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thesis entitled

MASPIN, A TUMOR SUPPRESSOR GENE, IS EXPRESSED IN
HUMAN BASAL BREAST EPITHELIAL CELLS BUT NOT IN
BREAST EPITHELIAL STEM CELLS AND BREAST CARCINOMA
CELLS

presented by

Maki Saitoh

has been accepted towards fulfillment
of the requirements for the

MS. degree in Genetics Program

 

 

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MASPIN, A TUMOR SUPPRESSOR GENE, IS EXPRESSED IN HUMAN BASAL
BREAST EPITHELIAL CELLS BUT NOT IN BREAST CARCINOMA CELLS

By
MakiSaitoh

ATHESIS
Submittedto
MidnTganStateUniversity
inparthlfulfillmentoftherequirements
forthedegreeof
MASTEROFSCIENCE
GemtimProgram

2004

ABSTRACT
MASPIN, A TUMOR SUPPRESSOR GENE, IS EXPRESSED IN HUMAN BASAL
BREAST EPITHELIAL CELLS BUT NOT IN BREAST EPITHELIAL STEM CELLS
AND BREAST CARCINOMA CELLS
By
Maki Saitoh

The phenotypes of cancer cells are very similar to stem cells as compared to
differentiated somatic cells. Both are described as undifferentiated cells and share the
common expression of many genes related to tumorigenesis, a basis for the concept that
cancer cells are primarily derived from stem cells. Maspin, a protease inhibitor known to
regulate not only invasion and metastasis, but also tumor growth and apoptosis, could be
a tumor suppressor gene not expressed in stem cells and tumor cells. To test this
hypothesis, I have examined the expression of maspin, using immunocytochemical and
western blot techniques, in two types of normal human breast epithelial cells (HBEC),
Type I HBEC show stem cell characteristics, whereas Type II HBEC expressed basal
epithelial cell phenotypes. The results clearly show that Type I HBECs, indeed, did not
express maspin in contrast to Type II HBECs which highly expressed the maspin. A
series of Type I HBEC lines, neoplastically transformed at different stages (immortal,
weakly and highly tumorigenic) and three breast carcinoma cell lines were also not found
to express maspin. Thus, the silencing of maspin expression in breast cancer cells is very
likely due to the continuous non-expression of a target stem cell phenotype. The results
provide additional evidence for the stem cell theory of carcinogenesis and indicate that

maspin could be used as a marker for early detection of breast cancer and for assessing

the efficacy of chemopreventive or chemotherapeutic agents.

ACKNOWLEDGEMENTS

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andwarmsupport. Ifisbdiefmmeamnagedmetokeepwoddngmdlfisdevotedmdflmghtfifl
attituletowardmeasaneduoatortaughtmegreataspeotsoflife.

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suggestions. Imndoeplyappredafiveoftfisvalmbleadvioafimeandmpport

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iii

TABLE OF CONTENTS

List ofTables and Figures

 

Literamre Review

 

L'lheRoleofStanCdlinCarcinogenesis

 

 

ILCanoerCellthotypes
1)Ijmitlelsreplimtivepotential

 

2) Self-sufficiency in growth signals

 

3) Insensitivityto growth-inh‘bitmy signals

 

 

4)Evasionofprogrmnnedoelldeath

 

S)Sustainedangiogams
@Tmmirwasimandmetaaas's

 

I. GmesAlta'odinBieastCamer

 

 

00mm
2)“!‘umorSuppmsorGenes

 

IL Maspin

 

 

1) Identification and location.
2) Fundim
3) Transcriptional Regilation

 

Irmotmdim

 

MataialsarxiMethods

 

CellCulture

 

1)NonnalenanBreaaEpithelialCefls

 

2)TmmigenicBreastEpiflieIialCells

 

 

WestanBlaAnalysisofMaqrinProteinExpression
hmnmncytodremimlDetedimofMawinEmmsim

 

Results

 

DiffuafialExprafionofMaspininTypeIandTypelIHBEC

 

EmessimofMaspinIanoTransfmnedHBECarxlCaerells
D' .

 

 

RefamoeList

 

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LIST OF TABLES

Table ICIIaIaaaistiqsonypeIaerTypeIII-IBECS

77

LISTOF FIGURES

FigmellsolafimsdmiemobtainZtypesomenalemBreastEpiflidialCdls .................. 23
Figure 2 In vitro immortalization and neoplastic transformation of Type I HBEC...25

Figme3MuflnlogiesonypeIaerTypelII—IBECS 79

 

 

Pigne4ExprmsimCytokeratinl4andl8inTypeIarflTypeHI-IBECS 30

Figme SaMmrinExpmsionbyhmnmostairfinginTypeIGefi)andTypeII (right) HBEC ofHME
30 32

FigueSbMasrfinExpmssimbyhmnmostairfinngypeIGefieriTypeH(right)I-IBECole\rIE
31 33

Figme6WrstanBlotAmlysisofM$pinExpresioninTypeIamiTypeIII—IBECofWGanes

 

 

1,2),I-ME300m3,4)andI-IME31(Im5,6) 34
Figure7MasphiEiqxesimoflnviuoTrmsfimnedTypeIHBECIjnesatDifi‘aanSmby

hmnmocytochanioalAnalysis 36
Figme8MmpinE>qxessiminBreastCardnmnaCdlLinesbyhmnmnstainh1g 37

 

Figm9Westm1BIdAmly§sofMaq3hE7qxessimmNommlHBECmvimlhmnmmhzodm
NeoplasfimflyTramfixmodTypeIHBECandBreastCm'cirnnaCdlfirm 38

vi

LITERATURE REVIEW

I. The Role of Stem Cell in Carcinogenesis

Several lines of evidence suggests that cancer cells are derived from the adult stem cell 4
pools of a tissue where initiating events of stem cells prevents cells from fully
differentiating and resulted in uncontrolled clonal expansion of these cells. The stem cell
theory of carcinogenesis proposes that stem cells are target cells in carcinogenesis and

certain developmental or differentiating genes are blocked or partially blocked so the

cells remain undifferentiated (Potter, 1978).

Stem cell research has received increasing attention because of its therapeutic application
and potential role as a target cell in carcinogenesis. Its unlimited or extensive self-
renewal capacity and ability to differentiate into multiple cell types (“pluri-potency”) are
highly valuable in various applications for medical treatments such as tissue engineering,
transplantation and gene therapy. These two unique properties of stem cell are also

important in understanding the role of stem cell in carcinogenesis.

