Irene Díez-Itza

Zn-α2-glycoprotein levels in breast cancer cytosols and correlation with clinical, histological and biochemical parameters

Abstract Zn- α 2 -glycoprotein (Zn-α 2 -gp), a protein present at high levels in breast cyst fluid, has been measured in 104 breast tumour cytosols by using an immunoenzymatic assay. Concentrations of Zn-α 2 -gp ranged from 0 to 23.5 μg/mg of total soluble protein, with an average value of 2.4 μg/mg. There was no significant correlation between Zn-α 2 -gp and menopausal status, tumour size or lymph node involvement, or between this protein and biochemical parameters such as oestrogen receptor, cathepsin D or pS2 levels. However, there was a significant association between Zn-α 2 -gp and histological grade of tumours, with higher Zn-α 2 -gp levels in well-differentiated tumours (mean 4.6 μg/mg) than in moderately (1.8 μg/mg) or poorly (0.9 μg/mg) differentiated tumours. On the basis of these results, we propose that Zn-α 2 -gp may be considered as a biochemical marker of differentiation in breast cancer.

Pepsinogen C is a new prognostic marker in primary breast cancer.

PURPOSE Here we evaluate in breast cancer patients the prognostic value of pepsinogen C, a proteolytic enzyme involved in the digestion of proteins in the stomach that is also synthesized by a significant percentage of breast carcinomas. PATIENTS AND METHODS Pepsinogen C expression was examined by immunoperoxidase staining in a series of 243 breast cancer tissue sections, and results obtained were quantified using the HSCORE system, which considers both the intensity and the percentage of cells staining at each intensity. Evaluation of the prognostic value of pepsinogen C was performed retrospectively in corresponding patients by multivariate analysis that took into account conventional prognostic factors. The mean follow-up period was 48.5 months. RESULTS A total of 113 carcinomas (46.5%) stained positively for this proteinase, but there were clear differences among them with regard to the intensity and percentage of stained cells. Pepsinogen C values were significantly higher in well differentiated (grade I, 89.1) and moderately differentiated (grade II, 88.5) tumors than in poorly differentiated (grade III, 27.7) tumors (P < .001). Similarly, significant differences in pepsinogen C content were found between estrogen receptor (ER)-positive tumors and ER-negative tumors (85.9 v 41.2, respectively; P < .05). Moreover, results indicated that low pepsinogen C content predicted shorter relapse-free survival duration and overall survival duration (P < .0001). Separate Cox multivariate analysis for relapse-free survival and overall survival in subgroups of patients as defined by node status showed that pepsinogen C expression was the strongest factor to predict both relapse-free survival and overall survival in node-positive patients (P < .0001 for both) and node-negative patients (P < .005 and P < .01, respectively). CONCLUSION Pepsinogen C is a new prognostic factor for early recurrence and death in both node-positive and node-negative breast cancer. In addition, and in contrast to most studies that concern the prognostic significance of proteolytic enzymes in cancer, pepsinogen C production by breast cancer cells is associated with lesions of favorable evolution.

Glucocorticoids and androgens up-regulate the Zn-alpha 2-glycoprotein messenger RNA in human breast cancer cells.

