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ALDEHYDE DEHYDROGENASE 1B1 IN ETHANOL METABOLISM, GLUCOSE HOMEOSTASIS AND COLON CANCER

by

SURENDRA SINGH

B.V.Sc. & A.H., Rajasthan Agricultural University, 2001 M.V.Sc., Rajasthan Agricultural University, 2004

A thesis submitted to the

Faculty of the Graduate School of the University of Colorado in partial fulfillment

of the requirements for the degree of Doctor of Philosophy

Toxicology Program 2014

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ii This thesis for the Doctor of Philosophy degree by

Surendra Singh has been approved for the

Toxicology Program by

David Thompson, Chair Vasilis Vasiliou, Advisor

Dennis Petersen David Orlicky

Ying Chen

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iii Singh, Surendra. (Ph.D., Toxicology)

Aldehyde Dehydrogenase 1B1 in Ethanol Metabolism, Glucose Homeostasis and Colon Cancer

Thesis directed by Professor Vasilis Vasiliou.

ABSTRACT

Aldehyde dehydrogenases (ALDHs) are a group of NAD(P)-dependent enzymes involved in the metabolism of a wide spectrum of aliphatic and aromatic aldehydes. ALDH1B1 is a mitochondrial homotetrameric enzyme which is 65% and 72% identical to ALDH1A1 and ALDH2 proteins, respectively. Our in vitro studies have shown that human ALDH1B1 metabolizes acetaldehyde with an apparent Km of 55 µM, indicating an important role of this protein in alcohol metabolism. ALDH1B1 is expressed only at the crypt base, along with stem cells, in human colon. It is highly expressed in all cancerous cells of human colonic adenocarcinomas. This pattern of expression corresponds closely to that observed for Wnt/β-catenin signaling activity in normal and cancerous colon. These findings suggest a potential role of ALDH1B1 in colon carcinogenesis. Recently we also found that ALDH1B1 is a marker for mice pancreatic progenitor cells and is required for maintenance and expansion of progenitor pools. To assess the in vivo role of ALDH1B1, we have generated transgenic Aldh1b1(-/-) mice; these mice are fertile and have a normal growth pattern. ALDH1B1

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iv examined expression of ALDH2 and ALDH1A1 in these organs and did not find compensatory up-regulation of these isozymes. Ethanol pharmacokinetics following a single intra peritoneal injection of ethanol 5g/kg revealed higher acetaldehyde levels in 3 and 24 hours in Aldh1b1(-/-) mice. At the 8-weeks of age, Aldh1b1(-/-) mice showed higher fasting blood glucose levels and

decreased glucose tolerance on intra-peritoneal glucose tolerance test. The shRNA-mediated knockdown of ALDH1B1 reduced the number and size of spheroids formed by SW480 colon cancer cells in 3-dimensional matrigel culture. ALDH1B1 knockdown depleted the highly carcinogenic ALDHbright cells and significantly decreased xenograft tumor formation in athymic mice. Protein and mRNA expression evaluation revealed down-regulation of Wnt/β-catenin, Notch and PI3K/Akt-signaling pathways in ALDH1B1-depleted colon cancer cells. In summary, our data demonstrate that ALDH1B1 is crucial for ethanol metabolism and glucose homeostasis and plays a functional role in colon cancer

tumorigenesis by modulating the Wnt/β-catenin, Notch and PI3K/Akt signaling pathways and could be a possible target for more effective treatments for this devastating condition.

The form and content of this abstract are approved. I recommend its publication.

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v I dedicate this thesis to

My father, late Shanker Singh Rajpurohit,

even though he did not live to share my achievement, I am sure he is delighted from his heavenly home

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vi ACKNOWLEDGEMENTS

It is not possible to express my sincere gratitude and appreciation in words to my advisor Vasilis Vasiliou for sharing his experience, scholastic guidance and scientific vision. He always been very patient and showed great confidence in me, I am especially thankful to him for constant encouragement, and personal interest in the research work right from its planning to its successful execution. The experience and knowledge I acquired here will continue to benefit me beyond my time in this lab.

I am grateful to have people with great scientific aptitude and helping nature in my advisory committee, their dedication to graduate education helped me immensely during my PhD research work. My sincere thanks to Dr. David Thompson, for his enthusiastic and creative support, Dr. Dennis Petersen for showing continuous interest in my work, Dr. David Orlicky for guiding me

throughout and for his great help with histopathological work. I can never thank enough to Dr. Ying Chen, who is not only a committee member but also been a great mentor, teacher, colleague, friend and a critic for me.

I also want to thank all the Vasiliou lab members for their cheerful company, constant encouragement and support during course of my research work, especially Chad Brocker, for his constructive critique. I am also grateful to members of Dr. Wells Messersmith lab, especially John Arcaroli for helping in planning and conducting experiments related with colon cancer cells.

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vii I extend my cordial thanks to my amazing and supportive friends Gaurav, Swetha and Sangeeta and their families for providing lot of memorable

experiences during this period.

Last but not least I express my deep sense of gratitude and love for my family especially my mother, Sharda Rajpurohit, who is there for me with her continuous encouragement, inspiration and support throughout all the ups and downs. It gives me immense pleasure to express my gratitude to my wife, Priti, and children, Srishti and Aditya, for their patience, understanding and

unconditional love during the years when this research work occupied a large chunk of my life, most of which originally belonged to them.

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viii TABLE OF CONTENTS

CHAPTER

I. BACKGROUND .. 1 Introduction HHHHHHHHHHHHHHHHHHH...HHH... 1 Acetaldehyde and retinaldehyde-metabolizing ALDHs in

colorectal cancer ...HHHHHHHHHHHHHH...HHHHH.. 2 Acetaldehyde: a carcinogen HHHHHHHHHHHHH.HHH 7 Opposing effects of retinoic acid on cancer cell proliferation H.H. 9 ALDH and cancer stem cells HHHHHHH...H..HHH...H... 11 Cancer stem cells in colorectal cancer HHHHHHHHHHHH 12 ALDH isozymes in pancreatic functions and progenitor cellsHH. 16 Summary HHHHHHHHHHHHHHHHHHHHHHHH.. 17 Objectives HHHHHHHHHH...HHHHHHHHHHHHH. 18 II. CHARACTERIZATION OF ALDEHYDE DEHYDROGENASE 1B1

KNOCKOUT MICE: PHYSIOLOGICAL IMPLICATION OF ALDH1B1 IN ETHANOL METABOLISM AND GLUCOSE HOMEOSTASIS ...... 20

IntroductionHHHHHHHHHHHHHHHHHHHHHHH.. 20 Materials and methods HHHHHHHHHHHHHHHHHH.. 22 Results HHHHHHHHHHHHHHHHHHHHHHHHH. 31 Discussion HHHHHHHHHHHHHHHHHHHHHHHH 40

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ix III. ALDH1B1 IS CRUCIAL FOR COLON TUMORIGENESIS BY

MODULATING WNT/β-CATENIN, NOTCH AND PI3K/AKT SIGNALING PATHWAYS .... 46

IntroductionHHH..HHHHHHHHHHHHHHHHHHHH 46

Materials and methods HHHHHHHHHHHHHHH..HH... 50 Results HHHHHHHHHHHHHHHHHHHHHHHHH.. 58 Discussion HHHHHHHHHHHHHHHHHHHHHHHH 71 IV. CONCLUSION .... 77 SummaryHHHHHHHHHHHHHHHHHHHHHHHH... 77 Significance HHHHHHHHHHHHHHHHHHHHHHH. 80 Future directions HHHHHHHHHHHHHHHHHHHHH. 80 REFERENCES . 83

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x LIST OF TABLES

TABLE

1.1. ALDH expression in various progenitor, stem and cancer cell types HH. 6 1.2. Affinity of ALDHs for acetaldehyde and retinaldehyde HHHHHHHH. 8 2.1. Primers for QPCR analysis HHHHHHHHHHHHHHHHHHHH 25

