Studies of Protein S-nitrosylation in Prostate Cancer focused on Integrin Alpha 6, Proliferating Cell Nuclear Antigen and Estrogen Receptor Beta
By Jared Isaac, B.Sc. Biochemistry, Lee University, Cleveland, TN 2005
For partial requirement of Doctorate of Philosophy in Biomedical Research degree from the University of Cincinnati College of Medicine
Thesis Committee:
Shuk-Mei Ho, PhD; Thesis Advisor
Robert Brackenbury, PhD; Thesis Committee Chair
Susan Waltz, PhD
Shao-Chun Wang, PhD
Ying-Wai Lam, PhD
i
ABSTRACT
Prostate cancer is the second most common cancer and cause of cancer related death of men in the United States of America. Given the proportion of men who will be entering the fifth decade, the number of prostate cancer cases will only increase in the next 10-20 years. Therefore, it is imperative to understand and develop treatment for prostate cancer.
Prostate cancers typically have the accumulation of free radicals such as reactive oxygen and nitrogen species. These free radicals can damage the cell and alter its normal cellular processes. The purpose of this body of work is to understand the affect of one free radical, nitric oxide (NO) and how it affects the function of proteins by post- translationally modifying them in a process called S-nitrosylation.
Chapter 1 gives a brief background on the prostate’s function in the reproductive system, diseases that affect the prostate, prostate cancer, the role of reactive nitrogen species in the prostate, S-nitrosylation, and a mass spectrometry based profiling study identifying S-nitrosylated proteins in the normal prostate epithelial cells. Chapter 2 focuses on integrin alpha 6, ITGα6, which becomes S-nitrosylated after exposure to iNOS. This chapter shows how S-nitrosylation of ITGα6 cysteine 86 by iNOS increases
ITGα6 mediated prostate cancer cell migration by increasing its heterodimerization with
ITGβ1 and decreasing its adhesion to laminin-β1 chain. Chapter 3 examines another identified S-nitrosylated protein, proliferating cell nuclear antigen, PCNA in prostate cancer. Doxorubicin causes PCNA S-nitrosylation in prostate cancer cells by
ii upregulating iNOS. When PCNA cysteine 81 becomes S-nitrosylated this causes
PCNA to bind to the chromatin and arrest DNA synthesis. Chapter 4 is preliminary work in which estrogen receptor beta is shown to be S-nitrosylated in androgen independent cells after treatment with estrogen. Cysteines 108, 209, and 512 were identified to be S- nitrosylated by mass spectrometry and iNOS expression inhibits expression of an ERβ regulated gene, vitellogenin. Chapter 5 summarizes the results, limitations, future directions and discusses the prospect of preventing S-nitrosylation in the treatment of prostate cancer.
This thesis shows for the first time how elevated levels of nitric oxide affect protein function by S-nitrosylation in the background of prostate cancer. The data presented here show that with different stimuli, three proteins important for prostate cancer progression, ITGα6, PCNA and ERβ can all become S-nitrosylated. ITGα6 S- nitrosylation lends support to similar studies by showing S-nitrosylation promotes prostate cancer progression by increasing prostate cell migration which has clear implications in prostate cancer metastasis. However, future experiments are needed to conclusively determine whether S-nitrosylation promotes or inhibits PCNA and ERβ function in prostate cancer. Animal studies and staining of patient biopsies are preferred before the development of potential clinical treatments modulating cellular S- nitrosylation. In conclusion, reducing the level of S-nitrosylation remains a potentially attractive target for the treatment of advanced stage prostate cancer.
iii
iv
ACKNOWLEDGEMENTS
Thesis Mentor
Dr. Shuk-Mei Ho:
Thank you for molding me into the scientist that I am today. I am truly thankful for the opportunity to learn all the techniques that were available in the lab and the freedom to pursue my research project in S-nitrosylation. The high standard you held me to taught me to reach my potential. You have molded my scientific thinking and training and I will take the lessons that I have learned with me forward in life. I will always remember my time in your lab fondly, and will miss the discussions, lunch-time parties, and lab meetings. I have been fortunate to have you to facilitate my experiment planning, troubleshooting, networking and access to resources in the department and across the university. In the beginning you warned me that my thesis project was high- risk/high-gain, but in retrospect, it was the best choice for me to develop my critical thinking skills to become an independent investigator. You have always been extremely supportive of new directions as long as I can defend my rationale.
Aside from my bench work, you developed my professionalism. My first test was to pass my PhD qualifying exam. This was a significant hurdle for me and you supported me through each step. Not long afterwards, I was awarded a departmental
NIEHS pre-doctoral training award. During this time, you encouraged me to think critically and apply for my own grant funding through outside funding agencies. My crowning achievement was being awarded my own funding which would not have happened without your support and encouragement. I owe my maturation as a young
v scientist to you, and for that I am thankful. As your student I have had extraordinary opportunities, allowing me to brush shoulders with leaders in biomedical research as well as to learn the latest techniques. In retrospect, the opportunities in your lab were tremendous. With my extensive training and specialization in proteomics and cell biology from Dr. Shuk-Mei Ho, I believe I am well prepared for my future career in science. I am grateful for the opportunity to pursue my doctoral thesis research under your guidance with your extensive experience in prostatic diseases.
Thesis Committee Chair
Dr. Robert Brackenbury:
Your expertise in cell-cell interactions in normal development and invasion of tumor cells has definitely been invaluable in my studies in cell migration in the ITGα6 S- nitrosylation project. You were one of the reasons I chose the Cancer and Cell Biology program because of your enthusiasm for the program and biomedical research in general. You have been one of the best teachers I have ever had and I enjoyed the classes that you taught at UC. You have been very encouraging and supportive throughout my thesis study in the classroom as well as serving as my thesis committee chair.
Thesis Committee Members
Dr. Susan Waltz:
Your expertise with endocrine related cancers provided a different prospective to my studies in protein S-nitrosylation in prostate cancer. Your expertise in tumor
vi formation and metastasis has been invaluable. Your planning and advice skills have provided clear solutions when problems, roadblocks and dead-ends were encountered.
Thank you for your support in the thesis committee meetings as well as being my co- mentor for my training grant.
Dr. Shao-Chun Wang:
I thank you for being approachable and available to discuss my data for the
ITGα6 and PCNA papers. Your honesty, helpfulness, and expertise of PCNA phosphorylation have been invaluable for my studies of PCNA S-nitrosylation. I have great respect for your accomplishments and your research is a model for my future scientific endeavors.
Dr. Ying Wai Lam:
Thank you for taking the time to teach me as your colleague. The highlights of my time at UC were definitely our conversations and working closely with you in the lab until late hours of the night. Thank you for showing me the importance of hard work, time management and literature review. I cannot express how thankful I am that you took the time to teach me mass spectrometry, the biotin switch technique and HPLC.
Your example of diligence and perseverance will lead me during my scientific career.
vii
Professors, Post-docs and Students
Dr. Pheruza Tarapore:
I thank you for your willingness to help with my research. I value your input and ideas for experiments. It is because of your shared ideas that the S-nitrosylation functional study papers are a reality. Your enthusiasm for research and critical thinking inspire my own motivation in the lab. In my future position, I will remember what you have shown me about cell biology, immunofluoresence, microscopy and scientific writing.
Dr. Jarek Meller, your expertise using bioinformatic analysis has been invaluable to predict functional aberrations of proteins induced by S-nitrosylation.
Dr. Xiang Zhang, thank you for your insights and discussion of how I should mutate ITGα6 and your mutation of PCNA.
Dr. Ricky Leung, thank you for sharing your expertise of ERβ functional assays to determine the role ERβ S-nitrosylation.
Dr. Neville Tam and Saikumar Karyala, M. Sc., thank you for your input and suggestions to better develop my projects.
Dr. Ming Lam, thank you for providing your reagents and experience of lentiviral production and cell migration and invasion assays.
Ming-Tsung Lee, M. Sc., thank you for your discussions and sharing your experience and reagents. I am especially thankful for you conducting the ERβ transactivation assays.
Dr. Yong Yuan, thank you for sharing your experimental parameters of how you identified the S-nitrosylation sites of ERβ.
viii
Cancer and Cell Biology and Biomedical Flex Programs
I thank the Biomedical Flex program for allowing me the opportunity to pick my lab rotations from every available lab and supporting me until choosing the Cancer and
Cell Biology program. I am thankful to the Cancer and Cell Biology program for my support and fostering an environment of scientific comradery.
Funding Agencies
I am indebted to the following U.S. agencies that provided my salary to carry out the studies outlined in this dissertation through the following training grants:
National Institute of Environmental Health Sciences Pre-doctoral training grant.
Army Department of Defense Congressionally Directed Medical Research Program predoctoral training grant.
Family and Friends
Catherine, Barbara, Donald, and Magdalene Isaac; Elaine and Jack Ginn; Andy and Bob Osborne; Matthew Loftspring; Nicholas Jury; Devikala Gurusamy; and Andrew
Paluch.
ix
CONTRIBUTIONS
Ying Wai Lam identified the S-nitrosylation sites of ITGα6 and PCNA (Figures
2.9A and 3.4A), modeled the S-nitrosylation sites of PCNA in Rasmol (Figure 3.4B), showed the iNOS mediated S-nitrosylation of PCNA (Figure 3.3D) and conducted
PCNA chromatin binding experiments in the presence of doxorubicin and GSNO and
CysNO (Figure 3.5A and 3.5B). Pheruza Tarapore did the immunofluoresence staining of PCNA in (Figure 3.5E). Xiang Zhang mutated cysteines 81, 135 and 162 in PCNA.
Three dimensional modeling of ITGα6 was done by Jarek Meller (Figure 2.9E). Liam
Lee conducted the ERβ reporter assays. (Figure 4.2D and 4.2E). Yong Yuan identified
ERβ to be S-nitrosylated by LC-MS. All other data presented here are the original work of Jared Isaac.
x
TABLE OF CONTENTS
Page Section
1 Chapter 1: General Introduction and Specific Aims
1 Prostate Location and Function in the Male Reproductive System
2 Prostate Development and Senescence
4 Diseases of the Prostate
4 Inflammatory Disorders
5 Benign Prostatic Hyperplasia
7 Prostatic Intraepithelial Neoplasia
7 Prostate Cancer
13 Prostate Cancer and Reactive Oxygen and Nitrogen Species
14 Nitric Oxide
18 Post-Translational Modifications of Proteins
S-nitrosylation
21 Comprehensive Identification and Modified Site Mapping of S-nitrosylated
Targets in Prostate Epithelial Cells.
23 Dissertation Projects and Specific Aims
26 Chapter 2: Site-specific S-nitrosylation of Integrin α6 Increases
Prostate Cancer Cell Migration by Increasing Integrin β1 Association
and Reducing Adherence to Laminin-1
26 Integrin α6 Literature Review
30 Abstract
30 Introduction
xi
32 Materials and Methods
35 Results
47 Discussion
51 Chapter 3: Proliferating Cell Nuclear Antigen is S-nitrosylated by
iNOS after Doxorubicin Treatment which Increases PCNA Interaction
with Chromatin.
52 Proliferating Cell Nuclear Antigen Literature Review
53 Proliferating Cell Nuclear Antigen Cointeracting Proteins
55 Proliferating Cell Nuclear Antigen PTMs
55 Abstract
56 Introduction
60 Materials and Methods
64 Results
73 Discussion
77 Chapter 4: Estrogen Receptor β S-nitrosylation in Prostate Cancer
77 Estrogen Receptor Function in the Prostate
78 Abstract
79 Introduction
80 Materials and Methods
81 Preliminary Results
83 Discussion
84 Chapter 5: General Discussion
84 Summary of Data
xii
85 Experimental Limitations
88 Future Directions
91 Discussion and Perspective
95 References
xiii
LIST OF FIGURES
Page Figure Description
16 Figure 1.1. iNOS is composed of oxygenase, calmodulin (CaM) and reductase domains
and requires cofactors FMN, FAD and NADPH.
17 Figure 1.2. iNOS signaling in the PCa microenvironment.
20 Figure 1.3. The biotin switch technique (BST) is used to detect labile S-nitrosylated
proteins.
27 Figure 2.1. Post-translational modifications of ITGα6.
28 Figure 2.2. Integrins α6 and β4 (ITGα6/β4) heterodimerize to form the hemidesmosome.
29 Figure 2.3. Signaling pathways downstream of ITGα6β4.
36 Figure 2.4. iNOS increases the migration of prostate cancer cells.
37 Figure 2.5. Nitrite increases in cell medium after iNOS transfection.
38 Figure 2.6. iNOS- and NO-mediated cell migration is blocked by 1400W or GoH3,
respectively.
39 Figure 2.7. ITGα6 interacts with iNOS and is S-nitrosylated with increased NO or iNOS
expression in cellullo.
40 Figure 2.8. ITGα6 is S-nitrosylated in LNCaP and DU-145 cells.
42 Figure 2.9. iNOS S-nitrosylates ITGα6 at Cys86.
44 Figure 2.10. NO mediates migration through ITGα6 Cys86 and promotes increased
ITGβ1 heterodimerization.
45 Figure 2.11. GoH3 does not block cell migration of PC-3/C86S cells in the presence of
100 μM GSNO.
46 Figure 2.12. Cys86 is responsible for NO destabilization of ITGα6-mediated cell
adherence.
xiv
50 Figure 2.13. Proposed model of how iNOS may affect ITGα6 binding to Laminin-1 and
heterodimerization to ITGβ4 and ITGβ1 resulting in decreased adherence and increased
cell migration.
65 Figure 3.1. PCNA is S-nitrosylated by doxorubicin in CRPC cells.
67 Figure 3.2. Doxorubicin induction of iNOS results in PCNA S-nitrosylation.
69 Figure 3.3. PCNA is S-nitrosylated on Cys81 and Cys135 by CysNO and doxorubicin,
respectively.
71 Figure 3.4. Doxorubicin induces PCNA chromatin binding.
72 Figure 3.5. PCNA Cys81 confers resistance to doxorubicin.
74 Figure 3.6. Doxorubicin induction of iNOS S-nitrosylates PCNA leading to increased
PCNA chromatin binding.
78 Figure 4.1. ERβ Functional Domains
82 Figure 4.2. ERβ is S-nitrosylated in-vitro by NO donor and 17-β-estradiol.
xv
LIST OF TABLES
Page Figure Description
11 Table 1.1. Current treatment regimen for prostate cancer (PCa).
54 Table 3.1. PCNA elicits divergent cellular responses through interaction with different proteins.
xvi
LIST OF ABBREVIATIONS
Abbreviation Expansion
Akt Protein kinase B mToR Mammalian target of rapamycin
AR Androgen receptor
BCa Breast cancer
Bcl-2 B-cell lymphoma 2
BPH Benign prostatic hyperplasia
BST Biotin switch technique
Cys L-cysteine
CysNO S-nitrosocysteine
DAF Diaminofluorescein
DU-145 Human prostate cancer cells metastasized to brain
E2 17- estradiol
EGF Epithelial growth factor
EGFR Epidermal growth factor receptor
ERK Extracellular signal-regulated kinase
FAD Flavin adenine dinucleotide
FMN Flavin mononucleotide
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
xvii
GSH Glutathione
GSNO S-nitrosoglutathione
GST Glutathione S-transferase
HEK-293 Human embryonic kidney cell line 293
HIF1-α Hypoxia inducible factor 1 alpha
IGF-1 Insulin growth factor 1
IHC Immunohistochemistry
ITGa6 Integrin alpha 6
MAPK Mitogen activated protein kinase
MAPK Mitogen-activated protein kinase
MKP-1 Mitogen-activated protein kinase phosphatase-1
NADPH Nicotinamide adenine dinucleotide phosphate-oxidase
NF B Nuclear factor kappa-light-chain-enhancer of activated B cells
NPrEC Normal prostate epithelial cell line
OS Oxidative stress
PAP Prostatic acid phosphatase
PC-3 Human prostate cancer metastasized to bone cell line 3
PCa Prostate Cancer
PCNA Proliferating cell nuclear antigen
PI3K Phosphatidylinositol 3-kinase
PIA Prostatic intraepithelial atrophy
PIN Prostatic intraepithelial neoplasia
PSA Prostate specific antigen
18
Raf RAF proto-oncogene serine/threonine-protein kinase
RNS Reactive nitrogen species
ROS Reactive oxygen species
SNO S-nitrosothiols
SNP Single-nucleotide polymorphism
TGFβ Transforming growth factor β uPA Urokinase plasminogen activator
19
Chapter 1: General Introduction and Specific Aims
Prostate Location and Function in the Male Reproductive System
The prostate is a male specific sexual organ, and its main function is secretion of seminiferous fluid comprising 20-30% of total ejaculate of which another 60% is produced by the adjacent seminal vesicles. The seminiferous fluid facilitates ovum fertilization and sperm delivery and survival. Included in seminiferous fluid is prostate specific antigen, PSA, an enzyme that loosens semen facilitating sperm migration and neutral epithelial mucins. Seminiferous fluid contains citrate and fructose which are important energy sources for sperm. The remaining ejaculate and sperm come from the testicles, which reside outside of the body in the scrotum. Even though it is a male sexual organ, the prostate can be removed without compromising male fertility.
The adult human prostate is a small, round secretory gland residing within the lower abdomen. It is compromised of a network of ducts that empty seminiferous fluid into the urethra and ejaculatory duct. The prostate is located in front of the rectum and directly underneath the bladder and seminal vesicles with the urethra running through prostate and penis from the bladder. Neurovascular bundles encircle the prostate and control penile erection. The structural organization of the prostate was historically divided into lobes: an anterior, middle, two lateral, and a posterior. However, separate lobes within the prostate were unable to be identified; this being supported by the fact that hormone induced histologic changes and diseases had no regard for the lobe
1 designations. Current medicine divides the prostate into five histologically distinct regions which are the proximal, peripheral, periurethral, dorsal, and ventral zones in addition to the anterior fibromuscular stroma, the ejaculatory duct and the veromontanum. The anterior zone is now the anterior lobe, which is principally composed of fibromuscular stroma and few prostatic glands. The peripheral zone is composed of the former lateral and posterior lobes and contains approximately 75% of the glandular component of the prostate and contains simple glands and loose stroma.
The central zone is located between the ejaculatory ducts and is separated from the peripheral zone by fibrous tissue. The periurethral glands are confined to a sleeve of the proximal urethra, and the transitional zone, contains glands that terminate in the proximal urethra and lies anterior to the central zone.
Prostate Development and Senescence
The human prostate develops from the ectoderm, and it arises from the epithelial layer from protrusions along the distal prostatic urethra during the third gestational month. These protrusions extend from the urethra as five independent bundles of tubules that form the prostate. During the postnatal growth period, prostatic glands become clustered in a fibromuscular stroma that is connected to the surrounding fibrous capsule. A single layer of epithelial cells encircle the secretory lumen. These epithelial cells are columnar and pseudostratified and are supported by adjacent basal cells layer and encapsulated by the basal lamina. This structure forms the prostatic acinus, of which there are 20-30 acini within the adult prostate. Simple epithelia line the prostatic
2 excretory ducts until reaching the urethra, where transitional epithelia of the urethra predominate. The fibromuscular stroma contains smooth muscle, collagen, and elastic fibers in which encircle the acini and ducts. Occasionally, skeletal muscle is found in the anterior and lateral zones of the prostate.
Starting in puberty and through adulthood, the prostate develops through the action of androgens, particularly of 5 -dihydrotestosterone (DHT) to an average weight of 20 grams. It is at puberty and not prior, that expression of prostate specific antigen occurs in the epithelial cells. Testosterone in the prostate is converted to DHT by 5α- reductase and binds to the androgen receptor (AR) which transcribes genes in the nucleus by binding to chromatin. DHT specific gene activation results in selective protein expression followed by increased cell proliferation and prostate cell growth.
Castration before puberty renders men immune to prostate cancer and causes prostatic atrophy, which can be reversed with exogenous DHT. Estrogen is also important in neonatal prostate development as estrogen receptor beta (ERβ) becomes expressed during early ductal morphogenesis 1, and is strongly associated with the squamous metaplasia in the distal periurethral ducts 2. Both androgens and estrogens are important in the developing prostate and thus are sensitive to exogenous endocrine disruptors which can increase the risk of developing PCa later in life.
However, DHT can be converted to estrogen through the expression of aromatase enzyme, and in the geriatric prostate as DHT levels decrease while 17- - estradiol (E2) increases 3. The ratio of estrogen to androgen in geriatric men is altered
3 by two metabolic processes androgen glucoronidation and increased aromatase activity
4. Changes in adiposity, testicular function, sex hormone binding globulins, and extragonadal aromatization contribute to exaggerating the ratio of estrogen to testosterone 5–9. Many newborn males display squamous metaplasia from maternal estrogen which regresses rapidly during infancy 2. The adult prostate also retains estrogen sensitivity because aberrant basal cell growth is witnessed after estrogen treatment for prostate cancer 1. However, as men age their testosterone levels drop and estrogen levels increase. At midlife the prostate undergoes either progressive atrophy or hyperplasia. The majority of elderly men have both atrophy and hyperplasia with half of their prostate acini obliterated. The remaining acini are reduced in size the epithelial cells are flattened or cuboidal and produce less seminiferous fluid. The collagen supporting the stromal cells becomes replaced with muscle. Acini which display hyperplasia are typically surrounded by atrophic cells. These glands lined with hyperplastic cells have been hypothesized as potential foci of prostate adenocarcinoma.
Diseases of the Prostate
Inflammatory Disorders
Acute prostatitis is commonly seen in older men and caused by gram-negative bacteria such as E. coli by reflux of infected urine into the prostatic ducts. Features of acute prostatitis include inflammatory infiltration of the prostatic acini and stroma.
Proliferative inflammatory atrophy (PIA) is a condition that is commonly seen in the
4 prostates of aging men 10. Atrophy involves the disorganization and shrinkage of the prostate. Chronic inflammation is commonly seen in the peripheral zone of the prostate, the site at which most prostatic carcinomas occur. Inflammation is being recognized more broadly as affecting the progression of many types of cancers 11–13. Inflammation can occur in the prostate through multiple agents such as viral or bacterial pathogens, urine reflux, dietary estrogenic compounds, and trauma 10. Hormones are also thought to cause chronic inflammation which could be an early step in prostate cancer progression 14–17. Macrophages and neutrophils in addition to extravasation to the site of damage can produce chemokines, cytokines and reactive oxygen and nitrogen species which can contribute to prostatic disease 10.
Benign Prostatic Hyperplasia
80% of men over 70 years of age experience abnormal swelling and growth of the prostate gland 18. This growth can cause difficulty in urination because of blockage or constriction of the urethra. However, this growth can be designated as a more insidious benign prostatic hyperplasia (BPH) 19. This growth is mainly comprised of the proximal prostate. The majority of men in the western world will develop BPH midway through life, which can progress to prostate cancer. BPH causes difficulty in urine clearance because the spreading prostatic lobes wrap around the urethra leading from the bladder. Age, race, and geography are the leading epidemiological factors related to the incidence of prostatic hyperplasia. The incidence of BPH progressively increases with age and 75% men at age 80 have BPH 20. Persons of Western European heritage
5 have the highest incidence of BPH compared to Asia with the United States being intermediate. In the United States, BPH is much more prevalent in African-Americans than Caucasians and Asians. However, the incidence of BPH in all groups is far greater at autopsy than is suggested, but the disorder is rarely observed in men younger than
40 years of age.
The etiology of BPH is unknown and was previously thought to be a neoplastic, inflammatory or vascular disorder. Interplay between the stromal-epithelial compartments may contribute to BPH, but it is unknown which compartment initiates the abnormal growth as pure hyperplasia is seen for both thus their relationship remains speculative. The role of testosterone in normal development is well documented but for
BPH its function is unclear as castration has limited efficacy in limiting BPH. Prostatic hyperplasia can be produced in canines through administration of DHT. BPH starts in the transitional zone of the prostate and compresses and distorts the urethra. In developed BPH, nodes are wrapped with a fibrous pseudocapsule and are composed of acinar epithelia, smooth muscle, and stromal fibroblast cells in varying proportions. In the most common fibromyoadenomatous nodules, the epithelial cells are tall, columnar and overlying the basal cell layer. The stromal cells of nodular hyperplasia all lack elastic tissue, and the ratio of epithelia to stroma widely varies with intermixed smooth muscle. In general, glandular acini are indiscriminately distributed in the stroma of a hyperplastic nodule. Histologic features of BPH are chronic inflammation, corpora amylacea within the glandular acini, and cystic dilation of ducts. Involved acini are often compressed and atrophic due to the expanding nodules. BPH will positively stain with
6
PSA and prostatic acid phosphatase, and 10% of operative BPH will also contain foci of prostatic adenocarcinoma.
