRA6 knockouts in zebra fish can result in vitamin A deficiency, leading to eye and embryonic malformations, and results in an excessive amount of retinoic acid production in certain tissues during development (Isken et al., 2008).

STRA6 knockout mouse models have been utilized to determine the role STRA6 plays in vitamin A homeostasis (Amengual et al., 2014; Berry et al., 2013; Terra et al.,

2013). One knockout mouse model was viable on a diet lacking vitamin A, however there were reduced retinoid levels and histological malformations in the retinas were reduced

(Amengual et al., 2014). Berry et al also produced a STRA6-null mouse model and similarly found reduced retinoids in the eyes, however modest effects of retinol uptake by peripheral tissues (Berry et al., 2013). The ablation of STRA6 expression was determined to have little to no physiologic effect on adult or embryonic retinoid homeostasis or on T- cell immune responses (Berry et al., 2013; Terra et al., 2013). These knockout studies

5 demonstrate that STRA6 expression does not play an important role in maintaining vitamin A levels in peripheral tissues, with the exception of the eye.

STRA6 influences cellular differentiation and proliferation (Muenzner et al.,

2013; Skazik et al., 2014). STRA6 expression increases the differentiation of adipose tissue (Muenzner et al., 2013). As pre-adipocytes mature, STRA6 mRNA decreases and its protein levels increase, suggesting post-translational regulation (Muenzner et al.,

2013). The presence of STRA6 in pre-adipocytes pushes differentiation into adipocytes

(Muenzner et al., 2013). Silencing STRA6 reduces differentiation, but this is rescued by a retinoic acid receptor alpha (RARA) antagonist and RPB4 repression, showing that

STRA6 is facilitating the efflux of retinol from the pre-adipocyte and leading to a reduction in retinoic acid signaling (Muenzner et al., 2013). A study utilizing STRA6 knockdown in human adult low calcium high temperature (HaCaT) cells showed that an absence of STRA6 increased cell proliferation (Skazik et al., 2014). A 3D skin model using these cells exhibited a thicker epidermis and an increase in proliferation, activation, and differentiation markers, which was reversed with treatment of free retinol (Skazik et al., 2014). It has been proposed that a lack of STRA6 causes retinoid deficiency in cells and that STRA6 is required for retinoid homeostasis and represses proliferation and differentiation in the skin (Skazik et al., 2014).

CRBP1 accepts retinol from STRA6 and binds it in the cytosol (D'Ambrosio et al., 2011; Kawaguchi et al., 2012; Ong, 1994). CRBP1 serves as a readily available source of retinol for the cell, as well as directs retinol either to LRAT to be stored or to dehydrogenases to be utilized for retinoic acid synthesis (Blomhoff and Blomhoff, 2006;

D'Ambrosio et al., 2011; Ong, 1994). Lecithin retinol acyltransferase (LRAT) catalyzes the esterification of retinol into retinyl esters for storage (D'Ambrosio et al., 2011; Kumar et al., 2012). It directly accepts retinol from CRBP1 and coordinates with CRBP1 and

STRA6 to increase retinol uptake (Berry et al., 2012b; D'Ambrosio et al., 2011;

Kawaguchi et al., 2007). A decrease in LRAT expression results in reduced retinol uptake through STRA6 and therefore decreased JAK2/STAT5 signaling (Berry et al., 2012b).

LRAT works independently of STRA6, but has an increased rate in the presence of

STRA6 (Kawaguchi et al., 2007). LRAT expression is under transcriptional control by retinoic acid and peaks about 48 hours after RA exposure in the skin (Pavez Loriè et al.,

2009a).

Retinol that is not stored as retinyl esters can be reversibly oxidized to retinaldehyde (Kumar et al., 2012; Napoli, 2012).This is the rate-limiting step in the retinoic acid synthesis pathway (Napoli, 2012) There are two families of enzymes that facilitate this conversion: The alcohol dehydrogenase family (ADH), consisting of

ADH1, ADH3, and ADH4, and the short-chain dehydrogenase/reductase family (SDR), consisting of RDH1, RDH10, and DHRS9 (Kumar et al., 2012; Napoli, 2012). These enzyme families differ in their structures and mechanisms of action (Kumar et al., 2012).

SDRs accept retinol from CRBP1, and thus the oxidation reaction is dependent on the amount of holo-CRBP1 present (Napoli, 2012). CRBP1-bound retinol is the main source of retinol for retinal production, and flux through the SDRs is the predominant pathway (Napoli, 2012). Three RDHs have been identified as being physiologically involved in retinal synthesis and belong to the SDR family: retinol dehydrogenase 1

(RDH1), retinol dehydrogenase 10 (RDH10), and dehydrogenase reductase 9 (DHRS9)

(Napoli, 2012). RDH1 is expressed in the liver and helps maintain retinyl ester levels

(Kumar et al., 2012). RHD1 knockout mice have a higher level of retinyl esters in the liver and kidneys (Zhang et al., 2007). RDH10 plays a major role in retinoic acid synthesis in specific tissues during development and is present in the epidermis (Lee et al., 2011; Sandell et al., 2012). RDH10 null mice have a loss of retinoic acid signaling during embryogenesis and die in utero (Sandell et al., 2007). DHRS9 is the most recently identified member of the SDR family and is present in epithelial tissues that are sensitive to retinoids, including the epidermis (Everts et al., 2005; Markova et al., 2003; Rexer and

Ong, 2002). Retinoic acid acts in a negative feedback loop and represses DHRS9 expression (Markova et al., 2003).

ADH1 is highly expressed in epithelial tissue and the liver and is argued by some, but not all, to be involved in the elimination of excess retinol (Kumar et al., 2012; Napoli,

2012). ADH1 knockout mice have increased retinyl esters, reduced retinoic acid metabolism, and are more susceptible to vitamin A toxicity (Molotkov et al., 2002a).

ADH1 and CRBP1 oppose each other in determining the fate of retinol (Kumar et al.,

2012). ADH1/CRBP1 double knockout mice have normal retinyl ester levels and are less sensitive to vitamin A deficiency than ADH1 knockout mice (Molotkov et al., 2004).

ADH3 is ubiquitously expressed and has the lowest catalytic activity of the ADHs

(Kumar et al., 2012). ADH3 knockout mice have a lower body mass than wild type mice

(Niederreither et al., 1999). This can be rescued by increasing retinol in the diet, suggesting that ADH3 has overlapping functions with other enzymes (Molotkov et al.,

2002b). ADH3 has overlapping function with ADH1 and ADH4 and assists in both the elimination of excess retinol and survival during vitamin A deficiency (Kumar et al.,

2012). ADH4 is expressed in epithelial tissues, but not the liver, and helps protect against vitamin A deficiency (Kumar et al., 2012). ADH4 knockout mice have no obvious defects but have decreased survival when fed a vitamin A deficient diet (Deltour et al.,

1999; Molotkov et al., 2002a). ADHs do not act on retinol that is bound to CRBP1

(Blomhoff and Blomhoff, 2006).

Retinaldehyde is further oxidized to retinoic acid by ALDH1A1, ALDH1A2, and

ALDH1A3 (alternatively named RALDH1, RALDH2, and RALDH3) (Kumar et al.,

2012). ALDH1A1 is expressed in epithelial tissues and the liver (Everts et al., 2005;

Kumar et al., 2012). ALDH1A1 knockout mice have a normal survival rate, suggesting that it is not the main enzyme required for retinoic acid synthesis in the embryo

(Blomhoff and Blomhoff, 2006; Fan et al., 2003). ALDH1A2 is required for retinoic acid signaling in specific stages of development (Kumar et al., 2012). A loss in ALDH1A2 results in embryonic lethality that can be rescued with retinoic acid treatment (Mic et al.,

2002; Niederreither et al., 1999). ALDH1A3 is involved in retinoic acid signaling in embryogenesis and the development of the nasal pit and eyes (Kumar et al., 2012).

ALDH1A3 knockout mice die at birth due to a nasal pit defect that prevents breathing

(Dupé et al., 2003). Another ALDH, ALDH8A1 (RALDH4), is expressed in the liver and kidney and facilitates the conversion of 9-cis-retinaldehyde to 9-cis-retinoic acid

(Blomhoff and Blomhoff, 2006). These knockout studies demonstrate the importance of

ALDH1A2 and ALDH1A3 in retinoic acid synthesis during development (Blomhoff and

Blomhoff, 2006). Disulfiram is a drug typically used in the treatment of alcoholism that inhibits alcohol and aldehyde dehydrogenases, including those involved in the synthesis of retinoic acid (Kulkarni et al., 2013).

Retinoic acid is metabolized by cytochrome P450 family members, CYP26A1,

B1, C1 (Kumar et al., 2012; Thatcher and Isoherranen, 2009). These cytochromes add hydrophilic groups to retinoic acid and produce 18-hydroxy retinoic acid, 4-hydroxy retinoic acid, retinoyl beta-glucuronide, and 4-oxo retinoic acid, which are more available for excretion (Blomhoff and Blomhoff, 2006; Kumar et al., 2012). These degradation enzymes have retinoic acid response element (RARE) promoter sequences and are transcriptionally induced by high levels of retinoic acid (Blomhoff and Blomhoff, 2006).

CYP26A1 and CYP26B1 are essential for development, evidenced by the lethality of knocking out these genes (Thatcher and Isoherranen, 2009). CYP26A1 knock-out mice died during gestation and exhibited effects commonly seen with excess retinoic acid

(Abu-Abed et al., 2001). CYP26B1 knockout mice die after birth due to respiratory failure and exhibited abnormal forelimb and hindlimb development, and have abnormal epidermal development and impaired barrier function (Okano et al., 2012; Yashiro et al.,

2004). CYP26C1 is most efficient at metabolizing 9-cis-retinoic acid (Blomhoff et al.,

1990). These cytochromes do not have common expression patterns among tissues, suggesting a unique role for each (Reijntjes et al., 2004). Their expression levels are capable of influencing retinoic acid signaling (Kumar et al., 2012).

Cellular retinoic acid binding proteins 1 and 2 (CRABP1 and 2) bind retinoic acid in the cytosol (Blomhoff and Blomhoff, 2006; Ong, 1994). CRABP1 directs retinoic acid

10 towards degradation (Blomhoff and Blomhoff, 2006; Ong, 1994). CRABP2 transports it to the nucleus, where it can exert its signaling (Blomhoff and Blomhoff, 2006). Both

CRABP1 and 2 are expressed in the skin (D'Ambrosio et al., 2011). Retinoic acid is the active metabolite of vitamin A and binds to the nuclear receptors retinoic acid receptor alpha, beta, and gamma (RARA, RARB, and RARG) to alter gene expression (Kumar et al., 2012). These RAR transcription factors each have a unique function (Petkovich,

1992). They function as heterodimers with retinoid X receptor (RXR) nuclear receptors and bind to retinoic acid response element (RARE) sequences in the promoter regions of genes (Blomhoff and Blomhoff, 2006; Kumar et al., 2012). RARs bind both at-RA and 9- cis-RA, but all-trans retinoic acid is the most abundant and main activator (Blomhoff and

Blomhoff, 2006). RXRs bind 9-cis-retinoic acid but do not require it to function in retinoic acid signaling (Kumar et al., 2012). Retinoic acid influences the expression of proteins involved in its metabolism, including STRA6, LRAT, and CYP26B1 (Wu and

Ross, 2010). Retinol is available to all cells but the expression of retinoic acid metabolism proteins is necessary for retinoic acid synthesis (Kumar et al., 2012).

Therefore, precise transcriptional and translational control over these enzymes is key in the regulation of retinoic acid signaling (Kumar et al., 2012).

