Peroxisome proliferator-activated receptor delta and gamma agonists differentially alter tumor differentiation and progression during mammary carcinogenesis

Peroxisome proliferator-activated receptor (PPAR) represents a ligand-dependent nuclear receptor family that regulates multiple metabolic processes associated with fatty acid beta-oxidation, glucose utilization, and cholesterol transport. These and other receptor-mediated actions pertain to their role in hypolipidemic and antidiabetic therapies and as potential targets for cancer chemopreventive agents. The present study evaluated the chemopreventive activity of two highly potent and selective PPARgamma and PPARdelta agonists in a progestin- and carcinogen-induced mouse mammary tumorigenesis model. Animals treated with the PPARgamma agonist GW7845 exhibited a moderate delay in tumor formation. In contrast, animals treated with the PPARdelta agonist GW501516 showed accelerated tumor formation. Significantly, tumors from GW7845-treated mice were predominantly ductal adenocarcinomas, whereas tumors from GW501516-treated animals were adenosquamous and squamous cell carcinomas. Gene expression analysis of tumors arising from GW7845- and GW501516-treated mice identified expression profiles that were distinct from each other and from untreated control tumors of the same histopathology. Only tumors from mice treated with the PPARgamma agonist expressed estrogen receptor-alpha in luminal transit cells, suggesting increased ductal progenitor cell expansion. Tumors from mice treated with the PPARdelta agonist exhibited increased PPARdelta levels and activated 3-phosphoinositide-dependent protein kinase-1 (PDK1), which co-associated, suggesting a link between the known oncogenic activity of PDK1 in mammary epithelium and PPARdelta activation. These results indicate that PPARdelta and PPARgamma agonists produce diverse, yet profound effects on mammary tumorigenesis that give rise to distinctive histopathologic patterns of tumor differentiation and tumor development.     

Various effects of hepatoma-derived growth factor on cell growth, migration and invasion of breast cancer and prostate cancer cells

Hepatoma-derived growth factor (HDGF) has been implicated in the growth and metastasis of various types of human cancer, but the role of HDGF expression in prostate cancer or breast cancer has not been documented. To assess the role of HDGF in the proliferation, migration and invasion by prostate and breast cancer cells, HDGF expression in DU145 and MCF7 cells was knocked down using siRNA, and the effect of such knockdown was assessed by MTS and [3H]-thymidine incorporation Transwell assays. Moreover, we identified differentially expressed genes that might mediate the HDGF-induced cellular effects. Our results demonstrate that down-regulation of HDGF expression significantly reduces the proliferation of both DU145 and MCF7 cells. However, down-regulation of HDGF expression in DU145 inhibited cell migration and invasion, but in MCF7 cells it stimulated cell migration and invasion. This differential effect might result from the differential induction of PIK3R1 or SERPINE1 in the two cell lines upon HDGF-siRNA treatment. In conclusion, HDGF may participate in the pathogenesis of prostate and breast cancer by promoting cell growth and it may be a therapeutic target for these cancers.     

Peroxisome proliferator-activated receptor-delta induces cell proliferation by a cyclin E1-dependent mechanism and is up-regulated in thyroid tumors

Peroxisome proliferator-activated receptors (PPARs) are lipid-sensing nuclear receptors that have been implicated in multiple physiologic processes including cancer. Here, we determine that PPARdelta induces cell proliferation through a novel cyclin E1-dependent mechanism and is up-regulated in many human thyroid tumors. The expression of PPARdelta was induced coordinately with proliferation in primary human thyroid cells by the activation of serum, thyroid-stimulating hormone/cyclic AMP, or epidermal growth factor/mitogen-activated protein kinase mitogenic signaling pathways. Engineered overexpression of PPARdelta increased thyroid cell number, the incorporation of bromodeoxyuridine, and the phosphorylation of retinoblastoma protein by 40% to 45% in just 2 days, one usual cell population doubling. The synthetic PPARdelta agonist GW501516 augmented these PPARdelta proliferation effects in a dose-dependent manner. Overexpression of PPARdelta increased cyclin E1 protein by 9-fold, whereas knockdown of PPARdelta by small inhibitory RNA reduced both cyclin E1 protein and cell proliferation by 2-fold. Induction of proliferation by PPARdelta was abrogated by knockdown of cyclin E1 by small inhibitory RNA in primary thyroid cells and by knockout of cyclin E1 in mouse embryo fibroblasts, confirming a cyclin E1 dependence for this PPARdelta pathway. In addition, the mean expression of native PPARdelta was increased by 2-fold to 5-fold (P < 0.0001) and correlated with that of the in situ proliferation marker Ki67 (R = 0.8571; P = 0.02381) in six different classes of benign and malignant human thyroid tumors. Our experiments identify a PPARdelta mechanism that induces cell proliferation through cyclin E1 and is regulated by growth factor and lipid signals. The data argue for systematic investigation of PPARdelta antagonists as antineoplastic agents and implicate altered PPARdelta-cyclin E1 signaling in thyroid and other carcinomas.     