Prostatic Intraepithelial Neoplasia
Increased epithelial cell proliferation surrounded by acini is called prostatic intraepithelial neoplasia (PIN), and is histologically graded into low and high grade PIN lesions 21. Low grade PIN does not have elevated PSA levels or normally show symptoms, signs or have any clinical significance. PIN is considered a malignant precursor because it shares cell morphology, genetics and molecular status with invasive PCa 22. However, it is unknown how high-grade PIN loses contact with the basal cells and becomes invasive to progress to PCa.
Prostate Cancer
Prostate cancer (PCa) causes an estimated 26,000 deaths yearly in American men 23. It is also the second most common cancer and cause of cancer-related death in the United States. One in six men will be diagnosed with this disease and 1 in 35 men will die from this cancer 22, 23. PCa incidence increases dramatically for men after the fifth decade, and the greatest risk factor is age 22. PCa is a disease of geriatric men with 75% of clinically apparent PCa diagnosed between the ages of 60-79. Patients younger than 50 years of age constitute less than 1% of cases of PCa in the United
States. However, at autopsy, the true frequency of developing prostatic carcinoma for
7 men 40 to 50 years is one in three and one in two for men over the age of 80 years.
Caucasian and African-American men past the age of 50 years have an estimated lifetime probability of developing clinically apparent PCa of 9.5% and 11.4%, respectively 24. The highest PCa mortality rates are reported in the United States and
Scandinavia, intermediate rates are in Western Europe and the lowest rates are in
Mexico, Greece, and Japan. Interestingly American groups of Polish, African or Asian descent have higher rates than their familial country of origin. The mortality rate from prostatic carcinoma among African-American men is among the highest in the world, significantly exceeding that of Caucasian and Asian-Americans 25.
Changes in hormone levels, andropause, cadmium, viral and bacterial infections, diet with charred meats, physical trauma, and urine reflux are all potential causes of
PCa 10, 26–28. Of all the potential etiological factors, much investigative research has been given to the influence of hormones on PCa production. Androgen control of normal prostatic growth and the responsiveness of PCa to physical castration and exogenous estrogens have well established the role of hormones in PCa progression 29. While a correlation between high androgen levels and PCa risk has not been established, several epidemiological studies have shown the importance of estrogen in PCa progression 4,22,26,30. Altered estrogen/testosterone ratios of up to 40% have been reported in the serum of PCa patients; however, tissue levels of estrogen may be a superior prognostic indicator to serum levels5, 31–33. Both epithelial and stromal compartments in the prostate can produce estrogen de novo during PCa progression 34,
8
35. Estrogen itself can be a carcinogen through its derivation to 2- and 4-hydroxyl catechol estrogens and their quinone/semiquinone intermediates 36.
Experimental animal models support the role of hormones in prostatic neoplasia and aberrant proliferation 22, 30 because estrogen and/or testosterone can cause permanent changes in hormone receptor expression as well as mimic the stromal atrophy and epithelial hypertrophy of geriatric humans37, 38. More importantly, these late stage effects were also seen by perinatal and neonatal exposure of rats 39 and mice 40.
Chronic exposure of rats and mice to DHT and 17 β estradiol (E2) can promote development and progression of prostatic disease that is similar to humans 41, 42.
Additionally, aromatase knockout mice have shown increased levels of DHT were elevated and they developed BPH but not PCa 43.
Several studies have identified tumorigenic changes at the molecular level which promote prostate carcinogenesis. TMPRSS2: ETS gene fusions and deletion of PTEN tumor suppressor are frequent in advanced PCa and metastases 44, 45. PCa promote their survival by the upregulation of anti-apoptotic mediators 46–48 and increase their proliferation by upregulating expression of IL-1B and TNF-α 49, 50. Increase in growth factor production involving estrogen 51, IGF-1 52, TGFβ 53, and EGF 54 as well as downstream signaling through MAPK 55 are also implicated in tumor promotion.
Even though PCa can be found around areas of nodular hyperplasia and atrophy a direct link between BPH, PIN or PIA and PCa is unsubstantiated. The current
9 hypothesis is that PCa arises from hyperplastic acini residing in areas of focal atrophy because these foci occasionally have cytological atypia. PSA and PAP IHC staining is used for identifying normal, hyper and neoplastic cells of prostatic origin and is extremely helpful for identifying distant PCa metastases. However if the PCa is of poorly differentiated origin these stains will be negative for primary and metastatic sites.
Prostate cancer which is organ-confined is treatable by surgery or brachytherapy or a combination of both (Table 1.1.). However, without early detection the cancer can metastasize resulting in higher patient morbidity. Metastases to distal organs and regional lymph nodes decrease patient survival. Most prostate cancer occurs in the peripheral zone which is the closest to the rectum. Initial detection of PCa by rectal examination is difficult, but detection of extraprostatic spread has been made easier using computed tomography scan. PSA and PAP screening has increased the detection of PCa but the number of false positives has also increased. Thus, a better serum marker besides PSA is needed. Prostate cancer is manageable if detected early enough and if it is localized to the prostatic capsule. PCa tumors are graded in clinical stages. The first stages, Stage A, are foci residing in clinically benign prostatic tissue and are detected with 25% frequency. Stage A1 is when the tumor foci are localized to a particular region; if there is spread to multiple prostatic zones, it is designated A2.
Tumors with clinically palpable nodules which are confined to prostatic capsule are designated Stage B. Tumor associated with clinically palpable nodules but still confined to the prostate is term clinical Stage B and represent 30% of all clinically detected PCa.
Focal lesions are Stage B1, and if one or more of the prostate zones is involved, the
10
Stage is B2. 15% of PCa patients present with local invasion outside of the prostatic capsule but not to regional lymph nodes or distant sites, this is designated Stage C.
The remaining of PCa patients present with pelvic lymph node metastasis or distant metastases are Stages D1 and D2, respectively. However, Stage D PCa may present as an “occult carcinoma” in which symptoms from distant metastases present first with their organ of origin identified afterward by IHC.
Table 1.1. Current treatment regimen for prostate cancer (PCa).
Due to frequent subcapsular location there is a high frequency of adenocarcinoma invading the prostatic capsule as well as peripheral nerves. Neural and seminal vesicle invasion commonly occurs through lapse in tissues thereby offering the path of least resistance. Direct invasion of the bladder occurs late in PCa progression and invasion of the rectum is uncommon. Distant metastases are commonly seen at autopsy. Clinical studies have shown that PCa metastases commonly occurring in the obturator lymph nodes extravasate to the iliac and periaortic lymph nodes. Metastases to bone (vertebral column, rib and pelvis) are osteoblastic and a common clinical problem resulting in extreme pain for patients. Metastases then progress through the lymphatic system and the inferior vena cava and prostatic venous plexus and ending in the lungs. At the time of autopsy, PCa metastases are found most commonly in the
11 lymph nodes, bones, lung, and liver. Widespread dissemination of the tumor
(carcinomatosis), frequently with terminal pneumonia or sepsis is the most common cause of death.
Prostatic adenocarcinoma commonly are multi-centric, well-differentiated, found within the peripheral zone and account for 98% of all primary prostatic tumors. These cells often have a high proliferative index 56, experience oxidative stress 57 and lose connection the extracellular matrix and basal membrane 58. Well differentiated tumors show medium- or small-sized glands lined by a single layer of uniform neoplastic glandular epithelium. Dedifferentiation of prostatic adenocarcinoma occurs by variability of gland size, configuration and intraglandular epithelial proliferation. Rarely is a tumor composed of small undifferentiated cells growing individually without structural organization. However, if this is the case, necrosis will be abundant in the tumor and surrounding normal tissue. Prostatic tumor differentiation is graded histologically by the dedifferentiation of the tumor glands which has prognostic value. Even though cytologic features are not included in the criteria for grading, tumor cells exhibit nuclear polymorphism and hyperchromatism. The most common feature is masses of chromatin localization in nucleoli. PCa cell cytoplasm may be vacuolated and stain slightly eosinophilic with distinct cell borders in well-differentiated tumors. When diagnosing
PCa, a single layer of cuboidal cells in neoplastic acini is the most frequently used method.
12
Even though 98% of PCa arise from acinar, glandular, epithelial cells there are other rare subtypes arising from transitional, ductal, squamous and mesenchymal subtypes. The transitional PCa arise from the distal prostatic ducts, proliferate in solid patterns, and are hormone insensitive, have a poorer prognosis and osteolytic metastases. Ductal PCa have a papillary arrangement and their natural history is unknown. Squamous PCa site of origin and histogenesis is unknown but are known to be estrogen insensitive. Rarely in PCa there is a mix of glandular, ductal or transitional cells. Mucinous carcinoma are composed of acini of varying size filled with extracellular mucin. PCa of mesenchymal origin contain smooth and skeletal muscle cells and usually are found in men less than 50 years of age. However, in the case of leiomyosarcoma the opposite is true with the majority of cases in men over 50 years of age. Other cancers can also metastasize to the prostate such as leukemia, melanoma and lung cancer.
Prostate cancer and Reactive Oxygen and Nitrogen Species
Oxidative species (OS) or free radicals are natural byproducts of cellular metabolism; however, if they are not properly reduced they can cause detrimental effects to the cell. OS can also be used by the immune system in warfare against microbial pathogens. However, recent studies have implicated that OS can promote cancer progression. OS are prevalent in cancer cells because of their high glycolytic rates and can promote angiogenesis, migration and proliferation. Estrogen metabolites and intermediates can also induce OS which can damage nucleic acids, proteins and
13 lipids 28, 59. Due to most cancer being highly proliferative and producing high levels of
OS, glutathione S-transferase (GST) is upregulated to protect from the side effects of
OS 60–62. Reactive oxygen and nitrogen species (ROS/RNS) compose OS and can be produced by inflammatory cells, the carcinoma and surrounding stroma 16. Release of these redox species can damage lipids, nucleic acids and proteins, and chronic exposure to OS is involved in metastasis of prostate cancer cells 63–65. On the other hand, ROS/RNS can also be created endogenously by disruption of the balance between OS metabolism 16. These reactive species can diffuse into the cell to modify signaling pathways 66. Chronic exposure to OS has been implicated in neoplastic transformation 67 and promotion of tumorigenesis 63. One of the OS in PCa is nitric oxide (NO) 68, 69.
Nitric Oxide
Nitrogen exists in cells as oxidized nitric oxide gas (NO), which is a polar, labile, membrane diffusible, free radical with a short half-life. NO can be detected using fluorescent probes such as diaminofluorescein (DAF-2 and DAF-2DA) 70 or indirectly by measuring its oxidized derivatives nitrate and nitrite by the Griess/Saltzman assay 71.
NO function in the cell was originally identified as a potent guanylate cyclase agonist of endothelial cells 72, but since has been shown to be a potent redox mediated signaling molecule comparable to the phosphate group in phosphorylation 73. Homeostatic production detoxification of NO is required for maintaining the steady state redox status of the cell; however, if this balance is perturbed by exaggerated levels of free NO then
14 this can result in a pathological condition called nitrosative stress. Aberrant production of NO can cause a host of detrimental effects to the cell; however, if the cell can tolerate nitrosative stress this could provide a niche for tumorigenesis. In addition to triggering guanylate cyclase signaling pathways, NO can cause apoptosis, proliferation, mutagenesis 74, and invasiveness 75 by damaging DNA and lipids and by post- translationally modifying proteins 12, 76. The extracellular and intracellular redox status of the cell, which includes NO, is an important contributor to prostate carcinogenesis 66,
77 because in the tumor microenvironment, NO can be generated exogenously from infiltrating macrophages 11 or endogenously in tumor cells 75.
In vivo and in vitro studies have shown the NO can exert either pro or anti- carcinogenic effects which is dependent upon cell background and NO concentration
78,79. NO can suppress apoptosis by inhibition of Bcl-2 cleavage and cytochrome c release in hepatocytes 80, and modulate lung cancer chemosensitivity to cisplatin 81.
Also, the kinase activity of epidermal growth factor receptor (EGFR) is inhibited by NO
82. For breast cancer (BCa) there is a NO gradient effect where nM quantities promote progression through the Raf/ERK pathway 83, p53 phosphorylation, and hypoxia inducible factor 1 alpha (HIF1-α) expression 84. In a murine BCa model NO was required for angiogenesis 85 and tumor growth could be inhibited in vivo by using specific iNOS inhibitors 86. Conversely, higher NO concentration in the μM range elicits
MKP-1 mediated apoptosis 87, 88 and inhibits angiogenesis 89–91 in BCa. Extensive study of intracellular/intracellular redox states of benign, non-invasive and aggressively metastatic PCa cell lines have concluded that NO can induce cell invasion 66, 92, 93. NO
15 effect on carcinogenesis is entirely dependent upon its concentration, enzymatic production and whether it is produced in tumor associated inflammatory cells, stromal compartments or cancerous tissue.
In addition to being a metabolic by product of cellular respiration, NO is produced from the catabolism of L-arginine to L-citrulline by NO synthases (NOS) 94, 95. Three
NOS enzymes exist which all contain oxygenase and reductase domains and require cofactors nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). However, these three isoforms of NOS are located on different chromosomes, and differ in their cellular expression, N-termini and level of NO produced. Type I or neuronal NOS (nNOS) is 135 kDa, constitutively expressed in neurons and produces nM levels of NO. Type II NOS
(iNOS) is a 131 kDa, inducible NOS, and produces μM levels of NO which macrophages use to kill microbial pathogens. Type III NOS (eNOS) is 131 kDa, constitutively produced in endothelial cells, and has an output of NO in the nM range
(Figure 1.1.). Interestingly iNOS is upregulated in a variety of cancers.
Figure 1.1. iNOS is composed of oxygenase, calmodulin (CaM) and reductase domains and requires cofactors FMN, FAD and NADPH.
16
iNOS has been shown to enhance angiogenesis and metastasis 96–98 and is expressed in carcinomas of the colon 99,100, prostate 94, breast 64,68,69,96,101, and gynecological tract 102. In BCa, iNOS has been shown to increase cell proliferation and invasion in vitro 68. In increasing grades of PCa biopsies, iNOS expression correlated with dedifferentiation and poor survival with a high number of iNOS positive stromal cells found in and around tumors 94,103. Importantly, iNOS expression promotes the proliferation of androgen independent PCa 104. In summary, iNOS promotes tumor progression and its inhibition is an attractive therapeutic target 105 (Figure 1.2.).
Figure 1.2. iNOS signaling in the PCa microenvironment.
While NO and iNOS levels are elevated in PCa their role in cancer progression is poorly understood. NO releasing drugs and iNOS inhibitors have been the subjects of clinical trials to treat the symptoms of BPH 106 and inhibit PCa vascularization 107. One of the ways NO could promote PCa progression is to post-translationally modify proteins
17 on tyrosine and cysteine residues which could modulate signaling pathways. Such post- translational modifications could promote increased cell proliferation or serve as prostatic biomarkers to identify premalignant prostate lesions and serve as potential targets for PCa specific redox modulation therapy 91,108.
Post-translational Modification of Proteins
Once a protein is translated it can be modified chemically in a process called post-translational modification (PTM). PTMs can take place as proteinase cleavage of propeptides or the covalent labeling of proteins with functional groups. Some of the known protein functional group modifications include glycosylation, acetylation, methylation, ubiquitination, sumoylation, phosphorylation and S-nitrosylation to name but a few 109. PTM is a mechanism developed by higher eukaryotes to control genes and is used to control all cellular functions including metabolism, proliferation, survival, apoptosis and necrosis. Protein PTMs can enhance the function of a protein by changing its inherent chemical nature, activity state, localization, turnover, protein: protein interactions, protein: nucleotide interactions, charge, enzymatic activity or its three dimensional structure 110. PTMs are typically detected, identified and quantified using mass spectrometry 111. Nitric oxide can alter protein function by two PTMs called tyrosine nitration and cysteine S-nitrosylation.
18
S-nitrosylation
S-nitrosylation is the PTM of the thiol group of cysteine residues with NO. Both chemical and enzyme derived NO has been shown to selectively post-translationally modify cysteines in a process called S-nitrosylation. NO production can cause S- nitrosylation via cysteine thiols, heme or non-heme containing proteins, and transnitrosylating Fe-S clusters 112. Intracellular glutathione (GSH) and L-cysteine (Cys) react with NO to form S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO) which act as physiological NO donors and trans-nitrosylate free cysteines or cysteines in polypeptides thus modifying protein function 112. It is important to note that extracellular GSNO and CysNO have different NO release half-lifes because of their different intracellular transport mechanisms. CysNO and GSNO both enter the cell using the amino acid transport system, but GSNO must transfer its NO group to cysteine 113.
Interestingly, depending on the cellular redox environment, protein S-nitrosothiols (S-
NO) can be reversible and reduced by redox enzymes like glutathione reductase, thioredoxin-1 or other yet to be discovered denitrosylases 114–116. S-nitrosylation is recognized as an important regulatory PTM akin to phosphorylation because it can modulate many protein classes including transcription factors, cell adhesion molecules and receptor tyrosine kinases 117. S-nitrosylation is extremely labile and light sensitive and thus a difficult PTM to detect until the development of the biotin switch technique which labels S-nitrosylated cysteines in proteins with biotin 118 (Figure 1.3.). Biotinylated proteins can then be isolated and detected by western blotted or identified by mass spectrometry (MS). Hence, S-nitrosylation can regulate protein activity, turnover,
19 subcellular localization, and molecular interactions (protein–protein or protein– nucleotide) 119. Since the coupling of the BST with MS, S-nitrosylation has been identified in a growing list of pathophysiological conditions, including cancer, multiple sclerosis, Parkinson's and Alzheimer’s diseases, cystic fibrosis and asthma 120. For example, Bcl-2, GAPDH, TRAIL receptor DR4, caspase-3, p21, protein disulfide isomerase and NF B have their function altered by S-nitrosylation 121–125. S-nitrosylation of GAPDH and EGFR inhibits their enzymatic activity 126–128. Also, ERα and AR are S- nitrosylated upon NO exposure which impairs their transactivation activity in BCa and
PCa cells, respectively 104,129. In a murine BCa model, angiogenesis was promoted following radiation therapy by the selective S-nitrosylation of HIF-1α by tumor associated macrophages 108. S-nitrosylation is one of the ways that cells respond to inflammatory cytokines and nitrosative stress with different subsets of proteins being S- nitrosylated 116,130.
Figure 1.3. The biotin switch technique (BST) is used to detect labile S-nitrosylated proteins.
20
In general, the majority of reports suggest that S-nitrosylation promotes disease under pathological conditions. Dependent on the concentration and localization of OS production distinct subsets of proteins will be S-nitrosylated. For cells with pathological levels of nitrosative stress, an understanding of which S-nitrosylated proteins promote disease could give insight into how to design targeted therapies. Unbiased profiling studies have identified many S-nitrosylated proteins in various tissues 119,131,132. Great strides have been made understanding the role of S-nitrosylation in many cell types but to date the protein S-nitrosylation in the prostate remains poorly understood. The next section details a profiling study to identify S-nitrosylated proteins in normal prostate epithelial cells exposed to NO.
Comprehensive Identification and Modified Site Mapping in Normal Prostate
Epithelial Cells
iNOS and eNOS have been associated with prostatic diseases 103,133,134; however, their function is unknown. Due to increasing levels of NO in the prostate because of NOS expression, one mechanism could be aberrant S-nitrosylation of proteins which promote the development of prostatic disease. The following summarizes a study 135 to detect S-nitrosylated proteins in immortalized normal prostate epithelial cell (NPrEC) cultures that were treated extracellularly with a NO donor, 1mM
CysNO, to enrich S-nitrosylated proteins. S-nitrosylated proteins were isolated using the
BST and identified with high confidence using a high accuracy (precursor mass error <5 ppm) LC-MS, LTQ-Orbitrap (Thermo-Electron, Thermo-Fisher, Boston, MA). Two MS
21 methods of isolating S-nitrosylated proteins were used, a protein pull-down and a peptide pulldown approach. The two approaches complemented each other by first identifying S-nitrosylated proteins, and in the second approach confirming the S- nitrosylated proteins and mapping their site of S-nitrosylation. Using this combined approach, 82 specific sites of S-nitrosylation were identified in a total of 116 S- nitrosylated proteins and 30% of this dataset were confirmed from previous publications.
The dataset of S-nitrosylated proteins were grouped mainly into 4 different protein classes: metabolism, cell structure, protein folding, and mRNA regulation.
Interestingly, several prominent proteins involved in disease development were identified: integrins alpha 6 and beta 4 (ITGα6 and ITGβ4), proliferating cell nuclear antigen (PCNA), maspin, epidermal growth factor receptor (EGFR) and α-catenin.
Unlike other PTMs, a S-nitrosylation motif in protein primary structure is unknown.
Combining the NPrEC S-nitrosylation site dataset with published datasets 131,132, no consensus sequence was found in either primary or secondary protein structure. 79% of the 141 SNO sites were found to be buried in tertiary structure with half of the cysteines residing in a charged microenvironment and the other half residing in hydrophobic pockets. For the S-nitrosylation sites of ITGα6, PCNA and EGFR, it is probable that S- nitrosylation could interfere with their ability to bind their respective ligands. As the first report of S-nitrosylated proteins in the prostate epithelial cells, this information reveals a distinct group of proteins which NO potentially controls in prostatic diseases. The identification of these S-nitrosylated proteins gave insight into which cellular pathways are being modulated by nitrosative stress and serve as the foundation of the following functional studies.
22
Dissertation Projects and Specific Aims
The identification of NO-sensitive signaling molecules in NPrEC provided insight into NO controlled pathways 135, but their contribution to PCa progression is unknown.
Since NO is important in other cancers 136, detailed study of the identified S-nitrosylated proteins 135in the prostate could give insight into how they affect cell migration, adhesion, proliferation and aggressiveness in PCa 93,137. ITGα6 and PCNA were identified by the NPrEC profiling study by both identification approaches (protein and peptide), and ERβ was identified to be S-nitrosylated in an independent study
(unpublished results). These proteins are important in PCa development and were chosen to study how S-nitrosylation affects their function in a PCa background. In the context of aberrant iNOS expression in the prostate 103,138,139, S-nitrosylation of ITGα6,
PCNA and ERβ could increase prostate cell migration, proliferation and hormone dependence/independence which are hallmarks of PCa.
Project 1: “Investigation of S-nitrosylation of ITGα6 in PCa Metastasis”
Hypothesis: “S-nitrosylation of ITG 6 could interfere with laminin-1 mediated cell adhesion leading to destabilization of ITG 6 ligand binding.
Specific Aims:
1. Determine role of S-nitrosylated ITG 6 in tumor cell migration/invasion.
2. Validate the physiological role of ITG 6 s-nitrosylation, using mutant ITG 6
stable metastatic prostate cancer cells in an orthotopic murine metastasis model.
23
3. Determine the clinical relevance of s-nitrosylation using anti-NO
immunohistochemistry on human tissue samples of increasing grades of PCa.
Project 2: “Genotoxic stimulation of PCNA S-nitrosylation in Castration Resistant PCa”
Hypothesis: “S-nitrosylation of PCNA at the interdomain loop interferes with protein- protein interactions thereby causing cell death. Specifically, through disassociation of
DNA polymerase δ and increased binding to p21”
Specific Aims:
1. Determine environmental stimulus to cause PCNA S-nitrosylation in CRPC cells
2. Determine Functional consequence of PCNA S-nitrosylation in CRPC cells
Project 3: “Investigation of ERβ S-nitrosylation in PCa”
Hypothesis: “S-nitrosylation of ERβ inhibits its E2 responsive gene transactivation.”
Specific Aims:
1. Determine physiological stimuli to cause ERβ S-nitrosylation.
2. Determine the effect of NO and E2 on ERβ transactivation activity.
24
Chapters 2 and 3 study the effect of S-nitrosylation on ITGα6 mediated cell migration and PCNA in the context of DNA synthesis while Chapter 4 outlines preliminary study of the S-nitrosylation of ERβ in the context of PCa.
25
Chapter 2: Site- specific S-nitrosylation of Integrin α6 Increases Prostate Cancer
Cell Migration by Increasing Integrin β1 Association and Reducing Adherence to
Laminin-1
The data found in this chapter is the result of work performed by:
Jared Isaac, Ying Wai Lam, Xiang Zhang, Jarek Meller, Pheruza Tarapore and
Shuk Mei Ho
Jared Isaac performed all experiments, data interpretation, and wrote the manuscript.
Ying Wai Lam provided experimental consultation performed the MS identification of
ITGα6 (Figures 2.8A) S-nitrosylation sites.
Xiang Zhang provided consultation about ITGα6 mutagenesis.
Jarek Meller modeled the structure of ITGα6 after ITGαV.
Pheruza Tarapore provided experimental consultation and wrote the manuscript.
Shuk Mei Ho provided experimental consultation and wrote the manuscript.
ITGα6 Literature Review
Previous work 135 had identified ITGα6 as a S-nitrosylated protein in prostate cells.