By definition, "toti-potent" cells can give rise to all cell types within the organism. They
can divide symmetrically or asymmetrically to produce partially committed organ
specific stem cells, namely "pluri-potent" stem cells. These pluri-potent stem cells can
give rise to several different cell types in different organs. Then, after further
development, some cells are finally restricted to giving rise to one or fewer cell types to

become "committed" progenitor cells. The committed progenitor cells eventually

develop and give rise to functionally and terminally differentiated cells. The stem cell
theory of carcinogenesis states that the carcinogenic process starts from a stem cell (most
likely a pluri-potent stem cell), assuming that a normal stem cell is "immorta " (Trosko,

2003)

As an alternative to the stem cell theory, the de—differentiation theory states that cancer
cells are derived from de-differentiation of differentiated of cells to explain the
undifferentiated state of tumor cells. The unlikelihood of dedifferentiation of
differentiated somatic cells to develop into tumor favors the stem cell theory. For
example, many differentiated hepatocytes, are tetraploid (Styles et al., 1985), while the
nodule that give rise to tumors are diploid (Schwarze et al., 1984). It is a rare chance,
therefore, that these cells can de-differentiate back to diploid state that characterize
precancerous cells and this fact seriously undermines the de-differentiation theory as a
possible explanation of less differentiated state of cancer cells. It has been documented
that the stem cells share a number of tumor cell characteristics: contact insensitivity
(Chang et al., 1987), extensive self-renewal capacity (Tang et al., 2001; Mathon et al.,
2001), anchorage- independent growth (Chang et al., 2001; Chang et al., 2004; Lin et al.,
2004), lack of gap junctional intercellular communication (6] 1C), (Y amasaki et al., 1987;
Chang et al., 1987; Kao et al., 1995; Matic et al., 2002; Grueterich and Tseng, 2002) and
activated telomerase (Hiyama et al., 1995) or high susceptibility of telomerase activation
(Sun et al., 1999) . This evidence also indicates the difficulty to de-differentiate back to

undifferentiated state to regain those characteristics described.

Recently, more evidence suggests the role of stem cells as target cells in carcinogenesis.
For example, McKay and his co-workers found a novel protein named nucleostemin,
from a subtractive hybridization study, that was expressed in the nucleoli of CNS stem
cells and embryonic stem cells, as well as in tumorigenic cells (Tsai and McKay, 2002).
Structural analysis and mutation study of nucleostemin indicated a role of the gene in cell
cycle regulation of cell growth, as well as the prevention of the cells from dying by
apoptosis. The expression of nucleostemin decreased rapidly prior to cell cycle exit when
the cells are differentiated. Furthermore, based on the evidence that activation or over-
expression of nucleostemin reduces cell proliferation in CNS stem cells and transformed
cells, nucleostemin seems to be correlated with cell proliferation or the lack of

differentiation in these cells.

Recently, the role of myoepithelial cells as a host defense cell against cancer received
some attention (Sternlicht and Barsky, 1997; Stemlicht et al., 1997). First, the mammary
myoepithelial cells express protease inhibitors, including maspin, which have various
tumor suppressive activities. Second, myoepithelial cells are rarely transformed and the
myoepithelial tumor tends to be benign or low-grade neoplasms. Third, the myoepithelial
cells promote differentiation and inhibit invasion in vitro. In terms of the stem theory of
carcinogenesis, the properties of myoepithelial cell show that they are differentiated cells
derived from stem cells and that they possess anti-cancer properties. The fact suggests
that the difierentiated breast epithelial cell type is unlikely to be a target cell and

indirectly supports the stem cell theory of carcinogenesis. Evidence supporting the stem

cell theory of carcinogenesis has been previously reviewed (Chang et al., 2001) and

mentioned in the Introduction.

11. Cancer Cell Phenotype

Hanahan and Weinberg summarized six essential capabilities of cancer cell or "hallmarks
of cancer": 1) limitless replicative potential, 2) self-sufficiency in growth signals, 3)
insensitivity to growth-inhibitory signals, 4) evasion of programmed cell death, 5)
sustained angiogenesis, and 6) tissue invasion and metastasis (Hanahan and Weinberg,
2000). Now, in view of the stem cell theory, I will briefly summarize them to re-
emphasize how the stem cell already possesses several important characteristics

commonly found in the tumor cell.

1) Limitless replicative potential

Normal human cells have limited life spans (Hayflick, 1997), however, almost all
malignant tumor cells can replicate indefinitely. In view of the stem cell theory, stem
cells theoretically have extensive self-renewing capacity. More specifically, the stem
cells or their partially differentiated daughter cells, that have not yet down-regulated their
telomerase activity, but are exposed to a carcinogenic "initiator," will remain immortal or
have limitless replication potential (Trosko et al., 2000). Thus, the first step of

carcinogenesis is to prevent the stem cells to become mortal or to terminally differentiate.

Although the stem cell has extensive self-renewal capacity, it is not all clear whether
stem cell maintains this capacity for an entire lifespan and if so, what type of intrinsic or
extrinsic factors are involved to maintain this capacity. Several in vitro studies indicate
that certain genetic alterations are required to maintain the self-renewal capacity. The
induction of so-called "immortalization" of human mammary epithelial cell (HMEC)
seems to involve two major events (Kiyono et al., 1998). First, the cell has to acquire an
extended lifespan (to overcome that first stage senescence or mortality stage, M1) by
altering the regulation of cell cycle genes especially the p16ink4/Rb pathway (Kiyono et
al., 1998). Inactivating this p16ink4/Rb pathway to extend the lifespan of HMEC has been
demonstrated by transfecting with SV40 large T-antigen (Kao et al., 1995) or papilloma
virus E6 and E7 (W azer et al., 1995). However, the cells that successfully evaded the
senescence soon stop growing when they reach the next plateau, called crisis (Kiyono et
al., 1998). As a second step in immortalization, the HBEC with extended lifespan may
become immortal after the activation of telomerase (to overcome the M2, or crisis).
HMEC with inactive p16/Rb may become immortal after transfecting with the human
telomerase gene hTERT (Kiyono et al., 1998). However, HMECs, growth on feeder
layers, have been immortalized without abrogating p16 (Herbert et al., 2002). It is
interesting to note that estrogen is an agent known to activate telomerase on the hTERT
promoter in a breast cancer cell line (Kyo et al., 1999). The capability of unlimited or
prolonged replicative potential is believed to be established in early stage of
carcinogenesis. This may be related to the characteristics of stem cells, the major target
cells for carcinogenesis. The unlimited self-renewal ability of stern/precursor cells has

been demonstrated for rodent cells (Tang et al., 2001; Mathon et al., 2001) but not for

human cells. However, breast epithelial stem cells have been shown to be more

susceptible to telomerase activation (Sun et al., 1999).