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xi LIST OF FIGURES

FIGURE

1.1. ALDHs modulate carcinogenesis by metabolizing acetaldehyde and

retinaldehyde HHHHHHHHHHHHHHHHHHHHHHHH.... 5 1. 2. ALDH-expressing cells are responsible for chemoresistance and

relapse of many tumors after chemotherapy HHHHHHHHHH... 12 1.3. ALDH1B1 expression pattern in normal colon and colon

adenocarcinomaHHHHHHHHHHHHHHHHHHHHHHH... 15 2.1. Generation of Aldh1b1(-/-) null mouse lineHHHHHHHHHHHH... 28 2.2. Expression analysis of ALDH1B1 mRNA and protein in Aldh1b1(-/-)

mice HHHHHHHHHHHHHHHHHHHHHHHHHHHHH 33 2.3. Growth curve of Aldh1b1(+/+) and Aldh1b1(-/-) mice HHHHHHHH 34 2.4. Aldh1b1(-/-) mice have normal cyto-architecture HHHHHHHHHH 35 2.5. Lack of ALDH1B1 does not affect number of proliferating cells in colon. 37 2.6. Lack of ALDH1B1 does not affect number of goblet cells in colon .H.... 38 2.7. Pharmacokinetics of ethanol and acetaldehyde in Aldh1b1(-/-) mice .H 39 2.8. Intra-peritoneal glucose tolerance test (GTT) in Aldh1b1(-/-) mice H... 41 2.9. Photomicrographs of mouse pancreas after immunostaining for insulin

and glucagonHHHHHHHHHHHHHHHHHHHHHHHH... 42 3.1. Illustration of Wnt/β-catenin signaling and distribution of ALDH1B1 in

wild type and ApcMin miceHHHHHHHHHHHHHHHHHHHH 60 3.2. Evaluation of human ALDH1B1 promoter activity in colon cancer cell

linesHHHHHHHHHHHHHHHHHHHHHHHHHHHHH. 62 3.3. ALDH1B1 promoter activity in colon cancer cell lines HHHHHHHH 63 3.4. ALDH1B1 knockdown in the SW480 cell line inhibits spheroid

formation and tumor growth HHHHHHHHHHHHHHHHHH... 65 3.5. ALDH1B1 knockdown in SW480 cells depletes ALDHbright cells HHH.. 68

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xii 3.6. ALDH1B1 positively regulate Wnt/β-catenin, Notch and PI3K/Akt

signaling pathways HHHHHHHHHHHHHHHHHHHHHH.. 70 3.7. Possible mechanism for the role of ALDH1B1 in colon tumorigenesis ... 76

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xiii ABBREVIATIONS

ADH Alcohol dehydrogenase ALDH Aldehyde dehydrogenase AODS Anti-oxidative defense systems APC Adenomatous polyposis coli ATRA All-trans-retinoic acid

AUC Area under the curve B2M β-2 microglobulin BAA Bodipy-aminoacetate

BAAA Boron dipyrromethene aminoacetaldehyde CRBPII Cellular retinoic acid binding protein

CRC Colorectal cancer CSC Cancer stem cell

CSL CBF-1/RBP-Jk, Su (H), Lag-1 CYP2E1 Cytochrome P4502E1 DEAB Diethylaminobenzaldehyde Dsh Dishevelled

ECM Extracellular matrix ESA Epithelial-specific antigen FABP5 Fatty acid binding protein 5

GC-MS Gas chromatography- Mass spectrometry GI Gastrointestinal

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xiv H&E Hematoxilin and eosin

H2O2 Hydrogen peroxide

HES Hairy and enhancer of split HSV-TK Thymidine kinase minicassete i.p. Intra-peritoneally

IACUC Institutional animal care and use committee IARC International agency for research on cancer ICN Intracellular Notch

iLBP Intracellular lipid binding protein IPGTT Intra-peritoneal glucose tolerance test LEF Lymphoid enhancer factor

LRP Lipoprotein receptor related protein MAPK Mitogen activated protein kinase Min Multiple intestinal neoplasia MS Mass spectrometry

MW Molecular weight marker

NAD(P) Nicotinamide adenine dinucleotide (phosphate) NEO Neomycin-resistance minicassette

NTP National toxicology program PCA Perchloric acid

PDTX Patient-derived tumor xenograft PI3K Phosphoinositide-3-kinase

PPAR Peroxisome proliferator-activated receptor

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xv RA Retinoic acid

RALDH Retinaldehyde dehydrogenase RAR RA receptor

ROS Reactive oxygen species RXR Retinoid X receptor

Sc Scramble

SIM Selected ion monitor

SNP Single nucleotide polymorphism

SPSS SigmaStat statistical analysis software TBE TCF/LEF binding element

TBST Tris-buffered saline with 0.1% tween 20 TCF T cell factor

TESS Transcription element search system TGFBR2 TGF-beta type II receptor

TGTC Transgenic and gene targeting core TIC Tumor initiating-cell

TSS Transcription start site

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1 CHAPTER I

BACKGROUND

Introduction

The aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)+ -dependent enzymes that oxidize a wide range of endogenous and exogenous aldehydes to their corresponding carboxylic acids [1]. The ability of ALDHs to act as ‘aldehyde scavengers’ is grounded in the observation that many have broad substrate specificities and can metabolize a wide range of chemically- and

structurally-diverse aldehydes. Many of the ALDH isozymes overlap in relation to substrate specificities, tissue distribution and subcellular localization but vary in their efficiency in metabolizing specific aldehydes [2-5]. The human genome contains 19 protein-coding ALDH genes. ALDH proteins are found in one or more subcellular compartments including the cytosol, mitochondria, endoplasmic

reticulum and nucleus [2]. Mutations and polymorphisms in ALDH genes are associated with various pathophysiological conditions in humans and rodents [6, 7] including alcohol-related diseases [8] and cancers [9]. ALDH1B1 is a

mitochondrial enzyme which was previously known as ALDHX or ALDH5 [10]. It

shares 72 and 65 percent peptide sequence identity with ALDH2 and ALDH1A1 respectively [11]. It shares some catalytic functions with ALDH2 by metabolizing acetaldehyde, lipid peroxidation-derived aldehydes and other short chain

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2 dehydrogenase (ADH1) isozymes into acetaldehyde which is then oxidized to acetic acid, primarily by mitochondrial ALDH2 [7]. ALDH1B1 has also been found to metabolize retinaldehyde [13] to retinoic acid (RA) and thereby could play crucial role in cell proliferation and differentiation. Recently ALDH1B1 has also been implicated in pancreatic development and regeneration [14].

Acetaldehyde- and retinaldehyde-metabolizing ALDHs in colorectal cancer Colorectal cancer (CRC) represents a serious health concern because of its very high morbidity and mortality. Each year, more than one million new CRC cases are diagnosed and over 500,000 deaths are associated with this condition worldwide [15]. The American Cancer Society estimated diagnosis of 136,830 new cases of CRC in the USA during 2014; of these, approximately 50,310 people are expected to die [16]. Although the exact mechanisms that promote CRC remain obscure, there is increasing evidence suggesting the involvement of lifestyle-related factors in addition to genetic predisposition. These factors include waist circumference, folate and multivitamins in the diet, high fat and high energy diet, physical exercise, tobacco smoking and alcohol consumption [17, 18].

According to dose-response meta-analysis and pooled results from cohort studies, chronic daily consumption of approximately 50 g alcohol increases the relative risk for colon cancer by 40 per cent [19, 20]. Various theories have been advanced regarding the mechanism by which alcohol induces cancer. For

example, ethanol may enhance mucosal penetration of a carcinogen by serving as a solvent. In addition, ethanol induces cytochrome P4502E1 (CYP2E1), an

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3 enzyme capable of generating reactive oxygen species (Figure 1.1). However, the most well accepted theory regarding ethanol-induced cancer involves acetaldehyde acting as a carcinogen [21, 22]. Ethanol is metabolized to acetaldehyde by ADH, CYP2E1 and catalase [23, 24] (Figure 1.1). Aldehydes covalently adduct proteins, nucleic acids and cellular biomolecules leading to DNA damage, altered cellular homeostasis and hyper-regeneration of colon mucosa, all of which can result in increased cancer risk [25-29]. In 2009, the International Agency for Research on Cancer (IARC) designated acetaldehyde (as associated with alcohol consumption) to be a group I human carcinogen [30]. Reactive aldehyde generation is elevated in all clinical stages of colorectal

cancer and the levels increase with, and are tightly correlated to, disease progression [31-33]. ALDH enzymes detoxify reactive aldehydes produced during oxidative stress, especially in metabolically-active tissues, such as

cancers [34-36]. Acetaldehyde is metabolized to acetate, a process catalyzed by ALDH2, ALDH1B1 and ALDH1A1 (Figure 1.1) [11]. The ability of these ALDHs to repress cellular acetaldehyde levels is consistent with a role for ALDHs in colon cancer and is supported strongly by the association of ALDH2 deficiency with high incidence of CRC in heavy ethanol drinkers [37]. In addition to metabolizing acetaldehyde, ALDH1 isozymes are the primary enzymes involved in the

metabolism of retinaldehyde to RA, a signaling molecule that plays a crucial role in cellular proliferation and differentiation [24]. Given their ability to affect cellular RA levels, it is likely that RA-generating ALDHs have a role in modulating

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4 ALDHs are implicated in cancer. First, ALDH activity has been used to identify and isolate normal and cancer stem cells of various lineages [38-40] (Table 1.1). Second, high ALDH expression has been found to be associated with poor

clinical outcome in leukemia [41], ovarian [42-44], prostate [45, 46], breast [47-49], colorectal [50] and pancreatic cancer [51, 52]. Third, ALDH+ cells (cells with very high ALDH expression) exhibit a greater tumorigenic capacity, as reflected in colony-forming capability in vitro and in xenograft-induced tumor formation in vivo [53]. We have found very strong up-regulation of ALDH1B1 expression in an animal model of colon polyps, specifically adenomatous polyposis coli

(Apc) multiple intestinal neoplasia (Apc Min) mice (our unpublished data). These mice have a point mutation in Apc, a tumor suppressor gene which when mutated leads to dysregulation of the Wnt-signaling pathway and results in up-regulation of oncogenes like c-Myc [54]. Overexpression of ALDH1B1 in polyps from these mice is suggestive of a possible relationship between Wnt-signaling and ALDH1B1 expression, a consideration that warrants further study.

