Endocrine disruptors (EDs) and hormone-dependent cancers: Correlation or causal relationship?

The selective increase in the incidence of hormone-dependent cancers (breast, prostate, testicular) in industrialized countries is associated with the increasing number of endocrine disruptors (EDs) in the environment and raises questions about the role of EDs in mammary carcinogenesis. Answering these questions is difficult because the number of EDs is large and varies with time. Moreover hormonal carcinogenesis is multifactorial and progresses slowly and in stages. This discussion will be limited to breast cancer and three EDs: distilbene, bisphenol A (BPA), and dichlorodiphenyltrichloroethane (DDT). All these three EDs bind estrogen receptors, albeit with widely different affinities. Several complementary approaches have been used: French cancer records, epidemiological studies on cohorts followed over several decades, numerous in vitro experimental studies using cell cultures and in vivo animal studies. These approaches all converge to the same result, strongly suggesting a causal relationship between EDs and precancerous lesions. Except for distilbene, the mechanisms and molecular targets involved are still unclear, which makes it difficult to look for substitute products that are just as efficient, but less toxic.

Cathepsin D stimulates the activities of secreted plasminogen activators in the breast cancer acidic environment.

Two proteases cathepsin D (cath D) and urokinase plasminogen activator (uPA) are tissue markers associated with an increased risk of metastasis in breast cancer. We investigated whether cath D, the major aspartyl protease overexpressed by breast cancer cells can trigger a proteolytic cascade via activation of plasminogens at the extracellular pH measured in hypoxic tumors. The effects of the aspartyl protease inhibitor pepstatin on the plasminogen activator (PA) system were analysed by conditioning media of human MDA-MB231 breast cancer cells at pH 6.6 and pH 7.4. Zymography analysis of culture media showed that pepstatin inhibited the secreted activity of tissue-type plasminogen activator (tPA) but not that of uPA. tPA was identified on the basis of the molecular weight, the immunoreactivity with relevant antibodies and the resistance to amiloride, a specific uPA inhibitor. The secreted tPA activity measured by a chromogenic assay in the presence of amiloride was also inhibited by pepstatin at pH 6.6. Surprisingly, pepstatin did not affect secreted tPA protein concentration but markedly increased the amount of the secreted plasminogen activator inhibitor-1 (PAI-1). We conclude that cath D overexpressed by these cells, stimulates at pH 6.6, but not at neutral pH, the extracellular PA proteolytic activity indirectly via PAI-1 proteolysis. This suggests that cath D at acidic pH close to the hypoxic regions of solid tumors, contributes to trigger a proteolytic cascade facilitating cancer cell invasion and metastasis.

[Bisphenol A and hormone-dependent cancers: potential risk and mechanism]. / Bisphénol A et cancers hormono-dépendants - risque potentiel et mécanisme d'action.

Being concerned by the increasing incidence of breast, prostate and testicular cancers, we overviewed the literature on the potential carcinogenic effect of endocrine disruptors (ED). It is extremely difficult to obtain the epidemiological proof of a carcinogenic effect of one ED in human for multi-factorial diseases and the high number of confusing factors. However, many experimental studies in rodents on bis-phenol A (BPA) and its assay in human blood and urine, strongly suggest that BPA might increase the risk of hormone dependant cancers. Contrary to the mitogenic effect of estradiol, directly mediated in mammary cells by the classical nuclear estrogen receptor α, the mechanisms of the deleterious effect of BPA at low doses and in utero remain unknown since the molecular and cellular target(s) have not been defined. Based on all deleterious effects of BPA, France has banned the use of BPA in all food packaging. This should force the food industry to collaborate with members of public research to rapidly find safer substitutes to BPA.

The role of sex steroid receptors on lipogenesis in breast and prostate carcinogenesis: a viewpoint.

