The LIFR Inhibitor EC359 Effectively Targets Type II Endometrial Cancer by Blocking LIF/LIFR Oncogenic Signaling.

Endometrial cancer (ECa) is the most common female gynecologic cancer. When comparing the two histological subtypes of endometrial cancer, Type II tumors are biologically more aggressive and have a worse prognosis than Type I tumors. Current treatments for Type II tumors are ineffective, and new targeted therapies are urgently needed. LIFR and its ligand, LIF, have been shown to play a critical role in the progression of multiple solid cancers and therapy resistance. The role of LIF/LIFR in the progression of Type II ECa, on the other hand, is unknown. We investigated the role of LIF/LIFR signaling in Type II ECa and tested the efficacy of EC359, a novel small-molecule LIFR inhibitor, against Type II ECa. The analysis of tumor databases has uncovered a correlation between diminished survival rates and increased expression of leukemia inhibitory factor (LIF), suggesting a potential connection between altered LIF expression and unfavorable overall survival in Type II ECa. The results obtained from cell viability and colony formation assays demonstrated a significant decrease in the growth of Type II ECa LIFR knockdown cells in comparison to vector control cells. Furthermore, in both primary and established Type II ECa cells, pharmacological inhibition of the LIF/LIFR axis with EC359 markedly decreased cell viability, long-term cell survival, and invasion, and promoted apoptosis. Additionally, EC359 treatment reduced the activation of pathways driven by LIF/LIFR, such as AKT, mTOR, and STAT3. Tumor progression was markedly inhibited by EC359 treatment in two different patient-derived xenograft models in vivo and patient-derived organoids ex vivo. Collectively, these results suggest LIFR inhibitor EC359 as a possible new small-molecule therapeutics for the management of Type II ECa.

Estrogen receptor coregulator binding modulator (ERX-11) enhances the activity of CDK4/6 inhibitors against estrogen receptor-positive breast cancers.

BACKGROUND: CDK4/6 inhibitors in combination with endocrine therapy (AE/AI/SERDs) are approved for the treatment of ER+ advanced breast cancer (BCa). However, not all patients benefit from CDK4/6 inhibitors therapy. We previously reported a novel therapeutic agent, ERX-11, that binds to the estrogen receptor (ER) and modulates ER-coregulator interactions. Here, we tested if the combination of ERX-11 with agents approved for ER+ BCa would be more potent. METHODS: We tested the effect of combination therapy using BCa cell line models, including those that have acquired resistance to tamoxifen, letrozole, or CDK4/6 inhibitors or have been engineered to express mutant forms of the ER. In vitro activity was tested using Cell Titer-Glo, MTT, and apoptosis assays. Mechanistic studies were conducted using western blot, reporter gene assays, RT-qPCR, and mass spectrometry approaches. Xenograft, patient-derived explants (PDEs), and xenograft-derived explants (XDE) were used for preclinical evaluation and toxicity. RESULTS: ERX-11 inhibited the proliferation of therapy-resistant BCa cells in a dose-dependent manner, including ribociclib resistance. The combination of ERX-11 and CDK4/6 inhibitor was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BCa cells, in vitro, in xenograft models in vivo, xenograft-derived explants ex vivo, and in primary patient-derived explants ex vivo. Importantly, the combination caused xenograft tumor regression in vivo. Unbiased global mass spectrometry studies demonstrated profound decreases in proliferation markers with combination therapy and indicated global proteomic changes in E2F1, ER, and ER coregulators. Mechanistically, the combination of ERX-11 and CDK4/6 inhibitor decreased the interaction between ER and its coregulators, as evidenced by immunoprecipitation followed by mass spectrometry studies. Biochemical studies confirmed that the combination therapy significantly altered the expression of proteins involved in E2F1 and ER signaling, and this is primarily driven by a transcriptional shift, as noted in gene expression studies. CONCLUSIONS: Our results suggest that ERX-11 inhibited the proliferation of BCa cells resistant to both endocrine therapy and CDK4/6 inhibitors in a dose-dependent manner and that the combination of ERX-11 with a CDK4/6 inhibitor may represent a viable therapeutic approach.

Significance of ER-Src axis in hormonal therapy resistance.

