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12-2006 The oler of prolactin and its antagonist in HER2/ neu tumorigenesis Michele Scotti Clemson University, [email protected]
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THE ROLE OF HUMAN PROLACTIN AND ITS ANTAGONIST IN HER2/NEU BREAST CANCER ______
A Dissertation Presented to the Graduate School of Clemson University ______
In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Microbiology ______
by Michele Lynn Scotti December 2006 ______
Accepted by: Dr. Wen Chen, Committee Chair Dr. Lesly Temesvari Dr. J. David Gangemi Dr. Lyndon Larcom
ii
ABSTRACT
Purpose: To study the role of prolactin (PRL) and its antagonist, G129R, in
HER2/Neu tumorigenesis. Specifically, to investigate the interaction between the
oncogene HER2 and the PRL receptor (PRLR) signaling pathways for designing
effective combinational therapeutics for breast cancer.
Experimental Design: The combination effects of G129R and an anti-HER2
antibody, Herceptin, were tested against HER2-overexpressing human breast cancer cell
lines, T-47D and BT-474, using cell based assays and xenografts established in athymic
mice. Furthermore, four different bitransgenic mouse lines co-expressing the murine
version of HER2 and PRL or G129R were generated. The mammary tumor incidence, characterization of mammary gland development, and alteration of the molecular biomarkers in the mammary glands were investigated in these transgenic mice.
Results: It was demonstrated that PRL was able to activate HER2 in human breast cancer cells and the addition of G129R competitively inhibited the stimulatory effect of hPRL. More importantly, G129R has synergistic/additive effects when used in combination with Herceptin in inhibiting HER2 activation. Results further demonstrate that Herceptin and G129R displayed a synergistic inhibitory effect on MAPK phosphorylation. Most importantly, the combinational treatment of Herceptin and
G129R significantly inhibited the growth of xenografts in athymic mice.
In the bitransgenic mouse study, it was found that co-expression of G129R in
MMTV-neu female mice had little effect on overall HER2 tumor incidence due to the
low expression levels of G129R. Surprisingly, co-expression of low levels of PRL in iii
MMTV-neu female mice demonstrated a drastic reduction in tumor incidence in both hPRL/neu bitransgenic lines. Suppressed expression of HER2, which lead to lower levels of MAPK activities and cyclin D1 expression were strong indications or evidence that support the significant change in the tumor incidence in these bitransgenic mice.
Conclusions: PRL possesses a dual role in HER2/neu tumorigenesis: as a tumor promoter in transformed cells and as a tumor suppressor when expressed early before tumor formation. To improve the outcome of breast cancer therapy, especially for HER2 positive breast cancer, G129R may be used together with Herceptin as a combinational therapy. On the other hand, data from the PRL bitransgenic mice suggests the possibility of using hPRL as a potential chemo-preventive agent in HER2/neu tumorigenesis. iv
DEDICATION
This work is dedicated to my loving parents, Dr. Ron and Sharon Scotti, for all their help, strength, love, support and motivation to help me in every way possible to get to where I am today. I admire both of you and love you so very much. Also to my big brothers, Mike and Jeff Scotti, who no matter what, support me in everything I do and believe in me. During my years of graduate work, Mike and his wife, Lee, have given me a beautiful niece, Kara, and a handsome nephew, TJ. Last and definitely not least, to
Jeff, who has been such an incredibly brave man and brother to fight for his country by serving in Iraq. Not one day has gone by that I didn’t think and pray for him while he was over there.
v
ACKNOWLEDGMENTS
The author wishes to thank all the people that have played an important role in my
graduate studies. This dissertation would not have been possible without the expert guidance of my advisor, Dr. Wen Y. Chen. He has always been extremely perceptive, helpful, and supportive in training me in the Molecular Biology field and I am gracious to have learned so much from him. I am also thankful for the cooperation and invaluable insights of the rest of my committee members: Dr. Lesly Temesvari, who also served on my Masters committee, Dr. Lyndon Larcom and Dr. J. David Gangemi.
I would also like to thank my lab colleagues and friends: Jang Pyo Park, John
Langenheim, Seth Tomblyn, Dr. Tom Scott (my Masters advisor), Dr. Michael Beck, Dr.
Karl Franek, Dr. Susan Peirce, Dr. Lori and Eric Holle, Blake Derrick, Eric Lee,
Guillermo DeAngulo, Dr. Alison Springs and Keneisha Burrell. Last but definitely not
least, I would like to thank my lab colleague and one of my best friends, Isabelle
Jacquemart, for being there by my side for all those years to help motivate, to help push,
and to help support one another as coworkers and as friends. I couldn't have done it
without you! Through our years of graduate work Isabelle and I have traveled the world
from Maryland and Washington, DC to Jacksonville, Orlando, Sea World and Miami,
Florida to Los Angeles, Santa Monica, Bakersfield and Anaheim, California all the way
to Brugge and Brussels, Belgium and Paris, France. I will never forget all our incredible
traveling memories!
vi
TABLE OF CONTENTS
Page
TITLE PAGE...... i
ABSTRACT...... ii
DEDICATION...... iv
ACKNOWLEDGEMENTS...... v
LIST OF TABLES...... ix
LIST OF FIGURES ...... x
CHAPTER
1. INTRODUCTION ...... 1
Cancer ...... 1 Breast cancer...... 3 PRL’s pathogenic role in mammary cancer...... 5 Prolactin gene, mRNA and protein...... 7 Multiple isoforms of PRLR ...... 9 Mechanism of PRL signaling...... 10 Anti-PRL approaches...... 13 ErbB2/HER2/neu oncogene...... 17 HER2/neu's role in mammary tumorigenesis ...... 19 Therapeutics targeting HER2/neu ...... 20 Cross talk between PRL and HER2/neu ...... 21 PRLR and ER...... 24 PRLR and EGFR...... 25 ER and EGFR ...... 26 Rational therapeutic combinations for breast cancer...... 26 PRLR antagonist and ER antagonist...... 27 anti-HER2 and ER antagonist...... 28 Transgenic models for breast cancer...... 29 PRL transgenics ...... 29 HER2/neu transgenics...... 31 Chemoprevention of human breast cancer...... 33 Overview and rationale of current work ...... 33
vii
Table of Contents (Continued)
Page
2. MATERIALS AND METHODS...... 35
Materials...... 35 Antibodies...... 35 Plasmid construction and cloning ...... 36 Cell culture...... 39 Anchorage-dependent cell growth ...... 40 Transient and stable transfection ...... 40 Isolation of proteins: SDS PAGE and Western blot ...... 42 Immunoprecipitation...... 42 Extraction of proteins from cells...... 42 Extraction of protein from tissues...... 43 Immunoblot analysis...... 43 Human breast cancer xenografts in nude mice ...... 44 Generation of multiparous MMTVneu transgenic mice...... 45 Generation of bitransgenic mouse models...... 45 Identification of transgenic mice ...... 47 Transgene screening, DNA: PCR ...... 47 Transgene expression confirmation, mRNA: RT-PCR ...... 47 Transgene expression confirmation, protein: IRMA, ELISA...... 48 Transgene sequencing confirmation ...... 48 Transgenic mouse mammary gland morphological assessment ...... 49 Mammary gland whole mount...... 49 Immunohistochemistry ...... 49 Statistical data analyses...... 50
3. RESULTS ...... 52
Co-expression of PRLR and HER2/Neu in human breast cancer cells...... 52 Effect of PRL and combination treatment of G129R and Herceptin on HER2 phosphorylation...... 53 Synergistic effect of Herceptin and G129R on inhibition of MAPK phosphorylation...... 58 Combination effects of G129R and Herceptin on other intracellular signaling pathways ...... 62 Effects of PRL and G129R overexpression on HER2 positive cell proliferation ...... 66 Effects of PRL, G129R, Herceptin or combinational treatments on anchorage-dependent cell growth ...... 68 Effects of G129R and Herceptin on growth of T-47D and BT-474 human breast cancer xenografts in nude mice...... 72 viii
Table of Contents (Continued)
Page
Generation of PRL or G129R and HER2 bitransgenic mouse models...... 75 Mammary tumor incidence in bitransgenic mice...... 78 HER2/Neu gene expression levels in the mammary gland...... 80 Molecular events in PRL bitransgenic mice ...... 82 Morphological comparison of the mammary gland of PRL bitransgenic mice and their littermates…………………………… 87 Proliferation indices in PRL bitransgenic mice...... 91 Neu phosphorylation levels in the tumors...... 93
4. DISCUSSION...... 95
Prolactin in transformed cells acts as a tumor promoter...... 95 Early expression of low levels of prolactin has a protective effect on HER2/Neu tumorigenesis ...... 102
5. CONCLUSION...... 114
APPENDICES ...... 115
A: Kinetics of tumor occurrence of nulliparous vs. early, multiparous MMTVneu transgenic mice...... 116 B: Comparison of transient hPRL expression driven by two promoters ...... 118 C: Primers used for PCR and RT-PCR identification of transgenic mice ...... 120
REFERENCES ...... 121
ix
LIST OF TABLES
Table Page
C1. Primers used for PCR and RT-PCR identification of transgenic mice.... 120
x
LIST OF FIGURES
Figure Page
1. PRLR expression level in breast cancer tissue compared to normal tissue ...... 7
2. Prolactin gene, mRNA and protein...... 8
3. Schematic representation of multiple isoforms of hPRL receptor...... 10
4. Sequential dimerization model of the hPRLR by hPRL...... 11
5. Schematic representation of the PRLR signaling pathways ...... 13
6. Schematic representation of the wild-type hPRL and the hPRLR antagonist (G129R) interaction with the PRLR...... 15
7. Endocrine and autocrine/paracrine ligands of the PRLR...... 17
8. Four members of the ErbB Family of receptor tyrosine kinases ...... 19
9. Anti-HER2/neu monoclonal antibody, Herceptin...... 21
10. Schematic representation of the potential cross-talk between hPRL and HER2/neu……. ………………………………………………….. 24
11. Schematic representation of the cloning and expression plasmids.... ….. 38
12. Schematic representation of the mammalian expression plasmids.... ….. 46
13. PRLR and HER2/Neu levels in multiple human breast cancer cells...... 53
14. Extended exposure of PRL on Neu phosphorylation in BT-474 ...... 55
15. Effect of PRL on phosphorylation of Jak2 and Neu in T-47D cells...... 56
16. Effect of G129R and Herceptin on phosphorylation of HER2 in BT-474 cells...... 57
17. Immunoblot analysis of MAPK activity in T-47D after PRL, G129R, Herceptin or in combination treatment...... 60
xi
List of Figures (Continued)
Figure Page
18. Immunoblot analysis of MAPK activity in BT-474 after PRL, G129R, Herceptin or in combination treatment...... 61
19. Immunoblot analyses of STAT5a/b, STAT3, and AKT in T-47D ...... 64
20. Immunoblot analyses of STAT5a/b, STAT3, and AKT in BT-474 ...... 65
21. Effects of PRL and G129R overexpression in HER2 positive cell proliferation...... 67
22. Dose response of the treatment of PRL, G129R and Herceptin in T-47D cells ...... 69
23. Growth response of T-47D and BT-474 cells to G129R, Herceptin or combinational treatments ...... 71
24. Effect of G129R and Herceptin on T-47D xenografts in nude mice ...... 73
25. Effect of G129R and Herceptin on BT-474 xenografts in nude mice ..... 74
26. Confirmation of transgenic mice ...... 76
27. Expression of PRL in mammary gland tissue in PRL bitransgenic mice ...... 77
28. Effects of PRL or G129R co-expression on Neu-induced tumorigenesis ...... 79
29. HER2/neu gene expression in mammary glands of PRL bitransgenics and their neu littermates...... 81
30. Cyclin D1 and phospho-MAPK levels in MT-hPRL/neu mice...... 84
31. Cyclin D1 and phospho-MAPK levels in MMTV-hPRL/neu mice...... 85
32. AKT and STAT levels in hPRL/neu mice ...... 86
33. Mammary glands of MT-hPRL/neu female mice...... 89
34. Mammary glands of MMTV-hPRL/neu female mice ...... 90
xii
List of Figures (Continued)
Figure Page
35. Proliferation indices in MT-PRL/neu mice and their neu littermates...... 92
36. HER2/Neu phosphorylation levels in tumors, which arise in the PRL bitransgenic mice and their neu littermates ...... 94
A1. Early, multiparous MMTVneu transgenic mice ...... 116
B1. hPRL expression driven by two promoters...... 118
1
CHAPTER ONE
INTRODUCTION
Cancer
Cancer is the second leading cause of death in the United States, behind heart disease. Over one million people get cancer each year. Approximately one out of every two American men and one out of every three American women will have some type of cancer at some point during their lifetime. Cancer is defined as a group of diseases that are characterized by the uncontrollable growth and spread of abnormal cells (Weinberg,
1996). Normal cells grow, divide and die in an orderly fashion, whereas, cancer cells are immortal and continue to grow and divide. Cancer cells can also travel to other parts of the body by spreading through the bloodstream and lymphatic system where they begin to grow and replace normal tissue, allowing the cancer to metastasize.
According to the American Cancer Society, cancer cells develop because of damage to DNA. DNA can become damaged many different ways such as exposure to something in the environment. Most of the time the body is able to repair its damaged
DNA; however, in cancer cells, the damaged DNA does not get repaired. People can also inherit damaged DNA, which also accounts for inherited cancers.
The exact cause of cancer is unknown. However, we do know that certain genes contain instructions for controlling when cells grow, divide, and die. A proto-oncogene is a normal gene whose protein product has the capacity to induce cellular transformation given it sustains some genetic insult. An oncogene is a gene that has sustained some genetic damage and, therefore, produces a protein capable of cellular 2 transformation. Genes that slow down cell division, or cause cells to die, are called tumor suppressor genes. It is known that cancers can be caused by DNA mutations that
"turn on" oncogenes or "turn off" tumor suppressor genes.
In Hanahan and Weinberg’s paper, The Hallmark of Cancer, they proposed that most if not all types of cancers acquire six alterations in their cell physiology. These different acquired capabilities of cancer are: 1. self-sufficiency in growth signals, 2.
insensitivity to growth-inhibitory (anti-growth) signals, 3. evasion of programmed cell
death (apoptosis), 4. limitless replicative potential, 5. sustained angiogenesis, and 6.
tissue invasion and metastasis. Over the years, three more provisions have been added to
this list including: stem cell source of tumor, energetic requirements for the tumor, and
lastly an immune system evasion.
Different types of cancer can behave very differently. They grow at different
rates and respond to different treatment. The major types of treatment for cancer are
surgery, radiation, chemotherapy, hormone therapy and immunotherapy. People with
cancer need treatment that is targeted for their particular type of cancer, location of
cancer, progression of the disease and the health status of the patient. Cancer treatments
are designed to either directly kill or remove the cancer cells or to lead to their eventual
death by depriving them of signals needed for cell division. Other treatments work by
stimulating the body's own defenses.
Often the first line of treatment for many solid tumors is surgery. Surgery may be
sufficient to cure the patient by removing all cancerous cells when the cancer is detected
at an early stage. Another cancer treatment that may be used in conjunction with surgery is radiation. The goal of radiation is to kill the cancer cells directly by damaging them 3
with high energy beams. Chemotherapy is a wide array of drugs used to kill cancer cells.
Chemotherapy drugs work by damaging the dividing cancer cells and preventing their
further reproduction. Hormone therapies are drugs that are designed to prevent cancer
cell growth by preventing the cells from receiving signals necessary for their continued
growth and division. Lastly, immunotherapy involves the use of naturally occurring,
normal proteins to stimulate the bodies own defenses against cancer.
According to the American Cancer Society, another class of drugs that is relatively new in the treatment of cancer is specific inhibitors. They work by targeting specific proteins and processes that are limited primarily to cancer cells or those that are more prevalent in cancer cells. Inhibition of these processes prevents cancer cell growth and division. Antibodies are another treatment that targets cancer cells. Antibodies used in the treatment of cancer have been manufactured for use as drugs. They may work by several different mechanisms, either depriving the cancer cells of necessary signals or causing the direct death of the cells. Antibodies may be thought of as a type of specific inhibitor due to their specificity. Finally, vaccines are another type of cancer treatment.
Cancer vaccines stimulate the body's defenses against cancer. By administering proteins found on or produced by cancer cells, the treatment increases the response of the body against the cancer cells.
Breast Cancer
According to the American Cancer Society, breast cancer is the most common cancer among women, other than skin cancer. It is the second leading cause of cancer death in women, after lung cancer. In 2006, about 212,920 women in the United States will be found to have invasive breast cancer. About 40,970 women will die from the 4
disease this year. The chance of a woman having invasive breast cancer during her life is about 1 in 8 and the chance of dying from breast cancer is about 1 in 33. Breast cancer death rates are declining due to earlier detection and improved treatment.
Breast cancer is a heterogeneous disease arising from multiple genetic changes in
oncogenes and tumor suppressor genes with pivotal roles in the homeostatic control of
mammary epithelial cell proliferation, differentiation and death (Sutherland and
Musgrove, 2002). The cause of breast cancer is not clear, and most likely cannot be
triggered by a single etiologic agent. Rather the genesis of breast cancer like many other
cancers results from the stepwise accumulation of mutations in genes (Chodosh, 2002).
Although many risk factors may increase the chance of developing breast cancer, it is not
yet known exactly how some of these risk factors cause cells to become cancerous. Like
improbable partners at a dance, the normal development of the breast goes hand in hand
with its susceptibility to cancer (Chodosh, 2002).
A study of population-attributable risks has estimated that at least 45% to 55% of
breast cancer cases in the United States may be explained by the following factors:
advanced age at the time of the first full-term pregnancy, nulliparity, family history of
breast cancer, higher socioeconomic status, earlier age at menarche, and prior benign
breast disease (Russo and Russo, 2004). Only 10% of human breast cancers are
considered to be the result of inherited mutations of the tumor suppressor genes, BRCA1
or BRCA2, while the remaining 90% of cases are related to the disruption of biochemical pathways involving growth factors and hormones and their receptors; such as the estrogen receptor, the progesterone receptor and more recently the c-ErbB-2 receptor
(Yamauchi, T. et al., 2000). The most common types of breast cancer are carcinoma in 5
situ, ductal or lobular carcinoma in situ, and infiltrative ductal or lobular carcinoma. The
cancer is confined to either the ducts or the lobules if it has not gone into the fatty tissues
in the breast nor spread to other organs in the body.
For decades, the primary therapy for women with breast cancer has been surgery,
radiation or a combination of both (Miller and Langdon, 1997; Forrest, 1997; Roses,
1999). Hormone therapy and chemotherapy are adjuvant treatments for breast cancer after surgery. Common hormone therapy for breast cancer is an anti-estrogen treatment such as tamoxifen. Tamoxifen blocks the actions of the hormone estrogen since certain types of breast cancer require estrogen to grow. It has been reported that tamoxifen is also effective as a preventive agent in reducing the incidence of breast cancer up to 45% in a group of healthy women over age 35 who have a higher-than-normal risk of getting the disease (Marshall, 1998). However, the heterogeneous nature of breast cancer translates to low response rate in many therapies. It has been reported that approximately half of the breast cancers are estrogen receptor (ER) negative, which partly explains why tamoxifen is not universally effective (Forrest, 1997).
It is obvious that the scope of the search for anti-breast cancer drugs should be expanded in order to effectively control tumor growth and/or recurrence. Also, combinations of proven effective methods, especially those intended to target different pathways or mechanisms should be further investigated.
PRL’s pathogenic role in mammary cancer
Prolactin (PRL) is the hormone intimately involved in puberty and pregnancy, acting both as a mitogen and a differentiation agent (Horseman, 1995; Vonderhaar, 1998;
Clevenger et al., 2002). The importance of PRL in pathological conditions such as 6
mammary tumor growth in women is well established. Several lines of evidence support
the involvement of PRL in breast cancer formation. PRL expression has been detected in
human mammary tumors and human mammary tumor cell lines can produce PRL (Ben-
Jonathon et al., 1996) suggesting that PRL is produced locally, thus serving as an autocrine and paracrine growth factor within the mammary glands. Second, transgenic
mice expressing lactogenic hormones such as PRL have a very high incidence of breast
cancer (Rose-Hellekant et al., 2003; Wennbo et al., 1997). Third, the level of expression
of PRL receptor is greatly elevated in human breast cancer cells compared to normal
(Reynolds et al., 1997), and in surgically removed breast cancer tissues compared to its
normal surrounding tissues as seen in Fig. 1 (Touraine et al., 1998). Fourth, PRLR
antagonists can inhibit the growth of established breast cancer cells in vitro (Fuh and
Wells, 1995) and in xenografts in nude mice (Chen et al., 1999; 2002). These findings
suggest that hPRL is an autocrine/paracrine growth factor that plays an important role in
breast cancer development and progression. Since both PRL and PRLR are expressed
widely in breast cancer, this provides a strong argument in support of using targeted
inhibition of signaling via the PRLR capable of blocking or inactivating the hPRLR
locally at the level of the breast cancer cell as therapeutics or chemopreventive agents. 7
(Touraine et al., J Clin Endo Metab 1998 83: 667-74)
Fig. 1. PRLR expression level in breast cancer tissue compared to normal tissue. In surgically removed breast cancer tissues PRLR expression level is greatly elevated compared to its normal surrounding tissues.