Ligand-activated PPARδ modulates the migration and invasion of melanoma cells by regulating Snail expression

Peroxisome proliferator-activated receptor (PPAR) δ is implicated in the carcinogenesis of several types of cancer. However, the therapeutic efficacy of PPARδ ligands against cancer progression is unclear. Here, we showed that PPARδ modulates the migration and invasion of melanoma cells by up-regulating Snail expression. Activation of PPARδ by {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516, a specific ligand for PPARδ, significantly increased the migration and invasion of highly metastatic A375SM cells, but not that of low metastatic A375P cells. The migration- and invasion-promoting effects of PPARδ on A375SM cells was associated with increased Snail expression, which was accompanied by a decrease in E-cadherin expression. Furthermore, a significant concentration- and time-dependent increase in the levels of Snail mRNA and protein was observed in A375SM cells (but not A375P cells) treated with {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516. The effects of {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516 were almost completely abrogated by a small interfering RNA against PPARδ, suggesting that PPARδ mediates the effects of {"type":"entrez-nucleotide","attrs":{"text":"GW501516","term_id":"289075981","term_text":"GW501516"}}GW501516. Activation of PPARδ in SK-MEL-2 and SK-MEL-5 (but not SK-MEL-3) melanoma cell lines also led to significant increases in the expression of Snail mRNA and protein, which mirrored the invasive and migratory potential of these cell lines. These results suggest that PPARδ promotes the aggressive phenotype observed in highly metastatic melanoma cells by up-regulating Snail.     

Synergistic inhibition of breast cancer cell lines with a dual inhibitor of EGFR-HER-2/neu and a Bcl-2 inhibitor

The epidermal growth factor receptor (EGFR) (ErbB1) and HER-2/neu (ErbB2) are members of the ErbB family of receptor tyrosine kinases. These receptors are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Lapatinib, a reversible inhibitor of both EGFR and HER-2/neu, has shown some success in achieving clinical responses in heavily pretreated advanced cancer patients. GW2974 is a reversible dual inhibitor similar to lapatinib, but GW2974 was not progressed to clinical trials due to pharmacokinetic issues. Bcl-2, an anti-apoptotic protein, is also overexpressed in a number of human tumors. Bcl-2 inhibitors induce apoptosis and sensitize cancer cells to other therapies. The purpose of this study was to assess the effects of combining ErbB and Bcl-2 inhibitors on the growth of human breast cancer cell lines. EGFR/HER-2/neu tyrosine kinase inhibitors (lapatinib and GW2974) were combined with Bcl-2 inhibitors (HA14-1 or GX15-070) and the anti-proliferative effects were determined by the MTT tetrazolium dye assay. Combinations were tested in MCF-7 human breast cancer cells, a HER-2/neu transfected MCF-7 cell line (MCF/18), and a tamoxifen-resistant MCF-7 cell line (MTR-3). A synergistic inhibitory effect was observed with the combination of inhibitors of EGFR-HER-2/neu (lapatinib or GW2974) and Bcl-2 (GX15-070 or HA14-1) on the growth of the MCF-7, MCF/18, and MTR-3 human breast cancer cell lines. This study suggests that simultaneously blocking the ErbB family of receptor tyrosine kinases and Bcl-2 family of proteins may be a benefit to breast cancer patients.     