Besides retinoic acid signaling, retinoids have alternative roles in the cell (Hoyos et al., 2012). Vitamin A is needed for mitochondrial function (Everts and Berdanier,

2002b). Retinol is a co-factor for protein kinase C delta (PKCδ) and participates in redox reactions in the intermembrane space in the mitochondria (Hoyos et al., 2012).

Anhydroretinol, a retinol metabolite and antagonist reduces ATP production, which

11 suggests that retinol may play a role in the generation of ATP (Hoyos et al., 2012). PKCδ associates with Src Homology 2 Domain Containing Transforming Protein 1 (p66Shc) and cytotochrome c and is thought to play a role in the donation of electrons to cytochrome c “via a retinol carrier” (Hoyos et al., 2012). RAR receptors B, G1, and G2, as well as CRABP1 and 2, have been found in the mitochondria (Everts and Berdanier,

2002a; Everts et al., 2002). RAREs are present in the mitochondrial genome and retinoic acid has been shown to increase expression of mitochondrial genes such as NADH dehydrogenase subunit 5 (ND5), cytochrome c oxidase, and ATPase 6 (Everts and

Berdanier, 2002a, b; Everts et al., 2002). Rats with a mutation in ATPase6 have reduced oxidative phosphorylation and require three times as much vitamin A in their diets to restore optimal mitochondrial functioning, suggesting that retinoic acid plays a role in oxidative phosphorylation (Everts and Berdanier, 2002a).

1.2 Retinoids in the Epidermis

Retinoids regulate differentiation, proliferation, and apoptosis in the epidermis and have been used in the treatment of many disorders of keratinization of the skin

(Eckert and Rorke, 1989; Huang et al., 2014; Roos et al., 1998). At-RA treatments induce hyperproliferation in the epidermis (Gericke et al., 2013). The effect of retinoids on differentiation may be dependent on the RAR receptor that gets activated (Gericke et al.,

2013). An RARG agonist induced a response similar to that seen with at-RA treatment: hyperproliferation, increased expression of retinoid target genes, including CRABP2 and

CYP26A1/B1, and increased expression of terminal differentiation genes, such as filaggrin and loricrin (LOR) (Gericke et al., 2013). However, an RARA agonist had an

12 opposing effect on the expression of these genes, suggesting that these receptors have opposing roles in skin homeostasis (Gericke et al., 2013). An in vivo study investigating keratin 6 (KRT6) expression, a marker of hyperproliferation, found that expression was increased 48 hours after retinoic acid treatment (Rothnagel et al., 1999). Retinoic acid treatments in reconstituted human epidermis, however, decrease the expression of terminal differentiation proteins, including LOR, transglutaminase K, and filaggrin

(Bernard et al., 2002). These discrepancies point to differences to consider between various tissue sources, which will be discussed later.

1.3 Ultraviolet Light

Two forms of ultraviolet (UV) enter our atmosphere and are physiologically relevant: UVA light, which has a wavelength between 315-400 nm and UVB light, which has a wavelength between 280-315 nm (Dupont et al., 2013). UVB light is the main UV light responsible for sunburns and penetrates deeper into the epidermis than does UVA light (Dupont et al., 2013; Sklar et al., 2013). Acute effects of UVB include thickening of the epidermis, erythema, vitamin D synthesis, immunosuppression, and pigment darkening (Polefka et al., 2012; Sklar et al., 2013). UVB exposure increases the production of inflammatory mediators such as prostaglandins, chemokines, cytokines, histamines, and nitric oxide (NO) (Polefka et al., 2012). Long term exposure to UVB can result in photoaging, immunosuppression, and cancer (Polefka et al., 2012; Sklar et al.,

2013). UVB exposure suppresses immune surveillance in the skin, allowing cancerous cells to evade immune detection and proliferate (Polefka et al., 2012). Chronic UVB

13 exposure results in constitutive expression of cyclooxygenase 2 (COX-2) in the skin, causing chronic inflammation and oxidation damage, which can promote cancer (Wilgus et al., 2003).

UVB can penetrate into the basal layer of the epidermis, and be absorbed by DNA or other molecules, such as NADH (Polefka et al., 2012) This leads to DNA damage and reactive oxygen species (ROS) formation, which can cause further DNA damage and lipid peroxidation (Dupont et al., 2013; Polefka et al., 2012; Reichrath et al., 2014; Sklar et al., 2013). Upon DNA damage, cyclobutane-pyrimidine and thymine dimers are formed and P53 accumulates (Dupont et al., 2013; Polefka et al., 2012; Sklar et al.,

2013).

Retinol and retinyl esters absorb light at around 325nm, which includes both

UVA and UVB light (Fu et al., 2007). UV exposure causes retinol and retinyl esters to degrade and photoexcite (Sorg et al., 2002). This photoexcitement forms photoproducts, as well as produces ROS that are capable of damaging DNA and lipids (Fu et al., 2007).

Retinyl esters comprise 70% of the vitamin A found in the skin and are more susceptible than retinol to this excitation (Fu et al., 2007). UV exposure decreases the concentration of retinoids in the skin (Takeda et al., 2003). UVA causes a greater depletion of vitamin

A than UVB and increases lipid peroxidation in the epidermis, whereas UVB does not

(Sorg et al., 2002). The degradation of these compounds could lead to vitamin A deficiency or toxicity through production of retinoid photoproducts (Fu et al., 2007).

Some retinoid metabolism proteins change expression in the skin after UV exposure (Sorg et al., 2002; Sorg et al., 1999; Takeda et al., 2003). UV treatment

14 decreases RARA expression, increases B-carotene dioxygenase activity, and UVA exposure increases CRBP1 expression (Sorg et al., 2002; Takeda et al., 2003). Our results add to these changes in expression and contribute to a better understanding of the effects of UV light on endogenous retinoids in the skin.

1.4 Cell Death

P53 has been termed the “guardian of the genome” (Lane, 1992), as it senses

DNA damage and arrests the cell cycle at G1 (Kastan et al., 1991). Because of this, it plays a key role in the prevention of cells with DNA damage from replicating and becoming cancerous (Hollstein et al., 1991). Its importance is highlighted by the fact that almost all human cancers show an inactivation of p53 (Hollstein et al., 1991), as do 50-

75% of ultraviolet radiation (UVR) induced cancers in the SKH-1 hairless (Hr) mouse model (Benavides et al., 2009). After UV exposure and DNA damage, p53 accumulates due to post-translational stabilization and halts the cell cycle to attempt to repair the damage (Kastan et al., 1991; Maltzman and Czyzyk, 1984). P53 directly binds to DNA in a sequence-specific manner and controls gene expression (Bargonetti et al., 1991; Kern et al., 1991; Kern et al., 1992). If the DNA cannot be repaired, p53 initiates the intrinsic apoptotic pathway (Yonish-Rouach et al., 1991).

There are two apoptotic pathways: the extrinsic, which initiates through ligand- binding death receptors on the cell surface and the intrinsic, which is initiated in the mitochondria (Khan et al., 2014). These pathways are both regulated by zymogens, cysteine aspartic acid specific proteases (caspases) (Khan et al., 2014). An activation

15 cascade leads to the activation of “executioner” caspases, which cleave proteins required for survival (Khan et al., 2014).

The extrinsic pathway utilizes receptors of the tumor necrosis factor (TNF) receptor superfamily, such as TNF-related apoptosis-inducing ligand receptors-1

(TRAIL), TNF-R1, and Fas/APO1 (Khan et al., 2014). Ligands such as APO2L/TRAIL,

FasL, and TNF bind these death receptors (Khan et al., 2014). There are both functional and decoy death receptors, but only the functional receptors contain death domains and are capable of relaying a signal (Khan et al., 2014). Upon ligand binding, the receptors trimerize and cluster (Khan et al., 2014). Fas-associated death domain protein (FADD) and caspases 8 & 10 are recruited and death-inducing signal complex (DISC) is formed

(Khan et al., 2014).DISC activates effector caspases 3, 6, and 7 (Khan et al., 2014).

The intrinsic pathway is initiated in response to cell stress such as from DNA damage, loss of survival factors, and defective cell cycle (Khan et al., 2014). Activation of pro-apoptotic proteins such as Bcl-2 antagonist of the cell death (BAD), BH3 interacting domain death agonist (BID), Bcl-2 interacting mediator of the cell death

(BIM), Bcl-2 modifying factor (BMF), and p53 upregulated modulator of apoptosis

(PUMA) bind and inhibit anti-apoptotic proteins (Khan et al., 2014). This includes inhibition of Bcl-2, which helps preserve the integrity of the mitochondrial membrane

(Khan et al., 2014). BCL2-antagonist/killer (BAK) and BCL2-associated X protein

(BAX) form homo-oligomers that form pores in the outer mitochondrial membrane

(Khan et al., 2014). This causes the release of pro-apoptotic factors such as cytochrome c and second mitochondria-derived activator of caspases/direct inhibitors of apoptosis

16 proteins-binding protein with low ph (Smac/DIABLO) (Khan et al., 2014). These activate caspase 9 and inhibitors of apoptosis proteins (IAPs) (Khan et al., 2014). Caspase 9 activates effector caspase 3 (Khan et al., 2014).

If p53 induces apoptosis, many changes occur within the cell, including changes in mitochondrial structure and function (Cosentino and García-Sáez, 2014). BAK and

BAX activation promotes the release of cytochrome c and other apoptotic factors that activate caspases (Cosentino and García-Sáez, 2014). Lipid transfer also occurs between the mitochondria and the endoplasmic reticulum (ER) (Cosentino and García-Sáez,

2014). The association of the mitochondria and ER is facilitated by mitofusin 2 (Mfn2)

(Cosentino and García-Sáez, 2014). Cardiolipin (CL) is one of the lipids involved in the transfer and a reduction of CL in the mitochondrial membrane reduces the binding ability of cytochrome c in the inner mitochondrial membrane, promoting its release into the intermembrane space (Cosentino and García-Sáez, 2014). The oxidation of CL by ROS is a key step in the release of cytochrome c and other factors from the mitochondria

(Cosentino and García-Sáez, 2014).

STRA6 expression is induced by p53 after DNA damage, independently of RA signaling (Carrera et al., 2013). This occurs in normal cells and cancer cell lines alike

(Carrera et al., 2013). STRA6 is a key player in p53 induced apoptosis and is involved in the cell’s decision on whether to live or die following DNA damage (Carrera et al.,

2013). STRA6 contributes to the increase in ROS and mitochondrial depolarization that promotes apoptosis (Carrera et al., 2013). This has been shown to be strictly through the intrinsic apoptotic pathway, as STRA6 shRNA has no effect on the extrinsic pathway

(Carrera et al., 2013). Silencing of STRA6 shows a similar result as the silencing of Bax

(Carrera et al., 2013).

Upon DNA damage, STRA6 changes its localization from the outer cellular membrane and moves into the cytosol (Carrera et al., 2013). This has been shown to be directly due to p53 induction, as treatment with RA only increased STRA6 in the outer membrane (Carrera et al., 2013).

1.5 Cell Death and Cornification

Programmed cell death is an important process in the formation of the dead, outer stratum corneum layer of the epidermis, which gives the skin its barrier function (Eckhart et al., 2013). This process is referred to as cornification, and involves three important steps: the replacement of intracellular organelles with cytoskeletal components, the cross- linking of proteins at the cell membrane to form the cornified cell envelope, and the intercellular adhesion of corneocytes to form a functional barrier (Eckhart et al., 2013).

Prior to the cornification process, differentiating keratinocytes receive pro- survival signals that prevent premature cell death that would cause inflammation and compromise the barrier function of the epidermis (Eckhart et al., 2013). Throughout differentiation and into cornification, keratins are produced at large volumes, especially in later stages (Eckhart et al., 2013). More than 85% of the proteins that make up corneocytes are keratins, which are key to forming the cytoskeleton that contributes to barrier function (Eckhart et al., 2013). These kertains remain intact while other cellular proteins undergo degradation and proteolysis in the early stages of cornification (Eckhart

18 et al., 2013). Along with proteolysis, three main intracellular events occur that lead to cell death: molecular machinery is degraded, the mitochondria stop producing energy, and the nucleus and its DNA are degraded (Eckhart et al., 2013). These processes are tightly regulated and ensure that proper cornification occurs to maintain the barrier function of the epidermis (Eckhart et al., 2013).

