Hormone-independent Variants Research Articles

The presence of androgen receptor elements regulates ZEB1 expression in the absence of androgen receptor.

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that plays a central role in the epithelial to mesenchymal transition (EMT) of cancer cell lines. Studies on its regulation have mostly focused on the negative 3'UTR binding of miR200c. Interestingly, it has been previously reported that androgen receptor (AR) regulates ZEB1 expression in breast and prostate cancers. In order to validate this, various ZEB1 promoter deletions were cloned into a luciferase reporter system to elucidate the contribution of two putative androgen response elements (AREs). The in vivo contribution of AR was also assessed in cell lines after R1881 treatment using qPCR with prostate specific antigen (PSA) as the positive control. We discovered that AR upregulates the levels of expression of ZEB1 10-fold on a luciferase promoter that only contains the distal ARE. However, when the proximal ARE is included, no additional activation is apparent with AR or its hormone independent variant, AR-V7. Furthermore, we demonstrate here that a promoter construct containing both AREs activates transcription of ZEB1 even in the AR-null cell lines DU145 and PC3. Incubation of the AR-positive cell line, LNCaP with R1881, failed to substantially increase the expression levels of ZEB1. Despite the presence of AREs in the promoter region, it appears that ZEB1 expression can be induced even without AR. In addition, the region around the distal ARE is a potent repressor in AR-null cell lines.

Associated expressions of FGFR-2 and FGFR-3: from mouse mammary gland physiology to human breast cancer

Fibroblast growth factor receptors (FGFRs) are tyrosine kinase receptors which have been implicated in breast cancer. The aim of this study was to evaluate FGFR-1, -2, -3, and -4 protein expressions in normal murine mammary gland development, and in murine and human breast carcinomas. Using immunohistochemistry and Western blot, we report a hormonal regulation of FGFR during postnatal mammary gland development. Progestin treatment of adult virgin mammary glands resulted in changes in localization of FGFR-3 from the cytoplasm to the nucleus, while treatment with 17-β-estradiol induced changes in the expressions and/or localizations of FGFR-2 and -3. In murine mammary carcinomas showing different degrees of hormone dependence, we found progestin-induced increased expressions, mainly of FGFR-2 and -3. These receptors were constitutively activated in hormone-independent variants. We studied three luminal human breast cancer cell lines growing as xenografts, which particularly expressed FGFR-2 and -3, suggesting a correlation between hormonal status and FGFR expression. Most importantly, in breast cancer samples from 58 patients, we found a strong association (P<0.01; Spearman correlation) between FGFR-2 and -3 expressions and a weaker correlation of each receptor with estrogen receptor expression. FGFR-4 correlated with c-erbB2 over expression. We conclude that FGFR-2 and -3 may be mechanistically linked and can be potential targets for treatment of estrogen receptor-positive breast cancer patients.

Abstract 3353: Hormone receptor and FGFR expression in mammospheres of a hormone dependent murine mammary carcinoma and its hormone independent variant