Prolactin gene, mRNA and protein
PRL is a 23-kDa single chain polypeptide hormone that is closely related to growth hormone (GH). PRL is synthesized in the anterior pituitary by hormone producing cells called lactotrophs and are involved in the growth and development of the mammary gland (Clevenger et al., 1995). It is also synthesized and secreted by a broad range of other cells in the body, most prominently various immune cells, the brain and the deciduas of the pregnant uterus. In addition to its role as a neuroendocrine hormone,
PRL is also produced by the breast epithelium as an autocrine/paracrine growth factor 8 and functions as a cytokine (Clevenger et al., 1995, 2003; Ginsburg and Vonderhaar,
1995; Ben-Jonathon et al., 2002).
The hPRL gene is located on chromosome 6 and is composed of 5 exons, which are separated by 4 large introns that together measure approximately 10kb (Fig. 2).
Human PRL's 1 kilobase mature mRNA codes for 227 amino acids that are composed of the signal peptide containing 28 amino acids and the mature protein containing 199 amino acids with a molecular weight of 23 kDa. The signal peptide is a short chain peptide that directs the post-translational transport of prolactin. Human PRL contains 3 disulfide bonds between amino acids 4-11, 58-174 and 191-199. This creates small loops at amino and carboxy terminal ends and a large loop in the middle of the molecule.
There is high homology (80-90% between hPRL and rPRL) in the 5' flanking region of the gene between human, rat and bovine indicating conserved regulatory element in the pituitary (Truong et al., 1984).
Transcription Initiation 12 3 4 5 Promoter AB C D Prolactin 84 176 108 180 310 1.3 kb 1.5 kb 1.9 kb 2.0 kb Gene bp bp bp bp bp 5’ 3’
Prolactin mRNA 5’ Untranslated 3’ Untranslated Region Region S-S S-S S-S Prolactin Signal Peptide Prolactin
Fig. 2. Prolactin gene, mRNA and protein. Prolactin gene consists of 5 exons which, when transcribed to the mRNA, will contain 5' and 3' untranslated region together with the signal and protein sequence. The prolactin protein contains a 28 amino acid signal peptide and 199 PRL amino acids. 9
Multiple isoforms of PRLR
In order for PRL to have an effect on a cell target, PRL must first bind to a
specific membrane receptor, prolactin receptor (PRLR), which belongs to the cytokine
receptor superfamily. The PRLR is composed of at least three separate regions: an
extracellular region with 5 cysteines that contain the prolactin binding site, a single
transmembrane region and a distinct intracellular region that lacks intrinsic catalytic
activity.
The hPRL receptor gene can create multiple isoforms resulting from alternative
splicing as seen in Fig. 3. These different PRLR isoforms are referred to as short (∆S1), intermediate or long isoforms in accordance with their sizes. While three PRLR isoforms have been characterized in the rat, studies have suggested the existence of several human isoforms in breast carcinoma species and normal tissues (Kline et al., 1999). Both the intermediate and the long form of the PRLR contain an identical extracellular domain
(ECD), which is critical for engagement with its ligand. Although the affinity for the intermediate hPRLR is similar to that of the long isoform, the intermediate isoform is truncated in its C-terminus as a consequence of an out-of-frame splicing event. The intermediate isoforms is capable of Jak2 activation, but is incapable of activating the Fyn tyrosine kinase. The short, ∆S1, isoform shares a similar intracellular portion of the hPRLR with the long isoform, but is missing the entire S1 domain of the extracellular portion of the receptor (Fig 3) causing the affinity of the ∆S1 isoform to be reduced by approximately 7-fold (Clevenger et al., 2003). The function within the mammary gland of the various PRLR isoforms remains a difficult question to address, as covariable expression of each of the PRLR isoforms is observed within mammary tissues and cell 10
lines (Clevenger et al., 2003). Multiple isoforms of PRLR exist that differ in their cytoplasmic domains and signaling capabilities (Brockman et al., 2002).
Intermediate Long ∆S1 S1
S2
Y237 Y237 Y237
Y28 Y28 Y28 Y290 Y290 Y290 Y31 Y31 Y35 Y35 Y38 Y38
Y40 Y40
Y48 Y48
Y522 Y522
Y58 Y58
Fig. 3. Schematic representation of multiple isoforms of hPRL receptor. Different PRLR isoforms are referred to as short (∆S1), intermediate or long.
Mechanism of PRL signaling
In order for the cell to respond to PRL, PRL has to bind to its receptor. Receptor dimerization for PRL has been shown to involve two different regions of the ligands referred to as binding sites I and II (Fig 4). PRL first binds to one PRLR via binding site I to form an inactive, intermediate 1:1 complex. PRL bound in this complex then binds to a second PRLR molecule via the site II binding site to create a 1:2 PRL-PRLR complex.
Upon binding of the extracellular PRL to the membrane bound homodimer receptor, the ligand/receptor complex dimerizes and activates a series of signaling cascades, triggered by phosphorylation, that can lead to cell proliferation (Ihle, 1996). However if the hormone concentration is too high, the hormone and the receptor will only form a 11
heterodimeric complex, possibly acting as an antagonist because it blocks signal
transduction.
(Fuh G. et al., JBC 1993 vol. 268, pp. 5376-81)
Fig. 4. Sequential dimerization model of the hPRLR by hPRL. At low concentrations, hPRL binds first at site1 and then site 2. At high concentrations, hPRL saturates the receptor through site 1 interaction and acts as an antagonist by preventing receptor dimerization.
Since, the PRLR is devoid of intrinsic tyrosine kinase activity, it relies on other kinases to phosphorylate the intracytoplasmic portion of the receptor and signal transduction molecules involved in PRL signaling events within the cell (Fig. 5). The function of the PRLR is mediated, at least in part, by two families of signaling molecules:
Janus kinases (Jak) and signal transducers and activators of transcription (STATs). Jak2 12
is constitutively associated with the PRLR (Lebrun et al., 1994; Goupille et al., 1997) and
autophosphorylation of Jak2 occurs upon PRL binding to the PRLR. Phosphorylated
tyrosine residues on the intracytoplasmic domain of the PRLR recruit and induce
phosphorylation of cytoplasmic transcription factors, mainly STAT-1, STAT-3, and
STAT-5 (Vonderhaar, 1999; DaSilva et al., 1996). STAT proteins are important transcriptional regulators in the cell and have two main functions that include signal transduction in the cytoplasm and activation of transcription in the nucleus (Cataldo et al., 2000). STAT proteins can either stimulate or inhibit gene transcription, depending on promoter context (Brockman et al., 2002). The PRLR utilizes the Jak-STAT pathway as its main signaling cascade (Ben-Jonathon et al., 2002).
In addition to the Jak/STAT pathway, PRL has been shown to utilize other pathways in some systems. By phosphorylation of the PRLR at the carboxy-terminal tyrosinases, Jak2 may enable the association of the signal adaptor protein SHC with the
PRLR, which in turn activates the Shc/Grb2/Vav/Sos/Ras/Rac/Raf/MEK/MAPK signaling cascade (Clevenger et al., 1998). A variety of growth factors and cytokines
mediate proliferation by activating the ras-MAP kinase pathway of signal transduction
(Vonderhaar, 1999). PRL also can activate the JNK group of MAPKs, which is
important for its mitogenic signaling and suppression of apoptosis in some cell types
(Brockman et al., 2002). The mitogen-activated protein kinase (MAPK) pathway controls the growth and survival of a broad spectrum of human tumors (Sebolt-Leopold and Herrera, 2004).
Both the long and the short form of the PRL receptor are able to activate MAP kinase, with the activity reaching a peak within 5 min of PRL treatment (Das & 13
Vonderhaar, 1995). Both Jak-STAT and MAPK pathways are activated upon PRL
treatment in several breast tumor cell lines, and may act in parallel, or may converge at
some point in the signaling pathway (Brockman et al., 2002).
(Bole-Feysot C. et al., Endo Rev 1998 19(3) : 225-268)
Fig. 5. Schematic representation of the PRLR signaling pathways. The major signal transduction pathway of the long PRLR isoforms is the JAK-STAT cascade. For the short isoforms, however, many components are still unknown except that it signals through MAPKK.
Anti-PRL approaches
Significant inhibition of tumor progression has been achieved in rodents by
removing their pituitary gland or by inhibiting the release of PRL from the pituitary using
a dopamine agonist, bromocriptine. Bromocriptine has been considered minimally
effective or ineffective in treating breast cancer thus causing PRL to be disregarded as a 14 major contributing factor in breast cancer progression (Bonneterre et al., 1988; Anderson et al., 1993; McMurray et al., 1995). Failure of treatments blocking pituitary PRL production, are now thought to be due to extrapituitary PRL production by many tissues throughout the body, including local production of PRL within the mammary gland.
Removal of the pituitary gland reportedly reduces the circulating levels of hPRL by 20-
70% whereas other pituitary hormones became undetectable, suggesting therapeutics should be designed that directly antagonize hPRLR signaling rather than its release.
Anti-hPRL antibodies and anti-sense RNA directed against the hPRL gene have been used to successfully inhibit T-47Dco cell proliferation in vitro, as have antibodies against hPRLR in multiple breast cancer cell lines. These findings validate that PRLR antagonists may be used to counteract the pro-tumor action of PRLR ligands by acting at the level of receptor activator as shown in Fig. 7 (Goffin et al., 2003).
It has previously been demonstrated that the third α-helix of GH is important for its growth promoting activity (Chen et al., 1990; 1991; 1994; 1995). It was also demonstrated that Gly 119 of bGH (Chen et al., 1991) or Gly 120 of hGH (Chen et al.,
1994) plays a critical structural role in growth enhancement. By substituting one, single amino acid with one that contains a bulky side chain such as Arg, Lys, Trp, or Pro at position 120 of hGH results in a GH antagonist. This antagonist prevented receptor dimerization and disrupted intracellular signal transduction. It was demonstrated that a single amino acid substitution at position 129 of hPRL from Gly to Arg (G129R) results in an hPRL antagonist (Chen et al., 1999; Goffin et al., 1996). Steric hindrance prevents binding to the second receptor in the dimmer and hence they act as hormone antagonists as demonstrated in Fig. 6 (Goffin & Kelly, 1997). 15
(Chen et al., Clin Can Res 1999 5: 3583-93)
Fig. 6. Schematic representation of the wild-type hPRL (left) and the hPRLR antagonist (G129R, right) interaction with the PRLR. The four helical regions of the ligand are labeled as I, II, III, and IV. The Gly to Arg substitution is in the third alpha- helix and blocks functional dimerization and signal transduction of the receptors.
It has been shown that G129R has a receptor binding affinity similar to that of
hPRL demonstrating that G129R has a competitive receptor antagonistic action. By
using cell proliferation assays it was shown that G129R exerts inhibitory effects on breast
cancer cell proliferation (Chen et al., 1999). G129R exerted its inhibitory effects through
apoptosis in four hPRLR positive breast cancer cell lines (T-47D, MCF-7, MDA-MB-134 and BT-474) in a dose dependent manner as determined by the Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) assay (Chen et al., 1999). To understand the molecular basis of how the antagonist induced apoptosis, differential gene expression profile using subtractive PCR combined with cDNA microarray techniques of four human breast cancer cell lines were tested following treatment with hPRL and G129R (Beck et al., 2001; 2002). The anti-apoptotic gene, bcl-
2, was differentially expressed in the PRL treated breast cancer cell lines, but was not expressed in the G129R treated breast cancer cell lines (Beck et al., 2001; 2002). To 16 confirm these results, real-time reverse transcription polymerase chain reaction (RT-
PCR) was used to measure bax and bcl-2 gene expression in eleven breast cancer cell lines after hPRL or G129R treatment. It was shown that Bcl-2 was up-regulated in response to hPRL and was competitively inhibited by G129R in the majority of the cell lines tested (Pierce & Chen, 2004). This suggested that hPRL might act as a survival factor by inducing genes involved in suppression of apoptosis further strengthening
G129R's potential anti-apoptotic, therapeutic role in breast cancer therapy.
The phosphorylation status of the oncogenes STAT-3 and STAT-5 were investigated since STAT-3 and STAT-5 have been shown to play a role in signal transduction of mammary gland development and human breast cancer cells (Li, 1997).
The inhibitory effects of G129R on human breast cancer cells are mediated, at least in part, through the inhibition of JAK2/STAT phosphorylation, in particular, oncogene
STAT-3 phosphorylation (Cataldo et al., 2000). hPRL was able to activate both STAT-3 and STAT-5 and G129R was capable of inhibiting STAT-3 and STAT-5 tyrosine phosphorylation activation in human breast cancer cells when induced by hPRL (Cataldo et al., 2000). These data further strengthen our speculation that activation of the
JAK2/STAT pathway is an hPRLR specific event.
G129R's anti-tumor effects were also tested in nude mice using two PRLR positive (T-47D and MCF-7) human breast cancer xenografts (Chen et al., 2001). Daily treatment of PRL exhibited enhanced tumor growth versus the control mice, whereas the treatment with G129R, inhibited tumor growth by approximately 50% compared to the control (Chen et al., 2001). In conclusion, both in vitro and in vivo results demonstrate that G129R has potential as a novel anti-breast cancer agent. G129R has been 17
demonstrated to antagonize every aspect of hPRL-induced effects analyzed in various
breast cancer cell lines (Llovera et al., 2000) including inhibition of hPRL-activated
JAK/STAT and MAPK pathways.
(Goffin et al., Endocr Rev 2005 3: 400-22)
Fig. 7. Endocrine and autocrine/paracrine ligands of the PRLR. Dopamine and somatostatin are inhibitors of pituitary synthesis, but G129R, a PRLR antagonist, can be used as a negative regulator of extrapituitary PRL production.
ErbB2/HER2/neu oncogene
The name ErbB-2, neu (rat homolog), or HER2 is derived from “Human
Epidermal growth factor Receptor” as it features substantial homology with EGFR (Ross 18 et al., 2001). In Fig. 8, HER2/ErbB-2 is one of four members in a family of tyrosine kinase growth factor receptors that also include HER1/EGFR/ErbB-1, HER3/ErbB-3, and
HER4/ErbB-4 (Watson, 2002). These four receptors form a complex array of homodimers and heterodimers in response to ligand binding, with HER2 serving as the preferred heterodimer binding partner (Watson, 2002). Heterodimerization of HER2 with other ErbB family members initiated through ligand binding leads to autophosphorylation, transphosphorylation, and finally activation of the receptor dimer as a kinase for cytoplasmic substrate (Ross et al., 2001; Stern, 2000). This horizontal network of interactions is crucial to the ErbB signaling pathway, since HER3 is devoid of intrinsic kinase activity and HER2 is a ligand-less receptor. Therefore, in isolation neither HER3 nor HER2 have the capacity to initiate down-stream signaling, and thus requires co-recruitment with another ErbB-member to be transactivated (Carraway and
Cantley, 1994). An exception to the rule is that HER2 can spontaneously form active ligand-less homodimers in cells overexpressing HER2 (Baselga et al., 1998) ErbB pathways become activated by several mechanisms including overproduction of ligands, overexpression of receptors, or constitutive activation of receptors (Baselga et al., 1998).
19
(Tzahar and Yarden. Biochim Biophys Acta. 1998; 1377:M25)
Fig. 8. Four members of the ErbB family of receptor tyrosine kinases: HER1, HER2, HER3, and HER4. HER2 has no known ligand and HER3 lacks tyrosine kinase activity. There are numerous ErbB-specific ligands that include: transforming growth factor-α (TGF-α), amphiregulin (AR), epidermal growth factor (EGF), heparin-binding EGF (HB- EGF), betacellulin (β-CEL), epiregulin (EPR) and neuregulins (NRG).
HER2/neu’s role in mammary tumorigenesis
Overexpression of the HER2 protein occurs in 25%-30% of human breast cancers and leads to a particularly aggressive form of the disease (Ciardiello and Tortora, 2001;
Miles, 2001; Shawver et al., 2002; Vogel et al., 2002). Women whose cancers overexpress HER2/neu have a relatively poor prognosis with a median survival of 3 years, compared with 6–7 years for HER2-negative cases (Slamon et al., 1987; Chazin et al., 1992). HER2 overexpression has also been associated with estrogen and progesterone receptor negativity. 20
Given the close correlation between neu overexpression and mammary carcinogenesis, a number of laboratories have been interested in directly testing the tumorigenic potential of the neu oncogene in the mammary epithelium of transgenic mice. Several lines of transgenic mice were generated carrying the activated rat neu oncogene under the transcriptional control of the mouse mammary tumor virus (MMTV) promoter/enhancer (Guy et al., 1992). The HER2/Neu transgenic mouse is an important preclinical model because it recapitulates the development of HER2/Neu positive human breast cancer, characterized by over-expression of HER2/Neu antigen and aggressiveness
(Ren, 2004) and results in the rapid development of multifocal mammary tumors that metastasize with high frequency (Davies et al., 1999). Overexpression of neu in the mammary tumors was also associated with elevated neu intrinsic tyrosine kinase activity
(Guy et al., 1992).
Therapeutics targeting HER2/neu
One successful anti-receptor strategy has been the development of trastuzumab
(Herceptin; Genetech, San Francisco, CA). Herceptin is a recombinant humanized monoclonal antibody directed at the extracellular domain of HER2, which is designed to block and neutralize HER2 protein (Fendly et al., 1990). Herceptin exerts its antitumor activity by several mechanisms including receptor down modulation, prevention of cleavage of the receptor’s extracellular domain (which leads to receptor constitutive activation), and by recruiting host’s immune effector cells (Baselga et al., 1998).
Herceptin was also shown to induce regression of HER2 overexpressing breast cancers, thus validating HER2 as a therapeutic target within the HER (erbB) network (Moulder et al., 2001). Herceptin has shown clinical activity against HER2 overexpressing breast 21
tumors and has been recently approved for clinical use given alone or in combination
with chemotherapy. However, not all women respond to these treatments, and the precise
role that HER2 plays in the etiology of breast cancer remains poorly understood. A
critical question that remains unknown is whether HER2 is involved directly in the
initiation of breast cancer or rather instead only helps to drive cancer progression
(Watson, 2002).
(http://www.herceptin.com/herceptin/professional/about/moa.jsp#references)
Fig. 9. Anti-HER2/neu monoclonal antibody, Herceptin. Herceptin is a recombinant humanized monoclonal antibody directed against the extracellular domain of the HER2 tyrosine kinase receptor.
Crosstalk between PRL and HER2/neu
Recent research in breast cancer is identifying many significant differences between cell lines derived from different breast tumors. These breast cancer cell lines have recently been found to differ not only in their response to treatment, but vary greatly in their utilization of different death/survival pathways. Researchers are also uncovering crosstalk between various cell surface receptor pathways, and discoveries such as the 22
interaction between hPRL and the ErbB-2 receptor (Yamauchi, 2000). This emphasizes
the importance of expanding our knowledge and better defining the role of prolactin in
human breast cancer.
It is believed that constitutive phosphorylation of HER2/neu contributes to poor prognosis and unsatisfactory clinical response of HER2/neu positive breast cancer to anti-
HER2/neu monoclonal antibody therapy. Despite encouraging reports regarding
Herceptin clinical trial results, there was only a 20 to 30% overall response rate
(Cobleigh et al., 1999; Miles, 2001; Slamon et al., 2001; Vogel et al., 2002). Herceptin's ineffectiveness in many cases many be explained by evidence that hPRL constitutively activates oncogene HER2/neu through activation of JAK2 kinase (Yamauchi et al.,
2000). Yamauchi et al. revealed that autocrine secretion of hPRL led to constitutive tyrosine phosphorylation of HER2/neu via Jak2 by providing docking sites for Grb2 and stimulating the Ras-MAP kinase cascade. This cross-phosphorylation leads to enhanced cellular proliferation, additive to the intrinsic tyrosine kinase activity of ErbB-2, in human breast cancer cell lines (Fig. 10).