The estrogen receptor (ER) is implicated in the progression of breast cancer. Despite positive effects of hormonal therapy, initial or acquired resistance to endocrine therapies frequently occurs. Recent studies suggested ERα-coregulator PELP1 and growth factor receptor ErbB2/HER2 play an essential role in hormonal therapy responsiveness. Src axis couples ERα with HER2 and PELP1, thus representing a new pathway for targeted therapy resistance. To establish the significance of ER-Src axis in PELP1 and HER2 mediated therapy resistance, we have generated model cells that stably express Src-shRNA under conditions of PELP1, HER2 deregulation. Depletion of Src using shRNA substantially reduced E2 mediated activation of Src and MAPK activation in resistant model cells. Pharmacological inhibition of Src using dasatinib, an orally available inhibitor substantially inhibited the growth of therapy resistant MCF7-PELP1, MCF7-HER2, and MCF7-Tam model cells in proliferation assays. In post-menopausal xenograft based studies, treatment with dasatinib significantly inhibited the growth of therapy resistant cells. IHC analysis revealed that the tumors were ERα positive, and dasatinib treated tumors exhibited alterations in Src and MAPK signaling pathways. Combinatorial therapy of tamoxifen with dasatinib showed better therapeutic effect compared to single agent therapy on the growth of therapy resistant PELP1 driven tumors. The results from our study showed that ER-Src axis play an important role in promoting hormonal resistance by proto-oncogenes such as HER2, PELP1, and blocking this axis prevents the development of hormonal independence in vivo. Since PELP1, HER2, and Src kinase are commonly deregulated in breast cancers, combination therapies using both endocrine agents and dasatinib may have better therapeutic effect by delaying the development of hormonal resistance.

PELP1: A novel therapeutic target for hormonal cancers.

Recent studies implicate that the estrogen receptor (ER) coregulator proline-, glutamic acid-, and leucine-rich protein (PELP) 1 as playing critical roles in ER-genomic, ER-nongenomic, and ER-signaling cross talk with growth factor signaling pathways. PELP1 expression is deregulated in hormonal cancers and recent studies further elucidated the molecular mechanisms by which PELP1 regulates hormone therapy response. Although PELP1 is important for normal functions of the ER, the possibility to target ER-PELP1 axis appears to be an effective strategy for preventing hormonal carcinogenesis and therapy resistance. Thus, PELP1 may be useful as prognostic marker for hormonal cancers and PELP1 signaling may be useful to generate targeted therapeutics to overcome hormonal therapy resistance.

Growth factor regulation of estrogen receptor coregulator PELP1 functions via Protein Kinase A pathway.

PELP1 (proline-rich, glutamic acid-rich, and leucine-rich protein-1) is a potential proto-oncogene that functions as a coregulator of estrogen receptor (ER), and its expression is deregulated during breast cancer progression. Emerging evidence suggests growth factor signaling crosstalk with ER as one possible mechanism by which breast tumors acquire resistance to therapy. In this study, we examined mechanisms by which growth factors modulate PELP1 functions, leading to activation of ER. Using in vivo labeling assays, we have found that growth factors promote phosphorylation of PELP1. Utilizing a panel of substrate-specific phosphorylated antibodies, we discovered that growth factor stimulation promotes phosphorylation of PELP1 that is recognized by a protein kinase A (PKA) substrate-specific antibody. Accordingly, growth factor-mediated PELP1 phosphorylation was effectively blocked by PKA-specific inhibitor H89. Utilizing purified PKA enzyme and in vitro kinase assays, we obtained evidence of direct PELP1 phosphorylation by PKA. Using deletion and mutational analysis, we identified PELP1 domains that are phosphorylated by PKA. Interestingly, site-directed mutagenesis of the putative PKA site in PELP1 compromised growth factor-induced activation and subnuclear localization of PELP1 and also affected PELP1-mediated transactivation function. Utilizing MCF-7 cells expressing a PELP1 mutant that cannot be phosphorylated by PKA, we provide mechanistic insights by which growth factor signaling regulates ER transactivation in a PELP1-dependent manner. Collectively, these findings suggest that growth factor signals promote phosphorylation of ER coactivator PELP1 via PKA pathway, and such modification may have functional implications in breast tumors with deregulated growth factor signaling.