1.6 Repair from Ultraviolet Light Exposure

Acute UV exposure results in epidermal thickening, erythema, Vitamin D synthesis, immunosuppression, and pigment darkening, and is the biggest risk factor for skin cancers (Polefka et al., 2012; Sklar et al., 2013). Many changes occur following UV exposure, including changes in gene expression and cellular processes involved in cell survival and apoptosis (Polefka et al., 2012; Sklar et al., 2013). Gene array analysis of in vivo human epidermis showed an early upregulation of genes involved in DNA repair, followed by an apoptotic response, and an upregulation of proliferation genes, peaking at

72h (Enk et al., 2006). In consistency, proliferation and terminal differentiation markers such as KRT6, Ki-67, and involucrin, increased in the epidermis following UVB exposure, with their expression peaking at 48h and most returning to baseline by 72 hours

(Lee et al., 2002). In contrast, an in vitro human skin model showed a decrease in early differentiation marker, K10, and some terminal differentiation markers, such as LOR, and filaggrin in the 3 days following UVB exposure (Bernerd and Asselineau, 1997). Other terminal differentiation markers, such as involucrin, did not change expression. The

19 differences in response between in vivo and in vitro experiments will be discussed further in the following section.

1.7 Comparison of Epidermal Models

In vitro models of human epidermis are often used as a replacement to in vivo studies but many differences exist that must be considered when evaluating data (Groeber et al., 2011). In vitro models have different metabolisms than normal human skin and monolayer cultures are limited due to the lack of inter-cellular and matrix interactions

(Groeber et al., 2011). 3-dimensional (3D) cell models, however, do allow for these interactions to occur and can serve as good models for examining epidermal processes such as wound repair (Groeber et al., 2011). While human epidermal skin models have many similarities to human skin, they have reduced barrier function (Netzlaff et al.,

2005).

Of the different skin models, reconstituted human epidermal models are the most relevant to normal human skin (Bernard et al., 2002). To elucidate what differences exist between the different models, Bernard et al compared a cDNA microarray between normal human epidermal biopsies, a reconstituted human epidermis skin model

(SkinEthic), and normal human epidermal keratinocyte monolayer cultures (NHEK)

(Bernard et al., 2002). While most of the genes expressed in the biopsies were also expressed in the other tissue sources, there were some important differences (Bernard et al., 2002). Terminal differentiation markers such as filaggrin and LOR were highly expressed in the biopsies and skin model, but were not significantly expressed in the

20 monolayer cultures (Bernard et al., 2002). The skin model was also treated with retinoids and exhibited a decrease in expression of some terminal differentiation markers including

LOR, showing retinoids as exerting a differentiation-limiting effect (Bernard et al.,

2002). Retinoid treatment had opposing effects on the expression of some genes, such as catalase (CAT) and CRABP2 between NHEK cultures and the skin model, further highlighting differences between the two models (Bernard et al., 2002).

In vivo and in vitro studies also differ in their responses to UV exposure. An in vivo study using human biopsies showed an increase in expression of proliferation and differentiation markers such as KRT6, Ki-67, LOR, and involucrin in the epidermis 48 hours after exposure (Lee et al., 2002). In contrast, an in vitro human skin model showed a decrease in early differentiation marker, K10, and some terminal differentiation markers, such as LOR, and filaggrin in the 3 days following UVB exposure (Bernerd and

Asselineau, 1997). Other terminal differentiation markers, such as involucrin, did not change expression (Bernerd and Asselineau, 1997). These results are different than those presented by Lee et al, which show an increase in involucrin, filaggrin, and other markers and yield more evidence for differences between tissue sources. Because the skin model used can yield varying responses, it is important to give close consideration to the tissue source used when examining results of studies.

1.8 Cutaneous Squamous Cell Carcinoma

Cutaneous Squamous Cell Carcinoma (cSCC) is the second most prevalent non- melanoma skin cancer and is most commonly found in sun-exposed regions of the body

(Lansbury et al., 2013; Parikh et al., 2014). It originates from epidermal keratinocytes and possibly from the hair follicle stem cells (Faurschou et al., 2007; Lansbury et al., 2013;

Morris, 2004). Exposure to UV light is the biggest risk factor for cSCC and organ transplantation patients are at an increased the risk for this cancer, due to their immunosuppressed state (Jones et al., 2004). cSCC is usually treated by surgical removal of the cancerous lesions, however this can sometimes cause cosmetic and functional impairments (Lansbury et al., 2013; Parikh et al., 2014). This type of cancer has a low rate of metastasis, similar to that of renal carcinomas, but has high rates of morbidity and mortality when metastasis does occur (Parikh et al., 2014). Invasive cSCC is characterized by the migration of cancer cells into the dermis, and has increased risk of recurrence and metastasis (Lansbury et al., 2013).

Retinoids are thought to have cancer preventing effects, as they are involved in the regulation of cell growth, apoptosis, differentiation, and immune function and altered expression of the retinoic acid nuclear receptors (RARs) is seen in many skin cancers, including cSCC (Jones et al., 2004; Marcato et al., 2011). Oral retinoids have been used to successfully treat and prevent some cancers, including squamous cell carcinomas

(Harwood et al., 2005; Jones et al., 2004). Low-dose systematic retinoids (0.2 to 0.4 mg/kg per day of acitretin) reduced the incidence of cSCC in organ transplant patients

(Harwood et al., 2005). Patients who stopped taking the treatment had a significant increase in the risk of cSCC (Harwood et al., 2005). All-trans (at)-retinoic acid has been used to treat cancers such as acute promyelocytic leukemia (APL) because of its ability to induce apoptosis and differentiation of cancer cells, including cancer stem cells (CSC)

(Ginestier et al., 2009; Huang et al., 1988; Marcato et al., 2011; Zito et al., 2014).

However, retinoid treatments are often accompanied by side effects and toxicity and have shown no effect in some studies (Brewster et al., 2007; Jones et al., 2004). A randomized controlled trial with patients at a high risk of cSCC recurrence were treated with isotetinoin and interferon alfa (IFNa) and showed no improvement in recurrence rate

(Brewster et al., 2007). Oral retinoids have been effective in treating skin cancer in mouse models as well. In SENCAR mice treated with 7,12-dimethylbenz[a]anthracene

(DMBA)/ 12-O-tetradecanoyl phorbol-13-acetate (TPA), a diet containing high amounts of retinoic acid (30 micrograms/g diet) inhibited skin carcinoma formation (Chen et al.,

1994). Mice fed the high retinoic acid diet had reduced carcinoma formation, even when the diet was started after carcinogenesis, suggesting retinoic acid helps prevent the propagation from benign to malignant carcinomas (Chen et al., 1994). A study examining cutaneous keratoacanthoma (KAs) showed that retinoic acid treatment can cause regression in malignant tumors that do not spontaneously regress (Zito et al., 2014). They demonstrate that these tumors have aberrant Wnt signaling that contributes to tumor growth, which is suppressed with retinoic acid treatment (Zito et al., 2014).

In SKH-1 mice, retinoids have also shown mixed success in treating and preventing SCC (Halliday et al., 2000). SKH-1 mice treated with 0.05% retinoic acid after receiving a 1/3 minimal edemal dose of UV light were at an increased risk of photocarcinogenesis (Halliday et al., 2000). In contrast, hairless mice that received topical tretinoin after exposure to 1.5 times the minimal erythema dose of UVA/UVB had a decreased risk for tumorigenesis (Kligman and Crosby, 1996). The same group found

23 that giving retinoic acid treatment along with the UV exposure and continuing the treatment for weeks after UV exposure did not affect photocarcinogenesis (Kligman and

Kligman, 1981). While retinoid treatments have been successful in many studies, their applications are limited and the mechanisms of action and which retinoid treatments are most effective remain unclear.

Alterations in the retinoid metabolism pathway occur in many carcinomas and are seen in both in vivo and in vitro models (Guo and Gudas, 1998). Increased STRA6 expression has been implicated in cancer formation and progression (Berry et al., 2014).

Elevated expression of STRA6, along with its ligand RBP, is seen in some cancers, including breast and colon cancers (Berry et al., 2014; Szeto et al., 2001). The increased expression of these proteins contributes to carcinogenesis, which is mediated through the activation of STAT3 via STRA6-JAK2 activation (Berry et al., 2014). STAT3, along with other STAT proteins, are highly activated in many cancers, including SCC, and promote oncogenic properties including cell survival and proliferation (Bromberg and

Darnell, 2000; Sano et al., 2008). Mice with constitutive epidermal activation of STAT3 developed SCC to a greater extent and more rapidly than control mice (Sano et al., 2008).

Berry et al. show that STRA6 is upregulated early in breast cancer formation and its expression continues to increase as the cancer progresses (Berry et al., 2014). However,

STRA6 expression may be involved in initiating apoptosis of cancer cells (Mittal et al.,

2014). Fenretinide, a synthetic retinoid, was used to treat endometrial cancer cells in vitro and induced apoptosis, independent of the retinoic acid signaling pathway. This increase in apoptosis may be mediated by influx of retinol via STRA6, as STRA6

24 silencing reversed this effect (Mittal et al., 2014). This suggests that a specific level of

STRA6 expression may be required to initiate apoptosis without extraneous activation of

STAT signaling.

Cultured human SCC cells have reduced LRAT expression and esterify retinol to a lesser degree than cells cultured from the normal epithelia (Guo and Gudas, 1998; Guo et al., 2000; Jurukovski and Simon, 1999). These studies suggest that some SCC lines have a reduced ability to store retinol and modulate retinoic acid levels, therefore potentially disrupting the regulation of proliferation and differentiation (Guo and Gudas,

1998; Jurukovski and Simon, 1999). In contrast, LRAT overexpression is evident in some cancers and its expression may be a prognostic marker for malignancy in melanoma, as its high expression is associated with lower survival rates (Brown et al., 2014; Hassel et al., 2013).

The dehydrogenases involved in retinoic acid synthesis are other enzymes that show alterations in cancers. Not much is known about what role DHRS9 may play in cancer or how its expression may be affected. One study found that DHRS9 expression is reduced in cells that possess cancer stem cell (CSC)-like properties in a squamous cell carcinoma line (Geng et al., 2013). ALDH expression is a marker of CSCs and has been used as a predictor of tumor progression and poor patient survival in many cancers, including lung, colon, head and neck , breast, pancreatic, and prostate cancers (Hou et al.,

2014; Liu et al., 2014; Marcato et al., 2011). The resistance to chemotherapy of CSC may be facilitated through increased ALDH activity, as this family of enzymes, in particular

ALDH1A1 and ALDH1A3, are capable of metabolizing chemotherapy drugs (Marcato et

25 al., 2011). Increased expression of ALDH1A1 is associated with increased invasiveness in nasopharyngeal carcinoma, and cells with high levels of ALDH1A1 expression also had higher expressions of stem cell markers (Hou et al., 2014). ALDH1A1 is involved in the maintenance of stemness of lung adenoma stem cells through the suppression of the

Notch pathway (Li et al., 2014). The ALDH isoform responsible for these cancer properties varies and is cancer-specific, as ALDH1A1 and ALDH1A3 are both associated as CSC markers in different cancers (Marcato et al., 2011). Alternatively, decreased

ALDH1A1 expression may contribute to pathogenesis of high-grade serous carcinoma

(Chui et al., 2014). The carcinoma expresses ALDH1A1 to a lesser degree than the normal epithelium and loss of expression occurs in tumor initiating cells, suggesting this early loss may contribute to carcinogenesis (Chui et al., 2014). This finding suggests that

ALDH1A1 may have multiple mechanisms that affect cancer pathology.