According to Yaumauchi et al., immunohistochemical analyses revealed that 76
human breast carcinoma tissues examined, 29% were positive for HER2/neu, 95%
positive for PRL and 100% were positive for PRLR. Together, 28% breast cancers
displayed HER2/neu and PRL immunoreactivity. This expression of HER2/neu or PRL
was then correlated to that of PCNA, a marker of proliferative activity. The mean
positive percentage of PCNA was 52.4% in the group of patients with both HER2/neu
and PRL-positive compared to 38.3% (p<0.013) in the patients with PRL-positive,
HER2/neu-negative breast cancer. These data suggest that breast cancers with HER2/neu 23 overexpression have higher proliferative and metastatic activity in the presence of autocrine/paracrine secretion of PRL. The same authors further demonstrated that the autocrine secretion of PRL in breast cancer cells stimulates tyrosine phosphorylation of
HER2/neu via activation of Jak2 kinase, which is one of the main reasons that HER2/neu is constitutively activated in breast cancer.
Since the oncogenic potential of HER2/neu appears to depend on the state of tyrosine phosphorylation (D’souza and Taylor-Papadimitriou, 1994; Clark et al., 1996),
Herceptin, which is designed to bind to the extracellular portion of HER2/neu (thus preventing it from receiving further stimulation), may not be effective in stopping the signal transduction from already the constitutively phosphorylated cytoplasmic tail of
HER2/neu protein. Therefore, in order to effectively reduce the oncogenic effects of
HER2/neu, inhibition of PRL pathway using hPRL antagonist should be considered.
Physiological events in vivo are regulated by a complex interplay of signals, including multiple growth factors and hormones (Gutzman et al., 2005). Although much is known about signaling by cytokines and growth factors, relatively little is known about how their signaling pathways may interact to exert biological effects (Huang Y et al.,
2006).
24
hPRL ?
hPRLR HER2/neu P P Jak2 Jak2 Grb2 P Src P STAT P SOS P PI3K Ras P P
STAT 1 P Raf STAT 3 P STAT P STAT 5 P P P P MAPKK AKT STAT STAT MAPK P
Proliferation Differentiation Cell survival
Target genes
nucleus
Fig. 10. Schematic representation of the potential cross-talk between hPRL and HER2/neu. Proposed signaling cascade of hPRLR and ErbB2 in the presence of autocrine secreted PRL.
PRLR and ER
The importance of mammogenic hormones such as PRL and 17β-estradiol (E2) in normal mammary growth and development has led to exploring their roles in the development and progression of breast cancer. Estrogen receptors and PRL receptors were found to be co-expressed and cross regulated in mammary tumor cell lines and in 25
primary breast cancers (Bonneterre et al., 1990; Murphy et al., 1984; Ormandy et al.,
1997). It has been reported that sex steroid hormones and PRL interact synergistically to control cancerous growth within the mammary gland (Ormandy et al., 1997). It has also been shown that endogenous PRL increases ERα levels and their responsiveness of cells to estrogens in vitro, suggesting that PRL amplifies the effects of estrogen (Gutzman JH, et al., 2004).
There is a hormonal crosstalk between PRL and E2 which is initiated primarily by
a rise in levels of phosphorylated ERK1/2 that occurs relatively late after exposure to both hormones and stimulates c-fos promoter activity (Gutzman et al., 2005). This receptor cross-regulation may provide a general mechanism allowing synergy among estrogens, progesterone, and PRL in many target tissues; which suggest that the use of antisteroid hormone therapy in breast cancer may be attacking only half of the synergistic equation (Ormandy CJ et al., 1997).
PRLR and EGFR
Recent studies suggest that PRL, growth hormone (GH) and other cytokines may
use multiple mechanisms to crosstalk with EGFR and/or ErbB-2 (Johnson et al., 1997;
Yamauchi et al., 1997, 1998, 2000; Qiu et al., 1998; Quijano and Sheffield, 1998; Kim et al., 1999; Maus et al., 1999; Badache and Hynes, 2001; Huang et al., 2003). It has been demonstrated that GH induces tyrosine-phosphorylation without the activation of EGFR kinase activity (Yamauchi et al., 1997; Kim et al. 1999). In several breast cancer cell lines, it has been demonstrated that hPRL and EGF synergize to enhance cell motility and in activating SHC and ERK signaling, suggesting a potentially important context-specific elements of crosstalk between these two factors (Huang et al., 2006). PRL has also been 26
shown to induce threonine phosphorylation of the EGFR in normal mammary epithelial
cells (Fenton and Sheffield, 1993; Johnson et al., 1996; Quijano and Sheffield, 1998).
Whereas, autocrine secretion of PRL is also able to stimulate tyrosine phosphorylation of
ErbB-2 by Jak2 and stimulate the Ras-MAP kinase cascade (Yamauchi et al., 2000). The identification of this novel cross-talk between growth factor receptors and cytokine receptors may provide new targets for therapeutics and preventative intervention.
ER and EGFR
The EGFR family proteins are receptor tyrosine kinases that, when bound to ligand through the extracellular ligand binding domain, result in receptor dimerization, kinase activation, and transphosphorylation on C-terminal regulatory tyrosinases.
Tumors typically depend on only one of these pathways and may overexpress either estrogen receptor (ER) or EGF receptor (EGFR) and related family members.
Intracellular mediators of these growth-stimulatory pathways are not completely defined, but one potential common mediator of EGF and ER signaling is the transcription factor signal transducer and activator of transcription 5 (STAT-5) (Boerner et al., 2005).
Estrogen was shown to negatively regulate EGF-mediated tyrosine phosphorylation of
STAT5, in human EGF family receptor-overexpressing breast cancer cells (Boerner et al., 2005).
Rational therapeutic combinations for breast cancer
Synergy, as it applies to drug-drug interactions, is defined as a combination of two or more drugs which achieves a therapeutic effect greater than that expected by the simple addition of the effects of the component drugs (Pegram et al., 1999). Such synergistic interactions between drugs may improve therapeutic results in cancer 27
treatment if the synergism is specific for tumor cells (Chou and Talalay, 1984). Analysis
of the nature of the interaction between two drugs (synergism, addition, or antagonism) may yield insight into the biochemical mechanisms of interaction of the drugs (Pegram et al., 1999).
PRLR antagonist and ER antagonist
The ability of PRL to stimulate ER expression provides evidence for this important physiological crosstalk between these two hormones. Anti-estrogens are compounds that compete with estrogens for binding to the estrogen receptors (ERs).
Anti-estrogens fail to induce receptor activation, thereby inhibiting hormonal actions on target cells. Their ability to interfere with the mitogenic activity of estrogens, anti- estrogens are widely used for the treatment of various hormone-dependent cancers, either alone or in combination. Tamoxifen (a selective ER modulator, and an ER antagonist) has been shown to down-regulate prolactin receptors in breast cancer cells (de Castillo B et al., 2004). A combined regimen using an anti-estrogen (tamoxifen) and an anti-PRL secretion drug (CV 205-502) has been reported to have significantly better clinical results
in metastatic breast cancer patients as compared with tamoxifen therapy alone (Bontenbal
M. et al., 1998). However, this regimen did not take into account that PRL is synthesized
by human mammary cells and could not block the autocrine/paracrine activity of PRL on
breast cancer. Another combination study was tested using tamoxifen with a complete
PRL blockade that acts at the receptor level. When anti-PRL (G129R) and tamoxifen were applied simultaneously, an additive inhibitory effect on breast cancer cell proliferation was observed (Chen et al., 1999) demonstrating the crosstalk between these two pathways. 28
anti-HER2 and ER antagonist
Although there is an inverse relationship between the degree of ER positivity and
HER-2/neu expression, a significant proportion of human breast cancers are both ER
positive and overexpress HER-2/neu (Ravdin et al., 1998; McCann et al., 1991; Berry et al., 2000). A large body of literature has demonstrated extensive interactions between the
ER signaling pathway and growth factor receptor (including HER-2/neu) signaling, both in terms of downstream effects as well as regulation of each other’s activity (Argiris et al., 2004). A combination approach using the anti-estrogen, tamoxifen, with an anti-
HER2, Herceptin, resulted in synergistic growth inhibition and enhancement of G0-G1 cell cycle accumulation. This data demonstrates that the combined inhibition of ER and
HER-2/neu signaling may be a powerful approach to the treatment of breast cancer.
However, preclinical data suggest that interactions between the ER and HER2
signaling pathways might be expected to result in clinical resistance of HER2-positive breast cancer to hormone therapy. It is also possible that women with HER2-positive,
ER-positive breast cancer could have a worse prognosis with tamoxifen therapy than with no therapy due to promotion of the agonist effects of tamoxifen through HER2-stimulated
signaling (Jones, 2003).
Herceptin and various other chemotherapy drugs have been tested to see which
are the most effective and safe. The most effective and safest combination was found in
a synergistic interaction of Herceptin plus carboplatin, which is an alkylating agent that
interferes with the growth of cancer cells, and docetaxel, which damages structures
involved in cell division. Aromatase inhibitors, indicated for use in postmenopausal 29
women, act by blocking the formation of estrogen, which fuels the growth of "hormone
receptor-positive" breast cancers.
Therefore, understanding the mechanism by which PRL stimulates mitogenesis
and how it interacts with other factors important in breast cancer may lead to improved
diagnostic assays and therapeutic approaches complementary to current available
methods (Schroeder et al., 2002). Important crosstalk mechanisms, such as those between PRL and HER2/neu, may be relevant for breast cancer behavior is important to investigate.
Transgenic models for breast cancer
Transgenic mice are used to test the effects of externally introduced genes. They
have allowed researchers to studying mammalian gene function and regulation on an
entire organism. Transgenic models are powerful tools used to assess the role of selected
growth factors and associated signaling pathways in mammary carcinogenesis (Rose-
Hellekant et al., 2003).
PRL transgenics
Prolactin transgenic mice were developed to understand the role of PRL on
mammary gland development in experimental models. A study by Wennbo et al., 1997
generated two lines of transgenic mice that systemically express rPRL using the
metallothionein (Mt) promoter in virgin, female mice. One line had expressed high
levels of rPRL (150ng/ml), which is four times higher than normal peak values, and the
other line expressed rPRL levels in the normal range of endogenous PRL (13ng/ml).
Both lines of female mice overexpressing the rPRL gene developed malignant mammary
adenocarcinomas (n=9). The tumors in the rPRL transgenic mice were not seen until 30
later in life (11-15 months of age). Cell lines were also established from a tumor from a
transgenic mouse expressing low serum levels of PRL produced rPRL and expressed
PRLR. The mammary tumors that developed in the PRL transgenic mice did not
metastasize. It was concluded that the tumor development was caused by the activation
of the PRLR.
To model the effects of local mammary production of PRL, Rose-Hellenkant et al. 2003 generated virgin, female mice under the control of a mammary selective hormonally nonresponsive promoter, neu related lipocalin (NRL). This promoter was useful to study the interaction of factors contributing to mammary carcinoma independent of hormonal changes in transgene expression. In two NRL-PRL transgenic lines generated, both lines of female mice lead to the development of mammary lesions and
ERα positive and ERα negative mammary tumors (Rose-Hellekant et al., 2003). This model provided a system to examine the roles of both local and circulating PRL in mammary oncogenesis in a defined transgenic system (Arendt et al., 2006).
Based on Wennbo's study, PRL transgenic mouse models revealed the pro- oncogenic effect of PRL over-expression in virgin mammary glands. To address whether
PRL tumorigenicity was maintained on differentiated mammary glands, Manhes et al.,
2006 generated mammary specific transgenic mice expressing hPRL under the control of the milk whey acidic protein (WAP) promoter. This promoter directs autocrine hPRL over-expression in late gestation throughout lactation. In this transgenic model, minimal levels of transgene expression (2-8ng/ml) were detected in the mammary glands of the virgin mice, but expression of mammary hPRL dramatically increased at the end of the first pregnancy (180ng/ml). Overexpression of hPRL led to morphological mammary 31
alterations, lactation defects and eventually to involution failure. Although some older,
multiparous females developed benign tumors, none of the mice developed mammary
carcinomas.
HER2/neu transgenics
Overexpression and amplification of the neu (c-erbB, ErbB2) proto-oncogene have been implicated in the development of aggressive human breast cancer (Guy et al.,
1992). To assess the effects of the expression of the neu proto-oncogene in the mammary gland, Guy et al. established transgenic mice carrying neu under the transcriptional
control of the mouse mammary tumor virus (MMTV) promoter. Expression of the neu
transgene resulted in the appearance of focal mammary adenocarcinomas, which
eventually metastasized to the lung. Tumorigenesis in these transgenic lines was
correlated with elevated expression of the neu transgene and an increase in neu-
associated tyrosine kinase activity (Guy et al., 1992)
Cyclin D1 proto-oncogene is an important cell cycle regulator of G1 to S- phase
transition. Binding of cyclin D1 to its kinase partners, the cyclin dependent kinases 4 and
6 (CDK4\6) results in the formation of active complexes that phosphorylate the
retinoblastoma tumor suppressor protein (Rb). Hyper-phosphorylation of Rb results in the
release of Rb-sequestered E2F transcription factors and the subsequent expression of
genes required for entry into S-phase. It has been reported that cyclin D1 plays an
important role in breast cancer formation and that cyclin D1 protein is over expressed in
over 50% of human mammary carcinomas. Wang et al. 1994 also demonstrated that
MMTV-cyclin D1 transgenic mice develop mammary hyperplasia and carcinomas. Yu et
al. investigated whether the ablation of cyclin D1 protects against breast cancers by 32 crossing cyclin D1 knockout mice with four different strains of breast-cancer-prone
MMTV-oncogene transgenic mice: Ras, c-neu, c-myc and Wnt-1. Cyclin D1 knockout mice were resistant to mammary cancers induced by the neu and ras oncogene, but not the c-myc or Wnt-1 oncogenes. Cyclin D1 knockout mice revealed that the ras and neu oncogene were dependent on cyclin D1 for malignant transformation of mammary glands, but not cyclin D2 or cyclin D3.
To address the role of cyclin-D1-dependent kinases in transformation of
HER2/neu, Yang et al. 2004 established a bitransgenic line expressing the MMTV-p16 and the MMTV-neu transgene. The expression of p16 tumor suppressor effectively blocked the formation of tumors caused by MMTV-neu. This demonstrated that deregulation of the cyclin-dependent kinase partner of cyclin D1 is an essential target of
ErbB2 (Yang et al., 2004). It was also demonstrated that MMTV-cyclin D1 transgene failed to accelerate tumor formation by erbB2 since cyclin D1 if fully downstream of erbB2.
These studies have demonstrated that transgenic mice expressing the neu oncogene in the mammary epithelium, mammary tumors are formed. However Neu- induced tumors were prevented in the breast in the absence of cyclin D1. If overexpression of cyclin D1 is an early event in mammary carcinogenesis and if blocking cyclin D1 in fully formed tumors is not effective, then inhibitors of cyclin D1 may be more useful as chemo preventative agents than as therapeutic agents (Chodosh, 2002).
33
Chemoprevention of Human Breast Cancer
The failure of conventional chemotherapy against advanced invasive disease
indicate that new approaches to the control/prevention of breast cancer are critically
needed (Arun and Hortobagyi, 2002; Greenwald, 2002). Cancer chemoprevention can be
defined as treatment of carcinogenesis, including cancer prevention, inhibition, or
reversal (Sporn & Suh 2000; 2001; Steele, 2003). Chemo preventative agents are drugs,
vitamins, diet, hormone therapy or any agent that delays the start of cancer, keep it from
starting, or stop it from coming back.
The major challenges facing investigators studying chemoprevention of breast
cancer are to develop alternative preventive therapies that have fewer side effects, and to
develop drugs that act independently of status (Cuzick, 2000; Arun and Hortobagyi,
2002; Greenwald, 2002). Five classes of potential chemoprevention agents are being
tested and have shown promise in clinical trials. These agents include selective estrogen
receptor modulators (SERMS) such as tamoxifen, and other hormonal agents;
nonsteroidal anti-inflammatroy (NSAIDS); calcium compounds; glucocorticoids
(compounds that are a type of steroid); and retinoids (chemical cousins of vitamin A).
Despite increasing evidence that hPRL is involved in breast cancer development, there are no chemopreventive or chemotherapeutic agents that target the hPRL receptor available.
Overview and rationale of current work
In this study we use hPRL antagonist (G129R) together with Herceptin to develop a more effective approach to block the HER2 signal transduction in breast cancer cells.
We will determine if G129R and Herceptin combination approach will effectively block 34
HER2 and PRL signaling pathways. We will also investigate whether the
autocrine/paracrine effects of hPRL stimulate and G129R blocks overexpressed HER2
protein phosphorylation via the interaction of JAK2 in breast cancer cells. Understanding the molecular mechanism by which PRL stimulates tumorigenesis and how it interacts with other factors are important in breast cancer and help develop new therapeutic approaches.
To investigate the relationship between PRL and HER2/neu in mammary tumorigenesis we generated bitransgenic mice co-expressing hPRL or G129R and neu.
These bitransgenic mice provide a unique model to study the relationship between PRL and ErbB2 signaling pathways and possibly gain insight into the molecular mechanisms that lead to tumorigenesis in mammary glands. Comparison of the mammary tumor incidence, rate of tumor formation, characterization of mammary gland whole mounts, and potential biomarkers will help us to identify the role of hPRL in HER2/neu tumorigenesis.
To conquer this diverse or heterogeneous disease, growing consensus in the field is to attack multiple key pathways at once using agents possessing entirely different inhibitory mechanisms. Designing novel combination strategies for each cancer type are essential to decrease cancer morbidity or as an effective preventive measure. In view of the prevalence of ErbB2 overexpression in human breast cancer and the ability of local mammary PRL to contribute to human breast cancer, it is critical to establish the consequences of the interaction between these two pathways in mammary cancer pathogenesis in the in vivo setting. 35
CHAPTER TWO
MATERIALS AND METHODS
Materials
Human PRL and G129R used in this study were produced using an E.coli protein production system. BL21 (DE3) cells (Invitrogen) were transformed with pET22b plasmids (Novagen; Madison, WI) which contain cDNAs encoding hPRL or G129R. A seed culture was grown overnight at 37oC. The following day a large-scale LB growth
culture was prepared and IPTG was added to induce expression of hPRL or G129R.
Bacteria was collected, resuspended in a solution containing 0.2M NaPO4 pH 8, 10mM
EDTA, and 0.5% Triton X-100, and lysed using a Sonic Dismembrator. The inclusion
bodies were collected and resuspended in 0.2M NaPO4 pH7, 1%v/v beta mercaptoethanol, and 8M urea for refolding. The refolding process consisted of dialyzing the protein against decreasing amounts of urea and beta-mercaptoethanol in the presence of 20mM Tris HCl, pH 8.0 for three consecutive days. The protein was then purified by a Q-Sepharose anionic exchange column using a FPLC system (Amersham).
Based on our previous experience, the purity of the products were typically 98% pure, as determined by SDS-PAGE and silver staining. Trastuzumab (HerceptinTM) was kindly
provided by the Cancer Center of Greenville Hospital System (Greenville, SC).
Antibodies
The following primary antibodies were used: 1:1000 dilution of anti-phospho-
HER2/neu for cell lysate (Lab Vision; Fremont, CA); 1:1000 dilution of anti-HER2/neu
for cell lysate (EMD Biosciences; Darmstadt, Germany); 1:1000 dilution of anti-cyclin 36
D1 (Invitrogen); 1:10,000 dilution of anti-β-actin (Sigma, St. Louis, MO); 1:10,000 dilution of anti-hPRL (Abcam; Cambridge, MA); 1:1000 dilution of anti-hPRLR (Zymed
Laboratories; South San Francisco, CA); 1:1000 dilution of anti-phospho-STAT5a/b, anti-phospho-STAT3, and a 1:10,000 dilution of anti-β-tubulin (Upstate Biotechnology;
Lake Placid, NY). Anti-phospho-Akt 1/2/3; anti-Akt1; anti-phospho-MAPK (Erk1/2);
MAPK (anti-Erk1); anti-STAT5; anti-phospho-HER2 (for tissue lysate), and anti-HER2
(for tissue lysate) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a dilution of 1:1000. The secondary antibodies, goat anti-mouse IgG- and goat anti-rabbit IgG-horseradish peroxidase-conjugates were obtained from Bio-Rad
Laboratories (Hercules, CA) and used at a dilution of 1:2000.
Plasmid construction and cloning
Primers corresponding to hPRL and G129R (with the sequences encoding the signal peptide and with and without the stop codon, plus restriction sites for NdeI: 5'-
CAT ATG AAC ATC AAA GGA TCG -3', NheI 5'- GCT AGC ATG AAC ATC AAA
GGA -3', NotI 5'- GCG GCC CGC ATG AAC ATC AAA -3', SpeI 5'- ACT AGT TTA
GCA GTT GTT -3', SpeI without the stop codon 5' ACT AGT GCA GTT GTT GTT -3',
XhoI 5'- CTC GAG TTA GCA GTT GTT -3' and XhoI without the stop codon 5'- CTC
GAG GCA GTT GTT GTT -3') were used to amplify the hPRL and G129R fragment
from a previous clone in our lab.
