PI-PLCβ1 gene copy number alterations in breast cancer

Article (PDF Available)inOncology Reports 27(2):403-8 · November 2011with28 Reads
DOI: 10.3892/or.2011.1529 · Source: PubMed
Abstract
Deregulation of signal transduction pathways frequently confers selective biological advantages to tumors. Phosphoinositides play an essential role in numerous cellular functions and, among the enzymes implicated in these processes, phosphoinositide-specific phospholipase C β1 (PI-PLCβ1) is one of the key regulators. In the present study, a fluorescence in situ hybridization (FISH) approach was used to investigate PI-PLCβ1 gene copy number alterations in various types of breast cancer differing in their invasiveness and proliferative activity, according to their mitotic index. At the molecular level, we also performed both real-time PCR and immunohistochemical analyses on PI-PLCβ1 to further investigate its expression in primary breast cancers. Finally, we analyzed the correlation between PI-PLCβ1 gene copy number and clinicopathological parameters. Our results show that most of our cases had aneusomies on the PI-PLCβ1 locus (20p12) and amplification of this specific region was the most frequent alteration observed. Our findings also indicate that the amplification of the region containing the PI-PLCβ1 gene was mostly related to the mitotic index, rather than to the invasion status. Finally, even though our case series is limited, PI-PLCβ1 gene amplification seems to be correlated to clinicopathological parameters.

Figures

ONCOLOGY REPORTS 27: 403-408, 2012
Abstract. Deregulation of signal transduction pathways
frequently confers selective biological advantages to tumors.
Phosphoinositides play an essential role in numerous cellular
functions and, among the enzymes implicated in these
processes, phosphoinositide-specific phospholipase C β1
(PI-PLCβ1) is one of the key regulators. In the present study,
a fluorescence in situ hybridization (FISH) approach was
used to investigate PI-PLCβ1 gene copy number alterations in
various types of breast cancer differing in their invasiveness
and proliferative activity, according to their mitotic index. At
the molecular level, we also performed both real-time PCR
and immunohistochemical analyses on PI-PLCβ1 to further
investigate its expression in primary breast cancers. Finally,
we analyzed the correlation between PI-PLCβ1 gene copy
number and clinicopathological parameters. Our results show
that most of our cases had aneusomies on the PI-PLCβ1 locus
(20p12) and amplication of this specic region was the most
frequent alteration observed. Our ndings also indicate that
the amplication of the region containing the PI-PLCβ1 gene
was mostly related to the mitotic index, rather than to the
invasion status. Finally, even though our case series is limited,
PI-PLCβ1 gene amplication seems to be correlated to clini-
copathological parameters.
Introduction
Breast cancer is one of the most frequent malignancies in
women, with the highest incidence rate reported in Western
industrialized countries (1). The widespread occurrence and
clinical heterogeneity of this disease, along with its signicant
social impact, has driven the continuous search for new biomole-
cular markers to better characterize this tumor. A number of
different factors are involved in breast carcinoma pathogenesis,
making a correct biopathological characterization essential to
determine tumor aggressiveness and to identify the most appro-
priate therapy. Moreover, as numerous genetic alterations are
preserved throughout the evolution of breast cancer and a strong
correlation exists between the presence of precursor lesions and
the risk of developing invasive carcinoma (2-5), the discovery of
new molecular targets could play an important role in the early
diagnosis and monitoring of this disease.
In breast cancer, as well as in other tumors, the deregulation
of signal transduction pathways frequently confers selec-
tive biological advantages to tumor cells. Phosphoinositide
signaling has been implicated in several cellular functions, and
one of the key regulators of this pathway is phosphoinositide-
specific phospholipase C β1 (PI-PLCβ1), an enzyme that
catalyzes the formation of inositol 1,4,5-trisphosphate (IP3)
and diacylglycerol (DAG), important second messengers that
control numerous cellular functions (6-10). There are two
alternative splicing variants of PI-PLCβ1, PI-PLCβ1a and
PI-PLCβ1b (9), each of which has a different cellular local-
ization (1a, both cytoplasmic and nuclear; 1b, predominantly
nuclear) and modes of activation (11-13).
Aneuploidy and aneusomy are often correlated with
low rates of cell differentiation and with proliferation (14).