Inhibition of adult liver progenitor (oval) cell growth and viability by an agonist of the peroxisome proliferator activated receptor (PPAR) family member gamma, but not alpha or delta

Multifaceted evidence links the development of liver tumours to the activation and proliferation of adult liver progenitor (oval) cells during the early stages of chronic liver injury. The aim of this study was to examine the role of the peroxisome proliferator activated receptors (PPARs): PPARalpha, delta and gamma, in mediating the behaviour of liver progenitor cells during pre-neoplastic disease and to investigate their potential as therapeutic targets for the treatment of chronic liver injury. We observed increased liver expression of PPARalpha and gamma in concert with expanding oval cell numbers during the first 21 days following commencement of the choline deficient, ethionine supplemented (CDE) dietary model of carcinogenic liver injury in mice. Both primary and immortalized liver progenitor cells were found to express PPARalpha, delta and gamma, but not gamma2, the alternate splice form of PPARgamma. WY14643 (PPARalpha agonist), GW501516 (PPARdelta agonist) and ciglitazone (PPARgamma agonist) were tested for their ability to modulate the behaviour of p53-immortalized liver (PIL) progenitor cell lines in vitro. Both PPARdelta and gamma agonists induced dose-dependent growth inhibition and apoptosis of PIL cells. In contrast, the PPARalpha agonist had no effect on PIL cell growth. None of the drugs affected the maturation of PIL cells along either the hepatocytic or biliary lineages, as judged by their patterns of hepatic gene expression prior to and following treatment. Administration of the PPARgamma agonist ciglitazone to mice fed with the CDE diet for 14 days resulted in a significantly diminished oval cell response and decreased fibrosis compared with those receiving placebo. In contrast, GW501516 did not affect oval cell numbers or liver fibrosis, but inhibited CDE-induced hepatic steatosis. In summary, PPARgamma agonists reduce oval cell proliferation and fibrosis during chronic liver injury and may be useful in the prevention of hepatocellular carcinoma.     

Ligand activation of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits cell growth in a mouse mammary gland cancer cell line

The effects of ligand activation of PPARβ/δ were examined in the mouse mammary tumor cell line (C20). Expression of PPARβ/δ was markedly lower in C20 cells as compared to the human non-tumorigenic mammary gland derived cell line (MCF10A) and mouse keratinocytes. Ligand activation of PPARβ/δ in C20 cells caused upregulation of the PPARβ/δ target gene angiopoietin-like 4 (Angptl4). Inhibition of C20 cell proliferation and clonogenicity was observed following treatment with GW0742 or GW501516, two highly specific PPARβ/δ ligands. In addition, an increase in apoptosis was observed in C20 cells cultured with 10 μM GW501516 that preceded the observed inhibition of cell proliferation. Results from this study show that proliferation of the C20 mouse mammary gland cancer cell line is inhibited by ligand activation of PPARβ/δ due in part to increased apoptosis.     

Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines

Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines in vitro. However, the replacement of the knocked down GHRH expression by exogenous GHRH (1-29)NH(2) re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1-29)NH(2) and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor.     

Effects of the EGFR/HER2 kinase inhibitor GW572016 on EGFR- and HER2-overexpressing breast cancer cell line proliferation, radiosensitization, and resistance

PURPOSE: Two members of the epidermal growth factor receptor family, EGFR and HER2, have been implicated in radioresistance in breast cancer and other malignancies. To gauge the potential clinical utility of targeting both EGFR and HER2 to control growth and radiosensitize human breast cancers, we examined the effect of a dual EGFR/HER2 inhibitor, GW572016, on the proliferation and radiation response of either EGFR- or HER2-overexpressing human breast cancer cell lines. METHODS AND MATERIALS: Primary human breast cancer cell lines that endogenously overexpress EGFR or HER2 and luminal mammary epithelial H16N2 cells stably transfected with HER2 were evaluated for the effect of GW572016 on inhibition of ligand-induced or constitutive receptor phosphorylation, proliferation, radiosensitization, and inhibition of downstream signaling. RESULTS: GW572016 inhibited constitutive and/or ligand-induced EGFR or HER2 tyrosine phosphorylation of all five cell lines, which correlated with the antiproliferative response in all but one cell line. GW572016 radiosensitized EGFR-overexpressing cell lines, but HER2-overexpressing cells were unable to form colonies after brief exposure to GW572016 even in the absence of radiation, and thus could not be evaluated for radiosensitization. One cell line was resistant to the antiproliferative and radiosensitizing effects of GW572016, despite receptor inhibition. Exploration of potential mechanisms of resistance in SUM185 cells revealed failure of GW572016 to inhibit downstream ERK and Akt activation, despite inhibition of HER2 phosphorylation. In contrast, sensitive HER2-overexpressing cell lines demonstrated inhibition of both ERK and Akt phosphorylation. CONCLUSION: GW572016 potently inhibits receptor phosphorylation in either EGFR- or HER2-overexpressing cell lines and has both antiproliferative and radiosensitizing effects. Resistance to GW572016 was not due to a lack of receptor inhibition, but rather with a lack of inhibition of ERK and Akt, suggesting that measurement of inhibition of crucial signaling pathways may better predict response than inhibition of receptor phosphorylation. The SUM185 cell line provides a valuable model for studying mechanisms of resistance of EGFR/HER2 inhibitor therapy.     