PCR was run on the GeneAmp 9700 (Perkin-Elmer Biosystems, Foster City, CA,
USA). hPRL and G129R PCR fragments were ligated into the TA cloning vector pCR2.1
(Invitrogen, Inc., Carlsbad, CA) using T4 DNA ligase supplied in the kit. After ligation
overnight, 1µl of the ligation was transformed in 25µl of TOP10 chemically competent 37
cells (Invitrogen) following manufacture's procedure for transformation. The
transformation was then plated on Luria-Bertani (LB) agar plates containing ampicillin
(500µg/ml) and X-galactosidase (50nM). X-galactosidase was used to blue-white screen the colonies for PCR insert where the transformants that contain vector with the insert will be a white color whereas the blue colonies will not have the insert. White colonies were picked from plates and grown in LB broth containing Amp (500µg/ml) and allowed
to grow overnight at 37° with continuous shaking. The cultures were then subjected to isolation of the plasmid DNA using the Qiagen Miniprep kit (Qiagen Corp., USA) following manufacture's instructions. The plasmid DNA was then digested with restriction enzymes to check for the proper size of insert in the vector.
hPRL and G129R was subcloned into pCR3.1 mammalian expression vector
(Invitrogen) and pET22b bacterial expression vector (Novagen) with NdeI and XhoI
restriction sites including the stop codon. The pCR3.1 vector was used for stable
transfections and the pET22b vector was used for protein production.
hPRL and G129R was subcloned into the puCIG mammalian expression vector
(Chen et al., 1990) and the PMSG mammalian expression vector (Amersham
Biosciences) using the NotI and SpeI ad the NheI and XhoI restriction sites, respectively.
38
LacZ NdeI hPRL
XhoI
pUC ori pCR2.1-hPRL 4587 bp
Kanamycin
Amp
(source: Invitrogen)
CMV T7 promoter NdeI f1 ori hPRL
pCR3.1-hPRL XhoI 6496 bp Amp
SV40 promoter Neomycin/kanamycin resistance gene (source: Invitrogen)
f1 origin XhoI hPRL Amp NdeI lac operator T7 promoter pET22b-hPRL 6180 bp
LacI
(source: Novagen)
Fig. 11. Schematic representation of the cloning and expression plasmids. The 600 bp hPRL cDNA was subcloned into the pCR2.1, pCR3.1, and pET22b expression plasmids. 39
Cell Culture
Several human breast cancer cell lines (T-47D, MDA-MB-134, MDA-MB-483,
BT-474, MCF-7, MDA-MB-453, MDA-MB-468, MDA-MB-436, SKBR-3 and MDA-
MB-231) were purchased from the American Type Culture Collection (ATCC, Rockville,
MD). All cell lines were grown in media supplemented with 10µg/ml Gentamicin
(GIBCO-BRL, Gaithersburg, MD). T-47D, MDA-MB-483, BT-474 and SKBR-3 cells
were maintained in phenol-red free RPMI 1640 medium (GIBCO-BRL, Gaithersburg,
MD) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan
UT). MDA-MB-483 cells were supplemented with 0.2mM sodium pyruvate, 2mM
HEPES buffer and 200 IU insulin (Sigma, St. Louis, MO). BT-474 cells were
supplemented with 1mM sodium pyruvate and 10µg/ml insulin. MCF-7 cells were
maintained in DMEM (GIBCO-BRL, Gaithersburg, MD) supplemented with 10% FBS.
MDA-MB 134, MDA-MB 453, MDA-MB-468, MDA-MB-436 and MDA-MB-231 cells
were maintained in Leibovitz L-15 medium. MDA-MB 453, MDA-MB-436, and MDA-
MB-231 were supplemented with 10% FBS, but the MDA-MB-436 also received 200 IU
insulin. MDA-MB-134 were supplemented 20% FBS and MDA-MB-468 were
supplemented with 15% FBS. Cell lines (T-47D, MDA-MB-483, BT-474, MCF-7,
SKBR-3 and MDA-MB-231) were maintained at 37°C in a humidified atmosphere in the
presence of 5% CO2 and MDA-MB-134, MDA-MB-453, MDA-MB-468, and MDA-MB-
436 were maintained in the absence of CO2.
MCNeu, an epithelial origin, and N202F3, a fibroblast origin, were isolated from a tumor of a MMTV-Neu transgenic mouse. MCNeu and N202F3 are maintained in
DMEM medium supplemented with 10% FBS, 10 µg/ml Gentamicin and 1mM sodium 40
pyruvate. L cells, a human fibroblast cell line, were maintained in DMEM medium
supplemented with 10% FBS and 10 µg/ml Gentamicin.
Anchorage-dependent cell growth
Breast cancer cells in exponential growth were harvested by trypsinization and seeded at a concentration of 1x104cells/100µl/well into 96-well culture plates in RPMI-
1640 medium supplemented with 10% CSS and 10µg/ml gentamicin. Cells were
incubated overnight for attachment. The medium was then removed and various
concentrations of hPRL, G129R, Herceptin or combination of the compounds were added
in serum-deprived RPMI 1640 medium in a volume of 200µl/well. Compounds were not renewed during the entire period of cell exposure and were incubated for an additional 48 h. After incubation, the culture medium was removed and replaced with fresh medium
(100ul/well) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium/phenazine methosulfate (MTS/PMS) solution diluted at a
1:6 ratio (Cell Titer 96 AQueous non-radioactive cell proliferation kit; Promega, Madison
WI). The relative viability of the cells was determined by colorimetric measurement of the reduction of MTS by the living cells using a Benchmark microplate reader (Bio-Rad).
Plates were read at 490 nM and all experiments were carried out in triplicate and repeated at least four times for each cell line. Cell viability was calculated as a percentage of control treatments.
Transient and stable transfection
A mouse and human epithelial cell line, MCNeu and T-47D and a mouse and human fibroblast cell line, N202F3 and L cells were used for transient transfection. Cells were transfected with hPRL cDNA using different plasmids; the pMSG mammalian 41
expression vector with MMTV promoter (source: Amersham Biosciences) and the
pUCIG mammalian expression vector with MT promoter (source: Chen et al.). T-47D,
and BT-474 were used for stable cell transfection using pcDNA3.1 and pCR3.1 plasmids
containing the G129R or hPRL cDNA respectively.
The day before transfection, cells were trypsinized and counted. Epithelial cells
were plated at a density of 1.75 x 105 cells/ml and fibroblasts were plated at a density of
0.5 x 105 cells/ml in 2 ml of complete growth medium. Cell density was ~50-80% confluent on the day of transfection. For each well of cells to be transfected, 2 µg of
DNA was diluted in 100 µl of Opti-MEM® I Reduced Serum Medium without serum.
For each well, 20 µl of Lipofectin® was diluted in 100 µl of Opti-MEM® I Reduced
Serum Medium without serum and incubated at room temperature for 30-45 min. The diluted DNA solution was combined with the diluted Lipofectin® solution and mixed gently and incubated for 10-15 min. at room temperature to form DNA-Lipofectin® complexes. Growth medium was removed from cells, washed, replaced with 0.8 ml of
Opti-MEM® I Reduced Serum Medium without serum combined with the DNA-
Lipofectin® complexes, and mixed gently by rocking the plate back and forth. Cells were incubated at 37°C in a CO2 incubator and medium was replaced with 1ml of growth medium containing serum 8h post-transfection for transient transfections and with 2ml of growth medium containing serum 24h post-transfections for stable transfections. For stable transfections, cells were passed into the selective medium (500 µg/ml Geneticin or
G418 (Invitrogen) 72h post-transfection to select for neo gene expression. The expression levels of the individual cell lines were determined and the cell lines with the 42
high expression levels were expanded. Transgene expression of hPRL was tested 48 hrs
after transfection using an IRMA kit.
Isolation of proteins: SDS PAGE and western blot:
Immunoprecipitation
Treatments were performed at 37° in serum starved media and were terminated by washing the cells with ice-cold phosphate-buffered saline (PBS) supplemented with
0.4mM sodium orthovanadate. The cells were then harvested by scraping in lysis buffer
(150mM NaCl, 50mM Tris-HCl (pH 8.0), 100mM sodium fluoride, 2mM EDTA, 1mM phenylmethylsulfonyl fluoride, 1mM sodium orthovanadate, 5 mg/ml aprotinin and 5 mg/ml leupeptin) containing 1% (v/v) NP-40 and were kept on ice for 15 min. Cell lysates were centrifuged at 15,000 g for 15 min. at 4°C. The detergent extracts
(supernatant) were collected and subjected to immunoprecipitation. Cell extracts (500-
1000µg) were mixed with 5µg anti-Jak2 (SC-278) or 4µg anti-Neu (SC-284) and incubated at 4° for 2 h with continuous agitation. Protein A-sepharose (GIBCO-BRL,
Gaithersburg, MD) (50µl) was added and incubated at 4° overnight. The beads were washed four times with lysis buffer and were pelleted by pulse centrifugation (5 seconds in the microcentrifuge at 15,000g). The sepharose beads were resuspended in 40 µl in 6 x sample buffer and mixed gently.
Extraction of protein from cells
Cells were washed with ice cold phosphate buffered saline (PBS) and lysed in
200µl of lysis buffer [20mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA (pH 8.0),
0.25% sodium deoxycholate, 1% Igepal CA630] containing protease inhibitors (1mM phenylmethylsulfonyl fluoride, 1µg/ml aprotinin, 1µg/ml leupeptin; and 1mM sodium 43
orthovanadate). Cells were incubated on an orbital rotator for 10 min. and then the lysate
was transferred to a 1.5ml eppendorf tube. Lysates were gently passed through a 21-
gauge needle 5-6 times to shear genomic DNA and then placed on ice for 10-20 min.
The lysates were cleared by centrifugation for 15 min. at 14,000 rpm. at 4oC.
Extraction of protein from tissues
Fresh mammary gland or tumor tissue was excised and frozen immediately on dry ice and stored at -80°C until use. Approximately 100-200 mg of frozen tissue was homogenized on ice using a Polytron PT1200 B in 1 mL of tissue homogenate buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.25% sodium deoxycholate, 1% Igepal CA630] containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 1 µg/ml pepstatin, 1 µg/ml aprotinin, 1
µg/ml leupeptin; and 1 mM sodium orthovanadate). Tissue homogenates were incubated
on ice for 20-30 min. and homogenates were cleared by centrifugation for 15 min. at
14,000 rpm. at 4oC.
Immunoblot analysis
Protein content was determined against a standardized control using the
Coomassie Blue Protein Assay Reagent (Rockford, IL). Equal amounts of protein were
heated in SDS sample buffer for 5 min. at 100oC, subjected to electrophoresis on a 10% or 4-15% SDS-PAGE (Biorad, Hercules, CA) and then transferred to Hybond nitrocellulose membrane (Amersham, Arlington Heights, IL). Non-specific binding on the nitrocellulose membrane was minimized by blocking for 1h at room temperature (RT) with TBS-T [25mM Tris-HCL (pH 7.5), 150mM NaCl and 0.05% Tween 20] containing
5% non-fat dry milk (Biorad, Hercules, CA). Membranes were then incubated overnight 44
at 4oC in TBS-T with 5% milk with specific primary antibodies at a concentration
recommended by the manufacturer with constant gentle agitation. Membranes were
washed in TBS-T, and then incubated with secondary antibodies for 2 hours at room
temperature (RT) with constant gentle agitation. After secondary antibody incubation, membranes were thoroughly washed with water for 5 min, TBS-T for 5 min, and water again for 5 min. Membranes were developed by enhanced chemiluminescence reagent
(Amersham) and then exposed to Kodak MR film (Fisher). Nitrocellulose membranes were stripped in stripping buffer (2% (w/v) sodium dodecylsulfate, 62.5 mM Tris-HCl pH 6.7, and 100 mM β-mercaptoethanol added fresh) and incubated at 50°C for 30 min with rotation. Membranes were washed 2x for 10 min in TBST at RT and then blocked in 5% milk TBST blocking solution for 1h at RT. Different primary and secondary antibodies were used as described.
Human breast cancer xenografts in nude mice
Six to eight-week-old, female athymic nude (nu/nu) mice (Jackson Lab, Bar
Harbor, ME) were inoculated into the mammary fat pad with 7 x 106 T-47D cells per
100µl. Mice were implanted with 17β-estradiol tablets (1.7mg/pellet and 60-day release;
Innovative Research of America; Sarasota, FL). Tumor measurements were performed
once or twice per week, and volumes were calculated using the formula use ½ [length
(mm)] X [width (mm)]2. Treatment with Herceptin, G129R, a combination of the both,
or a control was initiated one week after tumor inoculation. Purified G129R
(10mg/kg/daily) or Herceptin (10mg/kg/bi-weekly), or a combination of both was
injected intraperitoneal (i.p.) for 6 weeks. Control groups were given sterile phosphate
buffered saline (PBS). Each group consisted of 6-10 mice. 45
Generation of multiparous MMTVneu transgenic mice
Homozygous mice expressing rat neu under the control of the mouse mammary tumor virus promoter (MMTVneu) were purchased from the Jackson Laboratory (Bar
Harbor, ME). Female MMTVneu mice were bred at 5 weeks of age with male
MMTVneu mice. The breeding group contained 34 MMTVneu female mice. To prevent litters from overlapping, all surviving pups from each litter were weaned by 30 days of age. All female study mice experienced two cycles of pregnancy and lactation. They
were palpated for tumors twice weekly through 12 months.
Generation of bitransgenic mouse models
MMTVneu female mice were purchased from the Jackson Laboratory (Bar
Harbor, ME). Mice expressing either hPRL or G129R under the control of the MT
promoter (MT-hPRL, MT-G129R) came from existing FVB transgenic lines generated at
our facility (18). The MT promoter is regulated by heavy metal and directs expression of
the transgene in most tissues including the liver, kidney, spleen, and mammary gland.
The linear DNA fragment used for microinjection was a 1.4 kilobase (kb) molecule
located between the KpnI sites in the pUCIG plasmid.
MMTV-hPRL and MMTV-G129R cDNA were used to generate hPRL and
G129R transgenic founder mice from the FVB strain (Jackson) by microinjection of
DNA into the male pronucleus of fertilized mouse eggs as described previously (Chen
WY, Wright DC, Wagner TE and Kopchick JJ; Proc Natl Acad Sci USA, 1990). The
linear DNA fragment used for microinjection was a 3.22-kilobase (kb) molecule located
between the Hind III sites in pMSG, which includes the MMTV LTR promoter and the
SV40. The MMTV promoter directs expression locally in the mammary gland tissue. 46
MT or MMTV-hPRL and MT or MMTV-G129R heterozygotic male mice were
bred with MMTVneu homozygous females to produce four bitransgenic lines (MT-hPRL/ neu; MT-G129R/neu; MMTV-hPRL/neu; and MMTV-G129R/neu). All offspring were
heterozygous for MMTVneu and approximately 50% were positive for either hPRL or
G129R, respectively. They were palpated for tumors twice weekly through 12 months.
Mice were maintained in barrier facilities under IACUC approved protocols.
KpnI MT
NotI Amp hPRL pUCIG-hPRL 4487 bp SpeI Poly A KpnI
Ori
Kpn I MT SP hPRL Poly A Kpn I
XhoI hPRL PolyA NheI BamHI SV40 MMTV-LTR HindIII gpt pMSG-hPRL Hind III 8313 bp
Amp
Hind III MMTV-LTR SP hPRL Poly A Hind III
Fig. 12. Schematic representation of the mammalian expression plasmids. The 600 bp hPRL cDNA was subcloned into the puCIG or pMSG mammalian expression plasmids. The MT-hPRL or MMTV-hPRL cDNA fragment was digested, purified and microinjected into fertilized mouse eggs of FVB mice along with the tyrosinase gene, following standard protocol.
47
Identification of transgenic mice
Transgene screening, DNA: PCR
For DNA, approximately 0.5-0.6 cm of tail snip was used to confirm the presence
of transgene cDNA. For each 0.5-0.6 cm of tail, 200 µl of Direct PCR Lysis Reagent
(Viagen Biotech) was added along with 0.1 mg/mL of proteinase K (Invitrogen).
Eppendorf tubes were incubated at 55° for 3-16h, to ensure complete lysis. Lysates were then incubated at 85° for 45 min. and were cleared by centrifugation for 10 sec. To
confirm DNA expression of transgenes, PCR was performed using extracted DNA PCR
using the primer pairs listed in Table C1. Glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) primers were used as an internal control for PCR. PCR was run on the
GeneAmp 9700 (Perkin Elmer Biosystems, Foster City, CA). PCR for hPRL and
G3PDH used the following parameters: 30 cycles: 1 min.-94°C, 30 sec-94°C, 2 min.-
68°C, 1 min.-68°C, and final hold of 4°C. For HER2 primers the following parameters used were: 30 cycles: 1 min.-94°C, 30 sec-94°C, 1 min.-62°C, 1 min.-72°C, 6 min.-72°C, and final hold of 4°C.
Transgene expression confirmation, mRNA: RT-PCR
To confirm mRNA expression of transgenes, RNA was extracted from mammary gland tissue that had been frozen immediately following excision. Approximately 50-100 mg of frozen tissue was homogenized on ice using a Polytron PT1200 motorized homogenizer (Polytron; Bad Wildbad, Germany) in 1mL Trizol (Invitrogen; Carlsbad,
CA) and processed according to the manufacturer’s instructions. Female bitransgenic mice used for this study were confirmed by RT-PCR using the primer pairs listed in
Table C1. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) primers were used as 48
an internal control for RT-PCR. For RT-PCR the parameters used were: 25 cycles: 45
min.- 48°C, 1 min.-94°C, 30 sec-94°C, 1 min.-62°C, 1 min.-72°C, 6 min.-72°C, and final
hold of 4°C.
Transgene expression confirmation, protein: IRMA, ELISA
The expression level of hPRL (transgene product) from MT-hPRL and MT-
G129R transgenic and bitransgenic lines was quantitated in serum (blood harvested by tail vein) using a Coat-A-Count Prolactin Immunoradiometic Assay (IRMA) kit (DPC,
Inc., Los Angeles, CA) which measures the specific binding affinity between hPRL and anti-hPRL. This immunological assay does not cross-react with endogenous mouse PRL
(Bernichtein et al. 2003b) and was performed as per the manufacturer’s recommendations. The expression level of hPRL was measured in tissue homogenates, using the Active Prolactin ELISA kit (Diagnostic Systems Laboratories; Webster, Texas).
Approximately 100 mg of fresh mammary gland tissue isolated from the MT and
MMTV-hPRL and G129R transgenic and bitransgenic lines were homogenized and were assayed to quantitate the expression level of hPRL.
Transgene sequencing confirmation
Using hPRL primers, hPRL was PCR and RT-PCR amplified from DNA and
RNA isolated from the tail and mammary gland, respectively, from the MT-hPRL,
MMTV-hPRL, MT-hPRL/neu and MMTV-hPRL/neu mice. Reactions were ran on a 1% agarose gel for 45 min. at 100V. The hPRL fragment was gel extracted and purified.
DNA or cDNA was then ligated into pCR2.1 plasmid (Invitrogen) and plated on
LB/AMP/IPTG plates. Colonies were grown up and DNA was extracted and then 49
digested with restriction enzymes to confirm hPRL insert. DNA was sequenced using
M13 forward and reverse primers to confirm sequencing.
Transgenic mouse mammary gland morphological assessment
Mammary gland whole mount
The fourth and fifth inguinal mammary glands of bitransgenic female mice and
their female littermates were removed at 1, 3, and 6 months of age and used to prepare
mammary gland whole mounts. The isolated glands were spread on glass slides and
fixed overnight in freshly prepared Carnoy’s fixative at room temperature. The following day, the glands were gradually re-hydrated by decreasing the concentration of ethanol.
Glands were stained with Carmine Alum Stain overnight and were subsequently destained and dehydrated by incrementally increasing the concentration of ethanol. The fat pad was cleared with xylene (Sigma) for 30-60 minutes and mounted with Permount
(Fisher). Glands were documented via digital photography. Glands were documented via digital photography and Kodak 1D Image Analysis software (Eastman Kodak
Company’s Molecular Imaging Systems; Rochester, NY) was used to analyze 40x images of the whole mounts. Two researchers working independently evaluated the total numbers of side branches, terminal end buds (TEBs), and lobulo-alveolar structures within six different one mm2 grids chosen from each animal (n=6 for each transgenic group) and the results were combined.