Elevated aromatase expression correlates with cervical carcinoma progression.

OBJECTIVES: We have previously demonstrated that aromatase mRNA is induced in cervical carcinomas compared to normal tissue, suggesting that in situ aromatase expression leading to elevated local estrogen production may contribute to cervical carcinogensis. Our objectives are to examine 1) whether aromatase protein and activity are induced in cervical carcinomas, 2) aromatase expression correlates with disease stage, and 3) inflammatory cytokines (e.g., IL-6 and TNFalpha) may correlate with aromatase expression. METHODS: RNA and protein were isolated from human cervical carcinomas and normal cervical biopsies to examine aromatase expression, using real-time RT-PCR, Western blot analysis, and immunohistochemistry. Aromatase activity in tissue was measured using the tritiated water release method. IL-6 and TNFalpha expression was also examined. RESULTS: Aromatase protein and activity levels were increased in cervical carcinomas compared to normal tissue. RNA levels correlated significantly with disease progression, with highest aromatase expression detected in stage IV tumors (p<0.001, R(2)=0.77). Aromatase promoters 1.3 and 1.4 were elevated in cervical carcinomas and in cervical cancer cells. The expression of inflammatory cytokines IL-6 and TNFalpha, known to induce aromatase, significantly correlated with aromatase expression (R(2)>0.9). TNFalpha treatment induced aromatase expression in cervical cancer cells. CONCLUSION: Increased aromatase protein and activity in cervical carcinomas and the correlation of its expression with disease stage implicates it in cervical carcinogenesis. The correlation of IL-6 and TNFalpha expression with aromatase suggests that these inflammatory cytokines may induce aromatase expression, which is confirmed by induction of aromatase expression due to TNFalpha treatment of cervical cancer cells.

Modulation of in situ estrogen synthesis by proline-, glutamic acid-, and leucine-rich protein-1: potential estrogen receptor autocrine signaling loop in breast cancer cells.

In situ estrogen synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms especially in postmenopausal women. Several recent studies demonstrated activity of aromatase, an enzyme that plays a critical role in estrogen synthesis in breast tumors. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1/MNAR) is an estrogen receptor (ER) coregulator, and its expression is deregulated in breast tumors. In this study, we examined whether PELP1 promotes tumor growth by promoting local estrogen synthesis using breast cancer cells (MCF7) that stably overexpress PELP1. Immunohistochemistry revealed increased aromatase expression in MCF7-PELP1-induced xenograft tumors. Real-time PCR analysis showed enhanced activation of the aromatase promoter in MCF7-PELP1 clones compared with MCF7 cells. Using a tritiated-water release assay, we demonstrated that MCF7-PELP1 clones exhibit increased aromatase activity compared with control MCF-7 cells. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II, and growth factor signaling enhanced PELP1 activation of aromatase. PELP1-mediated induction of aromatase requires functional Src and phosphatidylinositol-3-kinase pathways. Mechanistic studies revealed that PELP1 interactions with ER-related receptor-alpha and proline-rich nuclear receptor coregulatory protein 2 lead to activation of aromatase. Immunohistochemistry analysis of breast tumor array showed increased expression of aromatase in ductal carcinoma in situ and node-positive tumors compared with no or weak expression in normal breast tissue. Fifty-four percent (n = 79) of PELP1-overexpressing tumors also overexpressed aromatase compared with 36% (n = 47) in PELP1 low-expressing tumors. Our results suggest that PELP1 regulation of aromatase represents a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.

Oncogenic potential of the nuclear receptor coregulator proline-, glutamic acid-, leucine-rich protein 1/modulator of the nongenomic actions of the estrogen receptor.