Cancer cells require both nutrients and mitogens to multiply and survive in the unfavorable microenvironment of solid tumors or metastases before angiogenesis. Most cancer cells do not use fatty acids (FA) from the circulation but synthesize them in situ especially to make membranes and lipid signals required for continuously dividing cells. Three lipogenic enzymes are overexpressed, induced by sex steroid hormones and responsible for the in situ increased lipogenesis in cancer cells. We propose that in the early stages of human breast and prostate carcinogenesis, an increased activity of sex steroid receptors is partly responsible for the overexpression of FA synthase (FASN) whose regulation in cancer cells has been particularly studied. Increased activity of androgen receptor (AR), via different mechanisms, was extensively reported in prostate cancer. An increased level/and or activity of progesterone receptors, correlated with an increased expression of FASN, was found both in early stages of breast carcinogenesis and during the hormone replacement therapy of menopausal women. While the majority of recent targeted therapies are based on inhibition of mitogenic pathways, the inhibition of cancer cell nutrition by interfering with lipid synthesis and hormone action should open the way to new therapeutic and preventive approaches of hormone-dependent cancers.

[Endocrine disruptors: are they carcinogens?]. / Environnement et cancers: perturbateurs endocriniens; existe-t-il un risque cancérigène?

Concerned with the high incidence of breast and prostate cancers in industrialized countries, including France, we reviewed the literature and national reports on the potential carcinogenic effects of several endocrine disruptors (ED) present in the environment. We examine why it is extremely difficult to obtain clear proof of a carcinogenic effect of ED in humans. Yet the results of several independent studies strongly point to such a carcinogenic effect, particularly in the case of hormone-dependent cancers. Such malignancies have been induced experimentally in rodents and have also been observed in humans. For example, a moderately elevated incidence of prostate cancer has been noted in U.S. farmers and, more recently in the French West Indian population exposed for more than 30 years to the insecticide chlordecone. We discuss the molecular mechanisms involved in this effect in prostate cancer Lessons from the observed trans-generational carcinogenic effect of the synthetic estrogen diethylstilboestrol also strongly suggests that future generations must be protected from widespread distribution of synthetic estrogens in the environment. We argue that a reduction in the use of some EDs in agriculture and the plastics industry would be much more beneficial in France than the prohibition of transgenic plants.

[How to reduce the incidence of breast cancer]. / Cancers du sein, incidence et prévention.

The incidence of breast cancer is rising in all industrialized countries. In France, with 41,000 new cases and 11,000 deaths per year, breast cancer is still the first cause of death from cancer in women. Genetic familial breast cancer is rare (only-2% due to mutated BRCA1/2 genes). Most of the increase is due to sporadic cases associated with hormonal, reproductive and nutritional factors, some of which can be avoided. Prevention should include: - avoidance of risk factors such as alcohol, obesity, smoking, sedentarity, and, when possible, late first pregnancies and absence of breast feeding. - Lesser use of hormone replacement therapy, in order to avoid the tumor-promoting action of ovarian hormones; this is supported by the strong fall in the incidence of breast cancer in U.S.A after 2002, following a drop in the use of HRT by post-menopausal women. - For very-high-risk women (BRCA 1/2 mutations, or in situ carcinoma with proliferative atypia), chemoprevention with SERM should be authorized, based on randomized trials of tamoxifen and raloxifen. New trials of other agents should be encouraged. - Risk indexes such as that developed by the NCI should be more widely used. - More research on the early steps of human mammary carcinogenesis should be encouraged, as this should lead to individual targeted prevention.

[Hormonal carcinogenesis in women: from mechanisms to prevention]. / Cancérogenèse hormonale chez la femme: des mécanismes à la prévention.

In spite of the explosion of basic knowledge, breast cancer remains a major problem of public health. Basic endocrinology was at the origin of the first targeted therapy in cancer with the anti-estrogens. Continuous breast cancer cell lines helped to specify the mechanism of the mitogenic activity of estrogens, the basis of their tumour promoter activity. They could not help to explain the deleterious effect of progestins after the menopause and to study the effect of ovarian hormones in the early steps of carcinogenesis. The variations of expression of ovarian hormone receptors in pre-malignant mammary lesions strongly suggest an increased sensitivity to these hormones. Moreover breast carcinogenesis is heterogeneous and the oestrogen receptor negative pathways require other targeted therapies. A better prevention of hormone-dependent cancers will need both increased basic researches translatable to human and good information of women on the avoidable risk factors.