ITGα6 consists of 1050 amino acids with a 991-amino-acid extracellular, a 23-amino- acid transmembrane and a 36-amino-acid cytoplasmic domain 140 (Figure 2.1.). The extracellular domain contains three putative divalent cation-binding sites and nine potential N-linked glycosylation sites. The extracellular portion contains seven IgG like domains. Two different isoforms for ITGα6 exist, α6A and α6B. The difference between the two being α6B has an unique cytoplasmic domain 18 amino acids longer than that of
α6A and their expression differs in cell lines. The ITGα6 subunits are similar to other α
26 subunits (26-31% identity with cleaved alpha subunits) of the integrin family but they are more similar to the α3 subunit (40% identity) 140. This high degree of similarity may be the basis for their functional resemblance since both α3 and α6 subunits, when associated with ITGβ1, function as laminin receptors and bind to the long β and γ laminin chains 141.
.
Figure 2.1. Post-translational modifications of ITGα6
The natural ligand of ITGα6 is the ECM protein laminin, which is a large (over 200 kDa) multimer of an α, β, and γ chain. In particular ITGα6 natural ligands are laminin-
511 (α5, β1 and γ1) and laminin-111(α1, β1 and γ1). Experimentally it was determined that ITGα6 binds specifically at β1 and γ1 142. ITGα6 heterodimerizes with ITGβ4 or
ITGβ1 in epithelial cells, and can mediate different functions dependent on its ITGβ binding partner. When ITGα6 associates with ITGβ4 this forms a special basal-side, adhesion structure called the hemidesmosome (Figure 2.2.).
27
Figure 2.2. Integrins α6 and β4 (ITGα6/β4) heterodimerize to form the hemidesmosome.
There are two types of hemidesmosomes, type I and II which differ in their function.
Type I are mainly involved in static adherence to the extracellular matrix, while type II are dynamic and are typically seen at the leading edge of motile cells 143. These types of hemidesmosomes also differ in their adapter protein composition besides containing
ITGα6β4. These components consist of CD150, BP 220 and BP230 proteins. ITGα6 can be activated through two pathways “outside-in” or inside-out” signaling. When
ITGα6 binds ligand this causes phosphorylation of ITGβ4 cytoplasmic tail by focal adhesion kinase (FAK), which in turn can phosphorylate four major downstream kinases, p120, CAS, RAF and PI3K (Figure 2.3.). These pathways lead to survival, proliferation, or migration/invasion 144. ITGα6 plays an important role in maintaining cell polarity, cell adhesion, and mediating normal and malignant cell migration through ECM and cell-cell contacts 145.
28
Figure 2.3. Signaling pathways downstream of ITGα6β4.
ITGα6 has a unique function in the prostate. ITGα6 is expressed in the basal membrane in normal tissues and BPH 146. However, its subcellular localization changes in HGPIN 147 and is overexpressed in cell undergoing metastasis 148. Also, there is an unexplained heterodimerization switch of ITGα6 from ITGβ4 to ITGβ1 during prostate cancer progression 149,150. In recent years a novel variant has been shown to be expressed called ITGα6p 151. This isoform is actually a urokinase plasminogen activator
(uPA) cleavage product of the full length ITGα6, and ITGα6 cleavage dramatically increases PCa cell migration in vitro/ in vivo 152.
29
Abstract
The increased mortality in prostate cancer is usually the result of metastatic progression of the disease from the organ-confined location. Major events in this progression cascade are increased cell migration and loss of adhesion. Moreover, elevated levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) found within the tumor microenvironment are hallmarks of progression for this cancer. To understand the role of nitrosative stress in prostate cancer progression, the effects of
NO/iNOS on prostate cancer cell migration and adhesion were investigated here. Our results indicate that ectopic expression of iNOS in prostate cancer cells increased cell migration, which could be blocked by selective ITGα6 and iNOS inhibitors. Furthermore, iNOS S-nitrosylated ITGα6 Cys86 in prostate cancer cells. Using ITGα6 wild-type and a
Cys86 mutant, we show that treatment of prostate cancer cells with NO increased ITGα6 heterodimerization with ITGβ1. Furthermore, S-nitrosylation of ITGα6 decreased its binding to laminin-β1, and reduced the prostate cancer cell adhesion to laminin-1. In conclusion, S-nitrosylation of ITGα6 increased the migration of prostate cancer cells, which could be a potential mechanism of NO/iNOS induced prostate cancer metastasis.
Introduction
To reduce mortality from this cancer, it is therefore imperative to understand how
PCa cells escape the primary tumor and spread to secondary sites. A loss of cellular adhesion and an increase in cell motility are major events in this metastatic cascade.
30
Integrins are expressed in all epithelial cells and have diverse functions in regulating cell morphology, cell-cell interaction, and signal transduction from extracellular matrix (ECM) 153. Altered expression or aberrant distribution of integrins disrupts the cell-substratum relationship, increases cell motility, and promotes progression of epithelial cancers including PCa 144. Using the biotin switch technique
(BST) 118, we recently conducted a site-specific mapping of the S-nitrosoproteome in an immortalized prostate epithelial cell line NPrEC 135and identified integrin α6 (ITGα6) as a target for S-nitrosylation at cysteines 86, 131 and 502. BST substitutes biotin for the labile and otherwise difficult to detect NO moiety making identification of S-nitrosylation more readily achievable. In normal epithelial cells, ITGα6 binds specifically to integrin
β1 (ITGβ1) or ITGβ4 to form α6β4 or α6β1 integrin. These are receptors of prostate acinar laminins, allowing cell adherence to the basement membrane 144,148. However, during PCa progression, while multiple integrins including ITGβ4 are downregulated, both ITGα6 and ITGβ1, subunits of α6β1, are overexpressed in PCa and in their corresponding lymph node metastases 146,147, suggesting that ITGα6 expression favors
PCa cell metastasis 154. This report is first to address if S-nitrosylation plays a role in regulating the function of ITGα6 in PCa progression. Here, we reported that (i) ectopic expression of iNOS or treatment with a NO donor stimulates PCa cell migration through
ITGα6, (ii) iNOS associates with ITGα6, (iii) substitution of Cys86 with serine, when compared to substitution at the other two sites, most significantly affected the iNOS/NO- induced S-nitrosylation of ITGα6 in PCa cells. We further demonstrated that S- nitrosylation of ITGα6 Cys86 enhanced binding of the integrin subunit to ITGβ1, but not
ITGβ4, and diminished its adhesion to laminin-β1. These data suggest site-specific S-
31 nitrosylation of ITGα6 is part of the mechanism underlying the iNOS/NO-mediated promotion of PCa cell migration
Materials and Methods
Cell culture- All cells were maintained in a humidified incubator at 37°C with 5%
155 CO2 as previously described .
Vector production- Plasmids expressing ITGα6 (BC136455) and iNOS
(BC130283) (Thermo Fisher, Boston, MA) were cloned into pcDNA3.1/TOPO expression vector according to manufacturer’s protocol (Invitrogen, Carlsbad, CA).
ITGα6 was C-terminally tagged with Flag epitope (DYKDDDDK). Mutagenesis of cysteines 86 (TGC->TCC), 131 (TGT->TCT), and 502 (TGT->TCT) to serine residues
(C86S, C131S and C502S) was accomplished with Stratagene QuikChange XL site directed mutagenesis (Agilent, Santa Clara, CA) of pcDNA3.1/ITGα6-Flag constructs.
Ectopic expression of iNOS or a control vector (LacZ) in PC-3 cells was accomplished using lentivirus constructs as previously described
Cell Transfection/Infection- HEK-293 cells were transfected using
Lipofectamine™ 2000 (Invitrogen), and PC-3 cells were transfected using Mirus
TransIT®-Prostate Transfection Kit (MirusBio Corp. Madison, WI) according to manufacturer’s protocol for 24 hours unless otherwise noted. PC-3 and DU-145 cells were infected as previously described 156.
32
NO donor preparation, nitrite measurement and the Biotin Switch Technique - S- nitroso-L-cysteine (CysNO) and S-nitroso-glutathione (GSNO) were freshly prepared as previously described 135. CysNO and GSNO were used for experiments with NO donors needed for short and long time points, respectively. The Griess/Saltzman assay was used to detect nitrite level in cell culture media according to manufacturer’s protocol
(Invitrogen) in triplicate. The BST 118 was performed as previously described 135. All experiments were performed in triplicate.
Gel electrophoresis and western-blot analysis- SDS-PAGE was performed with
8% gels under non-reducing conditions in triplicate, and were transferred onto a PVDF membrane (Immobilon FL, Millipore, Billerica, MA) with a blotting cell (Invitrogen).
Antibodies used were as follows: Flag (#2368, Cell Signaling, Danvers, MA), β-actin
(A2228, Sigma Aldrich, St. Louis, MO), ITGα6 (SC-6597, Santa Cruz Biotechnology
(SCBT), Santa Cruz, CA), ITGβ4 (SC-6629, SCBT), iNOS (SC-651, SCBT), Laminin-β1
(SC-17810, SCBT), ITGβ1 (MAB1987Z, Millipore), and rabbit IgG (SC-2027, SCBT).
The ITGα6 antibody (GoH3) blocks the interaction of ITGα6 with laminin, and was a generous gift from Dr. Arnoud Sonnenberg 142. For all inhibition studies, GoH3 was used at 10 μg/mL. Primary antibodies were used at 1:1000 dilutions overnight at 4oC or 1 hour followed by corresponding IRDye conjugated secondary antibodies (LICOR
Biosciences, Lincoln, NE) used at 1:15,000 dilutions as previously described 135.
33
Wound healing- Cells were allowed to reach confluence, serum starved for 24 hours, and wounded as previously described 157in triplicate. Specifically, cells were treated with NO donor and/or ITGα6 inhibiting antibody, GoH3 at indicated doses.
Pictures were taken at 0, 8, or 24 hours using a Zeiss microscope (Carl Zeiss
Microimaging, GmbH, Germany) at 40X magnification. Migratory distances were measured using Zeiss Axiovision software (Carl Zeiss Microimaging, GmbH, Germany).
Experiments were performed in triplicate.
Cell Adherence- Cell adhesion experiments were performed as previously described 158 in triplicate. Specifically, wells were coated with 10 μg/ml human laminin-1
(Sigma Aldrich) followed by blocking with 0.5% BSA/PBS. Wells were washed with
0.1% BSA/PBS prior to seeding 40,000 cells. Cells were allowed to adhere for 30 minutes at 37°C. Non-adherent cells were aspirated and wells were washed with rotation at 70 rpm four times. Adherent cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Plates were washed followed by methanol incubation.
Adherent cells were stained with crystal violet and read spectrophotometrically at 490 nm. Cells adherence was calculated as the ratio of cells adhered to laminin-1 to those adhered to poly-lysine. Experiments were performed in triplicate.
Co-immunoprecipitation - Lysates were co-immunoprecipitated according to a published protocol 159. Specifically, cells were seeded on plates coated with 10μg/mL laminin-1 and lysed with radio-immunoprecipitation assay (RIPA) buffer (Sigma Aldrich) with protease inhibitors. Lysates were incubated with 2 μg/mL primary or control
34 antibodies for 16 hours at 4°C. The primary antibodies were pulled down with protein
A/G Plus agarose beads (SC-651 SCBT). Laminin- 1 was chosen for laminin-1 coimmunoprecipitation because it is known to interact with ITG 6 142. Experiments were performed in triplicate.
Statistical Analyses- All statistical analyses were calculated using GraphPad
Prism software (GraphPad Software, La Jolla, CA). ImageJ software was downloaded from http://rsbweb.nih.gov/ij/index.html.
Results
NO/iNOS stimulates PCa cell migration via ITGα6 action. We first tested whether increased NO/iNOS contents enhance PCa cell migration, a key event in cancer progression. Using the wound healing assay, we measured cell migration in two PCa cell lines, PC-3 and DU-145, following treatment with GSNO or transfection with an iNOS expression plasmid. A significant increase in migration was observed in these cell lines after both treatments when compared with their respective untreated controls
(Figure 2.4A.). To confirm ectopic expression of iNOS increased NO production, the levels of nitrite were measured in cell culture media at the end of the wound healing assay (Figure 2.5A and 2.5B.). A significant increase in the levels of measurable nitrite was observed in PCa cell cultures 24 hours after transfection with the iNOS-expression- plasmid (Figure 2.5C). The nitrite levels in the culture media from these cells were
35 comparable to those obtained from cells treated with 100 μM GSNO. Furthermore, elevated levels of NO persisted for at least 48 hours after either treatment (Figure
2.5C). The observed increases in cell migration were not an artifact of an enhancement of cell proliferation because neither GSNO nor ectopic iNOS expression altered cell viability in the treated cultures (Figure 2.4B.).
Figure 2.4. iNOS increases the migration of prostate cancer cells. (A) PC-3 (left) and DU-145 (right) cells were subjected to the wound healing assay -/+ 100 μM GSNO, empty vector (EV) or iNOS expressing vector. (B) PC-3 and DU-145 cells were treated with -/+ 100 μM GSNO, and transfected with EV or iNOS for 24 hours followed by MTS cell viability assay (Promega, Madison, WI). All experiments were done three times. (*) represents p<0.05.
36
Figure 2.5. Nitrite increases in cell medium after iNOS transfection. Nitrite levels in empty vector (EV) or iNOS transfected PC-3 (A) and DU-145 (B) cell media after the wound healing assay. Nitrite levels are relative to that present in EV transfected cells. (C) Nitrite levels in HEK- 293 cells transfected with EV or iNOS and incubated for 24 and 48 hours. Nitrite measured by Griess-Salzmann assay. iNOS produces an appreciable μM nitrate level after 24 hours which increasees slightly with time. (*) represents p<0.05.
To demonstrate enhanced expression of iNOS is causally linked to increased cell migration, the wound healing assay was performed in PC-3 cultures transfected with a control or iNOS-expressing plasmid in the presence or absence of an iNOS inhibitor,
1400W. We found that iNOS-mediated enhancement of cell migration in PC-3 cells 8 hours after wounding was ameliorated upon treatment with 1400W (Figure 2.6A.). To test the hypothesis that NO induces PCa cell migration through ITGα6 action per se, the wound healing assay was performed in PC-3 cultures treated with GSNO in the presence or absence of an anti-ITGα6 antibody. The antibody was found to block the
GSNO-mediated enhancement of cell migration in these cultures (Figure 2.6B.).
37
Figure 2.6. iNOS- and NO-mediated cell migration is blocked by 1400W or GoH3, respectively. (A) PC-3 cultures infected with LacZ (control) or an iNOS-expression plasmid were wounded in the absence or presence (-/+) of 100 μM 1400W for 8 hours. (B) PC-3 cells were wounded and treated with 100 μM GSNO -/+ anti-ITGα6 antibody, GoH3, for 8 hours. All experiments were done three times. (**) represents p<0.01.
ITGα6 interacts with iNOS and is S-nitrosylated by increased levels of NO or iNOS expression. Co-immunoprecipitation experiments were carried out to examine whether iNOS associates with ITGα6. Cell lysates from PC-3 cells infected with the iNOS-expression virus or LacZ-expression virus were immunoprecipitated with anti- iNOS, anti-ITGα6, or IgG control antibodies and subjected to western blot analysis.
ITGα6 was found to coimmunoprecipitate with iNOS (Figure 2.7A., lane 3), suggesting the possibility of an association between these two proteins. To determine whether iNOS S-nitrosylates ITGα6, HEK-293 cells transiently transfected with a control or
ITGα6-Flag-expression plasmid were subjected to the BST in the presence or absence
38 of the NO donor CysNO or following the transfection of an iNOS-expression plasmid.
Western blot analyses revealed that S-nitrosylation of ITGα6 occurred in the presence of CysNO or ectopic expression of iNOS (Figure 2.7B, lanes 3 and 4), but not in their absence (lane 2). To demonstrate endogenous ITGα6 can be S-nitrosylated in PCa cells, PC-3 cells were infected with the iNOS-expression virus or treated with CysNO, and subjected to the BST (Figure 2.7C.). S-nitrosylated ITGα6 was detected only in
PC-3 cells treated with the NO donor or are ectopically expressing iNOS (Figure 2.7C., lanes 2 and 3), but not in control PC-3 cells (lane 1). ITG 6 was also S-nitrosylated in
LNCaP and DU-145 cells (Figure 2.8).
Figure 2.7. ITGα6 interacts with iNOS and is S-nitrosylated with increased NO or iNOS expression in cellullo. (A) iNOS was coimmunoprecipitated (Co-IP) with ITGα6 using from PC-3 lysates infected with LacZ (-) or the iNOS-expression plasmid. (+) indicates iNOS antibody immunoprecipitation. (B) The BST was conducted using lysates from HEK-293 transfected with or without an ITGα6-Flag plasmid following treatment with 1mM CysNO or transfected with an iNOS-expression plasmid (iNOS). (C) The BST was carried out in PC-3 cells with or without treatment with 1mM CysNO or transfection of an iNOS-expression plasmid. All experiments were done three times.
39
Figure 2.8. ITGα6 is S-nitrosylated in LNCaP and DU-145 cells. (A) LNCaP were treated with 1mM CysNO and (B) DU-145 cells were transfected with iNOS and both were subjected to the BST in the absence or presence of sodium ascorbate (NaAsc) followed by western blotting.
Cys86 is central in ITGα6 S-nitrosylation. We previously reported treatment of
NPrEC with CysNO induced S-nitrosylation of 172 proteins 135. ITGα6 was identified to be in this S-nitrosoproteome and the S-nitrosylation shown to occur at Cys86, Cys131 and
Cys502 by mass-spectrometry (Figure 2.9A., the mass spectra of Cys86 and Cys131 were not published previously 131). As indicated in the cartoon of ITGα6, these sites are located on the extracellular head (Cys86 and 131) and thigh (Cys502) regions of ITGα6
(Figure 2.9B.). To determine which of these three cysteines is the most important site mediating ITGα6 S-nitrosylation, PC-3 cells stably expressing the empty vector (PC-
3/EV), wild-type ITGα6-Flag (PC-3/WT), or ITGα6-Flag with a C86S (PC-3/C86S),
C131S (PC-3/C131S) or C502S (PC-3/C502S) substitution were generated. These cell lines were treated with CysNO and subjected to BST analysis (Figure 2.9C., upper panel). We observed a marked decrease in S-nitrosylated ITGα6 (Flag-pull down) only in PC-3/C86S (Figure 2.9C., lane 3) but not in PC-3/C131S or in PC-3/C502S (Figure
2.9C, lane 4 and 5). The chemical saturation of all possible cysteines might be
40 responsible for the background pull down observed in the C86S lane. These results indicate that biochemically, Cys86 is the most important site mediating S-nitrosylation of
ITGα6. To demonstrate the physiological significance of Cys86 in S-nitrosylation of
ITGα6, PC-3/WT and PC-3/C86S cells were transfected with the iNOS-expression plasmid and subjected to BST analysis (Figure 2.9D.). No S-nitrosylated ITGα6 was pulled down by the Flag-antibody in the PC-3/C86S cell line whereas such protein was readily detected in PC-3/WT.
To visualize how site-specific S-nitrosylation may affect the function of ITGα6, the tertiary structure of the protein including the three S-nitrosylation sites were modeled
(Figure 2.9E.) using the available crystal structure of ITGαV which shares significant similarity in its primary sequence with that of ITGα6. The S-nitrosylated cysteine residues Cys86, Cys131 and Cys502 are conserved in ITGαV cysteines and their positions are shown in red with their predicted cysteine binding partners shown in magenta in
(Figure 2.9E.). While Cys86 and Cys131 are found located within the head region of
ITGα6, Cys502 is located within the thigh region. Based on structural analysis, the Cys86-
Cys95 loop located within the head region is largely exposed to the solvent, suggesting that Cys86 could be transiently exposed on the surface of ITGα6. Approximate normal mode analysis performed using Elastic Network Model, as implemented in the NOMAD- ref server (http://lorentz.immstr.pasteur.fr/nomad-ref.php) 160, lends further support to the idea that the Cys86 residue contributes significantly to slow coordinated motions in
ITGα6 (data not shown). Taken together, experimental and modeling results indicate
41 that S-nitrosylation of ITGα6 at Cys86 may have significant functional relevance. Hence, subsequent functional studies were performed using PC-3/WT and PC-3/C86S cells.
Figure 2.9. iNOS S-nitrosylates ITGα6 at Cys86. (A) MS/MS spectra of ITGα6 biotinylated peptides indicating S-nitrosylated cysteines Cys86 (R.TGGLYSC#DITAR.G, XCorr: 2.769, ΔCn: 0.53, ΔM: 2.85), Cys131 (K.VVTC#AHR.Y, XCorr: 2.124, ΔCn: 0.2, ΔM: -0.48), and Cys502 ((K)ScFEYTANPAGYNPSISIVGTLEAEK(E), XCorr: 3.534, Cn: 0.35, M: 0.42). The spectrum of Cys502 was reported previously 135. # indicates HPDP-Biotin (428.191567 amu). (B) Cartoon model of ITGα6 indicating S-nitrosylation sites relative to homologous repeat domains I-VII in grey and transmembrane domain in dark grey. (C) PC-3 cells stably expressing ITGα6-Flag with cysteines 86, 131, and 502 mutated to serines were treated with 1mM CysNO and submitted to the BST. (D) WT/PC-3 and PC-3/C86S cells were transfected with the iNOS-expression plasmid and submitted to the BST. (E) ITGα6 was modeled using ITGαV crystal structure (PDB: 3IJE) to visualize S-nitrosylated Cys86, 131 and 502 (red) with their predicted cysteine binding partners (magenta) within the “head” and “thigh” tertiary domains of ITGα6. All experiments were done three times.
42
ITGα6 Cys86 is required for the NO-induced increase in PC-3 cell migration and enhanced association with ITGβ1. To determine the consequence of a C86S substitution on NO-induced enhancement of cell migration, wound healing assays were performed with ITGα6 PC-3/WT and PC-3/C86S cells treated with GSNO or the vehicle control (Figure 2.10A.). As shown previously (Figure 2.4A.), NO significantly increased migration of PC-3/WT cells but not that of PC-3/C86S (Figure 2.10A.) cells.
We then examined the effects of NO on the association of ITGα6 with ITGβ1 or
ITGβ4, and determined whether the process involves Cys86. PC-3/WT and PC-3/C86S cells were seeded onto laminin-1-coated plates and treated with GSNO for 8 hours.
Lysates obtained from PC-3/WT had more ITGα6-associated ITGβ1 following GSNO- treatment when compared to levels in lysates from vehicle-treated controls (Figure
2.10B., lane 1 versus lane 3, upper panel; histograms). This difference cannot be completely accounted for by the NO-induced increase in ITGα6 expression (lane 1 versus lane 3 bottom panel). In contrast, GSNO-treatment did not affect the amount of
ITGβ4 associated with ITGα6 (lane 1 versus lane 3, middle panel). Importantly, the amount of ITGα6, and the degree of ITGβ1 association with ITGα6 in PC-3/C86S cells were insensitive to GSNO-treatment (lane 5 versus lane 7, upper, middle and bottom panel).
43
Figure 2.10. NO mediates migration through ITGα6 Cys86 and promotes increased ITGβ1 heterodimerization. (A) PC-3/WT and PC-3/C86S cells were wounded for 8 hours -/+ 100 μM GSNO. (*) represents p<0.05. (B) PC-3/WT and PC-3/C86S cells were seeded onto plates pre- coated with 10 μg/ml laminin-1 and allowed to adhere for 8 hours with -/+ 100 μM GSNO. ITGα6 was immunoprecipitated using a Flag antibody and its associated ITGβ1 or ITGβ4 was detected by western blotting. Quantitation was done with ImageJ software by measuring ITGβ1 bands and subtracting from WT and C86S Flag immunoprecipitation bands. WT Flag/ITG 1 without NO bands was set to baseline. All experiments were done three times.
We next conducted the wound healing assay on PC-3/WT and PC-3/C86S cells with or without treatment with GSNO, and in the presence and absence of anti- ITGα6
GoH3 antibody, that blocks the interaction of integrin with laminin 142. The antibody was found to block the NO-induced enhancement of cell migration in PC-3/WT cells, but not in PC-3/C86S cells (Figure 2. 11.).
44
Figure 2.11. GoH3 does not block cell migration of PC-3/C86S cells in the presence of 100 μM GSNO. WT and C86S expressing PC-3/WT and PC-3/C86S cells were wounded and allowed to recover for 8 hours in the presence of 100 μM GSNO plus 10 μg/mL IgG or GoH3 antibodies. (**) represents p<0.01. PC-3/WT cells showed inhibition of cell migration in the presence of GSNO. While PC-3/C86S cells showed decreased cell migration compared to PC-3/WT, but did not show any further inhibition.