Immunohistochemistry
For histological analyses, the fourth and fifth inguinal mammary glands were
removed from bitransgenic female mice and their female littermates at 1, 3, and 6 months of age and fixed with 4% paraformaldehyde before embedding in paraffin. Five- 50
micrometer sections of the mammary glands were deparaffinized in xylene for 5 min and
rehydrated in a graded series of ethanol. For antigen retrieval, slides were brought to boiling in trisodium citrate buffer (10 mM trisodium citrate, 0.05% Tween-20; pH 6.0) and placed in a 95°C water bath for 30 min, then cooled to room temperature.
Endogenous peroxidase activity was blocked with a 10-minute hydrogen peroxide treatment and non-immune serum was added to prevent non-specific binding. Primary antibodies (2.5µg/mL) were diluted in non-immune serum and slides were incubated overnight in a moist chamber at 4° C. 3,3'-diaminobenzidine (DAB) was used as the detection reagent. For detection of Ki67 antigen, a standard kit was used (Novocastra
Laboratories; Newcastle upon Tyne, England) following the manufacturer’s instructions.
Sections were lightly counterstained in hematoxylin (Vector Laboratories; Burlingame,
CA), dehydrated in ethanol, cleared in xylene, and coverslipped with Cytoseal™ 280
(Richard-Allen Scientific; Kalamazoo, MI). Stained sections were observed microscopically and digitally photographed. To quantify the Ki67 staining intensity, H scores were calculated as the summation of the proportion of nuclei stained in each of four categories. The staining intensity (SI) ranged from 0 (no detectable staining) to 3
(most intense).
Statistical data analyses
For Kaplan-Meier survival curves, statistical differences were calculated using the
Wilcoxon rank sum test. The results for the number of side branches, number of lobules,
and Ki67 H-scores were expressed as the mean ± SE accompanied by the indicated
number of experiments. Statistical differences between the groups were determined
using Student's t-test and a two-tailed distribution with unequal variances. For 51
immunoblots, gel documents were scanned and analyzed using Kodak 1D Image
Analysis Software. Densitometric values of protein bands were quantified based on net
intensity and results for the fold change were expressed as the mean ± SD. Statistical
differences between the groups were determined using Student's t-test. Tumors were
measured along two major axes with calipers. Tumor volume was calculated as follows:
2 V= 4/3πR1 R2 where R1 is radius and R2 is radius 2. The results were presented as means
± SE. Statistical significance, for all in vivo experiments including the cumulative tumor volume and tumor weights, were determined using a one-way ANOVA, followed by
Tukey's Multiple Comparison Test. A value of p<0.05 and p<0.001 was considered significant or very significant.
52
CHAPTER THREE
RESULTS
Co-expression of PRLR and HER2/Neu in human breast cancer cells
The expression levels of PRLR, Neu, and phospho-Neu in ten human breast cancer cell lines were measured by Western blotting analysis to examine HER2/Neu expression with their relative hPRLR levels (Fig. 13). In six cell lines that express high levels of Neu (BT-474. SKBR3, MDA-MB453, MB134, BT483, and T-47D), they were found to also express relatively high levels of hPRLR. The highest HER2 overexpression
is seen in BT-474, SKBR3, and MDA-MB453 cell lines in decreasing order, which is
similar to what is reported by others (Cuello et. al., 2001). There are dual bands that are
apparent for the Neu and phospho-Neu which sequencing revealed that the upper band
was a wild-type sequence and the lower band contained a 51-bp in-frame deletion
(Campbell et al., 2002). On the other hand, in three cell lines (MDA-MB468, MDA-
MB436, and MDA-MB231) with relatively low hPRLR levels, the HER2 levels are also relative low or absent. This information suggests the correlation between PRLR and
HER2 systems.
53
BT-474 SKBR3 453 134 483 T-47D MCF-7 468 436 231 Neu
p-Neu
hPRLR
β-actin
Fig. 13. PRLR and HER2/Neu levels in multiple human breast cancer cells. Cell lysates from ten breast cancer cell lines were isolated to determine the expression level of Neu, phospho-Neu and hPRLR levels through western blot analysis. β-actin shows equal loading.
Effect of PRL and combination treatment of G129R and Herceptin on HER2 phosphorylation
BT-474 cells were cultured in the presence of 0.1 or 1µg/ml of PRL for 5, 10, and
14 days (Fig. 14). PRL was able to slightly activate Neu phosphorylation after 5 days.
After 10 days of incubation with PRL, Neu phosphorylation was maximally activated with increasing doses of PRL. After 14 days, Neu remained phosphorylated, in the presence of PRL. In T-47D cells, where constitutive phosphorylated HER2 was non- detectable, PRL is also able to induce HER2 phosphorylation determined by immunoprecipitation assay (Fig. 15). The exogenous PRL was able to induce tyrosine phosphorylation of Neu approximately by 1.5 fold in T-47D cells.
The effect of PRL on the tyrosine phosphorylation status of Jak2 in T47D cells was also examined by immunoprecipitation assay. PRL was able to stimulate tyrosine phosphorylation of Jak2 (Fig. 15), which was similar to what was reported (Yamauchi et al., 2000). 54
To test the effects of combinational treatments of G129R and Herceptin, BT-474 cells were used. There was little change in Neu phosphorylation status in all treatment regimens at 1h, and 8h (Fig. 16); this is probably due to the fact that BT-474 cells have high levels of constitutively phosphorylated Neu. After 24h, the inhibitory effects of
Herceptin on Neu phosphorylation became apparent.
PRL is able to cross-phosphorylate Neu by 52% compared to the control as detected through densitometry after 48h treatment (Fig. 16B). It is interesting to point out that although G129R alone had little effect on hPRL’s stimulatory activity at this time point, it exhibited significant additive effect with Herceptin in inhibition of Neu phosphorylation both in the absence (-66%) or presence (-70%) of PRL compared to
Herceptin alone in the absence (-34%) or presence (-42%) of hPRL.
55
-+ PRL 5 days 0.1 µg/ml 1µg/ml
p-Neu
β-actin
10 days p-Neu
β-actin
14 days
p-Neu
β-actin
Fig. 14. Extended exposure of PRL on Neu phosphorylation in BT-474 cells. BT-474 cells were cultured in the presence of hPRL (0.1µg/ml and 1µg/ml) for 5, 10, or 14 days as indicated. Cells were trypsinized and replaced with fresh media or PRL treatment every three days. Cell lysates (30µg) were electrophoresed and immunoblotted with proper antibodies. Beta-actin was shown as equal loading control.
56
A. IP: Jak2 - + 500ng/ml PRL
WB: 4G10
WB: Jak2
B. IP: Neu
WB: 4G10
WB: Neu
Fig. 15. Effect of PRL on phosphorylation of Jak2 and Neu in T-47D cells. Serum- starved T-47D cells were cultured in the presence of hPRL (500ng/ml) for 20 min. Detergent extracts (1000µg) were immunoprecipitated with anti-Jak2 (A) and anti-Neu (B), respectively. Eluted proteins were analyzed by immunoblotting with 4G10 (upper panels), anti-Jak2 (A, lower panel), and anti-Neu (B, lower panel) as indicated. The experiments shown are representative of three experiments. 57
-PRL + PRL A. BT-474, p-Neu - G H G+H - G H G+H
1 h
8 h
24 h
48 h
β-actin
B. BT-474, p-Neu
52 50 37 l l o o r r 0 1 0 ont ont C C of of -34 -50 % % -42
-66 -70
-100
Fig. 16. Effect of G129R and Herceptin on phosphorylation of HER2 in BT-474 cells. Serum-starved BT-474 cells (A.) were cultured in the presence of G129R (G, 10µg/ml), Herceptin (H, 10µg/ml), or combination of both (G+H) in the presence or absence of hPRL (100ng/ml) for the time indicated. Cell lysates (30µg) were electrophoresed and immunoblotted with p-Neu. The results shown are representative of multiple experiments performed under the same conditions. Beta-actin was shown as equal loading control. B. Densitometric scan of p-Neu on BT-474 cells after 48h; representative of single experiment.
58
Synergistic effect of Herceptin and G129R on inhibition of MAPK phosphorylation
To analyze the synergistic effects of combinational inhibition on MAPK
phosphorylation to G129R and Herceptin treatment, cellular responses after 15, 30, 60, and 90 min. treatments were tested in T-47D cells (Fig. 17A). After 15 min. treatment,
Herceptin activated the basal phosphorylation of MAPK in the BT-474 cells, which is reported in the literature (3). Treatment with hPRL, as expected, induced phosphorylation of MAPK 15 min. through 90 min. G129R had little effect on basal
MAPK activity, but it was able to competitively inhibit hPRL-induced phosphorylation of
MAPK throughout the whole time course. The combination of G129R and Herceptin was able to synergistically inhibit MAPK phosphorylation induced by hPRL as early as
30 min., but the best response was seen after 60 min. Based on densitometry, the combination of G129R and Herceptin at 1h was able to synergistically inhibit MAPK phosphorylation induced by hPRL (-64%) compared to G129R alone (-19%) or Herceptin alone (35%). The synergistic inhibitory effect of G129R and Herceptin on MAPK phosphorylation was prolonged to 48h (data not shown).
We further tested G129R at a low and high dose in combination with Herceptin after 1h treatment (Fig. 17B). Treatment with hPRL induced strong phosphorylation of
MAPK by approximately 62% compared to the control (based on densitometry scanning).
G129R was able to competitively inhibit hPRL-induced phosphorylation of MAPK at a low and high dose. The combination of G129R and Herceptin synergistically inhibited
MAPK phosphorylation induced by hPRL by -33% at G129R's low dose and by -64% at
G129R's high compared to Herceptin alone (2%). The combination of G129R and 59
Herceptin not only competitively inhibited PRL's effects, but together these two drugs
synergistically inhibited MAPK phosphorylation beyond the basal level.
The effects of combinational inhibition on MAPK phosphorylation in BT-474 was
also analyzed after 1 and 48 h (Fig. 18). After 1 h treatment, Herceptin alone activated
the phosphorylation of MAPK in the BT-474 cells as seen in the T-47D cells (Fig. 16A).
Treatment with hPRL and G129R had no effect on MAPK phosphorylation after 1 h.
However, PRL was able to activate MAPK phosphorylation after 48 h compared to the
control, and G129R was able to competitively inhibit hPRL-induced phosphorylation of
MAPK. Based on densitometry (Fig. 18B), the combination of G129R and Herceptin at
48 h was able to synergistically inhibit MAPK phosphorylation induced by hPRL (-86%)
compared to G129R alone (-19%) or Herceptin alone (-75%). The synergistic inhibitory
effect of G129R and Herceptin on MAPK phosphorylation was not as dramatic of an
effect in the BT-474 cells as it was in the T-47D cells most likely due to the level of
hPRLR. 60
A. T- 47D, p- MAPK -PRL + PRL - G H G+H - G H G+H 15 min
30 min
60 min
90 min
MAPK
B. T- 47D
[ +PRL ] [ -PRL ] - PRL G G+H H G H GH 1ug 10ug 1ug 10ug 10ug 10ug 10ug
p- MAPK
MAPK
80 62 33 20 l 30
o 0 2 3
-20 Contr -33 of -70 % -64 -74 -82 -120
Fig. 17. Immunoblot analysis of MAPK activity in T-47D cells after PRL, G129R, Herceptin or in combination treatment. A. T-47D cells were cultured in the presence of G129R (G, 10µg/ml), Herceptin (H, 10µg/ml), or combination of both (G+H) in the presence or absence of hPRL (100ng/ml) at the times indicated (Panel A) or after 1h (Panel B). Cell lysates were prepared as described in Materials and Methods and 30µg of protein were electrophoresed and immunoblotted with proper antibodies as indicated. The results shown are representative of multiple experiments performed under the same conditions. B. Densitometric scan of p-MAPK on T-47D cells after 1h; representative of single experiment. MAPK (panel A&B) was shown as equal loading control. 61
A. BT-474, p-MAPK -PRL + PRL - G H G+H - G H G+H
1 h
48 h
MAPK
B. BT-474
40 20 20 7 0 l l 0 o o r r -2 -20 ont ont C C
-40 of of % % -60
-80 -75 -86 -100 -88 -91
-120
Fig. 18. Immunoblot analysis of MAPK activity in BT-474 after hPRL, G129R, Herceptin or in combination treatment. A. BT-474 cells were cultured in the presence of G129R (G, 10µg/ml), Herceptin (H, 10µg/ml), or combination of both (G+H) in the presence or absence of hPRL (100ng/ml) for 1h (Panel A) or 48h (Panel B). Cell lysates were prepared as described in Materials and Methods and 30-50 µg of protein were electrophoresed and immunoblotted with proper antibodies. The results shown are representative of multiple experiments performed under the same conditions. MAPK was shown as equal loading control. B. Densitometric scan of p-MAPK on BT-474 cells after 48h; representative of single experiment. 62
Combination effects of G129R and Herceptin on other intracellular signaling pathways
To analyze the effects of combinational inhibition on hPRLR and HER2, the phosphorylation status of STAT5a/b, STAT3, and AKT, were also examined. As shown in Fig. 19A, treatment of T-47D cells with hPRL for 1h dramatically induced STAT5a/b and STAT3 phosphorylation. The phosphorylation of STAT5a/b induced by hPRL continued for 48h as seen in Fig. 19B. G129R was able to completely block the stimulatory effects of PRL on STAT activation. As expected, Herceptin had no effect on the inhibition of either STAT5a/b or STAT3 phosphorylation.
Treatment with PRL induced AKT phosphorylation in T-47D after 1h and continued for 48h (Fig. 19A & B, respectively). G129R alone had no effect on Akt phosphorylation, but was able to competitively inhibit the effects in the presence of PRL back down to basal level. Exposure to Herceptin decreased Akt phosphorylation in the absence or presence of PRL. As for the combination effects of G129R and Herceptin seen in the inhibition of Akt phosphorylation, were most likely due to the effect of
Herceptin.
The effects of G129R and Herceptin treatment on STAT5a/b, STAT3, AKT, and
MAPK phosphorylation were repeated in BT-474 cells (high HER2, but low hPRLR, Fig.
20). Despite the fact that BT-474 cells have considerable levels of hPRLR, hPRL had little effect on phosphorylation of STATs. The effects of G129R on AKT phosphorylation were also minimal, whereas the effects of Herceptin were apparent after
1h and remained after 48h. Herceptin was able to inhibit AKT phosphorylation to a greater extent at 48h than at 1h in the absence and presence of hPRL in BT-474 cells. 63
When G129R and Herceptin were combined, results were similar to that of Herceptin alone, showing that the BT-474 is responding only to Herceptin.
64
A. T-47D, 1 h -PRL + PRL - G H G+H - G H G+H
p-STAT5a/b
p-STAT3
p-AKT
β-actin
B. T-47D, 48 h -PRL + PRL - G H G+H - G H G+H
p-STAT5a/b
p-STAT3
p-AKT
β-actin
Fig. 19. Immunoblot analyses of STAT5a/b, STAT3, and AKT in T-47D cells. T- 47D cells were cultured in the presence of G129R (G, 10µg/ml), Herceptin (H, 10µg/ml), or combination of both (G+H) in the presence or absence of hPRL (100ng/ml) as indicated for 1h (Panel A) or 48h (Panel B). Cell lysates (30µg) of protein were electrophoresed and immunoblotted with proper antibodies as indicated. The results shown are representative of multiple experiments performed under the same conditions. Beta-actin was shown as equal loading control.
65
A. BT-474, 1 h -PRL + PRL - G H G+H - G H G+H
p-STAT5a/b
p-STAT3
p-AKT
β-actin
B. BT-474, 48 h -PRL + PRL - G H G+H - G H G+H
p-STAT5a/b
p-STAT3
p-AKT
β-actin
Fig. 20. Immunoblot analyses of STAT5a/b, STAT3, and AKT in BT-474. BT-474 were cultured in the presence of G129R (G, 10µg/ml), Herceptin (H, 10µg/ml), or combination of both (G+H) in the presence or absence of hPRL (100ng/ml) as indicated for 1h (Panel A) or 48h (Panel B). Cell lysates (30µg) of protein were electrophoresed and immunoblotted with proper antibodies as indicated. The results shown are representative of multiple experiments performed under the same conditions. Beta-actin was shown as equal loading control.
66
Effects of PRL and G129R overexpression on HER2 positive cell proliferation
To examine the effects of PRL or its antagonist on cell growth in HER2 and
PRLR positive cell lines, T-47D and BT-474 cells were transfected with PRL or G129R
(Fig. 21). The stable cells were confirmed by RT-PCR and the PCR products were further sequence confirmed for either PRL or G129R (data not shown). Stable cell lines with high expression levels of PRL or G129R were selected using an immunoradiometric assay (IRMA). For T-47D cells, two different clones with different expression levels for
PRL and G129R were tested. T-47D hPRL A expression was approximately 4ng/ml/48h and hPRL B expression 2ng/ml/48h. Whereas G129R A expression was 120ng/ml/48h and G129R B expression was 61ng/ml/48h. BT-474 cells expressed 11ng/ml/48h of hPRL and 41 ng/ml/48h of G129R expression.
Using a cell proliferation assay, stable cells and the vector transfected cells as control was monitored for cell growth over a five day period. Cells over-expressing PRL slightly increased cell proliferation in both T-47D (Fig. 21A) and BT-474 cells (Fig. 21B) depending on the PRL expression level. G129R stable cells were able to inhibit cell growth in both T-47D and BT-474 cell lines. The higher the expression level of G129R in T-47D cells, the more the cell growth was inhibited.
67
A.
350
300 Vector hPRL A (4ng/ml/48h) l 250 o hPRL B (2ng/ml/48h) 200 G129R A (120ng/ml/48h) ontr * G129R B (61ng/ml/48h) C 150 100 * % of * 50 0 12345 -50 Days B. 100 Vector 80 hPRL (11ng/ml/48h)
l G129R (41ng/ml/48h) o 60 ntr
o *
C 40 *
20 % of
0
-20 12345
Days
Fig. 21. Effects of PRL and G129R overexpression in HER positive cell proliferation. T-47D (A) and BT-474 (B) cells were transfected with vector, hPRL or G129R and cell growth was monitored over five days. * p < 0.05 vs control.
68
Effects of PRL, G129R, Herceptin or combinational treatments on anchorage- dependent cell growth
To study the proliferative and anti-proliferative effects of hPRL, G129R and
Herceptin, cells were treated with a wide dosing range to determine the EC50 or IC50 of
each agent. T-47D breast cancer cells were treated with hPRL for 48 h demonstrating a
bell shaped curve where 100ng of hPRL was a maximum dose (Fig. 22A). T-47D cells
were also treated with a range of doses (0.5µg-50µg) of G129R or Herceptin. Both
agents inhibited cell proliferation in a dose dependent manner (Fig. 22 B&C) with their
IC50 at approximately 10µg/ml, which was selected for combination studies. 69
A.
50 hPRL 40 l o r
t 30 n
o 20 C
f 10 % o 0 0.025 0.05 0.1 0.25 0.5 1
B.
0 l -5 o r nt
o -10 C
-15 of
% G129R -20 0.515102050 C.
0 l o
r -5 ont C -10 of
% Herceptin -15 0.515102050 [ug/mL]
Fig. 22. Dose response of the treatment of PRL, G129R and Herceptin in T-47D cells. T-47D cells were treated with increasing doses of hPRL (Panel A), G129R (Panel B), and Herceptin (Panel C) respectively for 48 h. After continuous exposure to each agent, MT-PMS colorimetric growth assay was performed. Results were expressed as % of control (cells without treatment) in each experiment. The mean value and SD of the mean value of three or more wells in at least three experiments was reported. 70
To study the anti-proliferative effects of 10µg/ml G129R, 10µg/ml Herceptin, or the combination of both agents with or without hPRL (100ng/ml), T-47D (Fig. 23A) and
BT-474 (Fig. 23B) cells were cultured continuously for 48 h with treatment. G129R or
Herceptin alone exhibited an inhibition on cell growth, whereas the combination of the two had a slight additive affect both in the presence or absence of hPRL in both cell lines.
In T-47D cells, G129R inhibited cell proliferation approximately -5%, Herceptin inhibited -16% (p<0.01) and the combination demonstrated an additive effect by inhibiting cell growth by -23% (p<0.01) compared to the control. However, when T-47D cells were cultured in the presence of 100ng/ml of hPRL, cell viability was increased to
+8%. The stimulatory effect of hPRL was reversed with G129R down to -1%, Herceptin to -10%. The combination of both agents significantly inhibited the rate of proliferation to -13% (p<0.01) compared to that of the control level.