In vitro cytotoxicity of Phortress against colorectal cancer

Phortress is a novel benzothiazole compound with activity concentrated in certain breast, ovarian and renal cancer cell lines. Its anti-angiogenic effects are unknown. In this study, the in vitro anti-angiogenic effects of Phortress were screened for and results compared with two control drugs, paclitaxel and fumagillin. in vitro anti-angiogenic activity was examined by MTS assays, growth curves and clonogenic survival assays on human umbilical vein endothelial cells (HUVEC). In addition and as a comparator, effects were examined on MRCV fibroblasts and also the MCF7 breast cancer cell line, shown to be sensitive on the NCI60 panel and 3 colorectal cancer cell lines (HT29, SW480 and SW620) that were reportedly insensitive. Effects on endothelial tube differentiation were assessed by the Matrigel assay. Phortress had no effect on HUVEC and MRCV cell proliferation and survival. Unlike paclitaxel and fumagillin, Phortress did not inhibit endothelial tube differentiation. Phortress therefore exhibits no in vitro anti-angiogenic activity. As expected, Phortress was cytotoxic to MCF7 breast cancer cells, but unexpectedly, Phortress was also potent against colorectal cancer cells in clonogenic survival and cell growth (growth curves but not MTS assay) end-points. The efficacy of Phortress against colorectal cancer cells in the current study confirms that the spectrum of activity of Phortress may be wider than previously thought.     

Prolactin and heregulin override DNA damage-induced growth arrest and promote phosphatidylinositol-3 kinase-dependent proliferation in breast cancer cells

Heregulin (HRG), a ligand of ErbB receptor tyrosine kinases, is a potent mitogenic factor for breast cancer cells. Prolactin (PRL) has also been reported to regulate proliferation in breast cancer cells through its receptor, a member of the type I cytokine receptor family. Cytokine receptors are potent mitogens in hematopoietic cells, where they also override DNA damage-induced growth arrest checkpoints through activation of a phosphatidylinositol-3 kinase (PI3K) signaling pathway. In this study, we assessed the effect of gamma-irradiation on the mitogenic activity of HRG and PRL in breast cancer cells. HRG and PRL enhanced the proliferation of non-irradiated breast cancer cell lines in association with their ability to activate PI3K signaling pathways. Both growth factors also overrode irradiation-induced growth arrest in T47D cells, which resulted in decreased viability after irradiation. An inhibitor of PI3K, LY294002, abrogated growth factor-induced proliferation and the activity of cell cycle-dependent kinases in non-irradiated and irradiated cells. Thus, growth factors acting through distinct receptor families share a similar PI3K-dependent ability to promote proliferation and override DNA damage-induced growth arrest in breast cancer cells. These observations also suggest that selective activation of PI3K-dependent signaling can enhance radiosensitivity in breast cancer cells.     

The Ret receptor tyrosine kinase pathway functionally interacts with the ERalpha pathway in breast cancer

A limited number of receptor tyrosine kinases (e.g., ErbB and fibroblast growth factor receptor families) have been genetically linked to breast cancer development. Here, we investigated the contribution of the Ret receptor tyrosine kinase to breast tumor biology. Ret was expressed in primary breast tumors and cell lines. In estrogen receptor (ER)alpha-positive MCF7 and T47D lines, the ligand (glial-derived neurotrophic factor) activated signaling pathways and increased anchorage-independent proliferation in a Ret-dependent manner, showing that Ret signaling is functional in breast tumor cells. Ret expression was induced by estrogens and Ret signaling enhanced estrogen-driven proliferation, highlighting the functional interaction of Ret and ER pathways. Furthermore, Ret was detected in primary cancers, and there were higher Ret levels in ERalpha-positive tumors. In summary, we showed that Ret is a novel proliferative pathway interacting with ER signaling in vitro. Expression of Ret in primary breast tumors suggests that Ret might be a novel therapeutic target in breast cancer.     

