Abhijit Mazumdar

Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor.

Activation of the heregulin/HER2 pathway in oestrogen receptor (ER)-positive breast-cancer cells leads to suppression of oestrogen-receptor element (ERE)-driven transcription and disruption of oestradiol responsiveness, and thus contributes to progression of tumours to more invasive phenotypes. Here we report the identification of metastatic-associated protein 1 (MTA1), a component of histone deacetylase (HDAC) and nucleosome-remodelling complexes, as a gene product induced by heregulin-β1 (HRG). Stimulation of cells with HRG is accompanied by suppression of histone acetylation and enhancement of deacetylase activity. MTA1 is also a potent corepressor of ERE transcription, as it blocks the ability of oestradiol to stimulate ER-mediated transcription. The histone-deacetylase inhibitor trichostatin A blocks MTA1-mediated repression of ERE transcription. Furthermore, MTA1 directly interacts with histone deacetylase-1 and -2 and with the activation domain of ER-α. Overexpression of MTA1 in breast-cancer cells is accompanied by enhancement of the ability of cells to invade and to grow in an anchorage-independent manner. HRG also promotes interaction of MTA1 with endogenous ER and association of MTA1 or HDAC with ERE-responsive target-gene promoters in vivo. These results identify ER-mediated transcription as a nuclear target of MTA1 and indicate that HDAC complexes associated with the MTA1 corepressor may mediate ER transcriptional repression by HRG.

A naturally occurring MTA1 variant sequesters oestrogen receptor-α in the cytoplasm

Oestrogen receptor (ER) is a good prognostic marker for the treatment of breast cancers. Upregulation of metastatic tumour antigen 1 (MTA1) is associated with the invasiveness and metastatic potential of several human cancers and acts as a co-repressor of nuclear ER-α. Here we identify a naturally occurring short form of MTA1 (MTA1s) that contains a previously unknown sequence of 33 amino acids with an ER-binding motif, Leu-Arg-Ile-Leu-Leu (LRILL). MTA1s localizes in the cytoplasm, sequesters ER in the cytoplasm, and enhances non-genomic responses of ER. Deleting the LRILL motif in MTA1s abolishes its co-repressor function and its interaction with ER, and restores nuclear localization of ER. Dysregulation of human epidermal growth factor receptor-2 in breast cancer cells enhances the expression of MTA1s and the cytoplasmic sequestration of ER. Expression of MTA1s in breast cancer cells prevents ligand-induced nuclear translocation of ER and stimulates malignant phenotypes. MTA1s expression is increased in human breast tumours with no or low nuclear ER. The regulation of the cellular localization of ER by MTA1s represents a mechanism for redirecting nuclear receptor signalling by nuclear exclusion.

P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-α and promotes hyperplasia in mammary epithelium

Stimulation of p21‐activated kinase‐1 (Pak1) induces cytoskeleton reorganization and signaling pathways in mammary cancer cells. Here, we show that inhibition of Pak1 kinase activity by a dominant‐negative fragment or by short interference RNA markedly reduced the estrogen receptor‐α (ER) transactivation functions. To understand the role of Pak1 in mammary glands, we developed a murine model expressing constitutively active Thr423 glutamic acid Pak1 driven by the β‐lactoglobulin promoter. We show that mammary glands from these mice developed widespread hyperplasia associated with apocrine metaplasia and lobuloalveolar hyperdevelopment during lactation. Mammary tissues with active Pak1 also exhibited an increased activation of mitogen‐activated protein kinase and stimulated transactivation functions of the ER and expression of endogenous ER target genes. Furthermore, Pak1 directly phosphorylated the activation function‐2 domain of the ER at the N‐terminal residue Ser305, and its mutation to Ala (S305A) abolished the Pak1‐mediated phosphorylation and transactivation functions of the ER, while its mutation to glutamic acid (S305E) promoted transactivation activity of ER. These findings reveal a novel role for the Pak1–ER pathway in promoting hyperplasia in mammary epithelium.

Growth of Triple-Negative Breast Cancer Cells Relies upon Coordinate Autocrine Expression of the Proinflammatory Cytokines IL-6 and IL-8

Triple-negative breast cancers (TNBC) are aggressive with no effective targeted therapies. A combined database analysis identified 32 inflammation-related genes differentially expressed in TNBCs and 10 proved critical for anchorage-independent growth. In TNBC cells, an LPA-LPAR2-EZH2 NF-κB signaling cascade was essential for expression of interleukin (IL)-6, IL-8, and CXCL1. Concurrent inhibition of IL-6 and IL-8 expression dramatically inhibited colony formation and cell survival in vitro and stanched tumor engraftment and growth in vivo. A Cox multivariable analysis of patient specimens revealed that IL-6 and IL-8 expression predicted patient survival times. Together these findings offer a rationale for dual inhibition of IL-6/IL-8 signaling as a therapeutic strategy to improve outcomes for patients with TNBCs.

