Indian Journal of Experimental Biology Vol. 38, July 2000, pp. 663-668

Adrenocorticotropic hormone and growth factor receptors in breast cancer

Amitabha Ray", Nitesh Ratnakarb, Nandagudi Srinivas Murthy" & Bhuvnesh Kumar Sharma "*

"Insti tute of Cytology and Preventi ve Oncology (ICMR), Maul ana Azad Medical Coll ege Campu s, New Delhi II 0 002, India

hDepartment of Cardio-thoracic and Vascul ar Surgery, G.B Pant Hospital, New Delhi II 0 002, Ind ia

Received 14 May 1999; revised 25 Febm ary 2000

Tissues from I 00 cases of breast cancer were analysed immunohistochemicall y fo r the presence of adrenocorticotropic hormone (ACTH) or ACTH-Iike peptides and expression of c-erbB-2 oncoprotein, epi dermal growth factor receptor (EGF­R) as well as oestrogen receptor (ER). Immunopositivity for ACTH was found in 15% cases of infiltrating duct carcinoma of the breast, whereas 38% and 36% breast tumours were positive for c-erbB-2 and EGF-R respect ively. While 27% cases were positive for ER. The immunoexpressions of all parameters were hi gher in breast cancer cases with upper age group (45 years or above) than the patients below 45 years of age. A significant correlati on was observed between the tu mour grade and the expression of c-erbB-2 oncoprotein. Further, a positive association between the immunoex pression of c-erbB-2 and EGF-R was noticed. Interestingly, a stat isticall y signiticant relationship was fo und between the immunopositivity of ACTH and ER. The study reflects a probable associat ion of ACTH or ACTH-Iike peptides in pathological process of breast cancer.

Carcinoma of the breast is the second most common cancer among Indi an women. The age adjusted rates vary between 2 I .9 and 28.3 per I 00,000 in the populations as well as hospital based urban cancer registries of the country (NCRP I 988-89) 1

• It is estimated that by the end of thi s century, annually 86,000 women would develop breast carc inoma.

Several lines of research suggest that steroid hormones are closely related with the pathological process of breast cancer. Positi ve assoc iati ons have been reported between breast cancer and semm levels of androgens2

. In females, adrenal androgens are important substrates during pregnancy and in post­menopausal women. Androgens may be converted into oestrogen (aromatization) e ither in the granulosa cell s of the ovarian follicle or in fat and other ti ssues. The factors which control aromatase activity in peripheral ti ssues are largely unknown. Glucocorticoids and cyclic AMP (cAMP) are capable of inducing aromatase in adipose ti ssue and, conversely, growth factors such as epidermal growth fac tor (EGF) and transforming growth factor alpha (TGFa) are inhibitor/.4. Since, many of these polypeptide factors are produced by breast cancecs, the potential ex ists for paracrine interac tion between tumour and adipose ti ssue within the breast5

. The most frequently implicated receptors and growth factors in human cancers, which may act in a paracrine fashi on6

, are members of the epidermal growth factor receptor sub-

*Correspondent author: Fax : l) 1-11-3233406.

family (like, EGF-R, c-erbB-2, etc.) and their ligands. It is reasonable to suggest that tumour cell s wh ich overexpress a given growth factor receptor will have a selective advantage over those which do noe.

All mammalian steroid hormones are formed fro m cholesterol via pregnenolone through a series of reactions that occur in either the mitochondria or endoplasmic reticulum of the adrenal cell. Upon stimul ation of the adrenal by ACTH (or cAMP), cholesterol is converted to pregnenol one. ACTH is single-chain polypeptide consisting of 39 amino acids. ACTH, like other peptide hormones, binds to a plasma membrane receptor. Within a few seconds of this interacti on, intracellular cAMP levels increase markedll.

Since hormones of the hypothalamic-pitui tary­adrenal system have an important role in the pathology of breast cancer9

-11

, the aim of this study was to search the presence of ACTH directly within the breast cancer ti ssue and its re lation, if any , with commonly assoc iated prognostic fac tors, i.e., nodal status , tumour grading, EGF-R, c-erbB -2 oncoprotein and ER.