Proline-, glutamic acid-, leucine-rich protein 1 (PELP1), a novel nuclear receptor coactivator, and its expression is deregulated in hormone-dependent cancers, including those of the breast, endometrium, and ovary. PELP1 interacts with estrogen receptor and modulates its genomic and nongenomic functions. In this study, we examined whether PELP1 functions as an oncogene. The overexpression of PELP1 in fibroblasts and epithelial model cells resulted in cellular transformation. PELP1 also enhanced the transformation potential of c-Src kinase in focus formation assays, and PELP1 overexpression potentiated estradiol-mediated cell migratory potential and anchorage-independent growth. Using PELP1-small interfering RNA, we provided evidence that endogenous PELP1 plays an essential role in E2-mediated anchorage-independent growth, cell migration, and cytoskeletal changes. When compared with control vector transfectants, breast cancer cells stably overexpressing PELP1 showed a rapid tumor growth in xenograft studies. Immunohistochemical analysis of PELP1 expression using a tumor progression array of 252 breast carcinomas and normal breast tissue specimens revealed that PELP1 expression is deregulated to a greater degree in higher grade node-positive invasive tumors than in normal breast tissue or ductal carcinoma in situ. Our data suggest that PELP1 is a potential oncogene, that its expression is deregulated during cancer progression, and that PELP1 may play a role in oncogenesis.

Elevated expression of the oncogene c-fms and its ligand, the macrophage colony-stimulating factor-1, in cervical cancer and the role of transforming growth factor-beta1 in inducing c-fms expression.

Cervical cancer is the third most common gynecologic cancer in the United States. The presence and possible involvement of several cytokines have been studied in cervical cancer; however, very little data, if any, are available on whether cervical tumors are responsive to stimulation by the macrophage colony-stimulating factor-1 (CSF-1). Given the involvement of c-fms and its ligand CSF-1 in gynecologic cancers, such as that of the uterus and the ovaries, we have examined the expression of c-fms and CSF-1 in cervical tumor (n = 17) and normal cervix (n = 8) samples. The data show that c-fms and its ligand are significantly higher in cervical carcinomas compared with normal samples. Immunohistochemistry not only showed that tumor cells expressed significantly higher levels of c-fms but also c-fms levels were markedly higher in tumor cells than tumor-associated stromal cells. Blocking c-fms activity in cervical cancer cells, which express CSF-1 and c-fms, resulted in increased apoptosis and decreased motility compared with control, suggesting that CSF-1/c-fms signaling may be involved in enhanced survival and possibly invasion by cervical cancer cells via an autocrine mechanism. Combined, the data show for the first time the induction of CSF-1 and c-fms in cervical carcinomas and suggest that c-fms activation may play a role in cervical carcinogenesis. Additionally, our data suggest that transforming growth factor-beta1 may be a factor in inducing the expression of c-fms in cervical cancer cells. The data suggest that c-fms may be a valuable therapeutic target in cervical cancer.

Up-regulation of VEGF, c-fms and COX-2 expression correlates with severity of cervical cancer precursor (CIN) lesions and invasive disease.

OBJECTIVES: To describe the expression of vascular endothelial growth factor (VEGF), proto-oncogene macrophage colony-stimulating factor receptor (c-fms) and cyclooxygenase-2 (COX-2) in cervical carcinogenesis and to analyze the correlation of VEGF with c-fms and COX-2 expression. METHODS: In this study, 26 cases of benign cervix, 28 low-grade cervical intraepithelial neoplasia (CIN; CIN 1), 30 high-grade CIN (CIN 2/3) and 28 squamous cervical carcinomas (SCC) were examined by immunohistochemistry (IHC) and analysis was performed separately for epithelium and stroma. RESULTS: Positive epithelial expressions in normal cervix, low-grade CIN, high-grade CIN and SCC were, respectively: VEGF - 11.5%, 39.3%, 53.3% and 75% (P<0.001); c-fms - 0%, 10.7%, 40% and 67.9% (P<0.001); COX-2 - 7.7%, 39.3%, 80% and 100% (P<0.001). Stromal VEGF expression was higher than epithelial expression in all CIN grades and was also associated with the lesion grade, while c-fms and COX-2 stromal expression was weak. VEGF expression was statistically correlated to c-fms and COX-2 expression in high-grade CIN (P=0.020 and P=0.027, respectively) and SCC (P=0.015 and P=0.005, respectively). CONCLUSIONS: On the basis of our findings, these factors may participate in the development and progression of CIN lesions, with possible interaction of c-fms and COX-2 on VEGF expression, and may be potential molecular targets for studies of cervical cancer prevention and treatment.