A causal relationship exists between alcohol consumption and CRC and this may be mediated, at least in part, by acetaldehyde [29, 52]. The significance of retinaldehyde and acetaldehyde in tumor formation, and very high expression of the ALDHs in colorectal cancer are suggestive of a crucial role for

acetaldehyde- and retinaldehyde- metabolizing ALDHs in these cancers. Lack of ALDH2 activity and resultant high acetaldehyde levels are linked with colon cancer initiation. By contrast, high ALDH1 activity (primarily ALDH1A1 and

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5 ALDH1B1) is required for the stemness and tumorigenic potential of cancer stem cells.

Figure 1.1. ALDHs modulate carcinogenesis by metabolizing acetaldehyde and retinaldehyde. Ethanol is metabolized by alcohol dehydrogenase (ADH), catalase and CYP2E1 to acetaldehyde. Acetaldehyde can interfere with anti-oxidative defense systems (AODS) and generate reactive oxygen species

(ROS); inhibits DNA repair and methylation; and forms DNA and protein adducts to promote tumor growth. Acetaldehyde is metabolized to acetate primarily by ALDH2, ALDH1B1 and ALDH1A1. Retinaldehyde, formed from retinol by ADH, is converted to retinoic acid (RA) by retinaldehyde-metabolizing ALDHs. RA exerts anti-carcinogenic activity by binding to cellular retinoic acid binding proteins (CRBPII) and activating the RA receptor (RAR). When RA binds to fatty acid binding protein 5 (FABP5), it activates orphan nuclear receptor peroxisome proliferator-activated receptor (PPAR)β/δ and acts as procarcinogenic agent. ALDH, aldehyde dehydrogenase; NAD+, NAD(P), nicotinamide adenine dinucleotide (phosphate); H2O2, hydrogen peroxide.

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6 Table 1.1: ALDH expression in various progenitor, stem and cancer cell types

S.No. Cell or tumor type ALDH isozyme(s) a Reference

1 Hematopoietic progenitor ALDH, ALDH1A3 [38-40, 55, 56]

2 Mesenchymal progenitors ALDH [55]

3 Endothelial progenitors ALDH [55]

4 Neural stem cells ALDH, ALDH1L1 [57, 58] 5 Normal mammary stem

cells

ALDH1A1 [47]

6 Breast cancer stem cells ALDH1A1,

ALDH1A3, ALDH2, ALDH6A1,

[47, 49, 56, 59, 60]

7 Prostate cancer ALDH, ALDH7A1 [45, 46, 61] 8 Ovarian cancer stem cells ALDH, ALDH1A1 [42-44, 62] 9 Ovarian cancer cells ALDH1A1,

ALDH1A3, ALDH3A2, ALDH7A1

[63]

10 Colon stem cells ALDH1A1, ALDH1B1

[12, 64]

11 Colon cancer stem cells ALDH1A1, ALDH1B1

[12, 42, 50, 53, 64, 65]

12 Leukemia stem cells ALDH [41]

13 Human lung cancer cells ALDH1A1 [42, 66, 67] 14 Head and neck cancer

stem cells

ALDH1A1 [68]

15 Pancreatic cancer ALDH, ALDH1A1, ALDH1A3

[51, 56, 69]

16 Liver cancer stem cells ALDH, ALDH1A1 [70, 71]

a

ALDH is designated for studies in which ALDH+ cells were identified and isolated using the Aldefluor® assay.

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7 Acetaldehyde: a carcinogen

Acetaldehyde is categorized as ‘carcinogenic to humans’ and ‘reasonably anticipated to be a human carcinogen according to IARC regulations and United States National Toxicology Program (NTP), respectively [30, 72]. Acetaldehyde has been shown to be a highly toxic, mutagenic and carcinogenic compound in a variety of in vitro and in vivo studies. Its effects range from damaging antioxidant defenses [24] to interfering with DNA methylation and repair mechanisms

through formation of adducts with DNA and proteins (Figure 1.1) [23, 29]. In the colon, acetaldehyde is primarily produced from ethanol by resident bacteria and, to a lesser extent, by mucosal ADHs. As a result of metabolism by intra-colonic microbes, large quantities (nine-fold higher than normal) of acetaldehyde

accumulate in the rat colon 2 hours after intra-peritoneal injection of ethanol [73]. Human colon mucosal cells harbor ADH1, ADH3 and ADH5, with the ADH1 and ADH3 isozymes being most active [74]. In an in vitro experiment, human colon contents were able to generate 60 to 250 µM acetaldehyde when incubated with a concentration of ethanol (10-100 mg%), which is known to be attained during normal ethanol drinking [75]. The high levels of acetaldehyde attained in the colon after drinking ethanol likely underlies the correlation between chronic, heavy ethanol consumption and CRC in humans. In ethanol-treated rats, a high concentration of acetaldehyde (50 to 350 µM) in the colon mucosa has been shown to correlate positively with hyper-proliferation of the colon crypt cells. Such a phenomenon would be anticipated to favor the development of CRC [27, 76].

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8 Acetaldehyde is metabolized primarily by mitochondrial ALDH2 and

ALDH1B1 and, to lesser extent, by cytosolic ALDH1A1 (Table 1.2) [11]. The most convincing evidence for a role of acetaldehyde in CRC initiation emanates from studies involving Asians who possess a polymorphism in their ALDH2 enzyme known as ALDH2*2. These subjects possess a single nucleotide polymorphism (SNP) that leads to a lysine to glutamate substitution at residue 487 that renders the enzyme functionally-inactive [77, 78]. Approximately 40 per cent of the Asian population carry an ALDH2*2 allele; this compromises their ability to metabolize acetaldehyde and increases their colon cancer risk 3.4 times [37].

Table 1.2: Affinity of ALDHs for acetaldehyde and retinaldehyde S.No. ALDH isozyme(s) Substrate Km Reference 1 ALDH1A1 Acetaldehyde 180 µM [11] All-trans Retinaldehyde 9-cis Retinaldehyde 11.6-26.8 µM 3.59 µM [79],[13] 2 ALDH1A2 All-trans Retinaldehyde 9-cis Retinaldehyde 0.66 µM 0.62 µM [80] 3 ALDH1A3 All-trans Retinaldehyde 0.2 µM [81] 4 ALDH1B1 Acetaldehyde 55 µM [11] Retinaldehyde 24.9 µM [13] 5 ALDH2 Acetaldehyde 3.2 µM [11]

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9 Opposing effects of retinoic acid on cancer cell proliferation

Retinoids exert many physiologically-important and diverse functions in relation to cellular proliferation and differentiation of normal and cancer cells. For example, retinoids are crucial for embryonic development and adult tissue

remodeling. The retinoids comprise all of the derivatives of retinol, including all-trans-, 9-cis- and 13-cis- retinoic acid. Retinol is oxidized to retinaldehyde by retinol dehydrogenases. The resultant retinaldehydes are further metabolized to their corresponding RA by retinaldehyde dehydrogenases which include

RALDH1 (ALDH1A1), RALDH2 (ALDH1A2), RALDH3 (ALDH1A3) and RALDH4 (ALDH8A1) (Table 1.2) [82-87]. Among the RAs, all-trans-RA (ATRA) is the most biologically potent retinoid. Abnormally low levels of ALDH1A2 have been

observed in breast and prostate cancers [88, 89]. Impaired RA formation and high levels of CYP26A1 (a RA-metabolizing enzyme) in human breast cancer are consistent with a protective role for RA in this cancer [88-90]. The physiological actions of the retinoids are mediated through binding of the RA receptor (RAR) and retinoid X receptor (RXR) heterodimer to the regulatory region of retinoid-responsive genes, known as RA response elements [91]. RARs and RXRs are ligand-dependent transcription factors and exist as α, β or γ isoforms. RAR isoforms interact with both ATRA and 9-cis RA, whereas RXR isoforms interacts only with 9-cis RA [92, 93]. The binding of RA with the RAR/RXR dimer recruits co-activator proteins and initiates transcriptional activation of the

retinoid-responsive genes [91]. Retinoids have been found to be effective for the

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10 prostate, skin and colon cancers [94-96]. In vivo studies involving rats have

revealed that retinoids added to the diet reduced colon cancer cell proliferation and prevented azoxymethane-induced aberrant crypt foci (putative precancerous lesions in colon) and colon tumor formation [96, 97]. A RXR-selective retinoid, AGN194204, has been found to inhibit the proliferation of human pancreatic cancer cells, an effect that can be reversed by a RXR-selective antagonist [98]. In addition to inhibiting the growth of pancreatic cancer cells, RA increases the sensitivity of pancreatic adenocarcinoma cells to the antineoplastic drugs gemcitabine and cisplatin [99].

