Hormone-independent Tumor Growth Research Articles

Abstract 1984: Anisotropy of collagen I matrix influences therapeutic response and secretome of Luminal A cells

Abstract High degree of collagen I fiber orientation has been associated with guided tumor cell invasion, but little is known about its impact in therapeutic response. Anisotropic substrates have been shown to influence the phenotype of stem cells which suggests that it can influence tumor cell behavior beyond cell migration. We hypothesized that the anisotropy of collagen I matrix impacts therapeutic response by modulating the secretome of breast cancer cells and this effect is mediated by integrin signaling. To test our hypothesis, the cell secretome, response to hormone ablation therapy and integrin inhibitors were evaluated in breast tumor cells cultured in collagen type 1 substrates of defined orientation. The orientation of electrospun collagen I fibrils was regulated by adjusting the syringe flow rate and collector velocity to obtain random (<70%) or aligned (>80%) oriented fibrils. Average porosity (45± 3.4 %), matrix stiffness (0.8 ±0.24 MPa) and fiber diameter (1.6 ± 0.86μm) were kept constant across collagen substrates. Estrogen-dependent Luminal A cells (MCF-7 and T-47D) cells were cultured on collagen substrates in a hormone-deprived environment for 3-6 days. Polycaprolactone (PCL) fibrous substrates of aligned and random orientation were used as negative fibrous controls for integrin signaling. Evaluation of cell growth showed that a fibrillar structure in combination with a high degree of fibril orientation significantly increased cell proliferation (>75%) as compared to gelatin-coated tissue culture plastic (TCP). Collagen fibers significantly supported hormone-independent tumor growth at levels equivalent to or above those observed in the estrogen-treated control. Increased proliferation levels were supported on collagen fibrous substrates treated with Tamoxifen and Fulvestrant, and this effect was enhanced in aligned collagen I. Inhibition of α2β1 integrin resulted in enhanced proliferation rates in random fibers and suppression of cell growth in aligned fibers. PCL aligned substrates where not sufficient to support hormone-independent cell growth. MCF-7 cells on collagen substrates significantly stimulated the growth (>7 fold) of adjacent cells on TCP in a dose-response manner as follows: aligned > random > gelatin. Expression levels of 1000 factors were quantified in conditioned medium to identify differences in the secretome signatures associated with cell-matrix signaling. MCF-7 cells cultured on collagen fibrous substrates displayed secretome signatures that were different across all substrates. Fourteen factors were identified to be upregulated in fibrous substrates by more than ten folds including apoptotic regulators RalA and BCL-2. Our results show for the first time the influence of collagen I fibril orientation in supporting hormone-independent growth and as a novel regulator therapeutic response in luminal A cells. Citation Format: Ana M. Reyes, Jorge Almodovar, Elaine Alarid, Maribella Domenech. Anisotropy of collagen I matrix influences therapeutic response and secretome of Luminal A cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1984.

Abstract 174: Collagen fiber orientation influences tumor cell growth and Hedgehog signaling in the breast tumor microenvironment

