Regulation Of Breast Cancer Cell Growth Research Articles

OR07-1 Estrogen-regulated Enhancer RNAs Contain a Functional Motif that Controls Enhancer Assembly and Function in Breast Cancer Cells

Abstract Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor. Upon binding to endogenous and exogenous ligands, ERα dimerizes and binds to regulatory regions across the genome known as enhancers. Enhancers act as nucleation sites for the recruitment of coregulatory proteins and ultimately regulate target gene transcription. We have previously used global run-on sequencing (GRO-seq) to detect transcription at active enhancers that generates enhancer RNAs (eRNAs). eRNAs are poorly characterized noncoding RNAs and are thought to regulate enhancer function and gene expression. To better understand the functions of these eRNAs, we annotated the estrogen-regulated eRNA transcriptome in ERα-positive breast cancer cells using precision nuclear run-on of capped RNA (PRO-cap) and polyA-depleted RNA-seq. We then cloned selected eRNAs, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9. eRNAs promoted expression of cognate, but not heterologous, target genes by increasing ERα recruitment and simulating p300-catalyzed H3K27 acetylation at the enhancer. We identified a ∼40 nucleotide functional eRNA regulatory motif (FERM) present in many estrogen-regulated eRNAs that is necessary and sufficient to promote gene expression by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation. The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers, to exert its effect on enhancer assembly and dynamics. Overexpression of a targeted eRNA controlling expression of an oncogene increased cell proliferation, implicating eRNAs in regulating breast cancer cell growth. Together, we provide a genome-wide annotation of estrogen-regulated eRNAs, demonstrate the mechanisms by which targeted eRNAs promote target gene expression in vivo, identify the FERM element within eRNAs that is critical for regulating target gene expression, and show the biological implications of oncogenic eRNAs in breast cancer growth. Collectively, our results demonstrate the biological and mechanistic roles of eRNAs in breast cancer. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K. Presentation: Saturday, June 11, 2022 11:30 a.m. - 11:45 a.m.

Rasal2 suppresses breast cancer cell proliferation modulated by secretory autophagy.

Rasal2, a Ras-GTPase-activating protein (RasGAP), is a tumor suppressor in Luminal B breast cancer, frequently metastatic and recurrent. Exosomes (Exos) are small membrane vesicles secreted by various cell types, including tumor cells, recognized as vehicles for cell-to-cell communication. Our study aimed to investigate whether Rasal2 regulates breast cancer cell growth via affecting this process. In this paper, we described that Rasal2 knockout (KO) in MCF-7 cells enhanced exosomal release and increased autophagy-related proteins in exosomal fraction, while attenuated by exosome release inhibitor GW4869. Moreover, MCF-7 cells with chloroquine (CQ) treatment boosted Rasal2 KO-induced secretory autophagy. In addition, we presented that exosomes derived from KO MCF-7 cells (KO-exo) significantly promoted breast cancer cell proliferation compared to those from MCF-7 cells transfected with an empty crispr-cas9 plasmid serving as controls (sgNT-exo); however, exosomes purified from KO MCF-7 cells co-cultured with 3-methyladenine ((3-MA + KO)-exo)/CQ ((CQ + KO)-exo) dramatically inhibited/facilitated MCF-7 cell proliferation in contrast to KO-exo group, separately. In conclusion, our findings revealed a new mechanism of Rasal2 in the regulation of breast cancer cell proliferation via autophagy-exo-mediated pathway.

Proteasome 26S subunit PSMD1 regulates breast cancer cell growth through p53 protein degradation.