Increased expression of fatty acid synthase and progesterone receptor in early steps of human mammary carcinogenesis.

Progestins increase the risk of breast cancer in the hormone therapy of menopause, and progesterone receptor-induced fatty acid synthase (FAS) is a potential therapeutical target of breast cancer. In a first attempt to specify in which lesions at risk of breast cancer progestins might be acting, we have compared the progesterone receptor (PR) and FAS expression in preinvasive breast lesions and in adjacent "normal" mammary glands. We used archive paraffin-embedded tissues from 116 patients, with 164 lesions of increasing histological risk from nonproliferative "benign" breast disease (BBD) to in situ breast carcinomas. Immunostaining using our FAS antibody and a PR antibody from Dako was quantified as continuous variables by computer-assisted image analysis. FAS level increased (p < 10(-3) by the Kruskall-Wallis test) in all lesions, starting from nonproliferative BBD, and was maximal in in situ carcinoma. The % of PR-positive cells increased from nonproliferative BBD and was higher in proliferative atypia (p < 10(-3)). It was very low in high-grade DCIS corresponding to a likely different carcinogenesis pathway. There was a trend for a positive correlation between FAS and PR in normal glands. However, the 2 markers increased independently in BBD and were negatively correlated in in situ carcinomas. FAS and PR were positively correlated with Ki67 in BBD. The increased PR level in premalignant steps of mammary carcinogenesis suggests an early increased responsiveness to progestins. The increased FAS expression, in lesions parallel to their increased breast cancer risk, suggests further studies to develop new markers of high-risk lesions and to prevent breast cancer.

Cathepsin D: newly discovered functions of a long-standing aspartic protease in cancer and apoptosis.

The lysosomal aspartic protease cathepsin D (cath-D) is over-expressed and hyper-secreted by epithelial breast cancer cells. This protease is an independent marker of poor prognosis in breast cancer being correlated with the incidence of clinical metastasis. Cath-D over-expression stimulates tumorigenicity and metastasis. Indeed it plays an essential role in the multiple steps of tumor progression, in stimulating cancer cell proliferation, fibroblast outgrowth and angiogenesis, as well as in inhibiting tumor apoptosis. A mutated cath-D devoid of catalytic activity still proved mitogenic for cancer, endothelial and fibroblastic cells, suggesting an extra-cellular mode of action of cath-D involving a triggering, either directly or indirectly, of an as yet unidentified cell surface receptor. Cath-D is also a key mediator of induced-apoptosis and its proteolytic activity has been involved generally in this event. During apoptosis, mature lysosomal cath-D is translocated to the cytosol. Since cath-D is one of the lysosomal enzymes which requires a more acidic pH to be proteolytically-active relative to the cysteine lysosomal enzymes, such as cath-B and -L, it is open to question whether cytosolic cath-D might be able to cleave substrate(s) implicated in the apoptotic cascade. This review summarises our current knowledge on cath-D action in cancer progression and metastasis, as well as its dual function in apoptosis.

Increased estrogen receptor betacx expression during mammary carcinogenesis.