Abstract Collagen matrix architecture is an important player in tumor transition to therapeutic resistance. Several studies have shown that high level of fiber orientation and density of collagen type I matrix enhances tumor cell invasion, decrease drug response and influence tumor metabolism, but the mechanisms of action that support transition to therapeutic resistance are not fully understood. We developed a 3D culture platform to evaluate the effects of collagen matrix architecture in the sensitivity of tumor and stromal cells to tumor promoting ligands and pharmacological inhibitors. Estrogen (E2) and Hedgehog (Hh) signaling pathways were selected for evaluation due to their relevance in breast cancer prognosis and tumor progression. In this 3D culture platform, electrospun collagen fiber sheets of random or aligned orientation are bound to double-sided medical grade tape to generate collagen fiber stickers. Automated cutting plotter allows razor-printing for precisely cutting shapes from collagen fiber sticker sheets. Round shapes were applied like a sticker to the bottom of culture well plates prior to cell seeding. The percentage of matrix porosity fiber orientation and diameter was quantified by Scanning Electron Microscopy (SEM) and Image J analysis. For estrogen signaling studies, MCF-7 cells were seeded in collagen fibers of random or aligned orientation using hormone free-culture conditions. Cells were treated +/- E2 (10nM) ligand and +/- estrogen receptor signaling inhibitors (Fulvestrant and Tamoxifen). Tumor cell proliferation was quantified at 48hrs via Image Cytometry. For Hh signaling studies, expression levels of GLI1, PTCH1 and SMO Hh target genes were monitored in triple negative breast cancer (MDA-MB-231 and MDA-MB-468) and mesenchymal cells. Cells were seeded on collagen fibers and treated with sonic hedgehog ligand for 24hrs. Tumor and mesenchymal cells were harvest for viability and qRT-PCR analysis respectively. Evaluation of cell growth in MCF-7 cultures shows that a fibrillar structure of collagen matrix in combination with high degree of fiber orientation can significantly increase cell proliferation as compared to gelatin or tissue culture plastic substrates. Collagen fibers significantly supported hormone-independent tumor growth at similar levels to those observed in E2 treatment. Increased proliferation levels were maintained in ER inhibition groups particularly when cultured in aligned collagen fibers. In Hh signaling studies, overexpression of GLI1 and SMO genes were observed for collagen fibers matrixes as compared to gelatin and tissue culture plastic. Overall our results show for the first time that collagen fiber architecture can promote hormone-independent growth dependence in breast cancer cells and activation of hedgehog signaling in tumor and mesenchymal cells. Citation Format: Ana M. Reyes, Jorge Almodovar, Maribella Domenech. Collagen fiber orientation influences tumor cell growth and Hedgehog signaling in the breast tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 174.

Inhibiting Notch Activity in Breast Cancer Stem Cells by Glucose Functionalized Nanoparticles Carrying γ-secretase Inhibitors

Cancer stem cells (CSCs) are a challenge in cancer treatment due to their therapy resistance. We demonstrated that enhanced Notch signaling in breast cancer promotes self-renewal of CSCs that display high glycolytic activity and aggressive hormone-independent tumor growth in vivo. We took advantage of the glycolytic phenotype and the dependence on Notch activity of the CSCs and designed nanoparticles to target the CSCs. Mesoporous silica nanoparticles were functionalized with glucose moieties and loaded with a γ-secretase inhibitor, a potent interceptor of Notch signaling. Cancer cells and CSCs in vitro and in vivo efficiently internalized these particles, and particle uptake correlated with the glycolytic profile of the cells. Nanoparticle treatment of breast cancer transplants on chick embryo chorioallantoic membranes efficiently reduced the cancer stem cell population of the tumor. Our data reveal that specific CSC characteristics can be utilized in nanoparticle design to improve CSC-targeted drug delivery and therapy.

The combination of the prodrugs perforin-CEBPD and perforin-granzyme B efficiently enhances the activation of caspase signaling and kills prostate cancer.

The survival of prostate cancer (PrCa) patients is associated with the transition to hormone-independent tumor growth and metastasis. Clinically, the dysregulation of androgen action has been associated with the formation of PrCa and the outcome of androgen deprivation therapy in PrCa. CCAAT/enhancer binding protein delta (CEBPD) is a transcription factor that has been reported to act as an oncogene or tumor suppressor, depending on the extra- and intracellular environments following tumorigenesis. We found that androgen can activate CEBPD transcription by direct binding of the androgen receptor (AR) to the CEBPD promoter region. Increases of suppressor of zeste 12 (SUZ12) and enhancer of zeste homolog 2 (EZH2) attenuated the androgen-induced transcription of CEBPD. Importantly, the increases in E2F1, SUZ12 and EZH2 as well as the inactivation of CEBPD were associated with the clinicopathological variables and survival of PrCa patients. We revealed that caspase 8 (CASP8), an apoptotic initiator, is responsive to CEBPD induction. Reporter and in vivo DNA-binding assays revealed that CEBPD directly binds to and activates CASP8 reporter activity. A prodrug system was developed for therapeutic application in AR-independent or androgen-insensitive PrCa to avoid the epigenetic effects on the suppression of CEBPD expression. Our results showed that the combination of a perforin (PF)-CEBPD prodrug (which increases the level of procaspase-8) and a PF-granzyme B prodrug (which activates CASP8 and caspase 3 (CASP3)) showed an additive effect in triggering the apoptotic pathway and enhancing apoptosis in PrCa cells.