Abstract It has been postulated that breast cancer stem cells (CSC) or progenitor cells express low levels of hormone receptors. Mouse mammary carcinomas induced by medroxyprogesterone acetate (MPA) are maintained by syngeneic transplantation and tumor variants with different hormone responsiveness have been generated. Tumors that only grow with MPA supply are named hormone dependent (HD), and tumors which grow without MPA were defined as hormone independent (HI). The main goal of our project is to study the evolution of the CSC compartment in mouse mammary carcinomas which transit through different stages of hormone dependence, focusing in the profiling of estrogen receptors alpha (ERα), progesterone receptors (PR), and fibroblast growth factor receptors 1-4 (FGFR1-4). Mammary cancer cells can be cultured using different methods: adhered to plastic, in matrigel, in soft agar or in suspension as non adherent spherical clusters of cells named mammospheres. It is accepted that there is an enrichment of the mammary CSC population when cells are grown as mamospheres. We performed primary cell cultures of the C4-HD tumor, a hormone-dependent mouse mammary carcinoma maintained by syngeneic transplantations in MPA-treated BALB/c mice, and of one of its hormone independent variants, C4-HI. Cells were cultured on plastic or as mammospheres. Using flow cytometry we analyzed the expression profile of the surface markers ALDH1 or CD44 and CD24, which have been reported to define the SC subpopulation (CD44High/CD24low/- cells). An increase of the CSC population was observed in both tumors when cultured as mammospheres, as compared with those growing in plastic. However, we did not detect differences between the HD and the HI tumors. Next, we compared the expression of ERα, PR and FGFR1-4 by western blot, using protein extracts of epithelial cells growing as mammospheres or monolayers. Although there were no differences in the expression of PR or ERα in extracts from both culture types, we did observe an increase in the expression of FGFR2 and 4 (p &amp;lt; 0,05) in mammospheres from both tumors. Flow cytometry analysis showed that most of the cells CD24low express PR. Taken together our results suggest that the CSC of this model are progenitor cells that express high levels of ERα, PR, FGFR2 and 4. The high levels of FGFR4 are almost exclusive of cells growing as mammospheres, suggesting that these receptors might be used as a CSC marker in this breast cancer model. It remains to be determined if this high expression is the consequence of the anchorage independent growth. Ongoing studies will reveal the evolution of these progenitor cells in hormone resistant variants. 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 3353. doi:10.1158/1538-7445.AM2011-3353

Abstract A27: Efficacy of estramustine + docetaxel in docetaxel-resistant human prostate cancer xenograft: a preclinical model of docetaxel resistance reversion

Abstract Introduction: Androgen-dependent prostate cancers (PC) are usually sensitive to docetaxel chemotherapy. Nevertheless, docetaxel-resistance frequently appears after several cycles of treatment, raising the treatment rescue of docetaxel-resistant PC patients. If the combination of estramustine to docetaxel prolongs the metastatic-free and overall survivals of patients with androgen-dependent PC, its use remains limited in patients because of unacceptable toxicity including venous thromboses. The aims of this study were (1) to evaluate the in vivo efficacy of estramustine combined to docetaxel since initial tumor growth and since appearance of docetaxel resistance in one androgen-dependent human PC xenograft, and (2) to evaluate the efficacy of estramustine in six human androgen-independent human PC variants. Experimental procedures: The preclinical models included the androgen-dependent PAC120 tumor obtained from a Gleason-9 primary PC of a 51-years old patient (De Pinieux et al, Am J Pathol, 2001), and six hormone-independent variants derived from PAC120. Docetaxel was administrated at a dosage of 20 mg/kg every three weeks by intraperitoneal injection for at most six cycles, and estramustine was given intraperitoneally at a dosage of 12 mg/kg days 1 to 5 every 3 weeks until mice sacrifice. Tumor volume was measured twice a week and Relative Tumor Volume (RTV) from start of treatment were then calculated. In the PAC120 model, escape to docetaxel was defined at the beginning of each next cycle (n + 1) as a ratio of RTVn+1/RTVn ≥ 2. Resistant mice were then randomized into two groups, one receiving estramustine alone, and the other treated by a combination of docetaxel + estramustine, with an evaluation of the tumor growth delay for a 2-fold tumor size increase from randomisation (TGD2). In the 6 androgen-independent PC xenografts (HID), estramustine administered alone was compared to a control group. Results: Estramustine alone did not induce significant tumor growth inhibition in both PAC120 and HID xenografts. In PAC120 model, tumor growth inhibition (TGI) after docetaxel alone and docetaxel + estramustine was 81% and 95%, with a mean response duration of 29 days [21–63] and 68 days [64–74], respectively (p &amp;lt; 0.05). In docetaxel-resistant tumor bearing mice, estramustine alone induced a TGD2 of 18 days, whereas estramustine + docetaxel induced a TGD2 of 49 days (p &amp;lt; 0.05). Metastatic lesions and venous thromboses into lung of tumor-bearing mice have also been evaluated by histopathological analyses and may be presented during the meeting. Conclusions: Estramustine alone was not efficient in both human androgen-dependent and -independent PC xenografts. Inversely, the combination of estramustine + docetaxel in first treatment line and estramustine added to docetaxel in docetaxel-resistant xenografts was highly effective. These data therefore suggest that estramustine should be combined to docetaxel in PC patients, but its use could be delayed, particularly in elderly patients, to docetaxel refractory cases. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A27.

