Tove Sivik

17β-Hydroxysteroid Dehydrogenase 14 Affects Estradiol Levels in Breast Cancer Cells and Is a Prognostic Marker in Estrogen Receptor–Positive Breast Cancer

Estrogens have an important role in the progression of breast cancer. The 17β-hydroxysteroid dehydrogenase (17HSD) family has been identified to be of significance in hormone-dependent tissues. 17HSD1 and 17HSD2 are the main 17HSD enzymes involved in breast cancer investigated this far, but it is possible that other hormone-regulating enzymes have a similar role. 17HSD5 and 17HSD12 are associated with sex steroid metabolism, and 17HSD14 is a newly discovered enzyme that may be involved in the estrogen balance. The mRNA expression of 17HSD5, 17HSD12, and 17HSD14 were analyzed in 131 breast cancer specimens by semiquantitative real-time PCR. The results were compared with recurrence-free survival and breast cancer-specific survival of the patients. The breast cancer cell lines MCF7, SKBR3, and ZR75-1 were transiently transfected with 17HSD14 to investigate any possible effect on estradiol levels. We found that high 17HSD5 was related to significantly higher risk of late relapse in estrogen receptor (ER)–positive patients remaining recurrence-free later than 5 years after diagnosis ( P = 0.02). No relation to 17HSD12 expression was found, indicating that 17HSD12 is of minor importance in breast cancer. Patients with ER-positive tumors with high expression levels of 17HSD14 showed a significantly better prognosis about recurrence-free survival ( P = 0.008) as well as breast cancer-specific survival ( P = 0.01), confirmed by multivariate analysis ( P = 0.04). Transfection of 17HSD14 in the human breast cancer cells MCF7 and SKBR3 significantly decreased the levels of estradiol, presenting an effect of high expression levels of the enzyme. (Cancer Res 2006; 66(23): 11471-7)

Expression Patterns of 17β-Hydroxysteroid Dehydrogenase 14 in Human Tissues

17βHSD enzymes catalyze the stereospecific oxidation/reduction at carbon 17β of androgens and estrogens, and are important players in intracrine sex hormone synthesis. The biological relevance of 17βHSD14, first named retSDR3, is largely unknown. We generated and validated an antibody targeting the 17βHSD14 antigen and used this for immunohistochemical evaluation of expression patterns in 33 healthy human tissues. Furthermore, sex steroid conversional activity in HSD17B14 overexpressing HEK293 and MCF10A cells was investigated by assessing interconversion products of estrone, estradiol, androstenedione, testosterone, and dehydroepiandrosterone. Immunohistochemical staining patterns of 17βHSD14 with the enzyme being primarily expressed in glandular epithelial tissue reveal an enzyme with possible implications in the secretion or conversion of externally derived compounds. A role for 17βHSD14 in sex steroid metabolism is supported by the finding that 17HSD14 oxidizes both estradiol and testosterone into less bioactive steroid metabolites estrone and androstenedione, respectively.

17β-Hydroxysteroid Dehydrogenase Type 14 Is a Predictive Marker for Tamoxifen Response in Oestrogen Receptor Positive Breast Cancer

Introduction 17β-hydroxysteroid dehydrogenases (17βHSDs) are important enzymes regulating the pool of bioactive steroids in the breast. The current study was undertaken in order to evaluate implications of 17βHSD14 in breast cancer, measuring 17βHSD14 protein expression in breast tumours. Methods An antibody targeting the 17βHSD14 antigen was generated and validated using HSD17B14-transfected cells and a peptide-neutralising assay. Tissue microarrays with tumours from 912 post-menopausal women diagnosed with lymph node-negative breast cancer, and randomised to adjuvant tamoxifen or no endocrine treatment, were analysed for 17βHSD14 protein expression with immunohistochemistry. Results Results were obtained from 847 tumours. Patients with oestrogen positive tumours with high 17βHSD14 expression had fewer local recurrences when treated with tamoxifen (HR 0.38; 95% C.I. 0.19–0.77, p = 0.007) compared to patients with lower tumoural 17βHSD14 expression, for whom tamoxifen did not reduce the number of local recurrences (HR 1.19; 95% C.I. 0.54–2.59; p = 0.66). No prognostic importance of 17βHSD14 was seen for systemically untreated patients. Conclusions Using a highly specific validated antibody for immunohistochemical analysis of a large number of breast tumours, we have shown that tumoural expression levels of 17βHSD14 can predict the outcome of adjuvant tamoxifen treatment in terms of local recurrence-free survival in patients with lymph node-negative ER+ breast cancer. The results need be verified to confirm any clinical relevance.

P1-06-06: The Importance of CXCL10 and CXCR3-A in Breast Cancer.