Proline-, glutamic acid-, and leucine-rich protein-1/modulator of nongenomic activity of estrogen receptor enhances androgen receptor functions through LIM-only coactivator, four-and-a-half LIM-only protein 2.

Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a coregulator of multiple nuclear receptors. Molecular mechanisms of PELP1 function are not completely understood, but its expression is up-regulated in hormonal-dependent cancers. Using a yeast two-hybrid screen, we found that four-and-a-half LIM-only protein 2 (FHL2) interacted with PELP1. FHL2 is a transcriptional regulator that associates with nuclear cofactors, including androgen receptors (ARs), and contains an intrinsic activation domain. PELP1 and FHL2 interact in vitro and in vivo and colocalize in the nuclear compartment. PELP1 interacts with FHL2 via LIM domains 3 and 4 and synergistically enhances the transcriptional activity of FHL2. Src kinase is required for PELP1-mediated enhancement of FHL2 functions because knockdown of Src kinase expression or function abolished PELP1-mediated FHL2 activation functions. PELP1 interacted with AR and enhanced FHL2-mediated AR transactivation functions. PELP1 knockdown by small interfering RNA or PELP1 mutant, which lacks an activation domain, reduced FHL2-mediated AR transactivation. Biochemical analyses revealed a complex consisting of PELP1, FHL2, and AR in prostate cancer cells. PELP1/MNAR expression was elevated in high-grade prostate tumors. Our results suggest that PELP1 functions as a molecular adaptor, coupling FHL2 with nuclear receptors, and PELP1-FHL2 interactions may have a role in prostate cancer progression.

Inhibition of estrogen-induced mammary tumor formation in MMTV-aromatase transgenic mice by 4-chlorophenylacetate.

Treatment of estrogen-sensitive breast cancer with selective estrogen selective modulators (SERMs) and, more recently, aromatase inhibitors has met with wide success. However, antagonism of estrogen receptor (ER) activity in breast carcinomas by SERMs such as tamoxifen has been associated with increased risk of cancer in other tissue such as the endometrium. Furthermore, current therapies using aromatase inhibitors have side effects on bone resulting in development of osteoporosis in some patients. We present in this paper the results of a study using 4-chlorophenylacetate (4-CPA), a compound which belongs to a family of small aromatic fatty acids that has been shown to possess anticancer properties, to treat DMBA exposed MMTV-aromatase mice. These animals exhibit elevated levels of estrogen in their mammary glands and develop estrogen-responsive tumors. Consistent with our earlier findings showing that 4-CPA inhibited the growth of ER positive breast cancer cells in vitro, we now demonstrate that this compound inhibits tumor formation in MMTV-aromatase mice. This effect was not associated with reduction of ER expression in their mammary tissue, or to alteration of aromatase levels or activity. The data suggest that 4-CPA is a novel therapeutic agent that could be used in the prevention or treatment of estrogen-sensitive breast cancer.

HER-2/neu x aromatase double transgenic mice model: the effects of aromatase overexpression on mammary tumorigenesis.

A majority of breast cancers are hormone-responsive, and require estrogen for growth, and respond to hormonal therapy that blocks estrogen receptor action. Breast tumors with low levels of or completely lacking estrogen receptor fail to respond to antiestrogen therapy yet require estrogen for tumor initiation. To address the importance of local estrogen in oncogene-mediated breast tumorigenesis, we have crossed MMTV-aromatase with MMTV-HER2/neu and examined the incidence of breast cancer in double transgenic mice in comparison with parental strains. Double transgenic mice show normal mammary development and express both transgenes at similar levels to that of parental strains. Tumor incidence in double transgenic mice (<5%) decreased compared to HER2/neu mice (>65%). In addition to a significant decrease in tumorigenesis, these mice expressed ERalpha as well as high levels of ERbeta along with decreased levels of cyclin D1 and phosphorylated pRb among other changes. Furthermore, experiments using THC (ERalpha-agonist and ERbeta-antagonist) clearly demonstrate the critical role of ERbeta in HER2/neu-mediated tumorigenesis. These studies provide the first genetic evidence that estrogen receptor, mainly ERbeta than ERalpha and its dependent changes play an important role in regulating mammary tumorigenesis. These findings provide further evidence for development and testing of novel therapeutic approaches based on selective regulation of estrogen receptors (ERalpha and beta)-dependent actions for the treatment and prevention of breast cancers.