Furthermore, DNA amplification at specific chromosomal
sites is a leading mechanism of oncogene overexpression and
has an important impact on the deregulation of cell growth
and survival (15). Several studies have focused on structural
and copy number changes of chromosome 20 as it is often
altered in prostate, ovarian, bladder, pancreatic, colon and
breast cancer, and may be involved in disease initiation and
progression. It is well known that amplications along the
long arm of chromosome 20 are present in 5-40% of breast
cancers and are associated with cell immortalization, genomic
instability and more aggressive phenotypes, although their
prognostic value remains controversial (16-21). In addition, the
short arm, where PI-PLCβ1 is located (20p12.3) (10), has often
been found to be altered in several solid tumors, including
breast cancer (20,21).
The aim of the present study was to investigate the status
of PI-PLCβ1 in distinct classes of breast cancer which differ in
their degree of invasiveness and proliferative activity.
PI-PLCβ1 gene copy number alterations in breast cancer
CHIARA MOLINARI
1
, LAURA MEDRI
2
, MATILDE Y. FOLLO
3
, MANUELA PIAZZI
3
,
GIULIA ADALGISA MARIANI
3
, DANIELE CALISTRI
1
and LUCIO COCCO
3
1
Biosciences Laboratory, Istituto Scientico Romagnolo per lo Studio e la Cura dei Tumori, I-47014 Meldola;
2
Pathology Unit, Morgagni-Pierantoni Hospital, I-47100 Forlì;
3
Department of Human Anatomical
Sciences, Cellular Signalling Laboratory, University of Bologna, I-40126 Bologna, Italy
Received August 12, 2011; Accepted September 20, 2011
DOI: 10.3892/or.2011.1529
Correspondence to: Dr Chiara Molinari, Biosciences Laboratory,
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori,
Via P. Maroncelli 40, I-47014 Meldola (FC), Italy
E-mail: c.molinari@irst.emr.it
Key words: breast cancer, PI-PLCβ1, fluorescence in situ hybridi-
zation, chromosome 20, mitotic activity index
MOLINARI et al: PI-PLCβ1 AND BREAST CANCER
404
Materials and methods
Case series. The present study included 52 patients with breast
cancer at rst diagnosis recruited at the Morgagni-Pierantoni
Hospital in Forlì, Italy, between 2000 and 2006. The study was
approved by the local ethics committee and each patient gave
informed consent in accordance with the Institutional guide-
lines. The median age of the patients at diagnosis was 61 years
(range 39-86). All patients underwent surgery for primary
breast cancer and none had previously received neoadjuvant
treatment or had distant metastases at that time. Histological
tumor sections were provided by the Pathology Unit of the
same hospital, and fresh biopsy samples were sent to the
Biosciences Laboratory of our institute (I.R.S.T., Meldola,
Italy) and immediately stored at -80˚C until use. Tumors were
classied according to the World Health Organization (WHO)
histological classication (22,23): 20 cases (38%) were ductal
carcinomas in situ (DCIS) and the remaining 32 (62%) were
invasive ductal carcinomas (IDC). Histological grading was
performed using the criteria of Holland et al for DCIS (24) and
of Bloom and Richardson for IDC (25) as modied by Elston
and Ellis (26). The proliferative activity was determined using
the mitotic activity index (MAI), calculated as the number of
mitotic gures per 10 consecutive elds, chosen in high density
areas (27). We used the same MAI cut-off criteria as those of
standard clinical practice to determine proliferation indices:
<10 mitotic gures/10 elds, ≥10 and ≤19, and ≥20.
Alexa Fluor 555 PI-PLCβ1 probe preparation. The PI-PLCβ1
FISH probe was prepared by digesting 4 µg of PAC Clone
HS881E24 DNA (obtained from the P de Jong RPCI-5 PAC
library) overnight at 37˚C with 80 units of RsaI endonuclease
(Promega Corp., Madison, WI, USA). The next day, after
enzyme inactivation at 65˚C for 20 min, digested DNA was
puried by the QIAprep Spin Miniprep kit (Qiagen, Hilden,
Germany) and about half of the product was labeled by random
priming (BioPrime Plus ArrayCGH Genomic Labeling
System, Invitrogen, Milan, Italy) using Alexa Fluor 555 labeled
primers and nucleotides, according to the manufacturer's
instructions. After a 2-h incubation at 37˚C, the reaction was
blocked on ice by adding 5 µl of Stop Buffer (BioPrime kit,
Invitrogen). To assess labeling efciency, 3 µl of DNA were
electrophoresed on a 2% agarose gel, obtaining fragments
between 100 and 500 bp. The labeled probe was then puri-
ed using a Purication Module (Invitrogen), according to the
supplier's instructions and precipitated with 30 µl of 1 mg/ml
COT-1 DNA (Roche Diagnostics Inc., Mannheim, Germany),
1/10 v/v of 3 M sodium acetate (pH 5.6) and 3 volumes of
100% ethanol. The probes were stored for 30 min at -80˚C and
then centrifuged at 13000 rpm for 30 min.