LIM kinase 1 increases tumor metastasis of human breast cancer cells via regulation of the urokinase-type plasminogen activator system

Mammalian LIM kinase 1 (LIMK1) phosphorylates and inactivates the actin‐binding and ‐depolymerizing factor cofilin and induces actin cytoskeletal changes. LIMK1 is reported to play an important role in cell motility, but the mechanism of induction of cell motility and the role of LIMK1 in tumor growth, angiogenesis and invasion are poorly understood. Here we show that expression of LIMK1 in MDA‐MB‐435 human breast cancer cells enhanced cell proliferation and cell invasiveness and promoted in vitro angiogenesis. Since tumor metastasis requires degradation of the extracellular matrix by the serine protease urokinase type plasminogen activator (uPA), we examined the role of LIMK1 in the regulation of uPA/uPAR system. LIMK1 overexpression in breast cancer cells upregulated the uPA system, increased uPA promoter activity, induced uPA and uPAR mRNA and protein expression and induced uPA secretion. In contrast, cells transfected with the catalytically inactive LIMK mutant D460N‐LIMK1 did not exhibit these phenotypic changes. Blocking antibodies against uPA and uPAR suppressed LIMK1‐induced cell invasiveness. In addition, LIMK1 overexpression increased tumor growth in female athymic nude mice, promoted tumor angiogenesis and induced metastasis to livers and lungs, possibly by increasing uPA expression in the tumors. Finally, LIMK1 and uPAR were coordinately overexpressed in human breast tumors. These results suggested an important role for LIMK1 signaling in breast cancer tumor growth, angiogenesis and invasion and a regulatory connection between LIMK1 and the uPA system.

Estrogen regulation of Pak1 and FKHR pathways in breast cancer cells

Stimulation of p21‐activated kinase‐1 (Pak1) and estradiol‐estrogen receptor‐α in mammary cancer cells promotes cell survival. We sought to determine whether estrogen stimulates the Pak1 pathway. We found that estrogen rapidly activated Pak1 kinase activity in a phosphatidylinositol 3‐kinase‐insensitive manner. Furthermore, estrogen induced phosphorylation and perinuclear localization of the cell survival forkhead transcription factor FKHR in the cytoplasm in a Pak1‐dependent manner. In addition, Pak1 directly interacted with FKHR and phosphorylated it. The noticed phosphorylation‐dependent exclusion of FKHR from the nucleus impaired the ability of FKHR to activate its target Fas ligand promoter containing the FKHR binding motif (FRE) in cells treated with estrogen or expressing catalytically active Pak1. In contrast, expression of the dominant‐negative auto‐inhibitory domain of Pak1 (Pak amino acids 83–149) promoted the ability of FKHR to activate transcription from FRE. Together, these results identify a novel signaling pathway linking estrogen action to Pak1 signaling, and Pak1 to FKHR, suggesting that Pak1 is an important mediator of estrogens cell survival functions.

Apoptotic effect of eugenol in human colon cancer cell lines

Eugenol, a natural compound available in honey and various plants extracts including cloves and Magnoliae flos, is exploited for various medicinal applications. Since most of the drugs used in the cancer are apoptotic inducers, the apoptotic effect and anticancer mechanism of eugenol were investigated against colon cancer cells. Antiproliferative effect was estimated using MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay]. Earlier events like MMP (mitochondrial membrane potential), thiol depletion and lipid layer break were measured by using flow cytometry. Apoptosis was evaluated using PI (propidium iodide) staining, TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling) assay and DNA fragmentation assay. MTT assay signified the antiproliferative nature of eugenol against the tested colon cancer cells. PI staining indicated increasing accumulation of cells at sub‐G1‐phase. Eugenol treatment resulted in reduction of intracellular non‐protein thiols and increase in the earlier lipid layer break. Further events like dissipation of MMP and generation of ROS (reactive oxygen species) were accompanied in the eugenol‐induced apoptosis. Augmented ROS generation resulted in the DNA fragmentation of treated cells as shown by DNA fragmentation and TUNEL assay. Further activation of PARP (polyadenosine diphosphate‐ribose polymerase), p53 and caspase‐3 were observed in Western blot analyses. Our results demonstrated molecular mechanism of eugenol‐induced apoptosis in human colon cancer cells. This research will further enhance eugenol as a potential chemopreventive agent against colon cancer.