Identification of proteins that markedly vary during early steps of mammary carcinogenesis may help to understand its pathophysiology and to develop a prevention strategy. The expression of total estrogen receptor beta (ERbeta) protein and of its COOH-terminally spliced variant ERbetacx (or ERbeta2) was compared in 43 invasive breast cancers and in 39 adjacent normal mammary glands and 26 ductal carcinoma in situ (DCIS). Thirty-six breast cancers were ER positive by radioligand binding assay. The analysis was done by immunohistochemistry on adjacent sections of formalin-fixed, paraffin-embedded tumors using polyclonal anti-ERbeta 503 IgY and sheep polyclonal ERbetacx antibodies that were previously validated. Nuclear staining was quantified using a computerized image analyzer in selected areas of normal and cancer epithelial cells. Total ERbeta expression was high in normal glands, decreased in DCIS (P = 0.0004), and increased from DCIS to invasive tumors (P = 0.029). In contrast, the ERbetacx expression was low in normal glands, increased significantly in DCIS (P = 0.0014), and continued to increase in invasive carcinomas (P = 0.0027) in both ERalpha-positive and ERalpha-negative tumors. This is the first study showing a significant increase of the ERbetacx variant protein in DCIS and invasive breast cancer compared with adjacent normal glands. This contrasts with the decrease of the total ERbeta level in the same patients and indicates different mechanisms to explain these variations during mammary carcinogenesis. It also suggests a role of the ERbetacx variant in carcinogenesis opposite to the protective effect of the wild-type ERbeta1.

Estrogen receptor beta (ER beta) level but not its ER beta cx variant helps to predict tamoxifen resistance in breast cancer.

The antiestrogen tamoxifen, a major endocrine therapy of estrogen receptor (ER)-positive breast cancer, is nevertheless inefficient in 30 to 40% of cases for unknown reasons. We retrospectively studied 50 ER-positive primary breast carcinomas. All of the patients had received tamoxifen as the only adjuvant therapy. They were divided into two groups depending on whether they relapsed within 5 years (16 tamoxifen-resistant cases) or did not relapse within 5 years (34 tamoxifen-sensitive cases). The expression of total ER beta protein, and of ER beta cx protein, was estimated anonymously in formalin-fixed, paraffin-embedded tumor sections, by using specific antibodies and quantifiying nuclear immunostaining with a computer image analyzer. All of the tumors were found to be HER-2/neu-negative by immunohistochemistry. Univariate analysis showed that Scarff-Bloom-Richardsson grade modified by Elston (SBR grade; P < 0.001), tumor size (P = 0.042), and MIB-1 proliferation index (P = 0.02) were significantly higher in tamoxifen-resistant tumors. A low level of total ER beta, whether in percentage of positive cells or in quantitative immunocytochemical (QIC) score, was also associated with tamoxifen resistance (P = 0.004). ER beta cx expression and lymph node status were similar between the two groups. The expression of ER beta in the total population was positively correlated with ER beta cx (r = 0.63, P < 0.001), and was independent of the other parameters. In a multivariate analysis, ER beta expression was the most important variable (P = 0.001), followed by SBR grade (I+II versus III; P = 0.008), and MIB-1 (P = 0.016). To conclude, tamoxifen resistance is associated with classical variables of aggressive tumors (high SBR grade, proliferation index, and tumor size) but not with node invasiveness. Low ER beta level is an additional independent marker, better than ER alpha level, to predict tamoxifen resistance.

Down-regulation of cathepsin-D expression by antisense gene transfer inhibits tumor growth and experimental lung metastasis of human breast cancer cells.

Overexpression of cathepsin-D in primary breast cancer has been associated with rapid development of clinical metastasis. To investigate the role of this protease in breast cancer growth and progression to metastasis, we stably transfected a highly metastatic human breast cancer cell line, MDA-MB-231, with a plasmid containing either the full-length cDNA for cathepsin-D or a 535 bp antisense cathepsin-D cDNA fragment. Clones expressing antisense cathepsin-D cDNA that exhibited a 70-80% reduction in cathepsin-D protein, both intra- and extracellularly compared to controls, were selected for further experiments. These antisense-transfected cells displayed a reduced outgrowth rate when embedded in a Matrigel matrix, formed smaller colonies in soft agar and presented a significantly decreased tumor growth and experimental lung metastasis in nude mice compared with controls. However, manipulating the cathepsin-D level in the antisense cells has no effect on their in vitro invasiveness. These studies demonstrate that cathepsin-D enhances anchorage-independent cell proliferation and subsequently facilitates tumorigenesis and metastasis of breast cancer cells. Our overall results provide the first evidence on the essential role of cathepsin-D in breast cancer, and support the development of a new cathepsin-D-targeted therapy.

