Effect of infertility treatment and pregnancy-related hormones on breast cell proliferation in vitro.

BACKGROUND: Breast cancer development involves a series of mutations in a heterogeneous group of proto-oncogenes/tumor suppressor genes that alter mammary cells to create a microenvironment permissive to tumorigenesis. Exposure to hormones during infertility treatment may have a mutagenic effect on normal mammary epithelial cells, high-risk breast lesions and early-stage breast cancers. Our goal was to understand the association between infertility treatment and normal and cancerous breast cell proliferation. METHODS: MCF-10A normal mammary cells and the breast cancer cell lines MCF-7 [estrogen receptor (ER)-positive, well differentiated] and HCC 1937 (ER-negative, aggressive, BRCA1 mutation) were treated with the weak ER activator clomiphene citrate and hormones that are increased during infertility treatment. Direct effects of treatment on cell proliferation and colony growth were determined. RESULTS: While clomiphene citrate had no effect on MCF-10A cells or MCF-7 breast cancer cells, it decreased proliferation of HCC 1937 versus untreated cells (P= 0.003). Estrogen had no effect on either MCF-10A or HCC 1937 cells but, as expected, increased cell proliferation (20-100 nM; P≤0.002) and colony growth (10-30 nM; P< 0.0001) of MCF-7 cells versus control. Conversely, progesterone decreased both proliferation (P= 0.001) and colony growth (P= 0.01) of MCF-10A cells, inhibited colony size of MCF-7 cells (P= 0.01) and decreased proliferation of HCC 1937 cells (P= 0.008) versus control. hCG (100 mIU/ml) decreased both proliferation (P ≤ 0.01) and colony growth (P ≤ 0.002) of all three cell lines. CONCLUSIONS: Although these data are preclinical, they support possible indirect estrogenic effects of infertility regimens on ER-positive breast cancer cells and validate the potential protective effect of pregnancy-related exposure to hCG.

Androgen deprivation induces human prostate epithelial neuroendocrine differentiation of androgen-sensitive LNCaP cells.

Neuroendocrine (NE) cells are the minor cell populations in normal prostate epithelial compartments. During prostate carcinogenesis, the number of NE cells in malignant lesions increases, correlating with its tumorigenicity and hormone-refractory growth. It is thus proposed that cancerous NE cells promote prostate cancer (PCa) cell progression and its androgen-independent proliferation, although the origin of the cancerous NE cells is not clear. To investigate the role of cancerous NE cells in prostate carcinogenesis, we characterized three NE subclone cell lines-NE-1.3, NE-1.8 and NE-1.9, which were transdifferentiated from androgen-sensitive human PCa LNCaP cells by culturing in an androgen-depleted environment, resembling clinical androgen-ablation therapy. These subclone cells acquire many features of NE cells seen in clinical prostate carcinomas, for example exhibiting a neuronal morphology and expressing multiple NE markers, including neuron-specific enolase, chromogranin B, neurotensin, parathyroid hormone-related peptide, and to a lesser degree for chromogranin A, while lacking androgen receptor (AR) or prostate specific antigen (PSA) expression. These cells represent terminally differentiated stable cells because after 3 months of re-culturing in a medium containing androgenic activity, they still retained the NE phenotype and expressed NE markers. Despite these NE cells having a slow growth rate, they readily developed xenograft tumors. Furthermore, media conditioned by these NE cells exhibited a stimulatory effect on proliferation and PSA secretion by LNCaP cells in androgen-deprived conditions. Additionally, we found that receptor protein tyrosine phosphatase alpha plays a role in upregulating multiple NE markers and acquiring the NE phenotype. These NE cells thus represent cancerous NE cells and could serve as a useful cell model system for investigating the role of cancerous NE cells in hormone-refractory proliferation of PCa cells.

Transcriptional activation of the human prostatic acid phosphatase gene by NF-kappaB via a novel hexanucleotide-binding site.