Taken together, to preserve the limitless self-renewal capacity of stem cells, alteration of
cell cycle gene regulation and activation of telomerase gene are required to occur during

the development and growth of cancer

2) Self-sufficiency in growth signals

Normal cells require mitogenic growth factors, whereas tumor cells produce and express
many of their own growth factors and growth factor receptors, thereby reducing their
dependence on exogenous or endogenous growth factors fi'om their normal tissue
microenvironment. The most important growth factor for breast cancer cells appears to
be the estrogen. In breast cancer patients, about two-thirds of tumors are ER-positive
(Lippman et al., 1988). Fifty percent of these ER-positive tumors are estrogen-dependent
and respond to endocrine therapy and anti-estrogen, Tamoxifen (Manni et al., 1980). The
estrogen induction of proliferation of breast cancer required the expression of Myc
(Watson et al., 1991) which is frequently amplified or overexpressed (Dickson, 1994).
The over- expression of the neu oncogene might enable the cancer cell to become hyper-
responsive to ambient level of growth factors that normally would not trigger
proliferation in normal cells (Fedi, 1997). In fact, amplification of the EGF-receptor-
related prooncogene c-erbB2/neu has been observed in 30% of primary human breast

carcinomas (Slamon et al., 1987) and another 10% over-expressed c-erbB2 without

amplification of the gene (Kraus et al., 1987). It should be noted that, in contrast to
breast epithelial cells, human breast epithelial stem cells express the estrogen receptors

(Kang et al., 1997).

3) Insensitivity to growth-inhibitory signals

In normal cells, a number of anti-proliferative signals, including grth inhibitors
(Hanahan and Weinberg, 2000), operate to maintain cellular quiescence and tissue
homeostasis. However, cancer cells have an ability to evade these anti-proliferation
signals and use various strategies to avoid being terminally differentiated. As an anti-
growth factor, TGF-beta, prevents the phosphorylation of Rb, subsequently blocks the
cell to advance through the GI phase of the cell cycle (Hanahan and Weinberg, 2000).
Endogenous TGF [3 signaling and growth suppression is lost during human mammary
epithelial cell transformation by benzo(a) pyrene (Ge and Stanpfer, 2004). Down-
regulation of GJIC is one of the mechanisms to prevent fiom being regulated by growth
inhibitory signals, such as cyclic AMP, which is considered as a reverse transformation
agent (Chan et al., 1989). It has been reported that Type I HBEC with stem cell
characteristics and most breast carcinomas are deficient in GJIC (Kao et al., 1995).
Another mechanism to avoid being terminally differentiated is the over-expression of the
survival factor c-myc, which encodes a transcription factor. During the normal
development, the grth stimulating action of Myc is associated with another factor,
Max. The association can be supplanted by alternative complex of Max with a group of

Mad transcription factors; the Mad-Max complexes elicit differentiation-inducing signals

(Foley and Eisenman, 1999). Overexpression of c-myc can reverse this process, shifting
the balance back to favor Myc-Max complex, thereby impairing differentiation and
promoting growth (Hanahan and Weinberg, 2000). In breast cancer cells, the
abnormalities in the c-myc gene were reported in about 6-32% of breast cancer (Van de
Vijver and Nusse, 1991 ). TGF-beta also suppresses the expression of the c-myc gene,
which regulated the 61 cell cycle (Moses et al., 1990). ER-positive breast cancer cells
are refractory to TGF-beta effects, whereas ER—negative breast cancer cells are often

TGF- beta sensitive (Arteaga and Moses, 1996).

4) Evasion of programmed cell death

The balance between cell proliferation rate and cell attrition rate also determines the
ability of tumor cell populations to expand in number. Apoptosis or programmed cell
death is an active, energy-dependent process of cell death which occurs during
development, in response to certain physiologic stimuli (Kerr et al., 1972). Unlike
necrosis which is a passive osmosis-driven process due to the loss of ion-pumping
activity, apoptosis shows characteristic morphologic changes i.e. cell shrinking and
blebbing, nuclear and chromatin condensation, nuclear fragmentation, and packaging of
cellular materials and organelles into mernbrane-bound vesicles termed apoptotic bodies
(Hahm, 1998). Once cells are triggered by a variety of physiologic signals, the cells
undergo apoptosis in a precisely choreographed fashion. There are two major protein
families involved in apoptosis: antiapoptotic protein family (Bel-2 relatives) and

proapoptotic protein family (Bax relatives) during induction stage of apoptosis. The

tumor suppressor protein p53 can elicit apoptosis by upregulating the expression of Bax

in response to sensing DNA damage.

5) Sustained angiogenesis

Angiogenesis, the growth of new capillary blood vessels, is critical for cancer cell to
grow, invade and metastasize. New blood vessels bring in fresh nutrients and grth
factors so that the tumor mass can expand. As a matter of fact, without forming new
blood vessels, solid tumors cannot exceed a size of about 1mm3 . Interaction between
angiogenic proteins (i.e. VEGF, FGFs) and angiogenic proteins (i.e. thrombospondin)
appear to be responsible for the angiogenic activity of malignant tumors. Interestingly,
analyses of histologically distinct stages of human and rodent tumors suggest that the
acquisition of an angiogenic phenotype occurs early in tumorigenesis and can be

considered the rate limiting step during tumor progression (Hanahan and Folkman, 1996).

6) Tissue invasion and metastasis

In order for cancer cells to spread, the cells need to acquire the ability to invade the tissue
and enter the blood stream to metastasize to other organs. These involve cell migration
and the expression of proteases. It has been shown that increased expression of
hepatocytes growth factor (HGF) and its receptor, met, occurs in invasive human breast

cancer (Tuck et al., 1996; Wang et al., 1994; J in, 1996) particularly at the migrating

tumor fi'ont (Tuck et al., 1996). The tissue invasion and metastasis involve the action of

adhesion molecules, metalloproteases and cllageneses (Akiyarna et al., 1996).

