Download Correlation of Heat Shock Protein 70 Expression With Estrogen

ANATOMIC PATHOLOGY
Original Article
Correlation of Heat Shock Protein 70
Expression With Estrogen Receptor Levels
in Invasive Human Breast Cancer
SHUJI TAKAHASHI, MD, 1 ' 2 TOSHIHIKO MIKAMI, MD, 3 YOSHIKI WATANABE, MD, 3
MINORU OKAZAKI, MD, 3 YUTAKA OKAZAKI, MD, 3 AKIRA OKAZAKI, MD, 3
TAKASHI SATO, MD, 3 KAZUAKI ASAISHI, MD, 3 KOICHI HIRATA, MD, 3
EIMEI NARIMATSU, MD, 1 MICHIO MORI, M D , ' NORIYUKI SATO, MD, 2
AND KOKICHI KIKUCHI, MD 2
The authors studied the role of 70-Kd heat shock protein (HSP70) in
the progression of breast cancer by examining the correlation between
the expression of HSP70 and epidermal growth factor receptor, c-erbB2, p53, and estrogen receptor in 124 cases of invasive primary human
breast cancers. Positivity of an anti-HSP70 monoclonal antibody, C92,
was closely associated with the elevation of estrogen receptor (P <
.008), whereas it inversely correlated with the expression of p53 (P <
.01). In addition, the expression of HSP70 correlated inversely with the
expression of epidermal growth factor receptor, although the correlation was not statistically significant (P = .06). These results suggest
that the expression of HSP70 plays a role in the progression of human
breast cancer. (Key words: Breast cancer; c-erbB-2; Epidermal growth
factor receptor; Estrogen receptor; Heat shock protein 70; lmmunohistochemistry; p53) Am J Clin Pathol 1994; 101:519-525.
Breast cancer is an important female neoplasm, and research is
being focused on its carcinogenesis and progression. Cancer
cells are thought to be monoclonal, although the phenotype of
hormone receptors in these cells may be heterogeneous.' Steroid hormones, which are important in growth regulation of
both normal breast tissues and their neoplastic counterparts,
facilitate the growth of mammary epithelial cells and breast
cancer cells.2 Estrogen has been thought to play a role in the
breast carcinogenesis via its receptor; therefore, endocrine
treatment has been used as an adjuvant therapy for breast
cancer. In the later stages of breast cancer progression, however, estrogen receptor-negative cells proliferate. Compared
with estrogen receptor-positive breast cancers, estrogen receptor-negative breast cancers have biologically more aggressive
characteristics, such as higher aneuploidy, higher histopathologic grade, enhanced proliferation, and higher tendency for
early recurrence.3"7
and heat shock protein 90 (HSP90) is important in receptor
function. Recently, other heat shock proteins, such as HSP70,
were also shown to be associated with steroid receptors.' 2 ' 3
Heat shock proteins have been implicated in many cellular
functions, such as chaperoning immature or denatured peptides and immunomodulation.' 4 In mammalian cells, two major members are found in the HSP70 family: an abundant,
constitutively expressed 73-kD protein (Hsc73), and a highly
stress-inducible 72-kD protein (Hsp72). Although these two
proteins are structurally related and have similar cellular functions, they are distinct gene products. 15 "' 7 Certain forms of
HSP70 are associated with mutant p53. 18 " 20 p53, a nuclear
phosphoprotein normally expressed at a low level, regulates
cell growth; however, its mutant form plays a role in malignant
transformation.21 According to previous immunohistochemical studies on human breast cancers, expression of p53 correlates with EGF-R and inversely correlates with estrogen receptor; however, the biologic background of such correlations
remains unclear.22"25 Another oncoprotein, c-erbB-2, which is
homologous to EGF-R, is also implicated in the progression of
breast cancer.26,27 Because members of the HSP70 family are
associated with steroid receptors and mutant p53, expression of
HSP70 is thought to play a role in the progression of breast
cancer. This hypothesis prompted us to examine the immunohistochemical expression of HSP70, EGF-R, c-erbB-2, and p53
in human breast cancer.