Background: CXCL10 is a chemokine with chemo attractant properties which signals through G protein-coupled receptor CXCR3. At least two reported isoforms exists; CXCR3-A and CXCR3-B. CXCL10 is involved in the recruitment of leucocytes, immune modulation and angiogenesis. In animal models CXCL10 inhibits tumour growth, metastase formation and inhibits angiogenesis. The receptors have opposite features; CXCR3-A mediates proliferative or anti-apoptotic response and CXCR3-B mediates growth inhibition and apoptosis in response to ligand binding. The aim of this study was to investigate the role of CXCL10 and CXCR3-A as prognostic or tamoxifen treatment predictive factors in breast cancer. Material and Methods: A randomized tamoxifen trial comprising 1,780 breast cancer patients was conducted in Stockholm, Sweden, 1976–1990. All patients had lymph node negative primary breast cancer and were postmenopausal at the time of diagnosis. The patients were randomized to tamoxifen or no endocrine treatment. We analyzed the protein expression of CXCL10 and the receptor CXCR3A with immunohistochemistry using tissue microarrays, which were constructed from paraffin blocks originating from 912 patients. CXCL10 and CXCR3A could be scored in 793 (87%) and 735 (81%), respectively. Results: High expression of CXCL10 was associated with significant benefit of tamoxifen treatment concerning local recurrence and breast cancer survival (P=0.02 and P=0.008). For patients with high CXCL10 expression, tamoxifen decreased the risk concerning local recurrence (rate ratio (RR) = 0.56, 95% C.I. 0.33−0.97, P=0.0029) and breast cancer survival (RR 0.6 95% C.I. 0.39−0.97, P=0.023). High expression of CXCR3-A was associated with significant benefit of tamoxifen treatment concerning total recurrence and breast cancer survival (P=0.00001 and P=0.004, respectively). For patients with high CXCR3-A expression, tamoxifen decreased the risk of total recurrence (RR 0.54 95% C.I. 0.35−0.83, P=0.005) and breast cancer survival (RR 0.49 95% C.I. 0.35−0.69, P=0.00003). On the opposite, in the cohort of patients with no endocrine treatment and high CXCR3-A expression, there was an increased risk of total recurrence (P=0.003). (RR 2.07 95% C.I. 1.18−3.61, P=0.011). Conclusions: This study indicates that high expression of CXCL10 can be used as predictive markers for tamoxifen treatment. Further, high expression of CXCR3-A is related to bad prognosis and predicts tamoxifen treatment in breast cancer patients. This needs to be further evaluated. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-06.

Progesterone and levonorgestrel regulate expression of 17βHSD-enzymes in progesterone receptor positive breast cancer cell line T47D.

The use of combined hormone replacement therapy (HRT) with oestrogens and progestins in postmenopausal women has been associated with an increased risk for developing breast cancer. The reasons are not fully understood, but influence of HRT on endogenous conversion of female sex hormones may be involved. The expression of 17β hydroxysteroid dehydrogenases (17βHSD), which are enzymes catalysing the conversion between more or less potent oestrogens, may partly be regulated by progestins. The breast cancer cell lines T47D, MCF7 and ZR75-1 were treated with progesterone, medroxyprogesterone acetate (MPA) or levonorgestrel for 48 and 72 h at 10(-7) and 10(-9)M to investigate influence on 17βHSD1, 17βHSD2 and 17βHSD5 mRNA expression measured by real time PCR. The expression of 17βHSD1 increased in progesterone and levonorgestrel treated T47D cells (48 h 10(-7)M P=0.002; P<0.001) and 17βHSD5 increased after progesterone treatment (48 h 10(-7)M P=0.003), whereas the expression of 17βHSD2 decreased after the (48 h 10(-7)M P=0.003; P<0.001). Similar, but less prominent effects were seen in MCF7 and ZR75-1. The progestin effects on 17βHSD-expression were lost when T47D cells were co-treated with progestins and the progesterone receptor (PgR) inhibitor mifprestone. We show that both reductive (17βHSD1 and 17βHSD5) and oxidative (17βHSD2) members of the 17βHSD-family are under control of progesterone and progestins in breast cancer cell lines. This is most clear in T47D cells which have high PgR expression. 17βHSD-enzymes are important players in the regulation of sex steroids locally in breast tumours and tumoural expression of various 17βHSD-enzymes have prognostic and treatment predictive relevance. We propose a mechanism for increased breast cancer risk after HRT in which hormone replacement affects the expression of 17βHSD-enzymes, favouring the expression of reductive enzymes, which in turn could increase levels of bioactive and mitogenic estrogens in local tissue, e.g. breast tissue.