S-nitrosylation of ITGα6 at Cys86 mediates the NO-induced reduction in cell adherence to laminin-1. We examined the effect of NO on ITGα6-mediated cell adhesion to laminin-1 which is composed of 1, 1, and 1 subunits and its other nomenclature is laminin-111. PC-3/WT and PC-3/C86S cells were seeded on laminin-1 coated plates in the presence or absence of GSNO for 1 hour. Treatment with GSNO caused a significant decrease in cell adherence in PC-3/WT but exerted no effect on the adherence of PC-3/C86S to laminin-β1 (Figure 2.12A). In concordance, while treatment of PC-3/WT cells with GSNO reduced ITGα6-laminin-1 interaction (Figure 2.12B, lane
1 versus lane 2) it had no effect on the PC-3/C86S cell line (lane 3 versus lane 4), as demonstrated in experiments using Flag-ITGα6 immuno-precipitation followed by
Western blotting for laminin-β1. These results indicate that the disruption of cell
45 adhesion after GSNO treatment is attended by a reduction in ITGα6-laminin-β1 interaction, with both processes likely mediated by S-nitrosylation of ITGα6 at Cys86.
Figure 2.12. Cys86 is responsible for NO destabilization of ITGα6-mediated cell adherence. (A) WT/PC-3 and C86S cells were seeded onto plates pre-coated with human laminin-1 or poly- lysine and allowed to adhere for 1 hour in the presence or absence of 500 μM GSNO. % Cell adherence was calculated as cell absorbance on laminin-1 divided by absorbance on poly- lysine x 100% with values observed in untreated WT/PC-3 arbitrarily set as 100%. (*) represents p<0.05. (B) WT/PCS and C86S/PC-3 cells adhered to laminin-1 for 1 hour in the presence 500 μM GSNO were used to prepare cell lysates for immunoprecipitation with Flag-antibody. Laminin- 1 was chosen for laminin-1 coimmunoprecipitation because it is known to mediate interactions with ITG 6. Quantitation of WT and C86S Flag IP bands relative to their laminin-β1 bands was done with ImageJ software. All experiments were done three times.
46
Discussion
Integrins are important mediators of cell adhesion and migration 153. In this study, we showed that NO and iNOS significantly increased PCa cell migration and decreased cell adhesion through S-nitrosylation of Cys86 on ITGα6. This post-translational modification was further showed to promote the interaction of ITGα6 with ITGβ1, but not
ITGβ4, and diminish its association with laminin-β1. These findings are consistent with reports demonstrating a positive association between iNOS expression and PCa progression and metastasis in clinical specimens 103,138,139 and the pivotal role of perturbations of cancer cell-matrix interaction in PCa progression 161.
Our findings provide the first evidence that S-nitrosylation of an integrin (ITGα6) promotes PCa cell migration in a site-specific manner. Although earlier studies have reported NO promotes cell migration in a variety of cells such as eosinophils 162 and mammary cancer cells 163few have definitively demonstrated S-nitrosylation of motility- related molecules at specific cysteines as a causative factor. This initial lack of progress was in part due to the very unstable nature of the S-nitrosylated proteins. With the development of the BST, the highly unstable protein-SNOs are replaced with biotin that can be readily detected, as well as be sequenced for the identification of the S- nitrosylation sites 118,120. Using BST, S-nitrosylation of actin in neutrophils was shown to alter actin polymerization, network formation, intracellular distribution and interaction with integrins 164. Similarly, both NO and estradiol-17β activates c-Src through S-
47 nitrosylation of its Cys498 in MCF-7 cells and promotes cancer cell invasion and metastasis 165.
Our data suggest iNOS may directly interact with ITGα6 and mediate S- nitrosylation of the integrin. This postulate is consistent with the general belief that the selectivity of S-nitrosylation is provided by protein-protein interaction such as that of the
S-nitrosylation of cyclooxygenase-2 166 and caspase-3 117. However, since Cys86, identified to be essential for S-nitrosylation, is located in the head region that normally resides outside the cell membrane, it raises the question of how intracellular iNOS can
S-nitrosylate this site. One probable mechanism is through the individual association of iNOS 167 and integrins 168 with caveolin-1, bringing these two molecules to the caveolae, the plasma membrane organelle responsible for recycling integrins and transducing matrix information during cell migration 169. Whether iNOS-induced S-nitrosylation of
ITGα6 occurs within the caveolae remains to be established. Alternatively, it is possible that the NO released by intracellular iNOS diffuses across the cell membrane and S- nitrosylates ITGα6 on the outside of the cell.
From modeling ITGα6, we recognize that Cys86 lies at the bottom of an easily exposed loop and, in contrast, Cys131 is fully buried. Therefore, the S-nitrosylation of
Cys86 might lead to exposure of Cys131 and its subsequent S-nitrosylation.
Consequently, in the absence of Cys86 S-nitrosylation, Cys131 may not be accessible for
S-nitrosylation. In this regard, Cys86 may be the first in a cascade of S-nitrosylation of a series of cysteines including Cys131 Cys502, analogous to the sequential phosphorylation
48 of multiple sites at the cytoplasmic tails of receptor tyrosine kinases 170. Thus, in silico predictions could explain the experimental observation that Cys86 is the most sensitive
S-nitrosylation site on ITGα6. However, future investigations are needed to ascertain whether sequential or cooperative S-nitrosylation of multiple cysteines in ITGα6 partakes in the regulation of the action of this integrin.
S-nitrosylation of Cys86 on ITGα6 was found to be essential and sufficient to mediate the NO/iNOS-induced enhancement of PCa cell migration and loss of matrix adhesion. These results are consistent with findings from a series of studies reporting
NO regulates the disulfide bonds in extracellular proteins including integrins 171–173. S- nitrosylation of specific cysteines in integrins induces breakage, reformation or reshuffling of disulfide bonds resulting in conformational changes in these molecules 174.
These changes ultimately lead to functional changes including alterations of ligand- binding/matrix-adhesion and cell motility for these integrins 174–176. Such structural and functional changes probably occur upon S-nitrosylation of Cys86 on ITGα6, resulting in decreased cell adhesion and increased cell migration.
At the molecular level, we showed that S-nitrosylation of Cys86 favors the association of ITGα6 with ITGβ1 (and not ITGβ4) and reduces its binding to laminin-
1/laminin-β1. These findings are in agreement with previous studies 148 reporting increased production/formation of integrin α6β1 and laminin as promoters of tumor progression and metastasis that is commonly attended with a loss of α6β4. One probable mechanism underlying the S-nitrosylation-induced alterations in heterodimer
49 preference and matrix protein-affinity could be the result of breaking/reshuffling of disulfide bonds at targeted cysteines as discussed above. Another likely mechanism may involve the cleavage of ITGα6 at Arg594, 595 that has previously been shown to play a significant role in PCa migration and metastasis 152. However, although Cys502 is in close proximity to Arg594, 595 it remains to be determined if S-nitrosylation of this site mediates or facilitates ITGα6 cleavage. In a preliminary experiment we did not find NO induces cleavage of ITGα6 (unpublished results).
In conclusion, iNOS induced increased migration of PCa cells via S-nitrosylation of ITGα6 at Cys86 that is likely the trigger of enhanced ITGα6/ITGβ1 heterodimerization and loss of ITGα6 binding to laminin-β1. These biochemical and molecular changes were accompanied by decreased cell adherence and increased motility in PCa cells. A schematic model has been proposed (Figure 2.13.). Given these observations, inhibiting S-nitrosylation has potential for use in prostate cancer therapy and prevention.
Figure 2.13. Proposed model of how iNOS may affect ITGα6 binding to Laminin-1 and heterodimerization to ITGβ4 and ITGβ1 resulting in decreased adherence and increased cell migration.
50
Chapter 3: PCNA is S-nitrosylated by iNOS after Doxorubicin treatment which increases PCNA interaction with chromatin.
The data found in this chapter is the result of work performed by:
Jared Isaac, Ying Wai Lam, Xiang Zhang, Jarek Meller, Pheruza Tarapore and
Shuk Mei Ho
Jared Isaac performed all experiments (unless otherwise specified), data interpretation, and wrote the manuscript.
Ying Wai Lam provided experimental consultation, performed the MS identification of
PCNA (Figure 3.3A), S-nitrosylation sites, modeled the S-nitrosylation sites of PCNA in
Rasmol (Figure 3.3B), showed the iNOS mediated S-nitrosylation of PCNA (Figure
3.2D) and conducted PCNA chromatin binding experiments in the presence of doxorubicin and GSNO and CysNO (Figure 3.4A and 3.4B).
Xiang Zhang mutated cysteines 81, 135 and 162 in PCNA.
Pheruza Tarapore did the immunofluoresence staining of PCNA in (Figure 3.4E), provided experimental consultation and wrote the manuscript.
Shuk Mei Ho provided experimental consultation and wrote the manuscript.
In addition to ITGα6, PCNA was identified to be S-nitrosylated in normal prostate epithelial cells. The following is a study of how S-nitrosylation affects PCNA function in
PCa.
51
PCNA Literature Review
PCNA is a 36 kDa acidic protein that resides within the nucleus. Even though the
PCNA homotrimer outer surface is predominantly negatively charged its inner surface is lined with positively charged arginines and lysines. This positively charged inner surface stabilizes PCNA loading upon DNA 177. It was originally discovered as an autoimmune antigen for lupus 178 prior to its discovery of its role in cell cycle progression as “cyclin” and as a proliferating tumor marker 179. As a replication processivity factor, PCNA is required for chromosomal DNA replication and repair and interacts with DNA polymerases and 180. PCNA exists as an evolutionarily conserved 181 monomer composed of two globular domains and an interdomain connecting loop. During S- phase, there is a functional transition of detergent soluble PCNA to detergent-insoluble chromatin-bound PCNA which is associated with the replication fork. PCNA is loaded onto primase–polymerase protein complex of the replication fork by replication factor C in an ATP-dependent manner, and wraps around the DNA as a toroidal homotrimer sliding clamp 182. PCNA stimulates DNA polymerases and other proteins in the protein complex enabling it to synthesize DNA efficiently. As cancer progression is usually characterized by high proliferation rates, PCNA is often overexpressed in aggressive cancer cells 183 and has been used as a marker for cell cycle and cell proliferation in various types of cancer, including prostate cancer 184–186. However, PCNA has no enzymatic activity; therefore its function comes from its ability to act as a scaffold to interact with other proteins.
52
PCNA Cointeracting Proteins
PCNA monomer is predicted to have a molecular mass of 30 kDa, however this is not the actual mass (36 kDa), probably due to a plethora of PTMs in response to environmental stimuli. In addition to facilitating DNA replication and repair, PCNA coordinates a host of other functions such as cell cycle progression, chromatin assembly and remodeling, chromatid cohesion and transcription by binding various proteins. Interestingly during S-phase only 20-30% of total PCNA is associated with chromatin at DNA replication foci underlining its important other functions 187. However, co-interacting proteins have only been shown to interact at the “face” of PCNA and not the “backside”. The specificity of these interactions could be due to the ability of PCNA to form a double homotrimer with each its faces facing outward 188. PCNA can interact with protein partners through the hydrophobic groove (41DSSH44) of the N-terminus, the interdomain-connecting loop (118LMDLDVEQLGIPEQEYSC135), and C-terminal tail
(254KIE257). Most of PCNA binding partners have a PIP (PCNA interacting protein)
QXX(L/M/I)XX(F/Y)(F/Y) or PIP-related (QLXLF) box (chromatin assembly factor, CAF-
1) 189 or KA motif in some proteins, although a few others do not bind PCNA through
PIP box (XRCC1 190). PCNA is thought to orchestrate different cellular events through binding of different protein partners in a competitive or a regulated manner by posttranslational modification in a time dependent fashion 191(Table 3.1).
53
Table 3.1. PCNA elicits divergent cellular responses through interaction with different proteins.
Notable proteins that PCNA interacts with are: DNA Ligase I, DNA polymerase p125, p66 and p12, Cdt1, RP-A, DNA polymerase , Replication factor C subunits 1-5,
FEN-1, KCTD13, p38, DNA topoisomerase 1 and 2, MCM10, Lamin A/C and Lamin-B1,
APEX nuclease 2, AP endonuclease 1, ADPG, ERCC-5, DNA polymerase , Cyclin-O,
DNPK1, Exonuclease 1, Gadd45 , and , Ku70, XRCC5, XRCC6, p15PAF, Msh2,
Msh3, Msh6, Werner syndrome ATP-dependent helicase, Terminal transferase, hMYH,
PARP1, Rad18, Rad5, DNA polymerase , , , and , XRCC-1, RAD9A, CDK1,
Cdc25C, CDC6, CDK6, Cyclin-A1, Cyclin-A2, Cyclin-B1, Cyclin-A, Cyclin-D1, Cyclin-D2,
Cyclin-D3, PARP1, CDK2, p21, CHK-1 CKD5, RAD1, RAD17, HUS1, ING1, CDN1C,
Mcl-1, MAD2B, PP-1G, p53, Mdm2, HDAC1, ECO1, CAF1A, CAF p90, p300, DCC1,
WALP2, Dnmt1, Prothymosin , SETD8, RRM3, PIF1, UBC9, MBB1A, RPC1, NF- B p65, YB-1, ECP-51, NDH-II, Annexin A2, T22D1, eEF1A-1, GAPDH, and I BKE.
54
PCNA PTMs
PCNA is extensively modified by a plethora of PTMs which include ubiquitination, sumoylation, phosphorylation, acetylation, methylation, and S-nitrosylation.
Interestingly, mono- or poly- ubiquitinylation at lysine164 of PCNA have been demonstrated to initiate distinct DNA repair pathways in response to DNA damage or replication stress 192,193. Mono-ubiquitination triggers error prone trans-lesion bypass of
DNA synthesis by TLS polymerase, while polyubiquitin induces error free mode of bypass repair. Phosphorylation of Tyr211 by a nuclear form of EGFR stabilizes chromatin-bound PCNA 194, and downregulation of Tyr211 -P inhibited cell growth and tumor development in a PCa xenograft model 195. PCNA can be acetylated by p300 and deacetylated by HDAC1 196, and Srs2p physically interacts with SUMOylated PCNA, which contributes to the recruitment of the helicase to replication forks 197. In the profiling study to identify S-nitrosylated proteins in NPrEC 135, we located on PCNA two novel sites of S-nitrosylation Cys81 and Cys135 and confirmed another site, Cys162 120. S- nitrosylation among the many other PCNA PTMs may have an unknown and dramatic function.
Abstract
Prostate cancer is often treated by DNA damaging drugs such as doxorubicin.
However, PCa resistance to doxorubicin quickly develops. Doxorubicin can also induce
NO production which damages DNA but also can post-translationally modify proteins.
55
We show that PCNA is S-nitrosylated by doxorubicin, NO donor and iNOS. These agents alter PCNA function by causing nuclear translocation and chromatin binding.
Mutation of Cys81 reduces PCa cell sensitivity to doxorubicin and PCNA chromatin binding. These findings suggest that PCNA Cys81 is S-nitrosylated by iNOS after doxorubicin treatment. This is an important addition to the catalog of PCNA post- translational modifications and future studies targeting PCNA for chemotherapy. This research provides an avenue of potential dual treatment of drugs and NO that may be advantageous to killing prostate cancer cells.
Introduction
Dependent on diagnosis, patients typically undergo radical prostatectomy in combination with androgen deprivation therapy 23. However, for many men their PSA levels spike over time after surgery indicating that their cancer has returned. At this stage, hormone based therapies are ineffective in halting the spread of PCa and chemotherapy is administered. Therefore, it is imperative to understand and circumvent these relapsed, hormone independent, metastatic PCa. Recurrent or malignant prostate cancer is usually hormone refractory and treated with chemotherapy.
Taxanes, platinum containing compounds, mitoxantrone, estramustine, actinomycin-D and anthracyclines are commonly used to treat PCa 198,199. Doxorubicin
(Adriamycin) is an anthracycline known to interchelate into DNA and stabilize
Topoisomerase II thereby inhibiting DNA synthesis and repair which leads to cell cycle
56 arrest and apoptosis 200. However, Doxorubicin efficacy in targeting rapidly proliferating cells such as cancers is limited because resistance to Doxorubicin ultimately develops.
It is known for colon cancer cells that Doxorubicin resistance is through increased expression of multidrug resistance proteins such as MRP3 which efflux Doxorubicin from the nucleus 201. However, Doxorubicin is unique because it causes calreticulum translocation to the plasma membrane rendering cancer cells susceptible to clearance by the immune system 202. Interestingly, Doxorubicin is the only known chemotherapeutic to significantly increase the level of nitric oxide, NO in treated cells.
In addition to being a free radical, NO can be produced from three isoforms of nitric oxide synthase, NOS. Endothelial and neuronal NOS are similar in that they both constitutively produce nM levels of NO. However another NOS can be induced under inflammatory stimulus, hence its nomenclature as inducible NOS or iNOS 203. After prolonged exposure to NO, tissues are often damaged and have increased potential to develop into cancers 204,205. NO promotes cancer initiation and development by increasing angiogenesis, metastasis and cell invasion 96–98.
In contrast to the other NOS isoforms, iNOS can contribute to pathological conditions because it produces excessive μM levels of NO 206,207. Several tissue types including the prostate 94 have pronounced upregulation of iNOS 69, 99,102. iNOS expression correlates with a number of parameters, including Gleason score, dedifferentiation and poor survival 103, and inhibition of iNOS by selective inhibitors suppressed tumor growth in vivo 86. For example, in comparison to benign prostatic
57 hyperplasia, high-grade prostatic intra-epithelial neoplasia (HGPIN) and adenocarcinoma were shown to have significant expression of iNOS 139. Both the development of the primary tumor and the process of metastasis seem to be influenced by the presence and the amount of NO. AR-independent cells are more resistant to NO than AR-dependent cells, suggesting that increased iNOS expression might favor prostate cancer progression to hormone independence in prostate cancer cells 104.
Hence, it is imperative to study the effects of pathological iNOS expression in prostate cancers.
iNOS production of NO modifies proteins post-translationally at cysteines which is called S-nitrosylation 116. Like phosphorylation, S-nitrosylation can regulate protein subcellular localization, redox signal transduction and interactions with nucleotides and other proteins 130,208. S-nitrosylation promotes the progression of diseases such as asthma, diabetes, neurodegenerative disorders and cancer 122,209,210. Protein S- nitrosylation alters intracellular signaling pathways and cellular phenotypes in a manner that promotes tumorigenesis 12, 76.
Previously, we located two novel sites of S-nitrosylation 135 Cys81 and 135 and confirmed another Cys162 115 in proliferating cell nuclear antigen (PCNA). As a 261 amino acid, 36 kDa replication processivity factor, PCNA ensures chromosomal DNA replication and repair fidelity. During DNA synthesis and repair, PCNA binds the DNA replication fork in the nucleus as a homotrimer wrapping around the DNA as a toroidal sliding clamp 182. PCNA interacts with a plethora of proteins allowing it to function in
58 divergent roles such as DNA synthesis (DNA polymerases δ and ε), cell cycle control
(p21 and cyclin D1), chromatin remodeling (CAF-1, HDAC, DNMT1, p300) and DNA repair (XP-G) 191. Mutations in PCNA diminish DNA repair mechanisms, demonstrating the important role of PCNA in such processes. PCNA orchestrates different cellular events through binding of protein partners in a competitive or regulated manner through regulatory, time-dependent, posttranslational modifications 191,196. Mono- or poly- ubiquitinylation at Lys164 of PCNA has been demonstrated to initiate distinct DNA repair pathways in response to DNA damage or replication stress 192,193; mono-ubiquitination triggers error prone trans-lesion bypass of DNA synthesis by TLS polymerase, while polyubiquitination induces error free mode of bypass repair. Other post-translational modifications also play a role in PCNA regulation, for example, phosphorylation of Tyr211 by the nuclear form of EGF stabilizes chromatin-bound PCNA 194,195.
In our study we used castrate resistant prostate cancer (CRPC) cell lines to investigate the effects of DNA damaging agents on PCNA S-nitrosylation. Interestingly, only Doxorubicin caused PCNA S-nitrosylation, which was accompanied by an increase in extracellular nitrite levels. Doxorubicin increased expression of iNOS, and transient overexpression of iNOS resulted in PCNA S-nitrosylation. Furthermore, using NOS inhibitors there was a significant decrease in the level of PCNA S-nitrosylation and nitrite levels in CRPC cells indicating that iNOS selectively S-nitrosylates PCNA.
Doxorubicin also causes increased PCNA chromatin binding which was inhibited using
NOS inhibitors. Mutation of PCNA Cys81 resulted in significant decrease in PCNA S- nitrosylation and Doxorubicin induced chromatin binding. Doxorubicin decreased DNA
59 synthesis of CRPC cells which was ablated using NOS inhibitors. Furthermore, PC-
3/Cys81 DNA synthesis was not inhibited with Doxorubicin treatment indicating iNOS S- nitrosylation at Cys81 is an important signal to inhibit DNA synthesis after inflammatory stimulus. These results suggested another mechanism of Doxorubicin mediated apoptosis of CRPC cells in addition to stabilization of Topoisomerase II 200and MDR nitration 201.
Materials and Methods
Cell culture- All cells were maintained in a humidified incubator at 37°C with 5%
156 CO2 as previously described .
Vector production- Plasmid expressing PCNA (BC000491) was purchased from
Open Biosystems and subsequently cloned into pcDNA3.1/TOPO expression vector according to manufacturer’s specifications (Invitrogen, Carlsbad, CA). PCNA was C- terminally tagged with Flag epitope (DYKDDDDK). Mutagenesis of cysteines 81 (TGC-
>TCC), 135 (TGT->TCT), and 162 (TGT->TCT) to serine residues (C81S, C135S and
C162S) was accomplished with Stratagene QuikChange XL site directed mutagenesis kit (Agilent, Santa Clara, CA) of pcDNA3.1/PCNA-Flag constructs to produce mutants.
Serine was chosen to replace cysteine because this mutation retains the overall charge of the cysteine and thus would not affect protein folding.
60
Cell Transfection- PC-3 cells were transfected using Mirus TransIT®-Prostate
Transfection Kit according to manufacturer’s protocol (MirusBio Corp. Madison, WI) for
24 hours prior to harvest unless otherwise noted. Cells were harvested with scraping using 1x PBS and the pellet was stored at -80 °C or lysed with MPER® (Pierce,
Rockford, IL) or RIPA buffer (Sigma-Aldrich, St. Louis, MO) plus protease inhibitors
(Calbiochem, Merck KgA, Darmstadt, Germany) for 15 minutes on ice. Lysates were centrifuged at 0.4g for five minutes and supernatants were stored at -20°C. siRNA transfections were done as follows, PC-3 and DU-145 cells were seeded for 24 hours followed by iNOS siRNA (5’-CAGGGTGGAAGCGGTAACAAA-3’, 5’-
CCACGGCATGTGAGGATCAAA-3’, 5’-CTGGGCCGTGCAAACCTTCAA-3’, and 5’-
ATCGAATTTGTCAACCAATAT-3’; Qiagen, Valencia, CA) transfection with Dharmafect
(Thermo Fisher, Boston, MA) reagent 1 and 2 for 48 hours in according to manufacturers protocol.
Stable cell line production- Stably expressing PCNA PC-3 cell lines were made by transfecting PC-3 cells with pcDNA3.1/PCNA-Flag constructs (wild type, C81S,
C135S, C162S) and adding Geneticin sulfate, G-418, (SCBT, Santa Cruz, CA) 40
μg/mL to transfectants and screening positive clones for 2-3 weeks. Stably expressing clones for each mutant were combined and maintained at 10 μg/mL G418 for experiments.
NO donor and drug preparation- S-nitroso-L-cysteine, CysNO, and S-nitroso- glutathione, GSNO, were freshly prepared as previously described 135. 1.0 mg/mL
61 stocks of doxorubicin hydrochloride (25316-40-9), Nω-Nitro-L-arginine methyl ester hydrochloride, L-NAME, (51298-62-5) and 1400W dihydrochloride (214358-33-5) (all from Sigma Aldrich, St. Louis, MO) were prepared, aliquoted and stored at -20°C.
Biotin switch technique - Cells were seeded for 24 hours and exposed to NO donor for 15 minutes in the dark. The dose of CysNO selected was based on our previous study of normal prostate epithelial cells. Cells were harvested by scraping as described. The cell pellets were washed twice with 1X PBS and stored frozen at -80°C until protein extraction. The biotin switch technique was performed as previously described 135. Biotinylated protein was then concentrated by 10,000 MWCO columns
(Microcon, Millipore, MA), heated at 95°C with reducing sample buffer, subjected to
SDS-PAGE, and followed by western-blot analysis.