Adrenomedullin Blockade Suppresses Growth of Human Hormone–Independent Prostate Tumor Xenograft in Mice

To study the role of the adrenomedullin system [adrenomedullin and its receptors (AMR), CLR, RAMP2, and RAMP3] in prostate cancer androgen-independent growth. Androgen-dependent and -independent prostate cancer models were used to investigate the role and mechanisms of adrenomedullin in prostate cancer hormone-independent growth and tumor-associated angiogenesis and lymphangiogenesis. Adrenomedullin and AMR were immunohistochemically localized in the carcinomatous epithelial compartment of prostate cancer specimens of high grade (Gleason score >7), suggesting a role of the adrenomedullin system in prostate cancer growth. We used the androgen-independent Du145 cells, for which we demonstrate that adrenomedullin stimulated cell proliferation in vitro through the cAMP/CRAF/MEK/ERK pathway. The proliferation of Du145 and PC3 cells is decreased by anti-adrenomedullin antibody (αAM), supporting the fact that adrenomedullin may function as a potent autocrine/paracrine growth factor for prostate cancer androgen-independent cells. In vivo, αAM therapy inhibits the growth of Du145 androgen-independent xenografts and interestingly of LNCaP androgen-dependent xenografts only in castrated animals, suggesting strongly that adrenomedullin might play an important role in tumor regrowth following androgen ablation. Histologic examination of αAM-treated tumors showed evidence of disruption of tumor vascularity, with depletion of vascular as well as lymphatic endothelial cells and pericytes, and increased lymphatic endothelial cell apoptosis. Importantly, αAM potently blocks tumor-associated lymphangiogenesis, but does not affect established vasculature and lymphatic vessels in normal adult mice. We conclude that expression of adrenomedullin upon androgen ablation in prostate cancer plays an important role in hormone-independent tumor growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis. Clin Cancer Res; 19(22); 6138-50. ©2013 AACR.

Phytoalexins, miRNAs and Breast Cancer: A Review of Phytochemical-mediated miRNA Regulation in Breast Cancer

There is growing interest in the diverse signaling pathways that regulate and affect breast tumorigenesis, including the role of phytochemicals and the emerging role of microRNAs (miRNAs). Recent studies demonstrate that miRNAs regulate fundamental cellular and developmental processes at the transcriptional and translational level under normal and disease conditions. While there is growing evidence to support the role of phytoalexin-mediated miRNA regulation of cancer, few reports address this role in breast cancer. Recent reports by our group and others demonstrate that natural products, including stilbenes, curcumin, and glyceollins, could alter the expression of specific miRNAs, which may lead to increased sensitivity of cancer cells to conventional anti-cancer agents and, therefore, hormone-dependent and hormone-independent tumor growth inhibition. This review will discuss how dietary intake of natural products, by regulating specific miRNAs, contribute to the prevention and treatment of breast cancer.

PI3K/AKT pathway regulates phosphorylation of steroid receptors, hormone independence and tumor differentiation in breast cancer

Using a model of medroxyprogesterone acetate (MPA)-induced mouse mammary tumors that transit through different stages of hormone dependence, we previously reported that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT (protein kinase B) pathway is critical for the growth of hormone-independent (HI) mammary carcinomas but not for the growth of hormone-dependent (HD) mammary carcinomas. The objective of this work was to explore whether the activation of the PI3K/AKT pathway is responsible for the changes in tumor phenotype and for the transition to autonomous growth. We found that the inhibition of the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway suppresses HI tumor growth. In addition, we were able to induce mammary tumors in mice in the absence of MPA by inoculating HD tumor cells expressing a constitutively active form of AKT1, myristoylated AKT1 (myrAKT1). These tumors were highly differentiated and displayed a ductal phenotype with laminin-1 and cytokeratin 8 expression patterns typical of HI tumors. Furthermore, myrAKT1 increased the tumor growth of IBH-6 and IBH-7 human breast cancer cell lines. In the estrogen-dependent IBH-7 cell line, myrAKT1 induced estrogen-independent growth accompanied by the expression of the adhesion markers focal adhesion kinase and E-cadherin. Finally, we found that cells expressing myrAKT1 exhibited increased phosphorylation of the progesterone receptor at Ser190 and Ser294 and of the estrogen receptor α at Ser118 and Ser167, independently of exogenous MPA or estrogen supply. Our results indicate that the activation of the PI3K/AKT/mTOR pathway promotes tissue architecture remodeling and the activation of steroid receptors.