Hormone escape is associated with genomic instability in a human prostate cancer model

Lack of hormone dependency in prostate cancers is an irreversible event that occurs through generation of genomic instability induced by androgen deprivation. Indeed, the cytogenetic profile of hormone-dependent (HD) prostate cancer remains stable as long as it received a hormone supply, whereas the profile of hormone-independent (HID) variants acquired new and various alterations. This is demonstrated here using a HD xenografted model of a human prostate cancer, PAC120, transplanted for 11 years into male nude mice and 4 HID variants obtained by surgical castration. Cytogenetic analysis, done by karyotype, FISH, CGH and array-CGH, shows that PAC120 at early passage presents numerous chromosomal alterations. Very few additional alterations were found between the 5th and 47th passages, indicating the stability of the parental tumor. HID variants largely maintained the core of chromosomal alterations of PAC120 - losses at 6q, 7p, 12q, 15q and 17q sites. However, each HID variant displayed a number of new alterations, almost all being specific to each variant and very few shared by all. None of the HID had androgen receptor mutations. Our study indicates that hormone castration is responsible for genomic instability generating new cytogenetic abnormalities susceptible to alter the properties of cancer cell associated with tumor progression, such as increased cell survival and ability to metastasize.

Potentiation of antitumour activity of docetaxel by combination with trastuzumab in a human prostate cancer xenograft model and underlying mechanisms

Antitumour activity of docetaxel (Taxotere®) in hormone-dependent (HD) and hormone-independent (HID) prostate cancer PAC120 xenograft model was previously reported, and its level was associated with HER2 protein expression. In the present study, we evaluate the antitumour effects of docetaxel combined with trastuzumab (Herceptin®), an anti-HER2 antibody. Although trastuzumab alone had no effect on tumour growth, it potentiated the antitumour activity of docetaxel in HD tumours and more strongly in HID variants. Using the HID28 variant, we show that docetaxel treatment of tumour-bearing mice induces an increased HER2 mRNA expression of the tyrosine kinase receptor of 25-fold 24 h after docetaxel treatment, while HER2 protein and p-AKT decreased. This was followed by an increase of HER2 protein 3 days (two-fold) after docetaxel treatment and by a strong HER2 release in the serum of treated mice; expression of phospho-ERK, p27, BCL2 and HSP70 concomitantly increased. Similar molecular alterations were induced by docetaxel plus trastuzumab combination, except for that there was a transient and complete disappearance of AR and HSP90 proteins 24 h after treatment. We show that in addition to its known effects on tubulin and mitotic spindles, docetaxel induces complex signalisation pathway mechanisms in surviving cells, including HER2, which can be pharmacologically targeted. This study suggests that the docetaxel/trastuzumab combination may prove an effective therapeutic approach for HER2-expressing hormone-refractory prostate cancer.

Mucinous differentiation features associated with hormonal escape in a human prostate cancer xenograft.

Many theories mention hypersensitive, promiscuous, outlaw or bypass signalling pathways to explain the acquisition of hormone independence in prostate cancer. Hormonal escape of prostate tumours is marked by many biological changes, including mucinous and neuroendocrine differentiation. Since expression of several mucins has been linked to carcinoma tumour progression, we have characterised the expression of mucins at both RNA and protein levels in an in vivo model of prostate cancer in hormonal escape. Using PAC120, a xenograft of a human hormone-dependent prostate tumour, and its hormone-independent variants, we analysed the expression of mucins (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC6) by immunohistochemistry or reverse transcriptase (RT)–PCR. While the parental PAC120 tumour was a compact poorly-differentiated tumour of Gleason score 9 (5+4), hormone-independent variants displayed mucinous, neuroendocrine-like or mixed histological changes; these changes were stable through serial transplantations or after testosterone supply. MUC1 mRNA was expressed in both PAC120 and the hormone-independent variants, although at variable levels. All tumours displayed a high and constant expression of MUC2 and no expression of MUC4 mRNA. While MUC1 was expressed in all xenografts whatever their hormone dependence status, MUC2, MUC5B and MUC6 were preferentially expressed in hormone-independent variants. The loss of hormone dependence in this prostate cancer xenograft model is therefore marked by irreversible histological alterations, mucinous or neuro-endocrine, associated with an expression of secretory MUC2, MUC5B and MUC6, independent of the histological differentiation subtype. These data point to mucinous differentiation as an important step in the acquisition of hormone independence in this cancer, and suggest that secretory mucins might participate in an unknown pathway of hormonal escape in prostate cancer.