Endocrine therapy using antiestrogens and aromatase inhibitors is usually efficient to treat patients with hormone-sensitive breast cancer. Many patients with endocrine therapy, however, often acquire resistance. In the present study, we performed functional screening using short hairpin RNA library to dissect genes involved in antiestrogen tamoxifen resistance in MCF-7 breast cancer cells. We identified seven candidate genes that are associated with poor prognosis of breast cancer patients based on clinical dataset. The expression levels of six out of seven genes were higher in 4-hydroxytamoxifen (OHT) resistant MCF-7 (OHTR) cells compared with parental MCF-7 cells. Among the six selected genes, siRNA-mediated knockdown of PSMD1 and TSPAN12 markedly reduced the proliferation of OHTR cells. Notably, the knockdown of proteasome 26S subunit PSMD1 exhibited cell cycle arrest and the accumulation of p53 protein through inhibiting p53 protein degradation. In accordance with p53 accumulation, its target genes p21 and SFN were also upregulated by PSMD1 silencing. Taken together, PSMD1 was identified as a potential gene that plays a role in the development of tamoxifen resistance in breast cancer cells. These findings will provide a new insight for the mechanism underlying endocrine therapy resistance and a prognostic and therapeutic molecular target for advanced breast cancer.

CCAAT enhancer binding protein β has a crucial role in regulating breast cancer cell growth via activating the TGF-β-Smad3 signaling pathway.

The aim of the present study was to examine the effect of CCAAT enhancer binding protein β (C/EBPβ) on human breast cancer cells. The plasmids pCDH-C/EBPβ and pLKO.1-shC/EBPβ were constructed and were infected into MDA-MB-468 cells, to provide C/EBPβ overexpressing and C/EBPβ knockdown cells, respectively. Cell viability, cell cycle and apoptosis were observed by MTT assay and flow cytometry analysis. Protein expression levels of C/EBPβ, TGF-β1, P-Smad3 and Smad3 were detected by western blotting. MTT assay showed that the absorbance of MDA-MB-468 cells in the pCDH-C/EBPβ group was increased, whereas that in the pLKO.1-shC/EBPβ group was decreased, compared with the respective control at 48 and 72 h. Flow cytometric analysis indicated that the percentage of cells in the G2 phase was significantly increased in the pCDH-C/EBPβ group (P<0.05) and decreased in the pLKO.1-shC/EBPβ group compared with the respective control group. The proportion of apoptotic cells was decreased in the pCDH-C/EBPβ group and increased in the pLKO.1-shC/EBPβ group compared with the controls. The scratch-wound assay revealed that MDA-MB-468 cells depleted of C/EBPβ exhibited reduced motility compared with the control cells. Moreover, western blotting demonstrated that pCDH-C/EBPβ increased transforming growth factor (TGF)β1 and P-Smad3 protein expression and decreased Smad3 protein expression, whereas pLKO.1-shC/EBPβ decreased TGFβ1 and P-Smad3 protein expression and increased Smad3 protein expression levels. The present study demonstrated that C/EBPβ has a crucial role in regulating breast cancer cell growth through activating TGF-β-Smad3 signaling. These findings suggest that C/EBPβ may be a potential therapeutic target for breast cancer; however, in vivo studies are required to confirm this.

Class II phosphoinositide 3-kinase C2β regulates a novel signaling pathway involved in breast cancer progression.

It is now well established that the enzymes phosphoinositide 3-kinases (PI3Ks) have a key role in the development and progression of many cancer types and indeed PI3Ks inhibitors are currently being tested in clinical trials. Although eight distinct PI3K isoforms exist, grouped into three classes, most of the evidence currently available are focused on one specific isoform with very little known about the potential role of the other members of this family in cancer. Here we demonstrate that the class II enzyme PI3K-C2β is overexpressed in several human breast cancer cell lines and in human breast cancer specimens. Our data indicate that PI3K-C2β regulates breast cancer cell growth in vitro and in vivo and that PI3K-C2β expression in breast tissues is correlated with the proliferative status of the tumor. Specifically we show that downregulation of PI3K-C2β in breast cancer cell lines reduces colony formation, induces cell cycle arrest and inhibits tumor growth, in particular in an estrogen-dependent in vivo xenograft. Investigation of the mechanism of the PI3K-C2β-dependent regulation of cell cycle progression and cell growth revealed that PI3K-C2β regulates cyclin B1 protein levels through modulation of microRNA miR-449a levels. Our data further demonstrate that downregulation of PI3K-C2β inhibits breast cancer cell invasion in vitro and breast cancer metastasis in vivo. Consistent with this, PI3K-C2β is highly expressed in lymph-nodes metastases compared to matching primary tumors. These data demonstrate that PI3K-C2β plays a pivotal role in breast cancer progression and in metastasis development. Our data indicate that PI3K-C2β may represent a key molecular switch that regulates a rate-limiting step in breast tumor progression and therefore it may be targeted to limit breast cancer spread.