In contrast to the anti-proliferative and anti-survival role of RA in cancer cells, dietary ATRA has been shown to enhance initiation and growth of intestinal tumors in the ApcMin mouse model in vivo [100]. RA can promote cell survival and hyperplasia in cells expressing high levels of fatty acid-binding protein 5 (FABP5) by activating an orphan nuclear receptor, peroxisome proliferator-activated

receptor (PPAR)β/δ [101]. PPARβ/δ mediates antiapoptotic properties partly by inducing the PDK1/Akt survival pathway [102]. RA binds to intracellular lipid binding proteins (iLBPs), including cellular retinoic acid-binding proteins

(CARBPII) and FABP5. CARBPII and FABP5 are selective for nuclear receptors RARα and PPARβ/δ, respectively [101]. Hence, RA induces CARBPII- or

FABP5- mediated activation of RAR or PPARβ/δ (respectively), depending on the ratio of FABP5/ CARBPII in the cells [101]. Human colorectal cancer cell lines (specifically, T84, COLO205, SW620, SW480, HCT116 and DLD-1) express ~30-fold higher levels of FABP5 relative to normal colorectal cells (CCD18-Co),

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11 suggesting the possibility of pro-proliferative and anti-apoptotic roles for RA in these cells [101, 103]. However, the expression levels of PPARβ/δ in colorectal cancer cells and its role in tumorigenesis are unresolved in various cancers, including CRC [104].

ALDH and cancer stem cells

In the gastrointestinal (GI) tract, tissue-specific stem cells are at the top of the cellular hierarchy and play a critical role in regulating tissue homeostasis. These specialized epithelial cells are characterized by their ability to self-renew and differentiate into a variety of cellular populations that perform specific functions within the GI tract. Currently, it is believed that these tissue-specific stem cells (or progenitor cells), when oncogenically transformed, become cancer stem cells (CSCs) or tumor initiating-cells (TICs) since they functionally possess the capacity to form tumors and maintain tumor growth. Accumulating evidence also suggests that CSCs are responsible for chemotherapeutic/radiation

resistance and tumor recurrence (Figure 1.2).

ALDH catalytic activity has been identified in many human cancers [49] and, as such, is used as a marker of CSCs, including colorectal cancer. The pathophysiological function of ALDH in CSCs remains unresolved. Intense research of ALDH enzymes is underway in order to elucidate the role of these proteins in the development and progression of cancer as well as drug

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12

Figure 1.2. ALDH-expressing cells are responsible for chemoresistance and relapse of many tumors after chemotherapy. Most current chemotherapy drugs are effective against the bulk of the tumor cells. However, the high ALDH-expressing (ALDH+) cancer stem cells are resistant to these treatments. As a result, during chemotherapy, the ALDH+cells proliferate and promote tumor growth. The resultant tumors contain an increased proportion of ALDH+cells, making them more resistant to chemotherapy than the original tumor.

Cancer stem cells in colorectal cancer

Although earlier stages of CRC are highly curable, therapeutic interventions in advanced disease have proven to be poorly effective at increasing the 5-year survival rate. Recent drug development has focused on targeting the CSC population as a potential therapy. In normal colon, CSC’s reside at the bottom of the crypt and generate upward, migrating and

differentiating into transit amplifying cells (in the middle of the crypt) which

become terminally differentiated cells as they move upward and eventually shed into the lumen (Figure 1.3A) [105].

In CRC, several different molecules, including the cell surface markers CD133 and CD44 as well as ALDH activity, have been proposed as biomarkers for identification and isolation of the CSC population [53, 64, 106-108]. CD133+ colon cancer cells were initially shown to be tumorigenic [107, 108]. However,

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13 subsequent studies identified that both CD133+ and CD133- cells possess

tumorigenic potential [109]. CD44+ (either with or without epithelial-specific antigen (ESA+)) was demonstrated to be a marker in colon CSCs [109]. However, additional studies showed that CD44+ cells reside throughout the entire crypt, including the proliferative compartment, suggesting that the CD44+ colon cells are not necessarily stem-like [64]. We have examined CD44 and ALDH together in one of our CRC patient-derived tumor xenograft (PDTX) models to determine if CD44+ cells had tumorigenic properties [110]. Despite ALDH+/CD44+ cells showing some tumorigenic growth, ALDH+/CD44- cells exhibited a higher incidence and faster growing tumors. In this same PDTX model, isolation and injection of ALDH+ and ALDH- cells in mice showed a significant difference with respect to tumor growth [110]. ALDH+ cells produced fast growing and large tumors when compared to ALDH- cells that either

produced very small tumors or no tumors in five separate PDTX models. Importantly, all ALDH+ tumors looked morphologically the same as the original tumor. Several other studies have shown that injection of ALDH+ cells from colitis and colon cancer patients facilitated spheroid formation (in vitro 3-dimensional spheroid cell culture that more closely resembles the in vivo environment) and tumor growth in a xenograft model, while ALDH- cells were incapable of tumor growth [53, 64]. These studies demonstrate that ALDH catalytic activity appears to be a robust marker of CSCs in CRC.

Given the apparent promise of ALDH activity as a potential biomarker of CSCs, many investigations are currently exploring the role of ALDH in CSC

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14 function. In particular, a great deal of focus is being placed on which ALDH

isoform(s) mediate the catalytic activity in the CSCs. In normal colon stem cells, ALDH1 has been demonstrated to be primarily expressed at the bottom of the crypt compartment in the colon (where colon-specific stem cells are located) and ALDH1 levels are significantly elevated in the development and progression of CRC [64]. Interestingly, ALDH1 protein levels are elevated in the colon of

patients with ulcerative colitis (a risk factor for colon cancer) compared to normal colon cells; such expression may be important in the transformation from colitis to colon cancer [53]. We have shown that ALDH1B1 protein is 5.6-fold higher when compared to ALDH1A1 in CRC patients and may be a potential biomarker in CRC (Figure 1.3B-C) [12]. Similarly, very high expression of ALDH1B1 was found in the colon polyps of ApcMin mice (our unpublished data). While these studies indicate elevations in individual ALDH isoforms in CRC, the contribution of these enzymes to the progression of CSCs and CRC remain to be clarified.

A common problem associated with standard chemotherapeutic regimens in CRC is treatment resistance. Although chemotherapy is effective at reducing tumor burden, many CRC patients will experience disease recurrence and ultimately succumb to their disease. CSCs are thought to be responsible for chemotherapy-resistance and disease recurrence [111]. Therefore, therapeutic elimination of this population would be predicted to reduce tumor recurrence and ultimately improve survival. In our CRC PDTX model, the effects of an inhibitor of the Notch pathway (considered to be important for self-renewal of colon stem cells) in combination with irinotecan was investigated on the ALDH+ cell

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15 population [110]. The combination therapy was effective at reducing the number of ALDH+ cells as well as tumor recurrence, even after treatment was

discontinued when compared to single agent Notch pathway inhibition and irinotecan. Administration of the combination therapy for 28 days prevented tumor growth in the ALDH+ cell xenograft model; this protection continued for 3 months after combination treatment was completed [110]. These data indicate that the ALDH+ population has the ability to self-renew, and significantly reducing this population of cells delays tumor recurrence (Figure 1.2). Whether specific ALDH isozymes contribute to chemotherapy resistance remains to be

determined.

Figure 1.3. ALDH1B1 expression pattern in normal colon and colon

adenocarcinoma. Location of various cell types in normal colon (A). ALDH1B1 expression (red arrows) is strictly localized to stem-like cells at the base of crypts in the normal human colon (B). ALDH1B1 is expressed at extremely high levels throughout all cells of human colon adenocarcinomas (C). In figures B and C (reproduced from Chen et al., 2011 [12]), lower panels are higher magnification of areas identified by squares in the upper panel.