Fluorescence in situ hybridization (FISH). PI-PLCβ1 and
human epidermal growh factor receptor 2 (HER2) genes were
analyzed by FISH using labeled PI-PLCβ1 (see above) and
LSI-HER2/neu-CEP17 probes (PathVysion™ HER-2 DNA
Probe kit; Vysis, Inc., Downers Grove, IL, USA). Briey, after
an overnight incubation at 56˚C, slides were deparafnized in
xylene for 30 min at 56˚C and 10 min at room temperature and
then dehydrated in two changes of 100% ethanol for 5 min. The
Parafn Pre-treatment kit (Vysis, Inc.) was used to prepare the
tumor sections: after a 10-min incubation in 0.2 M HCl solution
at room temperature, slides were washed in deionized water
for 3 min, wash buffer for 3 min, pre-treatment reagent (1 M
NaSCN) for 30 min at 80˚C and then rinsed once in deionized
water for 1 min and twice in wash buffer for 5 min. Samples
were then incubated in a solution of 0.2 M HCl per 4 mg/ml
protease at 37˚C for 25 min, rinsed twice in wash buffer for
5 min, incubated in neutral buffered formalin for 5 min, rinsed
twice in wash buffer for 5 min and then dehydrated. The HER2/
neu or PI-PLCβ1 probes (10 µl) were resuspended in 15 µl of
hybridization solution (50% formamide/2X SSC/10% dextran
sulphate), incubated for 5 min at 37˚C and then added to the
tumor sections. Slides were then coverslipped, sealed and
subjected to co-denaturation (85˚C for 5 min) and hybridiza-
tion (37˚C for 18 h in a humid atmosphere) in a Hybrite System
(Vysis, Inc.). The next day the rubber cement was removed and
the coverslips were oated off by soaking the slides at room
temperature in 2X SSC/0.3% Nonidet P-40. Slides were then
washed in the same solution at 7C for 2 min, dried in the
dark and counterstained with 4,6-diamidino-2-phenylindole
(DAPI) in antifade solution (Vysis, Inc.). The slides were
examined under an Axioscope 40 microscope (Carl Zeiss)
equipped with a triple lter (DAPI/Green/Orange; Vysis, Inc.).
At least 60 non-overlapping nuclei were counted in
contiguous x1000 microscopic elds to determine HER2 gene
copy number status. The number of signals per nucleus for the
HER2 gene and CEP17 region were counted on a cell-to-cell
basis, and tumors were dened as having HER2 gene ampli-
cation (HER2
+
) when the ratio between the total number of
HER2 and CEP17 signals was ≥2.0 (28,29). For PI-PLCβ1
evaluation, about 150-200 cells were counted. A sample was
dened as amplied for this gene when the percentage of cells
with more than two signals was at least 1.5-fold higher than the
percentage of cells with only one signal; otherwise, when this
percentage was lower, the sample was dened as deleted. All
remaining cases were dened as normal.
RNA extraction, retro-transcription and real-time polymerase
chain reaction (RT PCR). Total RNA was isolated from the
breast cell line SKBR3 and frozen tissues by the RNeasy Mini
kit (Qiagen) and cDNA was retrotranscribed from 500 ng of
total RNA in a nal volume of 20 µl using the iScript cDNA
Synthesis kit (Bio-Rad), according to the manufacturer's
instructions. The reaction was performed at 4C for 30 min,
followed by a 5-min incubation at 85˚C to block the enzyme.
Transcript levels of both PI-PLCβ1 splicing variants (1a and 1b)
were quantied using the MyiQ Single Color real-time PCR
Detection System (Bio-Rad) and SYBR-Green I approach.