LuCaP 35: a new model of prostate cancer progression to androgen independence.

Generation of suitable in vivo models is critical for understanding of processes associated with development and progression of prostate cancer (CaP). Lymph nodes containing metastatic androgen-independent CaP were implanted into athymic mice. A xenograft designated LuCaP 35 and its hormone-independent variant LuCaP 35V were established and characterized. LuCaP 35 is an androgen-sensitive, prostate-specific antigen (PSA)-producing xenograft. It expresses the wild-type androgen receptor and exhibits deletions in chromosome 8p, but not in chromosome 10. The response of LuCaP 35 to androgen ablation is similar to that observed in man. Using recurring LuCaP 35 tumors we have also established an androgen-insensitive variant of LuCaP 35. The availability of hormone-dependent and -independent variants of LuCaP 35, which exhibit many properties analogous to those of CaP in man, provides an excellent model system to study the processes associated with development of androgen independence and to evaluate new treatment modalities.

Growth inhibition by anti-estrogens and progestins in TGF-beta-resistant and -sensitive breast-tumor cells.

Transforming growth factor beta (TGF-beta) is a potent growth inhibitor of non-malignant breast tissue, and TGF-beta resistance could play a role in tumorigenesis. Treatment of breast-tumor cells with anti-estrogens and progestins has been shown to correlate with an increase in the levels of secreted TGF-beta, suggesting that the growth inhibition observed with these (anti)hormones is mediated by this growth factor. In the present study we have investigated the effects of anti-estrogens and progestins on breast-tumor cell lines, which are either resistant or sensitive to TGF-beta. A hormone-independent variant of the MCF7 cell line is shown to have lost its sensitivity to TGF-beta during its progression towards an autonomous phenotype, but has preserved its sensitivity to anti-estrogens. In addition, evidence is presented showing that progestins and anti-estrogens inhibit proliferation, irrespective of the sensitivity to TGF-beta in variants of the T47D cell line. Therefore, we conclude that, although TGF-beta seems an important growth inhibitor for mammary epithelial cells, both progestins and anti-estrogens can inhibit cell proliferation independent of induced TGF-beta production.

The metastatic potential of rat prostate tumor variant R3327-MatLyLu is correlated with an increased activity of N-acetylglucosaminyl transferase III and V

Enzyme activities of N-acetylglucosaminyltransferase (GlcNAc-Tase) I–V involved in N-linked complex-type carbohydrate synthesis were determined in a non-metastatic hormone-dependent rat prostate tumor (R3327-H) and a related, hormone-independent variant metastasizing to lymph nodes and lungs (R3327-MatLyLu). In the metastasizing variant a significantly increased activity of both GlcNAc-Tase III and GlcNAc-Tase V was observed, whereas the activities of GlcNAc-Tase I and II were essentially unchanged. The increase in activity of GlcNAc-Tase III is particularly noteworthy since it indicates that elevated expression of this enzyme cannot be considered as an exclusive marker of hepatic malignancy.