Herceptin was more effective in the inhibition of the proliferation of BT-474 cells
(-33%) (p<0.01) as compared to that of G129R’s (-5%), and the combination approach slightly increased to -38% (p<0.01) compared to the control due to the basal level of
HER2. This trend of response is also observed when BT-474 cells were cultured in the presence of hPRL, the inhibitory effect of Herceptin alone was -40% (p<0.01); whereas
G129R alone reached -11%; the combination of both had approximately -47% (p<0.01) inhibition of cell proliferation compared to the control. 71
-PRL + PRL A. - G H G+H - G H G+H 20
10 l o 0
Contr -10 of -20 % ** ** -30 ** -40
B. -PRL + PRL - G H G+H - G H G+H 20
10
0 l
o -10
-20
Contr -30
of -40 % ** -50 ** ** ** -60
Fig. 23. Growth response of T-47D and BT-474 cells to G129R, Herceptin or combinational treatments. T-47D (Panel A) and BT-474 cells (Panel B) were incubated with G129R (10µg/ml), Herceptin (10µg/ml), or combination of G129R and Herceptin in the absence or presence of hPRL (100ng/ml) for 48 h. After continuous exposure to treatment, MT-PMS colorimetric growth assay was performed. Results were expressed as a percentage of control (cells without treatment) in each experiment. The mean value and SD of the mean value of three or more wells in at least three experiments was reported. ** p < 0.01 vs control.
72
Effects of G129R and Herceptin on growth of T-47D and BT-474 human breast cancer xenografts in nude mice
To determine the effects of G129R and Herceptin combinational treatment on
tumor growth, T-47D and BT-474 cells were inoculated into the mammary fat pad of female nude mice. One week after tumor cell inoculation, purified G129R
(10mg/kg/daily) or Herceptin (10mg/kg/bi-weekly), or a combination of the two were intraperitoneal injected for at least 6 weeks.
After six-weeks of treatments, Herceptin was able to inhibit T-47D tumor growth
by 46%, whereas G129R inhibited tumor growth by 53.3% (p<0.05). The best response
was observed in the combination treatment group, in which the tumor growth was
significantly inhibited by as much as 76.5% (p<0.001) compared to the control group
(Fig. 24A). Similar results were apparent in the tumor weights when mice were
euthanized at the end of the study (Fig. 24B).
The results from the experiments conducted using BT-474 cells were similar to
that of from T-47D cells. Herceptin was able to inhibit BT-474 tumor growth by 62%
(p<0.05), whereas G129R inhibited tumor growth by 65% (p<0.05). The combination
treatment group inhibited tumor growth to a greater extent than single agents alone
(79.1%, p<0.01) (Fig. 25B).
73 ) 3 m
A. m (
e 400
m Control u l
o G129R 300 Herceptin r V G129R & Herceptin mo 200 u T
e * v i
t 100
a * l **
mu 0
Cu 3 10 17 24 31 45 Days
B. 225 )
g 200
(m 175
150 ht g i 125 e
W 100 *
r 75 o 50 **
Tum 25 0 Control G129R Herceptin G129R & Herceptin
Fig. 24. Effect of G129R and Herceptin on T-47D xenografts in nude mice. A. T- 47D cells (7 x106) were injected in the mammary fat pad of female nude mice. One week after tumor cell inoculation, purified G129R (10mg/kg/daily) or Herceptin (10mg/kg/bi- weekly), or a combination of both were injected i.p. for 6 weeks. Measurements of tumor growth were recorded once a week. B. Effects of G129R and Herceptin on the tumor weight of T-47D breast cancer xenografts in nude mice. Each value is a mean ± SD of 6- 8 mice/group. ** p < 0.01 and * p < 0.05 vs control.
74
A. ) 3 m 300 Control e (m G129R Herceptin olum 200
V G129R & Herceptin r o m
u 100 * *
ve T * ** ti a
l 0 u 0 10 20 30 40 50 Days Cum
B.
125 ) g
(m 100 ht
g 75 Wei
50 r o 25 Tum
0 Control G129R Herceptin G129R & Herceptin Fig. 25. Effect of G129R and Herceptin on BT-474 xenografts in nude mice. A. BT- 474 cells (7 x106) were injected in the mammary fat pad of female nude mice. One week after tumor cell inoculation, purified G129R (10mg/kg/daily) or Herceptin (10mg/kg/bi- weekly), or a combination of both were injected i.p. for 6 weeks. Measurements of tumor growth were recorded once a week. B. Effects of G129R and Herceptin on the tumor weight of BT-474 breast cancer xenografts in nude mice. Each value is a mean ± SD of 6- 8 mice/group. ** p < 0.01 and * p < 0.05 vs control.
75
Generation of PRL or G129R and HER2 bitransgenic mouse models
The MT- or MMTV-hPRL or MT- or MMTV-G129R single transgenic mice and
MT-hPRL/neu; MT-G129R/neu; MMTV-hPRL/neu; and MMTV-G129R/neu bitransgenic mice were confirmed by transgene expression (Fig. 26). Expression of the transgenes was confirmed by performing PCR on genomic DNA isolated from tail biopsies and by RT-PCR using RNA extracted from mammary tissues of the mice. All bitransgenic offspring were positive for the neu transgene and transcript and approximately 50% were positive for either hPRL or G129R (Fig. 26A&B). Based on
RT-PCR results, it does not appear that there is an interaction between any two pairs of transgenes; transcript levels were not affected by co-expression of Neu and hPRL or
G129R (Fig. 26B).
Expression of the MT-hPRL and MT-G129R transgenes were confirmed by measuring the levels of hPRL and G129R in the serum of the bitransgenic mice using an hPRL IRMA. Approximately 5-10 ng/ml of hPRL and G129R was detected in the serum of the bitransgenic mice, but was undetectable in the serum of the MMTV group.
Expression of the MT and MMTV hPRL transgenes were confirmed by measuring the levels of hPRL in the mammary gland tissue homogenates using a PRL ELISA kit.
Human PRL expression of the MT-hPRL/neu and the MMTV-hPRL/neu was approximately 78 ± 50.2 and 341 ± 237.0 pg/mg protein respectively in the mammary gland tissue homogenates (Fig. 27). 76
A. hPRL
neu
G3PDH
B. hPRL
neu
G3PDH
u e u e e u e B L R L R e u t e t u t t 9 9 e a a e a e a V R R 2 n n /n n n F P 2 P - / m m / m / m 1 1 V r R R -h -h LL/ e r L r r G -G T R t 9 e e 9 e T - V t 2 tt R tt 2 tt T V M PPR li 1 i P i 1 i M T h l h l l M T - u G - G M M T e - u u - u M T e V e V e M M n n T n n M M T M M M M
Fig. 26. Confirmation of transgenic mice. Transgene expression was confirmed in single and bitransgenic mice by (A) PCR and (B) RT-PCR using primers to amplify hPRL, G129R and neu sequences. (A) MT and hPRL primers (1002 bp), MMTV and hPRL primers (1203 bp) and neu primers (622 bp) were used for PCR to confirm the presence of the transgenes in the DNA; representative of n=2-3 mice. (B) hPRL and neu primers were used for RT-PCR to confirm the transcriptional expression of hPRL and neu in the mammary gland; representative of n=2-3 mice. G3PDH was used as an internal control for RNA quality and gel loading. Primers displayed in Table C1.
77
700 )
g 600 m / g
p 500 ( n o i
t 400 a r t 300 ncen 200 co L
R 100 hP
0 MT-hPRL/neu MMTV-hPRL/neu
Fig. 27. Expression of PRL in mammary gland tissue in PRL bitransgenic mice. Approximately 100 mg of fresh mammary gland tissue isolated from the MT-hPRL/neu and the MMTV-hPRL/neu bitransgenic lines and their neu littermates were homogenized and assayed for hPRL expression using the Active Prolactin ELISA kit. The neu littermates from both lines were averaged and subtracted out as background.
78
Mammary tumor incidence in bitransgenic mice
To investigate the effect of hPRL and its antagonist, G129R, on Neu-induced
mammary tumorigenesis, the four lines of bitransgenic mice that co-express neu under
the MMTV promoter and hPRL or G129R under transcriptional control of either the MT or MMTV promoter were monitored for palpable tumors. The total tumor incidence in the MT-hPRL/neu line (30/56, t50=438 days, p<0.005) was significantly lower than their neu littermates (34/37, t50=345 days) through 500 days (Fig. 28A). The total tumor incidence for the MMTV-hPRL/neu mice were even more drastically reduced (Fig. 28C)
with only 2 of 23 developing tumors (p<0.0001) at 385 days of age in comparison to their
littermates (32/35, t50=215 days). In contrast, co-expression of G129R with either promoter in neu female mice had little effect on overall tumor incidence (Fig. 28B): MT-
G129R/neu mice (49/53, t50=323 days) as well as in (Fig. 28D): MMTV-G129R/neu
mice (33/36, t50=241 days).
79
A. B. MT-hPRL/neu MT-G129R/neu 100 n=56 100 )
% n=53 ( 80 t50 = 438 80 t50 = 323 ee 60 60
Fr neu littermates neu littermates r o 40 n=37 40 n=29 m u
T t = 345 t = 370 20 50 20 50
0 0 150 200 250 300 350 400 450 500 150 200 250 300 350 400 450 500
Age (Days) Age (Days)
C. D.
100 100
) MMTV-G129R/neu 80 MMTV-hPRL/neu 80 (%
e n=36
e n=23
r 60 60 t = 241 neu littermates 50 r F neu littermates o 40 40
m n=20
u n=35 T 20 20 t50 = 233 t50 = 215 0 0 120 170 220 270 320 370 120 170 220 270 320 370 Age (Days) Age (Days)
Fig. 28. Effects of PRL or G129R co-expression on Neu-induced tumorigenesis. The data are plotted as the percentage of tumor-free animals versus age in days. All hPRL and G129R bitransgenic mice are represented by closed circles (●), and all neu littermates are represented by open circles (○). (A) MT-PRL/neu mice versus neu littermates. Mann- Whitney U test, p<0.005. (B) MT-G129R/neu mice versus neu littermates. p=0.19, n.s. (C) MMTV-PRL/neu mice versus neu littermates. p<0.0001. (D) MMTV-neu/G129R mice versus neu littermates. p=0.71, n.s.
80
HER2/Neu gene expression levels in the mammary gland
Because the transforming potential of the Neu protein is closely correlated with its intrinsic tyrosine kinase activity, we were interested in examining Neu tyrosine kinase activity and Neu expression levels in the mammary gland derived from the tumor resistant line MMTV-PRL/neu mice in comparison to their neu littermates (Fig. 29). We found that Neu expression in MMTV-PRL/neu mice was significantly altered. From 3 months (p=0.09) up to 6 month of age (p<0.001), MMTV-PRL/neu bitransgenic mice demonstrate non-detectable or significantly reduced levels of Neu gene expression as compared to their neu littermates. However, this difference is not obvious at phosphorylated Neu levels among animals of the same genotype in three or six months due to the large variation.
81
A. neu MMTV-hPRL/neu
#1 #2 #3 #4 #1 #2 #3 #4 pNeu 3M Neu
β-tubulin
6M pNeu
Neu
β-tubulin
B. C. 3.5 2.5 3 2 2.5 nge nge a a
h 1.5
2 h c c
d 1.5 d 1 1 Fol Fol 0.5 0.5 ** 0 0 3M 6M 3M 6M
Fig. 29. HER2/neu gene expression in mammary glands of PRL bitransgenics and their neu littermates. Immunoblot analyses of neu littermates versus MMTV-hPRL/neu mammary gland tissue isolated from three and six month old mice probed with the indicated antibodies. β-tubulin was used as the loading control. Panel B and C, Graphical representation of fold change of phospho Neu and Neu, respectively, based on densitometric scans of bands (representative of n=4 mice per group). Means +/- SD are presented and differences were evaluated using a two-tailed Student t-test. ** p < 0.001.
82
Molecular events in PRL bitransgenic mice
To determine the cellular events underlying the interaction between the transgenes or responsible for the tumor resistance in PRL bitransgenics, we examined the signaling molecules known to link to Her2 related tumorigenesis in the mammary gland.
At three and six months of age, the MT-hPRL/neu mice displayed significantly lower cyclin D1 levels (p<0.05) than their neu littermates (Fig. 30A). Two distinct molecular weight bands were observed for cyclin D1 as observed in other studies (19), with the higher molecular mass band corresponding to phosphorylated cyclin D1. Based on densitometry, cyclin D1 levels of three and six month old MT-PRL/neu mice were reduced approximately 20% and 40%, respectively (Fig. 30C).
The MMTV-PRL bitransgenic mice, on the other hand, exhibited lower cyclin
D1 levels earlier than the MT-PRL bitransgenic mice (Fig. 31). The MMTV-hPRL/neu mice reduced cyclin D1 levels by more than 50% at one month (p<0.001) and by approximately 40% at three months (p<0.05) compared to their neu littermates. By six months of age, the cyclin D1 levels of the MMTV-hPRL/neu mice were not significantly different from their neu littermates.
Differences were also seen in the MAPK activity between the MT-hPRL/neu mice and their neu littermates. One month old hPRL/neu mice demonstrated a slight decrease in MAPK phosphorylation and by three months of age, MAPK phosphorylation was significantly decreased by more than half (p<0.05) compared to their littermates (Fig.
30, B&D). By six months of age, MAPK phosphorylation levels were indistinguishable between the two groups. 83
Similar differences in the MAPK activity were also seen the MMTV-hPRL/neu mice and their neu littermates. There was slightly lower levels of MAPK in one month old MMTV-hPRL/neu mice (Fig. 31B), but significantly lower phosphorylated MAPK levels was observed at three months old (p<0.05). By six months of age, the difference was not obvious between these two groups. For both lines of PRL/neu bitransgenic mice, the decrease in cyclin D1 and MAPK levels was prominent at three month of age.
No detectable differences were found in the phosphorylation or basal levels of
Akt (Fig. 32A) and STAT5a/b (Fig. 32B) pathways at any age of the MT-PRL/neu mice compared to the neu littermates. There were also no measurable differences seen for Akt phosphorylation or expression in the MMTV-PRL/neu mice (Fig. 32C). As for the STAT signaling in the MMTV-PRL/neu, there were however slight differences seen in the basal and phosphorylated levels of STAT5a/b signaling (Fig. 32D). At one and three months of age, MMTV-PRL/neu mice demonstrated lower levels of STAT5 expression, but were not significantly different based on densitometry (data not shown). Also at three months of age, MMTV-PRL/neu mice induced higher levels of STAT5 phosphorylation, but were not significantly different from their neu littermates (p=0.08).
84
A. B. neu MT-hPRL/neu neu MT-hPRL/neu
# 1 #2 #1 #2 # 1 #2 #1 #2 Cyclin D1 p-MAPK 1M β-tubulin MAPK
Cyclin D1 P-MAPK 3M β-tubulin MAPK
Cyclin D1 p-MAPK 6M β-tubulin MAPK
C. D.
1.4 1.4 e g nge
n 1 a 1 a * h * h c c d
* d 0.6 l 0.6 Fol * Fo 0.2 0.2
1 M 3 M 6 M 1 M 3 M 6 M
Fig. 30. Cyclin D1 and phospho-MAPK levels in MT-hPRL/neu mice. Immunoblot of: (A) cyclin D1 (32/36 kD) and (B) phosphorylated MAPK (42/44 kD) in the mammary glands of MT-hPRL/neu mice and their neu littermates at 1, 3, and 6 months of age. Densitometric analyses of (C) cyclin D1 and (D) phosphorylated MAPK in the mammary glands of MT-hPRL/neu versus neu littermates at 1, 3, and 6 months of age. Graphical representation of fold change based on densitometric scans of bands from mammary gland tissue (representative of n=4-6 mice per group). β-tubulin was used as the loading control. Means +/- SD are presented and differences were evaluated using a two-tailed Student t-test. * p < 0.05.
85
A. B. neu MMTV-hPRL/neu neu MMTV-hPRL/neu
# 1 #2 #1 #2 # 1 #2 #1 #2 Cyclin D1 p-MAPK 1M β-tubulin MAPK
Cyclin D1 P-MAPK 3M β-tubulin MAPK
Cyclin D1 p-MAPK 6M β-tubulin MAPK
C. D.
1.6 2
e 1.6 1.2 g nge n a a h * h 1.2 c c
d 0.8 d ** l 0.8 ** Fol Fo 0.4 0.4
0 0 1M 3M 6M 1M 3M 6M
Fig. 31. Cyclin D1 and phospho-MAPK levels in MMTV-hPRL/neu mice. Immunoblot of: (A) cyclin D1 (32/36 kD) and (B) phosphorylated MAPK (42/44 kD) in the mammary glands of MMTV-hPRL/neu mice and their neu littermates at 1, 3, and 6 months of age. Densitometric analyses of (C) cyclin D1 and (D) phosphorylated MAPK in the mammary glands of MMTV-hPRL/neu mice versus neu littermates at 1, 3, and 6 months of age. Graphical representation of fold change based on densitometric scans of bands from mammary gland tissue (representative of n=4-6 mice per group). β-tubulin was used as the loading control. Means +/- SD are presented and differences were evaluated using a two-tailed Student t-test. * p < 0.05; * p < 0.001.
86
A. B. neu MT-hPRL/neu neu MT-hPRL/neu
#1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 P-AKT p-STAT5 1M AKT STAT5
P-AKT p-STAT5 3M AKT STAT5
P-AKT p-STAT5 6M AKT STAT5
A. B. neu MT-hPRL/neu neu MT-hPRL/neu
#1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 P-AKT p-STAT5 1M AKT STAT5
P-AKT p-STAT5 3M AKT STAT5
P-AKT p-STAT5 6M AKT STAT5
Fig. 32. AKT and STAT levels in hPRL/neu mice. Immunoblot of: (A&C) phosphorylated Akt (60 kD) and (B&D) phosphorylated Stat5a/b (97 kD) in the mammary glands of MT-hPRL/neu (A&B) and MMTV-hPRL/neu (C&D) mice respectively and their neu littermates at 1, 3, and 6 months of age. (representative of n=4-6 mice per group).
87
Morphological comparison of the mammary gland of PRL bitransgenic mice and their littermates
To determine the effects of hPRL on mammary gland development, we analyzed
mammary gland whole mounts obtained from the hPRL/neu mice and their neu
littermates. At one month of age, the MT-PRL/neu mice displayed a significant increase
in bud formation (p<0.001) relative to the neu littermates (Fig. 33H), whereas the
MMTV-PRL/neu mice displayed a significant decrease in bud formation (Fig. 34H;
p<0.05). However, there is no obvious difference in mammary ductal side branching and
the number of side branches in the MT (Fig. 33) and MMTV-PRL (Fig. 34) bitransgenics
as compared to their littermates. TEBs were clearly visible at the tips of the mammary
ducts in both groups indicating active ductal elongation.
By three months of age, the lobulo-alveolar structures in the MT-PRL/neu mice
were developing, and TEBs were much less apparent, indicating a reduction in major
ductal elongation. The mammary ducts had extended throughout the fat pad and become
well organized (Fig. 33B&E). The primary ducts of the MT-hPRL/neu mice contained
approximately 4.4 side branches per mm2 (Fig. 33G), representing a significant increase in the level of secondary and tertiary branching (p<0.05) in comparison to their neu littermates. A significant increase in the lobulo-alveolar structures was also observed in the MT-hPRL/neu mice (p<0.001) in comparison to their littermates (Fig. 33H).
At three months of age, the MMTV-PRL/neu mice demonstrated a phenotype
opposite to what was represented from the MT-PRL/neu mice. The primary ducts of the
MMTV-PRL/neu mice contained approximately 3.6 side branches per mm2 (Fig. 34E), representing a significant decrease in the level of secondary and tertiary branching 88
(p<0.05). MMTV-PRL/neu mice also have significantly less lobulo-alveolar structures
(p<0.001) in comparison to their littermates (Fig. 34H).
By six months of age, extensive lobulo-alveolar development had occurred in the
MT-hPRL/neu mice. At this stage, the mammary ducts of several MT-hPRL/neu mice
displayed secretory lobulo-alveolar products in their lumen, superficially resembling
lactation (data not shown). The MT-hPRL/neu mice displayed a significant increase in
numbers of lobulo-alveolar and TEB structures (Fig. 33H; p<0.001), however the number
of branches per duct did not significantly differ from their littermates, and had slightly
decreased from the age of three months (Fig. 33G). Simultaneously, the extent of alveolar
development was significantly decreased in the MMTV-PRL/neu bitransgenics compared
to their neu littermates by six months of age (Fig. 34H; p<0.001). This is in contrast to
the effects seen in the MT-PRL/neu bitransgenic mice, in which there was a significant
increase in lobular alveolar development relative to littermates. As for the neu littermates
for both lines of transgenic mice, they displayed very similar results in respect to the total
number of side branches and lobulo-alveolar development.
89
neu MT-hPRL /neu A. D.
1M 1M B. E.