A loss of RAR receptors, especially RARB is found in many cancers and have been considered as tumor suppressors (Duong and Rochette-Egly, 2011). Loss of RARB expression through deletion or silencing is associated with many cancers, including lung and breast cancers (Duong and Rochette-Egly, 2011). Loss of RARG predisposes keratinocytes to tumor development and its expression is lost in some head and neck cancers (Duong and Rochette-Egly, 2011). The tumor suppressor effect of RARs may be through suppression of AP-1, which regulates the expression of many genes involved in oncogenesis (Duong and Rochette-Egly, 2011). Conversely, RAR expression is associated with a tumor promoting effect in some cancers (Duong and Rochette-Egly,

2011). RARG expression is increased in human hepatocellular carcinoma and plays a role in cell growth through activation of PI3K and NFKB pathways (Yan et al., 2010).

1.9 SKH-1 Hairless Mouse Model

SKH-1 mice have a mutation in the hairless (Hr) gene and lack hair and pigmentation and are a common model used to study the epidermis (Benavides et al.,

2009). These mice have a normal first round of hair growth and then begin to lose their hair about 2 weeks after birth, resulting in eventual complete hair loss (Benavides et al.,

2009). Because of their lack of hair, this model does not require hair removal techniques that can cause inflammatory responses which may compromise experimental findings

(Benavides et al., 2009). Outbred SKH-1 mice are more commonly used than in-bred

SKH-1 mice and exhibit considerable mouse-to-mouse variation (Benavides et al., 2009).

Hr mice are commonly used to study effects of UV exposure and cSCC, as they are highly prone to UV-induced SCC (Benavides et al., 2009). UVB exerts more adverse and carcinogenic effects than UVA in SKH-1 mice, and the acute responses are comparable to those seen in humans (Benavides et al., 2009; Polefka et al., 2012). The minimal erythemal dose (MED) is the minimum dosage of UV required to induce inflammation in the skin and resulting dorsal epidermal thickening 48 hours after exposure (Benavides et al., 2009). The MED for SKH-1 mice is about 2240 J/m2 and peak inflammatory response is seen around 48 hours post-exposure (Benavides et al.,

2009). Chronic UV exposure induces skin tumors, which are easily identified in these mice (Benavides et al., 2009). The tumors begin as areas of hyperplasia and progress into

27 papillomas and eventually SCC (Benavides et al., 2009). There are three stages of tumor development: initiation, promotion, and progression (Benavides et al., 2009). Initiation of carcinogenesis is irreversible and characterized by mutated keratinocytes that are capable of forming tumors (Benavides et al., 2009). After initiation, promotion is characterized by the development of papillomas by the mutated keratinocytes and is reversible (Benavides et al., 2009). The progression of SCC involves additional genetic transformations that result in a malignant tumor (Benavides et al., 2009). These tumors have a low occurrence of metastasis (Benavides et al., 2009).

Tumors are classified as papillomas (1-3), microinvasive squamous cell carcinomas (MISCC 1-3), and fully invasive SCC (Benavides et al., 2009). Papillomas are the most juvenile stage and have not yet invaded the stroma (Benavides et al., 2009).

These are the only classes of tumors that are considered pre-malignant (Benavides et al.,

2009). The further progression to MISCCs is classified as tumors that have breached the basement membrane and invaded the dermis, often accompanied by an inflammatory response (Benavides et al., 2009). Fully invasive SCC are characterized as having invaded the panniculus carnosus (Benavides et al., 2009).

UV-induced cancerous lesions seen in SKH-1 mice highly resemble those seen in humans, making them a good model for examining the risks and mechanisms of SCC

(Benavides et al., 2009). However, some limitations do exist (Benavides et al., 2009).

SKH-1 mice acquire keratin-filled lesions that often develop into SCC (Benavides et al.,

2009). These lesions are similar to keratoacanthoma found in humans, however, in humans, these often regress and do not lead to SCC, pointing to a discrepancy between

28 the two species (Benavides et al., 2009). Another limitations of outbred SKH-1 mice is the high degree of variation in their genetic backgrounds, which can lead to a high degree of variation when comparing gene and protein expressions (Benavides et al., 2009)

Disparities exist between SKH-1 response to UVA and UVB (Benavides et al.,

2009). While high levels of UVA are capable of inducing cancer, UVA may also exert a protective effect against UVB damage (Benavides et al., 2009). This is an important consideration to keep in mind when examining studies that utilize UVA treatments.

In summary, SKH-1 mice are a commonly used mouse model for studying effects of UV exposure and cSCC, as their skin is readily accessible and they are prone to the development of cSCC. Retinoids are used in the treatment and prevention of many cancers, including cSCC, with limited success. Alterations in the expression of key enzymes in the metabolic pathway occur in cancers that may lead to differences in the effectiveness of these treatments. Here, we examine how acute UVB exposure alters the expression of retinoic acid metabolism proteins and explore the role retinoic acid may play in the repair of UVB damage. We further explore changes in these enzymes at different stages of cSCC. The results will increase our understanding of how retinoic acid metabolism changes in the development and progression of cSCC in SKH-1 mice and may improve prevention and treatment approaches for human use.

Chapter 2: Endogenous Retinoic Acid Required to Maintain the

Epidermis Following Ultraviolet Light Exposure in SKH-1 Hairless

Mice

2.1 Introduction

UVB has a wavelength between 280-315 nm and causes sunburns (erythema)

(Polefka et al., 2012; Sklar et al., 2013). Acute UVB exposure induces an inflammatory response characterized by increased neutrophils, monocytes, COX-2 activity, and PGE 2 levels (Benavides et al., 2009; Wilgus et al., 2003). Long-term exposure to UVB exposure results in: photoaging; immunosuppression; and skin cancers, such as basal cell carcinoma (BCC), squamous cell carcinoma (cSCC), and melanoma (Sklar et al., 2013).

UVB exposure causes: DNA damage; reactive oxygen species (ROS) formation; and induction of p53 (Kastan et al., 1991; Polefka et al., 2012; Sklar et al., 2013). During acute UVB exposure, p53 arrests the cell cycle such that cyclobutane-pyrimidine and thymine dimers can be repaired (Polefka et al., 2012; Sklar et al., 2013). When DNA cannot be repaired, p53 induces apoptosis (Hollstein et al., 1991). Aberrant DNA repair or mutations in p53 that fail to induce apoptosis lead to permanent DNA damage and cancer, especially cSCC (Polefka et al., 2012). UVB exposure also increases proliferation

30 and alters the expression and localization of differentiation markers (Del Bino et al.,

2004; Lee et al., 2002). Altered differentiation peaks 48 hours after UVB and is restored within 3-7 days. Very high doses of UVB also impair barrier function (Haratake et al.,

1997).

Figure 1. Retinoid Compounds. First generation retinoids include retinol, tretinoin, and isotretinoin. Second generation retinoids include etretinate and acitretin.

Retinoids are a class of natural compounds and synthetic drugs derived from vitamin A. Retinoids include: retinol; all-trans retinoic acid (atRA and the drug Retin A);

13-cis RA (isotretinoin); etretinate; and acretin (Figure 1). Exogenous retinoids produce variable effects on cSCC due to retinoid resistance. Both oral and topical RA reduce cSCC in the chemical carcinogenesis mouse model where 7,12-dimethylbenz[ a]anthracene (DMBA) initiates and 12- O-tetradecanoylphorbol-13-acetate (TPA) promotes tumorigenesis (Chen et al., 1994; Verma et al., 1979). These chemicals cause defects in the RAS signaling pathway that account for only 21% of cSCC in humans

(Ratushny et al., 2012). Retinoid treatments produced variable effects in the photocarcinogenesis model of cSCC using UVB or UVA&B treated hairless mice.

Topical RA accelerated (Halliday et al., 2000), inhibited (Kligman and Crosby, 1996), or had no effect (Kligman and Kligman, 1981) on photocarcinogenesis. These differential effects could be due to either the dose of RA or differences in the background strain.

Both oral retinol and etretinate also failed to alter photocarcinogenesis when given at two high doses (Kelly et al., 1989). In humans, low doses of oral retinoids prevent the reoccurrence of carcinomas in high-risk patients; but significant prevention by oral retinoids only occurs for a few years and relapse occurs upon stopping treatment

(Brewster et al., 2007; Harwood et al., 2005; Kadakia et al., 2012; Wu and Stern, 2012).

Topical RA failed to prevent carcinogenesis in humans (Stüttgen, 1986; Weinstock et al.,

2012; Weinstock et al., 2009). Therefore RA has the potential for chemoprevention; but something is blocking RA’s effectiveness.

Regulation of retinoid metabolism maintains precise levels of RA. Retinol enters the cell by either passive diffusion or the transport protein referred to as stimulated by

RA6 (STRA6) (Kawaguchi et al., 2007), and binds cellular retinol binding protein

(RBP1) within the cell (Ong, 1994). The majority of retinol that enters the keratinocyte is stored by the action of lectin:retinol acyltransferase (LRAT) (Kurlandsky et al., 1994;

MacDonald and Ong, 1988). The remaining retinol is reversibly oxidized to retinal by retinol dehydrogenases (Rexer and Ong, 2002; Wu et al., 2002). Retinal is subsequently oxidized to RA by retinal dehydrogenases 1-3 (ALDH1A1, 2, and 3) (Napoli, 1999). RA then binds either Cellular RA binding protein 1 or 2 (CRABP1, CRABP2). CRABP1 directs RA to degradation enzymes cytochrome P450 26A1, B1, and C1 (CYP26A1, B1, and C1) (Boylan and Gudas, 1992). In contrast, CRABP2 protects RA from degradation by CYP26 enzymes and directs the transport of cytosolic RA to the nucleus where it binds to its receptors (RARA, RARB and RARG) (Dong et al., 1999). These receptors are RA inducible transcription factors of the nuclear hormone family (Chambon, 1996;

Petkovich et al., 1987) that regulate the expression of >500 genes involved in: differentiation; cell cycle control; and apoptosis (Balmer and Blomhoff, 2002). RA also regulates its own metabolism and function by directly inducing LRAT, CRABP2,

CYP26A1, and RARB (de The et al., 1989; Giguère et al., 1990; Kurlandsky et al., 1996;

Matsuura and Ross, 1993; Pavez Loriè et al., 2009a; Pavez Loriè et al., 2009b; Pavez

Loriè et al., 2009c; Reijntjes et al., 2005).

UVA and UVB cause retinol and retinyl esters to degrade, leading to reduced concentration of these retinoids in the skin (Sorg et al., 2002; Sorg et al., 1999; Takeda et

33 al., 2003). In vitro, UVA/UVB causes retinoids to photoexcite and form reactive oxygen species (ROS) (Fu et al., 2007). But these effects are not seen in vivo. In cultured keratinocytes, UVB exposure also increases dehydroretinol synthesis (Tafrova et al.,

2012). Thus, UVB exposure alters the ratio of retinol to dehydroretinol and RA to dehydroRA. UVB and UVA also change the skin expression of retinoid metabolism proteins. UVA treatment decreases RXRA, increases B-carotene dioxygenase activity, and increases CRBP1 (Sorg et al., 2002; Takeda et al., 2003). Sun exposed skin and the precursor lesion actinic keratosis (AK) express higher levels of CYP26A1 (Osanai and

Lee, 2011). SCC cell lines contain reduced retinyl esters and LRAT activity (Guo and

Gudas, 1998; Guo et al., 2000; Jurukovski and Simon, 1999). Patients with cSCC express less message levels of Rara, Rarg, and Rarb1’ than patients with basal cell carcinoma

(Hartmann et al., 2010). To better understand retinoid resistance in UVB induced cSCC, we examined the effects of acute UVB exposure on key retinoid metabolism proteins. In the stratum granulosum, acute UVB STRA6, CRBP1, DHRS9, ALDH1A2, and increased

RARA in the stratum corneum.