10-Gingerol inhibits proliferation and invasion of MDA-MB-231 breast cancer cells through suppression of Akt and p38MAPK activity.

In the present study, we investigated the roles and molecular mechanism of 10-gingerol, a phenolic compound isolated from Zingiber officinale, in regulating cell proliferation and invasion of MDA‑MB‑231 breast cancer cells. 10-gingerol treatment inhibited cell proliferation through downregulation of cell cycle regulatory proteins such as cyclin-dependent kinases and cyclins, and subsequent induction of G1 phase arrest. In addition, 10‑gingerol treatment blocked cell invasion in response to mitogenic stimulation. These antitumor activities of 10‑gingerol were mediated through inactivation of Akt and p38MAPK activity, and suppression of epidermal growth factor receptor expression. Collectively, these findings demonstrate the pharmacological roles of 10-gingerol in regulating breast cancer cell growth and progression, and suggest further evaluation and development as a potential therapeutic agent for the prevention and treatment of breast cancer.

TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth

Breast cancer is a leading cause of cancer-related death, and it is important to understand pathways that drive the disease to devise effective therapeutic strategies. Our results show that Toll-like receptor 4 (TLR4) drives breast cancer cell growth differentially based on the presence of TP53, a tumor suppressor. TP53 is mutationally inactivated in most types of cancer and is mutated in 30-50% of diagnosed breast tumors. We demonstrate that TLR4 activation inhibits growth of TP53 wild-type cells, but promotes growth of TP53 mutant breast cancer cells by regulating proliferation. This differential effect is mediated by changes in tumor cell cytokine secretion. Whereas TLR4 activation in TP53 mutant breast cancer cells increases secretion of progrowth cytokines, TLR4 activation in TP53 wild-type breast cancer cells increases type I IFN (IFN-γ) secretion, which is both necessary and sufficient for mediating TLR4-induced growth inhibition. This study identifies a novel dichotomous role for TLR4 as a growth regulator and a modulator of tumor microenvironment in breast tumors. These results have translational relevance, demonstrating that TP53 mutant breast tumor growth can be suppressed by pharmacologic TLR4 inhibition, whereas TLR4 inhibitors may in fact promote growth of TP53 wild-type tumors. Furthermore, using data generated by The Cancer Genome Atlas consortium, we demonstrate that the effect of TP53 mutational status on TLR4 activity may extend to ovarian, colon, and lung cancers, among others, suggesting that the viability of TLR4 as a therapeutic target depends on TP53 status in many different tumor types.

TGF-beta receptor type-2 expression in cancer-associated fibroblasts regulates breast cancer cell growth and survival and is a prognostic marker in pre-menopausal breast cancer

Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with the capability to act as tumour suppressor or tumour promoter depending on the cellular context. TGF-beta receptor type-2 (TGFBR2) is the ligand-binding receptor for all members of the TGF-β family. Data from mouse model experiments demonstrated that loss of Tgfbr2 expression in mammary fibroblasts was linked to tumour initiation and metastasis. Using a randomised tamoxifen trial cohort including in total 564 invasive breast carcinomas, we examined TGFBR2 expression (n=252) and phosphorylation level of downstream target SMAD2 (pSMAD2) (n=319) in cancer-associated fibroblasts (CAFs) and assessed links to clinicopathological markers, prognostic and treatment-predictive values. The study revealed that CAF-specific TGFBR2 expression correlated with improved recurrence-free survival. Multivariate analysis confirmed CAF-TGFBR2 to be an independent prognostic marker (multivariate Cox regression, hazard ratio: 0.534, 95% (CI): 0.360-0.793, P=0.002). CAF-specific pSMAD2 levels, however, did not associate with survival outcome. Experimentally, TGF-β signalling in fibroblasts was modulated using a TGF-β ligand and inhibitor or through lentiviral short hairpin RNA-mediated TGFBR2-specific knockdown. To determine the role of fibroblastic TGF-β pathway on breast cancer cells, we used cell contact-dependent cell growth and clonogenicity assays, which showed that knockdown of TGFBR2 in CAFs resulted in increased cell growth, proliferation and clonogenic survival. Further, in a mouse model transfected CAFs were co-injected with MCF7 and tumour weight and proportion was monitored. We found that mouse xenograft tumours comprising TGFBR2 knockdown fibroblasts were slightly bigger and displayed increased tumour cell capacity. Overall, our data demonstrate that fibroblast-related biomarkers possess clinically relevant information and that fibroblasts confer effects on breast cancer cell growth and survival. Regulation of tumour-stromal cross-talk through fibroblastic TGF-β pathway may depend on fibroblast phenotype, emphasising the importance to characterise tumour microenvironment subtypes.

Downregulation of gas5 increases pancreatic cancer cell proliferation by regulating CDK6

Recent studies have revealed that long non-coding RNAs (lncRNAs) play important roles in cancer biology and that lncRNA gas5 (growth arrest-specific 5) regulates breast cancer cell growth. However, the role of gas5 in pancreatic cancer progression remains largely unknown. In the current study, we assay the expression level of gas5 in pancreatic cancer tissues and define the role of gas5 in the regulation of pancreatic cancer cell proliferation. We verify that the expression level of gas5 is significantly decreased in pancreatic cancer tissues compared with normal control. Overexpression of gas5 in pancreatic cancer cells inhibits cell proliferation, whereas gas5 inhibition induces a significant decrease in G0/G1 phase and an increase in S phase. We further demonstrate that gas5 negatively regulates CDK6 (cyclin-dependent kinase 6) expression in vitro and in vivo. More importantly, knockdown of CDK6 partially abrogates gas5-siRNA-induced cell proliferation. These data suggest an important role of gas5 in the molecular etiology of pancreatic cancer and implicate the potential application of gas5 in pancreatic cancer therapy.

PPARβ mRNA expression, reduced by n − 3 PUFA diet in mammary tumor, controls breast cancer cell growth

The effect of numerous anticancer drugs on breast cancer cell lines and rodent mammary tumors can be enhanced by a treatment with long-chain n−3 polyunsaturated fatty acids (n−3 PUFA) such as docosahexaenoic acid (DHA, 22:6n−3) which is a natural ligand of peroxisome proliferator-activated receptors (PPAR). In order to identify the PPAR regulating breast cancer cell growth, we tested the impact of siRNA, selected to suppress PPARα, PPARβ or PPARγ mRNA in MDA-MB-231 and MCF-7 breast cancer cell lines. The siPPARβ was the most effective to inhibit breast cancer cell growth in both cell lines. Using PPARα, PPARβ and PPARγ pharmacological antagonists, we showed that PPARβ regulated DHA-induced inhibition of growth in MDA-MB-231 and MCF-7 cells. In addition, the expressions of all 3 PPAR mRNA were co-regulated in both cell lines, upon treatments with siRNA or PPAR antagonists. PPAR mRNA expression was also examined in the NitrosoMethylUrea (NMU)-induced rat mammary tumor model. The expressions of PPARα and PPARβ mRNAs were correlated in the control group but not in the n−3 PUFA group in which the expression of PPARβ mRNA was reduced. Although PPARα expression was also increased in the n−3 PUFA-enriched diet group under docetaxel treatment, it is only the expression of PPARβ mRNA that correlated with the regression of mammary tumors: those that most regressed displayed the lowest PPARβ mRNA expression. Altogether, these data identify PPARβ as an important player capable of modulating other PPAR mRNA expressions, under DHA diet, for inhibiting breast cancer cell growth and mammary tumor growth.

