Immunocytochemistry In the Assessment of Ps2 Protein Expression In Fine Needle Aspiration Cytology From Breast Carcinoma

pS2 protein is a cysteine-rich polypeptide, of unknown function, the expression of which is induced in the human cancer cell line MCF-7 by oestrogen. the availability of a murine monoclonal antibody to human pS2 protein has prompted us to evaluate its expression in 47 cases of primary breast carcinoma. Using a double indirect immunoperoxidase technique, we compared the expression of pS2 protein in fine needle aspiration (FNA) cytology smears with that in formalin-fixed, paraffin-embedded sections from subsequently excised tumours from the same patients. We also compared the expression of pS2 protein and oestrogen receptor (ER) status using immunocytochemical assay (ER-ICA) in formalin-fixed, paraffin-embedded sections from 22 primary breast carcinomas. We found the application of immunocytochemistry in the assessment of pS2 protein expression in FNA cytology to be a reliable and cost-effective technique, having a sensitivity of 84% and a specificity of 100%. There was also a good correlation between the expression of pS2 protein and ER status.     

Specific expression of human pS2 gene in breast cancer

The hormone-dependence of some human breast cancers is well recognized. However, the molecular mechanisms responsible for the growth stimulation of these cancers by oestrogens are still poorly understood. With the hope of elucidating these mechanisms, we have recently cloned and studied the structure-function relationship of the human oestrogen and progestin receptors, and also undertaken a study aimed at characterizing genes whose expression is controlled by oestrogens in hormone-dependent breast cancers. We review here our findings concerning one of these genes and its expression products, the pS2 gene. We discuss also whether a systematic determination of pS2 gene expression in breast cancer biopsies could be useful to establish a new biochemical classification of these cancers which may be useful to improve the diagnosis of hormone-dependent cancers.     

Overexpression of insulin-like growth factor II (IGFII) in ZR-75-1 human breast cancer cells: higher threshold levels of receptor (IGFIR) are required for a proliferative response than for effects on specific gene expression

Previous transfection experiments using a zinc-inducible expression vector have shown that overexpression of insulin-like growth factor II (IGFII) in MCF7 human breast cancer cells can reduce dependence on oestrogen for cell growth in vitro (DALY RJ, HARRIS WH, WANG DY, DARBRE PD. (1991) Cell Growth Differentiation 2, 457-464.). Parallel transfections now performed into another oestrogen-dependent human breast cancer cell line (ZR-75-1) yielded three clones of transfected ZR-75-1 cells that produced levels of zinc-inducible IGFII mRNA and secreted mature IGFII protein similar to those found in the transfected MCF7 cells. However, unlike in MCF7 cells, no resulting effects were found on cell growth in the ZR-75-1 clones, even though the ZR-75-1 clones possessed receptors capable of binding 125I-IGFI and showed a growth response to exogenously added IGFII. Medium conditioned by the ZR-75-1 clones could stimulate growth of untransfected MCF7 cells, indicating that the secreted IGFII protein was bioactive. Furthermore, zinc-induced IGFII was capable of increasing both pS2 mRNA levels and CAT activity from a transiently transfected AP1-CAT gene in the ZR-75-1 clones. Constitutive co-overexpression of the protein processing enzyme PC2 resulted in reduced levels of large forms of zinc-inducible IGFII, but zinc treatment still produced no effect on cell growth rate. Finally, however, constitutive co-overexpression of the type I IGF receptor (IGFIR) did result in zinc-inducible increased basal cell growth and reduced dependence on oestrogen for cell growth. These results demonstrate that while overexpression of IGFII per se was sufficient to deregulate MCF7 cell growth, the ZR-75-1 cells are limited in their proliferative response by their intrinsic receptor levels. However, although the proliferative response was limited, molecular responses (expression of pS2 and AP1-CAT) were not limited, indicating that different cellular responses can have different threshold receptor level requirements.     

Urinary pS2/TFF1 levels in the management of hormonodependent breast carcinomas

pS2/TFF1 overexpression in breast carcinomas correlates with response to hormonotherapy. We evaluated the clinical relevance of urinary pS2/TFF1 in breast cancer patients. In healthy controls (100 cases), it represents an individual and relatively stable parameter. Although 24 out 83 pre-operative breast cancer patients showed elevated levels, both the sensitivity and specificity of the test were too low for breast cancer screening. However, neoadjuvant hormonotherapy decreased pS2/TFF1 levels in nine out of 20 patients. Furthermore, among 22 patients receiving long-term adjuvant hormonotherapy, four exhibited elevated levels, two of them at the time of relapse. Thus, urinary pS2/TFF1 quantification might be suitable as an in vivo diagnosis for tumor hormonodependency, and disease follow-up during hormonotherapy.     

Dissociation of Bax from a Bcl-2/Bax heterodimer triggered by phosphorylation of serine 70 of Bcl-2.

Serine 70 in the loop region of Bcl-2 is specifically phosphorylated by paclitaxel-treatment in tumor cells and BHK cells expressing Bcl-2. The phosphorylation of serine 70 of Bcl-2 (pS70-Bcl-2) peaks 24 to 48 h after paclitaxel treatment and accelerates apoptosis. Phosphorylation is effectively inhibited in the presence of actinomycin D or cycloheximide, which restore cell viability to the same level as control cells not expressing Bcl-2. These results indicate that paclitaxel-induced kinase(s) and/or its activator(s) are synthesized de novo and play an important role in paclitaxel-induced apoptosis by phosphorylating Bcl-2. In binding assays using the phosphorylation-specific antibody against pS70-Bcl-2, the induction of serine 70 phosphorylation 70 results in a loss of the binding ability of Bcl-2 to Bax, a pro-apoptotic partner, and induces subsequent cell death. When the pS70-Bcl-2 antibody was added to human breast cancer tissue, serine 70 phosphorylation was also detected, even prior to treatment with anticancer agents. Further study of breast cancers revealed 83% of tumors with high pS70-Bcl-2 expression responded to paclitaxel or docetaxel treatment, whereas 57% of those with low expression not respond. These findings suggest that pS70-Bcl-2 might be a predictive factor for prognosis and sensitivity to paclitaxel treatment for breast cancer.     