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16 ALDH isozymes in pancreatic functions and progenitor cells

Heavy alcohol consumption [112] and the inactivating polymorphism of ALDH2, resulting into impaired metabolism of ethanol have been found to be associated with increased risk of type 2 diabetes mellitus (T2D) [113]. ALDH2 has also been found to protect from diabetes induced cardiomyopathy and retinopathy possible by preserving cell survival and metabolizing reactive

aldehydes respectively [114, 115]. High ALDH expression has also been shown to be linked with diabetes associated large vessel disease [116]. These studies are indicative of the possible association of acetaldehyde metabolizing ALDH isozymes and T2D and associated complications. In the mouse, putative adult pancreas stem/progenitor cells have been isolated as cells with high ALDH activity from the centroacinar compartment and they demonstrate the capacity to generate endocrine and exocrine cells in culture [117]. Strikingly, experiments in a genetically engineered mouse model of pancreatic cancer indicated that cells in the same centroacinar location might be the cells of origin of pancreatic cancer [118]. We have recently found that in mice, ALDH1B1 is strongly expressed in the pancreas progenitor cells of the pancreatic primordia. As pancreas

differentiation proceeds by the growth and branching of the pancreatic

epithelium, strong expression persists in the tips and the trunks where tripotent and endocrine pancreas progenitor cells reside [14]. Aldh1b1 expression is subsequently restricted to exocrine progenitor pools and this expression is lost before birth. Inhibition of ALDH activity in explant cultures of embryonic

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17 together with Aldh1a1, the only other known ALDH gene expressed in the

developing epithelium, in maintaining the progenitor populations [14, 119]. Aldh1b1 expression persists in very rare centroacinar-like cells in the adult and the number of Aldh1b1+ cells increases dramatically following two different pharmacological treatments that induce pancreas regeneration [14]. These findings suggest a role of ALDH1B1 in maintenance and expansion of pancreatic progenitor pool as well as in pancreatic regeneration.

Summary

There is accumulating evidence that supports a role for ALDHs in cancer development and progression. The exact mechanisms by which ALDHs influence tumorigenesis remain to be defined. Certainly, metabolism of acetaldehyde and/or the generation of RA represent modalities by which ALDHs could

influence CRC. ALDH catalytic activity appears to be an excellent biomarker that can be utilized for the isolation and characterization of the CSC population in tumors obtained from patients with CRC. It is becoming apparent that the various ALDH isozymes may have different roles in tumorigenesis (from metabolism of the carcinogen to modulation of the proliferation-regulating retinoids) and that the timing and cellular localization of isozyme expression may be critical factors that influence how ALDHs modulate cancer development and progression. Further studies are needed that identify (i) the importance of ALDH catalytic activity in modulation of tumorigenesis, (ii) the specific ALDH isozymes involved (and that regulate CSCs), and (iii) ALDH-associated signaling pathways in cancer cells

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18 and CSCs. The results obtained from such studies should lead to the

development of novel therapies that may more effectively treat these devastating diseases.

Objectives

The goal of this project is to understand the role of mitochondrial ALDH1B1 in alcohol metabolism, glucose homeostasis and colon carcinogenesis. Chronic alcohol abuse elicits a plethora of pathological

outcomes, including damage to the liver and colon. Most of the adverse effects of ethanol are attributed to its metabolite acetaldehyde. Ethanol is primarily

metabolized via oxidation to acetaldehyde, which has recently been classified as a “Group 1” human carcinogen. Acetaldehyde is metabolized to acetate by aldehyde dehydrogenases (ALDHs), primarily by mitochondrial ALDH2 and, to a lesser extent, by cytosolic ALDH1A1. We have recently shown that ALDH1B1 has a high affinity for acetaldehyde and appears to be the second major enzyme in acetaldehyde elimination. Several ALDH1B1 polymorphisms also exist in humans, and recent studies suggest an association between these

polymorphisms and drinking aversion and more frequent alcohol hypersensitivity reactions in Caucasians. ALDH1B1 has also been found to be associated with maintenance and proliferation of pancreatic progenitor cells and pancreatic regeneration following injury. We have recently reported that ALDH1B1 protein is expressed specifically in the stem cell population of the normal human colon. In addition to detoxifying acetaldehyde, we found that ALDH1B1 metabolizes

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19 retinaldehyde to RA, a reaction that could explain its role in stem cells.

Interestingly, we also found that ALDH1B1 protein is expressed at high levels in colorectal cancer, making it a potential biomarker.

Objectives of this thesis are to: 1) determine the physiological implication of ALDH1B1 in ethanol metabolism and glucose homeostasis using Aldh1b1(-/-) knockout mice model 2) determine role of ALDH1B1 in colon tumorigenesis and the involved mechanism.

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20 CHAPTER II

CHARACTERIZATION OF ALDEHYDE DEHYDROGENASE 1B1 KNOCKOUT MICE: PHYSIOLOGICAL IMPLICATION OF ALDH1B1 IN ETHANOL

METABOLISM AND GLUCOSE HOMEOSTASIS

Introduction

The aldehyde dehydrogenases (ALDHs) are involved in metabolizing a wide range of endogenous aldehydes and xenobiotics [24]. ALDH1B1 is a mitochondrial enzyme which was previously known as ALDHX or ALDH5 [10].

ALDH1B1 is the second most efficient enzyme (Km= 55 µM) in metabolizing

acetaldehyde after ALDH2 (Km= 3.4 µM) [11]. ALDH2 is a polymorphic enzyme

with ALDH2*2 as an inactive variant, which is found in up to 50% of the Asian population [24, 120, 121]. These carriers accumulate high concentrations of acetaldehyde and show ethanol-induced hypersensitivity, hypertension and flushing syndrome [122]. The ALDH2*2 variant is nearly absent in Caucasians [21] but they carry inactive variants of ALDH1B1 [122]. In Caucasian populations the ALDH1B1 polymorphism is associated with the symptoms of acetaldehyde toxicity including ethanol hypersensitivity, hypertension and ethanol aversion [122, 123]. Together, these findings are suggestive of a crucial role for ALDH1B1 in ethanol metabolism.

ALDH1B1 also shares the physiological role of ALDH1A1 by metabolizing retinaldehyde[13] and could be associated with stem cells like properties in

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21 normal and cancer stem cells [124]. ALDH activity is used to identify and isolate normal and cancer stem cells [48, 53, 66]. Initially, human hematopoietic cells were found to be rich in ALDH activity but more recently, stem cells from other cancer types including bone marrow, ovary, breast, lung, pancreas, prostate and colon cancer have also been found to possess high ALDH activity [24, 38, 51, 66]. The Aldefluor® assay (method to determine ALDH activity) is used to identify cancer stem cells and tumor initiating cells in various cancer types [40, 42]. So far, ALDH1A1 is considered a marker for these cells [47, 64, 66]. However, the Aldefluor® assay is not specific for ALDH1A1 activity and other ALDHs also contribute to this phenotype [12, 63, 125]. We compared the expression of ALDH1A1 and ALDH1B1 in human colon cancer cells and found significantly high expression of the later suggesting ALDH1B1 as a potential biomarker for colon cancer[12]. High ALDH1B1 expression has also been found to correlate with poor prognosis in colorectal cancer patients in more recent studies [50, 125].

It has been shown that ALDH1B1 is strongly expressed in the early pancreatic buds in developing mice when compared to other retinaldehyde dehydrogenases (RALDHs) including ALDH1A1, ALDH1A2, ALDH1A3 and ALDH8A1 [14]. With further development and differentiation, strong ALDH1B1 expression remains confined exclusively to tips and the trunk of the pancreatic epithelium and persist only in centroacinar-like cells by the time of birth [14]. ALDH1B1+ cells expand dramatically in adult mice pancreas following acute caerulein-induced pancreatitis [14]. Together, these findings indicate the role of ALDH1B1 in pancreatic development and regeneration. The understanding of the

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22 molecular mechanism about the possible role of ALDH1B1 in restoring

pancreatic function would be important for the effective treatment of the diabetes. To delineate the in vivo role of ALDH1B1 in ethanol metabolism and glucose homeostasis, we have generated a mouse line with the global disruption of Aldh1b1 gene.