Computer-assisted immunohistochemical analysis of cervical cancer biomarkers using low-cost and simple software.

The study of biomarkers by immunohistochemistry (IHC) for cervical cancer and intraepithelial lesions is a promising field. However, manual interpretation of IHC and reproducibility of the scoring systems can be highly subjective. In this article, we present a novel and simple computer-assisted IHC interpretation method based on cyan-magenta-yellow-black (CMYK) color format, for tissues with diaminobenzidine cytoplasmatic staining counterstained with methyl green. This novel method is more easily interpreted than previous computer-assisted methods based on red-green-blue (RGB) color format and presents a strong correlation with the manual H-score. It is simple, objective, and requires only low-cost software and minimal computer skills. Briefly, a total of 67 microscopic images of cervical carcinoma, normal cervix, and negative controls were analyzed in Corel Photo Paint X3 software in CMYK and RGB color format, and compared with manual H-score IHC assessments. The clearest and best positive correlation with the H-score was obtained using the image in CMYK color format and crude values of magenta color (Spearman correlation coefficient=0.84; agreement of 93.33%, P<0.001). To obtain this value, only 3 steps were necessary: convert the image to CMYK format, select the area of interest for analysis, and open the color histogram tool to visualize the magenta value.

Induction of aromatase expression in cervical carcinomas: effects of endogenous estrogen on cervical cancer cell proliferation.

Epidemiologic studies have implicated estrogenic exposure as well as human papilloma virus (HPV) infection in cervical carcinogenesis, and some studies have suggested that estrogen and HPV may play synergistic roles in cervical tumorigenesis. In this study, we report a novel finding that approximately 35% of cervical carcinomas tested (n = 19) express aromatase, the enzyme responsible for converting androgen to estrogen, the rate-limiting and final step in estrogen biosynthesis. On the other hand, no aromatase expression was detected in precancerous (n = 42) or normal cervical (n = 17) tissue samples. Increased aromatase was associated with increases in estrogen receptors (ER-alpha and ER-beta) and a decrease in progesterone receptor levels, suggesting that in situ estrogen signaling via ER may be involved in tumor growth. Stable overexpression of aromatase in HPV+ cervical cancer cells resulted in increased cellular proliferation, anchorage-independent growth, and ER expression and activity. In contrast, little change in ER was observed in HPV- cells. Steroid hormone receptor expression observed in vitro paralleled that seen in cervical carcinomas expressing aromatase. Aromatase overexpression also induced the expression of cyclin D1, proliferating cell nuclear antigen, and the HPV oncogenes, E6 and E7. Furthermore, the data underscores the importance of steroid receptor (estrogen and progesterone receptors) regulation in cervical carcinogenesis. To our knowledge, this is the first report demonstrating the induction of aromatase expression in cervical carcinomas, and opens the possibility that aromatase inhibitors may be potential therapeutic agents in cervical carcinomas expressing aromatase.

Estrogen receptor coregulator binding modulators (ERXs) effectively target estrogen receptor positive human breast cancers.

The majority of human breast cancer is estrogen receptor alpha (ER) positive. While anti-estrogens/aromatase inhibitors are initially effective, resistance to these drugs commonly develops. Therapy-resistant tumors often retain ER signaling, via interaction with critical oncogenic coregulator proteins. To address these mechanisms of resistance, we have developed a novel ER coregulator binding modulator, ERX-11. ERX-11 interacts directly with ER and blocks the interaction between a subset of coregulators with both native and mutant forms of ER. ERX-11 effectively blocks ER-mediated oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant human breast cancer cells. ERX-11 is orally bioavailable, with no overt signs of toxicity and potent activity in both murine xenograft and patient-derived breast tumor explant models. This first-in-class agent, with its novel mechanism of action of disrupting critical protein-protein interactions, overcomes the limitations of current therapies and may be clinically translatable for patients with therapy-sensitive and therapy-resistant breast cancers.

Overexpression of the colony-stimulating factor (CSF-1) and/or its receptor c-fms in mammary glands of transgenic mice results in hyperplasia and tumor formation.