Are ‘omics of Estrogen Receptors Defining Potential Targets for Breast Cancer Treatment?

Among all neoplasias, breast cancer is certainly one of the worlds most frequently diagnosed malignancies in women. Estrogenic hormones have now been characterized for many years as being key players in regulating breast cancer cell growth. The identification in the 1990s of their cognate receptors (estrogen receptors [ERs]), characterized to act as ligand-dependent transcription factors, opened up a golden path in designing drugs targeting ERs and their downstream actions in order to cure cancer. Although some compounds identified exhibited pathological side effects, these approaches nevertheless led to the successful generation and use of active pharmacologicals including tamoxifen and aromatase – the enzyme which synthesizes estradiol – inhibitors [1,2]. Combined with other therapies, these antihormone treatments allowed an average 5-year relapse free survival rate to reach approximately 70%. However, some cancers do not respond to these treatments. A pioneering general comparative ana lysis of breast cancer-cell transcriptomes, published in 2000, rationalized some hypotheses that were raised from these observations of cancer’s estrogeninsensitivity: approximately 70% of breast cancer cells express ER [3]. Owing to the advances made in genomic technologies, our understanding of breast tumorigenesis has evolved significantly in recent years. What has become clear is that, similar to other cancer types, breast cancer is a complex disease involving many epigenetic mechanisms whose outcome is an alteration of transcriptional regulations. For example, one of the best described epigenetic modifications in breast cancer is an overall methylation of CpGs islands included within gene promoters, accompanied with specific hypomethylation of proto-oncogenes involved in cell proliferation and drug resistance [4,5]. Importantly, the loss or downregulation of ERs is linked to epigenetic

Abstract 1003: The inhibitory role of quercetin-induced Dickkopf-1 on the growth of 4T1 breast cancer cell line

Abstract Introduction: The highly conserved Wnt/β-catenin canonical signaling pathway plays a crucial role in the normal development and tumor development. Although β-catenin is stabilized in about 50% of breast cancer, mutations in the Wnt/β-catenin signaling pathway are relatively rare. Dickkopf-1 (Dkk-1) was first identified as a secreted inhibitor of the canonical Wnt/β-catenin signaling pathway and increased Dkk-1 results in the inhibition of β-catenin dependent target gene expression. In this study, we investigated the effect of quercetin, a flavonoid, to regulate 4T1 breast cancer cells by inducing the expression of Dkk-1. Methods: Using a MTT assay, the inhibition of cell proliferation by quercetin (0-40μM) on 4T1 cancer cells was measured. Effects of quercetin (20μM) on Dkk-1 mRNA expression were detected by quantitative real-time RT-PCR. To test the regulation of Wnt/β-catenin signaling activity, 4T1 cells were transiently transfected with β-catenin repoter gene (Topflash). After treatment of quercetin(20μM) for 48 hours, transcriptional activity of β-catenin was estimated by luciferase assay. The protein stability of β-catenin was analyzed by Western blotting. To confirm the inhibitory effect of Dkk-1, MTT assay was performed after treatment of human-recombinant Dkk-1 (h-r-Dkk-1) on 4T1 cells. Results: The growth of 4T1 cells, treated with the indicated amount of quercetin (0-40μM) for 48 hours, were reduced in a dose-dependent manner (p=0.0002). Real-time RT-PCR revealed that the expression of Dkk-1 mRNA was significantly increased by about five-fold at 20μM of quercetin. Within 48 hours of treatment, a 20μM concentration of quercetin reduced β-catenin transcriptional activity by approximately 2-fold (p=0.0037). Protein stability of β-catenin was down-regulated by quercetin (20μM) in a time-dependent manner. Also the addition of h-r-Dkk-1 to 4T1 cell line showed a similar role in the inhibition of cell proliferation (p=0.0320). Conclusion: The present study investigated the possible existence of autocrine Wnt/β-catenin signaling for regulating breast cancer cell growth. quercetin showed a potent inhibitory role in the Wnt/β-catenin signaling activity as well as the growth of 4T1 breast cancer cells. Moreover, Dkk-1, as a negative regulator for Wnt/β-catenin signaling, was confirmed to be induced by quercetin. Therefore the present study suggest that Wnt/β-catenin signaling may be a promising target pathway for innovative treatment strategies of breast cancer. Aknowledgement : This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-000-1266). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1003. doi:10.1158/1538-7445.AM2011-1003