Although the precise mechanism of such biologic alteration
has not been well elucidated, estrogen receptor-negative breast
cancer is thought to acquire an autocrine mechanism. 8 In concordance with this hypothesis, certain estrogen receptor-negative breast cancer cells with highly proliferative activity were
shown to express epidermal growth factor receptors (EGF-R)
and to produce cytokines, such as transforming growth factor
a. 9 "'' Steroid receptors are associated with heat shock proteins,
We demonstrated that expression of HSP70 correlated with
From the 'Division of Pathology, Sapporo Medical University Hospi- elevation of estrogen receptor and inversely correlated with ex2
3
tal; and Departments of Pathology and * Surgery, Sapporo Medical pression of both EGF-R and p53. These results suggest that
University. Sapporo. Japan.
HSP70 may be involved in the progression of breast cancer.
Manuscript received December 28, 1992; revision accepted April 15,
1993.
Address reprint requests to Dr. Takahashi: Division of Pathology,
Sapporo Medical University Hospital, SI, W16, Chuo-ku, Sapporo,
060 Japan.
MATERIALS A N D METHODS
Breast cancer tissues were obtained from mastectomy and
processed immediately after surgery. These samples were
519
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ANATOMIC PATHOLOGY
Original Article
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FIG. 1. Immunohistologic detection of HSP70 (A), p53 with PAb 1801 (B), EGF-R (C), and c-<?r6B-2 (D) in human breast cancer (original
magnification, X 80 each.)
soaked in Tissue-Tek compound (Miles, Inc., Elkhart, IN) and
frozen at - 8 0 °C. Mouse monoclonal antibody to EGF-R,
clone EGFR1 (Amersham Japan, Tokyo, Japan). Anti-p53 antibodies, clones PAb 1801 and PAb 240 (Oncogene Science,
Manhasset, NY), react with epitopes between amino acids 3279 and 156-214, respectively.28-29 Another anti-p53 antibody
used was clone 1620 (Oncogene Science), which preferentially
reacts with wild type p53. 30 Anti-c-<?r6B-2 antibody, clone
NCL-CB11 (Novocastra Laboratories, Newcastle, UK) was
also used. Anti-HSP70, clone C92 (Amersham) preferentially
reacts with Hsp72 of HSP70 family.31 An anticytokeratin antibody (clone KL1; Immunotech S.A., Marseille, France) was
used to identify epithelial components, and material that
stained poorly with this antibody was excluded from further
analysis. A mixture of class-matched negative control antibodies was used.
For immunoperoxidase staining, frozen sections (6 fim
thick) were placed on poly-L-lysine-coated glass slides and
fixed in chilled acetone. The slides were air-dried for 1 hour,
and primary antibodies were applied according to the standard
avidin-biotin system as recommended by the vendor (Nichirei,
Japan). Sections were stained with 3, 3'-diaminobenzidine
(Sigma Chemical, St. Louis, MO), and the nuclei were counterstained with methyl green. Other sections were used for hematoxylin-eosin staining.
Estrogen receptor content of the tissues was measured by
dextran-coated charcoal assay (Otsuka Assay Laboratories, Tokushima, Japan). The value (fmol/mg) obtained was closely
correlated with the expression of estrogen receptor in immunostaining.32
For statistical analysis, contingency tables were used to examine the relationship between expression of HSP70 and p53,
EGF-R, and c-erbB-2. The chi-squared test was performed to
determine whether the relationship was statistically significant.
The Mann-Whitney U (nonparametric) test was used to examine the relationship between estrogen receptor and HSP70,
p53, EGF-R, and c-erbB-2, because the distribution of estrogen
receptor was not normal. Correlations between estrogen receptor grade and patient's age were determined by the KruskalWallis test. P values less than .05 were considered significant.
Detection of estrogen receptor was not performed on all patients, because of limited material.