Human prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen. Cellular PAcP functions as a neutral protein tyrosine phosphatase and is involved in regulating androgen-promoted prostate cancer cell proliferation. Despite the fact that the promoter of the PAcP gene has been cloned, the transcriptional factors that regulate PAcP expression remain unidentified. This article describes our analyses of the promoter of the PAcP gene. Deletion analyses of the promoter sequence up to -4893 (-4893/+87) revealed that a 577 bp fragment (-1356/-779) represents the unique positive cis-active element in human prostate cancer cells but not in HeLa cervix carcinoma cells. Interestingly, the 577 bp fragment contains a non-consensus nuclear factor kappaB (NF-kappaB)-binding site that is required for NF-kappaB up-regulation in prostate cancer cells, while NF-kappaB failed to have the same effect in HeLa cells. Conversely, inhibition of the NF-kappaB pathway stopped p65 NF-kappaB activation of the p1356 promoter activity. Gel shift and mutation analyses determined that AGGTGT (-1254/-1249) is the core sequence for NF-kappaB-binding and activation. Biologically, tumor necrosis factor-alpha (TNF-alpha) activated endogenous PAcP expression in LNCaP human prostate cancer cells. The data collectively indicate that NF-kappaB up-regulates PAcP promoter activity via its binding to the AGGTGT motif, a novel binding sequence located inside the cis-active enhancer element in human prostate cancer cells.

Identification and characterization of regulatory elements of the human prostatic acid phosphatase promoter.

Human prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen. The cellular form of PAcP functions as a neutral protein-tyrosine phosphatase, and is involved in regulating prostate cell growth. Although some information on the PAcP gene structure has been obtained, little is known regarding the cis- and trans-acting factors that regulate its expression. Due to the biological importance of PAcP, we investigated the regulation of its expression. A region upstream of the PAcP gene from -2899 to +87 base pairs was linked to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene. Sequential deletions of the sequence between -2899 and -205 revealed that, in addition to the basic promoter, the region between -1258 and -779 represents a positive regulatory element. This -1258/-779 fragment could enhance the PAcP promoter activity in PC-3 and DU 145 human prostate cancer cells, but not in non-prostate cancer cells, including WI-38 lung diploid cells, A-431 epidermoid carcinoma cells, and HeLa cervix epitheloid carcinoma cells. Furthermore, this cis-element together with the promoter sequence could drive a high level of expression of green fluorescent protein (GFP) in PC-3 cells, but not in HeLa cells. The prostate-specific expression was further examined by injecting naked plasmid DNA into the prostate and the hamstring muscle of mice. The fluorescence pattern clearly showed that the level of GFP expression is consistently higher in prostate cells than in muscle cells of the intact animal. The data collectively indicate that region between -1258 and -779 is involved in governing the cell type-specific expression of the PAcP gene.

CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin.

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFß superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.

Impact of infertility regimens on breast cancer cells: follicle-stimulating hormone and luteinizing hormone lack a direct effect on breast cell proliferation in vitro.

OBJECTIVE: To examine the impact of hormones used for controlled ovarian hyperstimulation (COH) on normal and malignant breast cell growth and proliferation. DESIGN: In vitro study of cultured normal and malignant breast cell lines. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): Normal and malignant breast cell lines cultured in two- and three-dimensional (2D and 3D) systems and treated with follicle-stimulating hormone (FSH), luteinizing hormone (LH), or FSH with LH or human chorionic gonadotropin (hCG). MAIN OUTCOME MEASURE(S): Effects of treatment on cell proliferation in 2D culture using the MTS assay and on colony growth in 3D culture. RESULT(S): Compared with untreated cells, normal MCF-10A cells showed a decrease in proliferation and colony size when exposed to a combination of FSH and hCG. The HCC 1937 cells treated with FSH and LH also showed a decrease in colony growth but no change in proliferation. None of the treatments had an effect on the proliferation or colony size of the MCF-7 cells. CONCLUSION(S): Follicle-stimulating hormone, LH, and hCG do not appear to cause an increase in cell proliferation or colony growth in either normal or malignant mammary epithelial cell lines. The potential risk for mammary cell transformation associated with these agents may be related to indirect endocrine effects on breast cell physiology.

Cyclin-dependent kinase 4-mediated phosphorylation inhibits Smad3 activity in cyclin D-overexpressing breast cancer cells.