Materials and methods

Preparation of targeting construct and Generation of Aldh1b1 (-/-) mice A genomic 9.1 kb XbaI-KpnI fragment of mouse Aldh1b1 locus was isolated from C57BL/6J mouse genomic DNA using high-fidelity PCR and subcloned into a pBluescript(II)-KS vector. Using this clone, we constructed a targeting vector in which a LoxP-flanked NEO cassette carrying an extra NsiI site disrupts Aldh1b1 exon 2 and removes the complete coding region of Aldh1b1 gene; for counter-selection, a HSV-TK gene was placed at the 5’-end of the XbaI-KpnI fragment (Figure 2.1A). The Aldh1b1 gene was targeted in the EC7.1 hybrid (Sv129/C57Bl/6Bl6) ES cell line by the Transgenic and Gene Targeting

Core (TGTC) at the University of Colorado. Three homologous recombinant ES clones out of 280 clones resistant to both G418 and ganciclovir were identified by PCR and Southern blotting analysis (see below) (Figure 2.1B). These clones were subjected to karyotyping for detection of genetic alterations and the healthiest clone was selected for microinjection into non-agouti C57BL/6J

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23 blastocysts to generate chimeric mice. The chimeric mice were then mated with C57BL/6J female mice and resultant agouti Aldh1b1(+/-) heterozygous animals were intercrossed to generate Aldh1b1(-/-) homozygous knockout animals. The resulting offspring were of Sv129 and C57BL/6J mixed background. The

Aldh1b1(-) allele was then backcrossed into C57BL/6J background for 10 generations. All studies were carried out in accordance with the University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee (IACUC).

Southern blot and PCR analysis

Successful targeting in ES clones were identified first by PCR analysis and further confirmed by Southern blotting analysis (Fig. 2.1B). PCR screening in ES cells were performed for both short arm and long arm homologous

recombination. For Southern blotting analysis, genomic DNA was digested with NsiI, blotted, and hybridized with a 32P-labeled probe (600 bp) 5’ outside the region encompassed by the targeting construct. The band was visualized using a Storm 860 Phosphorimager (Molecular Dynamics; Sunnyvale, CA). Aldh1b1(+) and Aldh1b1(-) alleles gave rise to a 7.9-kb and 3.8-kb band, respectively.

Genotyping in offspring was performed by PCR analysis using tail genomic DNA (Fig. 2.1C). The Aldh1b1(+) allele was detected using forward primer 5’-

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24 ACACTGCAACAGGAGGACCAAGAA-3’) and reverse primer

5’-ACATGCCCAATGACCTCACCT-3’, generating a 429 bp product. The Aldh1b1(-) allele was detected using same forward primer as (+) allele and reverse primer 5’-TTAAACGCGGCCGCCAATTGT-3’, generating a 200 bp product.

Quantitative real time PCR (qRT-PCR)

Total cellular RNA from selected tissues was extracted using TRI reagent (Sigma, St.Louis) and further purified with an RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. For qPCR analysis, cDNA was produced from equal amount of total RNA using Maxima First-Strand cDNA Synthesis kit (Fermentas, K1641), following manufacturer’s instructions. The mRNA levels of Aldh1a1, Aldh1b1 and Aldh2 were quantified using Power SYBR Green Master Mix (Applied Biosystems) with β-2 microglobulin (B2M) as an endogenous control. QPCR analysis was performed on a 7500 Real Time PCR System (Applied Biosystems) using primers (given in table 2.1) using thermal cycling conditions of 95 °C for 4 min, followed by 40 cycles of 95 °C for 30 s, 61 °C for 30 s for Aldh1b1 (55 °C for Aldh1a1 and 66 °C for Aldh2), and 72 °C for 30 s. The relative mRNA expression levels were calculated by 2− ∆∆CT method [126], where (– ∆ ∆ C T )= – ( ∆ C Tsample – ∆ C Treference); (∆ C T ) = CT (gene) – CT

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25 Table 2.1: Primers for QPCR analysis

Gene name Primer sequence (5'-3') Tm/℃℃℃℃ ALDH1A1 F: AGGCCCTCAGATTGACAAGGA 60.3 57.6 R: GTTGCACTGGTCCAAATATCT ALDH1B1 F: AGCGCGATTCGGAGCCTCA 61.9 61.5 R: TGACCGCATCATGCCACTCGT ALDH2 F: AGGTCTTCTGCAACCAGATCT 58.0 58.8 R: AGATGCATCCATGCGGCG B2M F: CATGGCTCGCTCGGTGACC 61.2 60.0 R: AATGTGAGGCGGGTGGAACTG

F, forward primer; R, reverse primer; Tm, melting temperature of the primer

Western blot analysis

For Western blot analysis, mice were sacrificed using CO2 asphyxiation

and tissues were snap frozen immediately using liquid nitrogen. Frozen tissues samples (100 mg) were homogenized in lysis buffer and incubated on ice for 10 min followed by centrifugation at 10,000g for 10 min at 4°C. Supernatant was collected and total protein was estimated using Pierce® BCA protein assay (Thermo Scientific, Rockford, IL; 23223 and 23224) using the manufacturer’s protocol. Proteins were boiled in Lammeli׳s buffer for 5 min and were subjected to 5% SDS-PAGE gels, and then transferred to nitrocellulose membrane. Membranes were blocked in Tris-Buffered Saline with 0.1% Tween 20 (TBST) containing 5% (w/v) nonfat dry milk and probed overnight by incubation with primary antibodies according to the manufacturer's instructions and followed by

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26 washing in TBST. ALDH1B1 was detected using rabbit polyclonal anti-ALDH1B1 antibody (1:5,000 in 5% nonfat dry milk in TBST) [12] and β-actin was detected using mouse monoclonal anti-β-actin antibody (1:10,000; Sigma, St. Louis, MO). Membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:5,000; Sigma, St. Louis, MO). After washing in TBST, membranes were

incubated in Pierce® ECL Western blotting substrate (Thermo Scientific Rockford, IL; 32106), and exposed to X-ray film to visualize protein bands. Protein

expression was normalized to β-actin expression.

Histopathological and Immunohisotochemical analysis

Various tissue samples were dehydrated in ascending concentrations of ethanol and cleared in xylene. Tissues were embedded in paraffin, 5 µM sections were cut and sections were mounted on poly-L-lysine coated glass slides. These tissue sections were used for hematoxilin and eosin (H&E), periodic acid-Schiff (PAS) and immunohistochemical staining after deparaffinization using xylene, followed by rehydration in a graded series of ethanol. H&E stained slides were used to examine the histology of liver, lungs, kidneys, gastrointestinal tract, pancreas, ovaries, testes and uterus. For immunohistochemistry, antigen retrieval was performed in 10 mM/l citrate buffer (pH 6.0) and 3% hydrogen peroxide (v/v) was used to block endogenous peroxidase activity. The sections were incubated with TNB blocking buffer (PerkinElmer, Waltham, MA). Sections

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27 were incubated with rabbit polyclonal anti-ALDH1B1 antibody [11], rabbit

monoclonal anti-Ki67, anti-insulin and anti-glucagon antibody (Abcam,

Cambridge, MA) at a 1:750 dilution in TNB blocking buffer overnight in humidified chamber. TNB buffer was used as a negative staining control. HRP-conjugated anti-rabbit secondary antibody was used at a 1:500 dilution in TNB buffer for 60 min and TSA biotin signal amplification system (PerkinElmer, Waltham, MA) was used following manufacturer’s protocol. Sections were visualized by incubation in DAB working solution (Vector laboratories, Burlingame, CA) or AEC substrate kit (BD Pharmingen, San Jose, CA) for 10 min at room temperature and

counterstained with diluted hematoxylin (1:10 with distilled water) for 2 min. Finally sections were dehydrated and mounted with Permount (Sigma, St. Louis) for microscopic examination. Slides with AEC were mounted without dehydration using aqueous mounting medium (Sigma, St. Louis).

Ethanol administration and blood collection

Eight w old Aldh1b1 (-/-) and Aldh1b1 (+/+) littermate (Sv129 and C57BL/6J mixed background) mice (n=6) were matched for body weight and administered with a single i.p. dose of 20 percent ethanol (5g/kg). Blood was then collected at 0, 1, 3 and 24 h from right atrium using a heparin-coated ice cold syringe, the blood was mixed immediately with pre cooled 0.6 N perchloric acid (PCA) solution. Tubes were weighed before and after blood collection to measure the exact amount of blood samples and samples were processed for GCMS analysis soon after collection.

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28 Figure 2.1. Generation of Aldh1b1(-/-) mouse line.

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29 Figure 2.1. Generation of Aldh1b1(-/-) mouse line. (A) Structures of Aldh1b1 locus, targeting construct and of the mutant allele resulting from the homologous recombination. Although Aldh1b1 has two exons, the entire coding region is located on exon2, making this an intron-less gene. Neomycin-resistance minicassette (NEO) and herpes simplex virus thymidine kinase minicassete (HSV-TK) were used as selection markers. Arrow direction of the NEO and HSV-TK minicassetes represents the 5’ to 3’ orientation of those genes. (B) Screening of three ES clones for homologous recombination by PCR and

Southern blotting; NC, negative control, (C) PCR analysis of DNA extracted from tails of offspring from Aldh1b1 (+/-) heterozygous crossing. Molecular weight marker (MW), 1 Kb plus DNA ladder (Invitrogen, NY).

