The p38 mitogen activated protein kinase pathway (MAPK) is known to promote cell survival, endocrine therapy resistance and hormone independent breast cancer cell proliferation. Therefore, we utilized the novel p38 inhibitor RWJ67657 to investigate the relevance of targeting this pathway in the ER+ breast cancer cell line MCF-7. Our results show that RWJ67657 inhibits both basal and estrogen stimulated phosphorylation of p38α, resulting in decreased activation of the downstream p38α targets hsp27 and MAPAPK. Furthermore, inhibition of p38α by RWJ67657 blocks clonogenic survival of MCF-7 cells with little effect on non-cancerous breast epithelial cells. Even though p38α is known to phosphorylate ERα at residue within ER’s hinge region at Thr311, resulting in increased ERα transcriptional activation, our results suggest RWJ67657 inhibits the p38α-induced activation of ER by targeting both the AF-1 and AF-2 activation domains within ERα. We further show that RWJ67657 decreases the transcriptional activity of the ER coactivators SRC-1, SRC-2 and SRC-3. Taken together, our results strongly suggest that in addition to phosphorylating Thr311 within ERα, p38α indirectly activates the ER by phosphorylation and stimulation of the known ERα coactivators, SRC-1, -2 and-3. Overall, our data underscore the therapeutic potential of targeting the p38 MAPK pathway in the treatment of ER+ breast cancer.
p38; mitogen-activated protein kinase; estrogen receptor; breast cancer; SRC; drug discovery
Glyceollins are stress-induced compounds in soybeans with bioactive properties distinct from parent soy isoflavones. The goals of this study were to evaluate effects of dietary glyceollin-enriched and standard soy protein isolates and identify candidate target pathways of glyceollins on transcriptional profiles within mammary gland tissue. Thirty female postmenopausal cynomolgus monkeys were randomized to diets containing one of three protein sources for 3 weeks: (1) control casein / lactalbumin (C/L); (2) standard soy protein containing 194 mg/day isoflavones (SOY); and (3) glyceollin-enriched soy protein containing 189 mg/day isoflavones + 134 mg/day glyceollins (GLY). All diets contained a physiologic dose of estradiol (E2) (1 mg/day). All doses are expressed in human equivalents scaled by caloric intake. Relative to the control C/L diet, the GLY diet resulted in greater numbers of differentially regulated genes which showed minimal overlap with those of SOY. Effects of GLY related primarily to pathways involved in lipid and carbohydrate metabolism, including peroxisome proliferator-activated receptor (PPAR)-gamma and AMP-activated protein kinase (AMPK) signaling, adipocytokine expression, triglyceride synthesis, and lipase activity. Notable genes upregulated by the GLY diet included PPAR-gamma, adiponectin, leptin, lipin 1, and lipoprotein lipase. The GLY diet also resulted in lower serum total cholesterol, specifically non-high-density lipoprotein cholesterol, and increased serum triglycerides compared to the C/L diet. No effects of GLY or SOY were seen on serum insulin, adipocytokines, or vascular and bone turnover markers. These preliminary findings suggest that glyceollin-enriched soy protein has divergent effects from standard soy with some specificity for adipocyte activity and nutrient metabolism.
glyceollin; soy; isoflavone; estrogen receptor; metabolism
The estrogen receptor α (ERα) is a transcription factor that mediates the biological effects of 17β-estradiol (E2). ERα transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Gα protein subunits were tested for their ability to regulate ERα activity. Reporter assays revealed that overexpression of a constitutively active Gαo protein subunit potentiated ERα activity in the absence and presence of E2. Transient transfection of the human breast cancer cell line MCF-7 showed that Gαo augments the transcription of several ERα-regulated genes. Western blots of HEK293T cells transfected with ER±Gαo revealed that Gαo stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERα on serine 118. In summary, our results show that Gαo, through activation of the MAPK pathway, plays a role in the regulation of ERα activity.
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.