Procathepsin D interacts with prosaposin in cancer cells but its internalization is not mediated by LDL receptor-related protein.

The cell surface binding, endocytosis, and lysosomal routing of procathepsin D (procath-D) in cancer cells are mostly independent of the mannose-6-phosphate (M6P) receptors. In an attempt to define the receptor involved, we intracellularly cross-linked procath-D with a 68-kDa protein that we identified with specific antibodies as prosaposin in human breast and ovarian cancer cell lines. In cancer cells, this protein-protein interaction was resistant to ammonium chloride or M6P treatment, indicating that it was independent of the M6P receptors. A similar interaction also occurred in the breast cancer cell culture medium between the secreted prosaposin and procath-D. Since these two precursors can be endocytosed, we then determined whether they were interacting with the same cell surface receptor. In fibroblasts, we confirmed that the endocytosis of these two proteins was different since it was generally mediated by the M6P receptors for procath-D and mostly by LRP (LDL receptor-related protein) for prosaposin. In breast cancer cells, prosaposin endocytosis was not detected, in contrast to procath-D endocytosis, suggesting that the majority of procath-D is not internalized as a complex with prosaposin. Moreover, RAP (receptor-associated protein), a ligand inhibiting LRP-mediated endocytosis, prevented internalization of prosaposin in 49-F rat fibroblasts, but did not affect procath-D M6P-independent internalization in MDA-MB231 cells. We conclude that in breast cancer cells, even though procath-D interacts intracellularly and extracellarly with prosaposin, it is endocytosed independent of prosaposin by a receptor different from the M6P receptors and the LRP.

Estrogen induction and overexpression of fibulin-1C mRNA in ovarian cancer cells.

Fibulin-1 is an extracellular matrix protein induced by estradiol in estrogen receptor (ER) positive ovarian cancer cell lines. Alternative splicing of fibulin-1 mRNA results in four different variants named A, B, C and D that may have distinct biological functions. We studied the relative expression of fibulin-1 mRNA variants and their estrogen regulation in human ovarian cancer cells. In ovarian tissues and cancer cell lines, fibulin-1C and -1D are the predominant forms, whereas fibulin-1A and -1B are weakly expressed. We developed a competitive PCR assay based on coamplification of fibulin-1C and -1D to study the relative expression of these fibulin-1 variants in human ovarian samples. In ovarian cancer cell lines and ovarian cancer samples, there was a marked increase in the fibulin-1C:1D and fibulin-1C:HPRT mRNA ratios as compared to normal ovaries. In the BG1 estrogen receptor positive ovarian cancer cell line, fibulin-1C mRNA was induced by estradiol in a dose- and time-dependent manner. Since others and we have previously shown an increased expression of ERalpha as compared to ERbeta in ovarian cancer cells, we investigated whether ERalpha or ERbeta is involved in this induction. For this aim, MDA-MB-231 breast cancer cell line, which expresses both low basal levels of ERs and fibulin-1, was infected with recombinant ERalpha or ERbeta encoding adenovirus and treated with estradiol. Fibulin-1C was induced by estradiol in ERalpha- but not ERbeta-infected cells, suggesting that fibulin-1C induction is mediated through ERalpha. In ovarian tumors, a trend towards a correlation between fibulin-1C and ERalpha expression levels was noted. In conclusion, this study showed an increased fibulin-1C:-1D mRNA ratio in ovarian cancer cells as compared to normal ovaries. This finding suggests that the C variant may be involved in ovarian carcinogenesis. Fibulin-1C overexpression may thus be a clue for the understanding of a putative role of estrogens in ERalpha promoted ovarian tumor progression.