Abstract 3142: Expression of C-X-C motif chemokine 10 (CXCL10) in breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Chemokines are small cytokine-like secreted proteins with chemoattractant properties which function in the recruitment of leucocytes into inflamed tissue. Besides well studied roles in the immune system, chemokines and their receptors have also been shown to play critical roles in tumorigenesis. The present study focuses on C-X-C motif chemokine 10 (CXCL10), a potent chemoattractant for stimulated T-cells and NK-cells. Both CXCL10 and its receptor CXCR3 are expressed by breast tumor cells, and accumulating data suggests roles for this chemokine and its receptor in breast cancer development and proliferation. We have previously shown CXCL10 to be significantly upregulated in ER positive as well as negative breast cancer cell lines over-expressing 17β hydroxysteroid dehydrogenase 14 (17βHSD14). Using a validated anti-CXCL10 primary antibody, we examined the relationship between CXCL10 expression and clinicopathological features in tissue microarrays comprised of tumor material from 912 breast cancer patients participating in a randomized tamoxifen trial conducted in Stockholm, Sweden, 1976-1990. All patients had lymph node negative primary breast cancer and were postmenopausal at the time of diagnosis. Results were obtained from tumors of 793 patients, of which 6% were deemed negative, 23% weak, 28% moderate, and 42% strong. Staining intensity of CXCL10 was strongly correlated with expression of 17βHSD14 (p<0.0001), however no significant correlation was observed between CXCL10 and prognostic markers such as ER, PR or tumor size. No clear associations between CXCL10-expression levels and the clinical outcome of tamoxifen treatment were observed, and moreover no prognostic value of CXCL10 was seen in systemically untreated patients. In the present study we have investigated expression of CXCL10 in a breast tumour cohort. We have previously found a functional relationship between 17βHSD14 and CXCL10 suggesting a possible regulatory loop involving this 17βHSD enzyme. A strong association between CXCL10 and 17βHSD14 in this tumor cohort further strengthens this theory. We found CXCL10 to be ubiquitously expressed in the tumors however no significant relationship between CXCL10 expression and tamoxifen treatment prediction or prognosis among patients was noted. Previous data on the action of CXCL10 in cancer is conflicting as the chemokine has been suggested to act as a growth promoter as well as an inhibitor growth. The presence of two splice variants of the CXCL10 receptor CXCR3, of which CXCR3-A has been shown to promote proliferation and CXCR3-B- to induce apoptosis, has generally not been accounted for, and varying expression levels of these receptors may to some degree explain these conflicting findings. In order to elucidate the role of CXCL10 in breast cancer, tumoral distribution and expression levels of these two splice variants of the CXCL10 receptor need to be evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2011-3142

Significance of 17bHSD Type 14 as a Predictive Factor for Adjuvant Tamoxifen Treatment Response in Breast Cancer.

Background: Peripheral sex steroid synthesis in target organs from adrenally derived steroid precursors is believed to be an ongoing event throughout life, but becomes exceedingly important as the gonadal sources of sex steroids cease, as is the case for estrogen synthesis in women after menopause. 17β hydroxysteroid dehydrogenases (17bHSDs) comprise a family of 14 enzymes catalyzing the stereospecific oxidation/reduction at position C17 of estrogens and androgens. Due to their importance in fine-tuning estrogen levels, the 17bHSDs have been studied in breast cancer. In a previous study examining mRNA of 17bHSD type 14, an enzyme driving the oxidation of estradiol into less potent estrone, from a cohort of stage 2 tumors, high levels were associated with longer disease-free survival. The present study was conducted in order to investigate whether this result could be repeated in another group of patients examining protein expression and to investigate whether 17bHSD type 14 has any prognostic or predictive importance in breast cancer patients systemically treated or untreated with tamoxifen. Material and Methods: A peptide sequence corresponding to amino acids 255 to 269 of human 17bHSD type 14 was synthesized and used for immunization of rabbits with the subsequent purification of a polyclonal antibody towards the antigen. A randomized tamoxifen trial comprising 1780 breast cancer patients was conducted in Stockholm, Sweden, 1976-1990. All patients had lymph node negative primary breast cancer and were postmenopausal at the time of diagnosis. We analyzed the protein expression of 17bHSD type 14 with immunohistochemistry using tissue microarrays, which were constructed from paraffin blocks originating from 912 patients. Results: Results were obtained from 847 patients. Among patients with estrogen receptor positive tumors, high expression of 17bHSD type 14 was associated with a significantly better benefit from tamoxifen as shown by a higher local recurrence-free probability (RR 0.38; 95% C.I. 0.19-0.77, p=0.006) when compared to patients bearing tumors with intermediate, low or negative expression levels (RR 1.20; 95% C.I. 0.54-2.60; p=0.67). The interaction test was significant (p=0.03). No significant difference in tamoxifen response was found when looking at 17bHSD type 14 in relation to breast cancer specific mortality. Furthermore, for untreated patients no prognostic significance for 17bHSD type 14 was found. Discussion: In the current study we show that intratumoral levels of 17bHSD type 14, an enzyme potentially involved in lowering the levels of potent estradiol, is a predictive factor in determining the local response to adjuvant tamoxifen treatment. The fact that patients with ER positive tumors have a greater possibility of responding well to tamoxifen when the expression levels of 17bHSD type 14 are high suggests that some patients would gain better from other treatments than tamoxifen, e.g. aromatase inhibitors and/or inhibitors of reducing 17bHSD enzymes. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2015.