Phytoalexins; microRNA; breast cancer; estrogen
Triple negative breast cancer (TNBC) is subtype of breast disease devoid of the estrogen, progesterone, and Her2/neu receptors which are targets for pharmacological intervention. There is a need for novel anti-breast cancer agents that target TNBC. Therefore, novel isochalcone DJ52 was evaluated using the alamar blue dye exclusion assay, the luciferase colony assay, and xenograft models to determine its efficacy and potency. DJ52 significantly decreased proliferation of cells measured by using the alamar blue dye method and produced IC50 values of DJ52, DJ56, and DJ82 at 10-6M, 10-5M, and 10-5M, respectively. In vivo studies were conducted by injecting MDA-MB-231 cells into SCID mice to determine tumor regression was measured over 20 days. DJ52 at 50mg/kg caused significant decrease in tumor volume (p value <.05) by nearly 50% compared with the control with vehicle alone. These data suggest that DJ52 has merit for further evaluation as a novel anticancer agent.
Triple negative breast cancer; isochalcone; chalcone
Both estrogen, through the estrogen receptor (ER), and growth factors, through the phosphatidylinositol-3-kinase (PI3K)-AKT pathway, have been shown to independently promote cell survival. Here, we investigated the role of ER/PI3K-AKT crosstalk in the regulation of cell survival in MCF-7 breast carcinoma cells. The ER inhibitor ICI 182,780 was used to determine the requirement of the ER for estrogen in the suppression of tumor necrosis factor-α (TNFα) induced apoptosis. Gene reporter assays and Western blot analyses were used to determine the involvement of the pro-survival factor Bcl-2 and the coactivator GRIP1 in this survival crosstalk. We demonstrated that an intact ER signaling pathway was required for estrogen to suppress apoptosis induced by TNFα. Our gene reporter assays revealed that ERα, not ERβ, was targeted by AKT, resulting in transcriptional potentiation of the full-length Bcl-2 promoter, ultimately leading to increased Bcl-2 protein levels. AKT targeted both activation function (AF) domains of the ERα for maximal induction of Bcl-2 reporter activity, although the AF-II domain was predominately targeted. In addition, AKT also caused an upregulation of GRIP1 protein levels. Finally, AKT and GRIP1 cooperated to increase Bcl-2 protein expression to a greater level than either factor alone. Collectively, our study suggests a role for ER/PI3K-AKT crosstalk in cell survival and documents the ability of AKT to regulate Bcl-2 expression via differential activation of ERα and ERβ as well as regulation of GRIP1.
estrogen receptor; breast cancer; AKT; cell signaling; cell survival
Acquired chemoresistance and epithelial-to-mesenchymal transition (EMT) are hallmarks of cancer progression and of increasing clinical relevance. We investigated the role of miRNA and p38 mitogen-activated protein kinase (MAPK) signaling in the progression of breast cancer to a drug-resistant and mesenchymal phenotype. We demonstrate that acquired death receptor resistance results in increased hormone-independent tumorigenesis compared to hormone-sensitive parental cells. Utilizing global miRNA gene expression profiling, we identified miRNA alterations associated with the development of death receptor resistance and EMT progression. We further investigated the role of p38 MAPK in this process, showing dose-dependent inactivation of p38 by its inhibitor RWJ67657 and decreased downstream ATF and NF-κB signaling. Pharmacological inhibition of p38 also decreased chemoresistant cancer tumor growth in xenograft animal models. Interestingly, inhibition of p38 partially reversed the EMT changes found in this cell system, as illustrated by decreased gene expression of the EMT markers Twist, Snail, Slug and ZEB and protein and mRNA levels of Twist, a known EMT promoter, concomitant with decreased N-cadherin protein. RWJ67657 treatment also altered the expression of several miRNAs known to promote therapeutic resistance, including miR-200, miR-303, miR-302, miR-199 and miR-328. Taken together, our results demonstrate the roles of multiple microRNAs and p38 signaling in the progression of cancer and demonstrate the therapeutic potential of targeting the p38 MAPK pathway for reversing EMT in an advanced tumor phenotype.
p38 mitogen-activated protein kinase; epithelial-tomesenchymal transition; breast cancer; drug discovery
Background: The organochlorine dichlorodiphenyltrichloroethane (DDT), a known estrogen mimic and endocrine disruptor, has been linked to animal and human disorders. However, the detailed mechanism(s) by which DDT affects cellular physiology remains incompletely defined.