Gel electrophoresis and western-blot analysis- SDS-PAGE was performed with
8% gels, and were transferred onto a PVDF membrane (Immobilon FL, Millipore, MA) with a blotting cell (Invitrogen, Carlsbad, CA) at 27 V for 1.5 hours. Antibodies used were as follows: PCNA (SCBT, Santa Cruz, CA), Flag (#2368, Cell Signaling, Danvers,
MA), β-actin (A2228, Sigma Aldrich, St. Louis, MO), NOS-2 (SC-651 SCBT, Santa Cruz,
CA), and Lamin-B (SC-17810, SCBT, Santa Cruz, CA). Primary antibodies were used at
1:1000 dilutions overnight at 4oC or 1 hour at room temperature followed by corresponding IRDye conjugated secondary antibodies (LICOR Biosciences, Lincoln,
NE) used at 1:5000 dilutions. Blots were scanned using LICOR Odyssey scanner and software (LICOR Biosciences, Lincoln, NE).
62
PCNA chromatin extraction- Chromatin bound and unbound PCNA were isolated as previously described 194 with modification. Briefly, cultured cells were collected by scraping in 1X PBS and centrifuged at 0.5 g for 5 minutes and stored at -20°C. Prior to extraction, cells were placed on ice for 5 minutes, and then washed with 30 μL Buffer A
(100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES at pH 6.8, 1 mM EGTA,
0.2% Triton-X-100, 25 mM NaF, 2 mM Na3VO4,) with protease inhibitor cocktail
(Calbiochem, Merck, KgA) twice. Cytoplasmic washes were collected by centrifugation.
To release nuclear proteins, the washed pellet was lysed with RIPA buffer. Protein expression was analyzed by western blotting. The fractionation efficiency was assessed by antibodies against β-actin and Lamin B.
Cell viability- PC-3 and DU-145 cells were allowed to grow for 1 day in complete medium then incubated with indicated doses of Doxorubicin for 24 and 48 hours followed by cell viability assessment with CellTiter 96 AQueous Non-Radioactive Cell
Proliferation MTS assay (Promega, Madison, WI) as previously described 157.
DNA synthesis- BrDU incorporation was measured by colorimetric ELISA using the BrDU Cell Proliferation Kit (Millipore, Boston, MA).
Immunofluoresence- PCNA was stained with immunflourescent antibody as previously described 211 with modification. Cover slips with cells expressing Flag were incubated with 0.2 mL Buffer A for 5 minutes then aspirated. Following this 0.5 mL 10%
63 formalin (Electron Microscopy Sciences) was added and cells were incubated in the dark for 20 minutes. Cells expressing PCNA were fixed with ice cold methanol for 5 minutes (PCNA). After cells were fixed, they were washed with 1xPBS three times and permeabilized with 1% NP40 in PBS. Cover slips were blocked with normal chicken serum and stained with anti-PCNA, Flag or iNOS antibodies at a dilution of 1:500-1000.
Secondary chicken anti-mouse (Alexa Fluor 488) antibody (Invitrogen) was used at a dilution of 1:500, and cover slips were mounted using ProLong Gold Antifade Reagent
(Invitrogen).
Statistical Analyses- All statistical analyses were calculated using GraphPad
Prism software (GraphPad Software, La Jolla, CA). ImageJ downloaded from http://rsbweb.nih.gov/ij/index.html.
Results
PCNA is S-nitrosylated by doxorubicin in CRPC cells. Since we had previously identified PCNA as being S-nitrosylated in prostate cells 135, we investigated whether
NO producing agents 17-β-estradiol and doxorubicin could stimulate PCNA S- nitrosylation in DU-145 and PC-3 cells. Using NO donor as a positive control, we determined that both 17-β-estradiol and doxorubicin can induce PCNA S-nitrosylation, but doxorubicin induced the greatest amount of PCNA S-nitrosylation (Figure 3.1A.).
We collected the cell media from the cells treated with 17-β-estradiol and doxorubicin and measured the nitrite concentration by the Griess/Salzmann assay. Interestingly,
64 there was only a significant increase in nitrite level after doxorubicin treatment (Figure
3.1B.) and we therefore chose to study doxorubicin’s role in PCNA S-nitrosylation. We also tested the effect of other DNA damaging agents cisplatin and ultraviolet (UV) radiation, but these did not induce PCNA S-nitrosylation or nitrite production
(unpublished results). In DU-145 and PC-3 cells treated with increasing concentrations of doxorubicin (1 and 10 μM) there was a dose dependent decrease in cell viability after
24 hours. Interestingly, DU-145 and PC-3 sensitivity were inversely correlated with their level of PCNA S-nitrosylation (Figure 3.1C.).
Figure 3.1. PCNA is S-nitrosylated by doxorubicin in CRPC cells. (A) DU-145 (left) and PC-3 (right) cells were treated with NO producing agents: 1 mM nitrocysteine (CysNO) for 15 minutes, 100 μM 17-β-estradiol (E2) for 30 minutes and 50 μM doxorubicin (DOX) for 4 hours, and then subjected to BST. (B) Cell media from (A) were collected and analyzed for nitrite by the Griess/Salzmann assay. (C) DU-145 and PC-3 cells were treated with two concentrations of doxorubicin (DOX 1 and 10 μM) and cell viability was measured after 24 hours.
65
PCNA S-nitrosylation is through doxorubicin induction of iNOS. It has been previously shown that doxorubicin promotes expression of iNOS. Next, to determine if the increase in nitrite production was due to expression of iNOS we treated PC-3 cells with doxorubicin for 1, 2, 4, 8 and 24 hours and measured iNOS expression by western blot (Figure 3.2A.). Since doxorubicin was inducing the expression of iNOS we sought to determine if we could inhibit the doxorubicin mediated S-nitrosylation by inhibiting iNOS. To accomplish this we used a specific iNOS inhibitor, 1400W and pan-NOS inhibitor L-NAME. In the presence of 1400W there was a significant decrease in PCNA
S-nitrosylation (Figure 3.2B.) compared to doxorubicin treatment alone or in combination with L-NAME. Cell media was collected from cells treated with doxorubicin or doxorubicin/1400W and nitrite concentration was measured. 1400W significantly inhibited the nitrite production of cells treated with doxorubicin (Figure 3.2C.).
Furthermore with iNOS transfection PCNA was S-nitrosylated in PC-3 cells (Figure
3.2D.). PCNA S-nitrosylation could also be inhibited by transfection with iNOS siRNA
(Figure. 3.2E.). Proteins that interact with PCNA have PCNA Interacting Protein, PIP, motifs (QXX(L/M/I)XX(F/Y)(F/Y)) or PIP-related motif (QLXLF) and upon scanning of the primary sequence of iNOS there are 2 potential PIP motifs located at
478QEMLNYVLSPFYY490 and 1108QVEDYFF1114 (Figure 3.2F.). These results collectively show doxorubicin induces iNOS expression which S-nitrosylates PCNA.
66
Figure 3.2. Doxorubicin induction of iNOS results in PCNA S-nitrosylation. (A) PC-3 cells were treated with 50 μM for 0, 1, 2, 4, 8 and 24 hours followed by western blotting. (B) PC-3 cells were pretreated with 100 μM 1400W and 1mM L-NAME for 2 hours followed by 4 hour treatment with 50 μM doxorubicin. Cell lysates were subjected to the BST. (C) Cell media from (B) was measured for extracellular nitrite content by the Griess Saltzman assay. (D) PC-3 cells were transfected with iNOS for 24 hours followed by the BST. (E) PC-3 cells were treated with 1 mM CysNO for 15 minutes, 50 Μm DOX for 4 hours, or 48 hour iNOS siRNA transfection followed by 50 μM DOX treatment for 4 hours. Cell lysates were then subjected to the BST. (F) Search of iNOS primary structure reveals two potential PCNA interacting motifs (PIP): 478QEMLNYVLSPFYY496 and 1108 QVEDYFF1114.
PCNA is S-nitrosylated on Cys81 by Doxorubicin. From our profiling study 135, we identified three sites of PCNA S-nitrosylation Cys 81, 135, and 162 (Figure 3.3A.).
Interestingly, Cys81 resides within the trimer interface, Cys135 resides within the interdomain connecting region important for protein: protein interactions, and Cys162 resides close to Lys164 which becomes ubiquitinated in result to DNA damage (Figure
67
3.3B. and Table. 1). To determine which of these three sites is essential for PCNA S- nitrosylation, PC-3 cell lines stably expressing empty vector (PC-3/EV) , PCNA-Flag wild-type (PC-3/WT), C81S (PC-3/C81S), C135S (PC-3/C135S) or C162S (PC-
3/C162S) were created. Stable cells were treated with NO donor and subjected to the
BST (Figure 3.3C., upper panel). We observed a consistent significantly decreased pull down of S-nitrosylated PCNA only for the PC-3/C81S (Figure 3.3C, lane 3). PC-
3/C135S and PC-3/C162S did not show any significant changes in S-nitrosylation levels
(Figure 3.3C., upper and lower panels). These results indicate that biochemically,
Cys81 is most important for ITGα6 S-nitrosylation. To confirm the physiological relevance of Cys81 S-nitrosylation, PC-3/WT, PC-3/C81S, PC-3/C135S and PC-
3/C162S cells were treated with doxorubicin and subjected to the BST (Figure 3.3D).
We found that in the absence of Cys81 S-nitrosylation (C81S mutation), we did not pull down PCNA (Figure 3.3C and 3D.).
68
Figure 3.3. PCNA is S-nitrosylated on Cys81 and Cys135 by CysNO and doxorubicin, respectively. (A) (A) MS/MS spectra of PCNA biotinylated peptides indicating S-nitrosylated cysteines Cys81 ((K)C#AGNEDIITLR(A), XCorr: 3.68 Cn: 0.57 M: 0.023 ppm), Cys135, and Cys162 ((R)DLSHIGDAVVIScAK(D)), XCorr: 3.63 Cn: 0.57 M: 0.58 ppm). The spectrum of Cys81 and 162 were reported previously 135. # indicates HPDP-Biotin (428.191567 amu). (B) Rasmol modeling of PCNA showing where Cys81, Cys135 and Cys162 reside in 3-D structure. (C) Location of Cys 81, 135, and 162 in relation to known protein interaction domains. PC-3 cells stably expressing PCNA-Flag with cysteines 81, 135, and 162 mutated to serines were treated with 1mM CysNO (D) and 50 μM doxorubicin (E) then submitted to the BST.
Doxorubicin and NO increase PCNA chromatin binding. To determine the functional effect of S-nitrosylation on PCNA we conducted PCNA chromatin binding experiments in the presence of doxorubicin and NO donor. In the presence of doxorubicin (Figure 3.4A.) as well as NO donor (Figure 3.4B.) PCNA binding to
69 chromatin increased. Next, we sought to inhibit doxorubicin induced PCNA chromatin binding by treating with iNOS inhibitor, 1400W and general NOS inhibitor, L-NAME.
1400W had no effect on doxorubicin induced PCNA chromatin binding, whereas L-
NAME inhibited this process (Figure 3.4C.). This indicated that other NOSes besides iNOS may be compensating for doxorubicin induced PCNA chromatin binding, and that inhibiting all enzymatic NO production abolishes doxorubicin mediated PCNA chromatin binding.
Doxorubicin inhibits DNA synthesis by induction of NOS. To determine doxorubicin’s effect on PCNA mediated DNA synthesis we conducted BrDU incorporation experiments. In the presence of doxorubicin there was a significant decrease in DNA synthesis (Figure 3.4D), but this effect was diminished with NOS inhibitor L-NAME but not 1400W (Figure 3.4D). Once again this could be explained by another NOS compensating for specific iNOS inhibition. As expected, treatment of PC-3 cells induced PCNA foci, but surprisingly both NOS inhibitors reduced the number of
PCNA foci (Figure 3.4E and 4F). These results indicate that PCNA chromatin binding in response to doxorubicin is dependent on production of NO by NOS.
70
Figure 3.4. Doxorubicin induces PCNA chromatin binding. (A) PC-3 cells were treated with 150 μM doxorubicin (DOX) for 1.5, 5, and 12 hours, or (B) 1 mM CysNO and 500 M GSNO for 1.25 hours followed by enrichment of the chromatin-bound PCNA by chromatin extraction and western blotting. (C) PC-3 cells were pretreated with 100 μM 1400W and 1mM L-NAME for 2 hours followed by 4 hour treatment with 50 μM doxorubicin. Cell lysates fractionated and bound/ unbound chromatin fractions were western blotted. (D) PC-3 cells were completely serum starved for 24 hours, then pretreated with 100 μM 1400W and 1mM L-NAME for 2 hours followed by 4 hour treatment with 50 μM doxorubicin. 2 hours prior to the end of the experiment BrDU was added and % BrDU incorporation was detected by ELISA. (E) PC-3 cells were pretreated with 100 μM 1400W and 1mM L-NAME for 2 hours followed by 4 hour treatment with 50 μM doxorubicin. Cells were fixed with methanol stained with DAPI (blue) and PCNA (green). (F) The number of PCNA foci per 200 cells is represented graphically.
PCNA Cys81 is required for chromatin binding in response to doxorubicin. PC-
3/WT, PC-3/C81S, PC-3/C135S and PC-3/C162S cells were treated with two concentrations of doxorubicin (1 and 10 μM). PC-3/C81S cells were more resistant to doxorubicin treatment than the other stable PCNA clones (Figure 3.5A.). To determine
71 which cysteine if required for PCNA chromatin binding, PC-3/WT, PC-3/C81S, PC-
3/C135S and PC-3/C162S cells were treated with 50 μM doxorubicin (Figure 3.5B.).
PCNA in PC-3/C81S cells do not bind chromatin when exposed to doxorubicin.
However when PC-3/WT, PC-3/C81S, PC-3/C135S and PC-3/C162S cells were treated with 50 μM doxorubicin there was not a significant difference in DNA synthesis between cells (Figure 3.5C.). Collectively, these data suggest that S-nitrosylation of Cys81 is an important signal required for PCNA chromatin binding in response to doxorubicin treatment but not for DNA synthesis.
Figure 3.5. PCNA Cys81 confers resistance to doxorubicin. (A) Stable PCNA clones (EV, WT, C81S, C135S, and C162S) were treated with doxorubicin (1.0 and 10 μM) for 24 hours followed by the MTS assay. (B) Stable PCNA clones were treated with 50 μM doxorubicin for 4 hours followed by enrichment of the chromatin-bound PCNA by chromatin extraction and western blotting. (C) Stable PCNA clones were treated with 50 μM doxorubicin for 4 hours. 2 hours prior to the end of the experiment BrDU was added, and % BrDU incorporation was detected by ELISA.
72
Discussion
Resistance to doxorubicin is common to PCa. Doxorubicin resistance can come from the upregulation of multi-drug transporters which pump doxorubicin from its site of action in the nucleus and can be reversed by iNOS tyrosine nitration of MRP3 201. In this study, we have shown doxorubicin induces iNOS which site specifically S-nitrosylates
PCNA on Cys81. PCNA Cys81 S-nitrosylation results in PCNA increased binding to chromatin and interruption of DNA synthesis. These findings are consistent with doxorubicin’s ability to induce NO production through iNOS and post-translationally modify proteins such as MRP3 201. These data show an additional undiscovered pathway of how doxorubicin acts as a chemotherapeutic drug. Here we show for PCa another iNOS-mediated mechanism exists, S-nitrosylation of PCNA which causes
PCNA to bind to the chromatin and inhibit DNA synthesis
In addition to doxorubicin inhibiting DNA replication by stabilizing topoisomerase
II 200, we show that doxorubicin can cause PCNA chromatin binding by iNOS mediated
S-nitrosylation. PCNA is a 36 kDa nuclear protein that exists in two populations: a chromatin bound replication-competent form and a chromatin unbound form 187.
Phosphorylation of Tyr211 194 has been shown to cause PCNA stabilization and chromatin binding and loss of Tyr211 causes ubiquitin dependent degradation. Also
PCNA S-nitrosylation is reversible because after 24 hours, doxorubicin mediated S- nitrosylation of PCNA is gone (unpublished results). Several redox regulated denitrosylases exist and may be responsible for this event. Increased PCNA chromatin
73 binding is required for PCNA to function as a DNA polymerase δ processivity factor in repair of doxorubicin mediated DNA damage. It is important to note under doxorubicin treatment that S-nitrosylation of PCNA is required for this to happen. Under hyperoxic conditions in the lung, p21 inhibits cell growth by decreasing PCNA levels 212 which arrests cells in G1 phase of cell cycle. Loss of PCNA may be important under periods of oxidative stress to prevent low fidelity DNA repair which may incur mutations. However,
PCNA interactions with p21 and DNA polymerase δ are unknown, and in response to
NO, PCNA may be needed to help repair mutations from oxidative DNA damage
(Figure 3.6). Finally, the source of NO (hyperoxia or enzymatic) maybe important for determining whether PCNA becomes downregulated or S-nitrosylated.
Figure 3.6. Doxorubicin induction of iNOS S-nitrosylates PCNA leading to increased PCNA chromatin binding.
74
For this study doxorubicin treatment resulted in induction of iNOS. Riganti et al have shown in colon cancer that induction of iNOS is through the degradation of IKBα and NF-Kb expression 213. This pathway is the presumed pathway taking place in PCa cells however NF-Kb inhibitors are planned for future experiments. Owing to doxorubicin’s fungal source this could be an evolutionarily retained inflammatory response to fungal pathogens because doxorubicin elicits an immunological cell death different from other DNA damaging agents 202. However this stimulus could be a recapitulation of an unknown inflammatory signal within the prostate cancer microenvironment 75.
PCNA has many post-translation modifications which regulate its role dependent upon cell stimulus. We show here that PCNA is S-nitrosylated by iNOS on Cys81. This site has a potential to modulate PCNA function because it resides on the PCNA monomer interaction region that is important for forming the PCNA trimer on chromatin.
Our data shows that in response to doxorubicin there is site-specific S-nitrosylation which is required for PCNA chromatin binding, and mutation of Cys81 and NOS inhibition hinders the entire pool of PCNA from interaction with chromatin following doxorubicin treatment. Perhaps S-nitrosylation at Cys81 stabilizes PCNA binding by recruitment of an unknown interacting protein. Interestingly, many of the proteins which interact with
PCNA do so at the interdomain connecting region which is where Cys135 resides, but this site was not important for doxorubicin mediated S-nitrosylation 214. We have also identified two potential PIP motifs in iNOS which would indicate that iNOS would interact with PCNA at the interdomain connecting region. If this is the case, PCNA Cys135 and 162
75
S-nitrosylation may be a consequence of PCNA binding to iNOS. However, we have been unable to date to show their interaction by coimmunoprecipitation (unpublished results). Since doxorubicin induced PCNA to bind the chromatin we thought that this would involve the DNA repair machinery and Cys162 would be important because of its close proximity to Lys164 192,215. Even though Cys 135 and 162 were determined to not be important in PCNA chromatin binding in response to doxorubicin, they may have other roles in cells undergoing high levels of nitrosative stress.
PCNA has been identified to be S-nitrosylated 115 prior to our identification in prostate cells. Therefore, this modification may be a universal signal in all mammalian cells under inflammatory responses. S-nitrosylation of PCNA may serve as an environmental sensor of intracellular NO level which signals for the cell to stop synthesizing its DNA. PCNA S-nitrosylation may act as a nuclear redox sensor through which the cell senses increased levels of NO and therefore initiates cell arrest and protects itself from doxorubicin interchelation. Even without doxorubicin or inflammatory signals PCNA can become S-nitrosylated by high levels of NO within the cell and may be an indicator of metabolic dysregulation which would be harmful for a highly proliferating cancer cell. However, for this to happen there would have to be high levels of NO diffusing into the nucleus. Therefore, this modification is one that can be exploited for cancer therapy. If PCNA cannot be S-nitrosylated then DNA synthesis could continue therefore allowing the cell to be susceptible to DNA damaging therapies.
76
Chapter 4: Estrogen Receptor β S-nitrosylation in prostate cancer
The data found in this chapter is the result of work performed by:
Jared Isaac, Liam Lee, Yong Yuan, and Shuk Mei Ho
Jared Isaac performed all experiments (unless otherwise specified), data interpretation, and wrote the manuscript.
Liam Lee conducted the ERβ reporter assays. (Figure 4.2D and 4.2E).
Yong Yuan identified ERβ to be S-nitrosylated by LC-MS.
Shuk Mei Ho provided experimental consultation and wrote the manuscript.
Estrogen Receptor Function in the Prostate
Estrogen response in the prostate is under the control of two estrogen receptors
(ER), ERα and ERβ 27,216–218. ERs contain six domains, a highly conserved (97% homologous) DNA binding domain, a hinge domain, a ligand binding domain (56% homologous) and N terminal AF-1 and C terminal AF-2 domains which are highly divergent 219 (Figure 4.1). The structural differences of ERα and ERβ influence their recruitment of different cofactors which determines which DNA sequences they bind and is the basis for their functional differences 155,220–222. In addition to their function as transcription factors in the nucleus, ERα and ERβ can also cause signal events independent of their genomic actions 223–225. When ERα is localized to the membrane it can signal through EGFR/MAPK 226 or PI3K 227. Also, ERβ localizes to the mitochondria where it regulates mitochondrial gene expression 228, and during carcinogenesis travels from the mitochondria to the nucleus 229. Recently identified isoforms of ER show
77 different expression patterns various cell types and tissues which increase the complexity of estrogen control in the prostate 155,156,218,230. In normal prostate tissue,
ERα is mainly expressed in stromal cells and ERβ is expressed in the basal epithelial cells, but no ERs are expressed in the luminal epithelial cells in which 98% of PCa are derived 231. Men of Hispanic or Asian ancestry have increased levels of ERα in comparison to Americans of Caucasian or African descent 232. Interestingly, PCa risk increases by ERα polymorphisms in Japanese men 233, and a promoter single- nucleotide polymorphism (SNP) of ERβ 234,235.
Figure 4.1. ERβ Functional Domains
Abstract
In general, full-length ERβ (ERβ1) plays an anti-proliferative role in the prostate
220,236,237. During the course of PCa progression ERβ expression is gradually lost due to
DNA methylation of an AP2 site of the ERβ promoter 238–241. Paradoxically, once there is
78 metastasis to bone or lymph node ERβ is reexpressed, 231, which can be lowered using anti-estrogen therapies 242. ERβ knockout mice develop BPH which is not seen in ERα knockout mice 243,244. Neonatal exposure of rats to E2 causes 38 downregulation of ERβ and upregulation of ERα with a concordant increase of neoplastic lesion in the adult ventral prostate. In aromatase knockout mice treated with ERβ agonists there was increased apoptosis and decreased prostatic growth which was mediated by TNF-α signaling 220.
ERβ was identified to be S-nitrosylated on Cys108, 209, and 512 in vitro after treatment with 1 mM CysNO followed by LC-MS. ERβ is S-nitrosylated in HEK-293, PC-
3 and DU-145 cells with 1 mM CysNO and 100 μM E2 treatment. Furthermore, enzyme
(iNOS) produced NO can cause decreased ERβ mediated expression of vitellogenin.
Future studies are warranted to understand fully what function S-nitrosylation has in
PCa cells.
Introduction
Recently combined expression of ERβ, endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factor 2α (HIF-2α) was found to correlate with an aggressive phenotype of PCa 134. Also, when BCa cells were treated with NOS inhibitors in the absence of serum there was an increase in apoptosis 245. The interaction of eNOS and
ERα or ERβ has been shown to produce increased NO levels in response to estrogen in many cell types 246–250. Given that ERα has been shown to have its transcription
79 regulated by NO 129 and the functional impact phosphorylation has on ERβ 157, the possibility of ERβ S-nitrosylation was investigated.
Materials and Methods
Cell culture- All cells were maintained in a humidified incubator at 37°C with 5%
155 CO2 as previously described .
Vector production- Plasmid expressing ERβ was a generous gift from Ricky
Leung and was cloned into pcDNA3.1/TOPO expression vector according to manufacturer’s protocol (Invitrogen, Carlsbad, CA).
Cell Transfection/Infection- HEK-293 cells were transfected using
Lipofectamine™ 2000 (Invitrogen) 156.
NO donor preparation and the Biotin Switch Technique - S-nitroso-L-cysteine
(CysNO) and S-nitroso-glutathione (GSNO) were freshly prepared as previously described 135. CysNO and GSNO were used for experiments with NO donors needed for short and long time points, respectively. The BST 118was performed as previously described 135. All experiments were performed in triplicate.
Gel electrophoresis and western-blot analysis- SDS-PAGE was performed with
10% gels under non-reducing conditions in triplicate, and were transferred onto a PVDF
80 membrane (Immobilon FL, Millipore, Billerica, MA) with a blotting cell (Invitrogen).
Antibodies used were: β-actin (A2228, Sigma Aldrich, St. Louis, MO) and ERβ (SC-
8974, Santa Cruz Biotechnology (SCBT), Santa Cruz, CA). Primary antibodies were used at 1:1000 dilutions overnight at 4oC or 1 hour followed by corresponding IRDye conjugated secondary antibodies (LICOR Biosciences, Lincoln, NE) used at 1:15,000 dilutions as previously described 135.
ERE Luciferase Reporter assay- ERβ1 transactivation activity was performed as previously published 157.