A Kinome-Wide Screen Identifies the Insulin/IGF-I Receptor Pathway as a Mechanism of Escape from Hormone Dependence in Breast Cancer

Estrogen receptor α (ER)-positive breast cancers adapt to hormone deprivation and become resistant to antiestrogens. In this study, we sought to identify kinases essential for growth of ER(+) breast cancer cells resistant to long-term estrogen deprivation (LTED). A kinome-wide siRNA screen showed that the insulin receptor (InsR) is required for growth of MCF-7/LTED cells. Knockdown of InsR and/or insulin-like growth factor-I receptor (IGF-IR) inhibited growth of 3 of 4 LTED cell lines. Inhibition of InsR and IGF-IR with the dual tyrosine kinase inhibitor OSI-906 prevented the emergence of hormone-independent cells and tumors in vivo, inhibited parental and LTED cell growth and PI3K/AKT signaling, and suppressed growth of established MCF-7 xenografts in ovariectomized mice, whereas treatment with the neutralizing IGF-IR monoclonal antibody MAB391 was ineffective. Combined treatment with OSI-906 and the ER downregulator fulvestrant more effectively suppressed hormone-independent tumor growth than either drug alone. Finally, an insulin/IGF-I gene expression signature predicted recurrence-free survival in patients with ER(+) breast cancer treated with the antiestrogen tamoxifen. We conclude that therapeutic targeting of both InsR and IGF-IR should be more effective than targeting IGF-IR alone in abrogating resistance to endocrine therapy in breast cancer.

Effects of SDF-1–CXCR4 signaling on microRNA expression and tumorigenesis in estrogen receptor-alpha (ER-α)-positive breast cancer cells

The majority of breast cancer cases ultimately become unresponsive to endocrine therapies, and this progression of breast cancer from hormone-responsive to hormone-independent represents an area in need of further research. Additionally, hormone-independent carcinomas are characterized as being more aggressive and metastatic, key features of more advanced disease. Having previously shown the ability of the stromal-cell derived factor-1 (SDF-1)–CXCR4 signaling axis to promote primary tumorigenesis and hormone independence by overexpressing CXCR4 in MCF-7 cells, in this study we further examined the role of SDF-1/CXCR4 in the endogenously CXCR4-positive, estrogen receptor α (ER-α)-positive breast carcinoma cell line, MDA–MB-361. In addition to regulating estrogen-induced and hormone-independent tumor growth, CXCR4 signaling stimulated the epithelial-to-mesenchymal transition, evidenced by decreased CDH1 expression following SDF-1 treatment. Furthermore, inhibition of CXCR4 with the small molecule inhibitor AMD3100 induced CDH1 gene expression and inhibited CDH2 gene expression in MDA–MB-361 cells. Further, exogenous SDF-1 treatment induced ER-α-phosphorylation in both MDA–MB-361 and MCF-7–CXCR4 cells, demonstrating ligand-independent activation of ER-α through CXCR4 crosstalk. qPCR microRNA array analyses of the MDA–MB-361 and MCF-7–CXCR4 cell lines revealed changes in microRNA expression profiles induced by SDF-1, consistent with a more advanced disease phenotype and further supporting our hypothesis that the SDF-1/CXCR4 signaling axis drives ER-α-positive breast cancer cells to a hormone independent and more aggressive phenotype. In this first demonstration of SDF-1–CXCR4-induced microRNAs in breast cancer, we suggest that this signaling axis may promote tumorigenesis via microRNA regulation. These findings represent future potential therapeutic targets for the treatment of hormone-independent and endocrine-resistant breast cancer.