GM-CSF restoration of a differentiated (growth factor-regulated) phenotype in an anaplastic tumor

GM-CSF (granulocyte-macrophage-derived colony-stimulating factor) is a differentiation agent that stimulates bone marrow activity in patients receiving chemotherapy. GM-CSF (1 microgram/ml daily for 10 days), administered intralesionally, was evaluated to determine whether it would restore a more differentiated phenotype to an anaplastic, rapidly growing, hormone-independent variant (R3327 MAT-LyLu) of the Dunning prostatic adenocarcinoma. Immunohistology was used to quantitate the expression of epithelial growth factor receptors (rEGF) and the tissue testosterone content. GM-CSF therapy significantly (P less than 0.05) restored rEGF expression and tissue testosterone to levels associated with better differentiated, slower growing, androgen-dependent Dunning variants (R3327 H and G). GM-CSF may have a role in treatment of prostatic cancers by promoting androgen and epithelial growth factor regulation.

Activation of int-1 and int-2 mammary oncogenes in hormone-dependent and -independent mammary tumors of GR mice.

Mammary tumors in the GR mouse strain are caused by the expression of an endogenous provirus of the mouse mammary tumor virus (MMTV). The tumors progress from a hormone-dependent growth phase to autonomous, hormone-independent growth. We studied proviral insertion of MMTV at the int-1 and int-2 mammary oncogenes and the transcription of these genes during progression of a series of transplanted mammary tumors. During the hormone-dependent phase, 6 of 15 transplanted tumors were positive for proviral insertion at int-1 or int-2 or both. These tumors were oligoclonal with respect to the fraction of tumor cells with novel int-1 and int-2 restriction fragments and, apparently, consisted of different tumor cells with proviruses integrated at different oncogenes, including genes that are not yet known. In 10 tumors we detected expression of the int genes, indicating that most tumors contain minor populations of cells with int-1 or int-2 activations. On transplantation the tumors remained oligoclonal during the hormone-dependent phase. The hormone-independent variants of the tumors emerged as clonal outgrowths of cells with MMTV proviruses that could be traced back in the hormone-dependent tumors, but not always those of cells that were positive for insertions near int-1 or int-2. The maintenance of oligoclonality during the hormone-dependent phase suggests a growth-controlling effect of different populations of cells on each other. The clonal, hormone-independent tumors that arise later seem to be the result of mutations that are unrelated to int activation.

Characterization of progesterone receptors, estrogen receptors, and estrogen (type II)-binding sites in the hormone-independent variant of the MXT-3590 mouse mammary tumor.

A hormone-independent mouse mammary tumor line was derived by transplantation of a hormone-dependent line (MXT-3590) into ovariectomized-adrenalectomized animals and subsequent transplantation of surviving tumors. Although the tumor line is hormone independent, it contains a full complement of estrogen and progesterone receptors which are similar to those found in hormone-dependent tumors. This similarity includes the criteria of nuclear translocation of the estrogen receptor and subsequent stimulation of progesterone receptor synthesis. In addition to the estrogen receptor, a secondary estrogen-binding site (type II) is also present; this binding site was previously described in the hormone-dependent companion tumor line. Thus, while this variant tumor line contains the biosynthetic pathways that control estrogen and progesterone receptor synthesis, its growth does not depend on these steroids. Therefore, it may provide a good model for the study of this class of hormone-independent tumors.

Effects of thymidine analogs upon growth control in cultured hormone-dependent rat ovary cells

As described previously, a cultured line of rat ovary cells (31A), undergoes a transition in the presence of 5-fluorodeoxyuridine (FUdR); cells which had previously been dependent for growth upon hormones added to the culture medium became autonomous following exposure to FUdR, in a medium deficient in required growth factors. Working with 31Ac3, a clonal subline of 31A, I have found this transition to be due to two factors: (1) decreased FUdR sensitivity of the hormone-independent variant cells arising in a normal population; (2) a spontaneous transition to autonomy undergone by c3 cells in hormone-deficient medium, irrespective of the presence or absence of FUdR. However, other thymidine analogs, notably 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR) and 5-trifluoromethyldeoxyuridine (F 3TdR), can effect a transient modification of c3 cells to a hormone-independent state. This may result from incorporation of these analogs into cellular DNA, for simultaneous treatment with cytosine arabinoside or hydroxyurea, both inhibitors of DNA synthesis, prevents the transition of BUdR-treated cells to autonomy.