Objectives: We and others have shown that DDT activates cell-signaling cascades, culminating in the activation of estrogen receptor-dependent and -independent gene expression. Here, we identify a mechanism by which DDT alters cellular signaling and gene expression, independent of the estrogen receptor.
Methods: We performed quantitative polymerase chain reaction array analysis of gene expression in MCF-7 breast cancer cells using either estradiol (E2) or o,p´-DDT to identify distinct cellular gene expression responses. To elucidate the mechanisms by which DDT regulates cell signaling, we used molecular and pharmacological techniques.
Results: E2 and DDT treatment both altered the expression of many of the genes assayed, but up-regulation of vascular endothelial growth factor A (VEGFA) was observed only after DDT treatment, and this increase was not affected by the pure estrogen receptor α antagonist ICI 182780. Furthermore, DDT increased activation of the HIF-1 response element (HRE), a known enhancer of the VEGFA gene. This DDT-mediated increase in HRE activity was augmented by the coactivator CBP (CREB-binding protein) and was dependent on the p38 pathway.
Conclusions: DDT up-regulated the expression of several genes in MCF-7 breast cancer cells that were not altered by treatment with E2, including VEGFA. We propose that this DDT-initiated, ER-independent stimulation of gene expression is due to DDT’s ability to initiate crosstalk between MAPK (mitogen-activated protein kinase) signaling pathways and transcriptional coactivators.
breast cancer; CBP; coactivator; crosstalk; DDT; dichlorodiphenyltrichloroethane; endocrine-disrupting chemical; HIF-1α; MAPK; organochlorine; p38 kinase; vascular endothelial growth factor
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multi-kinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to Class III RTKs such as PDGFRβ and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
Endocrine therapy resistance is a primary cause of clinical breast cancer treatment failure. The p38 mitogen activated protein kinase (MAPK) signaling pathway is known to promote ligand independent tumor growth and resistance to endocrine therapy. In this study, we investigated the therapeutic potential of the p38 inhibitor RWJ67657 in the treatment of tamoxifen resistant MDA-MB-361 cells. RWJ67657 dose-dependently decreased both basal and stimulated activation of p38 MAPK signaling in this drug resistant cell system. Decreased activation of p38 by RWJ67657 resulted in inhibition of the downstream p38 targets hsp27 and MAPKAPK. Diminished p38 signaling resulted in inhibition of p38-medated gene transcription. Furthermore, pharmacological inhibition of p38 by RWJ67657 decreased biological effects of p38, including ER-mediated gene expression and clonogenic survival in a dose-dependent manner. Animal studies revealed significantly decreased p38 signaling in vivo following exposure to RWJ67657. Treatment with the inhibitor markedly decreased phosphorylation of p38 in MDA-MB-361 tumors, leading to decreased transcription of both Fra-1 and progesterone receptor. Utilizing well-established xenograft tumor models, we demonstrated that RWJ67657 exhibits potent anti-tumor properties. Treatment with RWJ67657 markedly decreased tamoxifen resistant tumor growth, both in the presence and absence of estrogen. Taken together, our findings demonstrate the therapeutic potential of targeting the p38-MAPK signaling cascade in the treatment of endocrine resistant breast cancer.
p38; mitogen-activated protein kinase; endocrine resistance; breast cancer; drug discovery; cancer biology; hormone independence; kinase inhibitors; estrogen receptor; gene transcription
Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo.
TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology.
Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype.
This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.