III. Genes Altered in Breast Cancer

As stated by Hanahan and Weinberg (2000), “tumors are the results of the accumulation
of a series of genetic alterations. Genetic studies over the years have identified a number
of oncogenes and tumor suppressor genes associated with the development of breast

cancer (See ref. In Chang et al., 2001).

i) Oncogenes

Three oncogenes (c-erb BZ/neu, c-myc and int-2) and the G. phase cyclins (D and E)
were frequently found to be amplified or overexpressed in breast cancers (See ref. In
Chang et al., 2001). In addition, many breast oncogenes have been identified. These
include Brk (breast tumor kinase) (Barker et al., 1997; Mitchell et al., 1994), srk (Jacobs
and Rubsamen, 1983; Luttrell et al., 1994), AKT3 (Nakatani et al., 1999) and ZN F217

(Nonet et al., 2001).

ii) Tumor Suppressor genes

10

As summarized (Chang et al., 2001 ), many tumor suppressor genes are involved in breast
cancer including those responsible for breast cancer syndromes (i.e. BRCAl, BECA2,
p53 in Li-Fraumeni syndrome, ATM gene in ataxia telangiectasia). In addition, PTEN (a
lipid phosphatase), P57klpz (a cyclin kinase inhibitor), F HIT (fragile histidine triad), Wnt-
5a, pl6ink4a (a cyclin-dependent kinase inhibitor) and maspin are also likely to be
involved in breast cancers. Loss of heterozygosity was frequently observed on many
chromosome arms in breast cancer (i.e. 1p, lq, 3p, 11p, 13q, 16q, 17p, 17q and l8q).
Except for chromosome 1, 16q and 18q, potential turner suppressor genes have been
identified (Chang et al., 2001). Subsequently, a senescence gene, SEN16 (Reddy et al.,
1999; Reddy et al., 2000)and a transcription repressor, CBFA2T3 (Kochetkova et al.,
2002) have been located on 16q24 and the maspin gene has been mapped to 18q21.3

(Schneider et al., 1995).

IV. Maspin

The mammary serpin protease inhibitor, maspin, is a candidate for being a tumor
suppressor gene whose expression is lost in many advanced breast tumors. Maspin has
drawn the attention due to the various proposed functions in breast carcinogenesis, such

as angiogenesis, metastasis, apoptosis and cell invasion.

1) Identification and location

The maspin gene was originally isolated from normal mammary myoepithelial cells by

subtractive hybridization on the basis of its expression at the m-RNA level (Sager et al.,

11

1994). This maspin gene encodes a 42-kDa protein (Sager et al., 1994) and is mapped at
18q21.3 (Schneider et al., 1995) where the loss of heterozygosity is frequently observed
in this region , not only breast cancer cells, but in a large number of human tumors
(Schneider et al., 1995).

Maspin m-RNA and protein were found in normal mammary myoepithelial cells, but
down-regulated in mammary carcinoma cells (Sager et al., 1994). The expression of
maspin is tissue-type and cell-type specific and it is expressed in skin keratinocytes and
prostate epithelial cells in addition to mammary epithelial cells (Futscher et al., 2002).
Immunohistochemical studies showed that maspin is expressed in pancreatic cancer cells
but not in normal pancreatic epithelium (Maass et al., 2001a). In contrast, in mammary
tissue, maspin is expressed in normal epithelial cells, but significantly less in mammary
tumor cells (Maass et al., 2001a). In contrast to p53, the maspin expression was higher in
ER-negative tissue than ER-positive tissue (Martin et al., 2000). Based on
immunohistochemical studies, the maspin is expressed in the nucleus, as well as in the

cytoplasm of breast myoepithelial cells (Reis-Filho et al., 2001; Lele et al., 2000).

2) Function

Although it has not been clearly understood the role of maspin in breast cancer, there are
many functions of maspin suggested since the discovery of the maspin gene in 1994.
Unlike the other serpins, maspin does not act as an inhibitor to an array of serine
proteases, including urokinase-type plasminogen activator (uPA) and tissue-type

plasminogen activator (tPA) (Martin et al., 2000; Pemberton et al., 1995). However,

12

there is evidence that tPA is a target of the tumor suppressor gene maspin (Sheng et al.,
1998). Exogenously introduced maspin gene expression in breast tumors rendered the
inhibition of cell invasion in vitro and metastasis in vivo (Sager et al., 1994).
Recombinant maspin protein blocks the motility of carcinoma cells (Sheng et al., 1996).
Neutralization of maspin by an anti-maspin antibody abolished the invasion suppressive
effect and increased apoptosis of conditioned medium from cultured breast myoepithelial
cells on tumor cells (Shao et al., 1998). Maspin overexpression increased a rate of
apoptosis and block angiogenesis in vivo and in vitro (J iang et al., 2002; Zhang et al.,
2000a; Zhang et al., 2000b). Maspin expression in breast tumor cells reduces tumor
induction and metastasis in nude mice (Sager et al., 1994; Shi et al., 2001; Shi et al.,
2003; Streuli, 2002), and also invasion of the basement membrane in vitro (Sager et al.,
1994). Moreover, treatment of human breast cancer cells with recombinant maspin

(rMaspin) inhibits cell motility (Sheng et al., 1996).

3) Transcriptional Regulation

The molecular and biological mechanisms of the functions of maspin still remain
unknown. However, there is evidence that maspin interacts with the p53 tumor
suppressor pathway (Maass et al., 2000). Zhou reported that p53 regulates the expression
of maspin in breast cancer cell lines (Zou et al., 2000). Maspin expression was up-
regulated when wild-type p53 gene was introduced through an adenoviral vector. P53
activates the maspin promoter by binding directly to the p53 consensus-binding site

present in the maspin promoter (Zou et al., 2000). Furthermore, they demonstrated that

13

DNA-damaging agents and cytotoxic drugs induced endogeneous maspin expression in
cancer cells containing wild-type p53, but not in cells containing mutant p53 (Zou et al.,
2000). These results indicate that the function of maspin in apoptosis might be regulated

through the p53 pathway.

Although the maspin gene is silenced in cancer cells, maspin gene deletions and
mutations have not been found (Barsky et al., 1997), indicating the involvement of an
epigenetic mechanism. The mechanism of the maspin gene regulation has not been
clearly understood. However, maspin is up-regulated by the treatment of gamma linoleic
acid, an essential fatty acid with anticancer properties (Jiang et al., 2002). Maspin reacts
with tissue type plasminogen activator in vivo (Sheng et al., 1998). Interestingly,
recombinant maspin exerts a biphasic effect on the activity of single-chain tissue
plasminogen activator, acting as a competitive inhibitor at low concentrations (<0.5 mM)
and as a simulator at higher concentrations (Sheng et al., 1998). Maspin gene expression
was induced by MnSOD (Manganese-containing superoxide dismutase cDNA)

transfectants of MCF-7. (Li et al., 1998).