Materials and Methods Patients - This study was conducted on breast

cancer ti ssues randomly collected from the Department of Pathology, Maulana Azad Medical College, taken over a period of I 994-97. A total of I 00 cases of histopathologically diagnosed primary infiltrating duct carcinoma (IDC) of breast were selected for the study. Data concerning clinical

664 INDI AN J EXP BIOL, JULY 2000

hi story, demography, marital status, family history , extent of di sease were also obtained. The mean age of the patients was 46.7 years (SD ± 12.3). All women included in thi s study were married and parous (mean

parity= 3.3 ± 0 .8) . None of them had a family history of breast cancer. All tumours were graded according to the criteri a of Bloom and Richardson

12

Immunohistochemistry - The immunohistochemical staining for the c-erbB -2 (neu) oncoprote in, EGF­receptor, oestrogen receptor (ER) and ACTH were carried out on 5 Jlm thick paraffin embedded ti ssue secti ons of breast cancer. Paraffin sections from fibroadenoma and lipoma were taken as negati ve controls. Tissues from 3 adrenal tumours were used as positi ve contro l for immunohi stochemical staining of ACTH. The secti ons were deparaffini zed by washing them in xylene and toluene, then put through success ive a lcohol washes, rehydrated and washed in phosphate buffe red saline (PBS). Endogenous perox idase enzy me ac t1 v1ty was blocked by incubati on of the secti ons in 6% H20 2 soluti on in methanol, fo ll owed by incubati on in appropri ate normal serum. Optima ll y diluted pri mary antibody was then applied to each secti on. The foll ow ing antibodies were used : mouse monoc lonal antibodies against c-erbB-2 (Boehringer Mannhe im, Germany), EGF-R (Sigma, USA), ER (ER-D5 anti gen, A mersham, UK) and rabb[t monoclona l antibody aga inst ACTH (Sigma, USA). Ti ssue secti ons were incubated with optimall y dilu ted primary antibody fo r 60 min at room temperature in a humid chamber. Secti ons were then rinsed in PBS and subsequentl y, the t issue secti ons were incubated with secondary an tibody fo r 60 mi n at room temperatu re. Arter three washing in PBS with Tween 20 (0.005%), the sections were incubated with peroxidase­antiperoxidase complex fo r 60 min at room temperature . The reaction was visualized by substrate diaminobenzidi ne (DAB, 0 .1% solut ion in PBS with 0.05% H20 2). After chromogen deve lopment, the sections were washed with double d ist illed water and then countersta ined wi th haematoxylin . T he sec ti ons were then moun ted and observed under microscope. Each sect ion was assessed independentl y and blindl y by 2 observers . There was 95% agreement between the observers.

Statistical analysis- The data were analysed on PC586. Initi a ll y, bi vari ate tables were tabul ated according to ACTH and othe.r va riabl es on which the inform<: tion was ava ilable. Similarl y, bi variate tables were also tabul ated with the vari ous combinati ons of

the variables studied . Prevalence of ACTH was calculated according to each level of the various factors studied. Odds ratio (OR) and 95% confidence intervals were estimated . Further, chi-square test of signifi cance was employed in order to test the differences in the prevalence rate of ACTH according to different levels of the variables. To test for the associ ation between the various combinati ons of the factors studied chi -square test of significance was also employed . For small samples F isher's exact test of s ignificance was used .

Results Out of I 00 spec imens of primary breast cancer, 38

(38%) specimens showed immunoexpression for c­erbB-2 oncoprote in , 36 (36%) cases were positi ve for EGF-R and 27 (27%) cases revealed immunopositi­vity for ER [Figs. I (A) to (C)]. The irnmunopositivity for ACTH was observed in 15 ( 15%) cases onl y [Fig I(D)]. Out of 15 cases of ACT H immunopos iti ve tumours, 3 cases (20%) belonged to low grade, 7 cases of moderate grade (47%), whil e 5 cases (33%) were of high grade. However, I I ou t of 15 ACT H pos itive tumours (73%) showed invo lvement of lymph nodes. The study subjects compri sed of low (n = 25), moderate (n = 49) and hi gh

0l ade (n = 26) of

tumours while 55 cases had metastasi s in lymph node (Table 1).

Overall , the immunopos iti vity of al l parameters in the present study showed a relati onsh ip with the age of the patients. Cases with age below 45 years (n = 42) revealed only I immunoposit ive tu mour (6.7%) for ACTH. Whereas 14 tumours (93.3%) showed immunopos iti vity fo r ACTH peptides among pa ti ents (n = 58) with age 45 or above . Similarl y, 10 cases (26.3%) \..'ere pos iti ve fo r c-erbB-2 in the age group be low 45 years and 28 cases (73.7%) were pos iti ve in the age group of 45 years or above. A simil ar trend of immunoexpress ion was observed fo r EG F-R and ER. The express ion in the lower age group (be low 45 yea rs) be ing onl y 9 for EGF-R (25 .0%) as compared to the express ion in the upper age group (45 years or above) where it was fo und to be immu nopos iti ve fo r 27 tumours (75.0%). T he immunopos iti vity of ER was fou nd in 8 cases (29.6 %) among patients be low 45 years of age while 19 cases (70.3 %) belonged to hi gher age group.