To better understand the role of this UVB-induced RA, we inhibited RA synthesis immediately after UVB exposure. Reducing RA damaged the suprabasal epidermis in a dose dependent manner. These results suggest that UVB-induced RA is important for the repair process after acute UVB exposure. In addition, some of these changes in retinoid metabolism and signaling proteins may contribute to retinoid resistance well before cancers arise in the UVB exposed skin.

2.2 Materials and Methods

Female SKH-1 Hairless (Hr) mice were exposed to or not exposed to one minimal erythemic dose of UVB (2240 J/m2) and treated with acetone, 1.5 mg/g BW disulfiram, or 2 mg/g BW disulfiram. Mice were sacrificed 48 hours post-treatment and dorsal skin samples were collected and fixed overnight, embedded in paraffin and sectioned at 5-6

μm, and placed on microscope slides. The Ohio State University Institutional Animal

Care and Use Committee approved all procedures*.

Immunohistochemistry (IHC) was performed on the skin samples using antibodies directed against STRA6, CRBP1, LRAT**, DHRS9, ALDH1A1, ALDH1A2,

CRABP2**, RARA, CYP26A1**, loricrin (LOR), and keratin 6 (KRT6). IHC in the epidermis and hair follicle remnants was scored blinded on a 4-point scale for both intensity (negative (0), weak (1), moderate (2), strong (3), or very strong (4)) and percent of cells (0 = 0, 1-25 = 1, 26-50 = 2, 51-75 = 3, 76-100 = 4). An IHC level (IHCL) was calculated as the sum of intensity plus percent of cells on a scale of 0-8. Lesions and abnormally thin areas of the epidermis were scored on a similar scale of 0-8 for IHCL.

The epidermis was grouped as upper epidermis (stratum corneum and stratum granulosum) and lower epidermis (stratum spinosum and stratum basale).

Mann Whitney tests and independent T-tests were performed with IBM SPSS software. A p value of less than 0.05 was considered significant.

* Mouse experiment performed by F Jason Duncan

** IHC run by Dr. Everts

2.3 Results

2.31 Altered expression of retinoid metabolism proteins in the upper epidermis and hair follicle remnants 48 hours after UVB treatment

The expression of key enzymes in the retinoic acid pathway were examined via

IHC and semi-quantitatively scored to generate an IHCL. In the upper epidermis, consisting of the stratum corneum and stratum granulosum layers, UVB significantly decreased the expression of LRAT * and increased the expression of STRA6, CRBP1,

DHRS9, ALDH1A2, CRABP2*, and CYP26A1* (Figure 2 a-d, data not shown). These proteins localized in the upper region of the stratum granulosum, closer to the stratum corneum. RARA was significantly increased in the corneum (Figure 2e). The expressions of DHRS9 and ALDH1A1 decrease and CRBP1 increases in the infundibulum and

DHRS9 significantly decreases in the bulge following UVB treatment (data not shown).

* Results obtained by Dr. Everts

Figure 2. RA metabolism proteins increase expression in the upper epidermis following UVB treatment. Immunohistochemistry was performed using antibodies against STRA6 (a), CRBP1 (b), DHRS9 (c), ALDH1A2 (d), and RARA (e) and scored on a scale of 0-8. The expression of all proteinsproteins increased in the upper epidermis 48 hours after UVB treatment. * indicates significant p values (p<0.(p<0.05).05). Bar = 25.15 μM. Arrows point to the infundibulum (inf) and stem cells (sc).

2.32 Reduced retinoic acid synthesis damaged the upper epidermis after UVB treatment

To better understand how these alterations in the retinoic acid pathway may be affecting the UVB repair process, we examined the expression of LOR and KRT6 via

IHC in UVB and disulfiram treated samples.

The Following paragraph was written by Drs. Helen Everts and Krista La Perle and is included as a detailed explanation of the pathological changes we observed:

Disulfiram treatment caused focal clefting of the epidermis between the spinous and granular layers in a dose dependent manner (Figure 3 a-d, green arrows). Other areas of the epidermis were reduced to a single layer of keratinocytes. Disulfiram significantly reduced skin fold thickness and the minimal epidermal thickness (Figure 3 e-f). The minimal number of keratinocyte layers ranged from 1-5, but the average of all 6 sites only trended to decrease with disulfiram (p = 0.074, data not shown). Disulfiram also did not alter the maximum epidermal thickness or number of cell layers, which ranged from

2-8 (data not shown). UVB treatment significantly increased all measures of epidermal thickness and cell layers (p < 0.01, Figure 3 e-f and data not shown). UVB treatment also significantly increased clefting between the basal layer and the dermis (Figure 3g, arrowhead) and intracellular edema (p < 0.001); but disulfiram did not alter either of these lesions (data not shown). Disulfiram also dose dependently increased the number of hypereosinophilic keratinocytes with pyknotic nuclei (Figure 3 g, black arrow, i). Both doses of disulfiram caused clefting with retention of viable keratinocytes subjacent to the stratum corneum (Figure 3 g-h, blue arrow).

LOR expression was assessed as a marker of terminal differentiation and KRT6 was assessed as a marker of hyperplasia. UVB significantly increased LOR and KRT6 expression in the stratum spinosum (Figure 4c and 5c) and stratum basale (Figure 4d and

5d), and KRT6 expression in the stratum granulosum (Figure 5b). Disulfiram treatments significantly increased LOR in the stratum corneum (Figure 4a). LOR expression in the stratum corneum significantly increased with disulfiram treatment. However, the areas of the stratum corneum that retained live cells had less LOR expression than the stratum corneum and stratum granulosum (Figure 6 a and b). In the non-UVB treated samples, disulfiram trended to decrease KRT6 expression in the thin areas of epidermis (p=0.06), however disulfiram did not change KRT6 expression in these areas in UVB treated samples (Figure 6c).

Figure 3. Reducing RA synthesis damages the epidermis. Clefting (green arrows) between the spinous and granular layers was scored as 1 (a), 2 (b) or 3 (c) and a 6-point scale was developed based on the amount of involvement (d). Skin fold thickness was measured on whole mice (e), while epidermal thickness was measured from photomicrographs (f). Retention of viable keratinocytes associated with clefts (g blue arrow) were scored on a 3-point scale based on skin involvement (h). Pyknotic keratinocytes (g black arrow) were counted (i). Arrowhead, sub-basilar clefts. Bar = 10.1 μM. *P < 0.05, **P < 0.005 disulfiram verses control within UVB treatment group. #P < 0.001 all UVB treatment groups verses control groups.

Figure 4. LOR IHCL changes with disulfiram and UVB treatments. IHC was performed using an antibody against LOR and scored on a scale of 0-8 in the stratum corneum (a), stratum granulosum (b), stratum spinosum (c), and stratum basal (d). Effect of disulfiram: * p<0.05, ** p<0.005, *** p<0.001; Effect of UVB: # p<0.05, ## p<0.005, ### p<0.001.

Figure 5. KRT6 IHCL changes with disulfiram and UVB treatments. IHC was performed using an antibody against KRT6 and scored on a scale of 0-8 in the stratum corneum (a), stratum granulosum (b), stratum spinosum (c), and stratum basal (d). Effect of disulfiram: * p<0.05, ** p<0.005, *** p<0.001; Effect of UVB: # p<0.05, ## p<0.005, ### p<0.001.

Figure 6. LOR and KRT6 IHCL changes in the lesions and thin epidermis. IHCL was performed using an antibody against LOR and scored on a scale of 0-8 and its expression in the lesion was compared to the normal stratum cornuem (a) and stratum granulosum (b). IHC was run using an antibody against KRT6 in the areas of the thin epidermis for disulfiram treated samples and scored on a scale of 0- 8 (c). *p<0.05, #P < 0.001 all UVB treatment groups verses control groups.

2.4 Discussion

This study aimed to explore the role endogenous retinoic acid plays in the epidermis following UVB exposure. We found that 48 hours following UVB exposure the expression of all proteins examined increased in the upper epidermis except

ALDH1A1. UVB exposure also decreased the expression of DHRS9 and ALDH1A1 in the infundibulum and DHRS9 in the bulge stem cells. Inhibiting retinoic acid synthesis with disulfiram treatments caused damage to the upper epidermis, suggesting that endogenous retinoic acid plays an important role in the UVB repair process.

No earlier studies, to our knowledge, have examined the entire retinoid pathway following UVB exposure. Previously published data found that CRBP1 expression increased in the epidermis upon UVA treatment (Sorg et al., 2002). The same group found a slight decrease in CRBP1 expression with UVB treatment (Sorg et al., 1999).

These findings, however, used homogenized epidermis and protein extractions, which are less sensitive and do not account for the differences in expression between the skin layers. Here, we used IHC and were able to separately score the epidermal layers. We found that the expression of CRBP1 in the upper epidermis and infundibulum was increased in UVB-treated mice.

There are many interactions among the key enzymes involved in retinoic acid synthesis. We show that UVB exposure changes the expression of many of these, and by examining the whole pathway we are able to get a good idea of how the changes in these proteins effect the flux of the pathway. CRBP1 increased expression and directly interacts with STRA6, LRAT, and DHRS9 (Berry et al., 2012b; Napoli, 1999). These proteins also changed expression in the upper epidermis 48 hours after UVB treatment:

STRA6 and DHRS9 expression increased and LRAT expression decreased (Gressel et al., 2015). A decrease in LRAT would reduce the rate of retinol storage, while increases in STRA6, CRBP1, DHRS9, ALDH1A2, and RARA increase the influx, oxidation, and signaling of retinol (Berry et al., 2012b; Napoli, 1999). The results obtained by Dr. Everts that show CRABP2 and CYP26A1 also increase in the upper epidermis further support the increase in retinoic acid synthesis, as these enzymes are directly inducible by retinoic acid (Blomhoff and Blomhoff, 2006).

Retinoic acid regulates epidermal proliferation, differentiation, and apoptosis

(Eckert and Rorke, 1989). The damage accompanied by the inhibition of its synthesis with disulfiram treatment demonstrates its role in regulating these processes after UVB exposure. Higher doses of disulfiram reduced the upper layers of the epidermis, an effect

43 similar to that seen in dominant negative RARA mice in the epidermis (Saitou et al.,

1995). Our results expand on the role retinoic acid plays in skin maintenance and demonstrate its additional role in epidermal recovery after UVB exposure.

To investigate how retinoic acid may be regulating epidermal recovery from UVB exposure, we examined markers of terminal differentiation and hyperplasia, LOR and

KRT6 respectively (Coulombe, 2003; Eckhart et al., 2013). LOR expression was decreased in the viable cells found in the corneum, suggesting that these cells are not terminally differentiated. Similar retention of viable cells was also seen in a CYB26B1 null mice in which the mice had defective epidermal barrier formation during development (Okano et al., 2012). This demonstrates that precise levels of retinoic acid synthesis and degradation are required for normal skin development, both in utero and in response to damage. Disulfiram treatment increased LOR expression in the corneum, which is consistent with previous studies showing that retinoic acid acts to decrease differentiation (Bernard et al., 2002; Törmä, 2011; Törmä et al., 2014). Disulfiram also increased KRT6 expression in the stratum granulosum and spinosum, suggesting increased cellular proliferation in these cell layers.