Smad3, a component of the transforming growth factor ß signaling cascade, contributes to G(1) arrest in breast cancer cells. Cyclin D1/cyclin-dependent kinase 4 (CDK4) promotes G(1)-S-phase transition, and CDK phosphorylation of Smad3 has been associated with inhibition of Smad3 activity. We hypothesized that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through CDK4-mediated phosphorylation and inhibition of Smad3 and release of G(1) arrest. Real-time quantitative reverse transcription-PCR and immunoblotting were used to evaluate expression of study proteins in cyclin D1-overexpressing breast cancer cells. Smad3 transcriptional activity and cell cycle control were examined in cells transfected with wild-type (WT) Smad3 or Smad3 with single or multiple CDK phosphorylation site mutations (M) in the presence or absence of the CDK4 inhibitor or cotransfection with cdk4 small interfering RNA (siRNA). Transfection of the Smad3 5M construct resulted in decreased c-myc and higher p15(INK4B) expression. Compared with WT Smad3, overexpression of the Smad3 T8, T178, 4M, or 5M mutant constructs resulted in higher Smad3 transcriptional activity. Compared with cells transfected with WT Smad3, Smad3 transcriptional activity was higher in cells overexpressing Smad3 mutant constructs and treated with the CDK4 inhibitor or transfected with cdk4 siRNA. Cells transfected with Smad3 T8 or T178 and treated with the CDK4 inhibitor showed an increase in the G(1) cell population. Inhibition of CDK-mediated Smad3 phosphorylation released cyclin D1-regulated blockade of Smad3 transcriptional activity and recovered cell cycle arrest in breast cancer cells. Targeted inhibition of CDK4 activity may have a role in the treatment of cyclin D-overexpressing breast cancers.

Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines.

Smad3, a component of the TGFß signaling pathway, contributes to G1 arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild type (WT) Smad3, or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1), or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers.

Suppression of ErbB-2 in androgen-independent human prostate cancer cells enhances cytotoxic effect by gemcitabine in an androgen-reduced environment.

We examined the efficacy of combination treatments utilizing cytotoxic drugs plus inhibitors to members of the ErbB-ERK signal pathway in human prostate cancer (PCa) LNCaP C-81 cells. Under an androgen-reduced condition, 50nM gemcitabine caused about 40% growth suppression on C-81 cells. Simultaneous treatment of gemcitabine plus 10microM AG825 produced 60% suppression (p<0.03); while, 85% growth inhibition (p<0.02) was seen if AG825 was added to gemcitabine-treated cells after a 24h-interval. Our data thus showed that in androgen-reduced conditions, inhibition of ErbB-2 increases the cytotoxic efficacy of gemcitabine in PCa cells. This finding has significant implications in the choice of drugs for combination therapy as well as the order of administration for treating cancer patients.

ERK inhibitor PD98059 enhances docetaxel-induced apoptosis of androgen-independent human prostate cancer cells.

Anticancer drugs docetaxel and vinorelbine suppress cell growth by altering microtubule assembly and activating the proapoptotic signal pathway. Vinorelbine and docetaxel have been approved for treating several advanced cancers. However, their efficacy in the management of advanced hormone-refractory prostate cancer remains to be clarified. Microtubule damage by some anticancer drugs can activate the ERK survival pathway, which conversely compromises chemotherapeutic efficacy. We analyzed the effect of ERK inhibitors PD98059 and U0126 on vinorelbine- and docetaxel-induced cell growth suppression of androgen-independent prostate cancer cells. In androgen-independent C-81 LNCaP cells, inhibition of ERK by PD98059, but not U0126, plus docetaxel resulted in enhanced growth suppression by an additional 20% compared to the sum of each agent alone (p < 0.02). The combination treatment of docetaxel plus PD98059 also increased cellular apoptosis, which was in part due to the inactivation of Bcl-2 by increasing phosphorylated Bcl-2 by more than 6-fold and Bax expression by 3-fold over each agent alone. At these dosages, docetaxel alone caused only marginal phosphorylation of Bcl-2 (10%). Docetaxel plus U0126 had only 20% added effect on Bcl-2 phosphorylation compared to docetaxel alone. Nevertheless, both U0126 and PD98059 exhibited an enhanced effect on docetaxel-induced growth suppression in PC-3 cells. No enhanced effect was observed for vinorelbine plus PD98059 or U0126. Thus, the combination therapy of docetaxel plus PD98059 may represent a new anticancer strategy, requiring lower drug dosages compared to docetaxel monotherapy. This may lower the cytotoxicity and enhance tumor suppression in vivo. This finding of a combination effect could be of potential clinical importance in treating hormone-refractory prostate cancer.