FHL1 interacts with oestrogen receptors and regulates breast cancer cell growth

Four and a half LIM protein 1 (FHL1) belongs to the Lin-1, Isl-1 and Mec-3 (LIM)-only protein family and plays important roles in muscle growth and carcinogenesis. However, the biological function of FHL1 remains largely unknown. Here, we show that FHL1 physically and functionally interacted with oestrogen receptors (ERs), which are involved in breast cancer development and progression. FHL1 bound specifically to the activation function-1 domain of ER. Physical interaction of FHL1 and ER is required for FHL1 repression of oestrogen-responsive gene transcription. FHL1 affected recruitment of ER to an oestrogen-responsive promoter and ER binding to an oestrogen-responsive element. Overexpression of FHL1 in breast cancer cells decreased expression of oestrogen-responsive proteins, whereas knockdown of endogenous FHL1 with FHL1 small interfering RNA increased the expression of these proteins. Further analysis of 46 breast cancer samples showed that FHL1 expression negatively associated with oestrogen-responsive gene expression in breast cancer cells. FHL1 inhibited anchorage-dependent and -independent breast cancer cell growth. These results suggest that FHL1 may play an important role in ER signalling as well as breast cancer cell growth regulation.

The liver receptor homolog-1 regulates estrogen receptor expression in breast cancer cells

Estrogen receptor-α (ER) is expressed in the great majority of breast cancers, and the inhibition of ER action is a key part of breast cancer treatment. The inhibition of ER action is achieved using anti-estrogens, primarily tamoxifen, and with aromatase inhibitors that inhibit estrogen biosynthesis, thereby preventing ER activation. However, resistance to these therapies is common. With the aim of identifying new molecular targets for breast cancer therapy, we have identified the liver receptor homolog-1 (LRH-1) as an estrogen-regulated gene. RNA interference and over-expression studies were used to investigate the role of the LRH-1 in regulating breast cancer growth and to identify the targets of an LRH-1 action. Promoter recruitment was determined using reporter gene and chromatin immunoprecipitation (ChIP) assays. We show that LRH-1 regulates breast cancer cell growth by regulating the ER expression. Reporter gene and in vitro DNA-binding assays identified an LRH-1-binding site in the ER gene promoter, and ChIP assays have demonstrated in vivo binding at this site. We also provide evidence for new LRH-1 variants in breast cancer cells arising from the use of alternative promoters. Previous studies have shown that LRH-1 functions in estrogen biosynthesis by regulating aromatase expression. Our findings extend this by highlighting LRH-1 as a key regulator of the estrogen response in breast cancer cells through the regulation of ER expression. Hence, inhibition of LRH-1 could provide a powerful new approach for the treatment of endocrine-resistant breast cancer.