The involvement of DNA methylation was first suggested by Domann in 2000 (Domann
et al., 2000). Maspin promoter is unmethylated and is an open chromatin structure in
normal, maspin-positive HMECs. In contrast, this chromatin structure is often aberrantly
methylated and associated with a closed chromatin structure in maspin-negative human
breast cancer cell lines (Domann et al., 2000). Methylation-associated silencing is found

with a high frequency, as high as 80% in human breast cancers (Domann et al., 2000).

14

Additional experiments confirmed that maspin expression is regulated by DNA
methylation and/or histone deacetylation in maspin-negative breast cancer cell lines

(Domann et al., 2000; Futscher et al., 2002; Mass et al., 2002).

15

INTRODUCTION

Cancer cells are generally known to be similar to stem cells in phenotypes. The extreme
case is exemplified by human embryonic stem cells (ES) which formed teratomas in
SCID-beige mice (Thomson et al., 1998). Of all the common characteristics, the most
notable description is the undifferentiated state of cancer and stem cells. The
undifferentiated state of cancer cells could be due to the de-differentiation of
differentiated somatic cells or due to the blocked differentiation of stem cells which give
rise to tumor cells (Varmus and Weinberg, 1993; Sell, 1993). The latter is similar to
other concepts of cancer as a disease of cell differentiation (Markert, 1968), a disease of
stem cells (Sawyers et al., 1991) or as an “oncogeny as blocked or partially blocked
ontogeny” (Potter, 1978). Cancer cells are known to be deficient in homologous or
heterologous junctional gap intercellular communication (GJIC) (Y amasaki et al., 1987).
Several human adult stem cells were also found to be deficient in GJIC (Chang et al.,
1987; Kao et al., 1995; Matic et al., 2002; Grueterich and Tseng, 2002; Chang et al.,
2004). Some characteristic tumor cell phenotypes such as contact-insensitive growth and
the ability of anchorage independent growth (AIG) are shared by stem cells. The former
has been reported for human kidney epithelial stem cells (Chang et al., 1987) and the
latter were shown in human breast epithelial, human liver and mesenchymal stem cells
(Chang et al., 2001; Chang et al., 2004; Lin et al., 2004). Nucleosternin, a nucleolar
protein that interacts with p53, is present in CNS stem cells, ES cells and cancer cells but
not in the differentiated cells of adult tissues (Tsai and McKay, 2002). Similarly, Oct-4,

a transcription factor previously reported to be exclusively expressed in pluripotent early

16

embryo cells, ES cells and undifferentiated tumor cells, has been recently shown to be
expressed in many human adult stem cells (Trosko et al., 2004). Furthermore, the
expression of ot-fetoprotein and vimentin, collectively termed the “oval cell phenotypes”
(Alison et al., 1997), was found in adult human liver stem cells and hepatomas (Chang et

al., 2004).

Mammary cancers primarily originated from the relatively undifferentiated mammary
gland, i.e. the rodent terminal end bud (TEB) or the human lobule 1 where most
mammary epithelial stem cells reside. For example, it has been demonstrated that the
carcinogen acts on TEB and that the structure is the one that evolves to intraductal
proliferation, carcinoma in situ and invasive carcinoma (Russo and Russo, 1996). The
study of pathogenesis of breast cancer in relation to the lobular composition of breast also
identified lobule 1 as the site of origin of the most frequent breast malignancy, the ductal
carcinoma (Russo and Russo, 1997). Consistent with this correlation is the observation
that lobule 1 contains high frequency of estrogen-receptor (ER) positive cells (14%)
compared to lobule 2 (4%) and lobule 3 (0.5%) (Russo and Russo, 1998), and the fact

that two thirds of breast cancer is ER-positive (Lippman and Allegra, 1980).

At the cellular level, the mammary gland contains two types of epithelial cells, the
lurninal and the basal (or myoepithelial in alveoli) epithelial cells besides the epithelial
stem cells. Our laboratory has developed a cell culture method to grow two types of
human breast epithelial cells (HBEC) from reduction marnmoplasty tissues (Kao et al.,

1995). Type II HBEC expressed basal epithelial cell markers whereas Type I HBEC

l7

showed luminal epithelial and stem cell phenotypes. The stem cell characters of Type 1
cells include 1) deficiency in GJIC; 2) the ability to differentiate into Type II HBEC; 3)
the expression of Oct-4 (Trosko et al., 2004) and 4) the ability to form ductal and budding
or lobule l-like structures on Matrigel (Chang et al., 2001). Furthermore, Type I HBEC
have been shown to be more susceptible to telomerase activation, immortalization and
neoplastic transformation (Sun et al., 1999; Kang et al., 1998). When the phenotypes of
breast carcinomas or in vitro neoplastically transformed Type I HBEC are compared with
Type I and Type II HBEC, it is clear that tumorigenic breast cells are similar to Type I
HBEC rather than Type II HBEC. These similarities include deficiency in GI 1C, the
expression of estrogen receptor, Oct-4 and luminal epithelial cell markers (i.e. epithelial
membrane antigen, cytokeratin 18) and ability of AIG (Kao et al., 1995; Kang et al.,

1997; Chang et al., 2001; Trosko et al., 2004).

Since stem cells and differentiated cells are substantially different in phenotypes as
shown in Type I and Type II HBEC, and if cancer cells are derived fi‘om and preserve
many stem cell characteristics, it is expected that the phenotypes of the early stage cancer
cells are already very different fiom differentiated normal cells. Indeed, it was found that
the most dramatic and consistent phenotypic change occurred at the normal-to-in situ
carcinoma transition (Porter et al., 2001). Another implication is the doubt about genetic
alterations in cancer cells based on differential display or microarray studies of cancer
and normal cells since the great majority of the cells in normal tissues are not the stem
cells from which most cancers arise. For example, Cx26 and a6 integrin and maspin

have been considered as tumor suppression genes based on differential display studies

18

showing their expression in normal cells but not in tumor cells (Lee et al., 1991; Sager et
al., 1993; Zou et al., 1994). In fact, our results show that Cx26 and a6 integrin were
expressed in Type II HBEC but not in Type I cells (Kao et al., 1995; Chang et al., 2001).
Bemards and Weinberg (Bemards and Weinberg, 2002) hypothesized that certain
combination of genetic alterations that are selected relatively early in tumorigenesis for
proliferation advantage they confer will, incidentally, also confer invasive/metastatic
phenotype. Since maspin is known to be a protease inhibitor which regulates not only
invasion and metastasis but also tumor growth, angiogenesis and apoptosis (Zhang et al.,
2000b; Jiang et al., 2002; Streuli, 2002; Shi et al., 2003), it is hypothesized that this gene
could be the kind of tumor suppressor gene not expressed in stem cells and tumor cells.