PCR amplifications were carried out using the following
oligonucleotide primers, designed using the Beacon Designer
software (Version 4, Bio-Rad): PI-PLCβ1a forward primer,
5'-TGGATAAAAAGAGGCAGGAGAAGA-3' and reverse
primer, 5'-GCAGCTTGGGCTTTTCATCC-3'; PI-PLCβ1b
forward primer, 5'-GAAGAGGAGAAGACAGAGATG-3'
and reverse primer, 5'-TGGCAAGTTTCCGACAAG-3'.
A pool of 3 housekeeping genes was amplied as internal
controls using the following primers: β-actin forward primer,
5'-CGCCGCCAGCTCACCATG-3' and reverse primer,
5'-CACGATGGAGGGGAAGACGG-3'; β
2
-microglobulin
forward primer, 5'-CATTCCTGAAGCTGACAGCATTC-3'
ONCOLOGY REPORTS 27: 403-408, 2012
405
and reverse primer, 5'-TGCTGGATGACGTGAGTAAACC-3';
hypoxanthine ribosyltransferase (HPRT) forward primer,
5'-AGACTTTGCTTTCCTTGGTCAGG-3' and reverse
primer, 5'-GTCTGGCTTATATCCAACACTTCG-3'. The
cDNA of each tumor sample was added to a PCR reaction
mix containing 1X SYBR-Green mix (Bio-Rad) and primers
in a 25l reaction volume. Each gene was tested in duplicate
within the same PCR run. The reactions were carried out as
follows: an initial run of 90 sec at 95˚C, followed by 40 cycles
consisting of 30 sec at 95˚C and an annealing step specic
for each gene: 30 sec at 60˚C for the housekeeping genes and
45 sec at 60˚C for the two PI-PLCβ1 isoforms. For PI-PLCβ1
amplification, an additional step of 72˚C for 45 sec was
carried out to offset the small amounts of mRNA obtained.
Melting curve analysis was performed after each experiment
to verify amplication product specicity. Semi-quantitative
analysis was performed by the ∆Ct method using the Gene
Expression Macro Software (Version 1.1, Bio-Rad). The
amount of mRNA expression in each sample was normalized
to the pool of housekeeping transcripts and expressed as n-fold
in relation to the calibrator level, i.e., cDNA of breast cancer
cell line SKBR3, at known concentrations.
Immunohistochemical analysis. Protein expression levels
were determined by immunohistochemistry (IHC) using the
following primary antibodies: PI-PLCβ1 (sc-5291, Santa Cruz
Biotechnology, Santa Cruz, CA, USA); estrogen receptor
(ER), clone 6F11 (Novocastra Laboratories, Newcastle upon
Tyne, UK) and progesterone receptor (PgR), clone 636 (Dako,
Copenhagen, Denmark). Briey, 5-µm sections of formalin-
fixed and paraffin-embedded breast cancer samples were
deparafnized by xylene and rehydrated through graded alco-
hols, according to standard protocols. Tissue sections were then
quenched for endogenous peroxidases with 6% (v/v) H
2
O
2
for
10 min, incubated in citrate buffer 10 mM (pH 6.0) at 98.5˚C
for 40 min and left to cool in the solution for 20 min at room
temperature. Next, the slides were washed in PBS for 5 min and
incubated for 30 min at room temperature in specic pre-diluted
primary antibody: 1:20 PI-PLCβ1, 1:200 ER and 1:200 PgR.
Hormone receptors were identied using the Polymer Detection
kit (Novocastra Laboratories), while PI-PLCβ1 expression levels
were determined with the LSAB
+
System-HRP kit (Dako) using
an avidin-streptavidin reaction, according to the manufacturer's
instructions. Peroxidase activity was detected by diaminoben-
zidine reaction for 10 min before counterstaining with Mayer's
hematoxylin and each tissue sample was analyzed using an
optical microscope (Axioscope, Carl Zeiss). The primary anti-
body was omitted to obtain negative controls.
ER and PgR staining was considered positive when >10% of
tumor cells were distinctly stained. Semi-quantitative analysis of
PI-PLCβ1 expression was performed by counting the percentage
of stained cells and by scoring immunostaining intensity
(ranging from 0 to 3), reecting the intensity as follows: 0, no
staining; 1, weak staining; 2, moderate staining; and 3, intense
staining. The nal mean score was then obtained by multiplying
the intensity score by the percentage of stained cells.
Statistical analysis. Statistical analyses were carried out with
SPSS software (SPSS Inc., Chicago, IL). χ
2
and Fisher's exact
tests were performed to identify the association between patient
characteristics and PI-PLCβ1 status. Multivariate analysis was
conducted by running a backward stepwise regression.