RESULTS
Immunostaining patterns of HSP70, EGF-R, p53, and cerbB-2 are shown in Figure 1. Expression of HSP70 was detected in the nuclei and perinuclear regions of the cancer cells,
in contrast to the surrounding fibrous tissues, which showed
indistinct immunoreactivity with the anti-HSP70 antibody
A.J.C.P.-April 1994
AMERICAN JOURNAL OF CLINICAL PATHOLOGY
-am
Erratum
In the article, Correlation of Heat Shock Protein 70 Expression With Estrogen Receptor Levels in Invasive Human Breast
Cancer, by Takahashi and colleagues, in the April 1994 issue ofAmerican Journal ofClinical Pathology, the art for Figures 1 and 2
did not match their corresponding legends.
We are printing the correct Figures 1A-D and 2A-H and their correct legends.
We regret any inconvenience this error may have caused.
FIG. 1. Immunohistologic detection of HSP70 (A), p53 with PAb 1801 (B), EGF-R (C), and c-erbB-2 (D) in human breast cancer (original
magnification, X80 each.)
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FIG. 2. Comparative immunostaining of HSP70
(A,C,E,G)andp53(B,D,
F, H). Case 1 was a 55year-old patient with invasive ductal carcinoma.
The estrogen receptor
level was 19.4 fmol/mg.
Heat shock protein 70 was
negative in the cancer
cells, whereas expression
of p53 was distinct in the
nuclei of the cancer cells
(A, B). Case 2 was a 55year-old patient with medullary carcinoma. The estrogen receptor was undetectable. Like case 1, distinct p53 expression was
demonstrated, whereas
HSP70 was undetectable
(C, D). Case 3 was a 58year-old patient with invasive ductal carcinoma (estrogen receptor: 31.4 fmol/
mg). Reactivity of HSP70
was found in both the cytoplasm and nucleus, although p53 was not detected (E, F). Similarly,
case 4 was a 61 -year-old patient with invasive ductal
carcinoma (estrogen receptor, 292 fmol/mg). Evident HSP70 expression
was demonstrated in most
of the cytoplasms, but was
negative for p53 (G, H)
(original
magnification,
X80 each.)
FIG. 2. Comparative immunostaining of HSP70 (A, C, E, G) and p53 (B, D, F, H). Case 1 was a 55-year-old patient with invasive ductal carcinoma. The estrogen receptor level was 19.4
fmol/mg. Heat shock protein 70 was negative in the cancer cells, whereas expression of p53 was distinct in the nuclei of the cancer cells (A, B). Case 2 was a 55-year-old patient with
medullary carcinoma. The estrogen receptor was undetectable. Like case 1, distinct p53 expression was demonstrated, whereas HSP70 was undetectable (C, D). Case 3 was a 58-year-old
patient with invasive ductal carcinoma (estrogen receptor, 31.4 fmol/mg). Reactivity of HSP70 was found in both the cytoplasm and the nucleus, although p53 was not detected (E, F).
Similarly, case 4 was a 61 -year-old patient with invasive ductal carcinoma (estrogen receptor, 292 fmol/mg). Evident HSP70 expression was demonstrated in most of the cytoplasms but
was negative for p53 (G, H). (Original magnification, X 80 each.)
522
ANATOMIC PATHOLOGY
Original Article
TABLE 1. RELATIONSHIP AMONG HISTOLOGIC TYPES OF BREAST CANCER AND EXPRESSION OF HSP70, P53, EGF-R,
AND C-ERBB-2 IN BREAST CANCER
HSP70
-
+
Invasive ductal
Invasive lobular
Mixed*
Medullary
Mucinous
Apocrine
58(53)
0(0)
1(33)
2(50)
0(0)
1(50)
52 (47)
2(100)
2(67)
2(50)
3(100)
1(50)
Total
62 (50)
62 (50)
c-erbB-2
EGF-R
p53
Histological
Type
-
+
92 (87)
1(50)
3(100)
1(25)
3(100)
2(100)
18(16)
1(50)
0(0)
3(75)
0(0)
0(0)
102 (82)
22(18)
-
+
97 (88)
2(100)
3(100)
2(50)
3(100)
1(50)
108 (87)
13(12)
0(0)
0(0)
2(50)
0(0)
1(50)
16(13)
-
+
90 (82)
2(100)
3(100)
4(100)
3(100)
2(100)
104 (83)
20(18)
0(0)
0(0)
0(0)
0(0)
0(0)
20(17)
Values arc expressed as numbers (percent).