MPA-induced gene expression and stromal and parenchymal gene expression profiles in luminal murine mammary carcinomas with different hormonal requirements

Over the past several years, we have been interested in understanding the mechanisms by which mammary carcinomas acquire hormone independence. We demonstrated that carcinoma associated fibroblasts participate in the ligand-independent activation of progesterone receptors inducing tumor growth. In this study, we used DNA microarrays to compare the gene expression profiles of tumors from the MPA mouse breast cancer model, one hormone-dependent (C4-HD) and one hormone-independent (C4-HI), using whole tumor samples or laser-captured purified stromal and epithelial cells obtained from the same tumors. The expression of selected genes was validated by immunohistochemistry and immunofluorescence assays. We identified 413 genes specifically expressed in tumor stroma. Eighty-five percent of these genes were upregulated, whereas the remaining 15% were downregulated in C4-HI relative to their expression in the C4-HD tumor stroma. Several matrix metallopeptidases were overexpressed in the C4-HI tumor microenvironment. On the other hand, 1100 genes were specifically expressed in the tumor parenchyma. Among them, the 29% were upregulated, whereas the remaining 71% were downregulated in C4-HI relative to C4-HD tumor epithelium. Steap, Pdgfc, Runx2, Cxcl9, and Sdf2 were among the genes with high expression in the C4-HI tumor parenchyma. Interestingly, Fgf2 was one of the few genes upregulated by MPA in C4-HD tumors, confirming its pivotal role in regulating tumor growth in this model. In conclusion, we demonstrate herein a gene expression profile that distinguishes both the epithelial and the stromal cells in mammary tumors with different hormone dependence, supporting the hypothesis that the tumor-associated stroma may contribute to hormone-independent tumor growth.

Inoculated mammary carcinoma-associated fibroblasts: contribution to hormone independent tumor growth

BackgroundIncreasing evidence has underscored the role of carcinoma associated fibroblasts (CAF) in tumor growth. However, there are controversial data regarding the persistence of inoculated CAF within the tumors. We have developed a model in which murine metastatic ductal mammary carcinomas expressing estrogen and progesterone receptors transit through different stages of hormone dependency. Hormone dependent (HD) tumors grow only in the presence of progestins, whereas hormone independent (HI) variants grow without hormone supply. We demonstrated previously that CAF from HI tumors (CAF-HI) express high levels of FGF-2 and that FGF-2 induced HD tumor growth in vivo. Our main goal was to investigate whether inoculated CAF-HI combined with purified epithelial (EPI) HD cells can induce HD tumor growth.MethodsPurified EPI cells of HD and HI tumors were inoculated alone, or together with CAF-HI, into female BALB/c mice and tumor growth was evaluated. In another set of experiments, purified EPI-HI alone or combined with CAF-HI or CAF-HI-GFP were inoculated into BALB/c or BALB/c-GFP mice. We assessed whether inoculated CAF-HI persisted within the tumors by analyzing inoculated or host CAF in frozen sections of tumors growing in BALB/c or BALB/c-GFP mice. The same model was used to evaluate early stages of tumor development and animals were euthanized at 2, 7, 12 and 17 days after EPI-HI or EPI-HI+CAF-HI inoculation. In angiogenesis studies, tumor vessels were quantified 5 days after intradermal inoculation.ResultsWe found that admixed CAF-HI failed to induce epithelial HD tumor growth, but instead, enhanced HI tumor growth (p < 0.001). Moreover, inoculated CAF-HI did not persist within the tumors. Immunofluorescence studies showed that inoculated CAF-HI disappeared after 13 days. We studied the mechanisms by which CAF-HI increased HI tumor growth, and found a significant increase in angiogenesis (p < 0.05) in the co-injected mice at early time points.ConclusionsInoculated CAF-HI do not persist within the tumor mass although they play a role during the first stages of tumor formation promoting angiogenesis. This angiogenic environment is unable to replace the hormone requirement of HD tumors that still need the hormone to recruit the stroma from the host.

Abstract 542: Inoculated carcinoma associated fibroblasts and hormone independent murine mammary tumor growth