Results
Tumor characteristics. Complete tumor characteristics are
summarized in Table I. Tumors were divided into four classes
according to their invasive status and proliferative activity:
DCIS, low proliferation rate with a mitotic activity index
(MAI)<10 (n=10); DCIS, high proliferation rate (MAI20;
Table I. Tumor characteristics.
A, Histotype and proliferation
Histological grade
a
% (n)
----------------------------------------------------------------------- E R
+
PgR
+
HER2
+
I II III % (n) % (n) % (n)
DCIS low proliferation (n=10) - 70 (7) 30 (3) 90 (9) 80 (8) 30 (3)
DCIS high proliferation (n=10) - - 100 (9) 30 (3) 10 (1) 60 (6)
IDC low proliferation (n=16) 40 (6) 60 (9) - 94 (15) 56 (7) 6 (1)
IDC high proliferation (n=16) 7 (1) 27 (4) 66 (10) 50 (8) 31 (5) 44 (7)
B, Phenotype % (n)
Luminal-like A: ER
+
/PgR
+
/HER2
-
56 (29)
Luminal-like B: ER
+
/PgR
+
/HER2
+
11.5 (6)
HER2-like: ER
-
/PgR
-
/HER2
+
21 (11)
Triple negative basal-like 11.5 (6)
a
Histological grade was not available for tumors <1 cm. DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; ER, estrogen receptor,
PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2.
MOLINARI et al: PI-PLCβ1 AND BREAST CANCER
406
n=10); IDC, low proliferation rate (MAI<10; n=16); and IDC,
high proliferation rate (MAI≥20; n=16). As Table I shows, low
proliferation DCIS were classi ed as grades II (70%) or III
(30%) and were mainly ER
+
and PgR
+
, with only 30% (n=3)
HER2
+
. For high proliferation DCIS, which were all grade III,
30% (n=3) were ER
+
, 10% (n=1) were PgR
+
and 60% (n=6)
were HER2
+
. In the IDC low proliferation class, 40% (n=6)
were grade I and 60% (n=9) were grade II tumors: 94% (n=15)
were ER
+
and 56% (n=7) were PgR
+
, while only 6% (n=1)
were HER2
+
. Finally, for high proliferation IDC, 7% (n=1)
were grade I, 27% (n=4) were grade II and 66% (n=10) were
grade III: 50% (n=8) were ER
+
, 31% (n=5) were PgR
+
and 44%
(n=7) were HER2
+
.
Hormone receptors and HER2 status were also used to
build four distinct phenotypes: ER
+
and/or PgR
+
/HER2
-
,
homologous to the luminal-like A phenotype; ER
+
and/or
PgR
+
/HER2
+
, homologous to the luminal-like B phenotype;
ER
-
/PgR
-
/HER2
+
, homologous to the HER2-like phenotype;
and ER
-
/PgR
-
/HER2
-
, homologous to the triple-negative basal-
like phenotype. On the basis of this classication, 56% of cases
(n=29) were homologous to the luminal-like A, 11.5% (n=6) to
the luminal-like B, 21% (n=11) to the HER2-like and 11.5%
(n=6) to the triple-negative basal-like phenotype.
PI-PLCβ1 copy number status in breast cancer samples. We
used the FISH assay to evaluate PI-PLCβ1 copy number status
in different classes of breast cancer (Table II). On the basis
of the previously reported criteria, the vast majority of cases
exhibited aneusomies of the locus containing the PI-PLCβ1
gene (deletion or amplification). The percentage of cases
showing copy number alterations in DCIS and IDC with the
same proli ferative index was similar for both deletions and
amplications, suggesting that the PI-PLCβ1 gene status could
be independent of invasiveness. Conversely, the percentage
of deleted or amplified cases in the distinct proliferative
classes differed substantially, especially for amplification.
Interestingly, the association between PI-PLCβ1 amplication
and the MAI was statistically signicant (p=0.001).