While KRT6 is commonly used as a marker of hyperplasia, it may also be associated with impaired barrier function (Imakado et al., 1995). In RARA dominant- negative mice that exhibited a loss of barrier function, KRT6 expression increased independent of increased proliferation (Imakado et al., 1995). In the thin areas of the epidermis, disulfiram treatment alone decreased KRT6 expression, but when paired with

UVB treatment, this decrease in expression did not occur. This is unexpected, as retinoic

44 acid increases KRT6 expression. This suggests that the mechanisms inducing its expression in response to barrier malformation are more potent than retinoic acid’s influence. However, further experiments are required to confirm that barrier function was disrupted in these areas.

In summary, our results indicate UVB exposure causes significant changes in enzymes involved in retinoic acid metabolism. Retinoic acid is a key regulator of differentiation, proliferation, and barrier function in the skin, and its synthesis may be vital for appropriate repair to occur. More studies are needed to elucidate the exact role retinoids play in the repair of UVB in regards to the regulation of differentiation and barrier function.

Chapter 3: Alterations in Endogenous Retinoid Metabolism in the

Progression of Cutaneous Squamous Cell Carcinoma

3.1 Introduction

Cutaneous Squamous Cell Carcinoma (cSCC) is the second most prevalent non- melanoma skin cancer and is most commonly found in sun-exposed regions of the body

(Lansbury et al., 2013; Parikh et al., 2014). Exposure to UV light is the biggest risk factor for cSCC and organ transplantation patients are at an increased the risk for this cancer, due to their immunosuppressed state (Jones et al., 2004). cSCC is usually treated by surgical removal of the cancerous lesions, however this can sometimes cause cosmetic and functional impairments (Lansbury et al., 2013; Parikh et al., 2014). This type of cancer has a low rate of metastasis, similar to that of renal carcinomas, but has high rates of morbidity and mortality when metastasis does occur (Parikh et al., 2014).

SKH-1 hairless mice are commonly used to study effects of UV exposure and cSCC, as they are highly prone to UV-induced SCC (Benavides et al., 2009). There are three stages of tumor development: initiation, promotion, and progression (Benavides et

46 al., 2009). Initiation of carcinogenesis is irreversible and characterized by mutated keratinocytes that are capable of forming tumors (Benavides et al., 2009). After initiation, promotion is characterized by the development of papillomas by the mutated keratinocytes and is reversible (Benavides et al., 2009). The progression of SCC involves additional genetic transformations that result in a malignant tumor (Benavides et al.,

2009). These tumors have a low occurrence of metastasis (Benavides et al., 2009).

These are the only classes of tumors that are considered pre-malignant (Benavides et al.,

2009). The further progression to MISCCs is classified as tumors that have breached the basement membrane and invaded the dermis, often accompanied by an inflammatory response (Benavides et al., 2009). Fully invasive cSCC is characterized by the migration of cancer cells into the panniculus carnosus, and has increased risk of recurrence and metastasis (Lansbury et al., 2013).

Retinoids are thought to have cancer-preventing effects, as they are involved in the regulation of cell growth, apoptosis, differentiation, and immune function (Altucci and Gronemeyer, 2001; Zito et al., 2014); Altered expression of RARs are seen in many skin cancers, including cSCC (Altucci et al., 2007; Jones et al., 2004). Retinoids have been used to successfully treat and prevent some cancers, including squamous cell carcinomas (Hartmann et al., 2010; Jones et al., 2004; Marcato et al., 2011). Low-dose systematic retinoids (0.2 to 0.4 mg/kg of acitretin per day) reduced the incidence of cSCC

47 in transplant patients (Harwood et al., 2005). Patients who stopped taking the treatment had a significant increase in the risk of cSCC (Harwood et al., 2005). At-retinoic acid has been used to treat cancers such as acute promyelocytic leukemia (APL) because of its ability to induce apoptosis and differentiation of cancer cells, including cancer stem cells

(CSC) (Ginestier et al., 2009; Huang et al., 1988; Marcato et al., 2011; Zito et al., 2014).

13-cis-retinoic acid (13cRA/isotetinoin) (1 mg/kg/d orally) and interferon alfa (IFNa) (3 x 106 U subcutaneously three times per week) and showed no improvement in recurrence rate (Brewster et al., 2007). While retinoid treatments have been successful in many studies, their applications are limited and the mechanisms of action and which retinoid treatments are most effective remain unclear.

Elevated expression of STRA6, along with its ligand RBP, is seen in some cancers, including breast and colon cancers (Berry et al., 2014; Szeto et al., 2001). The increased expression of these proteins contributes to carcinogenesis, which is mediated through the activation of STAT3 via STRA6-JAK2 activation (Berry et al., 2014). STAT3, along with other STAT proteins, are highly activated in many cancers and promote oncogenic properties including cell survival and proliferation (Bromberg and Darnell, 2000). Berry

48 et al. show that STRA6 is upregulated early in breast cancer formation and its expression continues to increase as the cancer progresses (Berry et al., 2014). However, STRA6 expression may be involved in the initiation of apoptosis in cancer cells (Mittal et al.,

2014). Fenretinide, a synthetic retinoid, was used to treat endometrial cancer cells in vitro and induced apoptosis independent of the retinoic acid signaling pathway. This increase in apoptosis may be mediated by influx of retinol via STRA6, as STRA6 silencing reversed this effect (Mittal et al., 2014). This suggests that a specific level of STRA6 expression may be required to initiate apoptosis without extraneous activation of STAT signaling.

2014; Liu et al., 2014; Marcato et al., 2011). The resistance to chemotherapy of CSC may be facilitated through increased ALDH activity, as this family of enzymes, in particular

ALDH1A1, are capable of metabolizing chemotherapy drugs (Marcato et al., 2011).

Increased expression of ALDH1A1 is associated with increased invasiveness in nasopharyngeal carcinoma, and cells with high levels of ALDH1A1 expression also had higher expressions of stem cell markers (Hou et al., 2014). ALDH1A1 is involved in the

49 maintenance of stemness of lung adenoma stem cells through the suppression of the

Notch pathway (Li et al., 2014). The ALDH isoform responsible for these cancer properties varies and is cancer-specific (Marcato et al., 2011). Alternatively, decreased

ALDH1A1 expression may contribute to pathogenesis of high-grade serous carcinoma

ALDH1A1 may have multiple mechanisms that affect cancer pathology.

While the precise changes in endogenous retinoid levels that occur in SCC are not clear, it is evident that alterations in the retinoid pathway occur in the formation and progression of this and other cancers. Here we show that STRA6, DHRS9, and

ALDH1A1 have altered expressions in various tumor stages throughout the progression of SCC. These changes may be affecting the levels of retinoic acid synthesis and signaling, contributing to aberrant regulation of key oncogenic processes such as proliferation, differentiation, and apoptosis.

3.2 Materials and Methods

Tumor samples were obtained from Dr. Oberyszyn’s lab from female SKH-1 mice that were exposed to one minimal erythemic dose of UVB (2240 J/m2) UVB light three times a week for 25 weeks and then sacrificed.

Lesions greater than 1mm in size were harvested and staged by a pathologist using hematoxyliln and eosin (H&E)-stained sections of tumors. All stages (papillomas

(Pap 1-3), micro-invasive squamous cell carcinoma (MISCC 1-3), and squamous cell carcinoma (SCC)) were collected and analyzed as dictated below.

IHC was performed on tumor samples using antibodies against STRA6, DHRS9, and ALDH1A1 and scored semi-quantitatively using the guidelines described in chapter

2: A scale of 0-8 based on percent cells (0-4) and intensity (0-4). For this study, we included an additional category of 0.2 for percent cells, indicating a percentage of 0-5% cells. We scored the following areas: differentiated and undifferentiated areas of the tumor, uninvolved epidermis (SC/SG and SS/SB). Tumors scored for DHRS9 and

ALDH1A1 were also given an overall score of 0-3 (0=absent; 1=weak; 2=moderate;

3=strong).

ANOVA statistical tests were performed using the above criteria. The tumor data was run both grouped into specific stages and general stages using IBM SPSS statistical software. To examine how the protein expression changed as cSCC progresses, IHCL was compared among the tumor stages. To examine how different amounts of UVB affect the protein expressions we compared the IHCL of non-exposed and acutely exposed epidermis, chronically exposed uninvolved epidermis, and tumor cells. We compared undifferentiated tumor cells to the uninvolved basal cells and lower epidermis

(SB/SS) of non-exposed and acutely exposed skin, as well as the differentiated tumor cells to the uninvolved suprabasal cells and upper epidermis (SG/SC) of non-exposed and acutely exposed skin. Tumor IHCL was converted to the scale used for the acute UVB study (no 0-5% category, see chapter 2).

3.3 Results

3.31 Retinoid Metabolism proteins change expression throughout the progression of cSCC

STRA6 expression significantly decreased with tumor progression in the uninvolved basal and suprabasal cells, differentiated tumor cells, and undifferentiated tumor cells (Figure 7 a-f, Table 1-4- appendix). STRA6 expression increased in the tumor corneum as the cancer progressed (Table 1-4- appendix).

Figure 7. STRA6 IHCL changes during the progression of cSCC. IHC was performed using an antibody against STRA6 for papilloma (a), micro-invasive SCC (b), and fully-invasive SCC (c). IHCL was scored on a scale of 0-8 in the differentiated tumor cells (d), and undifferentiated tumor cells (e/f). Different letters and * indicate significant (p<0.05) differences. Bar = 25.15 μM. Arrows point to undifferentiated tumor cells (Undif).

DHRS9 was significantly decreased in cSCC for the overall tumor, uninvolved suprabasal cells, outside corneum of the tumor, and undifferentiated tumor cells (Figure 8 d-f, Table 5-8-appendix). But there were significant increases in expression at the pap3 and MISCC2 stages in the uninvolved suprabasal and outer corneum (Figure 8e and 8f).

We also observed DHRS9 had strong focal (Figure 9a) and nuclear expression (Figure

9b) within some tumors, but we did not quantify this observation.

Figure 8. DHRS9 IHCL changes during the progression of cSCC. IHC was performed using an antibody against DHRS9 and scored on a scale of 0- 8. IHCL significantly changed in the uninvolved suprabasal cells(a), outer corneum (b), and undifferentiated tumor cells (c). DHRS9 expression in PAP (d), MISCC (e), and SCC (f). Different letters and * indicate significant (p<0.05) differences. Bar = 25.15 μM. Arrows point to undifferentiated tumor cells (Undif).

Figure 9. Focal and nuclear DHRS9 expression. DHRS9 was expressed focally (a) and in the nucleus (b) in some stages of cSCC. Bar = 25.15 μM.

ALDH1A1 expression significantly decreases in the overall tumor and outer corneum between the papilloma and MISCC stages when grouped generally (Figure 10c,

Tables 9-12- appendix). ALDH1A1 expression significantly decreases in undifferentiated tumor cells in MISCC and cSCC stages compared to papillomas (Figure 10a and 10b,

Tables 9-12- appendix) and in differentiated tumor cells in cSCC (Tables 9-12- appendix). There is a significant increase in ALDH1A1 expression at MISCC1 and a significant decrease in expression in MISCC3 in the uninvolved basal cells (Tables 9-12- appendix). ALDH1A1 expression was also observed around blood vessels and in immune cells surrounding the tumors.

Figure 10. ALDH1A1 expression changes throughout the progression of cSCC. IHC was performed using an antibody against ALDH1A1 for all tumor stages and scored on a scale of 0-8: Pap1 (a) Pap2 (b) Pap3 (c), MISCC 1 (d) MISCC2 (e), MISCC3 (f), and SCC (g). ALDH1A1 expression is significantly decreased in the later stages of cSCC in the undifferentiated tumor cells (a/b) and in the outer corneum (c). Different letters and * indicate significant (p<0.05) differences. Bar = 25.15 μM. Arrows point to undifferentiated tumor cells (Undif) and outer corneum (corn).

3.32 Retinoid metabolism proteins change expressions between no exposure, acute UVB exposure, and in cSCC and surrounding epidermis.