Abstract Murine mammary carcinomas from the MPA-murine breast cancer model express estrogen and progesterone receptors and transit through different stages of hormone dependence. Hormone dependent (HD) tumors only grow in the presence of medroxyprogesterone acetate (MPA) and hormone independent (HI) variants are those that grow without hormone supply. In co-culture studies we have demonstrated that carcinoma associated fibroblasts (CAF) from HI tumors (CAF HI) can stimulate epithelial cell proliferation better than those from HD tumors (CAF HD). However, in vivo, CAF-HI inoculated together with purified epithelial cells (EPI) only increased tumor growth of EPI HI and they were not able to replace the hormone requirement of EPI HD. Thus, the aim of this study was to investigate the role of CAF HI enhancing HI tumor growth and to elucidate why CAF HI failed to stimulate HD cells in vivo. For this purpose murine C4-HI tumors were transplanted in BALB/c-wt (wt) or in BALB/c-GFP (GFP) mice. Mice were subcutaneously inoculated with EPI HI alone or co-mingled with CAF HI-wt (in GFP mice) or CAF HI-GFP (in wt mice) and the resulting tumors were excised at different time points. Fluorescence analysis of frozen sections indicated that inoculated CAF HI-GFP were detected 7 days after their inoculum in wt mice whereas they were no longer detected at later time points. To elucidate how CAF HI are able to enhance tumor growth when co-inoculated with EPI HI cells, we quantified polymorphonuclear (hematoxilin and eosin) and mast cells (toluidine blue staining) in the EPI HI + CAF HI group and in the EPI HI group and no significant differences were observed. In similar experiments when cells were inoculated intradermically to evaluate angiogenesis, and animals were sacrificed after different time points, we observed that on day 5, there was a significantly higher number of vessels in the co-inoculated group (Control 1.1±0.1; CAF HI 1.8± 0.8; EPI HI 2.0±0.7; EPI HI + CAF HI 3.3±0.5 p&amp;lt;0.05). In summary, we conclude that the inoculated CAF fail to induce HD growth because they do not persist in the tumor mass, although they may participate during the first stages of tumor growth favoring angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 542.

Abstract 3170: Interaction between FGFR-2, progesterone receptor and STAT5 in breast cancer models

Abstract We have developed a murine model of breast cancer in which hormone-dependent (HD) carcinomas transit through different stages of hormone responsiveness retaining the expression of estrogen and progesterone receptors (PR). Unlike HD carcinomas, hormone-independent (HI) tumors do not need the exogenous administration of medroxyprogesterone acetate (MPA) to grow. However, when tumor cells are cultured, there are no differences in hormone responsiveness between both types, suggesting the involvement of host factors regulating in vivo tumor growth. We have previously reported that carcinoma associated fibroblasts (CAF) stimulate HI cells inducing PR activation and that FGFR-2 plays a key role mediating this effect. In addition, MPA and FGF2 induce a direct interaction between FGFR-2 and PR in the nuclei of C4-HI and T47D cells. We have postulated that the stroma of HI tumors regulates HI tumor growth by releasing FGF-2, that acting as a paracrine growth factor, can activate PR in epithelial cells. It is also known that STAT5 can be activated either by FGF2 or by progesterone. Thus, the main goal of this study was to study the mechanisms by which FGF2 stimulates HI growth evaluating the interaction between PR, FGFR-2 and STAT5 using our murine tumor model and the human T47D breast cancer cells. C4-HI primary cultures or T47D cells were grown in chamber slides, starved using 1% charcoalized fetal calf serum for 24 hours and treated with FGF-2 (50 ng/ml), MPA (10 nM), FGF1 (20 ng/ml) or vehicle for 24 hs. Confocal imaging suggested an intimate interaction between STAT5 and PR when the cells were treated with FGF-2 or MPA rather than with FGF1. The interaction of both proteins in response to MPA or FGF2 was confirmed by co-immunoprecipitation assays using C4-HI tumor samples or T47D cells. Next, we performed gene transcription reporter assays in both, C4-HI and T47D cells using PRE or GAS-Luc vectors. Interestingly, in C4-HI cells, FGF2 was able to activate PRE and GAS reporter genes (p&amp;lt;0.05) while progestins activated mainly the PRE reporter gene. In T47D cells, however, MPA and FGF-2 activated both reporter genes (p&amp;lt;0.05). These results corroborate the crosstalk between both pathways. We also studied the expression of proteins coded from endogenous genes which have PRE or GAS sequences in their promoters such as Bcl-xl, CyclinD1, Tissue Factor and STAT5 itself. The expression of all these proteins was similarly increased after treatment with FGF2 or MPA (p&amp;lt;0.05) supporting the previous in vitro data. In summary, these results show for the first time that PR interact with FGFR-2 and with STAT5 in the nuclei of MPA- or FGF2-treated cells, and this is followed with the expression of target genes. These data support our hypothesis where FGF2 released by the tumor stroma activates FGFR-2 in the epithelial cells inducing PR and STAT5 activation, providing a possible mechanism for the acquisition of hormone independency. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3170.