PI-PLCβ1 protein expression analysis in breast cancer
samples. Immunohistochemical analyses, performed in 50
patient samples, showed that breast cancer tissue expressed
higher levels of PI-PLCβ1 than normal breast tissue, which was
generally PI-PLCβ1 negative. An example of various staining
intensities, together with their relative scores, is presented in
Fig. 1. We used a cut-off value of 120 for PI-PLCβ1 protein
analysis, thus identifying a class that weakly or moderately
expressed PI-PLCβ1 (44%, n=22) and another that strongly
expressed the protein (56%, n=28). In contrast to copy number
status, PI-PLCβ1 protein levels seemed to be more strongly
associated with invasiveness than the proliferation index. In
fact, the number of low or high proliferating tumors showing
high protein expression was similar, while the difference in
protein expression levels between DCIS and IDC was greater,
albeit not statistically signicant.
PI-PLCβ1 mRNA expression analysis in breast cancer samples.
Quantification of PI-PLCβ1 mRNA splicing variants was
performed only on 12 samples of IDC with different prolife-
rative activities (50% low and 50% high proliferation) for
which frozen neoplastic tissue was available. Material was not
available for DCIS. The β-actin, β
2
-microglobulin and HPRT
genes were chosen as reference standards, while cDNA from
the breast cancer cell line SKBR3 was used as a calibrator.
Interestingly, our results showed that the relative amount of
the PI-PLCβ1b transcript was higher than that of PI-PLCβ1a.
Taking into consideration the proliferative classes, no apparent
differences were found in the amount of mRNA obtained for
PI-PLCβ1 splicing variants. However, a slight difference was
observed between tumors with PI-PLCβ1 gene amplication
and those with a normal copy number prole, becoming more
evident when the sum of both the transcripts was considered.
Furthermore, using the median expression value as a cut-off,
an association between PI-PLCβ1 gene amplification and
expression level was observed. In fact, a higher expression of
the PI-PLCβ1a and PI-PLCβ1b splicing variants was found in
Table II. Association between PI-PLCβ1 copy number status
and clinicopathological characteristics.
n (%)
a
--------------------------------------------------
Not amplied Amplied p-value
Mitotic activity index 0.001
<10 (low proliferation) 16 (62) 10 (38)
≥10 (high proliferation) 4 (15) 22 (85)
Histological grade 0.032
G1 4 (57) 3 (43)
G2 10 (50) 10 (50)
G3 5 (23) 17 (77)
ER (% positive nuclei) 0.049
<10 3 (19) 13 (81)
≥10 17 (47) 19 (53)
PgR (% positive nuclei) 0.07
<10 8 (28) 21 (72)
≥10 12 (52) 11 (48)
HER2 0.123
Not amplied 16 (46) 19 (54)
Amplied 4 (24) 13 (76)
Phenotype 0.161
Luminal-like A 15 (52) 14 (48)
Luminal-like B 1 (17) 5 (83)
HER2-like 3 (27) 8 (73)
Triple negative 1 (17) 5 (83)
basal-like
Lymph node status 0.365
N
-
12 (36) 21 (64)
N
+
3 (60) 2 (40)
Histotype 0.857
DCIS 8 (40) 12 (60)
IDC 12 (37) 20 (63)
a
Indicates the n and % of breast cancer samples with an amplied or
not amplied PI-PLCβ1 gene.
ONCOLOGY REPORTS 27: 403-408, 2012
4 07
amplied tumors. Moreover, considering both isoforms, 82% of
samples showed gene amplication and mRNA overexpression
(p= 0.061).
PI-PLCβ1 gene copy number and clinicopathological para-
meters. The association between PI-PLCβ1 status and the
main tumor characteristics (histological grade, ER or PgR
expression, HER2 or lymph node status, phenotype) is reported
in Table II. Considering all analyzed samples, independently
from MAI or histotype, univariate analysis highlighted
a significant correlation between gene amplification and
histological grade (p=0.032), with 77% (n=17) grade III
tumors exhibiting PI-PLCβ1 amplification. Conversely, in
our case series, the majority of samples that were ER
-
or PgR
-
displayed concomitant PI-PLCβ1 gene amplication (81 and
72%, respectively). However, statistical signicance was not
reached, probably due to the small number of cases analyzed.
Although there was no clear association between HER2 and
PI-PLCβ1 gene status, 13 cases showed concomitant HER2
and PI-PLCβ1 gene amplication. Furthermore, taking into
account the four phenotype classications, a higher number
of PI-PLCβ1-amplified tumors was observed in the most
aggressive phenotypes. No signicant association was found
between the PI-PLCβ1 gene and lymph node status. Overall,
multivariate analysis indicated that the proliferative index was
the only independent variable (p=0.003).