Differentiated and undifferentiated tumor cell IHCL was compared to the basal and suprabasal cells of the uninvolved skin, acutely exposed UVB skin, and non- exposurd skin. STRA6 expression was higher in unexposed basal cells compared to uninvolved basal cells and undifferentiated tumor cells (Figure 11a).STRA6 expression was significantly lower in the non-exposed suprabasal cells compared to all other suprabasal and differentiated cells exposed to UVB (Figure 11b). Differentiated and undifferentiated tumor cells had significantly lower expression of STRA6 than acutely exposed epidermis (Figure 11 a and b).

DHRS9 expression is decreased in the non and acutely exposed epidermis and of the undifferentiated tumor cells than the noninvolved basal cells (Figure 11c). DHRS9 expression is lower the uninvolved suprabasal cells compared to the suprabasal cells exposed to acute UVB (Figure 11d). As discussed in Chapter 2, DHRS9 expression was significantly higher in the suprabasal cells after acute UVB exposure (Figure 11d).

The only significant alteration of ALDH1A1 expression was a decreased expression in the differentiated tumor cells compared to the uninvolved suprabasal cells

(data not shown). This was only significant when comparing IHCLs taken for the representative intensity of the tumor.

Figure 11. Comparison of undifferentiated and differentiated epidermis and tumors with varying UVB exposures. IHC was performed using antibodies against STRA6 and DHRS9 and IHCL was scored on a scale of 0-8. IHCL for non-exposed epidermis, acutely exposed epidermis, uninvolved epidermis, and tumors were compared for STRA6 IHCL in the unidfferentiate cells (a) and differentiated cells (b) and for DHRS9 in the undifferentiated cells (c) and differentiated cells (d). Different letters indicate significant differences.

3.4 Discussion

Retinoids are used in the treatment of many cancers, including cSCC, with mixed effectiveness (Brewster et al., 2007; Harwood et al., 2005; Jones et al., 2004).

Additionally, the expression of some retinoid metabolism enzymes are altered in some carcinomas, including oral cavity SCC, breast, and colon cancers (Berry et al., 2014; Guo and Gudas, 1998). While the precise changes in endogenous retinoid levels that occur in cSCC are not clear, it is evident that alterations in the retinoid pathway occur in the formation and progression of this and other cancers. Here we show that STRA6, DHRS9, and ALDH1A1 have altered expressions in various tumor stages throughout the progression of cSCC in SKH-1 mice. These changes may be affecting the levels of retinoic acid synthesis and signaling, contributing to aberrant regulation of key oncogenic processes such as proliferation, differentiation, and apoptosis.

We show that DHRS9 decreases expression in the uninvolved epidermis and undifferentiated tumor cells in MISCC and SCC compared to the papillomas. We currently have little knowledge of the role DHRS9 may play in cancer formation and progression. Its expression was decreased in CSC-like cells in SCC, suggesting that the loss of DHRS9 may contribute to CSC properties (Geng et al., 2013). The decrease in its expression may contribute to a decrease in retinoic acid synthesis, and therefore may decrease differentiation and apoptosis, and increase proliferation (Eckert and Rorke,

1989). We also noted nuclear expression of DHRS9 in some tumors. DHRS9 may be acting as a transcriptional repressor in the nucleus, which may influence the many genetic changes that occur in cSCC (Markova et al., 2006).

We further show that DHRS9 expression is decreased in chronically exposed undifferentiated, uninvolved epidermis compared to non-exposed and acutely exposed epidermis and cSCC tumors. In differentiated cells, DHRS9 is significantly increased with acute UVB, which decreases expression in the tumors and uninvolved epidermis.

This suggests that chronic UVB exposure alters DHRS9 expression, which is regained at the tumor stages. Further experiments examining chronic UVB on DHRS9 expression are needed to see how this expression change progresses, and whether this occurs in chronically exposed epidermis that is not directly near the tumors.

ALDH expression is implicated in the pathology of many carcinomas. High

ALDH expression is commonly used as a marker of cancer stem cells (CSC) and as a prognostic tool for progression and poor survival in some cancers (Hou et al., 2014; Liu et al., 2014; Marcato et al., 2011). However, not all carcinomas exhibit this increase in

ALDH1A1 expression, and in fact, a decrease in its expression may occur in the initiation of some cancers such as high grade serous carcinoma (Chui et al., 2014). In the development of this cancer, tumor initiating cells and serous tubal intraepithelial carcinoma, precursors to high-grade serous carcinoma, lack ALDH1A1 expression and the loss of expression may be an important early event in carcinogenesis (Chui et al.,

2014). Here, we show that ALDH1A1 expression significantly decreases at certain stages of tumor progression. While ALDH1A1 expression increases in many cancers and has been implicated in metastasis and increased malignancy (Hou et al., 2014; Liu et al.,

2014; Yang et al., 2014), we show that ALDH1A1 expression in the undifferentiated tumor cells and corneum decrease in the MISCC and cSCC stages compared to the

60 papillomas. This is similar to the decrease in ALDH1A1 expression seen in high-grade serous carcinoma and suggests that a decrease in ALDH1A1 may be contributing to malignancy, as papillomas are the only stages considered pre-malignant (Benavides et al.,

2009; Chui et al., 2014). Our results also suggest that ALDH1A1 may not be a good CSC or prognostic marker for cSCC. More experiments need to be done that examine

ALDH1A1 co-localization with other CSC markers, as well as examine other ALDH enzymes, as ALDH1A1 is not the only isoform known to possess these qualities (Marcato et al., 2011).

We also found that while ALDH1A1 expression does not vary in the undifferentiated cells across the differing UVB exposures, suprabasal cells of the uninvolved epidermis have significantly increased ALDH1A1 expression compared to the normal and acutely exposed suprabasal cells, and expression is decreased in the tumor compared to the epidermis of all three UVB exposures. This suggests that chronic UVB may increase the expression of ALDH1A1, which is then decreased in the cSCC tumors.

This suggests that decreased expression of ALDH1A1 distinguishes chronically exposed epidermis from tumors in cSCC.

Changes in STRA6 expression occur in some cancers, including breast and colon cancers (Berry et al., 2014). Here, we show that STRA6 increases expression in the stratum corneum in cSCC compared with the papillomas. STRA6 expression decreases in the undifferentiated and differentiated tumor cells in MISCC and cSCC compared with the papillomas. While increased STRA6 expression contributes to carcinogenesis in some cancers, its expression may also be needed to induce apoptosis in cancerous cells (Berry

61 et al., 2014; Mittal et al., 2014; Szeto et al., 2001). We see a decrease in STRA6 expression in the malignant-staged tumors. This decrease may be hindering the ability to induce apoptosis and differentiation, both of which are shown to reduce malignancy, and are processes that STRA6 plays a role in (Carrera et al., 2013; Ginestier et al., 2009;

Huang et al., 1988; Mittal et al., 2014; Muenzner et al., 2013).

We further show that STRA6 expression in undifferentiated cells is decreased with chronic UVB exposure, in both the uninvolved epidermis and in the tumors, compared to non-exposed and acutely exposed epidermis. This decreased expression may contribute to cancer formation by reducing apoptosis and differentiation (Mittal et al.,

2014; Muenzner et al., 2013). Similarly to DHRS9 and ALDH1A1, future experiments should examine chronically UVB-exposed epidermis that is not near the tumors to decipher how STRA6 expression may be contributing to tumorgenesis.

Taken together, we see fluctuations in DHRS9, ALDH1A1, and STRA6 expression throughout the progression of cSCC. Many areas of the tumors decrease expression of all three enzymes in the MISCC and SCC stages. This indicates that endogenous retinoic acid synthesis may be changing throughout this progression, with decreased levels in the more advanced and malignant stages. The expression of these proteins is different in epidermal samples exposed to different amounts of UVB light, as well as in the tumors, suggesting that the expression changes may contribute to tumor formation, as well as tumor progression and malignancy.This may be why retinoid treatments for cSCC have had some success (Jones et al., 2004). However, we need to

62 examine more key enzymes in the pathway to get a better picture of how the entire pathway is changing.

Chapter 4: Epilogue

Cutaneous Squamous Cell Carcinoma (cSCC) is the second most prevalent non- melanoma skin cancer and is most commonly found in sun-exposed regions of the body

(Lansbury et al., 2013; Parikh et al., 2014). Exposure to UV light is the biggest risk factor for cSCC and organ transplantation patients are at an increased the risk for this cancer, due to their immunosuppressed state (Dupont et al., 2013; Jones et al., 2004). Retinoids are thought to have cancer preventing affects, as they are involved in the regulation of cell growth, apoptosis, differentiation, and immune function and altered expression of the retinoic acid nuclear receptors (RARs) and pathway enzymes are seen in many skin cancers, including cSCC (Jones et al., 2004). However, the changes in endogenous retinoid metabolism proteins is not known for cSCC and the mechanism by which endogenous retinoic acid affects cancer development remains allusive.

Here, we investigate how acute UVB exposure affects endogenous retinoids. We found that UVB increases the expression of retinoic acid metabolism proteins in the upper epidermis, suggesting the increased synthesis of retinoic acid, which we found is critical for normal epidermal repair. We also suggest that KRT6 expression is induced with UVB exposure in abnormally thin regions of the epidermis as a result of improper

64 barrier function, however, we need to examine hyperproliferation using a different marker, such as Ki-67, and examine the integrity of the barrier, perhaps looking at lipid components using Nile red staining. As UVB is the largest risk factor for cSCC and changes retinoic acid synthesis, examination of KRT19 expression may also be of interest. Its expression is induced by retinoic acid and it is only expressed in the epidermis following malignant transformation (Kim et al., 1984; Kopan et al., 1987; Wu and Rheinwald, 1981). Our findings of the expression changes and role of retinoic acid in UVB repair may be helpful in the development of cancer prevention treatments, and may provide insight as to what changes occur in this pathway as SCC develops.

To compare how the retinoic acid pathway is affected in cSCC, we examined the expression of key enzymes involved in the pathway in all tumor stages. We found that key retinoid metabolism proteins change expression throughout the progression of cSCC.

These results increase our knowledge of alterations of the endogenous retinoic acid pathway in the development and progression of cSCC in SKH-1 mice. We see that

STRA6, DHRS9, and ALDH1A1 expression is altered in different tumor stages, and decrease in later stages of SCC. While more proteins, such as CRBP1, LRAT, CRABP2, and the RARs, should be examined to gain a more complete understanding of the entire pathway, the expression changes seen here suggest that retinoic acid synthesis may be reduced in cSCC. This could have important implications for prevention and treatment approaches and may help explain why retinoid treatments have been effective. The expression changes should also be examined in humans to investigate whether this is an alteration that is of importance for our species.

The results from both the acute UVB and cSCC studies give us a better understanding of the changes in endogenous retinoids that occur in these states. We also discovered that endogenous retinoids play an essential role in recovery from UVB exposure, and may be future targets for cancer prevention treatments. Alterations of these proteins in the progression of cSCC may also improve our treatment approaches and give us a better understanding of how retinoids are involved in the development and progression of cSCC.

Appendix: Tables

Table 1. Highest intensity IHCL for STRA6 in uninvolved skin and tumor for specific stages. IHC was run using an antibody against STRA6 and scored for the highest intensity and percent cells in each tumor stage on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences.