Carcinoma‐associated fibroblasts activate progesterone receptors and induce hormone independent mammary tumor growth: A role for the FGF‐2/FGFR‐2 axis

The mechanisms by which mammary carcinomas acquire hormone independence are still unknown. To study the role of cancer-associated fibroblasts (CAF) in the acquisition of hormone-independence we used a hormone-dependent (HD) mouse mammary tumor and its hormone-independent (HI) variant, which grows in vivo without hormone supply. HI tumors express higher levels of FGFR-2 than HD tumors. In spite of their in vivo differences, both tumors have the same hormone requirement in primary cultures. We demonstrated that CAF from HI tumors (CAF-HI) growing in vitro, express higher levels of FGF-2 than HD counterparts (CAF-HD). FGF-2 activated the progesterone receptors (PR) in the tumor cells, thus increasing cell proliferation in both HI and HD tumors. CAF-HI induced a higher proliferative rate on the tumor cells and in PR activation than CAF-HD. The blockage of FGF-2 in the co-cultures or the genetic or pharmacological inhibition of FGFR-2 inhibited PR activation and tumor cell proliferation. Moreover, in vivo, the FGFR inhibitor decreased C4-HI tumor growth, whereas FGF-2 was able to stimulate C4-HD tumor growth as MPA. T47D human breast cancer cells were also stimulated by progestins, FGF-2 or CAF-HI, and this stimulation was abrogated by antiprogestins, suggesting that the murine C4-HI cells respond as the human T47D cells. In summary, this is the first study reporting differences between CAF from HD and HI tumors suggesting that CAF-HI actively participate in driving HI tumor growth.

Establishment and characterization of two breast cancer xenografts in immunodeficient mice for studies on hormone-dependent and hormone-independent tumor growth, progression and invasion

Establishment and characterization of two breast cancer xenografts in immunodeficient mice for studies on hormone-dependent and hormone-independent tumor growth, progression and invasion

Progression of metastatic human prostate cancer to androgen independence in immunodeficient SCID mice.

Prostate cancer mortality results from metastasis to bone and hormone-independent tumor growth. Models to study these progressive changes are lacking. Here we describe the propagation of advanced human prostate cancer by direct transfer of surgical samples from patients into immune-deficient male SCID mice. Explants from six of eight patients formed prostate tumors and two showed unique cytogenetic, biologic and molecular features that were retained through six or more passages. One grew in an androgen-independent fashion, whereas the second formed tumors that regressed following castration then regrew. Micrometastatic disease was detected in the hematopoietic tissues of half of the recipient mice. Thus selected specimens of advanced human prostate cancer can be propagated in SCID mice in a manner that recapitulates the clinical transition from androgen-sensitive to androgen-independent growth, accompanied by micrometastasis.

A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo.

MCF-7 human breast cancer cells provide a useful in vitro model system to study hormone-responsive breast cancer as they contain receptors for estrogen and progesterone, and estrogen both induces the synthesis of specific proteins in these cells and increases their rate of proliferation. An MCF-7 cell line which was selected for resistance to adriamycin (MCF-7/AdrR) exhibits the phenotype of multidrug resistance (MDR), and displays multiple biochemical changes. MDR in MCF-7/AdrR is also associated with a loss of mitogenic response to estrogen and the development of cross-resistance to the antiestrogen 4-hydroxytamoxifen. In addition, while the parental MCF-7 cell line responds to estrogen with increased levels of progesterone receptors and the secretion of specific proteins, these estrogen responses are lost in MCF-7/AdrR. Furthermore, while the formation of tumors in nude mice by wild-type MCF-7 cells is dependent upon the presence of estrogen, MCF-7/AdrR cells form tumors in the absence of exogenous estrogen administration. These changes in hormonal sensitivity and estrogen-independent tumorigenicity of the multidrug-resistant MCF-7 cell line are associated with a loss of the estrogen receptor and a concomitant increase in the level of receptors for epidermal growth factor. Thus, in MCF-7/AdrR cells, the development of MDR is associated with alterations in the expression of both cytosolic and membrane receptors, resulting in resistance to hormonal agents and the expression of hormone-independent tumor formation.