Preliminary Results
To test whether ERβ is S-nitrosylated, HEK-293 cells were transfected with ERβ constructs and treated with NO donor. S-nitrosylated ERβ was detected after the BST
(Figure 4.2A.) in the presence of NO donor. Recent reports have shown that high level of estrogens are correlated with NO production 246, and in two castrate resistant cell types, PC-3 and DU-145, ERβ becomes S-nitrosylated by estrogen as well as NO.
(Figure 4.2B.). Next through the work of Yong Yuan and Ying-Wai Lam, ERβ was shown to be S-nitrosylated on Cys108, 209 and 512 by BST followed by LC-MS identification (unpublished findings). ERβ identification was identified to be S- nitrosylated using previously published methodologies of biotinylated protein purification, tryptic digestion, LC-MS identification and data analysis 135. Interestingly
ERβ S-nitrosylation sites reside with domains critical for ERβ function the AF-1 (Cys108),
81
DNA binding domain (Cys209), AF-2 ligand binding (Cys512) domains (Figure 4.2C.). To determine the functional effect of ERβ, Ming-Tsung Lee conducted ERβ transactivation assays in HEK-293 with transfected ERβ luciferase reporters in the presence or absence of estrogen. iNOS transfections were chosen because of its high production of
NO, and caused decreased Vitellogenin expression in the presence of E2 (Figure
4.2D.). However in the absence of E2, iNOS caused increased AP-1 expression (Figure
4.2E.), and no effect was seen with GSNO (0.5 and 1.0 mM) treatment (Figures 4.2D and 2E.).
Figure 4.2. ERβ is S-nitrosylated in-vitro by NO donor and 17-β-estradiol. (A) HEK-293 cells were transfected with ERβ constructs for 24 hours then treated with 1 mM CysNO for 15 minutes. Cells were lysed and the BST was performed followed by western blotting. (B) PC-3 cells were treated with 1mM CysNO for 15 minutes or DMSO or 100 μM 17-β-estradiol for 30 minutes. Cells were lysed and the BST was performed followed by western blotting. Inputs 100 μg from confluent 10 cm plates ERββ blotting with H-150. (C) Recombinant ERβ was treated with 1mM CysNO for 30 minutes, quenched and tryptic digested followed by the BST and MS identification. Schematic of identified S-nitrosylation sites and where they reside within ERβ: A/B (AF-1 domain, Cys108), C (DNA binding domain Cys209), and F (AF-2 ligand binding domain Cys512). (D) ERβ transactivation reporter assays were done with estrogen, iNOS transfection or GSNO (0.5 and 1.0 mM) treatment.
82
Discussion
ERβ can be S-nitrosylated in response to exogenous NO exposure as well as high concentration of E2 in PC-3 and DU-145. ERβ can be S-nitrosylated on three sites
Cys108, 209 and 512 however, which of these sites is a physiologically S-nitrosylated in response to stimuli such as estrogen remains to be seen. Furthermore, enzyme (iNOS) produced NO can cause decreased ERβ mediated expression of vitellogenin.
Many studies have published on the interrelation between NO, NOS, ER, and estrogen 236–240, therefore it does not come as a surprise that estrogen can cause ERβ
S-nitrosylation. iNOS does not cause ERβ S-nitrosylation (unpublished results); therefore it is probable that eNOS causes ERβ S-nitrosylation and gives credibility to their interrelation in PCa patient prognosis 246. ERβ S-nitrosylation is probably a negative regulation given the Vitellogenin reporter data (Figure. 1D) and a response to elevated exposure to estrogens (Figure. 1B). This hypothesis is supported by ERβ1 anti-proliferative role and antagonistic nature of ERα in the prostate 220,236,237. Recently, it was shown that phosphorylated ERβ Ser105 inhibits BCa cell migration and invasion
157. Therefore, given its close proximity to Ser105, S-nitrosylated Cys108 may either antagonize or agonize ERβ mediated cell migration and invasion. This preliminary data should be used in future studies to better understand the role of ERβ S-nitrosylation in
PCa.
83
Chapter 5: General Discussion
Summary of Data
The data presented in this dissertation seeks to understand the role of a RNS molecule, NO, in PCa progression. Furthermore, this dissertation has focused on one
NO mediated PTM, S-nitrosylation, and how this affects the functions of three proteins,
ITGα6, PCNA and ERβ in the context of PCa. The major findings of this work are summarized as follows:
NO and iNOS can increase the migration of PCa cells (PC-3 and DU-145), and inhibition of iNOS and ITGα6 blocks NO-mediated PCa cell migration. ITGα6 is S- nitrosylated on Cys86, 131 and 502 with site specific, iNOS mediated S-nitrosylation taking place on ITGα6 Cys86. NO increases cell migration through ITGα6 Cys86 and promotes increased ITGβ1 heterodimerization. Cys86 is responsible for NO destabilization of
ITGα6-mediated cell adherence to laminin-1 by interrupting ITGα6 interaction with laminin β1 chain. Collectively, these results indicate that in PCa cells, iNOS can associate with ITGα6 S-nitrosylated Cys86 thereby causing decreased interaction with laminin β1 chain and increased interaction with ITGβ1. ITGα6 Cys86 therefore, causes decreased cell adhesion to laminin-1 and increased cell migration which may be important for causing PCa metastasis in humans.
84
In contrast to other DNA damaging chemotherapy drugs, only doxorubicin increases extracellular nitrite levels and causes PCNA S-nitrosylation in PC-3 and DU-
145 cells. DU-145 cells are more sensitive to doxorubicin and have a lower PCNA S- nitrosylation level compared to PC-3 cells. Doxorubicin induces rapid iNOS expression which in turn S-nitrosylates PCNA possibly through physical interaction. PCNA is S- nitrosylated on Cys81, 135 and 162 with Cys81 being most important for doxorubicin mediated S-nitrosylation. Doxorubicin induced PCNA chromatin binding can be inhibited by blocking NOS and doxorubicin inhibition of DNA synthesis can be partly restored using NOS inhibitors. Mutation of Cys81 inhibits PCNA chromatin binding and may confer resistance to doxorubicin. Overall, doxorubicin induction of iNOS causes PCNA
S-nitrosylation and chromatin binding in PCa cells.
In response to estrogen treatment and exogenous NO exposure ERβ is S- nitrosylated in PC-3 and DU-145 cells. Using MS, ERβ has three putative sites of S- nitrosylation Cys108, 209 and 512; however their function in PCa is unknown. Lastly, iNOS inhibits ERβ-mediated vitellogenin expression in response to estrogen in HEK-293 cells.
Experimental Limitations
In general, the major limitation of the work in this thesis is that many of the experiments are performed mainly in PC-3 cells and no correlations were made to primary PCa tumor samples. Even though there is some data in DU-145 and LNCaP cells, work involving stable clones was only successful using PC-3. Cells stably
85 expressing either ITGα6 or PCNA in both DU-145 and LNCaP did not survive antibiotic selection and was not the result of transfection efficiency. Approximately 50% transfection efficiency was seen for PC-3, DU-145 and LNCaP cells (data unpublished).
Satisfactory results were seen for PC-3 stable clones; therefore the use of an inducible expression system was not warranted. Not all functional assays dealing with S- nitrosylated cysteine mutants were performed with all the mutants. Showing that no effect was seen with other mutants besides ITGα6 Cys86 and PCNA Cys81 would bolster the conclusions presented in this thesis. Unlike phosphorylation, there exists no mutation that can mimic S-nitrosylation of cysteines. The mutation of cysteine to serine was chosen because serine retains a polarity similar to cysteine.
The contribution of endogenous ITGα6 was a potential confounding factors, however, sufficient functional changes negated the need for gene knockdown and rescue experiments. Cell adhesion on laminin-1 and wound healing assays were performed with PC-3/C131S cells in the presence and absence of 500 and 100 μM
GSNO respectively and showed similar results to PC-3/C86S cells (data unpublished).
ITGα6 Cys502 mutation caused PC-3 loss of adherence and down-regulation of β-actin expression and therefore, could not be cultured for more than one month (data unpublished). Even with conclusive cell migration data using the wound healing assay, the role of ITGα6 S-nitrosylation in cell invasion needs to be investigated using the
Boyden chamber assay. The 100 μM GSNO dose was chosen as the highest GSNO dose that did not adversely affect cell viability by first testing a range of doses (0, 100,
250 and 500 μM). The MTT assay which measures mitochondrial activity was used to
86 show that 100 μM GSNO and iNOS did not increase or decrease cell viability. Cell proliferation assays by BrDU incorporation were not warranted because no significant effects were seen with the MTS assay. Since invasive PCa cells are known to migrate on their own ECM proteins 251, coating of plates with laminin-1 was not done.
Even though PC-3 and DU-145 cells are both castrate-resistant cell lines they were chosen for the wound healing assay to demonstrate that NO mediated increase in cell migration was independent of wound closing pattern (single cells versus sheet of cells). iNOS transfection and 100 μM GSNO are used interchangeably because iNOS mediated production of NO is known to be in the μM range203,252. Discrepancy between
NO and iNOS mediated migration is related to because iNOS production of NO is targeted compared to GSNO. The Griess/Salzmann assay is used to detect extracellular nitrite and is used in this study as an indirect means to quantitate NO concentration. For more accurate detection of NO, before it is oxidized to nitrate and nitrite, fluorescent DAF probes should be used. Differences in detected levels of nitrite between PC-3/DU-145 and HEK-293 cells are because the PCa cell lines were grown in media containing phenol red and fibroblasts were grown in media without phenol red.
100 μM dose of 1400W was chosen to inhibit iNOS enzymatic function from several published studies 166,253,254.
It is unknown what region of ITGα6 (head or thigh) is responsible for binding to laminin-1, however, it is known that ITGα6 binds to laminin β1 and γ1 chains 142,158 by using ITGα6 inhibiting antibody, GoH3. S-nitrosylation of ITGα6 may be a physiological
87 signal for ITGα6 detachment from the basement membrane because low level of S- nitrosylation was seen without enrichment with NO. iNOS expression has been shown to be degraded in p53 expressing cells; however this was not a problem for PC-3 and
DU-145 cells which p53 null and p53 mutated, respectively. iNOS may bind to ITGα6β4 or ITGα6β1 complex, however blotting was only done for ITGα6 in the coimmunoprecipitations. In Figures 3.2B and 3.4C, there is an apparent contradiction as 1400W treatment results in decreased doxorubicin PCNA S-nitrosylation but L-
NAME treatment inhibits doxorubicin mediated PCNA chromatin binding. A potential reason for this may be that eNOS and nNOS may compensate during specific iNOS inhibition with 1400W, which is why a pan-NOS inhibitor is more successful. However this reason remains to be proven. Unsuccessfully cytokine treatments 125 of PCa cells did not result in iNOS induction, unlike doxorubicin treatment 213. Expression analysis of cytokine receptors in prostate cancer cell lines could give insight into which cells may be responsive to cytokine induction of iNOS. Also, eNOS expression was not shown for either PC-3 or DU-145 cells (data unpublished), but may not be true for AR positive cells such as LNCaP. In concluding, bioinformatic modeling of how S-nitrosylation may alter ERβ function or structure was deemed inconclusive without mutational data for support.
Future Directions
For ITGα6, live cell imaging and Boyden chamber assays of NO-mediated cell invasion will give further support to the findings presented here. The determine fully that
88 iNOS S-nitrosylates ITGα6 on Cys86 PC-3/EV, PC-3/WT, PC-3/C86S, PC-3/C131S and
PC-3/C502S cells should be infected with iNOS and submitted to the BST as in Figure
2.8D. Performing the wound healing assay with antibodies directed against ITGβ1,
ITGβ4 and ITGαV on laminin-1 and fibronectin coated plates would also show the specificity of S-nitrosylation of ITGα6. Validation of the physiological role of ITG 6 S- nitrosylation is needed, using mutant ITG 6 stable metastatic prostate cancer cells in an orthotopic murine metastasis model. Site identification and study of ITGβ4 S- nitrosylation may reveal as the mechanism of ITGβ4 loss in PCa progression.
For PCNA, RNA expression of iNOS in doxorubicin time course may be a better way to detect iNOS expression. It is also of importance to conclusively show that doxorubicin is acting through NF-κB to promote NO production. It will also be interesting to see if PCNA S-nitrosylation results in aberrant binding to its protein interacting partners p21, iNOS, p53, and DNA polymerase δ by immunofluoresence. Preliminary data suggests that NO alone affect PCNA trimer information (data unpublished) and the role of doxorubicin in PCNA trimer formation should be investigated. Using NOS inhibitors gave seemingly conflicting results Figures 3.2B and 3.4C, so future studies should be conducted using siRNA targeted toward eNOS, iNOS and nNOS. Due to the fact that doxorubicin, has other functions besides inducing NO production the DNA synthesis experiments should be conducted using iNOS expression alone without doxorubicin treatment. All experiments should be replicated in DU-145 cells to confirm the results found in PC-3.
89
For ERβ, ectopic expression of eNOS may be needed for ERβ enzymatic S- nitrosylation. Mutational analysis of each identified S-nitrosylated cysteine will reveal which site or sites are important in E2-mediated ERβ S-nitrosylation. Functional assays such as ERβ dimerization and E2 binding should be investigated as well as determining which ERβ isoforms are S-nitrosylated.
With the S-nitrosylation proteome identified in normal prostate epithelial cells 135, it is of interest to perform more MS-based profiling studies to determine which subsets of proteins are S-nitrosylated in a PCa background and in patient samples. It will be interesting to see if ITGα6, PCNA and ERβ S-nitrosylation are retained in advanced stages of PCa. Secondly, correlation of iNOS/eNOS expression with total cell S- nitrosylation by IHC staining of human tissue samples of increasing grades of PCa will give further insight into the role of S-nitrosylation in PCa progression. Also, correlating
ITGα6, PCNA and ERβ S-nitrosylation with PCa stage may be helpful in determining
PCa prognosis. Knowledge of specific protein s-nitrosylation using PCa could potentially impact the prediction of early stage and recurrent PCa metastasis and drug development targeting S-nitrosylated proteins involved in PCa metastasis. Previous studies have shown that iNOS expression increases as PCa grade increases. In theory the amount of S-nitrosylation should increase with prostatic disease progression. These results would show that S-nitrosylation correlates with PCa development. It is expect that S-nitrosylation will be highest in metastatic PCa samples. These data would provide a foundation to investigate further the role of S-nitrosylation in PCa. Future studies potentially can address effective treatments for advanced PCa in which the level of NO
90 within the prostate is controlled thus halting PCa progression. Also NO staining could be a useful technique to distinguish lethal from indolent disease in prostate biopsies.
Discussion and Perspective
Increased OS is common in cancer and causes DNA damage, lipid peroxidation and protein PTM. As a component of OS, NO is thought to both inhibit or promote cancer progression through nitrosative stress, which is determined by the concentration and localization of NO, genetic and epigenetic status and whether the cell is the recipient or host of NO production. In this body of work, three separate proteins that are all important in PCa progression, have their function modulated by a NO-mediated PTM,
S-nitrosylation. In agreement with the literature, S-nitrosylation causes different effects on each studied protein as expected for such divergent proteins: a cell membrane receptor, a DNA replication processivity factor, and an estrogen receptor. S-nitrosylation increases ITGα6-mediated cell migration and PCNA chromatin binding and decreases
ERβ gene expression. S-nitrosylation of ITGα6 in cell migration clearly has implications in cancer progression; however, its role in PCNA chromatin binding may either be a mechanism of overcoming doxorubicin resistance (MRP3 tyrosine nitration) 201,255 or proceeding with DNA replication. For ERβ S-nitrosylation more experiments are required to determine whether this PTM is anti or pro-tumorigenic.
It would be interesting to determine how S-nitrosylation affects ITGα6, PCNA and
ERβ in cancers in other tissues as well as normal tissue. ITGα6, PCNA, ERβ should
91 have the same S-nitrosylated cysteines because of their S-nitrosylation by overexpression in HEK-293 cells. It is expected that S-nitrosylation of ITGα6 in breast cancer and melanoma would increase cell migration as in PCa because of ITGα6 important role in these tissues. PCNA S-nitrosylation is readily detectable in normal as well as cancerous prostate tissue and is expected to occur in all cell types sensitive to doxorubicin with functional NF-κB pathways. Given the antiproliferative role that ERβ plays in the prostate it is expected that ERβ S-nitrosylation will be unique to the prostate.
From iNOS staining in PCa biopsies, iNOS is expressed at a high level in metastases 139 thus for these samples iNOS production of NO is assumed to be sustained. However for the case of ITGα6 iNOS S-nitrosylation may not need to be prolonged, but only long enough to cause detachment to laminin-β1. S-nitrosylation of
Cys86 may only occur at the leading edge of the cell during cell migration and then be removed by denitrosylases, reducing enzymes and proteasome dependent degradation.
Doxorubicin induction of iNOS occurs after 1 hour and is maintained for at least 4-8 hours (data unpublished) however PCNA is no longer S-nitrosylated after 24 hours of doxorubicin treatment (data unpublished). 100 μM E2 for 30 minutes results in ERβ S- nitrosylation but future experiments will determine whether this affects ERβ protein half- life.
Since NO is produced from tumor associated macrophages 108, S-nitrosylation of
ITGα6, PCNA, ERβ and HIF-1α may all be mechanisms of how cancers escape and
92 survive after radiation therapy. In fact, it has been shown that NOS, ERβ and HIF-1a expression in PCa patients is correlated with a poorer prognosis 134, so it would be of interest to determine if ERβ and HIF-1α are S-nitrosylated as well in these patients.
Perhaps ITGα6, PCNA and ERβ are all targets for cancer promoting signaling generated from inflammation-generated NO. Blocking of ITGα6, PCNA and ERβ S- nitrosylation could be effective by using antibodies specific for the S-nitrosylation sites, but better results are expected by targeting exogenous NO produced by the tumor associated macrophages. Therefore, NF-κB and NOS inhibitors could be used in combination with radiation to block inflammation induced PCa progression as well as increase the efficacy and lower the dose of radiation therapy. Treatment with NF-κB inhibitors could decrease PCa patient ability to fight infections and NOS inhibitors would have significant cardiovascular side effects, so these treatments would be most efficacious by specifically targeting the prostate. This could be accomplished by direct injection of NF-κB and NOS inhibitors into the prostate or using RNA aptamer delivery targeting prostate specific membrane antigen, PSMA. Reducing ROS, RNS and inflammation with diet and lifestyle are potential avenues to prevent pathological S- nitrosylation in the prostate. Prevention of chronic prostatitis, use of non-steroidal anti- inflammatory drugs, NSAIDS and reducing agents such as vitamins may provide options for curbing high levels of NO and therefore reduce nitrosative stress.
In conclusion, the data in this thesis is the first to show that S-nitrosylation can have a significant effect on protein function in prostate cancer. Using an unbiased approach, ITGα6 and PCNA were identified to S-nitrosylated by exogenous NO in
93 normal prostate cells and were the focus of functional studies. Specifically, this thesis sheds light on how NO produced by iNOS can affect PCa cell migration by S- nitrosylation of ITGα6 which has implications into understanding how PCa metastasizes from the prostate. However, it is unclear whether S-nitrosylation of PCNA contributes to doxorubicin cytotoxicity, acts as a redox sensor or is a mechanism of chemoresistance.
This is also the first report of estrogen induced S-nitrosylation of ERβ in prostate cancer cells. Given that S-nitrosylation affects ITGα6, PCNA and ERβ which all have important functions in prostate cancer progression inhibition of S-nitrosylation may have potential clinical value.
94
References
1. Adams JY, Leav I, Lau KM, Ho SM, Pflueger S. Expression of estrogen receptor beta in the fetal, neonatal, and prepubertal human prostate. Prostate 2002; 52(1): 69-
81.
2. Shapiro E, Huang H, Masch RJ, McFADDEN DE, Wilson EL, Wu XR.
Immunolocalization of estrogen receptor α and β in human fetal prostate. J Urol 2005;
174(5): 2051-3.
3. Krieg M, Nass R, Tunn S. Effect of aging on endogenous level of 5 alpha- dihydrotestosterone, testosterone, estradiol, and estrone in epithelium and stroma of normal and hyperplastic human prostate. Journal of Clinical Endocrinology &
Metabolism 1993; 77(2): 375.
4. Orwoll ES, Nielson CM, Labrie F, Barrett-Connor E, Cauley JA, Cummings SR, et al. Evidence for geographical and racial variation in serum sex steroid levels in older men. Journal of Clinical Endocrinology & Metabolism 2010; 95(10): E151.
5. Feldman HA, Longcope C, Derby CA, Johannes CB, Araujo AB, Coviello AD, et al. Age trends in the level of serum testosterone and other hormones in middle-aged men: longitudinal results from the Massachusetts male aging study. Journal of Clinical
Endocrinology & Metabolism 2002; 87(2): 589.
6. Gapstur SM, Gann PH, Kopp P, Colangelo L, Longcope C, Liu K. Serum
Androgen Concentrations in Young Men: A Longitudinal Analysis of Associations with
Age, Obesity, and Race. Cancer Epidemiology Biomarkers & Prevention 2002; 11(10):
1041.
95
7. Kaufman JM, Vermeulen A. The decline of androgen levels in elderly men and its clinical and therapeutic implications. Endocr Rev 2005; 26(6): 833-76.
8. Moretti C, Frajese GV, Guccione L, Wannenes F, De Martino MU, Fabbri A, et al.
Androgens and body composition in the aging male. J Endocrinol Invest 2005; 28(3
Suppl): 56-64.
9. Strka L, Pospilov H, Hill M. Free testosterone and free dihydrotestosterone throughout the life span of men. J Steroid Biochem Mol Biol 2009; 116(1): 118-20.
10. De Marzo AM, Platz EA, Sutcliffe S, Xu J, Grönberg H, Drake CG, et al.
Inflammation in prostate carcinogenesis. Nature Reviews Cancer 2007; 7(4): 256-69.
11. DeNardo DG, Coussens LM. Inflammation and breast cancer. Balancing immune response: crosstalk between adaptive and innate immune cells during breast cancer progression. Breast Cancer Res 2007; 9(4): 212.
12. Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis. International Journal of
Cancer 2007; 121(11): 2381-6.
13. Lu H, Ouyang W, Huang C. Inflammation, a key event in cancer development.
Molecular Cancer Research 2006; 4(4): 221.
14. Yatkin E, Bernoulli J, Talvitie EM, Santti R. Inflammation and epithelial alterations in rat prostate: impact of the androgen to oestrogen ratio. Int J Androl 2009; 32(4): 399-
410.
15. Bernoulli J, Yatkin E, Konkol Y, Talvitie EM, Santti R, Streng T. Prostatic inflammation and obstructive voiding in the adult Noble rat: impact of the testosterone to estradiol ratio in serum. Prostate 2008; 68(12): 1296-306.
96
16. Tam NNC, Leav I, Ho SM. Sex hormones induce direct epithelial and inflammation-mediated oxidative/nitrosative stress that favors prostatic carcinogenesis in the noble rat. Am J Pathol 2007; 171(4): 1334.
17. Yatkin E, Bernoulli J, Lammintausta R, Santti R. Fispemifene [Z-2-{2-[4-(4-chloro-
1, 2-diphenylbut-1-enyl)-phenoxy] ethoxy}-ethanol], a novel selective estrogen receptor modulator, attenuates glandular inflammation in an animal model of chronic nonbacterial prostatitis. J Pharmacol Exp Ther 2008; 327(1): 58-67.
18. McConnell JD. Benign prostatic hyperplasia. Curr Opin Urol 1998; 8(1): 1.
19. Barry MJ, Roehrborn CG. Benign prostatic hyperplasia. BMJ 2001; 323(7320):
1042-6.
20. De Jong F, Oishi K, Hayes R, Bogdanowicz J, Raatgever J, Van Der Maas P, et al. Peripheral hormone levels in controls and patients with prostatic cancer or benign prostatic hyperplasia: results from the Dutch-Japanese case-control study. Cancer Res
1991; 51(13): 3445.
21. Epstein JI. Prostatic intraepithelial neoplasia. Adv Anat Pathol 1994; 1(3): 123.
22. Bostwick DG, Burke HB, Djakiew D, Euling S, Ho S, Landolph J, et al. Human prostate cancer risk factors. Cancer 2004; 101(S10): 2371-490.
23. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA: A Cancer
Journal for Clinicians 2012; .
24. Morton Jr RA. Racial differences in adenocarcinoma of the prostate in North
American men. Urology 1994; 44(5): 637.
97
25. Hoffman RM, Gilliland FD, Eley JW, Harlan LC, Stephenson RA, Stanford JL, et al. Racial and ethnic differences in advanced-stage prostate cancer: the Prostate
Cancer Outcomes Study. J Natl Cancer Inst 2001; 93(5): 388-95.
26. Ho S, Leung YK, Chung I. Estrogens and antiestrogens as etiological factors and therapeutics for prostate cancer. Ann N Y Acad Sci 2006; 1089(1): 177-93.