Tumor Uninvolved Uninvolved Undifferentiated Differentiate Outside Stage Supra- Basal Tumor Cells d Tumor Corneum basal Cells Pap1 6.1 ±0.3 6.4 ±0.3a 7.0 ±0.4a 6.2 ±- 0.0 ±0a Pap2 5.0 ±0.6 5.0 ±0.7ab 4.3 ±0.5b 4.7 ±0.3 1.7 ±0.8ab Pap3 5.8 ±0.2 6.0 ±0.4a 5.3 ±0.3ab 5.4 ±0.8 0.0 ±0a MISCC1 6.5 ±0.4 5.3 ±0.6ab 5.5 ±0.4ab 5.4 ±0.3 0.0 ±0a MISCC2 4.7 ±1.0 3.5 ±0.8b 3.6 ±0.8b 3.6 ±0.3 2.6 ±0.9ab MISCC3 4.7 ±0.3 2.5 ±1.4b 4.0 ±0.6b 3.2 ±- 1.0 ±1.0ab SCC 4.3 ±0.5 2.6 ±0.6b 4.2 ±0.3b 3.9 ±0.3 2.7 ±0.7b

Table 2. Representative intensity IHCL for STRA6 in uninvolved skin and tumor for specific stages. IHC was run using an antibody against STRA6 and scored for the representative intensity and percent cells in each tumor stage on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences.

Tumor Uninvolved Uninvolved Undifferentiated Differentiated Outside Stage Supra- Basal Tumor Cells Tumor Cells Corneum basal Pap1 6.3 ±0.3 6.4 ±0.3a 6.6 ±0.4a - 0.0 ±0 Pap2 5.1 ±0.6 5.0 ±0.7abc 4.7 ±0.5ab 4.3 ±0.7 1.2 ±0.7 Pap3 5.8 ±0.2 5.9 ±0.4ac 5.3 ±0.4ab 6.0 ±0.4 0.0 ±0 MISCC1 6.2 ±0.6 5.4 ±0.6ac 5.4 ±0.5ab 5.5 ±0.3 1.0 ±1.0 MISCC2 5.4 ±0.8 3.5 ±0.8bc 3.8 ±0.9b 4.7 ±0.6 2.4 ±0.8 MISCC3 4.4 ±0.3 2.2 ±1.3b 2.7 ±0.6b 4.0 ±- 4.0 ±- SCC 4.6 ±0.6 2.5 ±0.6b 4.3 ±0.4b 5.0 ±0.4 3.0 ±0.7

Table 3. Highest Intensity IHCL for STRA6 in uninvolved and tumor for general stages. IHC was run using an antibody against STRA6 and scored for the highest intensity and percent cells in general tumor stages on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences.

Tumor Uninvolved Uninvolved Undifferentiated Differentiated Outside Stage Supra-basal Basal Tumor Cells Tumor Cells Corneum

Pap 5.7 ±0.2a 5.9 ±0.3 5.6 ±0.3a 5.2 ±0.5 0.5 ±0.3a MISCC 5.5 ±0.4b 3.9 ±0.6 4.2 ±0.4b 4.5 ±0.4 1.3 ±0.5ab SCC 4.3 ±0.5b 2.6 ±0.6 3.9 ±0.3b 4.2 ±0.3 2.7 ±0.7b

Table 4. Representative Intensity IHCL for STRA6 in uninvolved and tumor for general stages. IHC was run using an antibody against STRA6 and scored for the representative intensity and percent cells in general tumor stages on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences.

Tumor Uninvolved Uninvolved Undifferentiated Differentiated Outside Stage Suprabasal Basal Tumor Cells Tumor Cells Corneum

Pap 5.8 ±0.2 5.8 ±0.3a 5.5 ±0.3a 5.6 ±0.4 0.4 ±0.3a MISCC 5.5 ±0.4 3.9 ±0.6b 4.2 ±1.9b 5.0 ±0.3 1.9 ±0.6ab SCC 4.6 ±0.6 2.5 ±0.6b 4.3 ±0.4ab 5.0 ±0.4 3.0 ±0.7b

Table 5. Highest intensity IHCL for DHRS9 in uninvolved skin and tumor for specific stages. IHC was run using an antibody against DHRS9 and scored for the highest intensity and percent cells in each tumor stage on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences.

Tumor Uninvolved Uninvolved Undifferentiated Differentiated Outside Stage Supra-basal Basal Tumor Cells Tumor Cells Corneum

Pap1 5.3 ±0.2ab 3.1 ±0.3 4.6 ± 0.3 - 5.6 ±0.4 Pap2 5.0 ±0.2ab 2.6 ±0.3 4.1 ±0.2 - 4.7 ±1.1 Pap3 5.9 ±0.5a 2.7 ±0.4 4.2 ±0.3 - 6.1 ±0.5 MISCC1 4.8 ±0.2ab 2.7 ±1.3 4.7 ±0.1 5.4 ±0.8 5.4 ±0.4 MISCC2 4.9 ±0.2ab 3.4 ±0.5 4.7 ±0.4 5.5 ±0.7 5.2 ±0.3 MISCC3 4.9 ±0.3ab 2.7 ±0.7 4.1 ±0.3 6.0 ±1.0 4.7 ±0.4 SCC 4.1 ±0.3b 2.2 ±0.6 4.1 ±0.5 5.4 ±1.0 5.0 ±0.6

Table 6. Representative intensity IHCL for DHRS9 in uninvolved skin and tumor for specific stages. IHC was run using an antibody against DHRS9 and scored for the representative intensity and percent cells in each tumor stage on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences.

Tumor Uninvolved Uninvolved Undifferentiated Differentiated Outside Stage Supra-basal Basal Tumor Cells Tumor Cells Corneum

Pap1 5.3 ±0.1ab 3.0 ±0.3 5.0 ±0.3a - 6.1 ±0.3a Pap2 5.2 ±0.3ab 3.0 ±0.2 4.3 ±0.3ab - 4.7 ±0.5ab Pap3 6.3 ±0.4a 2.7 ±0.2 4.3 ±0.3ab - 6.2 ±0.4a MISCC1 5.2 ±0.3ab 3.0 ±0.3 3.9 ±0.4ab 6.3 ±0.3 5.0 ±0.5ab MISCC2 5.0 ±0.3ab 3.7 ±0.3 4.7 ±0.3ab 5.6 ±0.7 5.1 ±0.3ab MISCC3 4.2 ±0.6b 3.0 ±0.5 4.0 ±0.5ab 5.5 ±1.5 4.5 ±0.4ab SCC 4.4 ±0.3b 2.1 ±0.4 3.6 ±0.2b 5.3 ±0.4 4.3 ±0.2b

Table 7. Highest Intensity IHCL for DHRS9 in uninvolved and tumor for general stages. IHC was run using an antibody against DHRS9 and scored for the highest intensity and percent cells in general tumor stages on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences.

Tumor Uninvolved Uninvolved Undifferentiated Differentiated Outside Stage Supra- Basal Tumor Cells Tumor Cells Corneum basal Pap 5.4 ±0.2a 2.8 ±0.2 4.3 ±0.2 - 5.5 ±0.3 MISCC 4.8 ±0.1ab 2.9 ±0.3 4.5 ±0.2 5.6 ±0.4 5.1 ±0.2 SCC 4.1 ±0.3a 2.2 ±0.6 4.1 ±0.1 5.3 ±0.3 5.0 ±0.2

Table 8. Representative Intensity IHCL for DHRS9 in uninvolved and tumor for general stages. IHC was run using an antibody against DHRS9 and scored for the representative intensity and percent cells in general tumor stages on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences.

Tumor Uninvolved Uninvolved Undifferentiated Differentiated Outside Stage Supra- Basal Tumor Cells Tumor Cells Corneum basal Pap 5.6 ±0.2a 2.9 ±0.1ab 4.6 ±0.2a - 5.7 ±0.3a MISCC 4.9 ±0.2b 3.2 ±0.2a 4.2 ±0.2ab 5.8 ±0.4 4.9 ±0.2ab SCC 4.4 ±0.3b 2.1 ±0.4b 3.6 ±0.2b 5.3 ±0.4 4.3 ±0.2b

Table 9. Highest intensity IHCL for ALDH1A1 in uninvolved skin and tumor for specific stages. IHC was run using an antibody against ALDH1A1 and scored for the highest intensity and percent cells in each tumor stage on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences. Tumor Uninvolve Uninvolved Undifferentiated Differentiated Outside Stage d Supra- Basal Tumor Cells Tumor Cells Corneum basal Pap1 4.2 ±0.1 4.2 ±0.2 4.3 ±0.2a - 4.1 ±0.3 Pap2 4.4 ±0.2 4.6 ±0.3 4.3 ±0.2a - 4.6 ±0.5 Pap3 4.8 ±0.4 4.1 ±0.2 4.3 ±0.3a - 4.8 ±0.4 MISCC1 4.9 ±0.5 4.1 ±0.2 4.1 ±0.2ab 3.7 ±0.6 4.3 ±0.2 MISCC2 4.3 ±0.4 4.0 ±0.3 3.7 ±0.3ab 3.9 ±0.5 3.6 ±0.4 MISCC3 4.3 ±0.4 2.9 ±0.7 3.1 ±0.3b 4.1 ±0.1 3.9 ±0.2 SCC 4.5 ±0.1 3.9 ±0.4 4.1 ±0.1ab 4.7 ±0.3 4.5 ±0.1

Table 10. Representative intensity IHCL for ALDH1A1 in uninvolved skin and tumor for specific stages. IHC was run using an antibody against ALDH1A1 and scored for the representative intensity and percent cells in each tumor stage on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences. Tumor Uninvolved Uninvolved Undifferenti- Differenti- Outside Stage Suprabasal Basal ated Tumor ated Tumor Corneum Cells Cells Pap1 5.7 ±0.2ab 4.4 ±0.4 5.1 ±0.7ab - 5.5 ±0.2a Pap2 5.5 ±0.2ab 4.7 ±0.4 5.4±0.3a - 5.4 ±0.3a Pap3 5.7 ±0.2ab 4.6 ±0.5 5.6 ±0.3a - 5.5 ±0.3a MISCC1 5.9 ±0.3a 4.5 ±0.4 4.4 ±0.3ab 5.0 ±0.6 4.5 ±0.3ab MISCC2 4.9 ±0.3ab 3.7 ±0.3 4.4 ±0.5ab 5.2 ±0.2 4.1 ±0.3b MISCC3 4.6 ±0.4ab 3.8 ±0.6 3.5 ±0.6b 4.3 ±0.9 4.0 ±0.5b SCC 4.7 ±0.3b 4.5 ±0.3 4.2 ±0.4ab 4.2 ±0.4 4.0 ±0.4b

Table 11. Highest Intensity IHCL for ALDH1A1 in uninvolved and tumor for general stages. IHC was run using an antibody against ALDH1A1 and scored for the highest intensity and percent cells in general tumor stages on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences. Tumor Uninvolved Uninvolved Undifferentiated Differentiated Outside Stage Suprabasal Basal Tumor Cells Tumor Cells Corneum

Pap 4.5 ±0.2 4.3 ±0.1 4.3 ±0.1a - 4.5 ±0.2 MISCC 4.5 ±0.3 3.7 ±1.1 3.7 ±0.2b 3.9 ±0.2a 3.9 ±0.2 SCC 4.3 ±1.0 3.9 ±1.4 4.1 ±0.1ab 4.7 ±0.3b 4.5 ±0.1

Table 12. Representative Intensity IHCL for ALDH1A1 in uninvolved and tumor for general stages. IHC was run using an antibody against ALDH1A1 and scored for the representative intensity and percent cells in general tumor stages on a scale of 0-8. Mean scores ± SEM are shown. Different letters indicate significant differences. Tumor Uninvolved Uninvolved Undifferentiated Differentiated Outside Stage Suprabasal Basal Tumor Cells Tumor Cells Corneum

Pap 5.6 ±0.1a 4.6 ± 0.2 5.4 ±0.2a - 5.5 ±0.1a MISCC 5.2 ±0.2ab 4.0 ±0.2 4.2 ±0.3b 4.9 ±0.3 4.2 ±0.2b SCC 4.7 ±0.3b 4.5 ±0.3 4.2 ±0.4b 4.2 ±0.4 4.0 ±0.4b

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