A s igni ficant assoc iat ion was observed between th hi stological grade of the tumou r and the immune­express ion of c-erbB-2 (? <0.05). Further, stat isticall y s ignificant correlati on was fo und betw en the

RAY et al.: ADRENOCORTICOTROPIC HORMONE & GROWTH FACTOR RECEPTORS IN BREAST CANCER 665

immunoexpression of c-erbB-2 and EGF-R (P < 0.05) . 19 cases showed concomitant expression of c-erbB-2 and EGF-R. However, 12 cases revealed immunoexpression for both e-erbB-2 and ER; while, II cases showed the co-expression of both EGF-R and ER.

The concomitant immunopositivity of c-erbB-2 and ACTH was found in 6 cases, whereas both EGF-R and ACTH were present in 8 tumours (Table 2). It is most interesting to observe a significant association of concomitant immunopositivity between ACTH and ER (P < 0.05). Out of 15 cases which showed immunopositi vi ty for ACTH peptides, II cases (73.3%) were also found to be positive for ER.

Discussion The c-erbB-2 gene has been reported to be

amplified in most types of human adenocarcinomas including breast cancer 13

. The present study has revealed an overall expresston of c-erbB-2 oncoprotein in 38% cases of breast cancer. The complex oestrogenic actions on breast cancer are concerned with interactions between several

oncogenic products, tumour suppressor genes and growth factors 14

•15

• A significant number of breast cancer is oestrogen-dependent5

. Even in case of oestrogen-independent breast cancer, pathogenesis may start initially as oestrogen-dependent tumour (i.e., positive receptor status) and the cancer progression is related to a gradual loss of oestrogen requirement for tumour growth . The escape from hormonal control indicates a poor prognosis, and it may be associated with c-erbB-2 oncogene

. 16 17 H. h . f bB 2 overexpresston · . . tg concentrattons o c-er -protein are found more frequently in primary breast

. . h . 18 19 tumour ttssues wtt a negattve receptor status · . High grade carcinoma demonstrated c-erbB-2 more frequentl/0

. Our study has revealed a significant correlationship between the tumour grade (Bloom­Richardson's grading) and the expression of c-erbB-2 oncoprotein. 61.5% of high grade tumours were found to be immunoposi tive for c-erbB-2 oncoprotein.

The c-erbB-2 is located on chromosome 17 at q21 and specifies a transmembrane receptor like phospho­glycoprotein that is closely related in structure but is still biologically di stinct from the EGF-R21

. Gullick22

Fi g. I -lmmunoposilivily for (A) c-erbB-2, (B) EGF-R, (C) ER and (D) ACTH .

666 INDIAN 1 EXP BIOL, JULY 2000

observed that c-erbB-2 and EGF-R are often overexpressed together in human breast cancer. In our study, we have noticed a significant association between the immunoexpression of c-erbB-2 oncoprotein and EGF-R. Thi s study has shown the expression of EGF-R in 36% (36/ I 00) of cases.

c-erbB-2, EGF-R and ER. 93.3 % (14/15) of ACTH immunopositive cases belonged to upper age group. The higher immunopositivity for ACTH peptides in patients having 45 years of age or more indicates a probable relation between the presence of ACTH or ACTH-Iike peptides within breast cancer ti ssue and postmenopausal status of the patients. The immunopositivity for ACTH peptides was

markedly higher among patients of upper age group (45 years or more) as compared to other proteins, i.e.,

The major sites of oestrogen biosynthesis are the ovarian granulosa cells in premenopausal women and

Table I - lmmu nopositivity of c-erbB-2, EGF-R, ER and ACTH in various tumour grades of breast cancer

Tumour grade Lymph node involvement Low Moderate High Positive Negative

(n=25) (n=49) (n=26) (n=55) (n=45) c-erbB-2 Positive 11/25 11/49 16/26 22/55 16/45

(n=38) (44.0%) (22.4%) (61.5 %) (40.0%) (35.6%) Negative 14/25 38/49 10/26 33/55 29/45 (n=62) (56.0%) (77.6%) (38.5%) (60.0%) (64.4%)