Expression of genes coding for pS2, c-erbB2, estrogen receptor and the H23 breast tumor-associated antigen. A comparative analysis in breast cancer

Expression of the gene coding for a new breast tumor-associated antigen, H23, was compared to expression of genes coding for pS2, c-erbB2 and estrogen receptor (ER). Comparison involved mRNA expression in normal and malignant breast tissues as well as in non-breast tumors. Results obtained by RNA dot blot and Northern hybridizations showed that expression of the H23 antigen coding gene is a discriminatory marker in human breast cancer. It is expressed in 92% of breast tumors whereas 69%, 62% and 56% of breast tumors demonstrate significant mRNA levels of c-erbB2, ER and pS2, respectively. Non-malignant or normal breast tissue expresses much lower levels of the H23 antigen mRNA. From the comparative analysis presented here it is concluded that the gene coding for H23 antigen furnishes a most useful marker for human breast cancer.     

Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. Results. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone‐deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and α6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44^+/high/CD24^?/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial‐to‐mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX‐2 (cyclo‐oxygenase‐2) through a CD44‐mediated mechanism. Conclusion. An oestrogen‐responsive cell line with ERα and CD44⁺/CD24^?/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ERα‐positive breast cancers, (2) CD44⁺/CD24^?/low breast tumour stem cells and (3) the metastatic ability of breast cancer.     

Expression of the pS2 gene in human gastric cancer cells derived from poorly differentiated adenocarcinoma

NH2-terminal amino acid sequence of the pS2 protein produced and secreted by human gastric cancer cells, MKN-45, was determined to be identical to that of MCF-7 cells. A clone encoding pS2 protein was isolated from the cDNA library constructed from MKN-45 cells. The nucleotide sequence was identical to that of pS2 cDNA previously isolated from human breast cancer cells, MCF-7, except for one nucleotide in the 3' untranslated region. Thus, in this cell line, the pS2 gene product is translated and secreted as in MCF-7 cells. RNA blot hybridization analysis revealed that pS2 gene was expressed well in two (MKN-45 and KATO-III; derived from poorly differentiated adenocarcinoma) but not in three cell lines (MKN-1, MKN-28 and MKN-74; from well differentiated adenocarcinoma), suggesting that expression of the pS2 gene depends on the state of cell differentiation. These results suggest that pS2 is expressed in human gastric cancer cells in an estrogen-independent manner and is possibly associated with the malignant state of cells.     

Pilot and feasibility study: comparative proteomic analysis by 2-DE MALDI TOF/TOF MS reveals 14-3-3 proteins as putative biomarkers of response to neoadjuvant chemotherapy in ER-positive breast cancer

Neoadjuvant chemotherapy is used to treat oestrogen receptor-positive breast cancer however chemo-resistance is a major obstacle in this molecular subtype. The ability to predict tumour response would allow chemotherapy administration to be directed towards patients who would most benefit, thus maximising treatment efficacy. We aimed to identify protein biomarkers associated with response to neoadjuvant chemotherapy, in a pilot study using comparative 2-DE MALDI TOF/TOF MS proteomic analysis of breast tumour samples. A total of 3 comparative proteomic experiments were performed, comparing protein expression between chemotherapy-sensitive and chemotherapy-resistant oestrogen receptor-positive invasive ductal carcinoma tissue samples. This identified a list of 132 unique proteins that were significantly differentially expressed (≥ 2 fold) in chemotherapy resistant samples, 57 of which were identified in at least two experiments. Ingenuity? Pathway Analysis was used to map the 57 DEPs onto canonical signalling pathways. We implicate several isoforms of 14-3-3 family proteins (theta/tau, gamma, epsilon, beta/alpha and zeta/delta), which have previously been associated with chemotherapy resistance in breast cancer. Extensive clinical validation is now required to fully assess the role of these proteins as putative markers of chemotherapy response in luminal breast cancer subtypes.     

Aromatization inhibition alone or in combination with GnRH agonists for the treatment of premenopausal breast cancer patients.

Aromatase inhibition in postmenopausal women causes a marked fall in the plasma levels of oestrogens and is an effective treatment for breast cancer, however, trials with aminoglutethimide found that this aromatase inhibitor was ineffective in suppressing plasma oestrogen levels in premenopausal breast cancer patients. We found that the more potent inhibitor, 4-hydroxyandrostenedione (4-OHA), which can suppress oestrogen synthesis in rodents and non-human primates with intact ovarian function, was also unsuccessful as an oestrogen suppressant in premenopausal women at its maximum tolerated dose (500 mg/week i.m.). GnRH agonists are effective suppressants of ovarian oestrogen synthesis but oestrogen production from peripheral sites is unaffected. Our studies of a combination of the GnRH agonist goserelin and 4-OHA demonstrated that the combination caused greater oestrogen suppression than goserelin alone and led to objective clinical response in 4/6 breast cancer patients after their relapse from treatment with goserelin as a single agent. The combination of a GnRH agonist and an aromatase inhibitor should be subjected to clinical trials.