3M 3M C. F.
6M 6M
G. 2
m H. 6 20 m
/ 5 2 ** s * m 16 e m
4 / ** 12 anch 3 es br 8
e ** obul
d 2 l f si f 1 4 # o # o 0 0 1M 3 M 6 M 1M 3 M 6 M
Fig. 33. Mammary glands of MT-hPRL/neu female mice. The fourth inguinal mammary glands were dissected, stained in carmine alum stain and digitally photographed. Panels A-F are the mammary glands of neu littermates at 1, 3 and 6 month of age (A to C) versus age-matched hPRL/neu mice (D, E, F, respectively, representative of n=6 mice/group). Quantification of morphological features of 1, 3 and 6 month old neu littermates versus hPRL/neu mice was compared either by total number of side branches per mm2 (panel G) or total number of TEB and lobulo-alveoli per mm2 (panel H). Means +/- SE are presented (n=6 per group) and differences were evaluated using a two-tailed Student t-test. *, p < 0.05; **, p <0.001
90
neu MMTV-hPRL /neu A. D.
1M 1M B. E.
3M 3M C. F.
6M 6M
G. H. 2 ^ 7 2 16 ^ m m m
6 m 5 r 12 pe es per
4 * s e ch 8
an 3
r ** b obul
l
2 e 4 d
of * ** #
si 1 f 0 0 # o 1M 3M 6M 1M 3M 6M
Fig. 34. Mammary glands of MMTV-hPRL/neu female mice. The fourth inguinal mammary glands were dissected, stained in carmine alum stain and digitally photographed. Panels A-F are the mammary glands of neu littermates at 1, 3 and 6 month of age (A to C) versus age-matched hPRL/neu mice (D, E, F, respectively, representative of n=6 mice/group). Quantification of morphological features of 1, 3 and 6 month old neu littermates versus hPRL/neu mice was compared either by total number of side branches per mm2 (panel G) or total number of TEB and lobulo-alveoli per mm2 (panel H). Means +/- SE are presented (n=6 per group) and differences were evaluated using a two-tailed Student t-test. *, p < 0.05; **, p <0.001.
91
Proliferation indices in PRL bitransgenic mice
Reduced activation of extracellular signal-regulated kinases is indicative of reduced cellular proliferation and transformation. To examine proliferation we immunohistochemically detected the Ki67 marker in the mammary glands of mice at 1, 3 and 6 months of age in the MT-PRL/neu and their littermates. The levels of Ki67 in three month old MT-hPRL/neu mice had a significantly lower H-score than their littermates
(Fig. 35B). However, both the one month and six month old hPRL/neu mice were not significantly different from their littermates (data not shown). Obvious differences were apparent in acinar and lobulo-alveolar features between hPRL/neu mice and their littermates (Fig. 35A). The neu littermates had a more disorganized appearance and appeared to have lost the normal polar organization of basal and luminal epithelial cells as evidenced by the smaller luminal spaces. On the other hand, the MT-hPRL/neu mice had larger luminal spaces evident of less proliferating cells (Fig. 35A). 92
A. neu MT-hPRL/neu
B. 1.2 e r 1 o
c ** S
- 0.8
0.6
Ki67 H 0.4
0.2
0 neu MT-hPRL/neu
Fig. 35. Proliferation indices in MT-hPRL/neu mice and their neu littermates. (A) Ki67 detection in paraffin embedded mammary glands from 3 month old neu littermates (left panel) and MT-hPRL/neu mice (right panel). Arrowhead indicates brown nuclear staining. (B) Graphical representation of Ki67 H scores of neu littermates (n=4) versus MT-hPRL/neu mice (n=4). **, p < 0.001. H scores were calculated as the summation of the proportion of nuclei staining in each category. Means +/- SE are presented.
93
Neu phosphorylation levels in the tumors.
It was reported that the neu transgene is expressed in normal mammary epithelium, salivary gland and lung and higher expression was detected in tumor tissue.
Because the transforming potential of the c-Neu protein is closely correlated with its intrinsic tyrosine kinase activity, we were interested in examining Neu tyrosine kinase activity in tumor tissue samples derived from the hPRL/neu mice in comparison to their neu littermates. A prominent band representing phosphorylated Neu was observed in immunoblots of tumor extracts from neu littermates at 9 month (Fig. 36A&B). In contrast, no comparable phosphorylation of Neu was detected in the tumors that did develop of the MT- and MMTV-PRL/neu mice. Similarly, the tumor tissues of the neu littermates reacted to the phospho-Neu antibody through IHC analyses, but the tumor tissues of the PRL/neu mice demonstrated very little reactivity (Fig. 36C).
94
A. neu MT-hPRL/neu # 1 #2 #1 #2 pNeu Neu 9M (tumor) β-tubulin
B. neu MMTV-hPRL/neu # 1 #2 #1 #2
pNeu
Neu 9M (tumor)
β-tubulin
C.
Fig. 36. HER2/Neu phosphorylation levels in tumors, which arise in the PRL bitransgenic mice and their neu littermates. Panel A, immunoblot analyses of neu versus MT-hPRL/neu tumor tissue isolated from 9 month old mice probed with the indicated antibodies. Panel B, immunoblot analyses of neu versus MMTV-hPRL/neu tumor tissue isolated from 9 month old mice probed with the indicated antibodies. β- tubulin was used as the loading control. Panel C, Phosphorylated Neu IHC analyses from paraffin embedded tumor tissue from neu littermate (left panel) and MT- hPRL/neu mice (right panel).
95
CHAPTER FOUR
DISCUSSION
Prolactin in transformed cells acts as a tumor promoter
One of the most studied female hormones involved in mammary gland biology is
PRL. PRL is a polypeptide hormone intimately involved in the regulation of normal breast growth, development, and differentiation (Vonderhaar, 1999; Kelly et al., 2002).
The role of hPRL in breast cancer is still controversial, but most recent studies favor its role as a pathological agent in breast cancer with the following evidence. Studies suggest that hPRL acting as an autocrine/paracrine growth factor may be involved in breast cancer initiation/development (Ginsburg and Vonderhaar, 1995; Kelly et al., 2002;
Clevenger et al., 2003). The importance of hPRL in mammary tumor is supported by evidence that: hPRL is produced locally by breast cancer cells (Ginsburg and
Vonderhaar, 1995; Clevenger and Plank, 1997); hPRL receptor expression is up- regulated in breast cancers (Reynolds et al., 1997; Touraine et al., 1998); and hPRL acts as an anti-apoptotic agent whereas anti-hPRL agents such as the hPRL receptor antagonist, G129R, induce apoptosis of the breast cancer cells (Chen et al., 1999).
The human epidermal growth factor receptor 2 (HER2), also known as ErbB2 receptor tyrosine kinase, plays an important role in human malignancies (Lee et al.,
2002). Amplification of the HER2 gene with overexpression of HER2 protein occurs in
20-30% of primary human invasive breast carcinomas and is correlated with poor prognosis (DiGiovanna et al., 2002; Argiris et al., 2004; McKenzie et al., 2004; Witters et al., 1997; Merlin et al., 2002; Gilbert et al., 2003; Lu et al., 2004). The finding that 96
HER2 promotes aggressive breast cancer led to the first rationally designed cancer therapeutic, Herceptin (trastuzumab), a recombinant humanized monoclonal antibody directed against the HER2 extracellular domain (Nahta et al., 2004). Treatment with
Herceptin results in down regulation of receptor expression, internalization of the
antibody-HER2 complex and a decrease in both the association of HER2 with its
heterodimeric partners and HER2 tyrosine phosphorylation (Ropero et al., 2004), inducing regression of HER2 overexpressing breast cancers. It has been reported that autocrine/paracrine secretion of hPRL in breast cancer is able to phosphorylate HER2 through activation of JAK2 signaling (Yamauchi et al., 2000). Clinical data further demonstrates a strong correlation between hPRL expression and proliferative and metastatic activity in HER2 positive breast cancer patients (Yamauchi et al., 2000).
Since the oncogenic potential of HER2 largely depends on the state of its tyrosine phosphorylation, the finding of co-expression and cross-phosphorylation of HER2 via hPRL leads to the notion that in order to effectively block the oncogenic effects of HER2, inhibition of the hPRLR should be considered.
Therefore, to effectively block PRL’s effects as a tumor promoter on HER2 signaling and its ability to cross-activate HER2, we investigated the potential benefit of a combinational therapeutic approach using an anti-hPRLR (G129R) and an anti-HER2
(Herceptin) for HER2 positive breast cancer as presented in the first section of this dissertation. Two representative breast cancer cell lines, T-47D (high hPRLR, low
HER2) and BT-474 (high HER2, low hPRLR), were selected as study models. We were able to show that PRL was able to stimulate tyrosine phosphorylation of Jak2 (Fig. 15) in agreement with Yamauchi et al., 2000. PRL was also able to cross-phosphorylate HER2 97
in T-47D cells and BT-474 cells (Fig. 14 & 15, respectively). Since the oncogenic
potential of HER2 largely depends on the state of its tyrosine phosphorylation, the
finding of co-expression and cross-phosphorylation of HER2 via hPRL suggests PRL’s
role as a tumor promoter in HER2/neu tumorigenesis. By inhibiting the hPRLR to more
effectively block HER2 activation, we were able to demonstrate that combination of
G129R and Herceptin treatment was able to further inhibit the phosphorylation of HER2
better than that of using Herceptin alone (Fig. 16). This combined effect of G129R and
Herceptin demonstrated on inhibition of HER2 phosphorylation may be clinically
important since the tumorigenic potential is directly linked to the phosphorylation status of HER2. Our finding of Herceptin’s activation of the phosphorylation of MAPK in T-
47D (Fig. 17A) and BT-474 cells (Fig. 18A) is consistent with what has been reported regarding Herceptin in the literature (Argiris et al., 2004), which demonstrated Herceptin as being a partial HER2 agonist.
PRL’s role in mitogenesis was also evident when we demonstrated that hPRL
treatment induced MAP kinase phosphorylation in both T-47D (Fig. 17) and BT-474
cells (Fig. 18). This PRL induced activation of MAPK activity was attenuated by the
addition of G129R similar to what has been reported (Llovera et al., 2000). Since PRL induced phosphorylation of MAPK and MAPK is also a signaling molecule for HER2, we tested the effect between G129R and Herceptin. The combination treatment of
G129R and Herceptin synergistically reduced levels of hPRL induced MAPK phosphorylation to a greater degree as compared to either agent alone, suggesting cross talk between the hPRLR and HER2 pathways was mainly through regulation of MAPK.
PRL was shown to induce MAPK activation in both cases of high and low PRLR level 98
cell lines and using an anti-PRL and anti-HER2 therapeutic can cooperatively work
together in inhibiting the stimulation effects of PRL on MAPK phosphorylation.
The phosphorylation status of STAT5a/b, STAT3, and AKT were also used as
indications of cellular responses to PRL. Related to STAT signaling, we were able to demonstrate that both STAT5a/b and STAT3 was only responsive to hPRL and G129R as previously reported (Cataldo et al., 2000), whereas Herceptin had no effect in STAT signaling activities in T-47D cells (Fig. 19). Both STAT3 and STAT5 are involved in
PRL activation of the cyclin D1 promoter, suggesting at least one target of PRL through this pathway could contribute to tumorigenesis (Clevenger et al., 2003). In BT-474 cells,
however, there was no STAT response to PRL or G129R treatment, suggesting the
phosphorylated STATs were somehow dissociated from hPRLR regulation. Levels and
activities of the STATs are altered by multiple hormones, growth factors, and signaling
cascades, pointing to an obvious role they may play in cross-talk with many other agents important in mammary carcinogenesis (Clevenger et al., 2003). Another signaling pathway which is activated by HER2 is the phosphatidylinositol 3-kinase (PI3K)/AKT cascade (Nahta et al., 2004). Despite the fact that both T-47D and BT-474 cells exhibited a constitutively phosphorylated AKT, hPRL was able to further stimulate the phosphorylation status to a certain degree (Fig. 19 & 20). Phosphorylation of AKT was significantly inhibited with the treatment of Herceptin, whereas G129R had minimal effects in AKT phosphorylation. Based on the use of a hPRL antagonist, G129R, in combination with anti-HER2, it has been shown that MAPK, JAK/STAT, and AKT pathways play a critical role in PRL-induced proliferation of mammary epithelial cells. 99
Through the production of stable cells (Fig. 21), we demonstrated that
overexpression of hPRL increased cell proliferation implicating hPRL's activity as a
tumor promoter in both high and low HER2 expression level cell lines. The notion that
PRL acts as a mitogenic cytokine is supported by the suppression of breast cancer cell
proliferation with a PRL antagonist, G129R. Overexpression of G129R suppressed or
retarded cell growth, blocking the proliferative action of endogenous PRL in HER2
driven human breast cancer cells. This data supports the idea that PRL plays an
autocrine-paracrine proliferative role in the mammary gland and thereby may be involved
in breast tumor progression.
The breast cancer cells were also treated with increasing doses of hPRL. The
response of the cells exhibited a bell shape curve (Fig. 22) reflecting the sequential dimerization mechanism/ self-antagonism for activation as what has been reported (Fuh and Wells, 1995). Both T-47D and BT-474 cells stimulated breast cancer cell growth in the presence of PRL and marginally inhibited cell growth in the presence of G129R (Fig.
23). T-47D breast cancer cells showed significant resistance to either G129R or
Herceptin alone with the maximum inhibition response at less than 20%. In both cell
lines, an additive inhibitory effect was observed by the combination of G129R and
Herceptin, insisting that the combination of the two agents were better than single agent
alone. One possible explanation for the somewhat refractory responses from these two
cell lines in terms of proliferation compared to the signaling may be due to the fact that
these selected cells express extremely high levels of at least one receptor type, which
imposes a significant challenge for complete blockage of the surface receptors. T-47D
cells have more hPRL receptors than other cells lines and to antagonize their growth 100
would require that the antagonist occupy many more receptors at a time (Fuh and Wells,
1995). Previous experiments suggest that only a minimal activation of the hPRL
receptors was needed to signal a maximal response (Ashkenazi et al, 1987; Fuh et al.,
1993). This observation may also be true for HER2 activation. However, this argument was weakened when the G129R or Herceptin dose reached beyond physiological concentrations (50ug/ml) and the inhibitory effects of cell proliferation were still not improved. This leads to an alternative explanation, which points to the presence of constitutively activated intracellular signaling pathways in each of the selected cell lines
(p-MAPK and p-AKT in T-47D and BT-474 cells). We postulate that high levels of phospho-MAPK and phospho-AKT levels play a dominant role in cell proliferation.
More importantly, these constitutively activated molecules are for some reason
dissociated (insensitive) from receptor regulation and are therefore responsible for the
relative refractory state of these cells in growth regulation.
We further tested the effects of inhibiting the hPRLR and HER2 pathways with
G129R and Herceptin treatment on the growth of T-47D and BT-474 xenografts in
athymic nude mice. After a six-week period of treatments, the Herceptin treated mice
were able to inhibit T-47D tumor growth by 46%, whereas the G129R treated mice
inhibited tumor growth by 53.3%. When treating the mice with Herceptin and G129R
together, the tumor growth was significantly inhibited by as much as 76.5% (p < 0.01).
BT-474 cells responded to treatments in a similar fashion as T-47D cells. Herceptin was
able to inhibit BT-474 tumor growth by 62%, whereas G129R inhibited tumor growth by
65%. The combination treatment group inhibited tumor growth to a greater extent than
single agents alone (79.1%). The tumor growth in all treatment groups were significantly 101
inhibited compared to that of control group after six weeks of treatment. The
combination of G129R and Herceptin treatment demonstrated the best outcome in
inhibition of the tumor growth in both T-47D and BT-474 xenografts.
Taken together, these findings strongly suggest that PRL’s proliferative effect on
HER2/neu tumorigenesis exemplifies the reasoning for simultaneous blockade of different growth factor-driven signal-transduction pathways might result in a more significant antitumor effect (Normanno et al., 2002). Our results provide a rationale for
the combined treatment of both Herceptin and G129R on breast carcinoma patients
whose tumors co-express HER2 and hPRLR. However, caution has to be taken to
extrapolate the results from cultured breast cancer cell lines when applied to clinical
settings since each cell line may represent a unique case in terms of its response to
various stimuli or inhibitors. It has been reported that interactions between PRL receptor
and EGFR pathways depend on the physiological state of the cells and that divergent
results may reflect spurious differences in cell lines, limited sampling of pathways and
end points, or alternatively, modulation of receptors and available downstream pathways
by physiological context and/or accumulating neoplastic changes (Arendt et al., 2006).
In summary of the first section, we have demonstrated that there is significant
cross talk between hPRLR and HER2 in these two breast cancer cell lines. Each receptor
has its preference in utilizing intracellular signaling pathways, at least in the cell lines
tested, with MAPK being the shared signaling molecule, suggesting its value as a
potential biomarker for combinational treatment approach. It is generally accepted that
breast cancer, or cancer in general, is a mixture of multiple cell populations or a
heterogeneous disease. Our data further demonstrates a strong correlation between hPRL 102
expression and proliferative activity in HER2 positive breast cancer cell lines. Therefore,
it is crucial to develop effective combinational approaches with multiple targets to
maximize the therapeutic impact of each individual agent and at the same time reduce the
potential side effect.
Early expression of low levels of prolactin has a protective effect on HER2/Neu tumorigenesis
The human epidermal growth factor receptor 2 (HER2, ErbB2) plays an important role in human malignancies. Amplification of the HER2 gene with overexpression of
HER2 protein occurs in approximately 20-30% of primary human invasive breast carcinomas and is correlated with poor prognosis (Slamon et al., 1987). The decisive role of HER2 in mammary tumors has been supported by decades of transgenic studies using the rat homologue, neu, driven by the mouse mammary tumor virus (MMTV) promoter
(Guy et al., 1992). The MMTV-neu mouse is an important preclinical model because it recapitulates the development of HER2 positive human breast cancer, characterized by over-expression of HER2 protein and tumor aggressiveness (Ren et al., 2004; Davies et al., 1999). Based on the first section, our data suggests the involvement of hPRL in breast cancer development via its interaction with HER2/neu oncogene. To investigate the effect of PRL and it's antagonist on HER2/neu-induced mammary tumorigenesis, we generated
bitransgenic mice co-expressing neu and hPRL or G129R. These bitransgenic mice
provide a unique model to study the relationship between PRL and ErbB2 signaling
pathways that lead to tumorigenesis.
In the second section of our study, transgenic hPRL or G129R mice under
transcriptional control of either the metallothionein (MT) promoter (a systemic expression
model) or MMTV promoter (a mammary specific expression model) were cross-bred to 103
MMTV-neu resulting in four lines of bitransgenic mice (MT-hPRL/neu; MT-G129R/neu;
MMTV-hPRL/neu; and MMTV-G129R/neu) as seen in Fig. 28. To our surprise, co- expression of low levels of hPRL in MMTV-neu mice drastically reduced the incidence of mammary tumors in both the MT-hPRL and MMTV-hPRL/neu bitransgenic lines. The total tumor rate was only 2 out of 36 (5%) in the MMTV-hPRL/neu line and was reduced to 30 out of 56 (53%) in the MT-hPRL/neu line. In contrast, co-expression of G129R in
MMTV-neu female mice had no significant effect on overall tumor incidence (>90% tumor rates after one year which is similar to their MMTV-neu littermates). Our working hypothesis is that this drastic drop in tumor incidence in both hPRL bitransgenic lines is due to the transgenic expression of low physiological concentrations of hPRL induced early mammary gland differentiation, which were then resistant to the oncogenic insults of
HER2/neu.
Our first attention was on HER2 because the transforming potential of the Neu protein is closely correlated with its intrinsic tyrosine kinase activity. We examined Neu tyrosine kinase activity and Neu expression levels in the mammary gland derived from the
MMTV-PRL/neu mice in comparison to their neu littermates (Fig. 29). Mammary glands from MMTV-hPRL/neu at six months of age, the age when mammary glands from transgenic females begin to develop morphological abnormalities, revealed significant reduction in HER2 expression. It has been reported previously that elevated expression of neu may be an important step for tumorigenesis (Guy et al., 1992) implicating the role of
PRL playing a protective effect in neu tumorigenesis by decreasing HER2 gene expression. On the other hand, PRL had no significant effect on phosphorylation of 104
HER2/neu in the bitransgenic mice, unlike in vitro results where PRL displayed an
induction of HER2/neu phosphorylation.