* Mixed ductal and lobular carcinoma.
was not found in the estrogen receptor-high-positive group (estrogen receptor grades 3 and 4). Similar to p53, expression of
EGF-R demonstrated an inverse correlation with the estrogen
receptor grade (P < .03). Positivity of c-erbB-2 also showed an
inverse correlation with the estrogen receptor grade, although it
was not significant (P = .08). In concordance with previous
reports, our results demonstrated a significant correlation between the estrogen receptor level in breast cancer and patient
age (P < .004; data not shown). However, the age distribution
did not show any correlation with HSP70 (mean age of
HSP70-positive patients was 53.6 years; mean age of
HSP70-negative patients was 50.1), p53 (positive, 50.3; negative, 52.2), or EGF-R (positive, 50.9; negative, 52). These results suggest that the markers are specifically correlated with
estrogen receptor expression.
We further analyzed the coincident expression of p53 and
EGF-R. As shown in Table 3, a significant correlation between
p53 and EGF-R was observed (P < .002). Despite the structural
similarity between EGF-R and c-erbB-2, expression of c-erbB2 did not show any correlation with expression of p53 (P = .54,
Table 3).
Because HSP70 correlated with estrogen receptors, and the
expression of both p53 and EGF-R inversely correlated with
estrogen receptors, we speculated that the positivity of HSP70
might inversely correlate with both p53 and EGF-R. Reactivity
of p53 was detected in only 8% of HSP70-positive breast cancer
tissues (Table 4). In contrast, HSP70-negative cases showed a
(Figs. I A, 2E and G). Approximately half the cases expressed
HSP70 (Table 1). A positive reaction for p53 was detected in
the nucleus of each cell with various degrees of intensity (Figs.
IB, 2B and D). Twenty-two of 124 (18%) cases in this study
demonstrated detectable p53 (Table 1), which is comparable
with previous reports.21"24 Epidermal growth factor receptor
was localized in the cytoplasm or membrane and homogeneously expressed in most cancer cells (Fig. 1C). Myoepithelial
cells in normal breast tissues were positive for EGF-R, as previously reported.27 Thirteen percent of breast cancers were positive for EGF-R (Table 1), and c-erbB-2 was diffusely detected
in the cytoplasm of cancer cells in 17% of cases (Fig. ID and
Table 1).
As shown in Table 1, no significant relationship was found
between the histologic type of the tumor and the frequency of
expression of oncogene products or HSP70 positivity. In medullary carcinoma, however, p53 expression showed a significantly higher incidence (75%) than the others (18%). Positivity
of HSP70 in breast cancer increased significantly as the estrogen receptor grade increased, from 39% in the estrogen receptor-negative group to 80% in the estrogen receptor-high-positive group (P < .008, Table 2). In contrast to HSP70, the
highest incidence of p53 expression was observed in the estrogen receptor-negative and estrogen receptor-low-positive
groups (estrogen receptor grade 0 and estrogen receptor grade
1), and the expression of p53 decreased significantly as the
estrogen receptor grade advanced (P < .003). p53 expression
TABLE 2. RELATIONSHIP BETWEEN ESTROGEN RECEPTOR EXPRESSION AND HSP70, P53, EGF-R,
AND C-ERBB-2 IN BREAST CANCER
HSP70
ER Grade'
0
1
2
3
4
Total
23(61)
6(60)
19(53)
7(44) •
4(20)
+
-
+
15(39)
4(40)
17(47)
9(56)
16(80)
27(71)
7(70)
28 (78)
16(100)
20(100)
11 (29)
3(30)
8(22)
0(0)
0(0)
61(51)
59 (49)
(P < 0.008)
c-erbB-2
EGF-R
P53
98 (82)
22(18)
(P < 0.003)
+
28 (74)
9(90)
33 (92)
15(92)
19(94)
104 (87)
(P
10 (26)
1(10)
3(8)
1(8)
1(6)
16(13)
< o.o3;)
Values arc expressed as numbers (percent).