Activity of tamoxifen and its metabolites on endocrine-dependent and endocrine-independent breast cancer cells

To better understand the mechanism of action of antiestrogens, the growth-inhibitory effect of tamoxifen and its main metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, was studied in 6 breast cancer cell lines characterized by different steroid receptor contents. On the basis of the results, our cell lines could be classified into three groups: a first group, including 734B and ZR-75.1 cell lines, characterized by a clear endocrine-dependent behavior, in which cells were sensitive to antiestrogens although to different degrees; a second group, including MDA-MB 231 and BT20 cell lines, characterized by a clear endocrine-insensitive behavior, in which cells were affected only by the highest (10(-6) M) antiestrogen concentration; a third group, including MCF7 and T47D cell lines, characterized by a peculiar behavior. The T47D cell line displayed an increased growth rate after treatment with all three antiestrogens considered. Despite the positive receptor content in the MCF7 cell line, only 4-hydroxytamoxifen showed a clear antiestrogen dose-dependent effect, whereas tamoxifen decreased the cell growth rate only at lower concentrations (10(-8) and 10(-7) M). These results and the well-known heterogeneity of human breast tumors explain the failure of antiestrogen treatment in a certain percentage of patients with breast cancer with a positive estrogen receptor status.     

Development of an autocrine neuregulin signaling loop with malignant transformation of human breast epithelial cells

Neuregulin (NRG) is a heparin-binding factor that activates members of the epidermal growth factor family of tyrosine kinase receptors including erbB2 that is overexpressed in more aggressive types of breast cancer. The exact role that NRG plays in breast cancer is complicated by the fact that NRG has been shown to have both proliferative and antiproliferative effects, depending on the breast cancer cell line used. Using an isogenic series of breast epithelial cell lines (MCF10A) ranging from benign to malignant, we found that the actions of NRG changed from antiproliferative to proliferative as the cells progress to cancer. This correlated with a progressive inability of NRG to down-regulate a group of proliferation genes identified previously using cDNA microarrays. As the cells progress to malignancy, they expressed higher levels of erbB2 and lower levels of erbB3 and secreted high levels of NRG into the culture media, resulting in high basal levels of erbB receptor phosphorylation. Disruption of this autocrine signaling loop by blocking ligand-induced receptor activation inhibited cancer cell proliferation. These results demonstrate that the transition of MCF10A cells from normal to premalignant to malignant correlates with the development of a constitutively active autocrine NRG signaling loop that promotes cell proliferation and suggest that disrupting this autocrine loop may provide an important therapeutic measure to control breast cancer cell growth.     

Predictors of distress in female breast cancer survivors: a systematic review

Purpose

Breast cancers have a poorer prognosis if estrogen receptor expression was lost during recurrence. It is unclear whether this conversion is cell autonomous or whether it can be promoted by the microenvironment during cancer dormancy. We explored the ability of marrow-derived stromal cell lines to arrest co-cultured breast cancer cells and suppress estrogen receptor alpha (ER) expression during arrest, facilitating the emergence of estrogen-independent breast cancer clones.

Methods

Cancer cell growth, ER protein, microRNA, and mRNA levels were measured in breast cancer cell lines exposed to conditioned medium from marrow stromal lines in the presence and absence of estrogen and of signaling pathway modulators.

Results

We demonstrate that paracrine signaling from the stromal cell line HS5 downregulated ER in T47D and MCF7 breast cancer cells. This occurred at the mRNA level and also through decreased ER protein stability. Additionally, conditioned medium (CM) from HS5 arrested the breast cancer cells in G0/G1 in part through interleukin-1 (IL1) and inhibited cancer cell growth despite the activation of proliferative pathways (Erk and AKT) by the CM. Similar findings were observed for CM from the hFOB 1.19 osteoblastic cell line but not from two other fibroblastic marrow lines, HS27A and KM101. HS5-CM inhibition of MCF7 proliferation could not be restored by exogenous ER, but was restored by the IL1-antagonist IL1RA. In the presence of IL1RA, HS5-CM activation of AKT and Erk enabled the outgrowth of breast cancer cells with suppressed ER that were fulvestrant-resistant and estrogen-independent.

Conclusions

We conclude that marrow-derived stromal cells can destabilize estrogen receptor protein to convert the ER status of growth-arrested ER+ breast cancer cell lines. The balance between stromal pro- and anti-proliferative signals controlled the switch from a dormant phenotype to estrogen-independent cancer cell growth.