Beta protein 1 homeoprotein induces cell growth and estrogen-independent tumorigenesis by binding to the estrogen receptor in breast cancer.

Expression of Beta Protein 1 (BP1), a homeotic transcription factor, increases during breast cancer progression and may be associated with tumor aggressiveness. In our present work, we investigate the influence of BP1 on breast tumor formation and size in vitro and in vivo. Cells overexpressing BP1 showed higher viability when grown in the absence of serum (p \u3c 0.05), greater invasive potential (p \u3c 0.05) and formed larger colonies (p \u3c 0.004) compared with the controls. To determine the influence of BP1 overexpression on tumor characteristics, MCF-7 cells transfected with either empty vector (V1) or overexpressor plasmids (O2 and O4) were injected into the fat pads of athymic nude mice. Tumors grew larger in mice receiving O2 or O4 cells than in mice receiving V1 cells. Moreover, BP1 mRNA expression levels were positively correlated with tumor size in patients (p = 0.01). Interestingly, 20% of mice injected with O2 or O4 cells developed tumors in the absence of estrogen, while no mice receiving V1 cells developed tumors. Several mechanisms of estrogen independent tumor formation related to BP1 were established. These data are consistent with the fact that expression of breast cancer anti-estrogen resistance 1 (BCAR1) was increased in O2 compared to V1 cells (p \u3c 0.01). Importantly, O2 cells exhibited increased proliferation when treated with tamoxifen, while V1 cells showed growth inhibition. Overall, BP1 overexpresssion in MCF-7 breast cancer cells leads to increased cell growth, estrogen-independent tumor formation, and increased proliferation. These findings suggest that BP1 may be an important biomarker and therapeutic target in ER positive breast cancer

Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

<p>Abstract</p> <p>Background</p> <p>Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR) are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers.</p> <p>Methods</p> <p>Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses.</p> <p>Results</p> <p>We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76%) of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72%) and 27% had only low levels of expression.</p> <p>Conclusions</p> <p>Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in treatment of this disease by providing new reagents to study the protein expression of the human PRLR.</p

Progesterone Receptor Activates Msx2 Expression by Downregulating TNAP/Akp2 and Activating the Bmp Pathway in EpH4 Mouse Mammary Epithelial Cells

Previously we demonstrated that EpH4 mouse mammary epithelial cells induced the homeobox transcription factor Msx2 either when transfected with the progesterone receptor (PR) or when treated with Bmp2/4. Msx2 upregulation was unaffected by Wnt inhibitors s-FRP or Dkk1, but was inhibited by the Bmp antagonist Noggin. We therefore hypothesized that PR signaling to Msx2 acts through the Bmp receptor pathway. Herein, we confirm that transcripts for Alk2/ActR1A, a non-canonical BmpR Type I, are upregulated in mammary epithelial cells overexpressing PR (EpH4-PR). Increased phosphorylation of Smads 1,5, 8, known substrates for Alk2 and other BmpR Type I proteins, was observed as was their translocation to the nucleus in EpH4-PR cells. Analysis also showed that Tissue Non-Specific Alkaline Phosphatase (TNAP/Akp2) was also found to be downregulated in EpH4-PR cells. When an Akp2 promoter-reporter construct containing a ½PRE site was transfected into EpH4-PR cells, its expression was downregulated. Moreover, siRNA mediated knockdown of Akp2 increased both Alk2 and Msx2 expression. Collectively these data suggest that PR inhibition of Akp2 results in increased Alk2 activity, increased phosphorylation of Smads 1,5,8, and ultimately upregulation of Msx2. These studies imply that re-activation of the Akp2 gene could be helpful in downregulating aberrant Msx2 expression in PR+ breast cancers