" Expression of estrogen receptor was graded as follows: 0. = <5 (fmol/mg); 1. 5< = < I 0 : 2, 10< = <50; 3, 50< - <100; 4. 100<.
A.J.C.P. 'April 1994
29 (76)
7(70)
31 (86)
16(100)
17(85)
+
9(24)
3(30)
5(14)
0(0)
3(15)
20(17)
100(83)
(P = 0.08)1
523
TAKAHASHI ET AL.
HSP70 in Breast Cancer
TABLE 3. RELATIONSHIP BETWEEN P53 EXPRESSION
AND EGF-R AND C-ERBB-2 IN BREAST CANCER
EGF-R
c-erbB-2
-
-
+
+
Total
P53
94 (92)
14 (64)
+
Total
8(8)
8(36)
87 (85)
17(77)
15(15)
5(23)
102
22
108 (87)
16(13)
(P< 0.002)
104(84)
20(16)
124
(/> =0.54)
Values are expressed as numbers (percent).
higher incidence (27%) of p53 expression (P < .01), suggesting
an inverse correlation between HSP70 and p53 in human
breast cancer.
A higher incidence of EGF-R expression was observed in
HSP70-negative tumors; EGF-R was observed in 19% of the
HSP70-negative group, in contrast to 6% of the HSP70-positive group, although these incidences were statistically insignificant (P = .06, Table 4). We did not find an association between
HSP70 expression and c-erbB-2 (P = .46).
Representative immunohistochemical findings, with a combination of anti-p53 and anti-HSP70, are shown in Figure 2.
Case 1 was invasive ductal carcinoma with estrogen receptorlow-positive expression (Fig. 2A and B). Case 2 was an invasive
medullary carcinoma with negative estrogen receptor (Fig. 2C
and D). Both cases were negative for HSP70 and positive for
p53. Unlike these cases, case 3 was an invasive ductal carcinoma with a positive estrogen receptor level (Fig. 2E and F).
Case 4 was an invasive ductal carcinoma with a highly positive
estrogen receptor expression (Fig. 2G and H). Both cases were
positive for HSP70 and negative for p53.
The immunohistochemical results obtained in this study indicated that positivity for HSP70 in breast cancer cells correlated with estrogen receptor levels and inversely correlated with
both EGF-R and p53.
DISCUSSION
We immunohistochemically examined the relationship between expression of HSP70 and the levels of estrogen receptor,
EGF-R, and p53 in 124 cases of invasive human breast cancer.
Estrogen receptor levels are significantly implicated in breast
cancer biologically and clinically. Most tumor cells are estrogen-dependent in the early stages. In the later stages, however,
the estrogen receptor-negative population dominates, which
raises major obstacles to endocrine treatment of breast cancer.
Estrogen receptor-negative breast cancer often expresses oncogene products, such as EGF-R, p53, and c-erbB-2.9'022'2'' Epidermal growth factor receptor, which is expressed in 20% to
60% of human breast cancers, inversely correlates with estro1
In an experimental study, overgen receptor expression.
produced EGF-R and transforming growth factor-a induced
transformation of immortalized mouse fibroblasts.33 The
proto-oncogene p53 (wild type), a nuclear phosphoprotein normally expressed at a low level in all human cells, regulates cell
growth and division. Mutant p53 forms a complex with wild
type p53 and eliminates the function of the wild type.34 p53
expression is observed in 15% to 45% of breast cancer cells in
correlation with EGF-R expression, which suggests that these
oncoproteins play a cooperative role in malignant transformation.21-22 p53 correlates with established prognostic factors: age,
stage, metastatic involvement, concentration of estrogen and
progesterone receptors, and proliferative index.24 Thus, p53
expression is also considered a prognostic factor in human
breast cancer. However, the biologic significance of p53 in estrogen receptor-negative breast cancer has not been elucidated.