A number of recent studies have suggested that the colony-stimulating factor (CSF-1) and its receptor c-fms may be involved in the development of mammary glands during lactation and breast cancer. To study the role of CSF-1 or its receptor in initiation of mammary tumorigenesis, we have generated two independent lines of transgenic mice that overexpress either CSF-1 or c-fms under the control of the mouse mammary tumor virus promoter. Mammary glands of the virgin CSF-1 transgenic mice show increased ductal branching, hyperplasia, dysplasia, and other preneoplastic changes, which are indicative of increased cellular proliferation. Similar changes were also evident in the mammary glands of the c-fms transgenic mice. These changes became more prominent with age and resulted in mammary tumor formation. Moreover, secondary events like dimethylbenz(a)anthracene treatment accelerated mammary tumor formation in these mice. Although the expression of estrogen receptor alpha was not significantly changed in either of the transgenic mouse strains, progesterone receptor levels was higher in both transgenic lines as compared with the nontransgenic littermates. Expression of G1 cyclins was prominently increased in the mammary glands of both the CSF-1 and c-fms transgenic lines, suggesting increased cell cycle progression in these strains. In addition, the proliferation marker proliferating cell nuclear antigen (PCNA) and the mitogen-responsive transcription factor c-jun were also increased as compared with the nontransgenic controls. These findings, along with the histological data, support the hypothesis that CSF-1 and its receptor are involved in the etiology of breast cancer.

Selective Estrogen Receptor ß Agonist LY500307 as a Novel Therapeutic Agent for Glioblastoma.

Glioblastomas (GBM), deadly brain tumors, have greater incidence in males than females. Epidemiological evidence supports a tumor suppressive role of estrogen; however, estrogen as a potential therapy for GBM is limited due to safety concerns. Since GBM express ERß, a second receptor for estrogen, targeting ERß with a selective agonist may be a potential novel GBM therapy. In the present study, we examined the therapeutic effect of the selective synthetic ERß agonist LY500307 using in vitro and in vivo GBM models. Treatment with LY500307 significantly reduced the proliferation of GBM cells with no activity on normal astrocytes in vitro. ERß agonists promoted apoptosis of GBM cells, and mechanistic studies using RNA sequencing revealed that LY500307 modulated several pathways related to apoptosis, cell cycle, and DNA damage response. Further, LY500307 sensitized GBM cells to several FDA-approved chemotherapeutic drugs including cisplatin, lomustine and temozolomide. LY500307 treatment significantly reduced the in vivo tumor growth and promoted apoptosis of GBM tumors in an orthotopic model and improved the overall survival of tumor-bearing mice in the GL26 syngeneic glioma model. Our results demonstrate that LY500307 has potential as a therapeutic agent for GBM.

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice.

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor alpha and beta (ERalpha, ERbeta) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ERalpha in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase x ERalpha knockout cross (referred as aromatase/ERKO). Even though ERbeta is expressed in aromatase/ERKO mice, lack of ERalpha leads to impaired mammary growth in these mice. The data suggest that ERalpha plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ERalpha expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFbeta relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ERalpha results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ERalpha-mediated actions are critical for mammary development and carcinogenesis.

Use of letrozole as a chemopreventive agent in aromatase overexpressing transgenic mice.

Our recent studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in mammary glands, and gynecomastia and testicular cancer in male aromatase transgenic mice. Our studies also have shown that aromatase overexpression-induced changes in mammary glands can be abrogated with very low concentrations of letrozole, an aromatase inhibitor without any effect on normal physiology. In the present study, we have examined the effect of prior low dose letrozole treatment on pregnancy and lactation. We have also investigated the effect of low dose letrozole treatment on subsequent mammary growth and biochemical changes in these animals. There was no change in the litter size, birth weight and no visible birth defects in letrozole-treated animals. Although, there was an insignificant increase in mammary growth in aged animals after 6 weeks of letrozole treatment, the levels of expression of estrogen receptor, progesterone receptor and genes involved in cell cycle and cell proliferation remained low compared to control untreated animals. These observations indicate that aromatase inhibitors such as letrozole can be used as chemopreventive agents without effecting normal physiology in aromatase transgenic mice.