Chi-square= 11.53 Chi-square=0.06 P<O.OI * P=0.80

EGF-R Positive 8/25 15/49 13/26 22/55 14/45 (n=36) (32.0%) (30.6%) (50.0%) (40.0%) (31.1 %) Negati.ve 17/25 34/49 13/26 33/55 3 1/45 (n=64) (68.0%) (69.4%) (50.0%) (60.0%) (68.9 %)

Chi-square=3.00 Chi-square=0.51 P=0.22 P=0.48

ER Positive 6/25 14/49 7/26 13/55 14/45 (n=27) (24.0%) (28 .6%) (26.9%) (23.6%) (31. 1%)

Negative 19/25 35/49 19/26 42/55 3 1/45 (n=73) (76.0%) (7 1.4%) (73 .1 %) (76.4%) (68.9%)

Chi-square=0.18 Chi-square=0.37 P=0.92 P=0.54

ACTH Positive 3/25 7/49 5/26 11/55 4/45 (n=l5) (12.0%) (14.3%) (19.2%) (20.0%) (8.9%) Negative 22/25 42/49 21/26 44/55 41/45 (n=85) (88.0%) (85.7%) (80.8%) (80.0%) (91. 1 %)

Chi-square=0.56 Chi-square= 1.60 P=0.78 P=0.21

*Stati stical significance was observed between moderate and hi gh tumour grade.

Table 2 -Interrelationship of immunopositivity of ACTH with c-erbB-2 oncoprotein , EGF-receptor, oestrogen receptor (ER) in breast cancer

lmmunoexpression c-erbB-2 oncoprotein

EGF-receptor

Oestrogen receptor

Positive (n=38) Negative(n=62)

Positive (n=36) Negative (n=64)

Positive (n=27) Negative (n=73)

ACTH lmmunopositivity Positive (n=l5) Negative (n=85) 6/38 (15.8%) 32/38 (84.2o/c) 9/62 (14.5%) 53/62 (85.5~) Chi-square= 1.33, P=0.91

8/36 (22.2%) 7/64 (10.9%) Chi-square= 1.50, P = 0.22

11127 (40.7%) 4173 (5.5%) Chi-square= 16.55, P<O.OO I

28/36(77.8%) 57/64(89.1 %)

16/27 (59.3%) 69173 (94.5~)

RAY eta /.: ADRENOCORTICOTROPIC HORMONE & GROWTH FACTOR RECEPTORS IN BREAST CANCER 667

extraovarian ti ssues in postmenopausal women. The conversion of androgens to oestrogens is catalyzed by the enzyme complex aromatase (aromatization) . It has been demonstrated that certain breast carcinomas have the ability to produce oestrogen through . I ?1 24 . d. . I h mtratumoura aromatase-· · , m tcatmg t 1at t ese tumours may posses a possible autocrine or paracrine function in which locally produced oestrogen may be significant in stimulating the growth by interacting with ER25

. This may be particularly important in postmenopausal women in whom the ovarian contribution of oestrogen is markedly diminished or abolished.

Growth factors or other factors may act in a paracrine fashion to influence aromatase in the presence of glucocorticoids6 which itself can cause induction of aromatase activit/. Besides, glucocorti­coids up-regulate the prostate-specific antigen (PSA) gene expression in nonprostatic ti ssue like breast and it is speculated that PSA may be invol ved in the regulation of growth factors 26

. Zarghami et at?1

observed in the ir studies that 30-40% of female breast tumours produced PSA which was associated with the positive receptor status (ER). They also found that the breast carcinoma cell line T-470 produced PSA when stimulated by glucocorticoids/ mineralocorticoids and other steroid hormones, like human glandular kallikrein (hK2/8

. Further, glucocorticoids inhibit the matrix metalloproteinase (MMP) synthes is. MMP and disruption of the basement membrane may play key roles in induction and progression of breast cancer11

•

ACTH, an anterior pituitary hormone, responsible for the synthes is and release of adrenal steroids, viz. glucocorticoids, mineralocorticoids and precursor of androgen as we ll as other sex-hormones. It is the most significant finding of thi s study that 15 % ( 15/ I 00) breast tumours revealed the presence of ACTH or ACTH-Iike peptides and its significant association withER, which clearly reflects a complex re lationship between different steroid substances and ACTH in the pathological process of breast carci noma. Presence of ACTH in breast cancer ti ssue also suggests its paracrine behaviour. An elaborative study compris ing of various steroid hormones related factors in relation with ACTH will be useful to evaluate the precise role of ACTH or ACTH-Iike peptides in mammary carcinogenes is.

Acknowledgement The authors thank Mr. S. L. Dayalu Naik and Mrs.

Sudershan Kaur for their technical help .

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