The experiments of this study were carried out to investigate this hypothesis.

l9

MATERIALS and METHODS

Cell Culture

a) Normal Human Breast Epithelial Cells

Breast tissues were obtained from healthy women who underwent reduction
mammoplasty. After removing most adipose tissues, the tissues were minced into small
pieces and digested with collagenase for overnight before culturing in the MSU-l
medium (Kao et al., 1995). The initial primary culture developed in one week were

aliquoated in small vials and stored at —80°C liquid nitrogen storage until use.

To recover cells from liquid nitrogen storage, the cells were thawed at 37°C and
transferred to a 15 ml centrifuge tube containing MSU-1 medium. The tube was then
centrifuged at 1000 rpm for 10 min. After removing the freezing solution and the
medium fiom the tube, the cell pellet was plated in 6 ml of MSU-I medium with 5% fetal
bovine serum (PBS) (the Type I medium) in a 10 cm cell culture dish (Plate A) and
incubated for two hours. After the incubation, the supernatant containing unattached
cells was removed, transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for
10 min. The Plate A containing Type I HBEC and fibroblast cells was further incubated
after adding 6 ml of fresh MSU-I medium. The cell pellet was suspended in 6 ml of
MSU-I medium with bovine pituitary extract (BPE) (50 til/ml) (the Type 11 medium),
plated in a 10 cm cell culture dish (Plate B) and incubated for overnight. Next day, the
supernatant containing unattached cells was carefully removed and transferred to a 15 m1

centrifuge tube and centrifuge at 1000 rpm for 10 min. After discarding the medium, the

20

cell pellet was suspended in 6 ml of MSU-I medium with 5% FBS and plated in a 10 cm
dish (Plate C), which contains mostly Type I HBEC and few fibroblasts. The few
colonies of fibroblasts that developed in a week can be marked and removed by scraping
with a rubber policeman. The Plate B after further incubation in the Type 11 medium
contains Type II HBEC. The procedure of obtaining these 2 types of HBEC is
diagrammatically depicted in Fig. 1. For this study, the primary HBEC cultures HME 29,
30 and 31 derived from 3 different women (age 26, 23, 21 respectively) were used. The
phenotypic difference between Type I and Type II HBEC fi'om previous studies (Kao et

al., 1995; Chang et al., 2001) are listed in Table l.

The culture medium was changed once every three days. Subculture of HBEC was

carried out by cell removal with 0.01% trypsin and 0.01% EDTA followed by the

inactivation of trypsin with 10% PBS.

21

Table 1

Characteristics of Type I cells and Type II HBEC (Modified from

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(Kao et al., 1995)

Plastic Type I Type II

Cell Morphology in Variable in shape Uniform in shape, cobble-

MSU-l Medium stone appearance

Colony Morphology in Boundary smooth and Boundary not smooth,

MSU-1 Medium restricted unrestricted

Attachment on Plastic After Late Early

Epsinization

Effect of FBS on Cell Growth Promoted Inhibited

EMA Expression + -

Keratin 18 Expression + —

Keratin 14 Expression - +

a6 Integrin Expression - +

Connexin 43 ~ +

Connexin 26 - +

Effect of Cholera Toxin Induction of Type I into Growth promotion
Type II HBEC

AIG on Soft Agar + -

Telomerase Activation and High Frequency Low Frequency

Irnmortalization by SV-40

Large T Antigen

Organoids Structures Formed Acini Hollow Spheres

on Matrigel Budding/ductal structure
in conjunction with Type
II HBEC

Gap Junctional Intercellular Deficient Proficient

Communication

 

 

 

22

 

Storage of HBEC, developed in MSU-l medium for
1 week, in liquid nitrogen

 

Thawi g HBEC
2 hours . Centrifugation of Supernatant
111911939011 in Growth of Pelleted Cells
Type I
Medium
Centrifugation of
Atta h ed Overnight Incubation in Supernatant
ecu:- Type 11 Medium Growth of Pelleted Cells
HBEC and ‘
fibroblasts \\\\\\\\\
Attached
Cells:
Type II
HBEC Attached Cells:
Type I
HBEC

Figure 1. Isolation scheme to obtain 2 types of Normal Human Breast
Epithelial Cells

23

b) Tumorigenic Breast Epithelial Cells

Immortal, weakly tumorigenic and highly tumorigenic breast epithelial cell lines have
been derived from Type I HBEC after step-wise treatment with SV40 large T-antigen, X-
rays and c—erbB2/neu oncogene (Kang et al., 1998) (Fig. 2). These in vitro derived cell
lines together with the well known breast carcinoma cell lines, MCF-7 (ER-positive) and

MDA-MB-231 (ER-negative), were also used in this study.

24

HME 13 Type I HBEC

Transfection with SV40 Large T-antigen

 

V
M13SV1 (Immortal)

X-ray irradiation
Selection of fast-growing colonies

M13SV1R2 (Weakly Tumorigenic)

Transfection with a mutated neu oncogene

‘M13SV1R2N1 (Highly Tumorigenic)

Figures 2 In vitro immortalization and neoplastic transformation of Type I HBEC
(Kang et al., 1998)

25

Western Blot Analysis of Maspin Protein Expression

Cells cultured in 10 cm dish, were washed with phosphate buffered saline (PBS) three
times, and lysed with 20% SDS lysis solution (500 pl) containing protease and
phosphatase inhibitors (Trosko et al., 2000). The viscous lysate was carefully collected
with a scraper and transferred to a tube on ice. The lysate in the tube was sonicated with
a sonicator (range~33) for 30 seconds 3 times. The proteins were aliquated in smaller

tubes and stored in -20°C freezer until use.

Protein concentrations were measured using Biorad Protein Quantification kit (Biorad,
California). Equal amount of protein (20 ug/lane) were separated by 12% SDS-PAGE
and transferred from the gel to PVDF membranes (Millipore Corp, Bedford, MA).
Irnmunoblotting was carried out using anti-hmnan maspin antibodies (BD Biosciences-
PharMingen, San Diego, CA, 1:1,000 dilution). Horseradish peroxidase-conjugated
secondary antibody was used for the chemiluminescent assay. The immunoreactive
protein complexes were detected by ECL-detection kit (Amersham, Life Science,

Denver, CO).