Panobinostat; LBH589; triple-negative breast cancer; xenograft; histone deacetylase inhibitor; E-cadherin; CDH1; epithelial-to-mesenchymal transition
Resistance to chemotherapy remains a significant obstacle in the treatment of hormone-independent breast cancer. Recent evidence suggests that altered sphingolipid signaling through increased sphingosine kinase activity may be an important mediator of breast cancer drug resistance. Sphingosine kinase-1 (Sphk1) is a proposed key regulator of breast cancer tumorigenesis, proliferation and resistance. There is, however, conflicting data on the role of sphingosine kinase-2 (Sphk2) in cancer biology and resistance, with some suggesting that Sphk2 has an opposing role to that of Sphk1. Here, we studied the effects of the novel selective Sphk2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) amide), on human breast cancer. ABC294640 blocked both viability and survival at low micromolar IC50 concentrations in the endocrine therapy-resistant MDA-MB-231 and chemoresistant MCF-7TN-R cell systems. Treatment with the inhibitor significantly reduced proliferation, as seen in immunofluorescence staining of Ki-67 in vitro. Interestingly, pharmacological inhibition of Sphk2 induced apoptosis through the intrinsic programmed cell death pathway. Furthermore, ABC294640 also diminished NFκB survival signaling, through decreased activation of the Ser536 phosphorylation site on the p65 subunit. Xenografts of MCF-7TN-R cells growing in immunocompromised mice were utilized to validate the therapeutic efficacy of the sphingosine kinase-2 inhibitor. Treatment with 50 mg of ABC294640/kg completely blocked tumor volume in this model. These results indicate that pharmacological inhibition of Sphk2 with the orally bioavailable selective inhibitor, ABC294640, has therapeutic potential in the treatment of chemoand endocrine therapy-resistant breast cancer.
sphingolipids; chemoresistance; sphingosine kinase; NFkappaB; breast cancer; ceramide; TNF; sphingosine-1-phosphate
The purpose of this study was to investigate the effects of glyceollins on the suppression of tumorigenesis in triple-negative breast carcinoma cell lines. We further explored the effects of glyceollins on microRNA and protein expression in MDA-MB-231 cells. Triple-negative (ER-, PgR- and Her2/neu-) breast carcinoma cells were used to test the effects of glyceollins on tumorigenesis in vivo. Following this procedure, unbiased microarray analysis of microRNA expression was performed. Additionally, we examined the changes in the proteome induced by glyceollins in the MDA-MB-231 cells. Tumorigenesis studies revealed a modest suppression of MDA-MB-231 and MDA-MB-468 cell tumor growth in vivo. In response to glyceollins we observed a distinct change in microRNA expression profiles and proteomes of the triple-negative breast carcinoma cell line, MDA-MB-231. Our results demonstrated that the glyceollins, previously described as anti-estrogenic agents, also exert antitumor activity in triple-negative breast carcinoma cell systems. This activity correlates with the glyceollin alteration of microRNA and proteomic expression profiles.
triple-negative breast cancer; microRNA; tumorigenesis; glyceollins
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRβ and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
pemetrexed; sorafenib; autophagy; apoptosis; PDGFR; ZMP; AMP; thymidylate synthase
Daidzein (1) is a natural estrogenic isoflavone. We report here that 1 can be transformed into antiestrogenic ligands by simple alkyl substitutions of the 7-hydroxyl hydrogen. To test the effect of such structural modifications on the hormonal activities of the resulting compounds, a series of daidzein analogues have been designed and synthesized. When MCF-7 cells were treated with the analogues, those resulting from hydrogen substitution by isopropyl (3d), isobutyl (3f), cyclopentyl (3g), and pyrano- (2), inhibited cell proliferation, estrogen-induced transcriptional activity, and estrogen receptor (ER) regulated progesterone receptor (PgR) gene expression. However, methyl (3a) and ethyl (3b) substitutions of the hydroxyl proton only led to moderate reduction of the estrogenic activities. These results demonstrated the structural requirements for the transformation of daidzein from an ER agonist to an antagonist. The most effective analogue, 2 was found to reduce in vivo estrogen stimulated MCF-7 cell tumorigenesis using a xenograft mouse model.
Daidzein analogues; antiestrogen; isoflavones; breast cancer; phytoestrogen; SERM; estrogen receptor; xenograft model
Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of α-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-β1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kD when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against α-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-β1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.
The pineal gland hormone, melatonin, has been shown by numerous studies to inhibit the proliferation of estrogen receptor α (ERα)-positive breast cancer cell lines. Here, we investigated the role of melatonin in the regulation of breast cancer cell invasion.