It has been suggested that treatments with protective hormones result in persistent
changes in the intracellular pathways which mediate proliferative responses to
carcinogens (Rajkumar et al., 2001; Sivaraman et al., 1998). Since cross talk between the hPRLR and HER2 pathways was demonstrated mainly through regulation of MAPK when using an anti-PRL and anti-HER2 therapeutic in combination, we examined the expression levels of MAPK and other biomarkers known to relate to hPRL function in the mammary gland. A significant difference in MAPK phosphorylation was observed between the MT-hPRL/neu and the MMTV-hPRL/neu mice compared to their neu-
littermates (Fig. 30 & 31, respectively). MAPK phosphorylation was down regulated at
three months of age in both lines of hPRL/neu mice, indicating that the mammary glands
of the bitransgenic mice were not highly proliferative. The decrease in MAPK
phosphorylation induced by PRL in both hPRL/neu mice demonstrates PRL as having a
dual role; here as a tumor suppressor and earlier as a tumor promoter demonstrated
through induction of MAPK phosphorylation. The decreased phosphorylated MAPK
levels at three months are consistent with the finding that the mammary gland tissue of the
hPRL/neu mice displays a lower proliferative index than their neu littermates at three
months (Fig. 35). The proliferative activity of the mammary epithelium varies as a
function of the degree of lobular differentiation, which, in turn, is often driven by
estrogens and progesterone as well as other hormones of pregnancy (Russo et al., 2005).
Thus, the lower proliferative activity in the hPRL/neu mammary tissue reflects the lower 105
phospho MAPK activity and Ki67 staining indicative of a higher degree or extent of
differentiation.
No significant changes were found in the expression levels of STAT5a/b and AKT
and their phosphorylation status, which suggest that these pathways are probably not
directly involved in the alteration of the HER2/neu tumorigenesis. We examined other
molecular events related to hPRL's suppressed expression of HER2 and mammary tumor
resistance in the hPRL/neu mouse model such as cyclin D1. Cyclin D1 expression has been shown to be regulated by PRL (Brockman et al., 2002) and has been demonstrated to be essential in the process of HER2/Neu-induced tumorigenesis (Yu et al., 2001). The striking dependence of HER2/Neu on cyclin D1 is highlighted by experiments in which the cyclin D1 gene was deleted (Yu et al., 2001), mutated (Landis et al., 2006), or a cyclin-dependent kinase inhibitor (p16) was overexpressed in MMTV-neu mice (Yang et al., 2004). In all cases, a complete resistance to tumorigenesis was observed when cyclin
D1 expression or activity was blocked (Yu et al., 2001; Landis et al., 2006; Yang et al.,
2004). We found that co-expression of hPRL and neu significantly lowered the cyclin D1 expression as compared to that of neu littermates at every age examined in both MT- hPRL/neu and MMTV-hPRL/neu mice (Fig. 30 & 31, respectively). By one month of age in the MMTV-hPRL/neu, there was almost a 60% reduction in cyclin D1 protein levels and by six months of age in the MT-hPRL/neu, there was almost a 40% reduction in cyclin D1 protein levels in comparison to their neu littermates. This finding strongly suggests that down-regulation of cyclin D1 through prolonged PRL exposure may be a key molecular event by which hPRL affords protection against Neu-induced tumorigenesis. It is also possible that the earlier reduction in cyclin D1 levels in the 106
MMTV-hPRL/neu may lead to earlier tumor resistance which was seen later in age in the
MT-hPRL/neu mice. Although the observation of reduced cyclin D1 expression in hPRL/neu mice offers a logical explanation for the lower tumor incidence, it raises the question as to how the prolonged exposure of low levels of hPRL leads to down- regulation of cyclin D1 expression. It is interesting to point out that PRL has been reported to have short-term stimulatory effects on cyclin D1 expression (Brockman et al.,
2002). However, it has also been demonstrated that PRL and EGF have no additive effects in cyclin D1 activation (Brockman et al., 2002), suggesting that there may be distinct regulatory pathways mediated via the PRLR and HER2/neu in terms of net epithelial response. Detailed studies are warranted in the future to dissect the molecular components directly involved in cyclin D1 regulation, such as pRB, p16 and p21 in response to the activation of the PRLR and EGFR/HER2/neu in hPRL/neu mice.
In an effort to identify the morphological features in the mammary glands of hPRL/neu mice related to the resistance of HER2/neu tumorigenesis, we analyzed mammary gland whole mounts (Fig. 33 & 34). In the MT-hPRL bitransgenics, as early as one month, the structure of the mammary glands of the hPRL/neu mice was more complex than that of their neu littermates. In addition to new ductal growth, the mammary gland of the one month old hPRL/neu female mice displayed an increase in alveolar budding indicative of early proliferation/differentiation (Fig. 33H). This phenotype is in good agreement with what was observed in young virgin WAP-hPRL transgenic mice (Manhes et al., 2006). By three months of age, the mammary glands from the hPRL/neu mice had significantly more branching and the alveoli were more highly differentiated per lobule than their neu littermates (Fig. 33G-H). In contrast, the acinar and lobular structures of 107
neu littermates had a more disorganized appearance at every age examined. The lobules
of the neu mice appeared to have lost the normal polar organization of basal and luminal
epithelial cells as evidenced by the smaller luminal spaces filled with proliferating cells
(Fig. 35A). A reduction in luminal cell death has also been observed in mammary
epithelial cells expressing the HER2/neu oncogene or activated Akt when grown in 3D
culture. The disruption of the normal mammary epithelial cell morphology is a hallmark
of an epithelial to mesenchymal transition (EMT), indicative of transformation in the early
stages of malignancy (Bundy LM & Sealy L, 2003; Debnath et al., 2003). In agreement
with these observations, a recent study by Nouhi et al., (2006) supports the role of PRL in
suppression of EMT in cultured breast cancer cells. They found that increasing levels of
Jak2 led to a reduction in MAPK phosphorylation and the induction of a more
differentiated epithelial phenotype (Nouhi et al., 2006). Therefore, it appears that expression of hPRL in these MT-hPRL/neu mice induced and maintained extensive mammary gland proliferation/differentiation throughout the critical pre-tumor phase, which may serve as the key to the resistance of HER2/neu tumorigenesis.
However, when we analyzed the whole mounts from the MMTV-PRL bitransgenic
mice we noticed a decrease in ductal branching and reduction in development of lobulo-
alveolar structures. According to Manhes et al., 2006, amplification of PRL activity
during the final stages of mammary differentiation revealed mammary structures that
tended to be less developed and completely prevented lobulo-alveolar formation. It may
be possible that the stages analyzed for whole mount analysis, the differentiation stage
was prior to the age we examined and the phenotype that was apparent was similar to
what is characteristic of during involution. In another study by Arendt et al., 2006, they 108 demonstrated transgenic PRL delayed ductal morphogenesis, perhaps reflecting PRL suppression of estrogen-induced ductal proliferation. This decrease in ductal branching and lobulo-alveolar development is representative of the phenotype of whole mounts that display a lack of tumor occurrence.
A plausible explanation for the drastic reduction in tumor incidence in both MT- hPRL and MMTV-hPRL/neu bitransgenic lines is that expression of low levels of hPRL induced early mammary gland differentiation and that the differentiated mammary glands became resistant to the HER2/neu oncogenic insults. This explanation is supported by well-established epidemiological observations that early full-term pregnancy is associated with breast cancer risk reduction. Russo and colleagues have suggested that parity- associated protective effects are due to the differentiation of target structures during pregnancy (Rajkumar et al., 2001; Russo et al., 2005). Mechanistically, both hormonal and genomic models have been put forth to explain pregnancy-induced protection from breast cancer. It is known that PRL plays a decisive role in the stimulation of lobulo- alveolar proliferation and differentiation and ultimately the promotion of milk production
(Vonderhaar, 1999; Horseman et al., 1995). It has also been postulated that undifferentiated epithelial cells are targets for carcinogens and are susceptible to neoplastic transformation and that parity-induced differentiation results in an epithelial cell population no longer susceptible to carcinogenesis (Russo et al., 2005). This has also been demonstrated recently where PRL over-expression in WAP-hPRL mice is not as tumorigenic as it is in the Met-PRL and NRL-PRL models, since it over-activates an already differentiated gland (Manhes et al, 2006). Therefore, co-expression of hPRL in
MMTV-neu mice may have induced early mammary gland differentiation, mimicking the 109
effects of early pregnancy, thus leading to a relatively refractory state of the mammary epithelium to the neu oncogene. Several studies have shown that the mammary glands of parous rats have a decreased proliferative ability, a higher capacity to repair DNA, bind lower levels of carcinogens, and are more differentiated in comparison to the mammary glands of age-matched virgin rats (Rajkumar et al, 2001; Russo et al., 2005).
To investigate if the protective effects exerted by hPRL expression were
mimicking the effects of early pregnancy, we examined a breeding group of MMTV-neu
female mice (n=34) that had undergone early and multiple pregnancies. In Appendix A,
we found that early and multiple pregnancies in MMTV-neu mice had little effect on total
tumor incidence and the median onset of tumor occurrence compared to that of virgin
MMTV-neu mice (t50= 189 days versus t50= 205 days, respectively). These data are
consistent with those of Jamerson et al. (2003), who reported that the mammary glands of
pregnant mice were more susceptible to oncogene-mediated transformation when
expression of the HER2/neu transgene was positively modulated. It has also been
reported that transgenic mice expressing oncogenes under steroid and peptide hormone-
responsive promoters exhibit pregnancy-associated mammary tumors or accelerated
tumorigenesis in parous females, and therefore may be inappropriate models to
recapitulate the protective effects of pregnancy on breast cancer (Henry et al., 2004). It is
possible that the over-expression of a potent oncogene, such as HER2/neu, overrides the
potential protective effects elicited by early pregnancy. In fact, the stimulatory effects of
the pregnancy hormones may act in synergy with constitutively high levels of HER2/neu
signaling, resulting in an increase in tumorigenicity. In any event, the drastic reduction in
breast tumor incidence in our hPRL/neu bitransgenic models suggest a mechanism that is 110 more complex than simply mimicking the protective effects exerted through early pregnancy.
Despite the similar levels of Neu phosphorylation in the mammary glands of hPRL/neu mice and their neu littermates, there was a high level of phosphorylated Neu found in the tumors of the neu mice suggesting a link between malignant growth and Neu activation. However, the tumors derived from a small percentage of the hPRL/neu mice contain greatly reduced phosphorylated Neu. The difference in the levels of phosphorylated Neu between tumors derived from hPRL/neu and neu littermates provides further evidence for the modulation of Neu-induced tumorigenesis by hPRL. It has been suggested that autocrine PRL is able to cross-phosphorylate HER2/neu through interaction with Jak2 in human breast cancer (Yamauchi et al., 2000). Therefore it is possible that prolonged exposure to low levels of hPRL led to a constant cross-phosphorylation of Neu at a sub-threshold level of tumorigenesis in the hPRL/neu mice. We further speculate that sub-threshold Neu phosphorylation was insufficient to initiate tumorigenesis but was effective at desensitizing the Neu activation as indicated by reduced levels of Neu phosphorylation, which translated to the lower tumor rate.
PRL transgenic mice have been generated to discern the role of PRL in mammary tumorigenesis. In previous reports, overexpression of PRL has been linked to breast tumor formation (Wennbo et al., 1997; Rose-Hellekant et al., 2003). However, in both of our MT-PRL and MMTV-PRL mouse models we have not observed a significant increase in the incidence of palpable tumors during the two year period of observation (data not shown). One possible explanation for this discrepancy points to the differences in the expression levels of the PRL transgene. In one study, the expression level of MT-rat 111
(r)PRL in female mice ranged upwards to 150 ng/ml level and the mice developed non-
metastatic mammary tumors at high rates (Wennbo et al., 1997). Similarly, most female mice expressing moderate (45 ng/ml) or high (253 ng/ml) levels of the rPRL transgene under the control of the hormonally non-responsive neu-related-lipocalin (NRL) promoter developed invasive mammary tumors (Rose-Hellekant et al., 2003). The hPRL transgene expression levels in this study were very low (5-10 ng/ml in serum for the MT line and at
341 pg/mg of tissue extract for the MMTV line). This is supported by a recent study by
Manhes et al., 2006 where very low levels of hPRL (2-8ng/ml) were detected in sera of virgin hPRL transgenic female and none of the mice developed mammary carcinomas.
Another, albeit less likely explanation, is that hPRL was used in this study whereas
rPRL was used in the others, suggesting a potential functional difference between the
hormones from two species. Rat and human PRL share only a 62% amino acid identity
although it is known that hPRL activates mouse PRLRs as effectively as rPRL (Hwang et
al., 1972; Utama et al., 2006). To our knowledge, there is no evidence in the literature that hPRL and rPRL play different roles in mouse mammary tumorigenesis.
In this study, the extent of the reduction of tumor incidence was also promoter- specific. MMTV-hPRL/neu mice were resistant to Neu-induced tumorigenesis to a far
greater extent than that of MT-hPRL/neu mice. PRLR mRNA is present in the stroma,
ductal and alveolar epithelium, although the regulation of expression is different between
the cell types (Rose-Hellekant at al., 2003). It has been reported that the MT promoter, a
ubiquitously expressed regulatory element, may be more active in the stroma than
epithelium within the mammary gland (Joseph et al., 1999). The differentiating actions of
PRL have been recently recognized as an important mechanism by which the hormone 112 could prevent progression and metastasis of differentiated, epithelial-like breast cancer cells, but not dedifferentiated, mesenchymal-like breast cancer cells (Nouhi et al., 2006).
Thus, in addition to the mammary gland specificity of the MMTV promoter, the epithelial versus stromal preferential expression driven by the two promoters may also contribute to the difference in the resistance to the tumorigenesis. We examined promoter expression by transiently over expressing hPRL under the MT and MMTV promoter in human and mouse epithelial and stromal cell lines. However, it was demonstrated that both the MT and MMTV promoter demonstrated increased hPRL expression in the epithelial cell lines rather than the stromal cell lines (Appendix B).
It is noteworthy that co-expression of G129R in MMTV-neu female mice had little influence on the breast tumor incidence. One explanation is that the expression levels of
G129R in the bitransgenic mice were too low to effectively counteract or change the course of HER2/neu driven tumorigenesis. G129R expression is only at physiological concentration (< 20ng/ml); the ideal therapeutic concentration would be at least at hundreds ng/ml based upon our experience. Nonetheless, the lack of the protective effects in both lines of G129R/neu bitransgenic mice further verifies the specificity of hPRL induced resistance to Neu-induced breast tumorigenesis.
In summary of the second section, our findings indicate that co-expression of hPRL possesses a unique protective role in MMTV-neu female mice through suppressing expression of HER2, which lead to reduced proliferative activities such as lower levels of
MAPK activities and cyclin D1 expression. This decrease in HER2 expression and alterations in molecular events are indicative of the significant change in the tumor incidence in these bitransgenic mice. The finding of reduced tumor incidence in both the 113
MT-hPRL/MMTV-neu and MMTV-hPRL/MMTV-neu lines supports the notion that co- expression of hPRL renders the mammary gland resistant to the oncogenic effects of Neu.
Future studies would be to test exogenous application of hPRL in MMTV-neu female mice to see if it has effects similar to those seen in the hPRL/neu bitransgenic mice. The key issues to be tested are the timing, concentration, and the duration of the administration. If our assessment is correct, hPRL could potentially become a chemopreventive agent for individuals at risk for HER2 breast cancer.
114
CHAPTER FIVE
CONCLUSION
The data presented demonstrate that hPRL induces tyrosine phosphorylation of
HER2/Neu and can stimulate mitogenesis in HER2 positive human breast cancer cells therefore serving as a tumor promoter. By using a PRL antagonist, G129R, in combination with Herceptin, stimulatory effects of hPRL are competitively inhibited.
Results further demonstrate that Herceptin and G129R display synergistic inhibitory effects on HER2 and MAPK phosphorylation. Most importantly, the combination treatment of Herceptin and G129R markedly inhibited the growth of xenografts in athymic mice.
Surprisingly, co-expression of low levels of PRL in MMTV-neu female mice demonstrated a drastic reduction in tumor incidence in two hPRL/neu bitransgenic lines.
Suppressed expression of HER2, which lead to lower levels of MAPK activities and cyclin D1 expression were strong indications of evidence that support the significant change in the tumor incidence in these bitransgenic mice.
Thus, PRL possesses a dual role in HER2/neu tumorigenesis: as a growth factor in transformed cells or as a survival factor when expressed early before tumor formation.
To improve the outcome of breast cancer therapy, especially for HER2 positive breast cancer, G129R should be further evaluated for use together with Herceptin as a combinational therapy. In addition, data from the PRL bitransgenic mice suggests the possibility of using hPRL as a potential chemo-preventive agent in HER2/neu tumorigenesis. 115
APPENDICES
116
Appendix A
Kinetics of tumor occurrence of nulliparous vs. early, multiparous MMTVneu transgenic mice
Focal mammary tumors of MMTVneu homozygous transgenic first appear at 4 month of age, with a median incidence of 205 days (50% mice develop first palpable tumor at approximately 200 days of age). It is also reported that 72% of tumor bearing mice that live 8 months or longer develop metastatic disease to the lung. We tested to see if early and multiple pregnancies in MMTVneu homozygous transgenic mice decreased tumor incidence since one of the risk factors for breast cancer include nulliparity or first full-term pregnancy after age 30.
Female MMTVneu homozygous mice (n=34) were bred at 5 weeks of age with male MMTVneu homozygous mice, Fig. A1. All female study mice experienced two cycles of pregnancy and lactation and all surviving pups from each litter were weaned at post-partum day 30. Female mice were palpated for tumors twice weekly through 12 months. It was established that median onset of tumor occurrence of MMTVneu homozygous mice experiencing early and multiple pregnancies was slightly decreased
(t50 = 189 days) compared to virgin MMTVneu homozygous mice (t50 = 205 days).
Parity status did not affect tumor incidence in MMTVneu homozygotes.
117
100 % %
, , 80 s s al al m m i i 60 an an t50 = 189 ee ee n=34 r r 40 r-f r-f o o m m u u T T 20
0 0 100 200 300 400
Age, days
Figure A1. Early, multiparous MMTVneu transgenic mice. MMTVneu homozygous mice (n=34) were bred at 5 weeks of age with male MMTVneu homozygous mice to examine the effect of early and multiple pregnancies in MMTVneu female, transgenic mice. The median onset of tumor occurrence of MMTVneu homozygous mice experiencing early and multiple pregnancies was slightly decreased (t50 = 189 days) compared to virgin MMTVneu homozygous mice (t50 = 205 days).
118
Appendix B
Comparison of transient hPRL expression driven by two promoters
To investigate if there is a cell type preference (stromal versus epithelial) between
MMTV and MT promoters in directing gene expression, transient transfections were
performed. A mouse (MCNeu) and human (T-47D) epithelial cell line and a mouse
(N202F3) and human (L-cells) fibroblast cell line was transfected with hPRL cDNA under MMTV and MT promoters from the PMSG and puCIG vectors respectively.
Expression of prolactin was assayed 72 hrs post transfection using an hPRL IRMA kit. In the epithelial cells, hPRL expression was expressed 100 fold higher and in the fibroblasts, the hPRL expression was approximately 20-30 fold higher under the MT promoter compared to the MMTV promoter (Fig B1).
119
140
-) Epithelial
T 120 Mouse M 100 Human of %
( 80
n o i 60 Fibroblasts 40 20
4 Epithelial hPRL Express 2 Fibroblasts 0 MMTV-promoter MT-promoter
Figure B1. hPRL expression driven by two promoters. MCNeu, mouse (■) and T- 47D, human (□) epithelial cells and N202F3, mouse (■) and L, human (□) fibroblast cells were transfected with hPRL cDNA under MMTV and MT promoters. Expression of prolactin was assayed 72 hrs post transfection using an hPRL IRMA kit.
120
Appendix C
Table C1. Primers used for PCR and RT-PCR identification of transgenic mice
Primers Name Sequences Product (bp)
MT-hPRL/G129R MT for 5’-CAC GCT GCG AAT GGG TTT ACG-3’ (1002) hPRL rev 5’-CAG GAT GAA CCT GGA TGA CT-3’
MMTV-hPRL/G129R MMTV for 5’-AGA CTC GCC AGA GCT AGA C-3’ (1203) hPRL rev 5’-CAG GAT GAA CCT GGA TGA CT-3’
hPRL/G129R hPRL for 5’-TGC TGC TGC TGG TGT CAA-3’ (465) hPRL rev 5’-CAG GAT GAA CCT GGA TGA CT-3’
neu neu for 5’-CGG AAC CCA CAT CAG GCC-3’ (622) neu rev 5’-TTT CCT GCA GCA GCC TAC GC-3’
G3PDH G3PDH for 5’-ACC ACA GTC CAT GCC ATC AC-3’ (453) G3PDH rev 5’-TCC ACC ACC CTG TTG CTG TA-3’
•Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) primers were used for both PCR and RT-PCR
121
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