Discussion
Breast cancers frequently show evidence of aberrations in
signal transduction pathways, which can lead to increased
cellular proliferation, angiogenesis and metastasis, along with
inhibition of apoptosis. In the present study, we focused on
nuclear phosphoinositide signaling, which plays a crucial role
in cell proliferation and differentiation in normal and patho-
logical conditions. Several phospholipase isoforms have been
associated with breast cancer cell motility and invasiveness,
even though their exact function in tumor transformation
and prognosis has yet to be fully explored. Amongst these,
PI-PLCγ1 and PI-PLCδ4 up-regulation is related to cell
migration, proliferation and EGFR-directed tumor progres-
sion (30,31). Moreover, the PI-PLCβ2 isoform is overexpressed
in a high percentage of breast cancers and is associated with
histological grade and proliferative index, suggesting that its
up-regulation could inuence tumor differentiation and patient
prognosis (32,33). More recently, a specic role of isoforms
PI-PLCδ1 and PI-PLCδ3 in the growth and migration of
normal and neoplastic mammary epithelial cells has been
identied (34).
Several studies have shown that PI-PLCβ1 could play an
important role in cancer progression. In fact, this enzyme has
been identied as the principal mediator of the nuclear phosho-
inositide cycle, which is involved in cell cycle progression
(35). Moreover, chromosome band 20p12, where PI-PLCβ1 is
mapped, is rearranged in numerous solid tumors, but the role
of this alteration in cancer progression is still unknown.
The majority of our breast cancer cases showed aneuso-
mies in the PI-PLCβ1 locus, and the most common genetic
alteration was amplication (n=32). To further investigate the
role of PI-PLCβ1 amplication in breast cancer, we analyzed
PI-PLCβ1 gene and protein expression. Quantification of
PI-PLCβ1 mRNA by real-time PCR in IDCs showed that
PI-PLCβ1b transcript levels were higher than those of
PI-PLCβ1a. Although no signicant association was found
between the mRNA levels of each splicing variant and gene
copy number or proliferative activity, the sum of the 1a and
1b transcripts seemed to be correlated with gene amplication
(p=0.061), even though we only analyzed a small number of
cases. As for PI-PLCβ1 protein expression, although several
tumors with PI-PLCβ1 amplication also showed high protein
levels by immunohistochemical analyses, the association
between PI-PLCβ1 gene amplication and protein overexpres-
sion was not signicant.
To determine whether this alteration could affect tumor
characteristics, we investigated the association between
PI-PLCβ1 copy number and clinicopathological parameters,
Figure 1. Immunohistochemical analysis of PI-PLCβ1 expression in sections of normal and cancerous human breast tissue. (A) Normal breast tissue with
negative staining. (B-D) Ductal tumors with respectively weak, moderate and strong staining. Magnication x20 (A-C). (D) Magnied section (x40) of a
strongly-stained ductal sample to note the specicity of the immunoreaction.
MOLINARI et al: PI-PLCβ1 AND BREAST CANCER
408
such as proliferative activity and invasiveness. However, the
lack of any apparent difference in PI-PLCβ1 gene ampli-
cation between DCIS and IDC within the same proliferative
classes suggests that PI-PLCβ1 allelic status is independent
from tumor invasiveness. In contrast, PI-PLCβ1 gene ampli-
fication seemed to be closely related to MAI (p=0.001),
supporting the theory that in breast cancer this enzyme could
be involved in cell cycle processes (35). In fact, our results
also highlighted a statistically significant correlation with
histological grading (p=0.032), conrming that altered DNA
content frequently results in rapid cell proliferation and/or low
differentiation (14). Moreover, there was a borderline correla-
tion with hormone receptor status (ER, p=0.049; PgR, p=0.07)
and an inverse association between PI-PLCβ1 gain and the
luminal-like A phenotype (p=0.027). The presence of low
PI-PLCβ1 amplication in luminal-like A type tumors, which
have a relatively good prognosis among the four phenotypes
(36), is consistent with the hypothesis that this enzyme or other
genes within the 20p12 locus could be related to breast cancer
aggressiveness, given that we cannot dene the amplication
as solely affecting the PI-PLCβ1 gene.
In conclusion, our preliminary results show that PI-PLCβ1
may be altered in breast cancer. Further studies are needed to
dene its real role in this tumor and to elucidate the molecular
mechanisms regulating its activity in breast cancer.
Acknowledgements
The authors would like to thank Gráinne Tierney for editing
the manuscript.
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