Three invasive MCF-7 breast cancer cell clones - MCF-7/6, MCF-7/Her2.1, and MCF-7/CXCR4 cells - were employed in these studies. All three cell lines exhibited elevated phosphorylation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) as determined by Western blot analysis. The effect of melatonin on the invasive potential of these human breast cancer cells was examined by matrigel invasion chamber assays. The expression and proteinase activity of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were analyzed by Western blot analysis and gelatin zymography, respectively.
Melatonin (10-9 M) significantly suppressed the invasive potential of MCF-7/6 and MCF-7/Her2.1 cells as measured by matrigel invasion chamber assays, and significantly repressed the proteinase activity of MMP-2 and MMP-9. In MCF-7/CXCR4 cells, melatonin significantly inhibited stromal-derived factor-1 (SDF-1/CXCL12) induced cell invasion and activity of MMP-9. Elevated expression of the MT1 melatonin receptor further enhanced, while luzindole, an MT1/MT2 antagonist, abrogated melatonin's anti-invasive effect, suggesting that melatonin's effect on invasion is mediated, principally, through the MT1 receptor. Furthermore, melatonin repressed the phosphorylation of p38 MAPK in MCF-7/Her2.1 cells and blocked stromal-derived factor-1 (SDF-1) induced p38 phosphorylation in MCF-7/CXCR4 cells. SB230580, a p38 inhibitor, was able to mimic, while transfection of the cells with a constitutively-active MKK6b construct blocked melatonin's effect on cell invasion, suggesting that the anti-invasive action of melatonin is mediated through the p38 pathway.
Melatonin exerts an inhibitory effect on breast cancer cell invasion through down-regulation of the p38 pathway, and inhibition of MMP-2 and MMP-9 expression and activity.
Adult human mesenchymal stem cells (hMSC) have been shown to home to sites of carcinoma and affect biological processes, including tumour growth and metastasis. Previous findings have been conflicting and a clear understanding of the effects of hMSCs on cancer remains to be established. Therefore, we set out to investigate the impact of hMSCs on the oestrogen receptor positive, hormone-dependent breast carcinoma cell line MCF-7.
In this study, we show the effects of hMSCs on cancer cells are mediated through a secreted factor(s) which are enhanced by cancer cell-hMSC contact/communication. In addition to enhanced proliferation when in co-culture with hMSCs, MCF-7 cells were found to have increased migration potential in vitro. Inhibition of ER signalling by the pure anti-oestrogen ICI 182,780 decreased the effect of hMSCs on MCF-7 cell proliferation and migration supporting a role for ER signalling in the hMSC/MCF-7 cell interaction. Additionally, hMSCs have been shown to secrete a wide variety of growth factors and chemokines including stromal cell-derived factor-1 (SDF-1). This coupled with the knowledge that SDF-1 is an ER-mediated gene linked with hormone-independence and metastasis led to the investigation of the SDF-1/CXCR4 signalling axis in hMSC-MCF-7 cell interaction. Experiments revealed an increase in SDF-1 gene expression both in vivo and in vitro when MCF-7 cells were cultured with hMSCs. SDF-1 treatment of MCF-7 cells alone increased proliferation to just below that seen with hMSC co-culture. Additionally, blocking SDF-1 signalling using a CXCR4-specific inhibitor decreased hMSC induced proliferation and migration of MCF-7. However, the combined treatment of ICI and AMD3100 reduced MCF-7 cell proliferation and migration below control levels, indicating targeting both the ER and CXCR4 pathways is effective in decreasing the hMSCs induction of MCF-7 cell proliferation and migration.
The sum of these data reveals the relationship between tumour microenvironment and tumour growth and progression. Better understanding of the mechanisms involved in this tumour stroma cell interaction may provide novel targets for the development of treatment strategies for oestrogen receptor positive, hormone-independent, and endocrine-resistant breast carcinoma.
Tamoxifen is the most commonly prescribed therapy for patients with estrogen receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical models of HER2 overexpression fail to recapitulate the clinical spectrum of endocrine resistance associated with HER2/ER-positive tumors. Here, we show that ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16, which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family pharmacological inhibitor ABT-737 restores tamoxifen sensitivity. Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen resistance. Our results suggest that HER2Δ16 expression promotes endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16 therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and suppression of miR-15a/16, provides a template for unique therapeutic interventions combining tamoxifen with modulation of microRNAs and/or ABT-737-mediated BCL-2 inhibition and apoptosis.