ADP-ribosylation Factor 1 Controls the Activation of the Phosphatidylinositol 3-Kinase Pathway to Regulate Epidermal Growth Factor-dependent Growth and Migration of Breast Cancer Cells

Activation of intracellular signaling pathways by growth factors is one of the major causes of cancer development and progression. Recent studies have demonstrated that monomeric G proteins of the Ras family are key regulators of cell proliferation, migration, and invasion. Using an invasive breast cancer cell lines, we demonstrate that the ADP-ribosylation factor 1 (ARF1), a small GTPase classically associated with the Golgi, is an important regulator of the biological effects induced by epidermal growth factor. Here, we show that this ARF isoform is activated following epidermal growth factor stimulation and that, in MDA-MB-231 cells, ARF1 is found in dynamic plasma membrane ruffles. Inhibition of endogenous ARF1 expression results in the inhibition of breast cancer cell migration and proliferation. The underlying mechanism involves the activation of the phosphatidylinositol 3-kinase pathway. Our data demonstrate that depletion of ARF1 markedly impairs the recruitment of the phosphatidylinositol 3-kinase catalytic subunit (p110alpha) to the plasma membrane, and the association of the regulatory subunit (p85alpha) to the activated receptor. These results uncover a novel molecular mechanism by which ARF1 regulates breast cancer cell growth and invasion during cancer progression.

The AP-1 transcription factor regulates breast cancer cell growth via cyclins and E2F factors

The activating protein-1 (AP-1) transcription factor transduces growth signals through signal transduction pathways to the nucleus, leading to the expression of genes involved in growth and malignant transformation in many cell types. We have previously shown that overexpression of a dominant negative form of the cJun proto-oncogene, a cJun dominant negative mutant (Tam67), blocks AP-1 transcriptional activity, induces a G(1) cell cycle block and inhibits breast cancer cell growth in vitro and in vivo. We found that AP-1 blockade by Tam67 in MCF-7 breast cancer cells downregulates cyclin D1 transcriptional activity by at least two mechanisms: by suppressing transcription at the known AP-1 binding site (-934/-928) and by suppressing growth factor-induced expression through suppressing E2F activation at the E2F-responsive site (-726/-719). AP-1 blockade also led to reduced expression of E2F1 and E2F2, but not E2F4, at the mRNA and protein levels. Chromatin immunoprecipitation and supershift assays demonstrated that AP-1 blockade caused decreased binding of E2F1 protein to the E2F site in the cyclin D1 promoter. We also found that Tam67 suppressed the expression of the E2F1 dimerizing partner, DP1 and E2F-upregulated cell cycle genes (cyclins E, A, B and D3) and enhanced the expression of E2F-downregulated cell cycle genes (cyclins G(2) and I). Reduced expression of other E2F-regulated genes was also seen with AP-1 blockade and E2F suppression. Thus, the AP-1 factor regulates the expression of cyclin D and E2F (the latter in turn regulates E2F-downstream genes), leading to cell cycle progression and breast cancer cell proliferation.

Hexachlorobenzene Is a Tumor Co-carcinogen and Induces Alterations in Insulin-Growth Factors Signaling Pathway in the Rat Mammary Gland