27. Prins GS, Korach KS. The role of estrogens and estrogen receptors in normal prostate growth and disease. Steroids 2008; 73(3): 233-44.
28. Singh PB, Matanhelia SS, Martin FL. A potential paradox in prostate adenocarcinoma progression: oestrogen as the initiating driver. Eur J Cancer 2008;
44(7): 928-36.
29. Bain J. Testosterone and the aging male: To treat or not to treat? Maturitas 2010;
66(1): 16-22.
30. Ho SM. Estrogens and anti‐estrogens: Key mediators of prostate carcinogenesis and new therapeutic candidates. J Cell Biochem 2004; 91(3): 491-503.
31. Ellem SJ, Risbridger GP. Aromatase and regulating the estrogen: androgen ratio in the prostate gland. J Steroid Biochem Mol Biol 2010; 118(4-5): 246-51.
32. Griffiths K. Estrogens and prostatic disease. Prostate 2000; 45(2): 87-100.
33. Roberts RO, Jacobson DJ, Rhodes T, Klee GG, Leiber MM, Jacobsen SJ. Serum sex hormones and measures of benign prostatic hyperplasia. Prostate 2004; 61(2): 124-
31.
34. Farnsworth WE. Roles of estrogen and SHBG in prostate physiology. Prostate
1996; 28(1): 17-23.
98
35. Ellem S, Risbridger G. Aromatase and prostate cancer. Minerva Endocrinol
2006; 31(1): 1.
36. Cavalieri EL, Rogan EG. A unified mechanism in the initiation of cancer. Ann N Y
Acad Sci 2002; 959(1): 341-54.
37. Prins GS, Sklarew RJ, Pertschuk LP. Image analysis of androgen receptor immunostaining in prostate cancer accurately predicts response to hormonal therapy. J
Urol 1998; 159(3): 641-9.
38. Prins GS, Birch L, Couse JF, Choi I, Katzenellenbogen B, Korach KS. Estrogen
Imprinting of the Developing Prostate Gland Is Mediated through Stromal Estrogen
Receptor α. Cancer Res 2001; 61(16): 6089.
39. Prins G. Neonatal estrogen exposure induces lobe-specific alterations in adult rat prostate androgen receptor expression. Endocrinology 1992; 130(6): 3703-14.
40. McLachlan JA, Simpson E, Martin M. Endocrine disrupters and female reproductive health. Best Pract Res Clin Endocrinol Metab 2006; 20(1): 63-75.
41. Ho S, Lee K, Lane K. Neoplastic transformation of the prostate. Prostate: Basic and Clinical Aspects 1997; : 73-113.
42. Ricke WA, Ishii K, Ricke EA, Simko J, Wang Y, Hayward SW, et al. Steroid hormones stimulate human prostate cancer progression and metastasis. International journal of cancer 2006; 118(9): 2123-31.
43. McPherson SJ, Wang H, Jones ME, Pedersen J, Iismaa TP, Wreford N, et al.
Elevated androgens and prolactin in aromatase-deficient mice cause enlargement, but not malignancy, of the prostate gland. Endocrinology 2001; 142(6): 2458.
99
44. Mehra R, Tomlins SA, Yu J, Cao X, Wang L, Menon A, et al. Characterization of
TMPRSS2-ETS gene aberrations in androgen-independent metastatic prostate cancer.
Cancer Res 2008; 68(10): 3584.
45. Wang S, Gao J, Lei Q, Rozengurt N, Pritchard C, Jiao J, et al. Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer.
Cancer cell 2003; 4(3): 209-21.
46. Ghatak S, Oliveria P, Kaplan P, Ho S. Expression and regulation of metallothionein mRNA levels in the prostates of noble rats: lack of expression in the ventral prostate and regulation by sex hormones in the dorsolateral prostate. Prostate
1996; 29(2): 91-100.
47. Ho SM, Leav I, Ghatak S, Merk F, Jagannathan VS, Mallery K. Lack of association between enhanced TRPM-2/clusterin expression and increased apoptotic activity in sex-hormone-induced prostatic dysplasia of the Noble rat. The American journal of pathology 1998; 153(1): 131-9.
48. Ouyang X, Wang X, Lee D, Tsao S, Wong Y. Up-regulation of TRPM-2, MMP-7 and ID-1 during sex hormone-induced prostate carcinogenesis in the Noble rat.
Carcinogenesis 2001; 22(6): 965.
49. Tam NNC, Szeto CYY, Sartor MA, Medvedovic M, Ho SM. Gene Expression
Profiling Identifies Lobe-Specific and Common Disruptions of Multiple Gene Networks in
Testosterone-Supported, 17β-Estradiol-or Diethylstilbestrol-Induced Prostate Dysplasia in Noble Rats. Neoplasia (New York, NY) 2008; 10(1): 20.
100
50. Thompson CJ, Tam NNC, Joyce JM, Leav I, Ho S. Gene expression profiling of testosterone and estradiol-17β-induced prostatic dysplasia in Noble rats and response to the antiestrogen ICI 182,780. Endocrinology 2002; 143(6): 2093-105.
51. Lau KM, Leav I, Ho SM. Rat estrogen receptor-α and-β, and progesterone receptor mRNA expression in various prostatic lobes and microdissected normal and dysplastic epithelial tissues of the Noble rats. Endocrinology 1998; 139(1): 424-7.
52. Wang Y, Wong Y. Sex hormone‐induced prostatic carcinogenesis in the Noble rat: The role of insulin‐like growth factor‐1(IGF‐1) and vascular endothelial growth factor(VEGF) in the development of prostate cancer. Prostate 1998; 35(3): 165-77.
53. Wong Y, Xie W, Tsao S. Structural changes and alteration in expression of
TGF‐β1 and its receptors in prostatic intraepithelial neoplasia (PIN) in the ventral prostate of noble rats. Prostate 2000; 45(4): 289-98.
54. Kaplan PJ, Leav I, Greenwood J, Kwan PWL, Ho S. Involvement of transforming growth factor α (TGFα) and epidermal growth factor receptor (EGFR) in sex hormone- induced prostatic dysplasia and the growth of an androgen-independent transplantable carcinoma of the prostate. Carcinogenesis 1996; 17(12): 2571-9.
55. Ling MT, Wang X, Ouyang XS, Lee T, Fan TY, Xu K, et al. Activation of MAPK signaling pathway is essential for Id-1 induced serum independent prostate cancer cell growth. Oncogene 2002; 21(55): 8498.
56. Leav I, Merk FB, Kwan PWL, Ho SM. Androgen‐supported estrogen‐enhanced epithelial proliferation in the prostates of intact noble rats. Prostate
1989; 15(1): 23-40.
101
57. Ho S, Roy D. Sex hormone-induced nuclear DNA damage and lipid peroxidation in the dorsolaterel prostates of Noble rats. Cancer Lett 1994; 84(2): 155-62.
58. Li S, Chen G, Chan PSF, Choi H, Ho SM, Chan FL. Altered expression of extracellular matrix and proteinases in noble rat prostate gland after long‐term treatment with sex steroids. Prostate 2001; 49(1): 58-71.
59. Yager JD. Endogenous estrogens as carcinogens through metabolic activation.
JNCI Monographs 2000; 2000(27): 67-73.
60. Liehr JG. Is Estradiol a Genotoxic Mutagenic Carcinogen? Endocr Rev 2000;
21(1): 40- 54.
61. Wright JS. Genotoxicity of Endogenous Estrogens. Endogenous Toxins 2010; :
859-79.
62. Liehr JG. Breast carcinogensis and its prevention by inhibition of estrogen genotoxicity. Endocrine therapy in breast cancer.Marcel Dekker, New York 2002; : 287-
301.
63. Arbiser JL, Petros J, Klafter R, Govindajaran B, McLaughlin ER, Brown LF, et al.
Reactive oxygen generated by Nox1 triggers the angiogenic switch. Proceedings of the
National Academy of Sciences 2002; 99(2): 715-20.
64. Loibl S, Strank C, von Minckwitz G, Sinn HP, Buck A, Solbach C, et al.
Immunohistochemical evaluation of endothelial nitric oxide synthase expression in primary breast cancer. The Breast 2005; 14(3): 230-5.
65. Stetler-Stevenson W, Aznavoorian S, Liotta L. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu Rev Cell Biol 1993; 9(1): 541-
73.
102
66. Chaiswing L, Zhong W, Cullen JJ, Oberley LW, Oberley TD. Extracellular redox state regulates features associated with prostate cancer cell invasion. Cancer Res
2008; 68(14): 5820.
67. Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, et al. Cell transformation by the superoxide-generating oxidase Mox1. Nature 1999; 401(6748):
79-82.
68. Bulut AS, Erden E, Sak SD, Doruk H, Kursun N, Dincol D. Significance of inducible nitric oxide synthase expression in benign and malignant breast epithelium: an immunohistochemical study of 151 cases. Virchows Archiv 2005; 447(1): 24-30.
69. Thomsen LL, Miles DW, Happerfield L, Bobrow LG, Knowles RG, Moncada S.
Nitric oxide synthase activity in human breast cancer. Br J Cancer 1995; 72(1): 41-4.
70. Planchet E, Kaiser WM. Nitric oxide (NO) detection by DAF fluorescence and chemiluminescence: a comparison using abiotic and biotic NO sources. J Exp Bot 2006;
57(12): 3043-55.
71. Tsikas D. Analysis of nitrite and nitrate in biological fluids by assays based on the
Griess reaction: appraisal of the Griess reaction in the L-arginine/nitric oxide area of research. Journal of Chromatography B 2007; 851(1-2): 51-70.
72. Furchgott RF. Endothelium-derived relaxing factor: discovery, early studies, and identification as nitric oxide. Biosci Rep 1999; 19(4): 235-51.
73. Lane P, Hao G, Gross SS. S-nitrosylation is emerging as a specific and fundamental posttranslational protein modification: head-to-head comparison with O- phosphorylation. Science's STKE 2001; 2001(86): re1.
103
74. Stępnik M. Roles of nitric oxide in carcinogenesis. Protumorigenic effects. Int J
Occup Med Environ Health 2002; 15(3): 219-27.
75. Lucia MS, Torkko KC. Inflammation as a target for prostate cancer chemoprevention: pathological and laboratory rationale. J Urol 2004; 171(2): S30-5.
76. Hirst DG, Robson T. Nitrosative stress in cancer therapy. Front Biosci 2007; 12:
3406-18.
77. Kumar B, Koul S, Khandrika L, Meacham RB, Koul HK. Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Res
2008; 68(6): 1777.
78. Lala PK, Orucevic A. Role of nitric oxide in tumor progression: lessons from experimental tumors. Cancer Metastasis Rev 1998; 17(1): 91-106.
79. Xie K, Huang S, Dong Z, Juang S, Gutman M, Xie Q, et al. Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells. J Exp Med 1995; 181(4): 1333-43.
80. Kim YM, Kim TH, Seol DW, Talanian RV, Billiar TR. Nitric Oxide Suppression of
Apoptosis Occurs in Association with an Inhibition of Bcl-2 Cleavage and Cytochrome cRelease. J Biol Chem 1998; 273(47): 31437-41.
81. Chanvorachote P, Nimmannit U, Stehlik C, Wang L, Jiang BH, Ongpipatanakul
B, et al. Nitric oxide regulates cell sensitivity to cisplatin-induced apoptosis through S- nitrosylation and inhibition of Bcl-2 ubiquitination. Cancer Res 2006; 66(12): 6353.
82. Estrada C, Gómez C, Martín-Nieto J, De Frutos T, Jiménez A, Villalobo A. Nitric oxide reversibly inhibits the epidermal growth factor receptor tyrosine kinase. Biochem J
1997; 326(Pt 2): 369.
104
83. Pervin S, Singh R, Hernandez E, Wu G, Chaudhuri G. Nitric oxide in physiologic concentrations targets the translational machinery to increase the proliferation of human breast cancer cells: involvement of mammalian target of rapamycin/eIF4E pathway.
Cancer Res 2007; 67(1): 289.
84. Thomas DD, Espey MG, Ridnour LA, Hofseth LJ, Mancardi D, Harris CC, et al.
Hypoxic inducible factor 1α, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide. Proc Natl Acad Sci U S A 2004;
101(24): 8894.
85. Jadeski LC, Lala PK. Nitric oxide synthase inhibition by NG-nitro-L-arginine methyl ester inhibits tumor-induced angiogenesis in mammary tumors. Am J Pathol
1999; 155(4): 1381.
86. Thomsen LL, Scott JMJ, Topley P, Knowles RG, Keerie AJ, Frend AJ. Selective inhibition of inducible nitric oxide synthase inhibits tumor growth in vivo: studies with
1400W, a novel inhibitor. Cancer Res 1997; 57(15): 3300.
87. Pervin S, Singh R, Freije WA, Chaudhuri G. MKP-1-Induced Dephosphorylation of Extracellular Signal-Regulated Kinase Is Essential for Triggering Nitric Oxide-Induced
Apoptosis in Human Breast Cancer Cell Lines Implications in Breast Cancer. Cancer
Res 2003; 63(24): 8853-60.
88. Pervin S, Singh R, Chaudhuri G. Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): potential role of cyclin D1.
Proceedings of the National Academy of Sciences 2001; 98(6): 3583.
105
89. Pipili-Synetos E, Sakkoula E, Haralabopoulos G, Andriopoulou P, Peristeris P,
Maragoudakis M. Evidence that nitric oxide is an endogenous antiangiogenic mediator.
Br J Pharmacol 1994; 111(3): 894.
90. Pipili-Synetos E, Papageorgiou A, Sakkoula E, Sotiropoulou G, Fotsis T,
Karakiulakis G, et al. Inhibition of angiogenesis, tumour growth and metastasis by the
NO-releasing vasodilators, isosorbide mononitrate and dinitrate. Br J Pharmacol 1995;
116(2): 1829.
91. Xie K, Fidler IJ. Therapy of cancer metastasis by activation of the inducible nitric oxide synthase. Cancer Metastasis Rev 1998; 17(1): 55-75.
92. Chaiswing L, Oberley TD. Extracellular/microenvironmental redox state.
Antioxidants & redox signaling 2010; 13(4): 449-65.
93. Chaiswing L, Zhong W, Liang Y, Jones DP, Oberley TD. Regulation of prostate cancer cell invasion by modulation of extra-and intracellular redox balance. Free
Radical Biology and Medicine 2011; .
94. Klotz T, Bloch W, Volberg C, Engelmann U, Addicks K. Selective expression of inducible nitric oxide synthase in human prostate carcinoma. Cancer 1998; 82(10):
1897-903.
95. Tong X, Li H. eNOS protects prostate cancer cells from TRAIL-induced apoptosis. Cancer Lett 2004; 210(1): 63-71.
96. Vakkala M, Kahlos K, Lakari E, Paakko P, Kinnula V, Soini Y. Inducible Nitric
Oxide Synthase Expression, Apoptosis, and Angiogenesis in in Situ and Invasive Breast
Carcinomas 1. Clinical Cancer Research 2000; 6(6): 2408-16.
106
97. Cianchi F, Cortesini C, Fantappie O, Messerini L, Sardi I, Lasagna N, et al.
Cyclooxygenase-2 activation mediates the proangiogenic effect of nitric oxide in colorectal cancer. Clinical Cancer Research 2004; 10(8): 2694-704.
98. Ekmekcioglu S, Ellerhorst J, Smid CM, Prieto VG, Munsell M, Buzaid AC, et al.
Inducible Nitric Oxide Synthase and Nitrotyrosine in Human Metastatic Melanoma
Tumors Correlate with Poor Survival 1. Clinical Cancer Research 2000; 6(12): 4768-75.
99. Ambs S, Merriam WG, Bennett WP, Felley-Bosco E, Ogunfusika MO, Oser SM, et al. Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression. Cancer Res 1998;
58(2): 334-41.
100. Kojima M, Morisaki T, Tsukahara Y, Uchiyama A, Matsunari Y, Mibu R, et al.
Nitric oxide synthase expression and nitric oxide production in human colon carcinoma tissue. J Surg Oncol 1999; 70(4): 222-9.
101. Loibl S, Buck A, Strank C, von Minckwitz G, Roller M, Sinn HP, et al. The role of early expression of inducible nitric oxide synthase in human breast cancer. Eur J
Cancer 2005; 41(2): 265-71.
102. Thomsen LL, Lawton FG, Knowles RG, Beesley JE, Riveros-Moreno V, Moncada
S. Nitric oxide synthase activity in human gynecological cancer. Cancer Res 1994;
54(5): 1352-4.
103. Aaltoma SH, Lipponen PK, Kosma VM. Inducible nitric oxide synthase (iNOS) expression and its prognostic value in prostate cancer. Anticancer Res 2001; 21(4B):
3101-6.
107
104. Cronauer M, Ince Y, Engers R, Rinnab L, Weidemann W, Suschek C, et al. Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression. Oncogene 2006; 26(13): 1875-84.
105. Crowell JA, Steele VE, Sigman CC, Fay JR. Is inducible nitric oxide synthase a target for chemoprevention? Molecular cancer therapeutics 2003; 2(8): 815.
106. Kedia GT, Ückert S, Jonas U, Kuczyk MA, Burchardt M. The nitric oxide pathway in the human prostate: clinical implications in men with lower urinary tract symptoms.
World J Urol 2008; 26(6): 603-9.
107. Ng QS, Goh V, Milner J, Stratford MR, Folkes LK, Tozer GM, et al. Effect of nitric-oxide synthesis on tumour blood volume and vascular activity: a phase I study.
The lancet oncology 2007; 8(2): 111-8.
108. Li F, Sonveaux P, Rabbani ZN, Liu S, Yan B, Huang Q, et al. Regulation of HIF-
1α stability through S-nitrosylation. Mol Cell 2007; 26(1): 63-74.
109. Walsh G, Jefferis R. Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 2006; 24(10): 1241-52.
110. Mann M, Jensen ON. Proteomic analysis of post-translational modifications. Nat
Biotechnol 2003; 21(3): 255-61.
111. Witze ES, Old WM, Resing KA, Ahn NG. Mapping protein post-translational modifications with mass spectrometry. Nature methods 2007; 4(10): 798-806.
112. Stamler JS, Toone EJ, Lipton SA, Sucher NJ. (S) NO signals: translocation, regulation, and a consensus motif. Neuron 1997; 18(5): 691-6.
113. Zhang Y, Hogg N. < i> S-Nitrosothiols: cellular formation and transport. Free radical biology and medicine 2005; 38(7): 831-8.
108
114. Benhar M, Forrester MT, Hess DT, Stamler JS. Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins. Science's STKE 2008; 320(5879): 1050.
115. Forrester MT, Thompson JW, Foster MW, Nogueira L, Moseley MA, Stamler JS.
Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture. Nat
Biotechnol 2009; 27(6): 557-9.
116. Hess DT, Stamler JS. Regulation by S-nitrosylation of protein post-translational modification. J Biol Chem 2012; 287(7): 4411-8.
117. Hess DT, Matsumoto A, Kim SO, Marshall HE, Stamler JS. Protein S- nitrosylation: purview and parameters. Nature Reviews Molecular Cell Biology 2005;
6(2): 150-66.
118. Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH. Protein S- nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Biol 2001; 3(2):
193-7.
119. Foster MW, McMahon TJ, Stamler JS. S-nitrosylation in health and disease.
Trends Mol Med 2003; 9(4): 160-8.
120. Foster MW, Hess DT, Stamler JS. Protein S-nitrosylation in health and disease: a current perspective. Trends Mol Med 2009; 15(9): 391-404.
121. Benhar M, Stamler JS. A central role for S-nitrosylation in apoptosis. Nat Cell Biol
2005; 7(7): 645-6.
122. Uehara T, Nakamura T, Yao D, Shi ZQ, Gu Z, Ma Y, et al. S-nitrosylated protein- disulphide isomerase links protein misfolding to neurodegeneration. Nature 2006;
441(7092): 513-7.
109
123. Marshall HE, Stamler JS. Inhibition of NF-κB by S-nitrosylation. Biochemistry (N
Y ) 2001; 40(6): 1688-93.
124. Kelleher ZT, Matsumoto A, Stamler JS, Marshall HE. NOS2 regulation of NF-κB by S-nitrosylation of p65. J Biol Chem 2007; 282(42): 30667.
125. Azad N, Vallyathan V, Wang L, Tantishaiyakul V, Stehlik C, Leonard SS, et al. S- nitrosylation of bcl-2 inhibits its ubiquitin-proteasomal degradation. J Biol Chem 2006;
281(45): 34124-34.
126. Hara MR, Snyder SH. Nitric oxide–GAPDH–Siah: a novel cell death cascade.
Cell Mol Neurobiol 2006; 26(4): 525-36.
127. Jenkins JL, Tanner JJ. High-resolution structure of human D-glyceraldehyde-3- phosphate dehydrogenase. Acta Crystallographica Section D: Biological
Crystallography 2006; 62(3): 290-301.
128. Murillo-Carretero M, Torroglosa A, Castro C, Villalobo A, Estrada C. S-
Nitrosylation of the epidermal growth factor receptor: A regulatory mechanism of receptor tyrosine kinase activity. Free Radical Biology and Medicine 2009; 46(4): 471-9.
129. Garbán HJ, Márquez-Garbán DC, Pietras RJ, Ignarro LJ. Rapid nitric oxide- mediated S-nitrosylation of estrogen receptor: regulation of estrogen-dependent gene transcription. Proceedings of the National Academy of Sciences 2005; 102(7): 2632-6.
130. Nakamura T, Lipton S. Redox modulation by S-nitrosylation contributes to protein misfolding, mitochondrial dynamics, and neuronal synaptic damage in neurodegenerative diseases. Cell Death & Differentiation 2011; 18(9): 1478-86.
131. Greco TM, Hodara R, Parastatidis I, Heijnen HFG, Dennehy MK, Liebler DC, et al. Identification of S-nitrosylation motifs by site-specific mapping of the S-
110 nitrosocysteine proteome in human vascular smooth muscle cells. Proceedings of the
National Academy of Sciences 2006; 103(19): 7420.
132. Hao G, Derakhshan B, Shi L, Campagne F, Gross SS. SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures.
Proc Natl Acad Sci U S A 2006; 103(4): 1012-7.
133. Wartenberg M, Schallenberg M, Hescheler J, Sauer H. Reactive oxygen species‐mediated regulation of eNOS and iNOS expression in multicellular prostate tumor spheroids. International journal of cancer 2003; 104(3): 274-82.
134. Nanni S, Benvenuti V, Grasselli A, Priolo C, Aiello A, Mattiussi S, et al.
Endothelial NOS, estrogen receptor β, and HIFs cooperate in the activation of a prognostic transcriptional pattern in aggressive human prostate cancer. J Clin Invest
2009; 119(5): 1093.
135. Lam YW, Yuan Y, Isaac J, Babu CVS, Meller J, Ho SM. Comprehensive
Identification and Modified-Site Mapping of S-Nitrosylated Targets in Prostate Epithelial
Cells. 2010; .
136. Iyer AKV, Azad N, Wang L, Rojanasakul Y. Role of< i> S-nitrosylation in apoptosis resistance and carcinogenesis. Nitric Oxide 2008; 19(2): 146-51.
137. Koutsioumpa M, Hatziapostolou M, Mikelis C, Koolwijk P, Papadimitriou E.
Aprotinin stimulates angiogenesis and human endothelial cell migration through the growth factor pleiotrophin and its receptor protein tyrosine phosphatase [beta]/[zeta].
Eur J Pharmacol 2009; 602(2-3): 245-9.
111
138. Uotila P, Valve E, Martikainen P, Nevalainen M, Nurmi M, Härkönen P. Increased expression of cyclooxygenase-2 and nitric oxide synthase-2 in human prostate cancer.
Urol Res 2001; 29(1): 25-8.
139. Baltaci S, Orhan D, Gögüs C, Türkölmez K, Tulunay Ö, Gögüs O. Inducible nitric oxide synthase expression in benign prostatic hyperplasia, low‐and high‐grade prostatic intraepithelial neoplasia and prostatic carcinoma. BJU Int 2001; 88(1): 100-3.
140. Hogervorst F, Kuikman I, Kesssel AG, Sonnenberg A. Molecular cloning of the human α6 integrin subunit. European Journal of Biochemistry 1991; 199(2): 425-33.
141. Sonnenberg A, Calafat J, Janssen H, Daams H, van der Raaij-Helmer L, Falcioni
R, et al. Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol 1991; 113(4):
907-17.
142. Aumailley M, Timpl R, Sonnenberg A. Antibody to integrin [alpha] 6 subunit specifically inhibits cell-binding to laminin fragment 8. Exp Cell Res 1990; 188(1): 55-60.
143. Frijns E, Sachs N, Kreft M, Wilhelmsen K, Sonnenberg A. EGF-induced MAPK
Signaling Inhibits Hemidesmosome Formation through Phosphorylation of the Integrin
β4. J Biol Chem 2010; 285(48): 37650.