When the membrane was needed to be reblotted, the membrane was washed in 100 ml of
0.1 M citrate, pH=3.5, for 10 minutes. Then, the membrane was further washed in 100
ml Tris-buffered Saline (TBS) containing 0.5 M tris (pH=7.5), 0.05M NaCl and NP40
(IGEPAL) (0.5% v/v) for 20 minutes. After repeating the above two steps twice, the

membrane was washed twice with 100 ml TBS without IGEPAL for 10 minutes. After

26

this step, the membrane was reblocked with 5% milk in PBS with 0.1% Tween before

initiating the re-probing.

Immunocytochemical detection of maspin expression

Cells were plated into 8-well chamber slide (LAB-TEK, Nalge Nunc Int., Naperville, IL;
12,000 cells/well) for 2-3 days. The cells were rinsed with PBS and fixed with 4%
paraformaldehyde in PBS for 30 min. After rinse with PBS, the cells were permealized
with 3% H202 in methanol at room temperature for 10 min and treated with 10% FBS in
PBS for 30 min for blocking. These cells were then treated with primary antibody (a
mouse antihuman maspin monoclonal antibody from BD Biosciences-Pharmingen, Cat.
#554292, 1:100 dilution) for overnight at 4°C on a rocking platform, following rinse with
PBS, the cells were incubated with the secondary antibody (i.e. FITC-conjugated sheep
anti-mouse IgG, 1:200 dilution in 1% FBS in PBS) for 45 min at 4°C. The cells were
thoroughly washed with PBS and mounted with aqua-poly mount (Polysciences Inc.
Cat#18606) and a cover glass. The image and fluorescence of cells were observed and
recorded using a Nikon Eclipse TB 300 microscope connected to a digital camera and

computer. Images in this thesis are presented in color.

27

RESULTS

Difl'erent'mlexpressionofmaspininTypeIand'I‘ypeIIHBEC

TypeImflTypeHHBECswaeobminedfimreducfimnmmplaayfimudngmesdrane
describedinFig 1 bytaldngadvantageofflredifi‘amcemfimereqtnredforceflanadnnaumnoflre
plasticdishesaftercellplating Fmflmnmeflresehvotypesofl—IBECmnbeselectivelygrownusing
difi‘aartmediaieTypeIMSU-l medimnwiflrFBSpromotedthegrowthonypeII-IBEthile
inhibitedthegrowflr onypeII HBEC;TypeII MSU-1 mediurnwith BPEpromotedthegrowth of

TypeIIHBECandtheconversiononypeItoTypeIII-IBEC.

TypeIandTypeHHBECrfisplayawholesalerfifi‘amcehrphmotypesmaodal,1995;Changetal.,

2001) as showninTablel andcanbereoognizedmorphologicallyas shownin Fig 3.

BefomshrdyhgflreerqxessionofmaspmmflresecdlsTypeImxlTypeHHBECwae
nmnmncytodrmfimflystanwdwimcytokaafinceflmmkasmrecmfinnflrdrcdltypes Asshownin
arepresartative figure (Fig4), Type I HBEC ispositive forcytokeratin18,butmtcytoka'atin 14,

whereasTypeIIHBECispositiveforcytoka'atin 14,butnotcytokeratinl8.

28

 

figure3 MorphologissonypeIflop)andTypeII(Bottom)HBECsofHME30

29

Cytokeratin
1 4

TI I HBEC T. [I HBEC
Cytokeratin
18

“gum Expressbnonymkeratinl4andl8in'I‘ypeIand'I‘ypeIIHBECs

 

 

TopRight: Cytokeralinl4Expre$ioninTypeIHME30
BounmRight: CymkmtinrsExpmaoninrypeIHMEso
Toplcfi: Cytolmtin14ExprsssioninTypeIIHlv1E30

TopRight: Cytokeratinl8ExpressioninTypeIIIm’IE30

30

ToammeflreaqxemimmflcdhflarmhmfimofmaspmpotdnofflreZtypesofHBEC,
irrnnmocytodremicalstairfingsofthesecellswerecaniedout. AsslnwninFigS,maspinexpression
wasnotobsa‘vedinTypeII~IBEC,butwasclearlyseeninflrecytoplasmonypeHHBEC. AllTypeI

andTypeHwflsfiomflretwodifi‘a'entI-IBECstestedaiMEwand31)similarlyslmwedthatthe

maspinwasexprsssedinTypeIII-IBEC (Fig 5a, 5b),butnotinTypeII-IBEC (Fig 5a, 5b).

chmfinnafimweflanbldmflyfiswasdmcmfiedommmflwmmmofmaqfin
inTypeIarxlTypelIHBECderivedfiorntIneedifi‘a'entinfividuals Theresultsslnwflratmaspin(42
kD)washighlyeaq)ressedinTypeIIHBECs,lmtnotinTypeIHBECs(Fig 6). Althoughthelevelsof
equsimmyvmymrmgdiflaancdlaflmflmommawngoppodmmessimofmaspinm
TypeIarxlTypeIIHBECwasoonsistentintheseoellarlum

31

Typel .-
Type“ - -

figureSa MaspinExprssu’onbyImnnmostaininginT‘ypeIflofiandTypeIl
(Bottom)HBECofHME30
'I‘hephaseconn'astirnagssofcellsareshownontheright.

32

 

 

FigureSb MaspinExpressionbyImrmmostnininginTypeIflop)andTypeII
(Bottom)HBECofHME3l
Thephmecontrmtimagesofoellsareshownonflreright.

 

Lane 1 2 3 4 5 6

Maspin 1‘ M W

Actin

MR6 WestemetAnalysisofMasp’mExpresdonhTypeIandTypeII
HBECofHME29(hnesl,2),HIVIIBO(hnes3,4)andHIVIE31(IanssS,

6)

34

Expreesionofmasphrinr'nmnansformedHBECandcanceroefllines

A series ofin vitro immortalized and neoplastically transformed Type I HBEC lines (Ml38V1,
Ml3SV1R2, Ml3SV1R2Nl) weredraracterized formaspin expression. As shown in Fig. 7, all these
Type I HBEC neoplastically transformed at different stages (immortal Ml3SV1, weakly tumorigenic
M13SV1R2 and highly tumorigenic M13SV1R2N1) were negative in maspin expression by
irmnmncytodremicalsuxliessimilartononnalTypeIHBEC. Asexpected,thebreastcanoercelllines,

MCF-7 (ER+) and MDA-MB-231 (ER-) were negtive in maspin expression (Fig 8)

hrwestarrblotarralySisafltheinviuouansfonnedTypeIHBEClmesCLe immortalMlBSVl,weakly
tumorigenic M13SV1R2 and highly trnnorigmic M13SV1R2N1) (Kang et al., 1998) as well as the

MCF-7,MDA-MB-231de47Dbreastcmoa'cdlhnesweelbmdrutoexprelsflrenmspinpotein

(Fig 9).