SummaryWe have studied the hormonal regulation of the gene encoding Zn-α2-glycoprotein (Zn-α2-gp), a human protein with a high degree of amino acid sequence similarity to class I histocompatibility antigens that is produced by a specific subset of breast carcinomas. Northern blot analysis revealed that dexamethasone and 5α-dihydrotestosterone strongly induced the accumulation of Zn-α2-gp mRNA in T-47D human breast cancer cells. Furthermore, the effect of these two hormones was shown to be additive, since the combination of both hormones produced a stimulation of Zn-α2-gp mRNA of at least 3-fold over that produced by either hormone alone. By contrast, the addition of 5β-dihydrotestosterone, 17β-estradiol, or progesterone failed to induce the expression of Zn-α2-gp. The stimulatory effect of glucocorticoids and androgens on Zn-α2-gp expression was produced in a time and dose dependent manner, without significantly affecting the cell proliferation rate. A time-course study demonstrated that the induction of Zn-α2-gp mRNA by androgens and glucocorticoids reached a level of 4 or 3.2-fold over the untreated control after seven days of incubation in the presence of a 10−7 M concentration of 5α-dihydrotestosterone or dexamethasone, respectively. A dose-response study showed that as little as 10−11 M of 5α-dihydrotestosterone or dexamethasone produced an accumulation of Zn-α2-gp mRNA of 2.4 or 2.1-fold over the control, respectively. On the basis of these results, we propose that Zn-α2-gp may be useful as a biochemical marker of breast carcinomas with a specific pattern of hormone responsiveness in whose development glucocorticoids and/or androgens may play a significant role.

Expression of pepsinogen C in human breast tumours and correlation with clinicopathologic parameters

We have examined by immunohistochemistry the ability of breast carcinomas to produce pepsinogen C, an aspartyl proteinase usually involved in the digestion of proteins in the stomach. A total of 113 out of 245 breast tumours (46%) were positive for pepsinogen C immunostaining. There was a significant association between pepsinogen C and oestrogen receptors with proteinase levels higher (HSCORE) in oestrogen receptor positive tumours than in oestrogen receptor negative. There was also a significant association between pepsinogen C and histological grade, pepsinogen C levels being higher in well and moderately differentiated breast carcinomas than in poorly differentiated tumours. On the basis of these results, we suggest that pepsinogen C may be useful as a marker of good prognosis in breast cancer.

Factors affecting protein composition of breast secretions from nonlactating women

SummaryBreast secretions can be classified into two types according to their major protein components. Type I fluids contain Zn-α2-glycoprotein, apolipoprotein D, and gross cystic disease fluid protein-15, while Type II fluids are characterized by the presence of lactoferrin, lysozyme, and α-lactalbumin. In this study, the polypeptide composition of breast secretions from 719 nonlactating women was evaluated by using polyacrylamide gel electrophoresis. The required amount for the analysis (1 µl) was obtained from 50% of control women and from 75% of women with mammary disease. There were more secretors in premenopausal than in postmenopausal women, as well as in parous than in nulliparous women. Evaluation of factors affecting protein composition of breast secretions revealed that Type II fluids were found in the majority of women who had given birth in the last four years and in a high proportion of oral contraceptive users. After excluding both of these groups, Type II fluids were detected in 47% of patients with breast cancer, but only in 8% of control women and in 16% of women with benign breast diseases. Taken together, these results suggest that protein analysis of breast secretions could be an useful tool for the study of breast pathologies.

Cathepsin D in breast secretions from women with breast cancer

A proteinase accumulated in breast secretions from women with breast cancer has been characterised. Inhibition of the proteolytic activity of breast secretions by pepstatin A showed that the main enzyme involved was an aspartyl proteinase. Determination of its cleavage specificity by SDS-PAGE and amino acid sequence analysis revealed that it was identical to that of cathepsin D, an aspartyl proteinase suggested to be involved in breast cancer development. The identity between both proteins was further confirmed by immunological analysis with monoclonal antibodies against cathepsin D. Quantification of cathepsin D in nipple fluids from 41 women with benign or malignant breast diseases and from 19 control women without breast pathology revealed the presence of variable amounts of this proteinase. The average concentration of cathepsin D in breast secretions from cancer-bearing breasts was 7.2 +/- 2.2 fmol micrograms of protein, which was significantly higher than those of nipple fluids from control women (2.9 +/- 0.6 fmol micrograms-1) (P = 0.04) or from patients with benign breast diseases (2.1 +/- 0.3 fmol micrograms-1) (P = 0.004). Though the number of cancer patients studied was small (n = 21), no correlations were found with cytosolic concentrations of cathepsin D or oestrogen receptors, neither with other parameters such as tumour size, histological grade, axillary node involvement or menopausal status.