144. Goel HL, Li J, Kogan S, Languino LR. Integrins in prostate cancer progression.
Endocr Relat Cancer 2008; 15(3): 657-64.
145. Margadant C, Frijns E, Wilhelmsen K, Sonnenberg A. Regulation of hemidesmosome disassembly by growth factor receptors. Curr Opin Cell Biol 2008;
20(5): 589-96.
112
146. Bonkhoff H, Stein U, Remberger K. Differential expression of alpha 6 and alpha 2 very late antigen integrins in the normal, hyperplastic, and neoplastic prostate: simultaneous demonstration of cell surface receptors and their extracellular ligands.
Hum Pathol 1993; 24(3): 243-8.
147. Knox J, Cress A, Clark V, Manriquez L, Affinito K, Dalkin B, et al. Differential expression of extracellular matrix molecules and the alpha 6-integrins in the normal and neoplastic prostate. Am J Pathol 1994; 145(1): 167-74.
148. Cress AE, Rabinovitz I, Zhu W, Nagle RB. The α6β1 and α6β4 integrins in human prostate cancer progression. Cancer Metastasis Rev 1995; 14(3): 219-28.
149. Knudsen BS, Miranti CK. The impact of cell adhesion changes on proliferation and survival during prostate cancer development and progression. J Cell Biochem
2006; 99(2): 345-61.
150. Rabinovitz I, Nagle RB, Cress AE. Integrin α6 expression in human prostate carcinoma cells is associated with a migratory and invasive phenotypein vitro andin vivo. Clinical and Experimental Metastasis 1995; 13(6): 481-91.
151. Pawar SC, Demetriou MC, Nagle RB, Bowden GT, Cress AE. Integrin [alpha] 6 cleavage: A novel modification to modulate cell migration. Exp Cell Res 2007; 313(6):
1080-9.
152. King TE, Pawar SC, Majuta L, Sroka IC, Wynn D, Demetriou MC, et al. The role of alpha 6 integrin in prostate cancer migration and bone pain in a novel xenograft model. PloS one 2008; 3(10): e3535.
153. van der Flier A, Sonnenberg A. Function and interactions of integrins. Cell Tissue
Res 2001; 305(3): 285-98.
113
154. Rabinovitz I, Nagle RB, Cress AE. Integrin α6 expression in human prostate carcinoma cells is associated with a migratory and invasive phenotypein vitro andin vivo. Clinical and Experimental Metastasis 1995; 13(6): 481-91.
155. Leung YK, Mak P, Hassan S, Ho SM. Estrogen receptor (ER)-β isoforms: a key to understanding ER-β signaling. Proceedings of the National Academy of Sciences
2006; 103(35): 13162.
156. Leung YK, Lam HM, Wu S, Song D, Levin L, Cheng L, et al. Estrogen receptor
β2 and β5 are associated with poor prognosis in prostate cancer, and promote cancer cell migration and invasion. Endocr Relat Cancer 2010; 17(3): 675-89.
157. Lam HM, Suresh Babu CV, Wang J, Yuan Y, Lam YW, Ho SM, et al.
Phosphorylation of human estrogen receptor-beta at serine 105 inhibits breast cancer cell migration and invasion. Mol Cell Endocrinol 2012; .
158. Sonnenberg A, Linders C, Modderman P, Damsky C, Aumailley M, Timpl R.
Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8. J Cell Biol 1990; 110(6): 2145.
159. Neumann M, Klar S, Wilisch-Neumann A, Hollenbach E, Kavuri S, Leverkus M, et al. Glycogen synthase kinase-3β is a crucial mediator of signal-induced RelB degradation. Oncogene 2011; 30(21): 2485-92.
160. Lindahl E, Azuara C, Koehl P, Delarue M. NOMAD-Ref: visualization, deformation and refinement of macromolecular structures based on all-atom normal mode analysis. Nucleic Acids Res 2006; 34(suppl 2): W52-6.
114
161. Chung LWK, Baseman A, Assikis V, Zhau HE. Molecular insights into prostate cancer progression: the missing link of tumor microenvironment. J Urol 2005; 173(1):
10-20.
162. Thomazzi SM, Ferreira HHA, Conran N, De Nucci G, Antunes E. Role of nitric oxide on in vitro human eosinophil migration1. Biochem Pharmacol 2001; 62(10): 1417-
21.
163. Orucevic A, Bechberger J, Green AM, Shapiro RA, Billiar TR, Lala PK.
Nitric‐oxide production by murine mammary adenocarcinoma cells promotes tumor‐cell invasiveness. International journal of cancer 1999; 81(6): 889-96.
164. Thom SR, Bhopale VM, Mancini DJ, Milovanova TN. Actin S-nitrosylation inhibits neutrophil β2 integrin function. J Biol Chem 2008; 283(16): 10822.
165. Rahman MA, Senga T, Ito S, Hyodo T, Hasegawa H, Hamaguchi M. S- nitrosylation at cysteine 498 of c-Src tyrosine kinase regulates nitric oxide-mediated cell invasion. J Biol Chem 2010; 285(6): 3806.
166. Kim SF, Huri DA, Snyder SH. Inducible nitric oxide synthase binds, S- nitrosylates, and activates cyclooxygenase-2. Science 2005; 310(5756): 1966.
167. Felley-Bosco E, Bender F, Quest AFG. Caveolin-1-mediated post-transcriptional regulation of inducible nitric oxide synthase in human colon carcinoma cells. Biol Res
2002; 35(2): 169-76.
168. Echarri A, Muriel O, Del Pozo MA. Intracellular trafficking of raft/caveolae domains: insights from integrin signaling. Seminars in cell & developmental biology;
Elsevier; 2007.
115
169. Salanueva IJ, Cerezo A, Guadamillas MC, Del Pozo MA. Integrin regulation of caveolin function. J Cell Mol Med 2007; 11(5): 969-80.
170. Miyamoto S, Teramoto H, Gutkind JS, Yamada KM. Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors. J Cell Biol 1996;
135(6): 1633-42.
171. Ahamed J, Versteeg HH, Kerver M, Chen VM, Mueller BM, Hogg PJ, et al.
Disulfide isomerization switches tissue factor from coagulation to cell signaling.
Proceedings of the National Academy of Sciences 2006; 103(38): 13932.
172. Stathakis P, Fitzgerald M, Matthias LJ, Chesterman CN, Hogg PJ. Generation of angiostatin by reduction and proteolysis of plasmin. J Biol Chem 1997; 272(33): 20641.
173. Lahav J, Gofer-Dadosh N, Luboshitz J, Hess O, Shaklai M. Protein disulfide isomerase mediates integrin-dependent adhesion. FEBS Lett 2000; 475(2): 89-92.
174. Walsh GM, Leane D, Moran N, Keyes TE, Forster RJ, Kenny D, et al. S-
Nitrosylation of Platelet αIIbβ3 As Revealed by Raman Spectroscopy. Biochemistry (N
Y ) 2007; 46(21): 6429-36.
175. Walsh GM, Sheehan D, Kinsella A, Moran N, O'Neill S. Redox modulation of integrin αIIbβ3 involves a novel allosteric regulation of its thiol isomerase activity.
Biochemistry (N Y ) 2004; 43(2): 473-80.
176. Lahav J, Wijnen EM, Hess O, Hamaia SW, Griffiths D, Makris M, et al.
Enzymatically catalyzed disulfide exchange is required for platelet adhesion to collagen via integrin α2β1. Blood 2003; 102(6): 2085.
116
177. Krishna TSR, Kong XP, Gary S, Burgers PM, Kuriyan J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 1994; 79(7): 1233-43.
178. Miyachi K, Fritzler MJ, Tan EM. Autoantibody to a nuclear antigen in proliferating cells. The Journal of Immunology 1978; 121(6): 2228.
179. van Diest PJ, Brugal G, Baak J. Proliferation markers in tumours: interpretation and clinical value. J Clin Pathol 1998; 51(10): 716.
180. Schurtenberger P, Egelhaaf SU, Hindges R, Maga G, Jónsson ZO, May RP, et al. The solution structure of functionally active human proliferating cell nuclear antigen determined by small-angle neutron scattering1. J Mol Biol 1998; 275(1): 123-32.
181. Majka J, Burgers PMJ. The PCNA-RFC families of DNA clamps and clamp loaders. Prog Nucleic Acid Res Mol Biol 2004; 78: 227-60.
182. Moldovan GL, Pfander B, Jentsch S. PCNA, the maestro of the replication fork.
Cell 2007; 129(4): 665-79.
183. Naryzhny SN, Lee H. Characterization of proliferating cell nuclear antigen
(PCNA) isoforms in normal and cancer cells: there is no cancer-associated form of
PCNA. FEBS Lett 2007; 581(25): 4917-20.
184. Stoimenov I, Helleday T. PCNA on the crossroad of cancer. Biochem Soc Trans
2009; 37(Pt 3): 605-13.
185. Czyzewska J, Guzinska-Ustymowicz K, Lebelt A, Zalewski B, Kemona A.
Evaluation of proliferating markers Ki-67, PCNA in gastric cancers. Rocz Akad Med
Bialymst 2004; 49 Suppl 1: 64-6.
117
186. Morell-Quadreny L, Clar-Blanch F, Fenollosa-Enterna B, Perez-Bacete M,
Martinez-Lorente A, Llombart-Bosch A. Proliferating cell nuclear antigen (PCNA) as a prognostic factor in renal cell carcinoma. Anticancer Res 1998; 18(1B): 677-81.
187. Bravo R, Macdonald-Bravo H. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell
Biol 1987; 105(4): 1549-54.
188. Naryzhny SN, Zhao H, Lee H. Proliferating cell nuclear antigen (PCNA) may function as a double homotrimer complex in the mammalian cell. J Biol Chem 2005;
280(14): 13888.
189. Gérard A, Koundrioukoff S, Ramillon V, Sergère JC, Mailand N, Quivy JP, et al.
The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit of chromatin assembly factor 1 with proliferating cell nuclear antigen. EMBO Rep 2006;
7(8): 817-23.
190. Fan J, Otterlei M, Wong HK, Tomkinson AE, Wilson III DM. XRCC1 co‐localizes and physically interacts with PCNA. Nucleic Acids Res 2004; 32(7): 2193-201.
191. Naryzhny S. Proliferating cell nuclear antigen: a proteomics view. Cellular and molecular life sciences 2008; 65(23): 3789-808.
192. Unk I, Hajdú I, Fátyol K, Hurwitz J, Yoon JH, Prakash L, et al. Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination.
Proceedings of the National Academy of Sciences 2008; 105(10): 3768.
193. Unk I, Hajdú I, Fátyol K, Szakál B, Blastyák A, Bermudez V, et al. Human
SHPRH is a ubiquitin ligase for Mms2–Ubc13-dependent polyubiquitylation of
118 proliferating cell nuclear antigen. Proceedings of the National Academy of Sciences
2006; 103(48): 18107.
194. Wang S, Nakajima Y, Yu Y, Xia W, Chen C, Yang C, et al. Tyrosine phosphorylation controls PCNA function through protein stability. Nat Cell Biol 2006;
8(12): 1359-68.
195. Zhao H, Lo YH, Ma L, Waltz SE, Gray JK, Hung MC, et al. Targeting tyrosine phosphorylation of PCNA inhibits prostate cancer growth. Molecular cancer therapeutics
2011; 10(1): 29-36.
196. Naryzhny SN, Lee H. The Post-translational Modifications of Proliferating Cell
Nuclear Antigen. J Biol Chem 2004; 279(19): 20194-9.
197. Papouli E, Chen S, Davies AA, Huttner D, Krejci L, Sung P, et al. Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase
Srs2p. Mol Cell 2005; 19(1): 123-33.
198. Berry W, Friedland D, Fleagle J, Jackson D, Ilegbodu D, Boehm KA, et al. A phase II study of weekly paclitaxel/estramustine/carboplatin in hormone-refractory prostate cancer. Clinical Genitourinary Cancer 2006; 5(2): 131-7.
199. Saxman S, Ansari R, Drasga R, Miller M, Wheeler B, McClean J, et al. Phase III trial of cyclophosphamide versus cyclophosphamide, doxorubicin, and methotrexate in hormone‐refractory prostatic cancer: A hoosier oncology group study. Cancer 1992;
70(10): 2488-92.
200. Fornari FA, Randolph JK, Yalowich JC, Ritke MK, Gewirtz DA. Interference by doxorubicin with DNA unwinding in MCF-7 breast tumor cells. Mol Pharmacol 1994;
45(4): 649-56.
119
201. Riganti C, Miraglia E, Viarisio D, Costamagna C, Pescarmona G, Ghigo D, et al.
Nitric oxide reverts the resistance to doxorubicin in human colon cancer cells by inhibiting the drug efflux. Cancer Res 2005; 65(2): 516.
202. De Boo S, Kopecka J, Brusa D, Gazzano E, Matera L, Ghigo D, et al. iNOS activity is necessary for the cytotoxic and immunogenic effects of doxorubicin in human colon cancer cells. Mol Cancer 2009; 8: 108.
203. Moncada S, Higgs A. The l-arginine-nitric oxide pathway. N Engl J Med 1993;
329(27): 2002.
204. Lala PK, Chakraborty C. Role of nitric oxide in carcinogenesis and tumour progression. The lancet oncology 2001; 2(3): 149-56.
205. Thomsen LL, Miles DW. Role of nitric oxide in tumour progression: lessons from human tumours. Cancer Metastasis Rev 1998; 17(1): 107-18.
206. Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J 2001; 357(Pt 3): 593.
207. Martínez-Ruiza A, Cadenas S, Lamas S. Nitric oxide signaling: classical, less classical and non classical mechanisms. Free Radical Biology and Medicine 2011; .
208. Fang J, Nakamura T, Cho DH, Gu Z, Lipton SA. S-nitrosylation of peroxiredoxin
2 promotes oxidative stress-induced neuronal cell death in Parkinson's disease.
Proceedings of the National Academy of Sciences 2007; 104(47): 18742.
209. Gaston B, Sears S, Woods J, Hunt J, Ponaman M, McMahon T, et al.
Bronchodilator S-nitrosothiol deficiency in asthmatic respiratory failure. The Lancet
1998; 351(9112): 1317-9.
120
210. Chung KKK, Thomas B, Li X, Pletnikova O, Troncoso JC, Marsh L, et al. S- nitrosylation of parkin regulates ubiquitination and compromises parkin's protective function. Science 2004; 304(5675): 1328.
211. Tarapore P, Shu Y, Guo P, Ho SM. Application of Phi29 motor pRNA for targeted therapeutic delivery of siRNA silencing metallothionein-IIA and survivin in ovarian cancers. Molecular Therapy 2010; 19(2): 386-94.
212. Gehen SC, Vitiello PF, Bambara RA, Keng PC, O'Reilly MA. Downregulation of
PCNA potentiates p21-mediated growth inhibition in response to hyperoxia. American
Journal of Physiology-Lung Cellular and Molecular Physiology 2007; 292(3): L716-24.
213. Riganti C, Doublier S, Costamagna C, Aldieri E, Pescarmona G, Ghigo D, et al.
Activation of nuclear factor-κB pathway by simvastatin and RhoA silencing increases doxorubicin cytotoxicity in human colon cancer HT29 cells. Mol Pharmacol 2008; 74(2):
476-84.
214. Oku T, Ikeda S, Sasaki H, Fukuda K, Morioka H, Ohtsuka E, et al. Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase δ and replication factor C. Genes to Cells 1998; 3(6): 357-69.
215. Hendel A, Krijger PHL, Diamant N, Goren Z, Langerak P, Kim J, et al. PCNA
Ubiquitination Is Important, But Not Essential for Translesion DNA Synthesis in
Mammalian Cells. PLoS genetics 2011; 7(9): e1002262.
216. Ricke WA, Wang Y, Cunha GR. Steroid hormones and carcinogenesis of the prostate: the role of estrogens. Differentiation 2007; 75(9): 871-82.
217. Risbridger GP, Ellem SJ, McPherson SJ. Estrogen action on the prostate gland: a critical mix of endocrine and paracrine signaling. J Mol Endocrinol 2007; 39(3): 183-8.
121
218. Warner M, Gustafsson JÅ. The role of estrogen receptor β (ERβ) in malignant diseases—A new potential target for antiproliferative drugs in prevention and treatment of cancer. Biochem Biophys Res Commun 2010; 396(1): 63-6.
219. Katzenellenbogen BS, Sun J, Harrington WR, Kraichely DM, Ganessunker D,
Katzenellenbogen JA. Structure‐Function Relationships in Estrogen Receptors and the
Characterization of Novel Selective Estrogen Receptor Modulators with Unique
Pharmacological Profiles. Ann N Y Acad Sci 2001; 949(1): 6-15.
220. McPherson SJ, Hussain S, Balanathan P, Hedwards SL, Niranjan B, Grant M, et al. Estrogen receptor–β activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNFα mediated. Proceedings of the National
Academy of Sciences 2010; 107(7): 3123-8.
221. Osborne CK, Schiff R, Fuqua SAW, Shou J. Estrogen receptor: current understanding of its activation and modulation. Cancer Res 2001; 7(12 Supplement):
4338s.
222. Tremblay GB, Giguere V. Coregulators of estrogen receptor action. Crit Rev
Eukaryot Gene Expr 2002; 12(1): 1-22.
223. Kampa M, Pelekanou V, Castanas E. Membrane-initiated steroid action in breast and prostate cancer. Steroids 2008; 73(9): 953-60.
224. Lösel R, Wehling M. Nongenomic actions of steroid hormones. Nature Reviews
Molecular Cell Biology 2003; 4(1): 46-55.
225. Hammes SR, Levin ER. Extranuclear steroid receptors: nature and actions.
Endocr Rev 2007; 28(7): 726-41.
122
226. Zhang Z, Maier B, Santen RJ, Song RXD. Membrane association of estrogen receptor [alpha] mediates estrogen effect on MAPK activation. Biochem Biophys Res
Commun 2002; 294(5): 926-33.
227. Song RX, Zhang Z, Santen RJ. Estrogen rapid action via protein complex formation involving ERalpha and Src Trends Endocrinol Metab 2005; 16(8): 347-53.
228. O’Lone R, Knorr K, Jaffe IZ, Schaffer ME, Martini PGV, Karas RH, et al. Estrogen receptors α and β mediate distinct pathways of vascular gene expression, including genes involved in mitochondrial electron transport and generation of reactive oxygen species. Molecular Endocrinology 2007; 21(6): 1281-96.
229. Chen JQ, Russo PA, Cooke C, Russo IH, Russo J. ER [beta] shifts from mitochondria to nucleus during estrogen-induced neoplastic transformation of human breast epithelial cells and is involved in estrogen-induced synthesis of mitochondrial respiratory chain proteins. Biochimica et Biophysica Acta (BBA)-Molecular Cell
Research 2007; 1773(12): 1732-46.
230. Barone I, Brusco L, Fuqua SAW. Estrogen receptor mutations and changes in downstream gene expression and signaling. Clinical Cancer Research 2010; 16(10):
2702.
231. Leav I, Lau KM, Adams JY, McNeal JE, Taplin ME, Wang J, et al. Comparative studies of the estrogen receptors β and α and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma. The American journal of pathology 2001; 159(1): 79-92.
123
232. Haqq C, Li R, Khodabakhsh D, Frolov A, Ginzinger D, Thompson T, et al. Ethnic and racial differences in prostate stromal estrogen receptor α. Prostate 2005; 65(2):
101-9.
233. Tanaka Y, Sasaki M, Kaneuchi M, Shiina H, Igawa M, Dahiya R. Polymorphisms of estrogen receptor alpha in prostate cancer. Mol Carcinog 2003; 37(4): 202-8.
234. Hedelin M, Bälter KA, Chang ET, Bellocco R, Klint Å, Johansson JE, et al.
Dietary intake of phytoestrogens, estrogen receptor‐beta polymorphisms and the risk of prostate cancer. Prostate 2006; 66(14): 1512-20.
235. Thellenberg-Karlsson C, Lindström S, Malmer B, Wiklund F, Augustsson-Bälter
K, Adami HO, et al. Estrogen receptor β polymorphism is associated with prostate cancer risk. Clinical cancer research 2006; 12(6): 1936-41.
236. Dondi D, Piccolella M, Ciana P, Maggi A, Locatelli A, Motta M, et al. Estrogen receptor beta and the progression of prostate cancer–role of 5alpha-androstane-3beta,
17beta-diol (3beta-Adiol). European Journal of Cancer Supplements 2008; 6(9): 80.
237. Mak P, Leav I, Pursell B, Bae D, Yang X, Taglienti CA, et al. ERbeta impedes prostate cancer EMT by destabilizing HIF-1alpha and inhibiting VEGF-mediated snail nuclear localization: implications for Gleason grading Cancer Cell 2010; 17(4): 319-32.
238. Pasquali D, Rossi V, Esposito D, Abbondanza C, Puca GA, Bellastella A, et al.
Loss of estrogen receptor β expression in malignant human prostate cells in primary cultures and in prostate cancer tissues. Journal of Clinical Endocrinology & Metabolism
2001; 86(5): 2051-5.
124
239. Pasquali D, Staibano S, Prezioso D, Franco R, Esposito D, Notaro A, et al.
Estrogen receptor [beta] expression in human prostate tissue. Mol Cell Endocrinol 2001;
178(1-2): 47-50.
240. Zhu X, Leav I, Leung Y, Wu M, Liu Q, Gao Y, et al. Dynamic regulation of estrogen receptor-β expression by DNA methylation during prostate cancer development and metastasis. The American journal of pathology 2004; 164(6): 2003-12.
241. Zhang X, Leung Y, Ho S. AP-2 regulates the transcription of estrogen receptor
(ER)-β by acting through a methylation hotspot of the 0N promoter in prostate cancer cells. Oncogene 2007; 26(52): 7346-54.
242. Lai JS, Brown LG, True LD, Hawley SJ, Etzioni RB, Higano CS, et al. Metastases of prostate cancer express estrogen receptor-beta. Urology 2004; 64(4): 814-20.
243. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, et al.
Generation and reproductive phenotypes of mice lacking estrogen receptor beta Proc
Natl Acad Sci U S A 1998; 95(26): 15677-82.
244. Jiang J, Chang H, Sugimoto Y, Lin YC. Effects of age on growth and ERβ mRNA expression of canine prostatic cells. Anticancer Res 2005; 25(6B): 4081-90.
245. Kastrati I, Edirisinghe PD, Wijewickrama GT, Thatcher GRJ. Estrogen-induced apoptosis of breast epithelial cells Is blocked by NO/cGMP and mediated by extranuclear estrogen receptors. Endocrinology 2010; 151(12): 5602-16.
246. Zhang H, Feng L, Wang W, Magness RR, Chen D. Estrogen‐responsive nitroso‐proteome in uterine artery endothelial cells: Role of endothelial nitric oxide synthase and estrogen receptor‐β. J Cell Physiol 2012; 227(1): 146-59.
125
247. Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul
PW. Estrogen receptor alpha mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 1999; 103(3): 401-6.
248. Pyo Kim H, Young Lee J, Kim Jeong J, Won Bae S, Kyu Lee H, Jo I.
Nongenomic Stimulation of Nitric Oxide Release by Estrogen Is Mediated by Estrogen
Receptor [alpha] Localized in Caveolae. Biochem Biophys Res Commun 1999; 263(1):
257-62.
249. Chambliss KL, Shaul PW. Estrogen modulation of endothelial nitric oxide synthase. Endocr Rev 2002; 23(5): 665-86.
250. Chambliss KL, Yuhanna IS, Mineo C, Liu P, German Z, Sherman TS, et al.
Estrogen receptor α and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res 2000; 87(11): e44-52.
251. Rabinovitz I, Cress AE, Nagle RB. Biosynthesis and secretion of laminin and
S‐laminin by human prostate carcinoma cell lines. Prostate 1994; 25(2): 97-107.
252. Isenberg JS. Inhibition of nitric oxide synthase (NOS) conversion of L-arginine to nitric oxide (NO) decreases low density mononuclear cell (LD MNC) trans-endothelial migration and cytokine output. J Surg Res 2003; 114(1): 100-6.
253. Lacza Z, Snipes JA, Zhang J, Horváth EM, Figueroa JP, Szabó C, et al.
Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS. Free Radical Biology and Medicine 2003; 35(10): 1217-28.
254. Park SW, Lee SG, Song SH, Heo DS, Park BJ, Lee DW, et al. The effect of nitric oxide on cyclooxygenase‐2 (COX‐2) overexpression in head and neck cancer cell lines. International journal of cancer 2003; 107(5): 729-38.
126
255. Riganti C, Voena C, Kopecka J, Corsetto PA, Montorfano G, Enrico E, et al.
Liposome-encapsulated doxorubicin reverses drug-resistance by inhibiting P- glycoprotein in human cancer cells. Molecular Pharmaceutics 2011; .
127