Estrogen Induces c-myc Gene Expression via an Upstream Enhancer Activated by the Estrogen Receptor and the AP-1 Transcription Factor

c-myc oncogene is implicated in tumorigenesis of many cancers, including breast cancer. Although c-myc is a well-known estrogen-induced gene, its promoter has no estrogen-response element, and the underlying mechanism by which estrogen induces its expression remains obscure. Recent genome-wide studies by us and others suggested that distant elements may mediate estrogen induction of gene expression. In this study, we investigated the molecular mechanism by which estrogen induces c-myc expression with a focus on these distal elements. Estrogen rapidly induced c-myc expression in estrogen receptor (ER)-positive breast cancer cells. Although estrogen had little effect on c-myc proximal promoter activity, it did stimulate the activity of a luciferase reporter containing a distal 67-kb enhancer. Estrogen induction of this luciferase reporter was dependent upon both a half-estrogen response element and an activator protein 1 (AP-1) site within this enhancer, which are conserved across 11 different mammalian species. Small interfering RNA experiments and chromatin immunoprecipitation assays demonstrated the necessity of ER and AP-1 cross talk for estrogen to induce c-myc expression. TAM67, the AP-1 dominant negative, partially inhibited estrogen induction of c-myc expression and suppressed estrogen-induced cell cycle progression. Together, these results demonstrate a novel pathway of estrogen regulation of gene expression by cooperation between ER and AP-1 at the distal enhancer element and that AP-1 is involved in estrogen induction of the c-myc oncogene. These results solve the long-standing question in the field of endocrinology of how estrogen induces c-myc expression.

ZD6474, a dual tyrosine kinase inhibitor of EGFR and VEGFR-2, inhibits MAPK/ERK and AKT/PI3-K and induces apoptosis in breast cancer cells.

Abnormalities in gene expression and signaling pathways downstream of the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) contribute to the progression, invasion, and maintenance of the malignant phenotype in human cancers, including breast. Consequently, the dual kinase inhibitor of EGFR and VEGFR ZD6474 represents a promising biologically-based treatment that is currently undergoing clinical trials for non-small cell lung cancer. Patients suffering from breast cancers have a poor prognosis because of the lack of effective agents and treatment strategies. We hypothesized that inhibition of phosphorylation of the EGFR and VEGFR by ZD6474 would inhibit breast cancer cell proliferation and induce apoptosis. This hypothesis was tested using human breast cancer cell lines. ZD6474 inhibited cell proliferation in a dose-dependent manner, by blocking cell progression at the G0-G1 stage, through down-regulation of expression of cyclin D1 and cyclin E. In vitro, ZD6474 inhibited growth factor-induced phosphorylation of EGFR, VEGFR-2, MAPK, and Akt. ZD6474 also down regulated anti-apoptotic markers including Bcl-2, up-regulated pro-apoptotic signaling events involving expression of bax, activation of caspase-3, and induction of poly (ADP-ribose) polymerase during apoptosis. ZD6474 inhibited anchorage independent colony formation using soft agar assays, and invasion of breast cancer cells in vitro using Boyden chamber assays. In a xenograft model using human MDA-MB-231 breast cancer cells, ZD6474 inhibited tumor growth and induced cancer-specific apoptosis. Collectively, these data imply that ZD6474 a dual kinase inhibitor has potential for the targeted therapy of breast cancer.

Celecoxib alleviates tamoxifen-instigated angiogenic effects by ROS-dependent VEGF/VEGFR2 autocrine signaling

BackgroundTamoxifen (TAM) is widely used in the chemotherapy of breast cancer and as a preventive agent against recurrence after surgery. However, extended TAM administration for breast cancer induces increased VEGF levels in patients, promoting new blood vessel formation and thereby limiting its efficacy. Celecoxib (CXB), a selective COX-2 inhibitor, suppresses VEGF gene expression by targeting the VEGF promoter responsible for its inhibitory effect. For this study, we had selected CXB as non-steroidal anti-inflammatory drug in combination with TAM for suppressing VEGF expression and simultaneously reducing doses of both the drugs.MethodsThe effects of CXB combined with TAM were examined in two human breast cancer cell lines in culture, MCF7 and MDA-MB-231. Assays of proliferation, apoptosis, angiogenesis, metastasis, cell cycle distribution, and receptor signaling were performed.ResultsHere, we elucidated how the combination of TAM and CXB at nontoxic doses exerts anti-angiogenic effects by specifically targeting VEGF/VEGFR2 autocrine signaling through ROS generation. At the molecular level, TAM-CXB suppresses VHL-mediated HIF-1α activation, responsible for expression of COX-2, MMP-2 and VEGF. Besides low VEGF levels, TAM-CXB also suppresses VEGFR2 expression, confirmed through quantifying secreted VEGF levels, luciferase and RT-PCR studies. Interestingly, we observed that TAM-CXB was effective in blocking VEGFR2 promoter induced expression and further 2 fold decrease in VEGF levels was observed in combination than TAM alone in both cell lines. Secondly, TAM-CXB regulated VEGFR2 inhibits Src expression, responsible for tumor progression and metastasis. FACS and in vivo enzymatic studies showed significant increase in the reactive oxygen species upon TAM-CXB treatment.ConclusionsTaken together, our experimental results indicate that this additive combination shows promising outcome in anti-metastatic and apoptotic studies. In a line, our preclinical studies evidenced that this additive combination of TAM and CXB is a potential drug candidate for treatment of breast tumors expressing high levels of VEGF and VEGFR2. This ingenious combination might be a better tailored clinical regimen than TAM alone for breast cancer treatment.