The normal breast microenvironment of premenopausal women differentially influences the behavior of breast cancer cells in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>Breast cancer studies frequently focus on the role of the tumor microenvironment in the promotion of cancer; however, the influence of the normal breast microenvironment on cancer cells remains relatively unknown. To investigate the role of the normal breast microenvironment on breast cancer cell tumorigenicity, we examined whether extracellular matrix molecules (ECM) derived from premenopausal African-American (AA) or Caucasian-American (CAU) breast tissue would affect the tumorigenicity of cancer cells <it>in vitro </it>and <it>in vivo</it>. We chose these two populations because of the well documented predisposition of AA women to develop aggressive, highly metastatic breast cancer compared to CAU women.</p> <p>Methods</p> <p>The effects of primary breast fibroblasts on tumorigenicity were analyzed via real-time PCR arrays and mouse xenograft models. Whole breast ECM was isolated, analyzed via zymography, and its effects on breast cancer cell aggressiveness were tested <it>in vitro </it>via soft agar and invasion assays, and <it>in vivo </it>via xenograft models. Breast ECM and hormone metabolites were analyzed via mass spectrometry.</p> <p>Results</p> <p>Mouse mammary glands humanized with premenopausal CAU fibroblasts and injected with primary breast cancer cells developed significantly larger tumors compared to AA humanized glands. Examination of 164 ECM molecules and cytokines from CAU-derived fibroblasts demonstrated a differentially regulated set of ECM proteins and increased cytokine expression. Whole breast ECM was isolated; invasion and soft agar assays demonstrated that estrogen receptor (ER)^-, progesterone receptor (PR)/PR^-cells were significantly more aggressive when in contact with AA ECM, as were ER⁺/PR⁺cells with CAU ECM. Using zymography, protease activity was comparatively upregulated in CAU ECM. In xenograft models, CAU ECM significantly increased the tumorigenicity of ER⁺/PR⁺cells and enhanced metastases. Mass spectrometry analysis of ECM proteins showed that only 1,759 of approximately 8,000 identified were in common. In the AA dataset, proteins associated with breast cancer were primarily related to tumorigenesis/neoplasia, while CAU unique proteins were involved with growth/metastasis. Using a novel mass spectrometry method, 17 biologically active hormones were measured; estradiol, estriol and 2-methoxyestrone were significantly higher in CAU breast tissue.</p> <p>Conclusions</p> <p>This study details normal premenopausal breast tissue composition, delineates potential mechanisms for breast cancer development, and provides data for further investigation into the role of the microenvironment in cancer disparities.</p

In-Session Involvement in Anxious Youth Receiving CBT with/without Medication

Although in-session factors of CBT for youth anxiety (e.g., youth involvement; therapist behaviors) have demonstrated significant associations with treatment outcomes, no study has examined the role of concurrent selective-serotonin reuptake inhibitors (SSRI) on in-session behavior affecting youth outcomes. The combination of SSRI and CBT have demonstrated robust outcomes over either treatment alone. Research has also neglected to examine in-session behavior based on treatment phase (i.e., psychoeducation, exposure) and the association between in-session factors and treatment outcome. Youth (N = 190) were participants in the Children/Adolescent Anxiety Multimodal Study (Walkup et al. New England Journal of Medicine, 359, 2753-2766, 2008) who completed CBT; of these, n = 94 received concurrent sertraline. Tapes of psychoeducation/skillbuilding (first half) and exposure sessions (second half) were rated by reliable coders for positive youth involvement (e.g., participation, understanding), negative youth involvement (e.g., safety-behaviors, negative affect), and therapist behaviors. Youth and therapist in-session behaviors were examined as predictors of the trajectory of anxiety outcomes using multilevel modeling. Medication (sertraline) was examined as a moderator. Results indicated that positive and negative youth involvement in CBT was significantly associated with outcomes. Positive youth involvement during psychoeducation and exposure sessions predicted better outcomes, and negative youth involvement during psychoeducation sessions predicted less favorable outcomes. Sertraline did not moderate these findings. Therapist behaviors were not significantly associated with outcomes, likely due to limited variability and low frequency of observed behaviors. Youth in-sessions behaviors are associated with treatment outcomes in anxiety treatment. However, medication does not appear to have a differential impact on youth in-session behaviors