Hexachlorobenzene (HCB) is a widespread environmental pollutant. Controversy still exists about the breast carcinogenic properties of organochlorines in humans. The ligands, receptors, and related signaling proteins of the insulin growth factor family are involved in the regulation of breast-cancer cell growth. The aims of this study were to determine: (1) whether HCB is co-carcinogenic in a medium term assay of N-nitroso N-methylurea (NMU)-induced mammary tumors in rats; (2) the effect of HCB on insulin receptor (IR), insulin-like growth factor-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) levels and on IRS-1 phosphorylation; (3) microsomal and cytosolic Protein Tyrosine Kinase (PTK) activities in mammary glands and NMU-induced tumors. Sprague Dawley rats were injected with 50 mg/kg body weight of NMU at 50, 80, and 110 days old. HCB (100 mg/kg body weight) was administered three times a week from 65 to 110 days of age. Rats were separated in four groups: control, NMU, HCB, and NMU-HCB. HCB alone did not induce tumor development. Parameters of tumor development were increased in NMU-HCB compared to NMU rats. A higher cellular undifferentiation was observed in NMU-HCB tumors. IR, IGF-IR, and IRS-1 levels were higher in HCB than in controls. Conversely IGF-IR levels decreased in NMU-HCB vs. NMU group. The IRS-1 phosphorylation increased in HCB rats; however, it decreased in NMU-HCB vs. NMU. HCB decreased microsomal PTK activity in tumors. This study showed for the first time that HCB is a co-carcinogenic agent in NMU-induced mammary tumors in rats. Our results suggest that the IR and/or IGF-IR signaling pathway may be involved in the mechanism of action of HCB.

Pyridoxal Supplementation Reduces Cell Proliferation and DNA Synthesis in Estrogen-Dependent and -IndependentMammary Carcinoma Cell Lines

In previous studies, decreased growth of tumor cells by vitamin B-6 treatment has been attributed to modulation of steroid hormone action. Therefore, the growth-inhibiting properties of pyridoxal (PL) supplementation were studied in estrogen receptor-positive, MCF-7 and T-47D, and estrogen receptor-negative, BT-20, breast cancer cell lines. Cell counting and [3H]thymidine incorporation into DNA were used to assess growth, and analysis of pS2 expression was used to determine whether PL supplementation affected estrogen action. Treatment with 100 or 300 mM PL resulted in dose-dependent decreases in total cell numbers in the absence (26-85% and 72-98%, respectively) and presence (38-42% and 88-98%, respectively) of estradiol in all cell lines studied compared with control cells cultured without PL supplementation. Similar decreases in DNA synthesis were observed in response to PL supplementation. Incorporation of [3H]thymidine into DNA of cells cultured with 100 or 300 μM PL was decreased by 30-90% and 96-99%, respectively, in the absence and by 32-40% and 82-99%, respectively, in the presence of estradiol. Northern analysis showed that expression of the estrogen-sensitive gene pS2 was not affected by either concentration of PL. These results indicate that PL supplementation regulates breast cancer cell growth in vitro via a mechanism that appears to be steroid independent.

Down-regulation of insulin-like growth factor-I receptor and insulin receptor substrate-1 expression in advanced human breast cancer.

The ligands, receptors and related signaling proteins of the insulin-like growth factor family are involved in the regulation of breast-cancer cell growth. We investigated the expression pattern of insulin-like growth factor-I receptor (IGF-IR), insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), a core downstream signaling protein, in 69 primary breast-cancer specimens of different grades and in 21 control tissues by immunohistochemistry. In addition, cell proliferation (percentage of Ki67(+) nuclei) and estrogen receptor (ER) expression were determined. IGF-IR, IRS-1 and IR were expressed mainly in epithelial cells. IRS-1 and IGF-IR were expressed at high levels in control tissues and in well and moderately differentiated carcinomas but at low levels in poorly differentiated breast cancers. IR expression did not show a significant correlation with the differentiation grade of the tissues investigated. Statistical analysis (ROC analysis for tumor grade) demonstrated that down-regulation of IGF-IR and IRS-1 correlated better with tumor progression than reduction of ER expression or increase in cell proliferation, IGF-IR showing the best correlation, followed by IRS-1 and, less significant, ER and Ki67. Our findings clearly show that progression of breast cancer is accompanied by a reduction of IGF-IR/IRS-1 expression and that IGF-IR/IRS-1 expression inversely correlates with high proliferation rate in dedifferentiated breast cancers. The strong correlation of IGF-IR and IRS-1 down-regulation with tumor progression suggests the use of IGF-IR and IRS-1 as a novel set of marker proteins for tumor grading.