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30 Ethanol and acetaldehyde concentration measurement

The PCA solution containing blood was centrifuged at 15000g for 10 min at 0°C and 300 µl of the supernatant was transferred into a 10 ml glass vial in the cold room and sealed with a gas-tight cap. Ethanol and acetaldehyde

concentrations were measured according to the previously described procedure by gas chromatography- mass spectrometry (GC-MS) [127, 128]. The vial with supernatant was placed in a heating block at 65°C for 10 min; the headspace gas was then transferred using a Combi PAL auto sampler (CTC Analytics, Zwingen, Switzerland) to the GCMS (QP2010, Shimadzu, Kyoto, Japan). Ethanol and acetaldehyde were separated on a 60 m x 0.25 mm inner diameter AQUATIC capillary column (GL Sciences, Tokyo) with 1 µM film thickness. The injection port temperature was kept at 200°C and column oven temperature was

programmed from 40°C to 50°C at the rate of 2°C per minute and from 50°C to 170°C at the rate of 40°C per minute. Helium was used as carrier gas at a flow rate of 1.0 ml per minute. For acetaldehyde, the split ratio of the carrier gas was 20:1 and mass spectrometry (MS) was carried out by electron impact ionization at 300 eV. For ethanol, split ratio was 100:1 and MS was carried out by electron impact ionization at 70 eV. The interface and ion chamber temperatures were maintained at 230°C and 210°C, respectively. A selected ion monitor (SIM) mode was set at 43 and 29 m/z for acetaldehyde and 45 and 46 m/z for ethanol.

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31 Glucose tolerance test

Six male mice (C57BL/6J background) from each group (Aldh1b1 (-/-) and Aldh1b1 (+/+)) were used for i.p. glucose tolerance test (GTT). Animals were fasted overnight with water available ad libitum. Mice were anesthetized with isoflurane anesthesia before collecting blood from the tail vein. Mice were injected with 15% sterile D-glucose (1.5 mg/g, i.p.) and blood samples were collected from a nick on the tail immediately prior to and 15, 30, 60 and 120 min after glucose administration. Blood glucose levels were determined using a glucometer (OneTouch Ultra, One Touch). Area under the curve (AUC) for glucose levels was calculated using trapezoidal estimation method using the results obtained in the GTT.

Statistical analysis

All quantitative experiments were performed at least in triplicate, and the data shown are the means ± S.E. Statistical significance was determined using Student’s t-test using SigmaStat Statistical Analysis software (SPSS Inc., Chicago, IL). P < 0.05 was considered to be significant.

Results Generation of Aldh1b1 (-/-) mice

The targeting construct was designed to disrupt the exon2 (2 Kb) of Aldh1b1 gene, resulting in the removal of the complete coding region of Aldh1b1 gene (Figure 2.1A). Independent ES clones harboring successful homologous

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32 recombination were confirmed by Southern blotting and PCR analysis (Figure 2.1B). Male chimeras generated from these ES clones were crossed with C57BL/6 females to generate heterozygotes for the Aldh1b1(-) mutant allele. Intercrossing of heterozygotes produced Aldh1b1 (+/+) wild-type, Aldh1b1 (+/-) heterozygous and Aldh1b1 (-/-) knockout littermates (Figure 2.1C). Offspring of the three genotypes were born with expected Mendelian frequencies, suggesting no embryonic lethality due to the global disruption of Aldh1b1 gene.

Phenotype of Aldh1b1 (-/-) mice

Mice homozygous for the disrupted Aldh1b1 (-) allele showed no expression of Aldh1b1 mRNA in liver and colon tissues by qRT-PCR analysis (Figure 2.2A). To see if there are any compensatory changes in other closely related ALDHs, we examined the mRNA expression of Aldh1a1 and Aldh2 in these organs. No difference in mRNA expressions of these ALDH enzymes was observed between Aldh1b1 (-/-) and Aldh1b1 (+/+) mice (Figure 2.2A). Loss of ALDH1B1 protein in Aldh1b1 (-/-) mice was confirmed by Western immunobloting (Figure 2.2B) and immunohistochemistry (Figure 2.2C). ALDH1B1

immunostaining was present in the bottom of the crypt of small intestine and colon from the wild-type (WT) animals but was lacking in those from the

Aldh1b1(-/-) mice (Figure 2.2C). This pattern of ALDH1B1 expression in intestinal tissues is consistent with what we reported in human tissues [12].

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33 Figure 2.2. Expression analysis of ALDH1B1 mRNA and protein in

Aldh1b1(-/-) mice. (A) Comparison of liver and colon Aldh1b1, Aldh1a1 and

Aldh2 mRNA expression levels by real time PCR analysis between Aldh1b1(+/+) and (-/-) mice. The level of mRNA expression was normalized to β-2

microglobulin (B2M) mRNA expression and expressed as ratio of that found in the Aldh1b1(+/+) mice (=1). Data are presented as mean ± SE; ND, not

detectable; n=4 mice. (B) Western blot analysis of liver, colon and small intestine lysates using anti-ALDH1B1 antibody revealed complete loss of ALDH1B1

protein. (C) Photomicrographs of mouse colon and small intestine after immunostaining with anti-ALDH1B1 antibody. Cells at the crypt base showed ALDH1B1 reactivity in Aldh1b1(+/+) but not in Aldh1b1(-/-) mice. Representative images are presented at two magnifications, with the squared field in the top panel (100x) enlarged in the bottom panel (400x).

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34 We observed these mice until 6 mo of age and found them to be overtly healthy. We also documented the growth curve of Aldh1b1 (-/-) and Aldh1b1 (+/+) WT littermates. Male Aldh1b1 (-/-) and Aldh1b1 (+/+) mice did not show any difference in body weight. However female Aldh1b1 (-/-) mice showed lower body weights between weeks 3 and 5: after that, they caught up the growth rate with their WT littermates (Figure 2.3). Both Aldh1b1 (-/-) male and female mice were fertile (data not shown). Histological analysis revealed no evidence for

morphological abnormalities among the organs examined including liver, lung, pancreas, ovaries, uterus, small intestine and large intestine. Photomicrographs of representative tissues are presented in Figure 2.4.

Figure 2.3. Growth curve of Aldh1b1(+/+) and Aldh1b1(-/-) mice. Body weight of Aldh1b1(+/+) and Aldh1b1(-/-) mice as a function of age. The data presented are mean ± SE. *, P < 0.05. Students t-test, compared to levels in WT mice at same time points (B) Representative H & E staining of the sections from small intestine, colon, rectum, lung, testes and uterus from Aldh1b1(+/+) (top panel) and Aldh1b1(-/-) (bottom panel) mice revealed similar cyto-architecture in both the groups.

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35 Figure 2.4. Aldh1b1(-/-) mice have normal cyto-architecture. Representative H & E staining of the sections from pancreas, liver, small intestine, colon, rectum, lung, testes and uterus from Aldh1b1(+/+) (left panel) and Aldh1b1(-/-) (right panel) mice revealed similar cyto-architecture in both the groups.

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36 Since ALDH1B1 was expressed selectively at the bottom of the crypt in the colon (i.e., putative stem cell compartment) and is a potential biomarker for colon cancer, we examined these organs for Ki-67 immunostaining, a marker for cellular proliferation. We did not find significant difference in intensity or extent of the immunopositivity for Ki-67 between colons from Aldh1b1(-/-) and WT mice (Figure 2.5). To examine the effect of ALDH1B1 knockdown on the

differentiation, we examined and quantified goblet cells following PAS staining. We did not find any difference in the number of goblet cells in colon crypts from Aldh1b1(+/+) and Aldh1b1(-/-) mice (Figure 2.6).

Loss of ALDH1B1 leads to decreased clearance of blood acetaldehyde After intra-peritoneal administration of 5 g/ Kg ethanol, the blood ethanol and acetaldehyde levels were determined by GC-MS at 0, 1, 3 and 24 h.

Representative chromatogram for blood ethanol and acetaldehyde are shown in Figure 2.7A. Ethanol and acetaldehyde were distinguished by different retention time and identical ions, ruling out their interference in each other’s measurement (Figure 2.7A). A best fit second order polynomial function was used to generate calibration curves for ethanol and acetaldehyde (Figure 2.7B). There were no differences in the blood ethanol levels in Aldh1b1(-/-) mice relative to their age- and weight-matched Aldh1b1(+/+) mice (Figure 2.7C, left panel). However, the blood acetaldehyde levels after 3 and 24 h of ethanol administration as well as area under the curve (AUC) for circulating acetaldehyde levels were significantly

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37 higher in the Aldh1b1(-/-) mice, indicating crucial role for this enzyme in the

acetaldehyde clearance (Figure 2.7C, right panel).