The 250 kDa P2P-R protein (also known as PACT and Rbbp6) was cloned over a decade ago and was found to bind both the p53 and Rb1 tumor suppressor proteins. In addition, P2P-R has been associated with multiple biological functions, such as mitosis, mRNA processing, translation and ubiquitination. In the current studies, the online GeneNetwork system was employed to further probe P2P-R biological functions. Molecular studies were then performed to confirm the GeneNetwork evaluations.
GeneNetwork and associated gene ontology links were used to investigate the coexpression of P2P-R with distinct functional sets of genes in an adipocyte genetic reference panel of HXB/BXH recombinant strains of rats and an eye genetic reference panel of BXD recombinant inbred strains of mice. The results establish that biological networks of 75 and 135 transcription-associated gene products that include P2P-R are co-expressed in a genetically-defined manner in rat adipocytes and in the mouse eye, respectively. Of this large set of transcription-associated genes, >10% are associated with hormone-mediated transcription. Since it has been previously reported that P2P-R can bind the SRC-1 transcription co-regulatory factor (steroid receptor co-activator 1, [Ncoa1]), the possible effects of P2P-R on estrogen-induced transcription were evaluated. Estrogen-induced transcription was repressed 50-70% by the transient transfection of P2P-R plasmid constructs into four different cell types. In addition, knockdown of P2P-R expression using an antisense oligonucleotide increased estrogen-mediated transcription. Co-immunoprecipitation assays confirmed that P2P-R interacts with SRC-1 and also demonstrated that P2P-R interacts with estrogen receptor α.
The findings presented in this study provide strong support for the value of systems genetics, especially GeneNetwork, in discovering new functions of genes that can be confirmed by molecular analysis. More specifically, these data provide evidence that the expression of P2P-R co-varies in a genetically-defined manner with large transcription networks and that P2P-R can function as a co-repressor of estrogen-dependent transcription.
The activity of nuclear transcription factors is often regulated by specific kinase-signaling pathways. We have previously shown that the organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) stimulates activator protein-1 activity through the p38 mitogen-activated protein kinase (MAPK). Here, we show that DDT and its metabolites also stimulate the transcriptional activity of cyclic adenosine monophosphate response element-binding protein and Elk1 and potentiate gene expression through cyclic adenosine monophosphate and hypoxia response elements. Because DDT stimulates gene expression through various transcription factors and hence multiple response elements, we hypothesized that p38 signaling targets a common shared transcriptional activator. Here, we demonstrate using both pharmacological and molecular techniques, the general coactivator p300 is phosphorylated and potentiated by the p38 MAPK signaling cascade. We further show that p38 directly phosphorylates p300 in its N-terminus. These results, together with our previous work, suggest that p38 stimulates downstream transcription factors in part by targeting the general coactivator p300.
We recently showed that the pyridinylimidazoles SB203580 and SB202190, drugs designed to block human p38 mitogen-activated protein kinase (MAPK) activation, also inhibited replication of the medically important intracellular parasite Toxoplasma gondii in cultured human fibroblasts through a direct effect on the parasite. We now show that additional pyridinylimidazole and imidazopyrimidine p38 MAPK inhibitors inhibit intracellular T. gondii replication in vitro and protect mice against fatal T. gondii infection. Mice surviving infection following treatment with p38 MAPK inhibitors were resistant to subsequent T. gondii challenge, demonstrating induction of protective immunity. Thus, drugs originally developed to block human p38 MAPK activation are useful for treating T. gondii infection without inducing significant immunosuppression. MAPK inhibitors combined with either of the approved anti-Toxoplasma drugs sulfadiazine and pyrimethamine resulted in improved survival among mice challenged with a fatal T. gondii inoculum. A MAPK inhibitor also treated mice infected with the Microsporidium parasite Encephalitozoon cuniculi, suggesting that MAPK inhibitors represent a novel class of agents that may have a broad spectrum of antiparasitic activity. Preliminary studies implicate a T. gondii MAPK homologue as the target of drug action, suggesting possibilities for more-selective agents.