We used monoclonal antibody C92 to detect immunohistochemically Hsp72 among the HSP70 family.31 Heat shock protein 72 is highly inducible in mammalian cells. The antibody
did not react with another member of the HSP70 family,
Hsc73, which is an abundant and constitutively expressed heat
shock protein. Although Hsp72 and Hsc73 are structurally related and assumed to perform similar functions in the cell, they
are products of different genes.14"16
At present, the nature of the "stress" that induces heat shock
protein in breast cancer cells is not known. Our results revealed
that the expression of HSP70 strictly correlated with estrogen
receptor (P < .008). Because steroid receptors are physiologically associated with various heat shock proteins, including
HSP70, to form a multimolecular complex in normal tissues,"12 the heat shock protein may be induced because of a
physiologic demand, such as that induced by estrogen. Supporting this notion, two other types of heat shock protein,
HSP27 and HSP89, were inducible by estrogen in breastcancer cell lines.3536 Alternatively, a stressful interaction might
be found between estrogen receptor-positive breast cancer cells
and surrounding tissues in the early stages of oncogenesis.
Emergence of mutant p53 may relieve the tumor cells from the
stress in the later stage of oncogenesis, because overproduction
of mutant p53 made established rat cells highly tumorigenic.37
In relation to expression of HSP70, recent reports have demonstrated that heat shock proteins are involved in immunologic
surveillance. A member of the HSP70 family binds to a peptide
fragment of cytochrome c on murine B lymphocytes and is
TABLE 4. RELATIONSHIP BETWEEN HSP70 EXPRESSION AND P53, EGF-R, AND C-ERBB-2 IN BREAST CANCER
EGF-R
p53
+
-
c-erbB-2
+
-
+
-
HSP70
+
Total
45 (73)
57 (92)
102(82)
(/><0.01)
Values are expressed as numbers (percent).
17(27)
5(8)
50(81)
58 (94)
22(18)
108(87)
(P = 0.06)
12(19)
4(6)
54 (87)
50(81)
16(13)
104(84)
(P = 0.46)
Total
8(13)
12(19)
62
62
20(19)
124
524
ANATOMIC PATHOLOGY
Original Article
recognized by specific T lymphocytes.38 The other type of heat
shock protein, HSP65, interacts with T cells bearing the y/b
type T cell receptor.39 Thus, expression of HSP70 may lead to
immunologic interaction with the host, which, in turn, may
provide a better prognosis for patients with estrogen receptorpositive breast cancer.
Our results demonstrated that an inverse correlation exists
between HSP70 and p53 expression in human breast cancer.
Expression of p53 was detected by PAbl801 antibody, which
reacted with both wild and mutant p53. The PAb 1801-positive
specimens readily reacted with the PAb240 antibody, which
binds solely with mutant p53, but not with the PAb 1620 antibody, which reacts preferentially with wild p53, but not mutant
human p53 by immunohistochemistry (data not shown).29'30
Thus, expressed p53 can be attributed to a p53 mutation,
which may reflect a high frequency (60%) of allelic depletion of
17p.40 This is seemingly antithetical to the fact that mutant p53
is physically associated with HSP70 proteins, which stabilize
p53 and give rise to a longer half life than that of the wild
type.17"1941 However, such HSP70 proteins consist mostly of
Hsc73, and a quantitative association of human Hsp72 with
mutant p53 has not been reported, except for an osteosarcoma
cell line.19 Physiologic supplementation of HSP70 may rarely
occur in such an unphysiologic situation as cancer, unlike steroid receptors. Our results also demonstrated an inverse correlation between HSP70 and EGF-R in human breast cancer.
Estrogen receptor levels correlated inversely with both p53 and
EGF-R. We therefore speculate that the inverse correlation between HSP70 and these oncogene products is produced by specific interaction of HSP70 with estrogen receptor.
Further biochemical and molecular investigations are necessary to elucidate the significance of expression of HSP70 correlated with estrogen receptor levels and the inverse correlation
of HSP70 with expression of both p53 and EGF-R in human
breast cancer. However, HSP70 might be an important element in the puzzling correlation between p53 expression and
negativity for estrogen receptor in human breast cancer.
Acknowledgment. The authors thank Mrs. Hiroko Asanuma, Mr.
Kyogo Azuma, Mr.Yosihiro Kishi, and Mr. Eihin Yamamoto for their
excellent technical assistance, and Dr. Pekka Klemi for reviewing the
manuscript.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
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