Figure 2.5. Lack of ALDH1B1 does not affect number of proliferating cells in colon. (A) Photomicrographs of mouse colon after immunostaining with anti-Ki-67 antibody revealed no difference in number of proliferating cells between Aldh1b1(+/+) and Aldh1b1(-/-) mice. Representative images are presented at two magnifications, top panel (100x) and bottom panel (400x). (B) quantification of Ki-67 positive cells in proximal and distal colon crypt from Aldh1b1(+/+) and Aldh1b1(-/-) mice.

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38 Figure 2.6 Lack of ALDH1B1 does not affect number of goblet cells in

colon. (A) Proximal and distal colonic crypts were stained for goblet cells using periodic acid-Schiff (PAS) stain that detect mucus secreting cells and these were quantified for Aldh1b1(+/+) and Aldh1b1(-/-) mice (B). Bars represent mean ± SEM (Student’s t-test), n = 3 mice per genotype.

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39 Figure 2.7. Pharmacokinetics of ethanol and acetaldehyde in Aldh1b1(-/-) mice. (A) Representative total ion chromatogram and selected ion monitoring profile of a blood sample for ethanol and acetaldehyde using GCMS. Ion structure was set at 45 and 46 m/z for ethanol and 29, and 43 m/z for

acetaldehyde. (B) Standard curve for ethanol (left panel) and acetaldehyde (right panel) measurement. (C) Blood ethanol and acetaldehyde levels in Aldh1b1(+/+) (closed symbol) and Aldh1b1(-/-) (open symbol) mice after ethanol administration (5 g/kg, i.p). Area under the curve (AUC) is presented at the upper right corner. *, P < 0.05; data represent Mean±SEM (n=6); **, P < 0.001, Student’s t-test,

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40

Aldh1b1(-/-) mice have compromised glucose homeostasis

Since ALDH1B1 is crucial for embryonic development of pancreas, we performed an i.p GTT to evaluate the effect of ALDH1B1 deletion on systemic glucose homeostasis. Aldh1b1(-/-) mice showed elevated fasting blood glucose levels accompanied by elevated blood glucose levels following i.p. glucose administration (Figure 2.8A-B). Higher AUC for glucose in the Aldh1b1(-/-) mice was recorded following i.p. GTT ( Figure 2.8 A and C). However, we did not observe any difference in the pancreatic cyto-architecture or immunostaining for insulin and glucagon in pancreatic islets of Aldh1b1(-/-) and Aldh1b1(+/+) mice (Figure 2.9).

Discussion

Aldh1b1 gene has two exons and an intronless coding region completely confined to exon2 [11, 129]. We have generated homozygous Aldh1b1(-/-) mice by deleting the entire coding region of the Aldh1b1 gene and backcrossed them into a C57BL/6 background. These mice did not show any reproductive,

developmental or anatomical abnormalities, indicating that ALDH1B1 is dispensable for development and survival. This may be explained by the overlapping physiological functions of ALDH1B1 with other ALDHs like ALDH2 (e.g., acetaldehyde metabolism) and other ALDH1 isozymes (e.g., retinaldehyde metabolism) [1, 24].

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41 Figure 2.8. Intra-peritoneal glucose tolerance test (GTT) in Aldh1b1(-/-)

mice. (A) Overnight fasted mice were given an i.p. injection of 15% glucose (1.5 mg/g of body weight). Blood samples were collected from tail vein before (0) and 15, 30, 60 and 120 min after glucose administration and analyzed for glucose concentration. Histograms showing fasting blood glucose (B) and area under the curve (AUC) for GTT (C) in Aldh1b1(-/-) and Aldh1b1(+/+) mice. Each data point represents mean ± SE (n=6 mice); *, P < 0.05; **, P < 0.001. Student’s t-test, compared to levels in Aldh1b1(+/+) mice at same time point.

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42 Figure 2.9. Photomicrographs of mouse pancreas after immunostaining for insulin and glucagon. Pancreatic islets of Aldh1b1(-/-) and Aldh1b1(+/+) mice showed similar staining for insulin and glucagon. Representative images are presented at 100x magnification.

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43 The lower body weight of female (but not male) Aldh1b1(-/-) mice without clinical or histological correlates is an intriguing finding, especially considering there was no weight difference in these female mice after 6 weeks of age. Further research is required to confirm whether the ALDH1B1 knockdown has any possible effects during early development in female mice or to rule this out as a spurious result. ALDH1B1 has been suggested to be a biomarker for colon cancer and Ki-67 is a nuclear protein found in proliferating cells [130], however we did not find any difference in the expression of this protein in colon of Aldh1b1(-/-) mice suggesting that there is no difference in the number and distribution of actively proliferating cells in the colon of these mice.

The mouse Aldh1b1 gene encodes a protein with 519 amino acids that has highest expression level in the liver and parts of small intestine (ileum and jejunum) and moderate expression in the colon [11]. The expression pattern of ALDH1B1 is similar to that of ALDH2, the most efficient acetaldehyde

metabolizing enzyme [11, 131]. Since ALDH1B1 is second only to ALDH2 in metabolizing acetaldehyde [11] and has been found to be associated with

ethanol-induced hypersensitivity [122] and ethanol-related hypertension [123] in humans, we hypothesized that Aldh1b1(-/-) mice may have reduced

acetaldehyde clearance. We did not find any difference in blood ethanol levels in Aldh1b1(-/-) mice relative to their Aldh1b1(+/+) littermates after i.p. ethanol. This was expected because this enzyme is not involved in conversion of ethanol to acetaldehyde. However, despite normal levels of ALDH2 (the principle

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44 acetaldehyde metabolizing enzyme), Aldh1b1(-/-) mice exhibited higher blood acetaldehyde levels after ethanol administration. These findings corroborate the important contribution of ALDH1B1 to acetaldehyde clearance and also helps explain the hypersensitivity observed in the human population with SNP

encoding ALDH1B1 (rs2228093) [122], which is a slow acetaldehyde metabolizer relative to WT ALDH1B1[13]. This raises an important question: why is it that ALDH1B1 does not offer compensation for the acetaldehyde metabolism in people carrying ALDH2*2 variant, i.e, those having the inactivating ALDH2 mutation [77, 132-135]? This could be explained by the recent finding based on computational molecular modeling that suggests that ALDH1B1 may form a heterotetramer with inactive ALDH2 mutant subunits, the result of which would be decreased ALDH1B1 catalytic activity [78].

High ALDH activity is used to isolate putative progenitor cells from the centroacinar compartment of adult mice pancreas; these cells are capable of generating endocrine and exocrine cells in culture [117]. Recently, we have found that ALDH1B1 is expressed in pancreatic progenitor cells of the developing embryo and in adult centroacinar cells in mice [14]. On the basis of these

findings, we anticipated Aldh1b1 (-/-) mice would have compromised glucose homeostasis. Our results revealed Aldh1b1 (-/-) mice to have higher fasting glucose and poor glucose tolerance, suggesting an important role for this enzyme in maintaining glucose homeostasis. However, pancreatic islets architecture in Aldh1b1 (-/-) mice was ostensibly similar to Aldh1b1(+/+) mice. The glucose intolerance could be caused by a change in the insulin levels. Also,

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45 overtly normal-looking islet with normal islet volume and β-cell area can have altered insulin secretion and/or islet microvascular morphology resulting into hyperglycemia and glucose intolerance [136, 137]. Further studies are required to ascertain the cause of compromised pancreatic function in Aldh1b1 (-/-) mice.

In summary, we showed that Aldh1b1(-/-) mice have slow acetaldehyde clearance and glucose intolerance. Slow acetaldehyde clearance support the contribution of ALDH1B1 in metabolizing acetaldehyde along with ALDH2. Glucose intolerance in Aldh1b1(-/-) corroborates our earlier finding suggesting role of ALDH1B1 in pancreatic development and confirms its role in the

Figure

Figure 1.1. ALDHs modulate carcinogenesis by metabolizing acetaldehyde  and retinaldehyde
Table 1.2: Affinity of ALDHs for acetaldehyde and retinaldehyde  S.No.  ALDH  isozyme(s)  Substrate K m Reference  1  ALDH1A1  Acetaldehyde  180 µM  [11]  All-trans  Retinaldehyde  9-cis Retinaldehyde  11.6-26.8 µM 3.59 µM  [79],[13]  2  ALDH1A2  All-trans
Figure 1.2. ALDH-expressing cells are responsible for chemoresistance and  relapse of many tumors after chemotherapy
Figure 1.3. ALDH1B1 expression pattern in normal colon and colon
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References

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