In the lactating breast, ERBB4 localizes to the nuclei of secretory epithelium while regulating activities of the signal transducer and activator of transcription (STAT) 5A transcription factor essential for milk-gene expression. We have identified an intrinsic ERBB4 NLS (residues 676–684) within the ERBB4 intracellular domain (4ICD) that is essential for nuclear accumulation of 4ICD. To determine the functional significance of 4ICD nuclear translocation in a physiologically relevant system, we have demonstrated that cotransfection of ERBB4 and STAT5A in a human breast cancer cell line stimulates β-casein promoter activity. Significantly, nuclear localization of STAT5A and subsequent stimulation of the β-casein promoter requires nuclear translocation of 4ICD. Moreover, 4ICD and STAT5A colocalize within nuclei of heregulin β1 (HRG)-stimulated cells and both proteins bind to the endogenous β-casein promoter in T47D breast cancer cells. Together, our results establish a novel molecular mechanism of transmembrane receptor signal transduction involving nuclear cotranslocation of the receptor intracellular domain and associated transcription factor. Subsequent binding of the two proteins at transcription factor target promoters results in activation of gene expression.
Some organochlorine pesticides and other synthetic chemicals mimic hormones in representatives of each vertebrate class, including mammals, reptiles, amphibians, birds, and fish. These compounds are called endocrine-disrupting chemicals (EDCs). Similarly, hormonelike signaling has also been observed when vertebrates are exposed to plant chemicals called phytoestrogens. Previous research has shown the mechanism of action for EDCs and phytoestrogens is as unintended ligands for the estrogen receptor (ER). Although pesticides have been synthesized to deter insects and weeds, plants produce phytoestrogens to deter herbivores, as attractant cues for insects, and as recruitment signals for symbiotic soil bacteria. Our data present the first evidence that some of the same organochlorine pesticides and EDCs known to disrupt endocrine signaling through ERs in exposed wildlife and humans also disrupt the phytoestrogen signaling that leguminous plants use to recruit Sinorhizobium meliloti soil bacteria for symbiotic nitrogen fixation. Here we report that a variety of EDCs and pesticides commonly found in agricultural soils interfere with the symbiotic signaling necessary for nitrogen fixation, suggesting that the principles underlying endocrine disruption may have more widespread biological and ecological importance than had once been thought.
Endocrine-disrupting organochlorines, such as the pesticide dichlorodiphenyltrichloroethane (DDT), bind to and activate estrogen receptors (ERs), thereby eliciting estrogen-like effects. Although ERs function predominantly through activation of transcription via estrogen-responsive elements, both ERs, alpha and ss, can interact with various transcription factors such as activator protein-1 (AP-1). Additionally, estrogens may regulate early signaling events, suggesting that the biological effects of environmental estrogens may not be mediated through classic ER (alpha and ss) activity alone. We hypothesized that known environmental estrogens, such as DDT and its metabolites, activate AP-1-mediated gene transactivation through both ER-dependent and ER-independent means. Using two Ishikawa human endometrial adenocarcinoma cell line variants that we confirmed to be estrogen responsive [Ishikawa(+)] and estrogen unresponsive [Ishikawa(-)], we generated stably transfected AP-1 luciferase cell lines to identify the role of an estrogen-responsive mechanism in AP-1-mediated gene expression by various stimuli. Our results demonstrate that DDT and dichlorodiphenyldichloroethane (DDD) were the most potent activators of AP-1 activity; 2,2-bis(p-chlorophenyl) acetic acid failed to activate. Although stimulated in both Ishikawa(+) and Ishikawa(-) cells by DDT and its congeners, AP-1 activation was more pronounced in the estrogen-unresponsive Ishikawa(-) cells. In addition, DDT, DDD, and dichlorodiphenyldichloroethylene (DDE) could also stimulate AP-1 activity in the estrogen-unresponsive human embryonic kidney 293 cells using a different promoter context. Thus, our data demonstrate that DDT and its metabolites activate the AP-1 transcription factor independent of ER (alpha or ss) status.