35

t
:‘i

 

FIgure7 MaspinExpreesionofInvitmTransfondeypeIHBECLinesat
Difi'erentStageshy' ‘ ' ' "mt“
Thephasecontrastimagesofoelkareshownonflreright.

 

Figure 8 Maspin Expression in Breast Carcinoma Cell Lines, MCF-7 (mp),
MDA-MB-231 (bottom) by Immunostaining
'I‘hephaseoontrastimagesofwlkareshownontheright.

37

Lane 1 2 3 4 5 6 7 8

Maspin

Actin h i I b» a fig art a r g .3 ‘3'

Figure 9 Western Blot Analysis of Maspin Expression in normal HBEC, in
Vitro Immortalized or Neoplastically Transformed Type I HBEC and Breast
Carcinoma Cell Lines

Lane 1: TypeIHME 30 Lane 5: M13SV1R2N1
Lane 2: Type II HME 30 Lane 6: MCF7
Lane 3: Ml3SV1 Lane 7: MDA-MB-231
Lane 4: M13SV1R22 Lane 8: T47D

38

DISCUSSION

This study using immunocytochemical staining and Western blot analysis has
demonstrated that the human breast epithelial cell type (Type I HBEC) with stem cell
characteristics did not express maspin, whereas Type II HBEC with basal epithelial
phenotypes expressed maspin. Furthermore, a series of step-wise neoplastically
transformed cell lines derived from a Type I HBEC (Kang et al., 1998), i.e. immortal M
13SV1, weakly tumorigenic M 13SV1R2 and highly tumorigenic M 13SV1R2N1, did
not show maspin expression. Previously, normal human breast epithelial cells
(myoepithelial or basal epithelial) were shown to express maspin (Zou et al., 1994; Mass
et al., 2001a; Maass et al., 2001b) whereas the majority of human breast cancer cell lines
including MCF-7 and MDA-MB-231 did not show maspin expression (6/7 or 6/8) (Zou
et al., 1994; Mass et al., 2002). Our experiment also confirmed that these two well-
known breast cancer cell lines, MCF-7 and MDA-MB-231, did not express maspin.
Therefore, the results are not consistent with the idea that maspin is expressed in all
normal breast epithelial cells and that the expression of this serine proteinase inhibitor is

down-regulated or mutated during tumor progression (Maass et al., 2001b).

These results have several implications. First, similar to Cx 26, a-6 integrin and Oct-4
(Lee et al., 1991; Sager et al., 1993; Trosko et al., 2004) maspin is a breast epithelial stem
cell marker whose expression is preserved from stem cells to tumorigenic cells as shown
in the different Type I HBEC lines transformed at different stages. Although maspin has
been clearly shown to possess tumor-suppressing activity (Zou et al., 1994), its down

regulation in breast cancer cell lines (Zou et al., 1994; Maass et al., 2001a) or loss of

39

expression in tumor tissues, in contrast to cells in normal tissues such as myoepithelial
cells (Maass et al., 2001b), does not mean a real change has occurred in gene expression
from the target cells (i.e.Type I HBEC) to tumor cells. The results provide an alternative
origin of a tumor suppressor gene and an additional mechanism explaining how stem

cells could be target cells for carcinogenesis.

Second, since maspin disappears when Type I HBECs differentiate into other cell types,
maspin expression might be used to screen for chemopreventive agents. Indeed,
sphingoid bases (sphinosine and sphinganine), which are potential chemopreventive
agents, have been shown to induce the differentiation of Type I HBEC into Type II
HBEC with the concomitant emergence of maspin expression in Type II HBEC (Ahn,
2003). The effect of this type of agent could reduce the target breast epithelial stem cells

for carcinogenesis.

Thirdly, since down-regulation of maspin could be a marker for breast cancer and its
target precursor cell, the non-expression of this gene is expected to be early in
precancerous cells, i.e., ductal carcinoma in situ. This gene could be used as a marker for

early detection of precancerous cells.

When Type I HBECs differentiate into Type II HBEC, there is a wholesale change in the
expression of may genes and functions {Kao, Nomata, et al. 1995 24865 /id}{Chang,
Sun, et al. 2001 19479 lid} {Chang, Tsai, et al. 2004 31265 /id} {Chang, Sun, et al. 2001

19479 lid} {Chang, Olson, et al. 2004 31314 /id}. These conversions are clearly due to

40

 

epi genetic changes during cell differentiation. For maspin, the re-expression of maspin in
maspin-negative breast cancer lines (MCF-7, T-47D and MDA-MB-23 1) can be effected
by treatment with 5-aza-2’deoxycytidine and/or trichostatin A, indicating that DNA
methylation and/or histone deacetylation is/are partially responsible for the silencing of
the maspin gene in breast cancer cells (Futscher et al., 2002). Furthermore, it has been
shown that, in normal cells expressing maspin, the maspin promoter is unmethylated and
the promoter region has acetylated histones (Maass et al., 2002; Futscher et al., 2002). In
contrast, normal cells that do not express maspin have a completely methylated maspin
promoter with hypoacetylated histones (Maass et al., 2002). These mechanisms are very
likely to be involved in the silencing of maspin expression in Type I HBEC and can be

examined in future studies.

Bemards and Weinberg (Bemards and Weinberg, 2002) hypothesized that the metastatic
behavior of cancer cells seems to be determined relatively early in tumorigenesis. They
suggest that a subset of the mutant alleles acquired by incipient tmnor cells early in
tumorigenesis confer not only the selected replicative advantage, but also, later in
tumorigenesis, the proclivity to metastasize. Since maspin has been shown not only to
inhibit the proteinase activity but also to reduce tumor growth through a combination of
reduced angiogenesis and increased apoptosis (Shi et al., 2003), it is a gene fitting that
description. However, the silencing of maspin in tumor cells might not be due to
mutation or epigenetic alteration dming tumor progression, but rather due to continuous

expression of a target stem cell phenotype.

41

 

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